WO2010115424A1 - Methanogenic microorganisms for generating biogas - Google Patents

Abstract

The invention relates to a method for generating biogas from biomass in a fermentation reactor, wherein a microorganism of the type Methanoculleus bourgensis is added to the biomass.

Description

 Methanogenic microorganisms for the production of biogas

Technical area

The invention relates to processes for the production of biogas from biomass using methanogenic microorganisms and methanogenic microorganisms per se.

State of the art

Biogas plants produce methane through a microbial decomposition process of organic substances. The biogas is produced in a multi-stage process of fermentation or digestion by the activity of anaerobic microorganisms, i. in the absence of air.

The organic material used as fermentation substrate has a high molecular structure from a chemical point of view, which is degraded in the individual process steps of a biogas plant by metabolic activity of microorganisms to low molecular weight building blocks. However, the populations of microorganisms which are active in the fermentation of the organic fermentation substrate have hitherto been insufficiently characterized.

According to the current state of knowledge, it is possible to distinguish between four consecutive, but also parallel and interlocking biochemical single processes which enable the degradation of organic fermentation substrates to the end products methane and carbon dioxide: hydrolysis, acidogenesis, acetogenesis and methanogenesis.

In hydrolysis, high molecular weight, often particulate, organic compounds are converted to soluble fission products by exoenzymes (e.g., cellulases, amylases, proteases, lipases) of fermentative bacteria. For example, fats in fatty acids, carbohydrates, e.g. Polysaccharides, decomposed into oligo- and monosaccharides and proteins in oligopeptides or amino acids. The gaseous products formed besides consist predominantly of carbon dioxide.

Optional and obligate anaerobic bacteria, often identical to the hydrolyzing bacteria, metabolize in acidification the hydrolysis products (eg mono-, disaccharides, di-, oligopeptides, amino acids, glycerol, long-chain fatty acids) intracellularly to short chain fatty or carboxylic acids such as butter , Propion and Acetic acid, short-chain alcohols such as ethanol and the gaseous products hydrogen and carbon dioxide.

In the subsequent acetogenesis, the short-chain fatty acids and carboxylic acids formed in acidogenesis and the short-chain alcohols are taken up by acetogenic bacteria and excreted again as acetic acid after β-oxidation. By-products of acetogenesis are CO ₂ and molecular hydrogen (H ₂ ).

The products of acetogenesis such as acetic acid but also other substrates such as methanol and formate are converted by methane-forming organisms in the obligate anaerobic methanogenesis to methane and CO ₂ . The resulting here CO ₂ and also during the other process steps such as hydrolysis, CO ₂ formed in turn can also be converted by microorganisms with the incurred H ₂ to methane.

Increasing the yield of end products from a given amount of starting materials, as in any chemical reaction, is an urgent goal of process control even in the case of biogas production. For biogas production from biomass, this means that from a given amount of organic fermentation substrate a large amount of methane should be formed. The average amount of methane produced (methane productivity in standard cubic meters per m ³ working volume and day) of biogas plants is between 0.25 and 1, 1 Nm ³ CH ₄ / m ³ d, with most plants having a methane productivity in the range from 0.50 to 0, 75 Nm ³ CH ₄ / m ³ d (from "Results of the Biogas Measurement Program", 2005, Hrsgb, Agency for Renewable Resources, Gülzow) Substrate-specific methane yields (expressed in standard cubic meters per ton fed substrate) have a very large value range between about 16 and 206 Nm ³ CH ₄ A, because the substrates used to operate biogas plants are very different (eg manure or animal excreta, waste from food production or green waste, renewable raw materials such as silage maize or sugar beet pulp) and consequently very different energy contents (from "Results of the Biogas Measurement Program", 2005, Hrsgb. Agency for Renewable Resources, Gülz ow). In order to assess the profitability of a biogas plant, one can, among other things, compare the amount of biogas actually produced with a biogas quantity theoretically expected depending on the substrate. The Board of Trustees for Engineering and Construction in Agriculture e. V. (KTBL) but also the Federal Agricultural Research Center and the Agency for Renewable Resources have issued guidelines that roughly indicate which amounts of biogas are to be expected depending on the substrate used with a stable fermentation. These theoretical guidelines can thus reflect the amount of theoretically generated biogas. Alternatively, benchmarks issued by other institutes in other countries may be used. For maize silage, whose shares of oTS generally vary between 22% and 40%, expected gas yields in the range of 533 NI / kgoTS and 650 NI / kgoTS were mentioned (in "Biogas Production and Utilization Guide", 2006, Hrsgb. Agency for Renewable Raw Materials eV, Gülzow and KTBL Homepage, www.ktbl.de) In addition, plant-specific parameters such as room load or hydraulic residence time play a role in the substrate-specific methane yields, in particular the space load plays an important role, since an increase in the space load With the same system a much larger amount of biogas can be generated.

As a rule, the highest possible volume loading of the fermenter should be achieved. The average room loads (expressed in kg of dry matter per cubic meter per day), under which biogas plants are operated, are 1 to 3 kg oTR / m ³ d, whereby one-stage systems usually have higher room loads than multi-stage systems and a room load of 5 , 7 kg oTR / m ³ d was the highest value that was measured (from "Results of the Biogas Measurement Program", 2005, Hrsgb. Agency for Renewable Resources, Gülzow).

The volume loading of a fermenter is the amount of substrate fed to the fermenter, expressed in kilograms of dry organic matter per cubic meter of fermenter volume and per day. The amount of biogas produced depends strongly on the volume load of the fermenter, with increasing space load an increasingly larger amount of biogas is generated. A high space load makes the process of biogas production increasingly economically viable, but on the other hand leads to an increasing destabilization of the biological processes of fermentation.

There continues to be a need for methods for producing biogas which enable a higher yield of methane over the prior art.

definitions

In the context of the present invention, the term "biogas" is understood to mean the gaseous product of the anaerobic biodegradation of organic substrates, which generally contains about 45-70% of methane, 30-55% of carbon dioxide, and small amounts of nitrogen, hydrogen sulphide and other gases. - A -

The terms "fermentation" or "fermentation" in the context of the present invention include both anaerobic and aerobic metabolic processes which, under the action of microorganisms in a technical process from the supplied substrate to produce a product, e.g. Lead biogas. A differentiation from the term "fermentation" is given insofar as it is exclusively an annaerobic process.

In the context of the present invention, a "fermenter" is understood to mean the container in which the microbiological degradation of the substrate takes place with simultaneous formation of biogas.The terms "reactor", "fermenter" and "digester" are used interchangeably.

The term "fermentation substrate" or "substrate" in the context of the present invention means organic and biodegradable material which is added to the fermenter for fermentation. Substrates can be renewable raw materials, farm fertilizers, substrates from the processing agricultural industry, organic municipal residues, slaughter residues or green waste. Examples of the above-mentioned substrates are maize silage, rye silage, sugar beet pulp, molasses, grass silage, cattle or pig manure, cattle, pig, chicken or horse dung, spent grains, apple, fruit or vine pomace, cereal, potato or fruit vat. Organic waste from bio-waste, leftovers, market wastes, grease from fat separators, rumen contents, pig stomach content.

The term "fermentation residue" or "digestate" is understood to be the residue of the biogas production leaving the fermenter and often stored in its own container.

In the context of the present invention, "volume loading" is the amount of organic dry matter (oTS) supplied to the fermenter per day and cubic meter (m ³ ) working volume in kg.

In the context of the present invention, "organic dry substance" (o-TS) is understood as meaning the anhydrous organic fraction of a substance mixture after removal of the inorganic constituents and drying at 105 ^° C. As a rule, the dry matter content is stated in% of the substrate. In the context of the present invention, the term "residence time" is understood to mean the average residence time of the substrate in the fermenter.

In the context of the present invention, the term "specific biogas yield" or "specific methane yield" is used to denote the amount of biogas or methane produced (stated in standard cubic meters of Nm ³ gas) divided by the amount (as a rule per tonne) of organic dry substance used or substrate understood.

The term "nucleotide sequence" encompasses both the DNA sequence and the corresponding RNA sequence, For example, RNA sequences given in the invention using bases A, U, C, G also refer to the corresponding DNA sequences using bases A, T, C, G and vice versa.

The determination of the nucleotide sequence of a microorganism by means of a standardized process by which the individual nucleotides of the DNA or RNA of the microorganism can be detected with high accuracy. In spite of the high accuracy of the sequencing methods, individual positions can repeatedly be present in a sequence for which the determination of the nucleotide present at the respective position was not possible with sufficient accuracy. In such cases, the letter "N" is indicated in the nucleotide sequence in the context of the present invention. If the letter "N" is subsequently used in a nucleotide sequence, this stands as an abbreviation for any conceivable nucleotide, that is to say A, U, G or C. in the case of a ribonucleic acid or for A, T, G or C in the case of a deoxyribonucleic acid.

The term "nucleotide mutation" as used herein means a change in the starting nucleotide sequence, whereby individual nucleotides or a plurality of nucleotide sequences which are directly consecutive or interrupted by unaltered nucleotides may be deleted, added (insertion or addition) or replaced by others (substitution) The term also includes any combination of individual variations called.

The term "deletion" as used herein means the removal of 1, 2 or more nucleotides from the respective starting sequence.

The term "insertion" or "addition" as used herein means the addition of 1, 2 or more nucleotides to the respective starting sequence. The term "substitution" as used herein means the replacement of a nucleotide present at a particular position by another.

The term "microorganism" is understood to mean microscopically small organisms, which as a rule are single-celled organisms but may also be multicellular organisms Examples of microorganisms are bacteria, microscopic algae, fungi or protozoa.

For the purposes of the present invention, the terms "genus" of microorganisms, "type" of microorganisms and "strain" of microorganisms are understood to mean the corresponding basic category of biological taxonomy, in particular the phylogenetic classification of genera, species and strains of

Microorganisms are u.a. identified and distinguished by their RNA sequences.

Among a certain genus, a certain species or a particular strain fall not only microorganisms with a very specific RNA sequence, but also to a certain extent their genetic variants, the genetic variance in the series strain, species, genus increases ,

The definition of a "species" of a bacterium is undergoing scientific change and is neither complete nor is the various concepts uncontroversial In the present invention, "species" is understood to be the species concept that relates to genomic and phylogenetic analyzes, and in the review of Staley (Philos. Trans. R. Soc.

Lond. Biol. Sci., 2006, 361, 1899-1909). It is widely recognized that two bacterial species having more than 70% DNA-DNA hydride or more than 94% average nucleotide identity (ANI) belong to the same species, respectively two species having less than 97% identity 16S rRNA, are assigned to two different types.

In this context, the term "culture" refers to an accumulation of microorganisms under established conditions which ensure the growth or at least the survival of the microorganisms, for example enrichment cultures or pure cultures, liquid cultures as well as cultures on solid media such as nutrient media also permanent crops such as frozen glycerin cultures, immobilized cultures such as gel cultures or highly concentrated cultures such as cell pellets.

The term "pure culture" of a microorganism is understood to mean the progeny of a single cell, which is produced by a multi-stage process from a mixture isolated from various microorganisms. The multi-step process involves the separation of a single cell from a cell population and requires that the colony resulting from the cell through growth and cell division also remain separate from other single cells or colonies. By careful separation of a colony, resuspension in liquid and repeated spreading, pure cultures of microorganisms can be selectively obtained. The isolation of a pure culture can also be carried out in liquid nutrient media, provided that the desired organism outnumbered in the starting material. By serial dilution of the suspension in the nutrient solution, it can finally be achieved that only one cell remains in the last dilution stage. This cell then provides the basis for a pure culture. The explanation of the term "pure culture" and methods for producing the same are given in "General Microbiology" by Hans G. Schlegel, 7th revised edition, 1992, Georg Thieme Verlag, p , which is hereby incorporated by reference.

The term "mixed culture" is understood to mean a mixture of different microorganisms, but natural populations of microorganisms are usually mixed cultures, but mixed cultures can also be produced artificially, for example by combining several pure cultures.

Presentation of the invention

The object of the invention is to provide a process for the production of biogas, which allows an increased compared to the prior art yield of methane.

This object is achieved by the method for producing biogas according to claim 1. Further advantageous details, aspects and embodiments of the present invention will become apparent from the dependent claims, the description and the examples.

The question of whether the production of biogas in a given fermenter under certain conditions is satisfactory, can not be judged solely on the basis of the absolute amount of biogas produced. The amount of biogas produced depends strongly on the amount of substrate supplied, especially on the amount of organic dry matter that is introduced into the fermenter. This is expressed in the measuring parameters "room load" and "specific gas yield", which thus constitute a particular importance for the efficiency of a plant. If instabilities of the fermentation process occur, such as a decrease in the pH, a strong increase in the formation of volatile fatty acids or the accumulation of inhibitors such as ammonia or hydrogen sulfide, the specific gas yield decreases.

The composition of the microbial populations in the various fermentation substrates as well as the development of the organism composition during the fermentation process is largely unknown, but very variable and subject to a complicated dynamic process, which is also influenced by the respective process conditions. To determine the microbial composition and the total number of microorganisms in a fermentation substrate as well as the determination of the proportions of various types of microorganisms in the fermentation substrate, various methods are known to those skilled in the art, for example, in the review article by Amann et al. (Microbiol. Review., 59, 143-169, 1995). For example, a preferred method of determining the microorganism composition independent of prior culture of the microorganisms is to construct a rDNA clone library (e.g., based on 16S rRNA) after nucleic acid extraction and PCR, which can then be sequenced. Using this clone library, the composition of the microbial population in the fermentation substrate can be determined, for example, by in situ hybridization with specific fluorescence-labeled oligonucleotide probes. Suitable rRNA-based oligonucleotide probes are known from the review mentioned above or may be prepared, for example, by probe base (Loy et al., 2003, Nucleic Acids Res. 31, 514-516, Loy et al., 2007 Nucleic Acids Res. 35: D800. D804) or the ARB program package (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). A quantitative determination of the proportion of individual microorganisms in the total population can be carried out in a suitable manner with the methods of quantitative dot blot, in situ hybridization or whole cell hybridization.

All of the microorganisms described below, added from a biomass in a process for producing biogas, are preferably added to the fermenter in the form of an enriched culture. The addition of the bacterial culture can be carried out in the form of a liquid culture suspension, preferably in an enrichment or selection medium or in the form of dry, freeze-dried or moist cell pellets. For methanogenic microorganisms, the cell concentrations achieved in enriched cultures are in a concentration range of about 10 ⁶ cells per ml of culture to about 10 ¹³ cells per ml of culture. A preferred embodiment is also an immobilized culture of microorganisms. For all microorganisms described below, natural or synthetic polymers can be used as carrier materials on which the particular microorganism is immobilized. Gel-forming polymers are preferably used. These have the advantage that bacteria can be taken up or stored within the gel structure. Preferably, those materials are used which dissolve slowly in water or are degraded, so that the release of the respective microorganism takes place over a longer period of time.

Examples of suitable polymers are polyaniline, polypyrrole, polyvinylpyrrolidone, polystyrene, polyvinyl chloride, polyvinyl alcohol, polyethylene, polypropylene, epoxy resins, polyethyleneimines, polysaccharides such as agarose, alginate or cellulose, ethylcellulose, methylcellulose, carboxymethylethylcellulose, cellulose acetates, alkali cellulose sulfate, copolymers of polystyrene and maleic anhydride , Copolymers of styrene and methyl methacrylate, polystyrenesulfonate, polyacrylates and polymethacrylates, polycarbonates, polyesters, silicones, cellulose phthalate, proteins such as gelatin, gum arabic, albumin or fibrinogen, mixtures of gelatin and water glass, gelatin and polyphosphate, gelatin and copolymers of maleic anhydride and methyl vinyl ether, Cellulose acetate butyrate, chitosan, polydialkyldimethylammonium chloride, mixtures of polyacrylic acids and polydiallyldimethylammonium chloride and mixtures thereof. The polymer material can also be crosslinked using conventional crosslinkers such as glutaraldehyde, urea / formaldehyde resins or tannin compounds.

Alginates as Immobilisate prove to be particularly advantageous because they do not have a negative influence on the activity of the microorganisms and because they are slowly degraded by other microorganisms. Due to the slow degradation of the alginate immobilizates, the trapped microorganisms are gradually released.

For immobilization, the microorganisms are mixed with a polymer gel and then cured in a suitable hardener solution. For this they are first mixed with a gel solution and then dropped into a hardener solution of suitable height. The exact procedures for immobilization are known to the person skilled in the art.

It has been found that the microorganisms described in more detail below have properties, as a result of which the addition of the microorganisms during a fermentation to produce biogas has a positive effect on the amount of biogas produced, especially biomethane, which significantly increases the profitability of the respective plants.

In principle, the addition of microorganisms suitable for this purpose can take place at any desired point in time of the fermentation process; in particular, the microorganisms can be used to inoculate fermentation substrate during initial startup or restart of a fermenter. All of the microorganisms described in more detail below can be used to inoculate fermentation substrate. The microorganisms according to the invention may be added in the form of a culture once or several times at regular or irregular intervals, but preferably weekly or monthly, more preferably daily or twice weekly, in a suitable concentration and amount. Suitable concentrations of microorganisms and amounts added are explained in the specific embodiments.

In a preferred embodiment, the microorganisms are added even in case of disturbances in the fermentation process to stabilize the fermentation. Such disorders can be detected early by monitoring certain characteristic parameters of the fermentation. Characteristic parameters provide information about the quality of an ongoing fermentation process for the production of biogas. Such characteristic parameters are not only the amount of biogas produced and the methane content of the biogas produced but also, for example, the hydrogen content of the biogas produced, the pH of the fermentation substrate, the redox potential of the fermentation substrate, the carboxylic acid content of the fermentation substrate, the proportions of various carboxylic acids in the fermentation substrate, the hydrogen content of the fermentation substrate, the proportion of dry matter in the fermentation substrate, the proportion of the organic dry matter in the fermentation substrate, the viscosity of the fermentation substrate and the volume loading of the fermentation reactor. All the microorganisms described in more detail below can be added in case of disturbances of the fermentation process to stabilize the fermentation.

According to a further preferred embodiment, additional biomass is added to the fermentation reactor in a timely manner to the addition of the microorganisms described below. Timely addition of additional biomass may occur within a period of 1 second to 3 days after addition of microorganisms, or it may occur simultaneously with the addition of microorganisms. The space load in the fermentation reactor by continuous The addition of new substrate continuously increased or kept approximately constant, the fermentation at all room loads, preferably at a space load of ≥ 0.5 kg of organic dry matter per m ³ and day [kg oTS / m ³ d], more preferably at a space load of ≥4.0 kg oTS / m ³ d and particularly preferably at a volume load of ≥8.0 kg oTS / m ³ d, which is more than double the increase in average room load compared to the current state of the art. According to a further preferred embodiment, therefore, the space load in the fermentation reactor is continuously increased by the continuous addition of biomass.

According to another embodiment, fermentation substrate and microorganisms are added continuously. The continuous operation of a fermentation reactor should result in a stable microbial biocenosis to a continuous production of biogas, the exposure of the substrate addition to the fermentation should be reduced as a result of a process disturbance. In this embodiment of the present invention, all the microorganisms described in more detail below can be used.

The fermentation process and the processes associated therewith are also conceivable in a discontinuous operation, for example batch fermentation, For example, according to a further embodiment, a fermentation microorganism may be added to the fermentation substrate at regular intervals At regular intervals leads to an increase in the Lebendzellzahl and thus to an improved course of methane formation.Also in this embodiment of the present invention, all microorganisms described in more detail below can be used.

According to a further embodiment, the production of biogas from biomass takes place with constant mixing of the fermentation substrate. Due to the constant mixing of the fermentation substrate, the added cultures of microorganisms can be better distributed in the fermentation substrate. In addition, the biogas formed can be better removed from the fermentation process. The stated advantages arise in connection with all the microorganisms described in more detail below.

The constant mixing of the fermentation substrate also leads to a uniform heat distribution in the fermentation reactor. Measurements of the temperature in the fermentation reactor, carried out at periodic intervals, but also continuously were showed that the fermentation substrate is fermented efficiently in a temperature range from 2O ⁰ C and 8O ⁰ C, preferably at about 35 ⁰ C to 60 ⁰ C, more preferably at 4O ⁰ C to 50 ⁰ C. These temperature ranges are therefore preferred in the context of the present invention in connection with all the microorganisms described in more detail below. In addition to the hydrolysis, in particular the last stage of the fermentation process, namely the formation of methane by methanogenic microorganisms, particularly efficient at elevated temperatures. For multi-stage biogas plants, it is also useful to operate the different reactors at different temperatures, eg the first stage under mesophilic conditions and the downstream stages, especially methanogenesis, under thermophilic conditions. Suitable conditions for this are known from the prior art (eg DE 10 2005 012367 A1).

All embodiments of the present invention are not limited to one-step processes for the production of biogas. The use of all microorganisms described in more detail below can also be carried out in two or more stages.

It should again be pointed out that all of the microorganisms described in detail below can be used individually or in any combination in all the embodiments of the present invention explained above. The above statements therefore relate to all subsequent microorganisms.

Methanoculleus bourgensis

The present invention provides a method for producing biogas from biomass in a fermentation reactor. The biomass is added according to the invention a microorganism of the species Methanoculleus bourgensis.

Surprisingly, it has been found that the addition of microorganisms of the species Methanoculleus bourgensis to the fermentation substrate, the amount of biogas formed can be significantly increased. At constant space load, the addition of microorganisms of the species Methanoculleus bourgensis causes a significant increase in the amount of biogas produced. Alternatively, the amount of methane formed can also be increased by increasing the volume load, with the addition of microorganisms of the species Methanoculleus bourgensis preventing the fermentation process from becoming unstable under the altered conditions. The use of microorganisms of the species Methanoculleus bourgensis therefore leads to an improvement in the efficiency and efficiency of biogas plants.

The person skilled in the art knows which strains of microorganisms fall under the term "species Methanoculleus bourgensis." In particular, the names "Methanoculleus olentangy" and "Methanoculleus oldenburgensis" (Asakawa & Nagaoka, Int Syst. And Evol. Microbiol., 2003, 53, 1551-1552) as well as the acronyms "Methanogenium bourgense" and "Methanogenium olentangyr" (Bacterial Nomenclature Up-To-Date, Approved List of the DSMZ - German Collection for Microorganisms and Cell Cultures GmbH, as of March 2010). In connection with the production of biogas by fermentation of organic substrates, microorganisms of the species Methanoculleus bourgensis are not yet known.

According to a preferred embodiment of the present invention, a microorganism of the species Methanoculleus bourgensis is added in the form of a culture of microorganisms consisting predominantly of microorganisms of the species Methanoculleus bourgensis. In fermentation substrates of biogas plants microorganisms of the species could Methanoculleus bourgensis only in trace amounts of less than 10 ^'4% share of the total number of detected present microorganisms. Since the amount of microorganisms isolated from their natural occurrence is insufficient for the addition of the microorganisms, it is usually propagated in the form of a culture. In practice, it is found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out directly in the form of a culture of microorganisms.

The addition of the culture of Methanoculleus bourgensis can be carried out in the form of a culture suspension or in the form of dry, freeze-dried or moist cell pellets.

Since the already mentioned various positive effects on the fermentation process are associated with the microorganism species Methanoculleus bourgensis, this type of microorganism should be present in the added culture in a concentration exceeding the natural abundance. Of course, mixed cultures of any composition can be used for the addition. The only prerequisite is that the species Methanoculleus bourgensis is present in a concentration enriched in relation to the natural occurrence. The determination of the proportions of different types of microorganisms in mixed cultures is not a problem for the expert. Thus, for example, using fluorescent-labeled oligosensors specifically the proportion of microorganisms of the species Methanoculleus bourgensis can be identified in a mixture. As already mentioned, microorganisms of the species Methanoculleus bourgensis are preferably added to the fermentation substrate in the form of cultures of microorganisms, the cultures of microorganisms consisting predominantly of microorganisms of the species Methanoculleus bourgensis. If, in addition to the determination of the number of microorganisms of the species Methanoculleus bourgensis, the total number of microorganisms is also determined, the percentage of microorganisms of the species Methanoculleus bourgensis in the culture can be stated as a percentage. Microorganisms of the species Methanoculleus bourgensis are in a mixed culture then the predominantly present type of microorganisms, if they have the highest percentage of the various types of microorganisms present in the mixed culture.

According to preferred embodiments of the present invention make microorganisms of the species Methanoculleus bourgensis at least 10 ^'4%, especially at least ^{10' 2%,} more preferably at least 1% of the total number of existing in the added to the fermentation substrate culture microorganisms. According to further preferred embodiments, microorganisms of the species Methanoculleus bourgensis account for at least 10%, in particular at least 50%, particularly preferably at least 90%, of the total number of microorganisms present in the culture.

According to a very particularly preferred embodiment, a pure culture of a microorganism of the species Methanoculleus bourgensis is added. Due to the specific metabolic processes and activities, the addition of a pure culture can especially contribute to an improved methane production.

According to another preferred embodiment of the present invention, a microorganism of the species Methanoculleus bourgensis is added as a constituent of at least one immobilized culture of microorganisms. Since the amount of microorganisms isolated from their natural occurrence is insufficient for the addition of the microorganisms, it is usually propagated in the form of a culture. In practice, it has been found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out in the form of an immobilized culture of microorganisms. According to a preferred embodiment of the present invention, the microorganism of the species Methanoculleus bourgensis is added to the fermentation substrate in an amount such that, after addition, the proportion of the microorganism of the species Methanoculleus bourgensis is between 1 CT ⁸ % and 50% of the total number of microorganisms present in the fermentation substrate accounts. In particular, depending on the fermenter size and thus as a function of the amount of fermentation substrate can be necessary to achieve the desired effect, an addition of very different amounts of microorganisms.

A microorganism of the species Methanoculleus is particularly preferred bourgensis in an amount added to the fermentation substrate that makes up after addition of the content of the microorganism of the species Methanoculleus bourgensis between ^{10 -6%} and 25% of the total number present in the fermentation substrate microorganisms. The microorganism of the species Methanoculleus is particularly preferred bourgensis in an amount to the fermentation substrate is added that, after addition, the proportion of the microorganism of the species Methanoculleus bourgensis between 10 ^"4% and 10% of the total number present in the fermentation substrate microorganisms accounts. Very particular preference is the microorganism of the species Methanoculleus bourgensis admitted that makes up after addition of the content of the microorganism of the species Methanoculleus bourgensis at present in the fermentation substrate of microorganisms between ^{10 -3%} and 1% of the total in an amount to the fermentation substrate.

According to a particularly preferred embodiment of the present invention, the microorganism of the species Methanoculleus bourgensis is a microorganism of the strain Methanoculleus sp. dm 2. In all embodiments described above, which deal with the addition of a microorganism of the species Methanoculleus bourgensis, so preferably a microorganism of the strain Methanoculleus sp. dm2 added.

According to a further particularly preferred embodiment of the present invention, the microorganism of the species Methanoculleus bourgensis is a microorganism of the strain krchaeon clone GZK7. In all the embodiments explained in more detail above, which deal with the addition of a microorganism of the species Methanoculleus bourgensis, a microorganism of the strain krchaeon clone GZK7 is therefore preferably added.

According to a further particularly preferred embodiment of the present invention, the microorganism of the species Methanoculleus bourgensis is a Microorganism of the strain Archaeon clone 5.5ft C121A. In all of the above-described embodiments, which deal with the addition of a microorganism of the species Methanoculleus bourgensis, so preferably a microorganism of the strain Archaeon clone 5.5ft C121A is added.

According to a further particularly preferred embodiment of the present invention, the microorganism of the species Methanoculleus bourgensis is a microorganism of the strain Archaeon clone 5.5ft C124A. In all of the above-described embodiments, which deal with the addition of a microorganism of the species Methanoculleus bourgensis, so preferably a microorganism of the strain Archaeon clone 5.5ft C124A is added.

According to a further particularly preferred embodiment of the present invention, the microorganism of the species Methanoculleus bourgensis is a microorganism of the strain Archaeon clone 5.5ft C141A. In all of the above-described embodiments, which deal with the addition of a microorganism of the species Methanoculleus bourgensis, so preferably a microorganism of the strain Archaeon clone 5.5ft C141A is added.

According to a further particularly preferred embodiment of the present invention, the microorganism of the species Methanoculleus bourgensis is a microorganism of the strain Archaeon clone A52. In all of the embodiments explained in more detail above, which deal with the addition of a microorganism of the species Methanoculleus bourgensis, a microorganism of the strain Archaeon clone A52 is therefore preferably added.

According to a further particularly preferred embodiment of the present invention, the microorganism of the species Methanoculleus bourgensis is a microorganism of the strain Archaeon clone MCSArc_B6. In all of the embodiments explained in more detail above, which deal with the addition of a microorganism of the species Methanoculleus bourgensis, a microorganism of the strain Archaeon clone MCSArc_B6 is therefore preferably added.

According to a further particularly preferred embodiment of the present invention, the microorganism of the species Methanoculleus bourgensis is a

Microorganism of strain Euryarchaeote clone BSA1A-03. In all embodiments explained in more detail above, which deals with the addition of a microorganism of the type Methanoculleus bourgensis, so preferably a microorganism of the strain Euryarchaeote clone BSA 1 A-03 is added.

According to a further particularly preferred embodiment of the present invention, the microorganism of the species Methanoculleus bourgensis is a microorganism of the strain Euryarchaeote clone BSA2A-02. In all of the above-described embodiments, which deal with the addition of a microorganism of the species Methanoculleus bourgensis, so preferably a microorganism of the strain Euryarchaeote clone BSA2A-02 is added.

According to a further particularly preferred embodiment of the present invention, the microorganism of the species Methanoculleus bourgensis is a microorganism of the strain Euryarchaeote clone MAA04. In all embodiments described above, which deal with the addition of a microorganism of the species Methanoculleus bourgensis, so preferably a microorganism of the strain Euryarchaeote clone MAA04 is added.

The present invention also encompasses the use of a microorganism of the species Methanoculleus bourgensis for the fermentative production of biogas from biomass. Preference is given to the fermentative production of biogas from biomass, a microorganism of the strain Methanoculleus sp. dm2 used. Also preferably, a microorganism of the strain Archaeon clone GZK7 is used for the fermentative production of biogas from biomass. Also preferred for the fermentative production of biogas from biomass is a microorganism of the strain Archaeon clone 5.5ft C121A used. Also preferred for the fermentative production of biogas from biomass is a microorganism of the strain Archaeon clone 5.5ft C124A used. Also preferred for fermentative production of biogas from biomass, a microorganism of the strain Archaeon clone 5.5ft Ci '41 A is used. Also preferably, a microorganism of the strain Archaeon clone A52 is used for the fermentative production of biogas from biomass. Also preferably, a microorganism of the strain Archaeon clone MCSArc_B6 is used for the fermentative production of biogas from biomass. Also preferred for fermentative production of biogas from biomass, a microorganism of the strain Euryarchaeote clone BSA 1A-03 is used. Also preferred for the fermentative production of biogas from biomass, a microorganism of the strain Euryarchaeote clone BSA2A-02 is used. Also preferably, a microorganism of the strain Euryarchaeote clone MAA04 is used for the fermentative production of biogas from biomass. The microorganisms Methanoculleus bourgensis SBG7 obtained from the fermentation substrate of a post-fermenter were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID NO: 4 comprises 974 nucleotides. Methanoculleus sp.dm2 was identified as the next relative. A comparison of the sequences revealed that there are a total of 26 exchanges of nucleotides or deletions. At a length of the specific nucleotide sequence of 974 nucleotides of methanoculleus bourgensis SBG7 and a length of the reference sequence of 968 nucleotides, a 97.31% match is calculated using the FASTA algorithm.

The present invention also includes microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having more than 97.31% sequence identity with the nucleotide sequence of SEQ ID NO: 4. More preferably, the nucleotide sequence contains a sequence range greater than 97.4% or greater than 97.5% or greater than 97.6% or greater than 97.7% or greater than 97.8% or greater than 97.9%. or more than 98.0% sequence identity with the nucleotide sequence SEQ ID NO: 4, and more preferably, the nucleotide sequence contains a sequence region having more than 98.5% sequence identity with the nucleotide sequence SEQ ID NO: 4.

According to further preferred embodiments, the microorganism has a nucleotide sequence containing a sequence region having more than 99.0% sequence identity with the nucleotide sequence SEQ ID NO: 4, and more preferably the nucleotide sequence contains a sequence region having more than 99.5% sequence identity having the nucleotide sequence of SEQ ID NO: 4. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 4.

In preferred embodiments of the present invention, SEQ ID NO: 4 may be compared to the starting nucleotide sequence at one or two positions or at three positions or at four positions or at five positions or at six positions or at seven positions or at eight positions or at nine positions or ten positions or eleven positions or twelve positions or 13 positions or 14 positions or 15 positions or 16 positions or 17 positions or 18 positions or 19 positions or 20 positions or 21 positions or on 22 positions or at 23 positions or at 24 positions or at 25 positions or at 26 nucleotide mutations. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text. The present invention also encompasses a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism having a nucleotide sequence containing a sequence region greater than 97.31 is present % Sequence identity with the nucleotide sequence of SEQ ID NO: 4, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 97.4% or greater than 97.5% or greater than 97 , 6% or more than 97.7% or more than 97.8% or more than 97.9% or more than 98.0% sequence identity with the nucleotide sequence SEQ ID NO: 4. More preferably, the nucleotide sequence contains a sequence region having more than 98.5% sequence identity with the nucleotide sequence SEQ ID NO: 4.

According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range exceeding 99.0% sequence identity with the nucleotide sequence SEQ ID NO. 4 has. Most preferably, the nucleotide sequence contains a sequence region having more than 99.5% sequence identity with the nucleotide sequence SEQ ID NO: 4.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 4.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 4 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 4 in a method described above in connection with microorganisms of the species Methanoculleus bourgensis for the fermentative production of biogas from biomass. The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, the culture of microorganisms comprising at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 4 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. It is preferable to use a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID No. 16 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in a method described above in connection with microorganisms of the species Methanoculleus bourgensis for the fermentative production of biogas from biomass.

The microorganisms Methanoculleus bourgensis SBG12 also obtained from the fermentation substrate of a postgrader were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID No. 9 comprises 1119 nucleotides. The next related sequence identified was the sequence of the non-cultured archaeon clone GZK7. A comparison of the sequences revealed that there are a total of 27 exchanges of nucleotides or deletions. At a length of the particular nucleotide sequence of Methanoculleus bourgensis SBG12 of 1 1 19 nucleotides and a length of the reference sequence of 1099 nucleotides calculated using the FASTA algorithm a match of 97.54%.

The present invention also encompasses microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region that exceeds 97.54%.

Sequence identity with the nucleotide sequence SEQ ID NO. 9 has. More preferably, the nucleotide sequence contains a sequence range greater than 97.6% or greater than

97.7% or more than 97.8% or more than 97.9% or more than 98.0% or more

98.1% or more than 98.2% sequence identity with the nucleotide sequence SEQ ID NO: 9, and more preferably, the nucleotide sequence contains a sequence region having more than 98.5% sequence identity with the nucleotide sequence SEQ ID NO: 9.

According to further preferred embodiments, the microorganism has a nucleotide sequence containing a sequence region having more than 99.0% sequence identity with the nucleotide sequence SEQ ID NO: 9, and more preferably the nucleotide sequence contains a sequence region having greater than 99.5% sequence identity having the nucleotide sequence of SEQ ID NO: 9. According to a most preferred In an embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 9.

In preferred embodiments of the present invention, SEQ ID NO: 9 may be compared to the starting nucleotide sequence at one or two positions or at three positions or at four positions or at five positions or at six positions or at seven positions or at eight positions or at nine positions or ten positions or eleven positions or twelve positions or 13 positions or 14 positions or 15 positions or 16 positions or 17 positions or 18 positions or 19 positions or 20 positions or 21 positions or on 22 positions or at 23 positions or at 24 positions or at 25 positions or at 26 positions or at 27 positions nucleotide mutations. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also encompasses a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism is present which has a nucleotide sequence containing a sequence region greater than 97.54 % Sequence identity with the nucleotide sequence of SEQ ID NO: 9, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 97.6% or greater than 97.7% or greater than 97 , 8% or more than 97.9% or more than 98.0% or more than 98.1% or more than 98.2% sequence identity with the nucleotide sequence of SEQ ID NO: 9. More preferably, the nucleotide sequence contains a sequence region having more than 98.5% sequence identity with the nucleotide sequence SEQ ID NO: 9.

According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range exceeding 99.0% sequence identity with the nucleotide sequence SEQ ID NO. 9 has. The nucleotide sequence most preferably contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 9. According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 9.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 9 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 9 in a method described above in connection with microorganisms of the species Methanoculleus bourgensis for the fermentative production of biogas from biomass.

The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO. 9 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. Preference is given to the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID No. 9 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in a method described above in connection with microorganisms of the species Methanoculleus bourgensis for the fermentative production of biogas from biomass.

The microorganisms Methanoculleus bourgensis SBG13 also obtained from the fermentation substrate of a postgrader were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID NO: 10 comprises 1 123 nucleotides. The next related sequence identified was the sequence of the uncultivated Archaeon clone 5.5ft C121A. A comparison of the sequences revealed that a total of 22 exchanges of nucleotides or deletions are present. At a length of the particular nucleotide sequence of Methanoculleus bourgensis SBG 13 of 1 123 nucleotides and a length of the reference sequence of 1 123 nucleotides, a match of 98.04% is calculated using the FASTA algorithm. The present invention also includes microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having greater than 98.04% sequence identity with the nucleotide sequence of SEQ ID NO: 10. More preferably, the nucleotide sequence contains a sequence range greater than 98.1% or greater than 98.2% or greater than 98.3% or greater than 98.4% or greater than 98.5% or greater than 98.6%. or more than 98.7% sequence identity with the nucleotide sequence SEQ ID NO: 10, and more preferably, the nucleotide sequence contains a sequence region having more than 98.8% sequence identity with the nucleotide sequence SEQ ID NO: 10.

According to further preferred embodiments, the microorganism has a nucleotide sequence containing a sequence region having more than 99.0% sequence identity with the nucleotide sequence SEQ ID NO: 10, and more preferably the nucleotide sequence contains a sequence region having greater than 99.5% sequence identity having the nucleotide sequence of SEQ ID NO: 10. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 10.

In preferred embodiments of the present invention, SEQ ID NO: 10 may be compared to the starting nucleotide sequence at one or two positions or at three positions or at four positions or at five positions or at six positions or at seven positions or at eight positions or at nine positions or ten positions or eleven positions or twelve positions or 13 positions or 14 positions or 15 positions or 16 positions or 17 positions or 18 positions or 19 positions or 20 positions or 21 positions or on There are 22 nucleotide mutations. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also encompasses a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, in which culture of microorganisms a microorganism is present which has a nucleotide sequence containing a sequence region greater than 98.04 % Sequence identity with the nucleotide sequence SEQ ID NO: 10, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present, which has a nucleotide sequence having a sequence range greater than 98.1% or greater than 98.2% or greater than 98.3% or greater than 98.4% or greater than 98.5% or greater than 98.6% or more than 98.7% has sequence identity with the nucleotide sequence SEQ ID NO: 10. More preferably, the nucleotide sequence contains a sequence region having more than 98.8% sequence identity with the nucleotide sequence of SEQ ID NO: 10.

According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range exceeding 99.0% sequence identity with the nucleotide sequence SEQ ID NO. 10 has. Most preferably, the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 10.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 10.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 10 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 10 in a method described above in connection with microorganisms of the species Methanoculleus bourgensis for the fermentative production of biogas from biomass.

The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, the culture of microorganisms comprising at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 10 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. It is preferable to use a culture of microorganisms for fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO. 10 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in one of the above - mentioned In connection with microorganisms of the species Methanoculleus bourgensis method described in more detail for the fermentative production of biogas from biomass.

The microorganisms Methanoculleus bourgensis SBG14 also obtained from the fermentation substrate of a secondary fermenter were subjected to a sequence analysis. The determined

16S rRNA sequence SEQ ID NO: 1 comprises 1 133 nucleotides. As the next related

Sequence was identified as a partial sequence of the uncultured Archaeon clone 5.5ft C121A. A comparison of the sequences revealed that a total of 28 exchanges of

Nucleotides or deletions. At a length of the particular nucleotide sequence of Methanoculleus bourgensis SBG 14 of 1 133 nucleotides and a length of the

Reference sequence of 1 132 nucleotides is calculated using the FASTA algorithm

Match of 97.53%.

The present invention also encompasses microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region that exceeds 97.53%.

Sequence identity with the nucleotide sequence SEQ ID NO. 1 1 has. More preferably, the nucleotide sequence contains a sequence range greater than 97.6% or greater than

97.7% or more than 97.8% or more than 97.9% or more than 98.0% or more

98.1% or more than 98.2% sequence identity with the nucleotide sequence SEQ ID NO: 1 1, and more preferably, the nucleotide sequence contains a sequence region having more than 98.5% sequence identity with the nucleotide sequence SEQ ID NO: 1 1.

According to further preferred embodiments, the microorganism has a nucleotide sequence which contains a sequence region which has more than 99.0% sequence identity with the nucleotide sequence SEQ ID No. 11, and more preferably the nucleotide sequence contains a sequence region which exceeds 99.5%. Sequence identity with the nucleotide sequence SEQ ID NO. 1 1 has. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 11.

In preferred embodiments of the present invention, SEQ ID NO: 1 can be compared to the starting nucleotide sequence at one or two positions or at three positions or at four positions or at five positions or at six positions or at seven positions or at eight positions or at nine Positions or ten positions or eleven positions or twelve positions or 13 positions or 14 positions or 15 positions or 16 positions or 17 positions or 18 positions or 19 positions or 20 positions or 21 positions or at 22 Positions or at 23 positions or at 24 positions or at 25 positions or at 26 positions or at 27 positions or at 28 positions nucleotide mutations. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also encompasses a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, in which culture of microorganisms a microorganism is present which has a nucleotide sequence containing a sequence region greater than 97.53 % Sequence identity with the nucleotide sequence SEQ ID NO: 1 1, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 97.6% or more

97.7% or more than 97.8% or more than 97.9% or more than 98.0% or more

98.1% or greater than 98.2% sequence identity with the nucleotide sequence SEQ ID NO: 1 1. Particularly preferably, the nucleotide sequence contains a sequence region which has more than 98.5% sequence identity with the nucleotide sequence SEQ ID No. 11.

According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range exceeding 99.0% sequence identity with the nucleotide sequence SEQ ID NO. 1 1 has. Most preferably, the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 11.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 11.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 1 1 for the fermentative production of biogas from biomass. It is preferable to use a Microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 1 1 in a method described in more detail above in connection with microorganisms of the species Methanoculleus bourgensis for the fermentative production of biogas from biomass.

The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, the culture of microorganisms comprising at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 1 1 and wherein the microorganism comprises at least 10 ^'4 % of the total number of microorganisms present in the culture. It is preferable to use a culture of microorganisms for fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 1 1 and wherein the microorganism is at least 10 ^'4 % of the total number of microorganisms present in the culture, in a process described in more detail above in connection with microorganisms of the species Methanoculleus bourgensis, for the fermentative production of biogas from biomass.

The microorganisms Methanoculleus bourgensis SBG15 also obtained from the fermentation substrate of a postgrader were subjected to a sequence analysis. The determined

16S rRNA sequence SEQ ID NO: 12 comprises 801 nucleotides. The next related sequence identified was the sequence of the uncultivated Archaeon clone 5.5ft C124A. One

Comparison of the sequences revealed that a total of 25 exchanges of nucleotides or

Deletions are present. At a length of the particular nucleotide sequence of Methanoculleus bourgensis SBG15 of 801 nucleotides and a reference sequence length of 804

Nucleotides are calculated using the FASTA algorithm to match

96.88%.

The present invention also encompasses microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region that exceeds 96.88%.

Sequence identity with the nucleotide sequence SEQ ID NO. 12 has. More preferably, the nucleotide sequence contains a sequence range greater than 96.9% or greater than

97.0% or more than 97.1% or more than 97.2% or more than 97.3% or more

97.4% or more than 97.5% has sequence identity with the nucleotide sequence SEQ ID NO: 12, and more preferably the nucleotide sequence contains a sequence region having more than 98.0% or more than 98.5% sequence identity with the nucleotide sequence SEQ

ID No. 12. According to further preferred embodiments, the microorganism has a nucleotide sequence containing a sequence region having more than 99.0% sequence identity with the nucleotide sequence SEQ ID NO: 12, and more preferably the nucleotide sequence contains a sequence region having greater than 99.5% sequence identity having the nucleotide sequence SEQ ID NO: 12. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 12.

In preferred embodiments of the present invention, SEQ ID NO: 12 may be compared to the starting nucleotide sequence at one or two positions or at three positions or at four positions or at five positions or at six positions or at seven positions or at eight positions or at nine positions or ten positions or eleven positions or twelve positions or 13 positions or 14 positions or 15 positions or 16 positions or 17 positions or 18 positions or 19 positions or 20 positions or 21 positions or on 22 positions or 23 positions or 24 positions or 25 positions nucleotide mutations. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also includes a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism is present which has a nucleotide sequence containing a sequence region greater than 96.88 % Sequence identity with the nucleotide sequence of SEQ ID NO: 12, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range exceeding 96.9% or more

97.0% or more than 97.1% or more than 97.2% or more than 97.3% or more

97.4% or greater than 97.5% sequence identity with the nucleotide sequence SEQ ID NO: 12. More preferably, the nucleotide sequence contains a sequence region having greater than 98.0% or greater than 98.5% sequence identity with the nucleotide sequence SEQ ID

No. 12 has. According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range exceeding 99.0% sequence identity with the nucleotide sequence SEQ ID NO. 12 has. Most preferably, the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 12.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 12.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 12 for fermentative purposes

Generation of biogas from biomass. It is preferable to use a

Microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 12 in a method described in more detail above in connection with microorganisms of the species Methanoculleus bourgensis for the fermentative production of biogas from biomass.

The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 12 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. It is preferable to use a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID No. 12 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in a method described above in connection with microorganisms of the species Methanoculleus bourgensis for the fermentative production of biogas from biomass.

The microorganisms Methanoculleus bourgensis SBG31 also obtained from the fermentation substrate of a postgrader were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID No. 28 comprises 556 nucleotides. As the next related sequence the sequence of the uncultured Archaeon clone 5.5ft C141A was identified. A comparison of the sequences revealed that there are a total of 5 exchanges of nucleotides or deletions. At a length of 556 nucleotides of the specific nucleotide sequence of Methanoculleus bourgensis SBG31 and a length of the reference sequence of 556 nucleotides, a 99.10% match is calculated using the FASTA algorithm.

The present invention also includes microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having greater than 99.10% sequence identity with the nucleotide sequence of SEQ ID NO: 28. More preferably, the nucleotide sequence contains a sequence region that is greater than 99.2% or greater than 99.3% or greater than 99.4% or greater than 99.5% or greater than 99.6% sequence identity to the nucleotide sequence of SEQ ID NO 28, and particularly preferably, the nucleotide sequence contains a sequence region which has more than 99.7% sequence identity with the nucleotide sequence SEQ ID No. 28.

In further preferred embodiments, the microorganism has a nucleotide sequence containing a sequence region having greater than 99.8% sequence identity with the nucleotide sequence SEQ ID NO: 28, and more preferably, the nucleotide sequence contains a sequence region having greater than 99.9% sequence identity having the nucleotide sequence of SEQ ID NO: 28. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 28.

In preferred embodiments of the present invention, nucleotide mutations may be present at one or two positions or at three positions or at four positions or at five positions relative to the starting nucleotide sequence SEQ ID NO: 28. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also includes a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism is present which has a nucleotide sequence containing a sequence region greater than 99.10 having% sequence identity with the nucleotide sequence SEQ ID NO. 28, wherein the microorganism makes up at least 10 ^'4% of the total number of microorganisms present in the culture. Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 99.2% or greater than 99.3% or greater than 99 , 4% or more than 99.5% sequence identity with the nucleotide sequence SEQ ID NO: 28. Most preferably, the nucleotide sequence contains a sequence region having greater than 99.6% or greater than 99.7% sequence identity with the nucleotide sequence of SEQ ID NO: 28.

According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence region having more than 99.8% sequence identity with the nucleotide sequence SEQ ID NO. 28 has. Most preferably, the nucleotide sequence contains a sequence region having more than 99.9% sequence identity with the nucleotide sequence of SEQ ID NO: 28.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 28.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 28 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 28 in a method described above in connection with microorganisms of the species Methanoculleus bourgensis for the fermentative production of biogas from biomass.

The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, the culture of microorganisms comprising at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 28 and wherein the microorganism is at least 10 ^{'. 4} % of the total number of microorganisms present in the culture. Preference is given to the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 28 and wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture, in one above in connection with microorganisms of the species Methanoculleus bourgensis described method for the fermentative production of biogas from biomass.

The microorganisms Methanoculleus bourgensis SBG30 also obtained from the fermentation substrate of a secondary fermenter were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID NO: 27 comprises 909 nucleotides. As a next related sequence, the sequence of the uncultured Archaeon clone A52 was identified. A comparison of the sequences revealed that there are a total of 38 exchanges of nucleotides or deletions. At a length of the specific nucleotide sequence of 909 nucleotides of Methanoculleus bourgensis SBG30 and a length of the reference sequence of 926 nucleotides, a 95.82% match is calculated using the FASTA algorithm.

The present invention also includes microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having more than 95.82% sequence identity with the nucleotide sequence of SEQ ID NO: 27. More preferably, the nucleotide sequence contains a sequence range greater than 95.9% or greater than 96.0% or greater than 96.1% or greater than 96.2% or greater than 96.3% or greater than 96.4%. or more than 96.5% sequence identity with the nucleotide sequence SEQ ID NO: 27, and most preferably, the nucleotide sequence contains a sequence region having greater than 97.0% or greater than 98.0% sequence identity with the nucleotide sequence SEQ ID NO: 27 ,

According to further preferred embodiments, the microorganism has a nucleotide sequence containing a sequence region having more than 99.0% sequence identity with the nucleotide sequence SEQ ID NO: 27, and more preferably the nucleotide sequence contains a sequence region having greater than 99.5% sequence identity having the nucleotide sequence of SEQ ID NO: 27. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 27.

In preferred embodiments of the present invention, SEQ ID NO: 27 may be compared to the starting nucleotide sequence at one or two positions or at three positions or at four positions or at five positions or at six positions or at seven positions or at eight positions or at nine positions or at ten Positions or eleven positions or twelve positions or 13 positions or 14 positions or 15 positions or 16 positions or 17 positions or 18 positions or 19 positions or 20 positions or 21 positions or 22 positions or at 23 positions or 24 positions or 25 positions or 26 positions or 27 positions or 28 positions or 29 positions or 30 positions or 31 positions or 32 positions or 33 positions or 34 positions or 35 Positions or at 36 positions or at 37 positions or at 38 positions nucleotide mutations. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also encompasses a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism is present which has a nucleotide sequence containing a sequence region greater than 95,82 % Sequence identity with the nucleotide sequence SEQ ID NO: 27, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 95.9% or more

96.0% or more than 96.1% or more than 96.2% or more than 96.3% or more

96.4% or more than 96.5% sequence identity with the nucleotide sequence SEQ ID NO: 27. Most preferably, the nucleotide sequence contains a sequence region having greater than 97.0% or greater than 98.0% sequence identity with the nucleotide sequence SEQ ID

No. 27 has.

According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range exceeding 99.0% sequence identity with the nucleotide sequence SEQ ID NO. 27 has. Most preferably, the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID NO: 27.

According to a very particularly preferred embodiment, the culture suitable for use in a process for the fermentative production of biogas from biomass is Microorganisms include a microorganism having a nucleotide sequence containing a sequence region corresponding to the nucleotide sequence of SEQ ID NO: 27.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 27 for fermentative

Generation of biogas from biomass. It is preferable to use a

Microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 27 in a method described above in connection with microorganisms of the species Methanoculleus bourgensis for the fermentative production of biogas from biomass.

The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 27 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. It is preferable to use a culture of microorganisms for fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 27 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in a method described above in connection with microorganisms of the species Methanoculleus bourgensis for the fermentative production of biogas from biomass.

The microorganisms Methanoculleus bourgensis SBG23, also obtained from the fermentation substrate of a post-fermenter, were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID NO: 20 comprises 801 nucleotides. The next related sequence identified was the sequence of the non-cultured archaeon clone MCSArc_B6. A comparison of the sequences revealed that there are a total of 33 exchanges of nucleotides or deletions. At a length of 801 nucleotides in length determined by the nucleotide sequence of Methanoculleus bourgensis SBG23 and a length of the reference sequence of 802 nucleotides, the FASTA algorithm gives a 95.88% match.

The present invention also includes microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having more than 95.88% sequence identity with the nucleotide sequence of SEQ ID NO: 20. Especially preferred For example, the nucleotide sequence contains a sequence range greater than 95.9% or greater than 96.0% or greater than 96.1% or greater than 96.2% or greater than 96.3% or greater than 96.4% or greater having 96.5% sequence identity with the nucleotide sequence SEQ ID NO: 20, and more preferably, the nucleotide sequence contains a sequence region having more than 97.0% or more than 98.0% sequence identity with the nucleotide sequence SEQ ID NO: 20.

According to further preferred embodiments, the microorganism has a nucleotide sequence containing a sequence region having more than 99.0% sequence identity with the nucleotide sequence SEQ ID NO: 20, and more preferably the nucleotide sequence contains a sequence region having greater than 99.5% sequence identity having the nucleotide sequence of SEQ ID NO: 20. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 20.

In preferred embodiments of the present invention, SEQ ID NO: 20 may be compared to the starting nucleotide sequence at one or two positions or at three positions or at four positions or at five positions or at six positions or at seven positions or at eight positions or at nine positions or ten positions or eleven positions or twelve positions or 13 positions or 14 positions or 15 positions or 16 positions or 17 positions or 18 positions or 19 positions or 20 positions or 21 positions or on 22 positions or 23 positions or 24 positions or 25 positions or 26 positions or 27 positions or 28 positions or 29 positions or 30 positions or 31 positions or 32 positions or 33 positions nucleotide mutations. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also includes a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism is present, which contains a

Having a nucleotide sequence containing a sequence range greater than 95.88%

Sequence identity with the nucleotide sequence SEQ ID NO. 20, wherein the

Microorganism accounts for at least 10 ^'4 % of the total number of microorganisms present in the culture. Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 95.9% or greater than 96.0% or greater than 96 , 1% or more than 96.2% or more than 96.3% or more than 96.4% or more than 96.5% sequence identity with the nucleotide sequence SEQ ID NO: 20. Most preferably, the nucleotide sequence contains a sequence region having greater than 97.0% or greater than 98.0% sequence identity with the nucleotide sequence SEQ ID NO: 20.

According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range exceeding 99.0% sequence identity with the nucleotide sequence SEQ ID NO. 20 has. Most preferably, the nucleotide sequence contains a sequence region that has more than 99.5% sequence identity with the nucleotide sequence SEQ ID NO: 20.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID NO.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 20 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 20 in a method described above in connection with microorganisms of the species Methanoculleus bourgensis for the fermentative production of biogas from biomass.

The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO. 20 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. Preference is given to the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 20 and wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture, in one above in connection with microorganisms of the species Methanoculleus bourgensis described method for the fermentative production of biogas from biomass.

The microorganisms Methanoculleus bourgensis SBG28 also obtained from the fermentation substrate of a secondary fermenter were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID NO: 25 comprises 801 nucleotides. The next related sequence identified was the non-cultured Euryarchaeote clone BSA 1A-03. A comparison of the sequences revealed that there are a total of 3 exchanges of nucleotides or deletions. At a length of the specific nucleotide sequence of 801 nucleotides of Methanoculleus bourgensis SBG28 and a length of the reference sequence of 801 nucleotides, a 99.63% agreement is calculated using the FASTA algorithm.

The present invention also includes microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having more than 99.63% sequence identity with the nucleotide sequence of SEQ ID NO: 25. More preferably, the nucleotide sequence contains a sequence region that is greater than 99.65% or greater than 99.67% or greater than 99.69% or greater than 99.70% or greater than 99.72% sequence identity to the nucleotide sequence of SEQ ID NO 25, and particularly preferably, the nucleotide sequence contains a sequence region having more than 99.75 sequence identity with the nucleotide sequence SEQ ID NO: 25.

According to further preferred embodiments, the microorganism has a nucleotide sequence containing a sequence region having greater than 99.8% sequence identity with the nucleotide sequence SEQ ID NO: 25, and more preferably the nucleotide sequence contains a sequence region having greater than 99.9% sequence identity having the nucleotide sequence SEQ ID NO: 25. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 25.

In preferred embodiments of the present invention, nucleotide mutations may be present at one or two positions or at three positions relative to the starting nucleotide sequence of SEQ ID NO: 25. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text. The present invention also includes a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism is present which has a nucleotide sequence containing a sequence region greater than 99.63 % Sequence identity with the nucleotide sequence SEQ ID NO: 25, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 99.65% or greater than 99.67% or greater than 99 , 69% or more than 99.70% sequence identity with the nucleotide sequence SEQ ID NO: 25. Most preferably, the nucleotide sequence contains a sequence region having greater than 99.72% or greater than 99.75% sequence identity with the nucleotide sequence SEQ ID NO: 25.

According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence region having more than 99.8% sequence identity with the nucleotide sequence SEQ ID NO. 25 has. Most preferably, the nucleotide sequence contains a sequence region having more than 99.9% sequence identity with the nucleotide sequence SEQ ID NO: 25.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 25.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 25 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 25 in a method described above in connection with microorganisms of the species Methanoculleus bourgensis for the fermentative production of biogas from biomass. The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO. 25 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. It is preferable to use a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID No. 25 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in a method described above in connection with microorganisms of the species Methanoculleus bourgensis for the fermentative production of biogas from biomass.

The microorganisms Methanoculleus bourgensis SBG29 also obtained from the fermentation substrate of a postgrader were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID NO: 26 comprises 679 nucleotides. The next related sequence identified was the sequence of the uncultivated Euryarchaeote clone BSA2A-02. A comparison of the sequences revealed that there are a total of 5 exchanges of nucleotides or deletions. At a length of the specific nucleotide sequence of 679 nucleotides of Methanoculleus bourgensis SBG29 and a length of the reference sequence of 680 nucleotides, the FASTA algorithm gives a 99.26% match.

The present invention also includes microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having greater than 99.26% sequence identity with the nucleotide sequence of SEQ ID NO: 26. More preferably, the nucleotide sequence contains a sequence region that is greater than 99.28% or greater than 99.3% or greater than 99.4% or greater than 99.5% or greater than 99.6% sequence identity to the nucleotide sequence of SEQ ID NO , 26, and more preferably, the nucleotide sequence contains a sequence region having more than 99.7% sequence identity with the nucleotide sequence SEQ ID NO: 26.

According to further preferred embodiments, the microorganism has a nucleotide sequence which contains a sequence range which exceeds more than 99.8%.

Sequence identity with the nucleotide sequence SEQ ID NO. 26, and more preferably, the nucleotide sequence contains a sequence region having more than 99.9% sequence identity having the nucleotide sequence of SEQ ID NO: 26. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 26.

In preferred embodiments of the present invention, nucleotide mutations may be present at one or two positions or at three positions or at four positions or at five positions relative to the starting nucleotide sequence SEQ ID NO: 26. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also encompasses a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism is present which has a nucleotide sequence containing a sequence region greater than 99,26 % Sequence identity with the nucleotide sequence SEQ ID NO: 26, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 99.28% or greater than 99.3% or greater than 99 , 4% or more than 99.5% sequence identity with the

Nucleotide sequence has SEQ ID NO: 26. Particularly preferably, the

Nucleotide sequence has a sequence region which has more than 99.6% or more than 99.7% sequence identity with the nucleotide sequence SEQ ID No. 26.

According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence region having more than 99.8% sequence identity with the nucleotide sequence SEQ ID NO. 26 has. Most preferably, the nucleotide sequence contains a sequence region having more than 99.9% sequence identity with the nucleotide sequence SEQ ID NO: 26.

According to a very particularly preferred embodiment, the culture suitable for use in a process for the fermentative production of biogas from biomass is Microorganisms include a microorganism having a nucleotide sequence containing a sequence region corresponding to the nucleotide sequence of SEQ ID NO: 26.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 26 for fermentative

Generation of biogas from biomass. It is preferable to use a

Microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 26 in a method described in more detail above in connection with microorganisms of the species Methanoculleus bourgensis for the fermentative production of biogas from biomass.

The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, the culture of microorganisms comprising at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO. 26 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. It is preferable to use a culture of microorganisms for fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID No. 26 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in a method described above in connection with microorganisms of the species Methanoculleus bourgensis for the fermentative production of biogas from biomass.

The microorganisms Methanoculleus bourgensis SBG32 also obtained from the fermentation substrate of a postgrader were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID No. 29 comprises 935 nucleotides. The next related sequence was a partial sequence of the uncultivated Euryarchaeote clone MAA04. A comparison of the sequences revealed that a total of 23 exchanges of nucleotides or deletions are present. With a length of the specific nucleotide sequence of 945 nucleotides of Methanoculleus bourgensis SBG32 and a length of the reference sequence of 939 nucleotides, a 97.54% match is calculated using the FASTA algorithm.

The present invention also includes microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having more than 97.54% sequence identity with the nucleotide sequence of SEQ ID NO: 29. Especially preferred For example, the nucleotide sequence contains a sequence range greater than 97.6% or greater than 97.7% or greater than 97.8% or greater than 97.9% or greater than 98.0% or greater than 98.1% or greater 98.2% has sequence identity with the nucleotide sequence SEQ ID NO: 29, and more preferably, the nucleotide sequence contains a sequence region having more than 98.5% sequence identity with the nucleotide sequence SEQ ID NO: 29.

According to further preferred embodiments, the microorganism has a nucleotide sequence containing a sequence region having more than 99.0% sequence identity with the nucleotide sequence SEQ ID NO: 29, and more preferably the nucleotide sequence contains a sequence region having greater than 99.5% sequence identity having the nucleotide sequence of SEQ ID NO: 29. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 29.

In preferred embodiments of the present invention, SEQ ID NO: 29 may be compared to the starting nucleotide sequence at one or two positions or at three positions or at four positions or at five positions or at six positions or at seven positions or at eight positions or at nine positions or ten positions or eleven positions or twelve positions or 13 positions or 14 positions or 15 positions or 16 positions or 17 positions or 18 positions or 19 positions or 20 positions or 21 positions or on 22 positions or at 23 positions nucleotide mutations. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also encompasses a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism is present which has a nucleotide sequence containing a sequence region greater than 97.54 having% sequence identity with the nucleotide sequence SEQ ID NO. 29, wherein the microorganism makes up at least 10 ^'4% of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 97.6% or greater than 97.7% or greater than 97 , 8% or more than 97.9% or more than 98.0% or more than 98.1% or more than 98.2% sequence identity with the nucleotide sequence SEQ ID NO: 29 having. Particularly preferably, the nucleotide sequence contains a sequence region which has more than 98.5% sequence identity with the nucleotide sequence SEQ ID No. 29.

According to further preferred embodiments, the culture suitable for use in a process for the fermentative production of biogas from biomass is

Microorganisms present a microorganism having a nucleotide sequence with a

Sequence region has more than 99.0% sequence identity with the nucleotide sequence

SEQ ID NO: 29. Most preferably, the nucleotide sequence contains a

A sequence region having greater than 99.5% sequence identity with the nucleotide sequence SEQ ID NO: 29.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 29.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 29 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 29 in a method described above in connection with microorganisms of the species Methanoculleus bourgensis for the fermentative production of biogas from biomass.

The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, the culture of microorganisms comprising at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 29 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. It is preferable to use a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID No. 29 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in a method described above in connection with microorganisms of the species Methanoculleus bourgensis for the fermentative production of biogas from biomass. According to further preferred embodiments, all make above, characterized with respect to their nucleotide sequence microorganisms at least 10 ^'2%, preferably to at least 1% of the total present in the culture of microorganisms. Particularly preferably, the microorganisms make up at least 10%, particularly preferably at least 25%, of the total number of microorganisms present in the culture. Most preferably, the microorganisms described make up at least 50%, in particular at least 75%, of the total number of microorganisms present in the culture.

Also preferred are embodiments according to which the above-mentioned microorganisms make up at least 90% of the total number of microorganisms present in the culture. Particularly preferably it is a pure culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, which is a pure culture of a microorganism, as has been characterized above with respect to its nucleotide sequence.

Most preferably, in the cases described above, it is an immobilized culture of microorganisms.

Methanoculleus chikugoensis

The present invention provides a method for producing biogas from biomass in a fermentation reactor. The biomass is added according to the invention a microorganism of the species Methanoculleus chikugoensis.

Surprisingly, it has been found that the amount of biogas formed can be significantly increased by the addition of microorganisms of the species Methanoculleus chikugoensis to the fermentation substrate. At constant space load, the addition of microorganisms of the species Methanoculleus chikugoensis causes a significant increase in the amount of biogas produced. Alternatively, the amount of methane formed can also be increased by increasing the volume load, with the addition of microorganisms of the species Methanoculleus chikugoensis preventing the fermentation process from becoming unstable under the altered conditions. The use of microorganisms of the species Methanoculleus chikugoensis therefore leads to an improvement in the efficiency and efficiency of biogas plants. The person skilled in the art knows which strains of microorganisms fall under the term "species Methanoculleus chikugoensis." In connection with the production of biogas by fermentation of organic substrates, microorganisms of the species Methanoculleus chikugoensis have hitherto not been known.

According to a preferred embodiment of the present invention, a microorganism of the species Methanoculleus chikugoensis is added in the form of a culture of microorganisms consisting predominantly of microorganisms of the species Methanoculleus chikugoensis. In fermentation substrates of biogas plants microorganisms of the species could be detected by Methanoculleus present microorganisms chikugoensis only in trace amounts of less than 10 ^'4% share of the total number. Since the amount of microorganisms isolated from their natural occurrence is insufficient for the addition of the microorganisms, it is usually propagated in the form of a culture. In practice, it is found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out directly in the form of a culture of microorganisms.

The addition of the culture of Methanoculleus chikugoensis can be carried out in the form of a culture suspension or in the form of dry, freeze-dried or moist cell pellets.

Since the already mentioned various positive effects on the fermentation process are associated with the microorganism species Methanoculleus chikugoensis, this type of microorganisms should be present in the added culture in a concentration exceeding the natural abundance. Of course, mixed cultures of any composition can be used for the addition. The only requirement is that the species Methanoculleus chikugoensis is present in a concentration enriched with respect to the natural occurrence.

The determination of the proportions of different types of microorganisms in mixed cultures is not a problem for the expert. Thus, for example, with the help of fluorescently labeled oligosensors specifically the proportion of microorganisms of the species Methanoculleus chikugoensis be identified in a mixture. As already mentioned, microorganisms of the species Methanoculleus chikugoensis are preferably added to the fermentation substrate in the form of cultures of microorganisms, the cultures of microorganisms consisting predominantly of microorganisms of the species Methanoculleus chikugoensis. In addition to the determination of the number of microorganisms of the species Methanoculleus chikugoensis also determines the total number of microorganisms, the proportion of microorganisms of the species Methanoculleus chikugoensis in the culture can be specified in percent. Microorganisms of the species Methanoculleus chikugoensis are in a mixed culture then predominantly present type of microorganisms, if they have the highest percentage of the various types of microorganisms present in the mixed culture.

According to preferred embodiments of the present invention make microorganisms of the species Methanoculleus chikugoensis at least 10 ^'4%, especially at least 10 ^"2%, more preferably at least 1% of the total number of the added to the fermentation substrate culture microorganisms present. According to further preferred embodiments make microorganisms of the species Methanoculleus chikugoensis at least 10%, in particular at least 50%, particularly preferably at least 90% of the total number of microorganisms present in the culture.

According to a very particularly preferred embodiment, a pure culture of a microorganism of the species Methanoculleus chikugoensis is added. Due to the specific metabolic processes and activities, the addition of a pure culture can especially contribute to an improved methane production.

According to another preferred embodiment of the present invention, a microorganism of the species Methanoculleus chikugoensis is added as a component of at least one immobilized culture of microorganisms. Since the amount of microorganisms isolated from their natural occurrence is insufficient for the addition of the microorganisms, it is usually propagated in the form of a culture. In practice, it has been found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out in the form of an immobilized culture of microorganisms.

According to a preferred embodiment of the present invention, the microorganism of the species Methanoculleus chikugoensis is added in an amount to the fermentation substrate, so that after addition of the proportion of the microorganism of the species Methanoculleus chikugoensis between 10 ^{~ 8} % and 50% of the total number of present in the fermentation substrate microorganisms accounts. In particular, depending on the fermenter size and thus as a function of the amount of fermentation substrate can be necessary to achieve the desired effect, an addition of very different amounts of microorganisms. A microorganism of the species Methanoculleus chikugoensis is particularly preferably added in an amount to the fermentation substrate that makes up after addition of the content of the microorganism of the species Methanoculleus chikugoensis between ^{10 -6%} and 25% of the total number present in the fermentation substrate microorganisms. More preferably, the microorganism of the species Methanoculleus chikugoensis is added in an amount to the fermentation substrate such that, after addition, the proportion of the microorganism of the species Methanoculleus chikugoensis is between 10 ^'4 % and 10% of the total number of microorganisms present in the fermentation substrate. The microorganism of the species Methanoculleus chikugoensis is very particularly preferably added in an amount to the fermentation substrate that makes up after addition of the content of the microorganism of the species Methanoculleus chikugoensis between 10 ^"3% and 1% of the total number present in the fermentation substrate microorganisms.

According to a particularly preferred embodiment of the present invention, the microorganism of the species Methanoculleus chikugoensis is a microorganism of the strain Methanoculleus chikugoensis MG62. In all of the above-described embodiments, which deal with the addition of a microorganism of the species Methanoculleus chikugoensis, so preferably a microorganism of the strain Methanoculleus chikugoensis MG62 is added.

The present invention also encompasses the use of a microorganism of the species Methanoculleus chikugoensis for the fermentative production of biogas from biomass. Preferably, a microorganism of the strain Methanoculleus chikugoensis MG62 is used for the fermentative production of biogas from biomass.

The microorganisms methanoculleus chikugoensis SBG5 obtained from the fermentation substrate of a post-fermenter were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID No. 2 comprises 1,124 nucleotides. Methanoculleus chikugoensis MG62 was identified as the next relative. A comparison of the sequences revealed that there are a total of 12 exchanges of nucleotides or deletions. At a length of the particular nucleotide sequence of 1 7 124 nucleotides of methanoculleus chikugoensis SBG5 and a length of the reference sequence of 1124 nucleotides, the 98.93% agreement is calculated using the FASTA algorithm.

The present invention also encompasses microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region of greater than 98.93%. Sequence identity with the nucleotide sequence SEQ ID NO. 2 has. More preferably, the nucleotide sequence contains a sequence range greater than 99.0% or greater than 99.1% or greater than 99.2% or greater than 99.3% or greater than 99.4% or greater than 99.5%. or more than 99.6% has sequence identity with the nucleotide sequence SEQ ID NO: 2, and more preferably, the nucleotide sequence contains a sequence region having more than 99.7% sequence identity with the nucleotide sequence SEQ ID NO: 2.

According to further preferred embodiments, the microorganism has a nucleotide sequence containing a sequence region having more than 99.8% sequence identity with the nucleotide sequence SEQ ID NO: 2, and more preferably the nucleotide sequence contains a sequence region having greater than 99.9% sequence identity having the nucleotide sequence of SEQ ID NO: 2. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 2.

In preferred embodiments of the present invention, SEQ ID NO: 2 may be compared to the starting nucleotide sequence at one or two positions or at three positions or at four positions or at five positions or at six positions or at seven positions or at eight positions or at nine positions or nucleotide mutations at ten positions or at eleven positions or at twelve positions. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also includes a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism is present, which contains a

Having a nucleotide sequence containing a sequence region greater than 98.93%

Sequence identity with the nucleotide sequence SEQ ID NO. 2, wherein the

Microorganism accounts for at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 99.0% or greater than 99.1% or greater than 99 , 2% or more than 99.3% or more than 99.4% or more than 99.5% or more than 99.6% sequence identity with the nucleotide sequence SEQ ID NO: 2 having. More preferably, the nucleotide sequence contains a sequence region having more than 99.7% sequence identity with the nucleotide sequence SEQ ID NO: 2.

According to further preferred embodiments, the culture suitable for use in a process for the fermentative production of biogas from biomass is

Microorganisms present a microorganism having a nucleotide sequence with a

Sequence region has more than 99.8% sequence identity with the nucleotide sequence

SEQ ID NO: 2. Most preferably, the nucleotide sequence contains a

Sequence region having more than 99.9% sequence identity with the nucleotide sequence SEQ ID NO: 2.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 2.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 2 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 2 in a method described above in connection with microorganisms of the species Methanoculleus chikugoensis for the fermentative production of biogas from biomass.

The present invention also includes the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 2 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. It is preferable to use a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID No. 2 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in a method described above in connection with microorganisms of the species Methanoculleus chikugoensis for the fermentative production of biogas from biomass. According to further preferred embodiments make said, characterized in terms of their nucleotide sequence microorganisms at least 10 ^'2%, preferably to at least 1% of the total present in the culture of microorganisms. Particularly preferably, the microorganisms make up at least 10%, particularly preferably at least 25%, of the total number of microorganisms present in the culture. Most preferably, the microorganisms described make up at least 50%, in particular at least 75%, of the total number of microorganisms present in the culture.

Also preferred are embodiments according to which the above-mentioned microorganisms at least 90% of the total number of present in the culture

Make up microorganisms. Particularly preferred is a pure culture of

Microorganisms suitable for use in a process for the fermentative production of

Biogas from biomass, which is a pure culture of a microorganism as characterized above with respect to its nucleotide sequence.

Most preferably, in the cases described above, it is an immobilized culture of microorganisms.

In particular, the present invention includes the following aspects:

1. A process for the production of biogas from biomass in a fermentation reactor, characterized in that the biomass is added to a microorganism of the species Methanoculleus chikugoensis.

2. The method according to claim 1, characterized in that the microorganism of the species Methanoculleus chikugoensis is added in the form of a culture of microorganisms, wherein microorganisms of the species Methanoculleus chikugoensis make up at least 10 ^'4 % of the total number of microorganisms present in the culture.

3. The method according to claim 2, characterized in that in the culture of microorganisms microorganisms of the species Methanoculleus chikugoensis make up at least 10 ^{" 2} % of the total number of microorganisms present in the culture.

4. The method according to claim 3, characterized in that in the culture of microorganisms microorganisms of the species Methanoculleus chikugoensis make up at least 1% of the total number of microorganisms present in the culture. 5. The method according to claim 4, characterized in that in the culture of microorganisms microorganisms of the species Methanoculleus chikugoensis account for at least 10% of the total number of microorganisms present in the culture.

6. The method according to claim 5, characterized in that constitute in the culture of microorganisms microorganisms of the species Methanoculleus chikugoensis at least 50% of the total number of microorganisms present in the culture.

7. The method according to claim 6, characterized in that in the culture of microorganisms microorganisms of the species Methanoculleus chikugoensis make up at least 75% of the total number of microorganisms present in the culture.

8. The method according to claim 7, characterized in that in the culture of microorganisms microorganisms of the species Methanoculleus chikugoensis make up at least 90% of the total number of microorganisms present in the culture.

9. The method according to at least one of claims 2 to 8, characterized in that a pure culture of the microorganism of the species Methanoculleus chikugoensis is added.

10. The method according to at least one of claims 2 to 9, characterized in that microorganisms of the species Methanoculleus chikugoensis the biomass are added as part of at least one immobilized culture of microorganisms.

1 1. Method according to at least one of claims 1 to 10, characterized in that in time for the addition of the microorganism of the species Methanoculleus chikugoensis additional biomass is added to the fermentation reactor.

12. The method according to at least one of claims 1 to 1 1, characterized in that the space load in the fermentation reactor by continuous addition of

Biomass is continuously increased.

13. The method according to at least one of claims 1 to 12, characterized in that the production of biogas from biomass at a space load of ≥ 0.5 kg oTS / m ³ d, preferably ≥ 4.0 kg oTS / m ³ d, especially preferably ≥ 8.0 kg oTS / m ³ d. 14. The method according to at least one of claims 1 to 13, characterized in that the production of biogas from biomass takes place under constant mixing of the fermentation substrate.

15. The method according to at least one of claims 1 to 14, characterized in that the production of biogas from biomass at a temperature of 20 ^ to 80 ⁰ C, preferably at a temperature of 40 ^< € to 50 ⁰ C is performed.

16. The method according to at least one of claims 1 to 15, characterized in that the addition of fermentation substrate and the addition of the microorganism of the type

Methanoculleus chikugoensis take place continuously.

17. The method according to at least one of claims 1 to 16, characterized in that the microorganism of the species Methanoculleus chikugoensis is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species

Methanoculleus chikugoensis accounts for between 10 ⁸ % and 50% of the total number of microorganisms present in the fermentation substrate.

18. The method according to claim 17, characterized in that the microorganism of the species Methanoculleus chikugoensis is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanoculleus chikugoensis between 10 ^"6 % and 25% of the total in Fermentation substrate present microorganisms.

19. The method according to claim 18, characterized in that the microorganism of the species Methanoculleus chikugoensis is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanoculleus chikugoensis between 10 ^'4 % and 10% of the total in Fermentation substrate present microorganisms.

20. The method according to claim 19, characterized in that the microorganism of the species Methanoculleus chikugoensis is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanoculleus chikugoensis between 10 ^'3 % and 1% of the total in Fermentation substrate present microorganisms. 21. Process according to at least one of Claims 1 to 20, characterized in that the microorganism of the species Methanoculleus chikugoensis is a microorganism of the strain Methanoculleus chikugoensis MG62.

22. Use of a microorganism of the species Methanoculleus chikugoensis for fermentative production of biogas from biomass.

23. Use according to claim 22, characterized in that the microorganism of the species Methanoculleus chikugoensis is a microorganism of the strain Methanoculleus chikugoensis MG62.

24. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 98.93% sequence identity with the nucleotide sequence SEQ ID No. 2.

25. Microorganism according to claim 24, characterized in that the nucleotide sequence contains a sequence region which has more than 99.2% sequence identity with the nucleotide sequence SEQ ID No. 2.

26. Microorganism according to claim 25, characterized in that the nucleotide sequence contains a sequence region which has more than 99.4% sequence identity with the nucleotide sequence SEQ ID No. 2.

27. Microorganism according to claim 26, characterized in that the nucleotide sequence contains a sequence region which has more than 99.6% sequence identity with the nucleotide sequence SEQ ID No. 2.

28. The microorganism according to claim 27, characterized in that the nucleotide sequence contains a sequence region which has more than 99.8% sequence identity with the nucleotide sequence SEQ ID No. 2.

29. Microorganism according to claim 28, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 2.

30. Culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, characterized in that in the culture of Microorganisms, a microorganism according to claims 24 to 29 is present, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

31. Culture of microorganisms according to claim 30, characterized in that the microorganism makes up at least ^{10 -2%} of the total number of microorganisms present in the culture.

32. Culture of microorganisms according to claim 31, characterized in that the microorganism at least 1% of the total number present in the culture

Constitutes microorganisms.

33. Culture of microorganisms according to claim 32, characterized in that the microorganism constitutes at least 10% of the total number of microorganisms present in the culture.

34. Culture of microorganisms according to claim 33, characterized in that the microorganism constitutes at least 50% of the total number of microorganisms present in the culture.

35. Culture of microorganisms according to claim 34, characterized in that the microorganism constitutes at least 90% of the total number of microorganisms present in the culture.

36. Culture of microorganisms according to claim 35, characterized in that it is a pure culture of a microorganism according to claims 24 to 29.

37. Culture of microorganisms according to at least one of claims 30 to 36, characterized in that it is an immobilized culture of microorganisms.

38. Use of a microorganism according to at least one of claims 24 to 29 for the fermentative production of biogas from biomass.

39. Use according to claim 38, characterized in that it is a process for the fermentative production of biogas from biomass according to at least one of claims 1 to 21. 40. Use of a culture of microorganisms according to at least one of claims 30 to 37 for the fermentative production of biogas from biomass.

41. Use according to claim 40, characterized in that it is a process for the fermentative production of biogas from biomass biomass according to at least one of claims 1 to 21.

In summary, a process for the production of biogas from biomass in a fermentation reactor is described, wherein the biomass is added to a microorganism of the species Methanoculleus chikugoensis.

Methanoculleus marisnigri

The present invention provides a method for producing biogas from biomass in a fermentation reactor. The biomass is added according to the invention a microorganism of the species Methanoculleus marisnigri.

Surprisingly, it has been shown that the addition of microorganisms of the species Methanoculleus marisnigri to the fermentation substrate, the amount of biogas formed can be significantly increased. At constant space load, the addition of microorganisms of the species Methanoculleus marisnigri causes a significant increase in the amount of biogas produced. Alternatively, the amount of methane produced can also be increased by increasing the volume load, and the addition of microorganisms of the species Methanoculleus marisnigri prevents the fermentation process from becoming unstable under the changed conditions. The use of microorganisms of the species Methanoculleus marisnigri therefore leads to an improvement in the efficiency and efficiency of biogas plants.

It is known to those skilled in the art which strains of microorganisms fall within the term "species Methanoculleus marisnigri". In particular, the term "Methanoculleus marisnigri" is also to be understood as meaning the "Methanogenium marisnigri" (Bacterial Nomenclature Up-To-Date, Approved List of the DSMZ - German Collection for Microorganisms and Cell Cultures GmbH, as of March 2010) of biogas by fermentation of organic substrates, microorganisms of the species Methanoculleus marisnigri are not yet known. According to a preferred embodiment of the present invention, a microorganism of the species Methanoculleus marisnigri is added in the form of a culture of microorganisms consisting predominantly of microorganisms of the species Methanoculleus marisnigri. In fermentation substrates of biogas plants microorganisms of the species could Methanoculleus marisnigri only in trace amounts of less than 10 ^'4% share of the total number of detected present microorganisms. Since the amount of microorganisms isolated from their natural occurrence is insufficient for the addition of the microorganisms, it is usually propagated in the form of a culture. In practice, it is found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out directly in the form of a culture of microorganisms.

The addition of the culture of Methanoculleus marisnigri can be carried out in the form of a culture suspension or in the form of dry, freeze-dried or moist cell pellets.

Since the already mentioned various positive effects on the fermentation process are associated with the microorganism species Methanoculleus marisnigri, this type of microorganism should be present in the added culture in a concentration exceeding the natural abundance. Of course, mixed cultures of any composition can be used for the addition. The only requirement is that the species Methanoculleus marisnigri is present in a concentration enriched in relation to the natural occurrence.

The determination of the proportions of different types of microorganisms in mixed cultures is not a problem for the expert. Thus, for example, using fluorescent-labeled oligosensors specifically the proportion of microorganisms of the species Methanoculleus marisnigri be identified in a mixture. As already mentioned, microorganisms of the species Methanoculleus marisnigri are preferably added to the fermentation substrate in the form of cultures of microorganisms, the cultures of microorganisms consisting predominantly of microorganisms of the species Methanoculleus marisnigri. If, in addition to the determination of the number of microorganisms of the species Methanoculleus marisnigri, the total number of microorganisms is also determined, the proportion of microorganisms of the species Methanoculleus marisnigri in the culture may be expressed as a percentage. Microorganisms of the species Methanoculleus marisnigri are in a mixed culture then predominantly present type of microorganisms, if they have the highest percentage of the various types of microorganisms present in the mixed culture. According to preferred embodiments of the present invention make microorganisms of the species Methanoculleus marisnigri at least 10 ^'4%, especially at least ^{10' 2%,} more preferably at least 1% of the total number of existing in the added to the fermentation substrate culture microorganisms. According to further preferred embodiments, microorganisms of the species Methanoculleus marisnigri make up at least 10%, in particular at least 50%, particularly preferably at least 90%, of the total number of microorganisms present in the culture.

According to a very particularly preferred embodiment, a pure culture of a microorganism of the species Methanoculleus marisnigri is added. Due to the specific metabolic processes and activities, the addition of a pure culture can especially contribute to an improved methane production.

According to another preferred embodiment of the present invention, a microorganism of the species Methanoculleus marisnigri is added as a constituent of at least one immobilized culture of microorganisms. Since the amount of microorganisms isolated from their natural occurrence is insufficient for the addition of the microorganisms, it is usually propagated in the form of a culture. In practice, it has been found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out in the form of an immobilized culture of microorganisms.

According to a preferred embodiment of the present invention, the microorganism of the species Methanoculleus marisnigri is added in an amount to the fermentation substrate, so that after addition of the proportion of the microorganism of the species Methanoculleus marisnigri between 10 ^"8 % and 50% of the total number present in the fermentation substrate

Constitutes microorganisms. In particular, depending on the fermenter size and thus as a function of the amount of fermentation substrate can be necessary to achieve the desired effect, an addition of very different amounts of microorganisms.

A microorganism of the species Methanoculleus marisnigri is particularly preferably added in an amount to the fermentation substrate that makes up after addition of the content of the microorganism of the species Methanoculleus marisnigri between ^{10 -6%} and 25% of the total number present in the fermentation substrate microorganisms. More preferably, the microorganism of the species Methanoculleus marisnigri is added to the fermentation substrate in an amount added that after addition of the proportion of the microorganism of the species Methanoculleus marisnigri is between 10 ^'4 % and 10% of the total number of microorganisms present in the fermentation substrate. Most preferably, the microorganism of the species Methanoculleus marisnigri is added to the fermentation substrate in an amount such that, after addition, the proportion of the microorganism of the species Methanoculleus marisnigri is between 10 ^-3 % and 1% of the total number of microorganisms present in the fermentation substrate.

According to a particularly preferred embodiment of the present invention, the microorganism of the species Methanoculleus marisnigri is a microorganism of the strain Methanoculleus marisnigri JRI. In all the embodiments explained in more detail above, which deal with the addition of a microorganism of the species Methanoculleus marisnigri, it is therefore preferable to add a microorganism of the strain Methanoculleus marisnigri JR1.

The present invention also encompasses the use of a microorganism of the species Methanoculleus marisnigri for the fermentative production of biogas from biomass. Preferably, a microorganism of the strain strain Methanoculleus marisnigri JR1 is used for the fermentative production of biogas from biomass.

The microorganisms Methanoculleus marisnigri SBG6 obtained from the fermentation substrate of a post-fermenter were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID No. 3 comprises 1440 nucleotides. Methanoculleus marisnigri JR1 was identified as the next relative. A comparison of the sequences revealed that there are a total of 38 exchanges of nucleotides or deletions. At a length of the particular nucleotide sequence of 1440 nucleotides of Methanoculleus marisnigri SBG6 and a length of the reference sequence of 1441 nucleotides, a 97.36% match is calculated using the FASTA algorithm.

The present invention also encompasses microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region exceeding 97.36%.

Sequence identity with the nucleotide sequence SEQ ID NO. 3 has. More preferably, the nucleotide sequence contains a sequence range greater than 97.4% or greater than

97.5% or more than 97.6% or more than 97.7% or more than 97.8% or more

97.9% or more than 98% has sequence identity with the nucleotide sequence SEQ ID NO: 3, and more preferably, the nucleotide sequence contains a sequence region having more than 98.5% sequence identity with the nucleotide sequence SEQ ID NO: 3. According to further preferred embodiments, the microorganism has a nucleotide sequence which contains a sequence region which has more than 99% sequence identity with the nucleotide sequence SEQ ID No. 3, and more preferably the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence Nucleotide sequence SEQ ID NO. 3 has. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 3.

In preferred embodiments of the present invention, SEQ ID NO: 3 may be compared to the starting nucleotide sequence at one or two positions or at three positions or at four positions or at five positions or at six positions or at seven positions or at eight positions or at nine positions or at ten

Positions or eleven positions or twelve positions or 13 positions or 14

Positions or 15 positions or 16 positions or 17 positions or 18 positions or 19 positions or 20 positions or 21 positions or 22

Positions or 23 positions or 24 positions or 25 positions or 26

Positions or at 27 positions or at 28 positions or at 29 positions or at 30

Positions or at 31 positions or at 32 positions or at 33 positions or at 34

Positions or at 35 positions or at 36 positions or at 37 positions or at 38 positions nucleotide mutations. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also encompasses a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein a microorganism having a nucleotide sequence containing a sequence region greater than 97.36 is present in the culture of microorganisms % Sequence identity with the nucleotide sequence of SEQ ID NO: 3, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 97.4% or greater than 97.5% or greater than 97 , 6% or more than 97.7% or more than 97.8% or more than 97.9% or more than 98% sequence identity with the nucleotide sequence SEQ ID NO: 3. More preferably, the nucleotide sequence contains a sequence region having more than 98.5% sequence identity with the nucleotide sequence SEQ ID NO: 3. According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence region having more than 99% sequence identity with the nucleotide sequence SEQ ID NO: 3 , Most preferably, the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 3.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 3.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 3 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 3 in a method described above in connection with microorganisms of the species Methanoculleus marisnigή for the fermentative production of biogas from biomass.

The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, the culture of microorganisms comprising at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 3 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. It is preferable to use a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID No. 3 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in a method described above in connection with microorganisms of the species Methanoculleus marisnigri for the fermentative production of biogas from biomass.

According to further preferred embodiments make said, characterized in terms of their nucleotide sequence microorganisms at least 10 ^'2%, preferably at least 1% of the total number of microorganisms present in the culture. Particularly preferably, the microorganisms make up at least 10%, particularly preferably at least 25%, of the total number of microorganisms present in the culture. Most preferably, the microorganisms described make up at least 50%, in particular at least 75%, of the total number of microorganisms present in the culture.

Also preferred are embodiments according to which the above-mentioned microorganisms make up at least 90% of the total number of microorganisms present in the culture. Particularly preferably it is a pure culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, which is a pure culture of a microorganism, as has been characterized above with respect to its nucleotide sequence.

Most preferably, in the cases described above, it is an immobilized culture of microorganisms.

In particular, the present invention includes the following aspects:

1. A process for the production of biogas from biomass in a fermentation reactor, characterized in that the biomass is a microorganism of the type

Methanoculleus marisnigri is added.

2. The method according to claim 1, characterized in that the microorganism of the species Methanoculleus marisnigri is added in the form of a culture of microorganisms, wherein microorganisms of the species Methanoculleus marisnigri at least 10 ^'4 % of

Total number of microorganisms present in the culture.

3. The method according to claim 2, characterized in that constitute in the culture of microorganisms microorganisms of the species Methanoculleus marisnigri at least 10 ^'2 % of the total number of microorganisms present in the culture.

4. The method according to claim 3, characterized in that in the culture of microorganisms microorganisms of the species Methanoculleus marisnigri make up at least 1% of the total number of microorganisms present in the culture. 5. The method according to claim 4, characterized in that in the culture of microorganisms microorganisms of the species Methanoculleus marisnigri make up at least 10% of the total number of microorganisms present in the culture.

6. The method according to claim 5, characterized in that in the culture of microorganisms microorganisms of the species Methanoculleus marisnigri account for at least 50% of the total number of microorganisms present in the culture.

7. The method according to claim 6, characterized in that in the culture of microorganisms microorganisms of the species Methanoculleus marisnigri make up at least 75% of the total number of microorganisms present in the culture.

8. The method according to claim 7, characterized in that in the culture of microorganisms microorganisms of the species Methanoculleus marisnigri make up at least 90% of the total number of microorganisms present in the culture.

9. The method according to at least one of claims 2 to 8, characterized in that a pure culture of the microorganism of the species Methanoculleus marisnigri is added.

10. The method according to at least one of claims 2 to 9, characterized in that microorganisms of the species Methanoculleus marisnigri the biomass are added as part of at least one immobilized culture of microorganisms.

1 1. Method according to at least one of claims 1 to 10, characterized in that in time for the addition of the microorganism of the species Methanoculleus marisnigri additional

Biomass is added to the fermentation reactor.

12. The method according to at least one of claims 1 to 1 1, characterized in that the space load in the fermentation reactor is continuously increased by the continuous addition of biomass.

13. The method according to at least one of claims 1 to 12, characterized in that the production of biogas from biomass at a space load of ≥ 0.5 kg oTS / m ³ d, preferably ≥ 4.0 kg oTS / m ³ d, especially preferably ≥ 8.0 kg oTS / m ³ d. 14. The method according to at least one of claims 1 to 13, characterized in that the production of biogas from biomass takes place under constant mixing of the fermentation substrate.

15. The method according to at least one of claims 1 to 14, characterized in that the production of biogas from biomass at a temperature of 20 ^ to 80 ⁰ C, preferably at a temperature of 40 ^< € to 50 ⁰ C is performed.

16. The method according to at least one of claims 1 to 15, characterized in that the addition of fermentation substrate and the addition of the microorganism of the type

Methanoculleus marisnigή take place continuously.

17. The method according to at least one of claims 1 to 16, characterized in that the microorganism of the species Methanoculleus marisnigri is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species

Methanoculleus marisnigri represents between 10 ^"8 % and 50% of the total number of microorganisms present in the fermentation substrate.

18. The method according to claim 17, characterized in that the microorganism of the species Methanoculleus marisnigri is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanoculleus marisnigri between 10 ^"6 % and 25% of the total in Fermentation substrate present microorganisms.

19. The method according to claim 18, characterized in that the microorganism of the species Methanoculleus marisnigri is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanoculleus marisnigri between 10 ^'4 % and 10% of the total in the Fermentation substrate present microorganisms.

20. The method according to claim 19, characterized in that the microorganism of the species Methanoculleus marisnigri is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanoculleus marisnigri between 10 ^'3 % and 1% of the total in Fermentation substrate present microorganisms. 21. Process according to at least one of Claims 1 to 20, characterized in that the microorganism of the species Methanoculleus marisnigri is a microorganism of the strain Methanoculleus marisnigri JR1.

22. Use of a microorganism of the species Methanoculleus marisnigri for the fermentative production of biogas from biomass.

23. Use according to claim 22, characterized in that the microorganism of the species Methanoculleus marisnigri is a microorganism of the strain Methanoculleus marisnigri JR1.

24. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 97.36% sequence identity with the nucleotide sequence SEQ ID No. 3.

25. Microorganism according to claim 24, characterized in that the nucleotide sequence contains a sequence region which has more than 98% sequence identity with the nucleotide sequence SEQ ID No. 3.

26. Microorganism according to claim 25, characterized in that the nucleotide sequence contains a sequence region which has more than 98.5% sequence identity with the nucleotide sequence SEQ ID No. 3.

27. Microorganism according to claim 26, characterized in that the nucleotide sequence contains a sequence region which has more than 99% sequence identity with the nucleotide sequence SEQ ID No. 3.

28. Microorganism according to claim 27, characterized in that the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 3.

29. Microorganism according to claim 28, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 3.

30. Culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, characterized in that in the culture of Microorganisms, a microorganism according to claims 24 to 29 is present, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

31. Culture of microorganisms according to claim 30, characterized in that the microorganism makes up at least ^{10 -2%} of the total number of microorganisms present in the culture.

32. Culture of microorganisms according to claim 31, characterized in that the microorganism at least 1% of the total number present in the culture

Constitutes microorganisms.

33. Culture of microorganisms according to claim 32, characterized in that the microorganism constitutes at least 10% of the total number of microorganisms present in the culture.

34. Culture of microorganisms according to claim 33, characterized in that the microorganism constitutes at least 50% of the total number of microorganisms present in the culture.

35. Culture of microorganisms according to claim 34, characterized in that the microorganism constitutes at least 90% of the total number of microorganisms present in the culture.

36. Culture of microorganisms according to claim 35, characterized in that it is a pure culture of a microorganism according to claims 24 to 29.

37. Culture of microorganisms according to at least one of claims 30 to 36, characterized in that it is an immobilized culture of microorganisms.

38. Use of a microorganism according to at least one of claims 24 to 29 for the fermentative production of biogas from biomass.

39. Use according to claim 38, characterized in that it is a process for the fermentative production of biogas from biomass according to at least one of claims 1 to 21. 40. Use of a culture of microorganisms according to at least one of claims 30 to 37 for the fermentative production of biogas from biomass.

41. Use according to claim 40, characterized in that it is a process for the fermentative production of biogas from biomass biomass according to at least one of claims 1 to 21.

In summary, a process for the production of biogas from biomass in a fermentation reactor is described, wherein the biomass is added to a microorganism of the species Methanoculleus marisnigri.

Methanoculleus thermophilus

The present invention provides a method for producing biogas from biomass in a fermentation reactor. The biomass is added according to the invention a microorganism of the species Methanoculleus thermophilus.

Surprisingly, it has been shown that the addition of microorganisms of the species Methanoculleus thermophilus to the fermentation substrate, the amount of biogas formed can be significantly increased. At constant space load, the addition of microorganisms of the species Methanoculleus thermophilus causes a significant increase in the amount of biogas formed. Alternatively, the amount of methane formed can also be increased by increasing the volume load, with the addition of microorganisms of the species Methanoculleus thermophilus preventing the fermentation process from becoming unstable under the altered conditions. The use of microorganisms of the species Methanoculleus thermophilus therefore leads to an improvement in efficiency and efficiency of biogas plants.

It is known to those skilled in the art which strains of microorganisms fall under the term "species Methanoculleus thermophilus." In particular, the "Methanoculleus thermophilus" species are also to be understood as meaning the "Methanogenium thermophilicum" and "Methanogenium frittonir" and microorganisms which have been identified by the orthographic incorrect name "Methanoculleus thermophilicus" (Bacterial Nomenclature Up-To-Date, Approved List of the DSMZ - German Collection for Microorganisms and Cell Cultures GmbH, as of March 2010). In connection with the Production of biogas by fermentation of organic substrates are microorganisms of the species Methanoculleus thermophilus not yet known.

According to a preferred embodiment of the present invention, a microorganism of the species Methanoculleus thermophilus is added in the form of a culture of microorganisms consisting predominantly of microorganisms of the species Methanoculleus thermophilus. In fermentation substrates of biogas plants microorganisms of the species could be detected by Methanoculleus present microorganisms only in trace amounts of less than 10 ^'4% share of the total number thermophilus. Since the amount of microorganisms isolated from their natural occurrence is insufficient for the addition of the microorganisms, it is usually propagated in the form of a culture. In practice, it is found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out directly in the form of a culture of microorganisms.

The addition of the culture of Methanoculleus thermophilus can be carried out in the form of a culture suspension or in the form of dry, freeze-dried or moist cell pellets.

Since the already mentioned various positive effects on the fermentation process are associated with the microorganism species Methanoculleus thermophilus, this type of microorganism should be present in the added culture in a concentration exceeding the natural abundance. Of course, mixed cultures of any composition can be used for the addition. All that is required is that the species Methanoculleus thermophilus is present in a concentration which is enriched in relation to the natural occurrence.

The determination of the proportions of different types of microorganisms in mixed cultures is not a problem for the person skilled in the art. For example, the proportion of microorganisms of the species Methanoculleus thermophilus in a mixture can be specifically identified with the aid of fluorescence-labeled oligopeptides. As already mentioned, microorganisms of the species Methanoculleus thermophilus are preferably added to the fermentation substrate in the form of cultures of microorganisms, the cultures of microorganisms consisting predominantly of microorganisms of the species Methanoculleus thermophilus. If, in addition to the determination of the number of microorganisms of the species Methanoculleus thermophilus, the total number of microorganisms is also determined, the percentage of microorganisms of the species Methanoculleus thermophilus in the culture can be stated as a percentage. Microorganisms of the species Methanoculleus thermophilus are in a mixed culture then the predominantly present type of microorganisms, if they have the highest percentage of the various types of microorganisms present in the mixed culture.

According to preferred embodiments of the present invention make microorganisms of the species Methanoculleus thermophilus is at least 10 ^'4%, especially at least ^{10' 2%,} more preferably at least 1% of the total number existing in the added to the fermentation substrate culture microorganisms. According to further preferred embodiments, microorganisms of the species Methanoculleus thermophilus account for at least 10%, in particular at least 50%, particularly preferably at least 90%, of the total number of microorganisms present in the culture.

According to a very particularly preferred embodiment, a pure culture of a microorganism of the species Methanoculleus thermophilus is added. Due to the specific metabolic processes and activities, the addition of a pure culture can especially contribute to an improved methane production.

According to a further preferred embodiment of the present invention, a microorganism of the species Methanoculleus thermophilus is added as a constituent of at least one immobilized culture of microorganisms. As for the addition of the

Microorganisms the amount of isolated from their natural occurrence

Microorganisms is insufficient, is usually made an increase in the form of a culture. In practice it has been found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily achieved in the form of an immobilized culture of

Microorganisms is made.

According to a preferred embodiment of the present invention, the microorganism of the species Methanoculleus thermophilus is added in an amount to the fermentation substrate, so that after addition of the proportion of microorganism of the species Methanoculleus thermophilus between 10 ^{~ 8} % and 50% of the total number of present in the fermentation substrate microorganisms accounts. In particular, depending on the fermenter size and thus as a function of the amount of fermentation substrate can be necessary to achieve the desired effect, an addition of very different amounts of microorganisms. A microorganism of the species Methanoculleus thermophilus is particularly preferably added in an amount to the fermentation substrate that makes up after addition of the content of the microorganism of the species Methanoculleus thermophilus between ^{10 -6%} and 25% of the total number present in the fermentation substrate microorganisms. More preferably, the microorganism of the species Methanoculleus thermophilus is added to the fermentation substrate in an amount such that, after addition, the proportion of the microorganism of the species Methanoculleus thermophilus is between 10 ^'4 % and 10% of the total number of microorganisms present in the fermentation substrate. The microorganism of the species Methanoculleus thermophilus is very particularly preferably added in an amount to the fermentation substrate that makes up after addition of the content of the microorganism of the species Methanoculleus thermophilus between 10 ^"3% and 1% of the total number present in the fermentation substrate microorganisms.

According to a particularly preferred embodiment of the present invention, the microorganism of the species Methanoculleus thermophilus is a microorganism of the strain Archaeon clone HDBW-WA02. In all of the above-described embodiments, which deal with the addition of a microorganism of the species Methanoculleus thermophilus, so preferably a microorganism of the strain Archaeon clone HDBW-WA02 is added.

The present invention also encompasses the use of a microorganism of the species Methanoculleus thermophilus for the fermentative production of biogas from biomass. Preferably, a microorganism of the strain Archaeon clone HDBW-WA02 is used for the fermentative production of biogas from biomass.

The microorganisms Methanoculleus thermophilus SBG27 obtained from the fermentation substrate of a secondary fermenter were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID No. 24 comprises 1124 nucleotides. The next related sequence identified was the non-cultured Archaeon clone HDBW-WA02. A comparison of the sequences revealed that there are a total of 1 1 exchanges of nucleotides or deletions. With a length of the determined nucleotide sequence of Archaeon clone HDBW-WA02, SBG27 of 1124 nucleotides and a length of the reference sequence of 1053 nucleotides, the FASTA algorithm gives a 98.96% match.

The present invention also encompasses microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region of greater than 98.96%. Sequence identity with the nucleotide sequence SEQ ID NO. 24 has. More preferably, the nucleotide sequence contains a sequence range greater than 99.0% or greater than 99.1% or greater than 99.2% or greater than 99.3% or greater than 99.4% or greater than 99.5%. or more than 99.6% sequence identity with the nucleotide sequence SEQ ID NO: 24, and more preferably, the nucleotide sequence contains a sequence region having more than 99.7% sequence identity with the nucleotide sequence SEQ ID NO: 24.

According to further preferred embodiments, the microorganism has a nucleotide sequence which contains a sequence region which has more than 99.8% sequence identity with the nucleotide sequence SEQ ID No. 24, and more preferably the nucleotide sequence contains a sequence region which has more than 99.9% sequence identity having the nucleotide sequence of SEQ ID NO: 24. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 24.

In preferred embodiments of the present invention, SEQ ID NO: 24 may be compared to the starting nucleotide sequence at one or two positions or at three positions or at four positions or at five positions or at six positions or at seven positions or at eight positions or at nine positions or nucleotide mutations at ten positions or eleven positions. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also encompasses a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, in which culture of microorganisms a microorganism is present which has a nucleotide sequence containing a sequence region greater than 98.96 % Sequence identity with the nucleotide sequence SEQ ID NO: 24, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 99.0% or greater than 99.1% or greater than 99 , 2% or more than 99.3% or more than 99.4% or more than 99.5% or more than 99.6% sequence identity with the nucleotide sequence SEQ ID NO: 24. More preferably, the nucleotide sequence contains a sequence region having more than 99.7% sequence identity with the nucleotide sequence SEQ ID NO: 24. According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence region having more than 99.8% sequence identity with the nucleotide sequence SEQ ID NO. 24 has. Most preferably, the nucleotide sequence contains a sequence region which has more than 99.9% sequence identity with the nucleotide sequence SEQ ID No. 24.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 24.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 24 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 24 in a method described above in connection with microorganisms of the species Methanoculleus thermophilus for the fermentative production of biogas from biomass.

The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, the culture of microorganisms comprising at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO. 24 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. Preference is given to the use of a culture of microorganisms for the fermentative production of biogas from biomass, the culture of microorganisms comprising at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID No. 24 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in a method described above in connection with microorganisms of the species Methanoculleus thermophilus for the fermentative production of biogas from biomass.

According to further preferred embodiments make said, characterized in terms of their nucleotide sequence microorganisms at least 10 ^'2%, preferably at least 1% of the total number of microorganisms present in the culture. Particularly preferably, the microorganisms make up at least 10%, particularly preferably at least 25%, of the total number of microorganisms present in the culture. Most preferably, the microorganisms described make up at least 50%, in particular at least 75%, of the total number of microorganisms present in the culture.

Also preferred are embodiments according to which the above-mentioned microorganisms make up at least 90% of the total number of microorganisms present in the culture. Particularly preferably it is a pure culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, which is a pure culture of a microorganism, as has been characterized above with respect to its nucleotide sequence.

Most preferably, in the cases described above, it is an immobilized culture of microorganisms.

In particular, the present invention includes the following aspects:

1. A process for the production of biogas from biomass in a fermentation reactor, characterized in that the biomass is a microorganism of the type

Methanoculleus thermophilus is added.

2. The method according to claim 1, characterized in that the microorganism of the species Methanoculleus thermophilus is added in the form of a culture of microorganisms, wherein microorganisms of the species Methanoculleus thermophilus at least 10 ^'4 % of

Total number of microorganisms present in the culture.

3. The method according to claim 2, characterized in that in the culture of microorganisms microorganisms of the species Methanoculleus thermophilus make up at least 10 ^{' 2} % of the total number of microorganisms present in the culture.

4. The method according to claim 3, characterized in that in the culture of microorganisms microorganisms of the species Methanoculleus thermophilus make up at least 1% of the total number of microorganisms present in the culture. 5. The method according to claim 4, characterized in that in the culture of microorganisms microorganisms of the species Methanoculleus thermophilus make up at least 10% of the total number of microorganisms present in the culture.

6. The method according to claim 5, characterized in that in the culture of microorganisms microorganisms of the species Methanoculleus thermophilus make up at least 50% of the total number of microorganisms present in the culture.

7. The method according to claim 6, characterized in that in the culture of microorganisms microorganisms of the species Methanoculleus thermophilus make up at least 75% of the total number of microorganisms present in the culture.

8. The method according to claim 7, characterized in that in the culture of microorganisms microorganisms of the species Methanoculleus thermophilus make up at least 90% of the total number of microorganisms present in the culture.

9. The method according to at least one of claims 2 to 8, characterized in that a pure culture of the microorganism of the species Methanoculleus thermophilus is added.

10. The method according to at least one of claims 2 to 9, characterized in that microorganisms of the species Methanoculleus thermophilus the biomass are added as part of at least one immobilized culture of microorganisms.

1 1. Method according to at least one of claims 1 to 10, characterized in that in time for the addition of the microorganism of the species Methanoculleus thermophilus additional biomass is added to the fermentation reactor.

12. The method according to at least one of claims 1 to 1 1, characterized in that the space load in the fermentation reactor by continuous addition of

Biomass is continuously increased.

13. The method according to at least one of claims 1 to 12, characterized in that the production of biogas from biomass at a space load of ≥ 0.5 kg oTS / m ³ d, preferably ≥ 4.0 kg oTS / m ³ d, especially preferably ≥ 8.0 kg oTS / m ³ d. 14. The method according to at least one of claims 1 to 13, characterized in that the production of biogas from biomass takes place under constant mixing of the fermentation substrate.

15. The method according to at least one of claims 1 to 14, characterized in that the production of biogas from biomass at a temperature of 20 ^ to 80 ⁰ C, preferably at a temperature of 40 ^< € to 50 ⁰ C is performed.

16. The method according to at least one of claims 1 to 15, characterized in that the addition of fermentation substrate and the addition of the microorganism of the type

Methanoculleus thermophilus take place continuously.

17. The method according to at least one of claims 1 to 16, characterized in that the microorganism of the species Methanoculleus thermophilus is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species

Methanoculleus thermophilus accounts for between 10 ^"8 % and 50% of the total number of microorganisms present in the fermentation substrate.

18. The method according to claim 17, characterized in that the microorganism of the species Methanoculleus thermophilus is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanoculleus thermophilus between 10 ^"6 % and 25% of the total in Fermentation substrate present microorganisms.

19. The method according to claim 18, characterized in that the microorganism of the species Methanoculleus thermophilus is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanoculleus thermophilus between 10 ^'4 % and 10% of the total in Fermentation substrate present microorganisms.

20. The method according to claim 19, characterized in that the microorganism of the species Methanoculleus thermophilus is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanoculleus thermophilus between 10 ^'3 % and 1% of the total in Fermentation substrate present microorganisms. 21. Process according to at least one of Claims 1 to 20, characterized in that the microorganism of the species Methanoculleus thermophilus is a microorganism of the strain Archaeon clone HDBW-WA02.

22. Use of a microorganism of the species Methanoculleus thermophilus for the fermentative production of biogas from biomass.

23. Use according to claim 22, characterized in that the microorganism of the species Methanoculleus thermophilus is a microorganism of the strain Archaeon clone HDBW-WA02.

24. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 98.96% sequence identity with the nucleotide sequence SEQ ID No. 24.

25. Microorganism according to claim 24, characterized in that the nucleotide sequence contains a sequence region which has more than 99.2% sequence identity with the nucleotide sequence SEQ ID No. 24.

26. Microorganism according to claim 25, characterized in that the nucleotide sequence contains a sequence region which has more than 99.4% sequence identity with the nucleotide sequence SEQ ID No. 24.

27. Microorganism according to claim 26, characterized in that the nucleotide sequence contains a sequence region which has more than 99.6% sequence identity with the nucleotide sequence SEQ ID No. 24.

28. A microorganism according to claim 27, characterized in that the nucleotide sequence contains a sequence region which has more than 99.8% sequence identity with the nucleotide sequence SEQ ID No. 24.

29. Microorganism according to claim 28, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 24.

30. Culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, characterized in that in the culture of Microorganisms, a microorganism according to claims 24 to 29 is present, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

31. Culture of microorganisms according to claim 30, characterized in that the microorganism makes up at least ^{10 -2%} of the total number of microorganisms present in the culture.

32. Culture of microorganisms according to claim 31, characterized in that the microorganism at least 1% of the total number present in the culture

Constitutes microorganisms.

33. Culture of microorganisms according to claim 32, characterized in that the microorganism constitutes at least 10% of the total number of microorganisms present in the culture.

34. Culture of microorganisms according to claim 33, characterized in that the microorganism constitutes at least 50% of the total number of microorganisms present in the culture.

35. Culture of microorganisms according to claim 34, characterized in that the microorganism constitutes at least 90% of the total number of microorganisms present in the culture.

36. Culture of microorganisms according to claim 35, characterized in that it is a pure culture of a microorganism according to claims 24 to 29.

37. Culture of microorganisms according to at least one of claims 30 to 36, characterized in that it is an immobilized culture of microorganisms.

38. Use of a microorganism according to at least one of claims 24 to 29 for the fermentative production of biogas from biomass.

39. Use according to claim 38, characterized in that it is a process for the fermentative production of biogas from biomass according to at least one of claims 1 to 21. 40. Use of a culture of microorganisms according to at least one of claims 30 to 37 for the fermentative production of biogas from biomass.

41. Use according to claim 40, characterized in that it is a process for the fermentative production of biogas from biomass biomass according to at least one of claims 1 to 21.

In summary, a method for producing biogas from biomass in a fermentation reactor is described, wherein the biomass is added to a microorganism of the species Methanoculleus thermophilus.

Methanobacterium formicicum

The present invention provides a method for producing biogas from biomass in a fermentation reactor. The biomass is added according to the invention a microorganism of the species Methanobacterium formicicum.

Surprisingly, it has been shown that the addition of microorganisms of the species Methanobacterium formicicum to the fermentation substrate, the amount of biogas formed can be significantly increased. At constant volume loading, the addition of microorganisms of the species Methanobacterium formicicum causes a significant increase in the amount of biogas produced. Alternatively, the amount of methane formed can also be increased by increasing the space load, with the addition of microorganisms of the species Methanobacterium formicicum preventing the fermentation process from becoming unstable under the altered conditions. The use of microorganisms of the species Methanobacterium formicicum therefore leads to an improvement in the efficiency and efficiency of biogas plants.

The person skilled in the art knows which strains of microorganisms fall under the term "species Methanobacterium formicicum." In connection with the production of biogas by fermentation of organic substrates, microorganisms of the species Methanobacterium formicicum are hitherto unknown.

According to a preferred embodiment of the present invention, a microorganism of the species Methanobacterium formicicum in the form of a culture of Microorganisms added, which consists predominantly of microorganisms of the species Methanobacterium formicicum. In fermentation substrates of biogas plants could microorganisms of the species Methanobacterium formicicum only in trace amounts of less than 10 ^'4% share of the total number of detected present microorganisms. Since the amount of microorganisms isolated from their natural occurrence is insufficient for the addition of the microorganisms, it is usually propagated in the form of a culture. In practice, it is found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out directly in the form of a culture of microorganisms.

The addition of the culture of Methanobacterium formicicum can be carried out in the form of a culture suspension or in the form of dry, freeze-dried or moist cell pellets.

Since the already mentioned various positive effects on the fermentation process are associated with the microorganism species Methanobacterium formicicum, this type of microorganism should be present in the added culture in a concentration exceeding the natural abundance. Mixed cultures containing Methanobacterium formicicum along with any other microorganisms may also be used for the addition. All that is required is that the species Methanobacterium formicicum is present in a concentration which is enriched in relation to the natural occurrence.

For the determination of the proportions of different types of microorganisms in mixed cultures, various methods known to the person skilled in the art are known. Thus, for example, with the aid of fluorescence-labeled oligosensors specifically the proportion of

Microorganisms of the species Methanobacterium formicicum can be identified in a mixture. As already mentioned, microorganisms of the species Methanobacterium formicicum are preferably added to the fermentation substrate in the form of cultures of microorganisms, the cultures of microorganisms consisting predominantly of microorganisms of the species Methanobacterium formicicum. In addition to the determination of the number of microorganisms of the species

Methanobacterium formicicum also determines the total number of microorganisms, the percentage of microorganisms of the species Methanobacterium formicicum in the culture can be stated in percent. Microorganisms of the species Methanobacterium formicicum in a mixed culture are then the predominant species of microorganisms when they have the highest percentage of the various species present in the mixed culture

Have microorganisms. According to preferred embodiments of the present invention make microorganisms of the species Methanobacterium formicicum at least 10 ^'4%, especially at least ^{10' 2%,} more preferably at least 1% of the total number of existing in the added to the fermentation substrate culture microorganisms. According to further preferred embodiments, microorganisms of the species Methanobacterium formicicum account for at least 10%, in particular at least 50%, particularly preferably at least 90%, of the total number of microorganisms present in the culture.

According to a further preferred embodiment, a pure culture of a microorganism of the species Methanobacterium formicicum is added. Due to the specific metabolic processes and activities, the addition of a pure culture can especially contribute to an improved methane production.

According to another preferred embodiment of the present invention, a microorganism of the species Methanobacterium formicicum is added as a constituent of at least one immobilized culture of microorganisms. Since the amount of microorganisms isolated from their natural occurrence is insufficient for the addition of the microorganisms, it is usually propagated in the form of a culture. In practice, it has been found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out in the form of an immobilized culture of microorganisms.

According to a preferred embodiment of the present invention, the microorganism of the species Methanobacterium formicicum is added in an amount to the fermentation substrate, so that after addition of the proportion of the microorganism of the species Methanobacterium formicicum between 10 ^'8 % and 50% of the total number present in the fermentation substrate

Constitutes microorganisms. In particular, depending on the fermenter size and thus as a function of the amount of fermentation substrate can be necessary to achieve the desired effect, an addition of very different amounts of microorganisms.

A microorganism of the species Methanobacterium formicicum is particularly preferred in an amount added to the fermentation substrate, that after addition the content of the microorganism of the species Methanobacterium formicicum between ^{10 -6%} and 25% of the total number of microorganisms present in the fermentation substrate by weight. Particularly preferably, the microorganism of the species Methanobacterium formicicum is added in an amount added to the fermentation substrate that after addition of the proportion of the microorganism of the species Methanobacterium formicicum accounts for between 10 ^'4 % and 10% of the total number of present in the fermentation substrate microorganisms. Most preferably, the microorganism of the species Methanobacterium formicicum is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanobacterium formicicum accounts for between 10 ^'3 % and 1% of the total number of present in the fermentation substrate microorganisms.

According to a particularly preferred embodiment of the present invention, the microorganism of the species Methanobacterium formicicum is a microorganism of the strain Methanobacterium sp., Clone SMS-sludge-11. In all of the above-described embodiments, which deal with the addition of a microorganism of the species Methanobacterium formicicum, so preferably a microorganism of the strain Methanobacterium sp., Cloned SMS sludge-11 is added.

According to a further particularly preferred embodiment of the present invention, the microorganism of the species Methanobacterium formicicum is a microorganism of the strain Methanobacterium sp. 169. In all of the embodiments explained in more detail above, which deal with the addition of a microorganism of the species Methanobacterium formicicum, a microorganism of the strain Methanobacterium sp. 169 added.

According to a further particularly preferred embodiment of the present invention, the microorganism of the species Methanobacterium formicicum is a microorganism of the strain Archaeon clone CG-4. In all embodiments described above, which deal with the addition of a microorganism of the species Methanobacterium formicicum, so preferably a microorganism of the strain Archaeon clone CG-4 is added.

The present invention also encompasses the use of a microorganism of the species Methanobacterium formicicum for the fermentative production of biogas from biomass. Preference is given to the fermentative production of biogas from biomass, a microorganism of the strain Methanobacterium sp. Clone SMS-sludge-11 used. Also preferred for fermentative production of biogas from biomass is a microorganism of the strain Methanobacterium sp. 169 used. Also preferably, a microorganism of the strain Archaeon clone CG-4 is used for the fermentative production of biogas from biomass. The microorganisms obtained from the fermentation substrate of a post-fermenter

Methanobacterium formicicum SBG4 were subjected to sequence analysis. The determined 16S rRNA sequence SEQ ID No. 1 comprises 1 133 nucleotides. As the next related 16S rRNA sequence, the sequence of uncultivated Methanobacterium sp. clone

SMS-sludge-11 identified. A comparison of the sequences revealed that a total of 26

Exchanges of nucleotides or deletions are present. At a length of the particular

Nucleotide sequence of Methanobacterium formicicum SBG4 of 133 nucleotides and one

Length of the reference sequence of 1 128 nucleotides is calculated using the FASTA algorithm a 97.70% agreement.

The present invention also includes microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having more than 97.70% sequence identity with the nucleotide sequence of SEQ ID NO: 1. More preferably, the nucleotide sequence contains a sequence range greater than 97.8% or greater than 97.9% or greater than 98.0% or greater than 98.1% or greater than 98.2% or greater than 98.3%. or more than 98.4% or more than 98.5% sequence identity with the nucleotide sequence SEQ ID NO: 1, and more preferably, the nucleotide sequence contains a sequence region having more than 99% sequence identity with the nucleotide sequence SEQ ID NO: 1.

According to further preferred embodiments, the microorganism has a nucleotide sequence containing a sequence region having more than 99.5% sequence identity with the nucleotide sequence SEQ ID NO: 1, and more preferably the nucleotide sequence contains a sequence region having greater than 99.8% sequence identity having the nucleotide sequence of SEQ ID NO: 1. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 1.

In preferred embodiments of the present invention, SEQ ID NO: 1 may be compared to the starting nucleotide sequence at one or two positions or at three positions or at four positions or at five positions or at six positions or at seven positions or at eight positions or at nine positions or ten positions or eleven positions or twelve positions or 13 positions or 14 positions or 15 positions or 16 positions or 17 positions or 18 positions or 19 positions or 20 positions or 21 positions or on 22 positions or 23 positions or 24 positions or 25 positions or 26 Positions nucleotide mutations present. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also includes a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism is present, which contains a

Nucleotide sequence containing a sequence range exceeding 97.70%

Sequence identity with the nucleotide sequence SEQ ID NO. 1, wherein the

Microorganism accounts for at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 97.8% or greater than 97.9% or greater than 98 , 0% or more than 98.1% or more than 98.2% or more than 98.3% or more than 98.4% or more than 98.5% sequence identity with the nucleotide sequence SEQ ID NO: 1. Particularly preferably, the nucleotide sequence contains a sequence region which has more than 99% sequence identity with the nucleotide sequence SEQ ID No. 1.

According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range of more than 99.5% sequence identity with the nucleotide sequence SEQ ID NO. 1 has. Most preferably, the nucleotide sequence contains a sequence region which has more than 99.8% sequence identity with the nucleotide sequence SEQ ID No. 1.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 1.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 1 for fermentative purposes

Generation of biogas from biomass. It is preferable to use a

A microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 1 has been described in a method described above in connection with microorganisms of the species Methanobacterium formicicum for the fermentative production of biogas from biomass.

The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, the culture of microorganisms comprising at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 1 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. Preference is given to the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID No. 1 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in a method described above in connection with microorganisms of the species Methanobacterium formicicum for the fermentative production of biogas from biomass.

The microorganisms Methanobacterium formicicum SBG8 obtained from the fermentation substrate of a post-fermenter were subjected to a sequence analysis. The 16S rRNA sequence SEQ ID NO: 5 comprises 903 nucleotides. The next relative was Methanobacterium sp. 169 identified. A comparison of the nucleotide sequences revealed that a total of 22 exchanges of nucleotides or deletions are present. At a length of the specific nucleotide sequence of 903 nucleotides of Methanobacterium formicicum SBG8 and a length of the reference sequence of 91 1 nucleotides, a 97.56% match is calculated using the FASTA algorithm.

The present invention also includes microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having more than 97.56% sequence identity with the nucleotide sequence of SEQ ID NO: 5. More preferably, the nucleotide sequence contains a sequence range greater than 97.6% or greater than 97.7% or greater than 97.8% or greater than 97.9% or greater than 98.0% or greater than 98.1%. or more than 98.2% or greater than 98.3% or greater than 98.4% or greater than 98.5% sequence identity to the nucleotide sequence of SEQ ID NO: 5, and more preferably the nucleotide sequence contains a sequence region greater than 99% sequence identity with the nucleotide sequence SEQ ID NO: 5. According to further preferred embodiments, the microorganism has a nucleotide sequence containing a sequence region having more than 99.5% sequence identity with the nucleotide sequence SEQ ID NO: 5 and more preferably the nucleotide sequence contains a sequence region having greater than 99.8% sequence identity having the nucleotide sequence of SEQ ID NO: 5. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 5.

In preferred embodiments of the present invention, SEQ ID NO: 5 can be compared to the starting nucleotide sequence at one or two positions or at three positions or at four positions or at five positions or at six positions or at seven positions or at eight positions or at nine positions or at ten

Positions or eleven positions or twelve positions or 13 positions or 14

Positions or 15 positions or 16 positions or 17 positions or 18 positions or 19 positions or 20 positions or 21 positions or 22

Positions nucleotide mutations present. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also includes a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism is present, which contains a

Having a nucleotide sequence containing a sequence region exceeding 97.56%

Sequence identity with the nucleotide sequence SEQ ID NO. 5, wherein the

Microorganism accounts for at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 97.6% or greater than 97.7% or greater than 97 , 8% or more than 97.9% or more than 98.0% or more than 98.1% or more than 98.2% or more than 98.3% or more than 98.4% or more than 98, 5% sequence identity with the nucleotide sequence SEQ ID NO: 5. Particularly preferably, the nucleotide sequence contains a sequence region which has more than 99% sequence identity with the nucleotide sequence SEQ ID No. 5.

According to further preferred embodiments, the culture suitable for use in a process for the fermentative production of biogas from biomass is Microorganisms, a microorganism having a nucleotide sequence having a sequence region having more than 99.5% sequence identity with the nucleotide sequence of SEQ ID NO: 5. Most preferably, the nucleotide sequence contains a sequence region which has more than 99.8% sequence identity with the nucleotide sequence SEQ ID No. 5.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 5.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 5 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 5 in a method described above in connection with microorganisms of the species Methanobacterium formicicum for the fermentative production of biogas from biomass.

The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO. 5 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. It is preferable to use a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID No. 5 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in a method described above in connection with microorganisms of the species Methanobacterium formicicum for the fermentative production of biogas from biomass.

The microorganisms Methanobacterium formicicum SBG25 obtained from the fermentation substrate of a postgrader were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID NO: 22 comprises 914 nucleotides. The closest related sequence identified was a 16S rRNA sequence of the uncultured Archaeon clone CG-4. A comparison of the sequences revealed that a total of 27 exchanges of Nucleotides or deletions. At a length of the specific nucleotide sequence of 914 nucleotides of Methanobacterium formicicum SBG25 and a length of the reference sequence of 928 nucleotides, a 97.05% match is calculated using the FASTA algorithm.

The present invention also includes microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having more than 97.05% sequence identity with the nucleotide sequence of SEQ ID NO: 22. More preferably, the nucleotide sequence contains a sequence range greater than 97.1% or greater than 97.2% or greater than 97.3% or greater than 97.4% or greater than 97.5% or greater than 97.6%. or more than 97.7% or more than 97.8% or more than 97.9% or more than 98.0% sequence identity with the nucleotide sequence SEQ ID NO: 22, and more preferably the nucleotide sequence contains a sequence region greater than 98.5% or greater than 99.0% sequence identity with the nucleotide sequence SEQ ID NO: 22.

According to further preferred embodiments, the microorganism has a nucleotide sequence containing a sequence region having more than 99.5% sequence identity with the nucleotide sequence SEQ ID NO: 22, and more preferably the nucleotide sequence contains a sequence region having greater than 99.8% sequence identity having the nucleotide sequence SEQ ID NO: 22. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 22.

In preferred embodiments of the present invention, SEQ ID NO: 22 may be compared to the starting nucleotide sequence at one or two positions or at three positions or at four positions or at five positions or at six positions or at seven positions or at eight positions or at nine positions or ten positions or eleven positions or twelve positions or 13 positions or 14 positions or 15 positions or 16 positions or 17 positions or 18 positions or 19 positions or 20 positions or 21 positions or on 22 positions or at 23 positions or at 24 positions or at 25 positions or at 26 positions or at 27 positions nucleotide mutations. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also includes a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism is present, which contains a A nucleotide sequence containing a sequence region having more than 97.05% sequence identity with the nucleotide sequence of SEQ ID NO: 22, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 97.1% or greater than 97.2% or greater than 97 , 3% or more than 97.4% or more than 97.5% or more than 97.6% or more than 97.7% or more than 97.8% or more than 97.9% or more than 98, 0% sequence identity with the nucleotide sequence SEQ ID NO: 22. Particularly preferably, the nucleotide sequence contains a sequence region which has more than 98.5% or more, ie 99.0% sequence identity, with the nucleotide sequence SEQ ID No. 22.

According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range of more than 99.5% sequence identity with the nucleotide sequence SEQ ID NO. 22 has. Most preferably, the nucleotide sequence contains a sequence region which has more than 99.8% sequence identity with the nucleotide sequence SEQ ID No. 22.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 22.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 22 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 22 in a method described above in connection with microorganisms of the species Methanobacterium formicicum for the fermentative production of biogas from biomass.

The present invention also includes the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID No. 22 and wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture. Preference is given to the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID No. 22 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in a method described above in connection with microorganisms of the species Methanobacterium formicicum for the fermentative production of biogas from biomass.

According to further preferred embodiments make said, characterized in terms of their nucleotide sequence microorganisms at least 10 ^'2%, preferably to at least 1% of the total present in the culture of microorganisms. Particularly preferably, the microorganisms make up at least 10%, particularly preferably at least 25%, of the total number of microorganisms present in the culture. Most preferably, the microorganisms described make up at least 50%, in particular at least 75%, of the total number of microorganisms present in the culture.

Also preferred are embodiments according to which the above-mentioned microorganisms make up at least 90% of the total number of microorganisms present in the culture. Particularly preferably it is a pure culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, which is a pure culture of a microorganism, as has been characterized above with respect to its nucleotide sequence.

Most preferably, in the cases described above, it is an immobilized culture of microorganisms.

In particular, the present invention includes the following aspects:

1. A process for the production of biogas from biomass in a fermentation reactor, characterized in that the biomass is added to a microorganism of the species Methanobacterium formicicum.

2. The method according to claim 1, characterized in that the microorganism of the species Methanobacterium formicicum is added in the form of a culture of microorganisms, wherein microorganisms of the species Methanobacterium formicicum account for at least 10 ^'4 % of the total number of microorganisms present in the culture.

3. The method according to claim 2, characterized in that in the culture of microorganisms of the species Methanobacterium formicicum at least 10 ^'2 % of

Total number of microorganisms present in the culture.

4. The method according to claim 3, characterized in that constitute in the culture of microorganisms of the species Methanobacterium formicicum at least 1% of the total number of microorganisms present in the culture.

5. The method according to claim 4, characterized in that constitute in the culture of microorganisms of the species Methanobacterium formicicum at least 10% of the total number of microorganisms present in the culture.

6. The method according to claim 5, characterized in that constitute in the culture of microorganisms of the species Methanobacterium formicicum at least 50% of the total number of microorganisms present in the culture.

7. The method according to claim 6, characterized in that constitute in the culture of microorganisms of the species Methanobacterium formicicum at least 75% of the total number of microorganisms present in the culture.

8. The method according to claim 7, characterized in that constitute in the culture of microorganisms of the species Methanobacterium formicicum at least 90% of the total number of microorganisms present in the culture.

9. The method according to at least one of claims 2 to 8, characterized in that a pure culture of the microorganism of the species Methanobacterium formicicum is added.

10. The method according to at least one of claims 2 to 9, characterized in that microorganisms of the species Methanobacterium formicicum biomass are added as part of at least one immobilized culture of microorganisms. 1 1. Method according to at least one of claims 1 to 10, characterized in that in time for the addition of the microorganism of the species Methanobacterium formicicum additional biomass is added to the fermentation reactor.

12. The method according to at least one of claims 1 to 1 1, characterized in that the space load in the fermentation reactor is continuously increased by the continuous addition of biomass.

13. The method according to at least one of claims 1 to 12, characterized in that the production of biogas from biomass at a space load of ≥ 0.5 kg oTS / m ³ d, preferably ≥ 4.0 kg oTS / m ³ d, especially preferably ≥ 8.0 kg oTS / m ³ d.

14. The method according to at least one of claims 1 to 13, characterized in that the production of biogas from biomass under constant mixing of

Fermentation substrate takes place.

15. The method according to at least one of claims 1 to 14, characterized in that the production of biogas from biomass at a temperature of 20 ^ to 80 ⁰ C, preferably at a temperature of 40 ^< € to 50 ⁰ C is performed.

16. The method according to at least one of claims 1 to 15, characterized in that the addition of fermentation substrate and the addition of the microorganism of the species Methanobacterium formicicum carried out continuously.

17. The method according to at least one of claims 1 to 16, characterized in that the microorganism of the species Methanobacterium formicicum is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanobacterium formicicum between 10 ^"8 % and 50% the total number of microorganisms present in the fermentation substrate.

18. The method according to claim 17, characterized in that the microorganism of the species Methanobacterium formicicum is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanobacterium formicicum between 10 ^"6 % and 25% of the total in Fermentation substrate present

Constitutes microorganisms. 19. The method according to claim 18, characterized in that the microorganism of the species Methanobacterium formicicum is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanobacterium formicicum between 10 ^'4 % and 10% of the total in Fermentation substrate present microorganisms.

20. The method according to claim 19, characterized in that the microorganism of the species Methanobacterium formicicum is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanobacterium formicicum between 10 ^"3 % and 1% of the total in Fermentation substrate present

Constitutes microorganisms.

21. Process according to at least one of Claims 1 to 20, characterized in that the microorganism of the species Methanobacterium formicicum is a microorganism of the strain Methanobacterium sp. Clone SMS-sludge-11 trades.

22. The method according to at least one of claims 1 to 20, characterized in that it is a microorganism of the strain Methanobacterium sp. 169 acts.

23. The method according to at least one of claims 1 to 20, characterized in that it is the microorganism of the species Methanobacterium formicicum is a microorganism of the strain Archaeon clone CG-4.

24. Use of a microorganism of the species Methanobacterium formicicum for the fermentative production of biogas from biomass.

25. Use according to claim 24, characterized in that it is in the microorganism of the species Methanobacterium formicicum to a microorganism of the strain Methanobacterium sp. Clone SMS-sludge-11 trades.

26. Use according to claim 24, characterized in that the microorganism of the species Methanobacterium formicicum is a microorganism of the strain Methanobacterium sp. 169 acts. 27. Use according to claim 24, characterized in that the microorganism of the species Methanobacterium formicicum is a microorganism of the strain Archaeon clone CG-4.

28. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 97.70% sequence identity with the nucleotide sequence SEQ ID No. 1.

29. Microorganism according to claim 28, characterized in that the nucleotide sequence contains a sequence region which has more than 98% sequence identity with the nucleotide sequence SEQ ID No. 1.

The microorganism according to claim 29, characterized in that the nucleotide sequence contains a sequence region having more than 98.5% sequence identity with the nucleotide sequence of SEQ ID NO: 1.

31. Microorganism according to claim 30, characterized in that the nucleotide sequence contains a sequence region which has more than 99% sequence identity with the nucleotide sequence SEQ ID No. 1.

32. The microorganism according to claim 31, characterized in that the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 1.

33. The microorganism according to claim 32, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 1.

34. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which is more than

97.56% sequence identity with the nucleotide sequence SEQ ID NO: 5.

35. The microorganism according to claim 34, characterized in that the nucleotide sequence contains a sequence region which has more than 98% sequence identity with the nucleotide sequence SEQ ID No. 5. 36. The microorganism according to claim 35, characterized in that the nucleotide sequence contains a sequence region which has more than 98.5% sequence identity with the nucleotide sequence SEQ ID No. 5.

37. Microorganism according to claim 36, characterized in that the nucleotide sequence contains a sequence region which has more than 99% sequence identity with the nucleotide sequence SEQ ID No. 5.

38. The microorganism according to claim 37, characterized in that the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 5.

39. Microorganism according to claim 38, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 5.

40. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 97.05% sequence identity with the nucleotide sequence SEQ ID No. 22.

41. Microorganism according to claim 40, characterized in that the nucleotide sequence contains a sequence region which has more than 98% sequence identity with the nucleotide sequence SEQ ID No. 22.

42. The microorganism according to claim 41, characterized in that the nucleotide sequence contains a sequence region which has more than 98.5% sequence identity with the nucleotide sequence SEQ ID No. 22.

43. The microorganism according to claim 42, characterized in that the nucleotide sequence contains a sequence region which has more than 99% sequence identity with the nucleotide sequence SEQ ID No. 22.

44. The microorganism according to claim 43, characterized in that the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 22. 45. Microorganism according to claim 44, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 22.

46. culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, characterized in that in the culture of microorganisms, a microorganism according to claims 28 to 45 is present, wherein the microorganism at least 10 ^'4 % of the total in the culture microorganisms present.

47. Culture of microorganisms according to claim 46, characterized in that the microorganism constitutes at least 10 ^'2 % of the total number of microorganisms present in the culture.

48. Culture of microorganisms according to claim 47, characterized in that the microorganism constitutes at least 1% of the total number of microorganisms present in the culture.

49. Culture of microorganisms according to claim 48, characterized in that the microorganism at least 10% of the total number present in the culture

Constitutes microorganisms.

50. Culture of microorganisms according to claim 49, characterized in that the microorganism constitutes at least 50% of the total number of microorganisms present in the culture.

51. Culture of microorganisms according to claim 50, characterized in that the microorganism constitutes at least 90% of the total number of microorganisms present in the culture.

52. Culture of microorganisms according to claim 51, characterized in that it is a pure culture of a microorganism according to claims 28 to 45.

53. Culture of microorganisms according to at least one of claims 46 to 52, characterized in that it is an immobilized culture of microorganisms. 54. Use of a microorganism according to at least one of claims 28 to 45 for the fermentative production of biogas from biomass.

55. Use according to claim 54, characterized in that it is a method for the fermentative production of biogas from biomass according to at least one of

Claims 1 to 23 is.

56. Use of a culture of microorganisms according to at least one of claims 46 to 53 for the fermentative production of biogas from biomass.

57. Use according to claim 56, characterized in that it is a process for the fermentative production of biogas from biomass biomass according to at least one of claims 1 to 23.

In summary, a method for producing biogas from biomass in a fermentation reactor is described, wherein the biomass is added to a microorganism of the species Methanobacterium formicicum.

Methanosarcina acetivorans

The present invention provides a method for producing biogas from biomass in a fermentation reactor. The biomass is added according to the invention a microorganism of the species Methanosarcina acetivorans.

Surprisingly, it has been shown that the addition of microorganisms of the species Methanosarcina acetivorans to the fermentation substrate, the amount of biogas formed can be significantly increased. At constant space load, the addition of microorganisms of the species Methanosarcina acetivorans causes a significant increase in the amount of biogas produced. Alternatively, the amount of methane formed can also be increased by increasing the space load, with the addition of microorganisms of the species Methanosarcina acetivorans preventing the fermentation process from becoming unstable under the altered conditions. The use of microorganisms of the species Methanosarcina acetivorans therefore leads to an improvement in the efficiency and efficiency of biogas plants. It is known to those skilled in the art which strains of microorganisms fall under the term "species Methanosarcina acetivorans." In connection with the production of biogas by fermentation of organic substrates, microorganisms of the species Methanosarcina acetivorans are hitherto unknown.

According to a preferred embodiment of the present invention, a microorganism of the species Methanosarcina acetivorans is added in the form of a culture of microorganisms consisting predominantly of microorganisms of the species Methanosarcina acetivorans. In fermentation substrates of biogas plants microorganisms of the species Methanosarcina acetivorans could be detected only in trace amounts of less than 10 ^"4% share of the total number of present microorganisms. Since the amount is not sufficient for the addition of the microorganism at isolated from their natural occurrence microorganisms is In practice, it is found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out directly in the form of a culture of microorganisms.

The addition of the culture of Methanosarcina acetivorans can be carried out in the form of a culture suspension or in the form of dry, freeze-dried or moist cell pellets.

Since the already mentioned various positive effects on the fermentation process are associated with the microorganism species Methanosarcina acetivorans, this type of microorganism should be present in the added culture in a concentration exceeding the natural abundance. Of course, mixed cultures of any composition can be used for the addition. The only prerequisite is that the species Methanosarcina acetivorans is present in a concentration enriched in relation to the natural occurrence.

The determination of the proportions of different types of microorganisms in mixed cultures is not a problem for the expert. Thus, for example, with the help of fluorescently labeled oligosensors specifically the proportion of microorganisms of the species Methanosarcina acetivorans be identified in a mixture. As already mentioned, microorganisms of the species Methanosarcina acetivorans are preferably added in the form of cultures of microorganisms to the fermentation substrate, wherein the cultures of microorganisms consist predominantly of microorganisms of the species Methanosarcina acetivorans. If, in addition to the determination of the number of microorganisms of the species Methanosarcina acetivorans, the total number of microorganisms is also determined, the proportion of microorganisms of the species Methanosarcina acetivorans in culture in percent. Microorganisms of the species Methanosarcina acetivorans are in a mixed culture then the predominant species of microorganisms, if they have the highest percentage of the various types of microorganisms present in the mixed culture.

According to preferred embodiments of the present invention make microorganisms of the species Methanosarcina acetivorans at least 10 ^'4%, especially at least ^{10' 2%,} more preferably at least 1% of the total number existing in the added to the fermentation substrate culture microorganisms. According to further preferred embodiments, microorganisms of the species Methanosarcina acetivorans account for at least 10%, in particular at least 50%, particularly preferably at least 90%, of the total number of microorganisms present in the culture.

According to a very particularly preferred embodiment, a pure culture of a microorganism of the species Methanosarcina acetivorans is added. Due to the specific metabolic processes and activities, the addition of a pure culture can especially contribute to an improved methane production.

According to another preferred embodiment of the present invention, a microorganism of the species Methanosarcina acetivorans is added as a component of at least one immobilized culture of microorganisms. Since the amount of microorganisms isolated from their natural occurrence is insufficient for the addition of the microorganisms, it is usually propagated in the form of a culture. In practice, it has been found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out in the form of an immobilized culture of microorganisms.

According to a preferred embodiment of the present invention, the microorganism of the species Methanosarcina acetivorans is added in an amount to the fermentation substrate, so that after addition of the proportion of the microorganism of the species Methanosarcina acetivorans between 10 ^'8 % and 50% of the total number present in the fermentation substrate

Constitutes microorganisms. In particular, depending on the fermenter size and thus as a function of the amount of fermentation substrate can be necessary to achieve the desired effect, an addition of very different amounts of microorganisms. More preferably, a microorganism of the species Methanosarcina acetivorans is added to the fermentation substrate in an amount such that, after addition, the proportion of the microorganism of the species Methanosarcina acetivorans is between 10 ⁶ % and 25% of the total number of microorganisms present in the fermentation substrate. More preferably, the microorganism of the species Methanosarcina acetivorans is added to the fermentation substrate in an amount such that, after addition, the proportion of the microorganism of the species Methanosarcina acetivorans is between 10 ^-4 % and 10% of the total number of microorganisms present in the fermentation substrate. Most preferably, the microorganism of the species Methanosarcina acetivorans is added to the fermentation substrate in an amount such that, after addition, the proportion of the microorganism of the species Methanosarcina acetivorans between 1 CT is ³ % and 1% of the total number of microorganisms present in the fermentation substrate.

According to a particularly preferred embodiment of the present invention, the microorganism of the species Methanosarcina acetivorans is a microorganism of the strain Archaeon clone 5.5ft C85A. In all of the above-described embodiments, which deal with the addition of a microorganism of the species Methanosarcina acetivorans, so preferably a microorganism of the strain Archaeon clone 5.5ft C85A is added.

The present invention also encompasses the use of a microorganism of the species Methanosarcina acetivorans for the fermentative production of biogas from biomass. Preferably, a microorganism of the strain strain Archaeon clone 5.5ft C85A is used for the fermentative production of biogas from biomass.

The microorganisms Methanosarcina acetivorans SBG19 obtained from the fermentation substrate of a post-fermenter were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID NO: 16 comprises 919 nucleotides. The next related sequence identified was the sequence of the uncultivated Archaeon clone 5.5ft C85A. A comparison of the sequences revealed that a total of 24 exchanges of nucleotides or deletions are present. At a length of the specific nucleotide sequence of 919 nucleotides of Methanosarcina acetivorans SBG19 and a length of the reference sequence of 926 nucleotides, a 97.39% match is calculated using the FASTA algorithm.

The present invention also includes microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having more than 97.39% sequence identity with the nucleotide sequence of SEQ ID NO: 16. Especially preferred For example, the nucleotide sequence contains a sequence range greater than 97.4% or greater than 97.5% or greater than 97.6% or greater than 97.7% or greater than 97.8% or greater than 97.9% or greater when 98% has sequence identity with the nucleotide sequence SEQ ID NO: 16, and more preferably, the nucleotide sequence contains a sequence region having more than 98.5% sequence identity with the nucleotide sequence SEQ ID NO: 16.

According to further preferred embodiments, the microorganism has a nucleotide sequence which contains a sequence region which has more than 99% sequence identity with the nucleotide sequence SEQ ID No. 16, and more preferably the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence Nucleotide sequence SEQ ID NO. 16 has. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 16.

In preferred embodiments of the present invention, SEQ ID NO: 16 may be compared to the starting nucleotide sequence at one or two positions or at three positions or at four positions or at five positions or at six positions or at seven positions or at eight positions or at nine positions or ten positions or eleven positions or twelve positions or 13 positions or 14 positions or 15 positions or 16 positions or 17 positions or 18 positions or 19 positions or 20 positions or 21 positions or on 22 positions or 23 positions or 24 positions nucleotide mutations. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also encompasses a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism having a nucleotide sequence containing a sequence region greater than 97.39 is present % Sequence identity with the nucleotide sequence SEQ ID NO: 16, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 97.4% or more

97.5% or more than 97.6% or more than 97.7% or more than 97.8% or more 97.9% or more than 98% sequence identity with the nucleotide sequence SEQ ID NO: 16. More preferably, the nucleotide sequence contains a sequence region having more than 98.5% sequence identity with the nucleotide sequence SEQ ID NO: 16.

According to further preferred embodiments, the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass comprises a microorganism having a nucleotide sequence with a sequence region having more than 99% sequence identity with the nucleotide sequence SEQ ID NO: 16 , Most preferably, the nucleotide sequence contains a sequence region having more than 99.5% sequence identity with the nucleotide sequence SEQ ID NO: 16.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 16.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 16 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 16 in a method described above in connection with microorganisms of the species Methanosarcina acetivorans for the fermentative production of biogas from biomass.

The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO. 16 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. It is preferable to use a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID No. 16 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in a method described above in connection with microorganisms of the species Methanosarcina acetivorans for the fermentative production of biogas from biomass. According to further preferred embodiments make said, characterized in terms of their nucleotide sequence microorganisms at least 10 ^'2%, preferably to at least 1% of the total present in the culture of microorganisms. Particularly preferably, the microorganisms make up at least 10%, particularly preferably at least 25%, of the total number of microorganisms present in the culture. Most preferably, the microorganisms described make up at least 50%, in particular at least 75%, of the total number of microorganisms present in the culture.

Also preferred are embodiments according to which the above-mentioned microorganisms make up at least 90% of the total number of microorganisms present in the culture. Particularly preferably it is a pure culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, which is a pure culture of a microorganism, as has been characterized above with respect to its nucleotide sequence.

Most preferably, in the cases described above, it is an immobilized culture of microorganisms.

In particular, the present invention includes the following aspects:

1. A process for the production of biogas from biomass in a fermentation reactor, characterized in that the biomass is added to a microorganism of the species Methanosarcina acetivorans.

2. The method according to claim 1, characterized in that the microorganism of the species Methanosarcina acetivorans is added in the form of a culture of microorganisms, wherein microorganisms of the species Methanosarcina acetivorans make up at least 10 ^'4 % of the total number of microorganisms present in the culture.

3. The method according to claim 2, characterized in that constitute in the culture of microorganisms microorganisms of the species Methanosarcina acetivorans at least 10 ^'2 % of the total number of microorganisms present in the culture.

4. The method according to claim 3, characterized in that constitute in the culture of microorganisms microorganisms of the species Methanosarcina acetivorans at least 1% of the total number of microorganisms present in the culture. 5. The method according to claim 4, characterized in that in the culture of microorganisms microorganisms of the species Methanosarcina acetivorans make up at least 10% of the total number of microorganisms present in the culture.

6. The method according to claim 5, characterized in that in the culture of microorganisms microorganisms of the species Methanosarcina acetivorans make up at least 50% of the total number of microorganisms present in the culture.

7. The method according to claim 6, characterized in that in the culture of microorganisms microorganisms of the species Methanosarcina acetivorans make up at least 75% of the total number of microorganisms present in the culture.

8. The method according to claim 7, characterized in that in the culture of microorganisms microorganisms of the species Methanosarcina acetivorans make up at least 90% of the total number of microorganisms present in the culture.

9. The method according to at least one of claims 2 to 8, characterized in that a pure culture of the microorganism of the species Methanosarcina acetivorans is added.

10. The method according to at least one of claims 2 to 9, characterized in that microorganisms of the species Methanosarcina acetivorans the biomass are added as part of at least one immobilized culture of microorganisms.

1 1. Method according to at least one of claims 1 to 10, characterized in that in time for the addition of the microorganism of the species Methanosarcina acetivorans additional biomass is added to the fermentation reactor.

12. The method according to at least one of claims 1 to 1 1, characterized in that the space load in the fermentation reactor by continuous addition of

Biomass is continuously increased.

13. The method according to at least one of claims 1 to 12, characterized in that the production of biogas from biomass at a space load of ≥ 0.5 kg oTS / m ³ d, preferably ≥ 4.0 kg oTS / m ³ d, especially preferably ≥ 8.0 kg oTS / m ³ d. 14. The method according to at least one of claims 1 to 13, characterized in that the production of biogas from biomass takes place under constant mixing of the fermentation substrate.

15. The method according to at least one of claims 1 to 14, characterized in that the production of biogas from biomass at a temperature of 20 ^ to 80 ⁰ C, preferably at a temperature of 40 ^< € to 50 ⁰ C is performed.

16. The method according to at least one of claims 1 to 15, characterized in that the addition of fermentation substrate and the addition of the microorganism of the type

Methanosarcina acetivorans take place continuously.

17. The method according to at least one of claims 1 to 16, characterized in that the microorganism of the species Methanosarcina acetivorans is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species

Methanosarcina acetivorans accounts for between 10 ⁸ % and 50% of the total number of microorganisms present in the fermentation substrate.

18. The method according to claim 17, characterized in that the microorganism of the species Methanosarcina acetivorans is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanosarcina acetivorans between 10 ^"6 % and 25% of the total in Fermentation substrate present microorganisms.

19. The method according to claim 18, characterized in that the microorganism of the species Methanosarcina acetivorans is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanosarcina acetivorans between 10 ^'4 % and 10% of the total in Fermentation substrate present microorganisms.

20. The method according to claim 19, characterized in that the microorganism of the species Methanosarcina acetivorans is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanosarcina acetivorans between 10 ^'3 % and 1% of the total in Fermentation substrate present microorganisms. 21. Process according to at least one of claims 1 to 20, characterized in that the microorganism of the species Methanosarcina acetivorans is a microorganism of the strain Archaeon clone 5.5ft C85A.

22. Use of a microorganism of the species Methanosarcina acetivorans for the fermentative production of biogas from biomass.

23. Use according to claim 22, characterized in that it is a microorganism of the strain Archaeon clone 5.5ft C85A in the microorganism of the species Methanosarcina acetivorans.

24. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 97.39% sequence identity with the nucleotide sequence SEQ ID No. 16.

25. Microorganism according to claim 24, characterized in that the nucleotide sequence contains a sequence region which has more than 98% sequence identity with the nucleotide sequence SEQ ID No. 16.

26. Microorganism according to claim 25, characterized in that the nucleotide sequence contains a sequence region which has more than 98.5% sequence identity with the nucleotide sequence SEQ ID No. 16.

27. Microorganism according to claim 26, characterized in that the nucleotide sequence contains a sequence region which has more than 99% sequence identity with the nucleotide sequence SEQ ID No. 16.

28. Microorganism according to claim 27, characterized in that the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 16.

29. Microorganism according to claim 28, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 16.

30. Culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, characterized in that in the culture of Microorganisms, a microorganism according to claims 24 to 29 is present, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

31. Culture of microorganisms according to claim 30, characterized in that the microorganism makes up at least ^{10 -2%} of the total number of microorganisms present in the culture.

32. Culture of microorganisms according to claim 31, characterized in that the microorganism at least 1% of the total number present in the culture

Constitutes microorganisms.

33. Culture of microorganisms according to claim 32, characterized in that the microorganism constitutes at least 10% of the total number of microorganisms present in the culture.

34. Culture of microorganisms according to claim 33, characterized in that the microorganism constitutes at least 50% of the total number of microorganisms present in the culture.

35. Culture of microorganisms according to claim 34, characterized in that the microorganism constitutes at least 90% of the total number of microorganisms present in the culture.

36. Culture of microorganisms according to claim 35, characterized in that it is a pure culture of a microorganism according to claims 24 to 29.

37. Culture of microorganisms according to at least one of claims 30 to 36, characterized in that it is an immobilized culture of microorganisms.

38. Use of a microorganism according to at least one of claims 24 to 29 for the fermentative production of biogas from biomass.

39. Use according to claim 38, characterized in that it is a process for the fermentative production of biogas from biomass according to at least one of claims 1 to 21. 40. Use of a culture of microorganisms according to at least one of claims 30 to 37 for the fermentative production of biogas from biomass.

41. Use according to claim 40, characterized in that it is a process for the fermentative production of biogas from biomass biomass according to at least one of claims 1 to 21.

In summary, a process for the production of biogas from biomass in a fermentation reactor is described, wherein the biomass is added to a microorganism of the species Methanosarcina acetivorans.

Methanosarcina barkeri

The present invention provides a method for producing biogas from biomass in a fermentation reactor. The biomass is added according to the invention a microorganism of the species Methanosarcina barkeri.

Surprisingly, it has been found that the amount of biogas formed can be significantly increased by the addition of microorganisms of the species Methanosarcina barkeri to the fermentation substrate. At constant volume loading, the addition of microorganisms of the species Methanosarcina barkeri causes a significant increase in the amount of biogas produced. Alternatively, the amount of methane produced can also be increased by increasing the volume load, with the addition of microorganisms of the species Methanosarcina barkeri preventing the fermentation process from becoming unstable under the altered conditions. The use of microorganisms of the species Methanosarcina barkeri therefore leads to an improvement in efficiency and efficiency of biogas plants.

It is known to those skilled in the art which strains of microorganisms fall under the term "Methanosarcina barkeri type". In connection with the production of biogas by fermentation of organic substrates, microorganisms of the species Methanosarcina barkeri are not yet known.

According to a preferred embodiment of the present invention, a microorganism of the species Methanosarcina barkeri is added in the form of a culture of microorganisms consisting predominantly of microorganisms of the species Methanosarcina barkeri. In Fermentation substrates of biogas plants were detected in microorganisms of the species Methanosarcina barkeri only in traces of less than 10 ^'4 % share of the total number of microorganisms present. Since the amount of microorganisms isolated from their natural occurrence is insufficient for the addition of the microorganisms, it is usually propagated in the form of a culture. In practice, it is found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out directly in the form of a culture of microorganisms.

The addition of the culture of Methanosarcina barkeri can be carried out in the form of a culture suspension or in the form of dry, freeze-dried or moist cell pellets.

Since the already mentioned various positive effects on the fermentation process are associated with the microorganism species Methanosarcina barkeri, this type of microorganism should be present in the added culture in a concentration exceeding the natural abundance. Of course, mixed cultures of any composition can be used for the addition. All that is required is that the species Methanosarcina barkeri is present in a concentration enriched with respect to the natural occurrence.

The determination of the proportions of different types of microorganisms in mixed cultures is not a problem for the expert. Thus, for example, with the help of fluorescently labeled oligosensors specifically the proportion of microorganisms of the species Methanosarcina barkeri be identified in a mixture. As already mentioned, microorganisms of the species Methanosarcina barkeri are preferably added to the fermentation substrate in the form of cultures of microorganisms, the cultures of microorganisms consisting predominantly of microorganisms of the species Methanosarcina barkeri. If, in addition to the determination of the number of microorganisms of the species Methanosarcina barkeri, the total number of microorganisms is also determined, the percentage of microorganisms of the species Methanosarcina barkeri in the culture can be stated as a percentage. Microorganisms of the species Methanosarcina barkeri are in a mixed culture then the predominantly present type of microorganisms, if they have the highest percentage of the different species of microorganisms present in the mixed culture.

According to preferred embodiments of the present invention, microorganisms of the species Methanosarcina barkeri make at least 10 ^'4 %, in particular at least

10 ^'2 %, particularly preferably at least 1% of the total number of microorganisms present in the culture added to the fermentation substrate. According to further preferred Embodiments make microorganisms of the species Methanosarcina barkeri at least 10%, in particular at least 50%, particularly preferably at least 90% of the total number of microorganisms present in the culture.

According to a very particularly preferred embodiment, a pure culture of a microorganism of the species Methanosarcina barkeri is added. Due to the specific metabolic processes and activities, the addition of a pure culture can especially contribute to an improved methane production.

According to another preferred embodiment of the present invention, a microorganism of the species Methanosarcina barkeri is added as a component of at least one immobilized culture of microorganisms. Since the amount of microorganisms isolated from their natural occurrence is insufficient for the addition of the microorganisms, it is usually propagated in the form of a culture. In practice, it has been found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out in the form of an immobilized culture of microorganisms.

According to a preferred embodiment of the present invention, the microorganism of the species Methanosarcina barkeri is added in an amount to the fermentation substrate, so that after addition of the proportion of the microorganism of the species Methanosarcina barkeri between 10 ⁸ % and 50% of the total number present in the fermentation substrate

Constitutes microorganisms. In particular, depending on the fermenter size and thus as a function of the amount of fermentation substrate can be necessary to achieve the desired effect, an addition of very different amounts of microorganisms.

A microorganism of the species Methanosarcina barkeri is particularly preferably added in an amount to the fermentation substrate that makes up after addition of the content of the microorganism of the species Methanosarcina barkeri between ^{10 -6%} and 25% of the total number present in the fermentation substrate microorganisms. More preferably, the microorganism of the species Methanosarcina barkeri is added to the fermentation substrate in an amount such that, after addition, the proportion of the microorganism of the species Methanosarcina barkeri is between 10 ^'4 % and 10% of the total number of microorganisms present in the fermentation substrate. Most preferably, the microorganism of the species Methanosarcina barkeri is added in an amount to the fermentation substrate, that after addition the proportion of the microorganism of the species Methanosarcina barkeri is between 10 ^'3 % and 1% of the total number of microorganisms present in the fermentation substrate.

According to a particularly preferred embodiment of the present invention, the microorganism of the species Methanosarcina barkeri is a microorganism of the strain Archaeon clone 5.5ft C140A. In all the embodiments explained in more detail above, which deal with the addition of a microorganism of the species Methanosarcina barkeri, it is thus preferable to add a microorganism of the strain Archaeon clone 5.5 .mu.ft Cl.sub.4OA.

According to a further particularly preferred embodiment of the present invention, the microorganism of the species Methanosarcina barkeri is a microorganism of the strain Archaeon clone 5.5ft C17A. In all of the embodiments explained in more detail above, which deal with the addition of a microorganism of the species Methanosarcina barkeri, a microorganism of the strain Archaeon clone 5.5ft C17A is therefore preferably added.

According to a further particularly preferred embodiment of the present invention, the microorganism of the species Methanosarcina barkeri is a microorganism of the strain Archaeon clone 5.5ft C38A. In all of the above-described embodiments, which deal with the addition of a microorganism of the species Methanosarcina barkeri, so preferably a microorganism of the strain Archaeon clone 5.5ft C38A is added.

According to a further particularly preferred embodiment of the present invention, the microorganism of the species Methanosarcina barkeri is a microorganism of the strain Archaeon clone ATB-KM1219. In all of the embodiments explained in more detail above, which deal with the addition of a microorganism of the species Methanosarcina barkeri, a microorganism of the strain Archaeon clone ATB-KMI 219 is therefore preferably added.

According to a further particularly preferred embodiment of the present invention, the microorganism of the species Methanosarcina barkeri is a microorganism of the strain Archaeon clone ATB-KM1253. In all the embodiments explained in more detail above, which deal with the addition of a microorganism of the species Methanosarcina barkeri, a microorganism of the strain Archaeon clone ATB-KM '1253 is therefore preferably added. According to a further particularly preferred embodiment of the present invention, the microorganism of the species Methanosarcina barkeri is a microorganism of the strain Archaeon clone MH1492 3E. In all of the above-described embodiments, which deal with the addition of a microorganism of the species Methanosarcina barkeri, so preferably a microorganism of the strain Archaeon clone MH1492 3E is added.

The present invention also encompasses the use of a microorganism of the species Methanosarcina barkeri for the fermentative production of biogas from biomass. Preference is given to the fermentative production of biogas from biomass, a microorganism of

Tribe tribal Archaeon clone 5.5ft C140A used. Also preferred for fermentative production of biogas from biomass is a microorganism of the strain

Archaeon clone 5.5ft C17A used. Also preferred for fermentative production of biogas from biomass, a microorganism of the strain Archaeon clone 5.5ft C38A is used. Also preferred for the fermentative production of biogas from biomass, a microorganism of the strain Archaeon clone ATB-KM1219 is used. Also preferably, a microorganism of the strain Archaeon clone ATB-KM1253 is used for the fermentative production of biogas from biomass. Also preferred for fermentative production of biogas from biomass is a microorganism of the strain

Archaeon clone MH1492 3E used.

The microorganisms Methanosarcina barkeri SBG 16 obtained from the fermentation substrate of a postgrader were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID NO: 13 comprises 933 nucleotides. The next related sequence identified was the sequence of the uncultivated Archaeon clone 5.5ft C140A. A comparison of the sequences revealed that there are a total of 37 exchanges of nucleotides or deletions. With a length of the specific nucleotide sequence of Methanosarcina barkeri SBG16 of 933 nucleotides and a length of the reference sequence of 946 nucleotides, a match of 96.03% is calculated using the FASTA algorithm.

The present invention also includes microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having more than 96.03% sequence identity with the nucleotide sequence of SEQ ID NO: 13. More preferably, the nucleotide sequence contains a sequence range greater than 96.1% or greater than 96.2% or greater than 96.3% or greater than 96.4% or greater than 96.5% or greater than 96.6%. or more than 96.7% sequence identity with the nucleotide sequence SEQ ID NO: 13 and particularly preferably, the nucleotide sequence contains a sequence region having more than 97.0% sequence identity with the nucleotide sequence SEQ ID NO: 13.

According to further preferred embodiments, the microorganism has a nucleotide sequence containing a sequence region greater than 97.5% or more

98.0% or more than 98.5% or more than 99.0% sequence identity with the

Nucleotide sequence SEQ ID NO. 13 and particularly preferably contains the

Nucleotide sequence has a sequence region that has more than 99.5% sequence identity with the

Nucleotide sequence SEQ ID NO. 13 has. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which is the

Nucleotide sequence SEQ ID NO. 13 corresponds.

In preferred embodiments of the present invention, SEQ ID NO: 13 may be compared to the starting nucleotide sequence at one or two positions or at three positions or at four positions or at five positions or at six positions or at seven positions or at eight positions or at nine positions or ten positions or eleven positions or twelve positions or 13 positions or 14 positions or 15 positions or 16 positions or 17 positions or 18 positions or 19 positions or 20 positions or 21 positions or on 22 positions or 23 positions or 24 positions or 25 positions or 26 positions or 27 positions or 28 positions or 29 positions or 30 positions or 31 positions or 32 positions or 33 positions or 34 positions or at 35 positions or at 36 positions or at 37 positions nucleotide mutations. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also encompasses a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, in which culture of microorganisms a microorganism is present which has a nucleotide sequence containing a sequence region greater than 96.03 % Sequence identity with the nucleotide sequence SEQ ID NO: 13, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 96.1% or more 96.2% or greater than 96.3% or greater than 96.4% or greater than 96.5% or greater than 96.6% or greater than 96.7% sequence identity to the nucleotide sequence of SEQ ID NO: 13. Most preferably, the nucleotide sequence contains a sequence region having greater than 97.0% or greater than 97.5% or greater than 98.0% or greater than 98.5% sequence identity with the nucleotide sequence of SEQ ID NO: 13.

According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range exceeding 99.0% sequence identity with the nucleotide sequence SEQ ID NO. 13 has. Most preferably, the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 13.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 13.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 13 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 13 in a method described above in connection with microorganisms of the species Methanosarcina barkeri for the fermentative production of biogas from biomass.

The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, the culture of microorganisms comprising at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 13 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. Preference is given to the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID No. 13 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in one of the above - mentioned In connection with microorganisms of the species Methanosarcina barkeri described method for the fermentative production of biogas from biomass.

The microorganisms Methanosarcina barkeri SßG77 also obtained from the fermentation substrate of a post-fermenter were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID NO: 14 comprises 1 128 nucleotides. The next related sequence identified was the sequence of the uncultured Archaeon clone 5.5ft C17A. A comparison of the sequences revealed that there are a total of 9 exchanges of nucleotides or deletions. At a length of the particular nucleotide sequence of Methanosarcina barkeri SBG 17 of 1 128 nucleotides and a length of the reference sequence of 1 127 nucleotides, a 99.20% match is calculated using the FASTA algorithm.

The present invention also includes microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having greater than 99.20% sequence identity with the nucleotide sequence of SEQ ID NO: 14. More preferably, the nucleotide sequence contains a sequence range greater than 99.25% or greater than 99.30% or greater than 99.35% or greater than 99.40% or greater than 99.45% or greater than 99.50%. or more than 99.55% sequence identity with the nucleotide sequence SEQ ID NO: 14, and more preferably, the nucleotide sequence contains a sequence region having more than 99.60% sequence identity with the nucleotide sequence SEQ ID NO: 14.

According to further preferred embodiments, the microorganism has a nucleotide sequence which contains a sequence region which has more than 99.70% or more than 99.80% sequence identity with the nucleotide sequence SEQ ID No. 14, and more preferably the nucleotide sequence contains a sequence region which greater than 99.90% sequence identity with the nucleotide sequence SEQ ID NO: 14. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 14.

In preferred embodiments of the present invention, SEQ ID NO: 14 may be compared to the starting nucleotide sequence at one or two positions or at three positions or at four positions or at five positions or at six positions or at seven positions or at eight positions or at nine positions Nucleotide mutations are present. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text. The present invention also encompasses a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, in which culture of microorganisms a microorganism is present which has a nucleotide sequence containing a sequence range greater than 99.20 having% sequence identity with the nucleotide sequence SEQ ID NO. 14, wherein the microorganism makes up at least 10 ^'4% of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 99.25% or greater than 99.30% or greater than 99 , 35% or more than 99.40% or more than 99.45% or more than 99.50% or more than 99.55% Sequence identity with the nucleotide sequence SEQ ID NO.

14 has. Particularly preferably, the nucleotide sequence contains a sequence region which has more than 99.60% sequence identity with the nucleotide sequence SEQ ID No. 14.

According to further preferred embodiments, the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass comprises a microorganism having a nucleotide sequence with a sequence range greater than 99.70% or greater than 99.80% sequence identity having the nucleotide sequence SEQ ID NO: 14. The nucleotide sequence most preferably contains a sequence region which has more than 99.90% sequence identity with the nucleotide sequence SEQ ID No. 14.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 14.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 14 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 14 in a method described above in connection with microorganisms of the species Methanosarcina barkeri for the fermentative production of biogas from biomass. The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, the culture of microorganisms comprising at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 14 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. Preference is given to the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID No. 14 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in a method described above in connection with microorganisms of the species Methanosarcina barkeri for the fermentative production of biogas from biomass.

The microorganisms Methanosarcina barkeri SBG18 also obtained from the fermentation substrate of a secondary fermenter were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID NO: 15 comprises 1 123 nucleotides. The next related sequence identified was the sequence of the uncultivated Archaeon clone 5.5ft C38A. A comparison of the sequences revealed that there are a total of 32 exchanges of nucleotides or deletions. At a length of the particular nucleotide sequence of Methanosarcina barkeri SBG18 of 1 123 nucleotides and a length of the reference sequence of 1 122 nucleotides, a 97.15% match is calculated using the FASTA algorithm.

The present invention also includes microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having more than 97.15% sequence identity with the nucleotide sequence of SEQ ID NO: 15. More preferably, the nucleotide sequence contains a sequence range greater than 97.2% or greater than 97.3% or greater than 97.4% or greater than 97.5% or greater than 97.6% or greater than 97.7%. or more than 97.8% sequence identity with the nucleotide sequence SEQ ID NO: 15, and more preferably, the nucleotide sequence contains a sequence region having more than 97.9% sequence identity with the nucleotide sequence SEQ ID NO: 15.

According to further preferred embodiments, the microorganism has a nucleotide sequence which contains a sequence region which has more than 98.0% or more than 98.5% or more than 99.0% sequence identity with the nucleotide sequence SEQ ID No. 15 and is particularly preferred For example, the nucleotide sequence contains a sequence region that has greater than 99.5% sequence identity with the nucleotide sequence SEQ ID NO: 15. According to In a very particularly preferred embodiment, the nucleotide sequence contains a sequence region that corresponds to the nucleotide sequence SEQ ID No. 15.

In preferred embodiments of the present invention, SEQ ID NO: 15 can be compared to the starting nucleotide sequence at one or two positions or at three positions or at four positions or at five positions or at six positions or at seven positions or at eight positions or at nine positions or ten positions or eleven positions or twelve positions or 13 positions or 14 positions or 15 positions or 16 positions or 17 positions or 18 positions or 19 positions or 20 positions or 21 positions or on 22 positions or at 23 positions or at 24 positions or at 25 positions or at 26 positions or at 27 positions or at 28 positions or at 29 positions or at 30 positions or at 31 positions or at 32 positions nucleotide mutations. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also encompasses a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism having a nucleotide sequence containing a sequence region greater than 97.15 is present % Sequence identity with the nucleotide sequence of SEQ ID NO: 15, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 97.2% or greater than 97.3% or greater than 97 , 4% or more than 97.5% or more than 97.6% or more than 97.7% or more than 97.8% sequence identity with the nucleotide sequence SEQ ID NO: 15. More preferably, the nucleotide sequence contains a sequence region having more than 97.9% sequence identity with the nucleotide sequence SEQ ID NO: 15.

According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 98.0% or greater than 98.5% or greater than 99.0% sequence identity with the nucleotide sequence SEQ ID NO: 15. Most notably Preferably, the nucleotide sequence contains a sequence region having more than 99.5% sequence identity with the nucleotide sequence SEQ ID NO: 15.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 15.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 15 for fermentative

Generation of biogas from biomass. It is preferable to use a

Microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 15 in a method described above in connection with microorganisms of the species Methanosarcina barkeri for the fermentative production of biogas from biomass.

The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, the culture of microorganisms comprising at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO. 15 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. Preference is given to the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID No. 15 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in a method described above in connection with microorganisms of the species Methanosarcina barkeri for the fermentative production of biogas from biomass.

The microorganisms Methanosarcina barkeri SBG33 also obtained from the fermentation substrate of a secondary fermenter were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID NO: 30 comprises 801 nucleotides. As the next related sequence, the sequence of the non-cultured krchaeon clone ATB-KM1219 was identified. A comparison of the sequences revealed that there are a total of 2 exchanges of nucleotides or deletions. At a length of the particular nucleotide sequence of Methanosarcina barkeri SBG33 of 801 nucleotides and a reference sequence length of 801 Nucleotides calculated using the FASTA algorithm, a match of 99.75%.

The present invention also encompasses microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region greater than 99.75%.

Sequence identity with the nucleotide sequence SEQ ID NO. 30 has. Particularly preferably, the nucleotide sequence contains a sequence region which has more than 99.80% sequence identity with the nucleotide sequence SEQ ID No. 30 and particularly preferably contains

Nucleotide sequence has a sequence region that has more than 99.85% sequence identity with the nucleotide sequence SEQ ID NO: 30.

According to further preferred embodiments, the microorganism has a nucleotide sequence containing a sequence region having more than 99.90% sequence identity with the nucleotide sequence SEQ ID NO: 30, and more preferably the nucleotide sequence contains a sequence region having more than 99.95% sequence identity having the nucleotide sequence SEQ ID NO: 30. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 30.

In preferred embodiments of the present invention, nucleotide mutations may be present at one position or at two positions relative to the starting nucleotide sequence SEQ ID NO: 30. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also encompasses a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, in which culture of microorganisms a microorganism is present which has a nucleotide sequence containing a sequence region greater than 99.75 having% sequence identity with the nucleotide sequence SEQ ID NO. 30, wherein the microorganism makes up at least 10 ^'4% of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range exceeding 99.80%.

Sequence identity with the nucleotide sequence SEQ ID NO. 30 has. Especially preferred The nucleotide sequence contains a sequence region which has more than 99.85% sequence identity with the nucleotide sequence SEQ ID No. 30.

According to further preferred embodiments, the culture suitable for use in a process for the fermentative production of biogas from biomass is

Microorganisms present a microorganism having a nucleotide sequence with a

Sequence region having more than 99.90% sequence identity with the nucleotide sequence

SEQ ID NO: 30. Most preferably, the nucleotide sequence contains a

A sequence region having greater than 99.95% sequence identity with the nucleotide sequence SEQ ID NO: 30.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region corresponding to the nucleotide sequence SEQ ID No. 30.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 30 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 30 in a method described above in connection with microorganisms of the species Methanosarcina barkeri for the fermentative production of biogas from biomass.

The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO. 30 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. Preference is given to the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID No. 30 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in a method described above in connection with microorganisms of the species Methanosarcina barkeri for the fermentative production of biogas from biomass. The microorganisms Methanosarcina barkeri SBG20 also obtained from the fermentation substrate of a postgrader were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID NO: 17 comprises 801 nucleotides. As the next related sequence, the sequence of the uncultured Archaeon clone ATB-KM1253 was identified. A comparison of the sequences revealed that a total of 4 exchanges of nucleotides or deletions are present. At a length of 801 nucleotides of the particular nucleotide sequence of Methanosarcina barkeri SBG20 and a length of the reference sequence of 803 nucleotides, the 99.50% match is calculated using the FASTA algorithm.

The present invention also includes microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having greater than 99.50% sequence identity with the nucleotide sequence of SEQ ID NO: 17. More preferably, the nucleotide sequence contains a sequence range greater than 99.55% or greater than 99.60% or greater than 99.65% or greater than 99.70% or greater than 99.75% or greater than 99.80%. SEQ ID NO: 17 has sequence identity with the nucleotide sequence, and more preferably, the nucleotide sequence contains a sequence region having more than 99.85 sequence identity with the nucleotide sequence SEQ ID NO: 17.

According to further preferred embodiments, the microorganism has a nucleotide sequence containing a sequence region having more than 99.90% sequence identity with the nucleotide sequence SEQ ID NO: 17, and more preferably the nucleotide sequence contains a sequence region having more than 99.95% sequence identity having the nucleotide sequence of SEQ ID NO: 17. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 17.

In preferred embodiments of the present invention, nucleotide mutations may be present at one or two positions or at three positions or at four positions relative to the starting nucleotide sequence of SEQ ID NO: 17. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also includes a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism is present which has a nucleotide sequence containing a sequence region greater than 99.50 % Sequence identity with the nucleotide sequence SEQ ID NO: 17, wherein the Microorganism accounts for at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 99.55% or greater than 99.60% or greater than 99 , 65% or more than 99.70% or more than 99.75% or more than 99.80% sequence identity with the nucleotide sequence SEQ ID NO: 17.

Particularly preferably, the nucleotide sequence contains a sequence region which has more than 99.85% sequence identity with the nucleotide sequence SEQ ID No. 17.

According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range of more than 99.90% sequence identity with the nucleotide sequence SEQ ID NO. 17 has. Most preferably, the nucleotide sequence contains a sequence region which has more than 99.95% sequence identity with the nucleotide sequence SEQ ID No. 17.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 17.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 17 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 17 in a method described above in connection with microorganisms of the species Methanosarcina barkeri for the fermentative production of biogas from biomass.

The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, the culture of microorganisms comprising at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 17 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. Preference is given to the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID No. 17 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in a method described above in connection with microorganisms of the species Methanosarcina barkeri for the fermentative production of biogas from biomass.

The microorganisms Methanosarcina barkeri SBG24 also obtained from the fermentation substrate of a secondary fermenter were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID No. 21 comprises 1 129 nucleotides. The next related sequence identified was the sequence of the uncultivated Archaeon clone MH1492 3E. One

Comparison of the sequences revealed that a total of 21 exchanges of nucleotides or

Deletions are present. At a length of the particular nucleotide sequence of Methanosarcina barkeri SBG24 of 1 129 nucleotides and a length of the reference sequence of 1 129

Nucleotides are calculated using the FASTA algorithm to match

98.14%.

The present invention also encompasses microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region greater than 98.14%.

Sequence identity with the nucleotide sequence SEQ ID NO. 21 has. More preferably, the nucleotide sequence contains a sequence region greater than 98.2% or greater than

98.3% or greater than 98.4% or greater than 98.5% or greater than 98.6% sequence identity with the nucleotide sequence SEQ ID NO: 21, and most preferably the nucleotide sequence contains a sequence region greater than 98.7 % Sequence identity with the

Nucleotide sequence SEQ ID NO. 21 has.

According to further preferred embodiments, the microorganism has a nucleotide sequence containing a sequence region having more than 99.0% sequence identity with the nucleotide sequence SEQ ID NO: 21, and more preferably the nucleotide sequence contains a sequence region having greater than 99.5% sequence identity having the nucleotide sequence of SEQ ID NO: 21. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 21.

In preferred embodiments of the present invention, as compared to the starting nucleotide sequence, SEQ ID NO: 21 may be attached at one position or at two positions or at three positions or four positions or five positions or six positions or seven positions or eight positions or nine positions or ten positions or eleven positions or twelve positions or 13 positions or 14 positions or 15 positions or at 16 positions or at 17 positions or at 18 positions or at 19 positions or at 20 positions or at 21 positions nucleotide mutations. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also includes a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism is present, which contains a

Nucleotide sequence containing a sequence region greater than 98.14%

Sequence identity with the nucleotide sequence SEQ ID NO. 21, wherein the

Microorganism accounts for at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 98.2% or greater than 98.3% or greater than 98 , 4% or more than 98.5% sequence identity with the nucleotide sequence SEQ ID NO: 21. More preferably, the nucleotide sequence contains a sequence region having greater than 98.6% or greater than 98.7% sequence identity with the nucleotide sequence SEQ ID NO: 21.

According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range exceeding 99.0% sequence identity with the nucleotide sequence SEQ ID NO. 21 has. Most preferably, the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 21.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 21. The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 21 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 21 in a method described above in connection with microorganisms of the species Methanosarcina barkeri for the fermentative production of biogas from biomass.

The present invention also includes the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms at least one microorganism as above with respect to its

Nucleotide sequence SEQ ID NO: 21 is defined and wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preference is given to the use of a culture of microorganisms for the fermentative production of biogas from biomass, the culture of microorganisms comprising at least one microorganism as described above with regard to its nucleotide sequence SEQ

ID No. 21 has been defined and wherein the microorganism is at least 10 ^'4 % of the

Total number of microorganisms present in the culture, in one of the above - mentioned

In connection with microorganisms of the species Methanosarcina barkeri described method for the fermentative production of biogas from biomass.

According to further preferred embodiments, all make above, characterized with respect to their nucleotide sequence microorganisms at least 10 ^'2%, preferably to at least 1% of the total present in the culture of microorganisms. Particularly preferably, the microorganisms make up at least 10%, particularly preferably at least 25%, of the total number of microorganisms present in the culture. Most preferably, the microorganisms described make up at least 50%, in particular at least 75%, of the total number of microorganisms present in the culture.

Also preferred are embodiments according to which the above-mentioned microorganisms make up at least 90% of the total number of microorganisms present in the culture. Particularly preferably it is a pure culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, which is a pure culture of a microorganism, as has been characterized above with respect to its nucleotide sequence. Most preferably, in the cases described above, it is an immobilized culture of microorganisms.

In particular, the present invention includes the following aspects:

1. A process for the production of biogas from biomass in a fermentation reactor, characterized in that the biomass is a microorganism of the type

Methanosarcina barkeri is added.

2. The method according to claim 1, characterized in that the microorganism of the species Methanosarcina barkeri is added in the form of a culture of microorganisms, wherein microorganisms of the species Methanosarcina barkeri account for at least 10 ^'4 % of the total number of microorganisms present in the culture.

3. The method according to claim 2, characterized in that constitute in the culture of microorganisms of the species Methanosarcina barkeri at least 10 ^'2 % of the total number of microorganisms present in the culture.

4. The method according to claim 3, characterized in that constitute in the culture of microorganisms of the species Methanosarcina barkeri at least 1% of the total number of microorganisms present in the culture.

5. The method according to claim 4, characterized in that make up in the culture of microorganisms of the species Methanosarcina barkeri at least 10% of the total number of microorganisms present in the culture.

6. The method according to claim 5, characterized in that make up in the culture of microorganisms of the species Methanosarcina barkeri at least 50% of the total number of microorganisms present in the culture.

7. The method according to claim 6, characterized in that make up in the culture of microorganisms of the species Methanosarcina barkeri at least 75% of the total number of microorganisms present in the culture.

8. The method according to claim 7, characterized in that make up in the culture of microorganisms of the species Methanosarcina barkeri at least 90% of the total number of microorganisms present in the culture. 9. The method according to at least one of claims 2 to 8, characterized in that a pure culture of the microorganism of the species Methanosarcina barkeri is added.

10. The method according to at least one of claims 2 to 9, characterized in that microorganisms of the species Methanosarcina barkeri the biomass are added as part of at least one immobilized culture of microorganisms.

1 1. Method according to at least one of claims 1 to 10, characterized in that in time for the addition of the microorganism of the species Methanosarcina barkeri additional biomass is added to the fermentation reactor.

12. The method according to at least one of claims 1 to 1 1, characterized in that the space load in the fermentation reactor is continuously increased by the continuous addition of biomass.

13. The method according to at least one of claims 1 to 12, characterized in that the production of biogas from biomass at a space load of ≥ 0.5 kg oTS / m ³ d, preferably ≥ 4.0 kg oTS / m ³ d, especially preferably ≥ 8.0 kg oTS / m ³ d.

14. The method according to at least one of claims 1 to 13, characterized in that the production of biogas from biomass takes place under constant mixing of the fermentation substrate.

15. The method according to at least one of claims 1 to 14, characterized in that the production of biogas from biomass at a temperature of 2O ⁰ C to 80 ⁰ C, preferably at a temperature of 40 ⁰ C to 5O ⁰ C is performed.

16. The method according to at least one of claims 1 to 15, characterized in that the addition of fermentation substrate and the addition of the microorganism of the species Methanosarcina barkeri carried out continuously.

17. The method according to at least one of claims 1 to 16, characterized in that the microorganism of the species Methanosarcina barkeri in an amount to the

Fermentation substrate is added that after addition of the proportion of the microorganism of the species Methanosarcina barkeri constitutes at present in the fermentation substrate of microorganisms between ^{10 -8%} and 50% of the total.

18. The method according to claim 17, characterized in that the microorganism of the species Methanosarcina barkeri is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanosarcina barkeri between

10 ^{~ 6} % and 25% of the total number of microorganisms present in the fermentation substrate.

19. The method according to claim 18, characterized in that the microorganism of the species Methanosarcina barkeri is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanosarcina barkeri between 10 ^'4 % and 10% of the total in Fermentation substrate present microorganisms.

20. The method according to claim 19, characterized in that the microorganism of the species Methanosarcina barkeri is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanosarcina barkeri between 10 ^'3 % and 1% of the total in the Fermentation substrate present microorganisms.

21. Process according to at least one of Claims 1 to 20, characterized in that the microorganism of the species Methanosarcina barkeri is a microorganism of the strain Archaeon clone 5.5ft C140A.

22. The method according to at least one of claims 1 to 20, characterized in that it is a microorganism of the strain Archaeon clone 5.5ft C17A in the microorganism of the species Methanosarcina barkeri.

23. The method according to at least one of claims 1 to 20, characterized in that it is a microorganism of the strain Archaeon clone 5.5ft C38A in the microorganism of the species Methanosarcina barkeri.

24. The method according to at least one of claims 1 to 20, characterized in that it is in the microorganism of the species Methanosarcina barkeri to a

Microorganism of the strain Archaeon clone ATB-KM1219 acts. 25. The method according to at least one of claims 1 to 20, characterized in that it is the microorganism of the species Methanosarcina barkeri to a microorganism of the strain Archaeon clone ATB-KM1253.

26. The method according to at least one of claims 1 to 20, characterized in that it is the microorganism of the species Methanosarcina barkeri to a microorganism of the strain Archaeon clone MH1492 3E.

27. Use of a microorganism of the species Methanosarcina barkeri for the fermentative production of biogas from biomass.

28. Use according to claim 27, characterized in that it is a microorganism of the strain Archaeon clone 5.5ft Cl 4OA in the microorganism of the species Methanosarcina barkeri.

29. Use according to claim 27, characterized in that it is a microorganism of the strain Archaeon clone 5.5ft C17A in the microorganism of the species Methanosarcina barkeri.

30. Use according to claim 27, characterized in that it is a microorganism of the strain Archaeon clone 5.5ft C38A in the microorganism of the species Methanosarcina barkeri.

31. Use according to Claim 27, characterized in that the microorganism of the species Methanosarcina barkeri is a microorganism of the

Strain Archaeon clone ATB-KM1219 trades.

32. Use according to claim 27, characterized in that the microorganism of the species Methanosarcina barkeri is a microorganism of the strain Archaeon clone ATB-KM1253.

33. Use according to claim 27, characterized in that the microorganism of the species Methanosarcina barkeri is a microorganism of the strain Archaeon clone MH1492_3E. 34. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 96.03% sequence identity with the nucleotide sequence SEQ ID No. 13.

35. The microorganism according to claim 34, characterized in that the nucleotide sequence contains a sequence region which has more than 97.0% sequence identity with the nucleotide sequence SEQ ID No. 13.

36. The microorganism according to claim 35, characterized in that the nucleotide sequence contains a sequence region which has more than 98.0% sequence identity with the nucleotide sequence SEQ ID No. 13.

37. The microorganism according to claim 36, characterized in that the nucleotide sequence contains a sequence region which has more than 99.0% sequence identity with the nucleotide sequence SEQ ID No. 13.

38. The microorganism according to claim 37, characterized in that the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 13.

39. Microorganism according to claim 38, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 13.

40. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 99.20% sequence identity with the nucleotide sequence SEQ ID No. 14.

41. Microorganism according to claim 40, characterized in that the nucleotide sequence contains a sequence range exceeding 99.30%

Sequence identity with the nucleotide sequence SEQ ID NO. 14 has.

42. The microorganism according to claim 41, characterized in that the nucleotide sequence contains a sequence region which has more than 99.50% sequence identity with the nucleotide sequence SEQ ID No. 14. 43. The microorganism according to claim 42, characterized in that the nucleotide sequence contains a sequence region which has more than 99.70% sequence identity with the nucleotide sequence SEQ ID No. 14.

44. The microorganism according to claim 43, characterized in that the nucleotide sequence contains a sequence region which has more than 99.90% sequence identity with the nucleotide sequence SEQ ID No. 14.

45. Microorganism according to claim 44, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID NO.

14 corresponds.

46. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 97.15% sequence identity with the nucleotide sequence SEQ ID No. 15.

47. The microorganism according to claim 46, characterized in that the nucleotide sequence contains a sequence region which has more than 98.0% sequence identity with the nucleotide sequence SEQ ID No. 15.

48. The microorganism according to claim 47, characterized in that the nucleotide sequence contains a sequence region which has more than 98.5% sequence identity with the nucleotide sequence SEQ ID No. 15.

49. The microorganism according to claim 48, characterized in that the nucleotide sequence contains a sequence region which has more than 99.0% sequence identity with the nucleotide sequence SEQ ID No. 15.

50. The microorganism according to claim 49, characterized in that the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 15.

51. Microorganism according to claim 50, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 15. 52. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 99.75% sequence identity with the nucleotide sequence SEQ ID No. 30.

53. Microorganism according to claim 52, characterized in that the nucleotide sequence contains a sequence region which has more than 99.80% sequence identity with the nucleotide sequence SEQ ID No. 30.

54. The microorganism according to claim 53, characterized in that the nucleotide sequence contains a sequence range which exceeds 99.85%.

Sequence identity with the nucleotide sequence SEQ ID NO. 30 has.

55. The microorganism according to claim 54, characterized in that the nucleotide sequence contains a sequence region which has more than 99.90% sequence identity with the nucleotide sequence SEQ ID No. 30.

56. The microorganism according to claim 55, characterized in that the nucleotide sequence contains a sequence region which has more than 99.95% sequence identity with the nucleotide sequence SEQ ID No. 30.

57. Microorganism according to claim 56, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 30.

58. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 99.50% sequence identity with the nucleotide sequence SEQ ID No. 17.

59. Microorganism according to claim 58, characterized in that the nucleotide sequence contains a sequence range which exceeds 99.60%

Sequence identity with the nucleotide sequence SEQ ID NO. 17 has.

60. Microorganism according to claim 59, characterized in that the nucleotide sequence contains a sequence region which has more than 99.70% sequence identity with the nucleotide sequence SEQ ID No. 17. 61. Microorganism according to claim 60, characterized in that the nucleotide sequence contains a sequence region which has more than 99.80% sequence identity with the nucleotide sequence SEQ ID No. 17.

62. The microorganism according to claim 61, characterized in that the nucleotide sequence contains a sequence region which has more than 99.90% sequence identity with the nucleotide sequence SEQ ID No. 17.

63. Microorganism according to claim 62, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID NO.

17 corresponds.

64. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 98.14% sequence identity with the nucleotide sequence SEQ ID No. 21.

65. The microorganism according to claim 64, characterized in that the nucleotide sequence contains a sequence region which has more than 98.5% sequence identity with the nucleotide sequence SEQ ID No. 21.

66. The microorganism according to claim 65, characterized in that the nucleotide sequence contains a sequence region which has more than 99.0% sequence identity with the nucleotide sequence SEQ ID No. 21.

67. The microorganism according to claim 66, characterized in that the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 21.

68. The microorganism according to claim 67, characterized in that the nucleotide sequence contains a sequence region which has more than 99.9% sequence identity with the nucleotide sequence SEQ ID No. 21.

69. Microorganism according to claim 68, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 21. 70. culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, characterized in that in the culture of microorganisms, a microorganism according to claims 34 to 69 is present, wherein the microorganism at least 10 ^'4 % of the total in the culture microorganisms present.

71. Culture of microorganisms according to claim 70, characterized in that the microorganism makes up at least ^{10 -2%} of the total number of microorganisms present in the culture.

72. Culture of microorganisms according to claim 71, characterized in that the microorganism constitutes at least 1% of the total number of microorganisms present in the culture.

73. Culture of microorganisms according to claim 72, characterized in that the microorganism constitutes at least 10% of the total number of microorganisms present in the culture.

74. Culture of microorganisms according to claim 73, characterized in that the microorganism at least 50% of the total number of existing in the culture

Constitutes microorganisms.

75. Culture of microorganisms according to claim 74, characterized in that the microorganism constitutes at least 90% of the total number of microorganisms present in the culture.

76. Culture of microorganisms according to claim 75, characterized in that it is a pure culture of a microorganism according to claims 34 to 69.

77. Culture of microorganisms according to at least one of claims 70 to 76, characterized in that it is an immobilized culture of microorganisms.

78. Use of a microorganism according to at least one of claims 34 to 69 for the fermentative production of biogas from biomass. 79. Use according to claim 78, characterized in that it is a method for the fermentative production of biogas from biomass according to at least one of claims 1 to 26.

80. Use of a culture of microorganisms according to at least one of claims 70 to 77 for the fermentative production of biogas from biomass.

81. Use according to claim 80, characterized in that it is a process for the fermentative production of biogas from biomass biomass according to at least one of claims 1 to 26.

In summary, a process for the production of biogas from biomass in a fermentation reactor is described, wherein the biomass is added to a microorganism of the species Methanosarcina barkeri.

Methanosarcina mazei

The present invention provides a method for producing biogas from biomass in a fermentation reactor. The biomass is added according to the invention a microorganism of the species Methanosarcina mazei.

Surprisingly, it has been shown that the addition of microorganisms of the species Methanosarcina mazei to the fermentation substrate, the amount of biogas formed can be significantly increased. At constant volume loading, the addition of microorganisms of the species Methanosarcina mazei causes a significant increase in the amount of biogas produced. Alternatively, the amount of methane produced can also be increased by increasing the volume load, with the addition of microorganisms of the species Methanosarcina mazei preventing the fermentation process from becoming unstable under the altered conditions. The use of microorganisms of the species Methanosarcina mazei therefore leads to an improvement in the efficiency and efficiency of biogas plants.

It is known to those skilled in the art which strains of microorganisms fall under the term "Methanosarcina mazei". In particular, the synonyms "Methanococcus frisius" and "Methanococcus mazei" and "Methanosarcina frisia" are to be understood by the "type Methanosarcina mazei" (Bacterial Nomenclature Up-To-Date, Approved List of the DSMZ - German Collection for Microorganisms and Cell Cultures GmbH, As of March 2010). In connection with the production of biogas by fermentation of organic substrates, microorganisms of the species Methanosarcina mazei are not yet known.

According to a preferred embodiment of the present invention, a microorganism of the species Methanosarcina mazei is added in the form of a culture of microorganisms consisting predominantly of microorganisms of the species Methanosarcina mazei. In fermentation substrates of biogas plants microorganisms of the species Methanosarcina mazei could be detected only in trace amounts of less than 10 ^'4% share of the total number of present microorganisms. Since the amount of microorganisms isolated from their natural occurrence is insufficient for the addition of the microorganisms, it is usually propagated in the form of a culture. In practice, it is found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out directly in the form of a culture of microorganisms.

The addition of the culture of Methanosarcina mazei can be carried out in the form of a culture suspension or in the form of dry, freeze-dried or moist cell pellets.

Since the already mentioned various positive effects on the fermentation process are associated with the microorganism species Methanosarcina mazei, this type of microorganism should be present in the added culture in a concentration exceeding the natural abundance. Of course, mixed cultures of any composition can be used for the addition. All that is required is that the species Methanosarcina mazei is present in a concentration enriched with respect to the natural occurrence.

The determination of the proportions of different types of microorganisms in mixed cultures is not a problem for the person skilled in the art. For example, the proportion of microorganisms of the species Methanosarcina mazei can be specifically identified in a mixture with the aid of fluorescence-labeled oligoprobes. As already mentioned, microorganisms of the species Methanosarcina mazei are preferably added to the fermentation substrate in the form of cultures of microorganisms, the cultures of microorganisms consisting predominantly of microorganisms of the species Methanosarcina mazei. If, in addition to the determination of the number of microorganisms of the species Methanosarcina mazei, the total number of microorganisms is also determined, the proportion of microorganisms of the species Methanosarcina mazei in the culture may be expressed as a percentage. Microorganisms of the species Methanosarcina mazei are in a mixed culture then the predominant species of microorganisms, if they have the highest percentage of the different types of microorganisms present in the mixed culture.

According to preferred embodiments of the present invention, microorganisms of the species Methanosarcina mazei make at least 10 ^'4 %, in particular at least

10 ^'2 %, particularly preferably at least 1% of the total number of microorganisms present in the culture added to the fermentation substrate. According to further preferred

Embodiments make at least microorganisms of the species Methanosarcina mazei

10%, in particular at least 50%, particularly preferably at least 90% of the total number of microorganisms present in the culture.

According to a very particularly preferred embodiment, a pure culture of a microorganism of the species Methanosarcina mazei is added. Due to the specific metabolic processes and activities, the addition of a pure culture can especially contribute to an improved methane production.

According to a further preferred embodiment of the present invention, a microorganism of the species Methanosarcina mazei is added as a constituent of at least one immobilized culture of microorganisms. Since the amount of microorganisms isolated from their natural occurrence is insufficient for the addition of the microorganisms, it is usually propagated in the form of a culture. In practice, it has been found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out in the form of an immobilized culture of microorganisms.

According to a preferred embodiment of the present invention, the microorganism of the species Methanosarcina mazei is added to the fermentation substrate in an amount such that, after addition, the proportion of the microorganism of the species Methanosarcina mazei is between 10 ⁸ % and 50% of the total number of microorganisms present in the fermentation substrate , In particular, depending on the fermenter size and thus as a function of the amount of fermentation substrate can be necessary to achieve the desired effect, an addition of very different amounts of microorganisms.

A microorganism of the species Methanosarcina mazei is particularly preferably added in an amount to the fermentation substrate, that after addition the content of the microorganism of the species Methanosarcina mazei between ^{10 -6%} and 25% of the total number of in the Fermentation substrate present microorganisms. More preferably, the microorganism of the species Methanosarcina mazei is added to the fermentation substrate in an amount such that, after addition, the proportion of the microorganism of the species Methanosarcina mazei is between 10 ^'4 % and 10% of the total number of microorganisms present in the fermentation substrate. Most preferably, the microorganism of the species Methanosarcina mazei is added to the fermentation substrate in an amount such that, after addition, the proportion of the microorganism of the species Methanosarcina mazei is between 10 ^-3 % and 1% of the total number of microorganisms present in the fermentation substrate.

According to a particularly preferred embodiment of the present invention, the microorganism of the species Methanosarcina mazei is a microorganism of the strain Methanosarcina mazei Goe1. In all of the above-described embodiments, which deal with the addition of a microorganism of the species Methanosarcina mazei, so preferably a microorganism of the strain Methanosarcina mazei Goe 1 is added.

The present invention also encompasses the use of a microorganism of the species Methanosarcina mazei for the fermentative production of biogas from biomass. Preferably, a microorganism of the strain strain Methanosarcina mazei Goe1 is used for the fermentative production of biogas from biomass.

The microorganisms Methanosarcina mazei SBG9 obtained from the fermentation substrate of a post-fermenter were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID No. 6 comprises 1 131 nucleotides. Methanosarcina mazei Goe1 was identified as the next relative. A comparison of the sequences revealed that a total of 7 exchanges of nucleotides or deletions are present. At a length of the particular nucleotide sequence of Methanosarcina mazei SBG9 of 1 131 nucleotides and a length of the reference sequence of 1 130 nucleotides, the FASTA algorithm gives a 99.38% match.

The present invention also includes microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having greater than 99.38% sequence identity with the nucleotide sequence of SEQ ID NO: 6. More preferably, the nucleotide sequence contains a sequence range greater than 99.40% or greater than 99.45% or greater than 99.50% or greater than 99.55% or greater than 99.60% or greater than 99.65%. Sequence identity with the nucleotide sequence SEQ ID NO. 6 and in particular Preferably, the nucleotide sequence contains a sequence region having more than 99.70% sequence identity with the nucleotide sequence SEQ ID NO: 6.

According to further preferred embodiments, the microorganism has a nucleotide sequence which contains a sequence range which exceeds 99.80%.

Sequence identity with the nucleotide sequence SEQ ID No. 6, and more preferably, the nucleotide sequence contains a sequence region having more than 99.90% sequence identity with the nucleotide sequence of SEQ ID NO: 6. According to a most preferred

In an embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 6.

In preferred embodiments of the present invention, nucleotide mutations may be present at one or two positions or at three positions or at four positions or at five positions or at six positions or at seven positions relative to the starting nucleotide sequence SEQ ID NO: 6. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also encompasses a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, in which culture of microorganisms a microorganism is present which has a nucleotide sequence containing a sequence region greater than 99.38 % Sequence identity with the nucleotide sequence SEQ ID NO: 6, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 99.40% or greater than 99.45% or greater than 99 , 50% or more than 99.55% or more than 99.60% or more than 99.65% sequence identity with the nucleotide sequence SEQ ID NO: 6. Particularly preferably, the nucleotide sequence contains a sequence region which has more than 99.70% sequence identity with the nucleotide sequence SEQ ID No. 6.

According to further preferred embodiments, the culture suitable for use in a process for the fermentative production of biogas from biomass is

Microorganisms present a microorganism having a nucleotide sequence with a

Sequence region has more than 99.80% sequence identity with the nucleotide sequence SEQ ID NO. 6 has. Most preferably, the nucleotide sequence contains a sequence region which has more than 99.90% sequence identity with the nucleotide sequence SEQ ID No. 6.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 6.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 6 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 6 in a method described above in connection with microorganisms of the species Methanosarcina mazei for the fermentative production of biogas from biomass.

The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO. 6 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. Preference is given to the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID No. 6 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in a method described above in connection with microorganisms of the species Methanosarcina mazei for the fermentative production of biogas from biomass.

According to further preferred embodiments make said, characterized in terms of their nucleotide sequence microorganisms at least 10 ^'2%, preferably to at least 1% of the total present in the culture of microorganisms. Particularly preferably, the microorganisms make up at least 10%, particularly preferably at least 25%, of the total number of microorganisms present in the culture. Most preferably, the microorganisms described make up at least 50%, in particular at least 75%, of the total number of microorganisms present in the culture. Also preferred are embodiments according to which the above-mentioned microorganisms make up at least 90% of the total number of microorganisms present in the culture. Particularly preferably it is a pure culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, which is a pure culture of a microorganism, as has been characterized above with respect to its nucleotide sequence.

Most preferably, in the cases described above, it is an immobilized culture of microorganisms.

In particular, the present invention includes the following aspects:

1. A process for the production of biogas from biomass in a fermentation reactor, characterized in that the biomass is added to a microorganism of the species Methanosarcina mazei.

2. The method according to claim 1, characterized in that the microorganism of the species Methanosarcina mazei is added in the form of a culture of microorganisms, wherein microorganisms of the species Methanosarcina mazei make up at least 10 ^'4 % of the total number of microorganisms present in the culture.

3. The method according to claim 2, characterized in that constitute in the culture of microorganisms of the species Methanosarcina mazei at least 10 ^'2 % of the total number of microorganisms present in the culture.

4. The method according to claim 3, characterized in that constitute in the culture of microorganisms of the species Methanosarcina mazei at least 1% of the total number of microorganisms present in the culture.

5. The method according to claim 4, characterized in that make up in the culture of microorganisms of the species Methanosarcina mazei at least 10% of the total number of microorganisms present in the culture.

6. The method according to claim 5, characterized in that make up in the culture of microorganisms of the species Methanosarcina mazei at least 50% of the total number of microorganisms present in the culture. 7. The method according to claim 6, characterized in that constitute in the culture of microorganisms of the species Methanosarcina mazei at least 75% of the total number of microorganisms present in the culture.

8. The method according to claim 7, characterized in that make up in the culture of microorganisms of the species Methanosarcina mazei at least 90% of the total number of microorganisms present in the culture.

9. The method according to at least one of claims 2 to 8, characterized in that a pure culture of the microorganism of the species Methanosarcina mazei is added.

10. The method according to at least one of claims 2 to 9, characterized in that microorganisms of the species Methanosarcina mazei the biomass are added as part of at least one immobilized culture of microorganisms.

1 1. Method according to at least one of claims 1 to 10, characterized in that in time for the addition of the microorganism of the species Methanosarcina mazei additional biomass is added to the fermentation reactor.

12. The method according to at least one of claims 1 to 1 1, characterized in that the space load in the fermentation reactor is continuously increased by the continuous addition of biomass.

13. The method according to at least one of claims 1 to 12, characterized in that the production of biogas from biomass at a space load of ≥ 0.5 kg oTS / m ³ d, preferably ≥ 4.0 kg oTS / m ³ d, especially preferably ≥ 8.0 kg oTS / m ³ d.

14. The method according to at least one of claims 1 to 13, characterized in that the production of biogas from biomass under constant mixing of

Fermentation substrate takes place.

15. The method according to at least one of claims 1 to 14, characterized in that the production of biogas from biomass at a temperature of 20 ^ to 80 ⁰ C, preferably at a temperature of 40 ^< € to 50 ⁰ C is performed. 16. The method according to at least one of claims 1 to 15, characterized in that the addition of fermentation substrate and the addition of the microorganism of the species Methanosarcina mazei carried out continuously.

17. The method according to at least one of claims 1 to 16, characterized in that the microorganism of the species Methanosarcina mazei is added in an amount to the fermentation substrate that after addition of the proportion of the microorganism of the species Methanosarcina mazei between 10 ^'8 % and 50% the total number of microorganisms present in the fermentation substrate.

18. The method according to claim 17, characterized in that the microorganism of the species Methanosarcina mazei is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanosarcina mazei between 10 ^"6 % and 25% of the total in Fermentation substrate present microorganisms.

19. The method according to claim 18, characterized in that the microorganism of the species Methanosarcina mazei is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanosarcina mazei between 10 ^{~ 4} % and 10% of the total in Fermentation substrate present microorganisms.

20. The method according to claim 19, characterized in that the microorganism of the species Methanosarcina mazei is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanosarcina mazei between

Represents 10 ^"3 % and 1% of the total number of microorganisms present in the fermentation substrate.

21. Method according to at least one of Claims 1 to 20, characterized in that the microorganism of the species Methanosarcina mazei is a

Microorganism of the strain Methanosarcina mazei Goe1 acts.

22. Use of a microorganism of the species Methanosarcina mazei for the fermentative production of biogas from biomass. 23. Use according to claim 22, characterized in that the microorganism of the species Methanosarcina mazei is a microorganism of the strain Methanosarcina mazei Goe1.

24. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 99.38% sequence identity with the nucleotide sequence SEQ ID No. 6.

25. Microorganism according to claim 24, characterized in that the nucleotide sequence contains a sequence region which has more than 99.40% sequence identity with the nucleotide sequence SEQ ID No. 6.

26. Microorganism according to claim 25, characterized in that the nucleotide sequence contains a sequence region which has more than 99.60% sequence identity with the nucleotide sequence SEQ ID No. 6.

27. Microorganism according to claim 26, characterized in that the nucleotide sequence contains a sequence region which has more than 99.80% sequence identity with the nucleotide sequence SEQ ID No. 6.

28. Microorganism according to claim 27, characterized in that the nucleotide sequence contains a sequence region which has more than 99.90% sequence identity with the nucleotide sequence SEQ ID No. 6.

29. Microorganism according to claim 28, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 6.

30. Culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, characterized in that in the culture of

Microorganisms, a microorganism according to claims 24 to 29 is present, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

31. Culture of microorganisms according to claim 30, characterized in that the microorganism makes up at least ^{10 -2%} of the total number of microorganisms present in the culture. 32. Culture of microorganisms according to claim 31, characterized in that the microorganism constitutes at least 1% of the total number of microorganisms present in the culture.

33. Culture of microorganisms according to claim 32, characterized in that the microorganism constitutes at least 10% of the total number of microorganisms present in the culture.

34. Culture of microorganisms according to claim 33, characterized in that the microorganism constitutes at least 50% of the total number of microorganisms present in the culture.

35. Culture of microorganisms according to claim 34, characterized in that the microorganism comprises at least 90% of the total number of existing in the culture

Constitutes microorganisms.

36. Culture of microorganisms according to claim 35, characterized in that it is a pure culture of a microorganism according to claims 24 to 29.

37. Culture of microorganisms according to at least one of claims 30 to 36, characterized in that it is an immobilized culture of microorganisms.

38. Use of a microorganism according to at least one of claims 24 to 29 for the fermentative production of biogas from biomass.

39. Use according to claim 38, characterized in that it is a process for the fermentative production of biogas from biomass according to at least one of claims 1 to 21.

40. Use of a culture of microorganisms according to at least one of claims 30 to 37 for the fermentative production of biogas from biomass.

41. Use according to claim 40, characterized in that it is a process for the fermentative production of biogas from biomass biomass according to at least one of claims 1 to 21. In summary, a process for the production of biogas from biomass in a fermentation reactor is described, wherein the biomass is added to a microorganism of the species Methanosarcina mazei.

Methanothermobacter wolfeii

The present invention provides a method for producing biogas from biomass in a fermentation reactor. The biomass according to the invention is added to a microorganism of the species Methanothermobacter wolfeii.

Surprisingly, it has been shown that the addition of microorganisms of the species Methanothermobacter wolfeii to the fermentation substrate, the amount of biogas formed can be significantly increased. At constant space load, the addition of microorganisms of the species Methanothermobacter wolfeii causes a significant increase in the amount of biogas produced. Alternatively, the amount of methane formed can also be increased by increasing the space load, with the addition of microorganisms of the species Methanothermobacter wolfeii preventing the fermentation process from becoming unstable under the altered conditions. The use of microorganisms of the species Methanothermobacter wolfeii therefore leads to an improvement in the efficiency and efficiency of biogas plants.

It is known to those skilled in the art which strains of microorganisms fall under the term "species Methanothermobacter wolfeii '. In particular, under the "Art

Methanothermobacter wolfeii 'also the synonyms "Methanothermobacter wolfef as well as

"Methanobacterium wolfei" to understand (Bacterial Nomenclature Up-To Date, Approved List of DSMZ - German Collection of Microorganisms and Cell Cultures GmbH, March

2010). In connection with the production of biogas by fermentation of organic substrates, microorganisms of the species Methanothermobacter wolfeii are not yet known.

According to a preferred embodiment of the present invention, a microorganism of the species Methanothermobacter wolfeii is added in the form of a culture of microorganisms consisting predominantly of microorganisms of the species Methanothermobacter wolfeii. In fermentation substrates of biogas plants microorganisms of the species could be detected from Methanothermobacter present microorganisms wolfeii only in trace amounts of less than 10 ^'4% share of the total number. There For the addition of microorganisms, the amount of microorganisms isolated from their natural occurrence is insufficient, usually a propagation is carried out in the form of a culture. In practice, it is found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out directly in the form of a culture of microorganisms.

The addition of the culture of Methanothermobacter wolfeii can be carried out in the form of a culture suspension or in the form of dry, freeze-dried or moist cell pellets.

Since the already mentioned various positive effects on the fermentation process are associated with the microorganism species Methanothermobacter wolfeii, this type of microorganism should be present in the added culture in a concentration exceeding the natural abundance. Of course, mixed cultures of any composition can be used for the addition. All that is required is that the species Methanothermobacter wolfeii is present in a concentration which is enriched in relation to the natural occurrence.

The determination of the proportions of different types of microorganisms in mixed cultures is not a problem for the expert. Thus, for example, with the help of fluorescence-labeled oligosensors specifically the proportion of microorganisms of the species Methanothermobacter wolfeii be identified in a mixture. As already mentioned, microorganisms of the species Methanothermobacter wolfeii are preferably added to the fermentation substrate in the form of cultures of microorganisms, the cultures of microorganisms consisting predominantly of microorganisms of the species Methanothermobacter wolfeii. If, in addition to the determination of the number of microorganisms of the species Methanothermobacter wolfeii, the total number of microorganisms is also determined, the proportion of microorganisms of the species Methanothermobacter wolfeii in the culture can be stated in percent. Microorganisms of the species Methanothermobacter wolfeii are in a mixed culture then the predominantly present type of microorganisms, if they have the highest percentage of the various types of microorganisms present in the mixed culture.

According to preferred embodiments of the present invention make microorganisms of the species Methanothermobacter wolfeii at least 10 ^'4%, especially at least 10 ^"2%, more preferably at least 1% of the total number of the added to the fermentation substrate culture microorganisms present. According to further preferred embodiments make microorganisms of the species Methanothermobacter wolfeii at least 10%, in particular at least 50%, more preferably at least 90% of the total number of microorganisms present in the culture.

According to a very particularly preferred embodiment, a pure culture of a microorganism of the species Methanothermobacter wolfeii is added. Due to the specific metabolic processes and activities, the addition of a pure culture can especially contribute to an improved methane production.

According to another preferred embodiment of the present invention, a microorganism of the species Methanothermobacter wolfeii is added as a component of at least one immobilized culture of microorganisms. As for the addition of the

Microorganisms the amount of isolated from their natural occurrence

Microorganisms is insufficient, is usually made an increase in the form of a culture. In practice it has been found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily achieved in the form of an immobilized culture of

Microorganisms is made.

According to a preferred embodiment of the present invention, the microorganism of the species Methanothermobacter wolfeii is added in an amount to the fermentation substrate, so that after addition of the proportion of the microorganism of the species Methanothermobacter wolfeii between 10 ^'8 % and 50% of the total number of present in the fermentation substrate microorganisms accounts. In particular, depending on the fermenter size and thus as a function of the amount of fermentation substrate can be necessary to achieve the desired effect, an addition of very different amounts of microorganisms.

A microorganism of the species Methanothermobacter is particularly preferred wolfeii in an amount added to the fermentation substrate that constitutes wolfeii after addition of the content of the microorganism of the species Methanothermobacter between ^{10 -6%} and 25% of the total number present in the fermentation substrate microorganisms. The microorganism of the species Methanothermobacter is particularly preferred wolfeii in an amount added to the fermentation substrate that constitutes wolfeii after addition of the content of the microorganism of the species Methanothermobacter between ^{10 -4%} and 10% of the total number present in the fermentation substrate microorganisms. Most preferably, the microorganism of the species Methanothermobacter wolfeii is added in an amount to the fermentation substrate such that, after addition, the proportion of the microorganism of the species Methanothermobacter wolfeii is between 10 ^{'. 3} % and 1% of the total number of microorganisms present in the fermentation substrate.

The present invention also encompasses the use of a microorganism of the species Methanothermobacter wolfeii for the fermentative production of biogas from biomass.

The microorganisms Methanothermobacter wolfeii SBG10 obtained from the fermentation substrate of a postgrader were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID No. 7 comprises 1130 nucleotides. Methanothermobacter wolfeii was identified as the next relative. A comparison of the sequences revealed that there are a total of five exchanges of nucleotides or deletions. At a length of the particular nucleotide sequence of Methanothermobacter wolfeii SBG10 of 1130 nucleotides and a length of the reference sequence of 1 129 nucleotides, the FASTA algorithm gives a 99.56% match.

The present invention also includes microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having greater than 99.56% sequence identity with the nucleotide sequence of SEQ ID NO: 7. More preferably, the nucleotide sequence contains a sequence range greater than 99.58% or greater than 99.60% or greater than 99.62% or greater than 99.64% or greater than 99.66% or greater than 99.68%. Sequence identity with the nucleotide sequence SEQ ID NO. 7, and more preferably, the nucleotide sequence contains a sequence region having more than 99.70% sequence identity with the nucleotide sequence SEQ ID NO: 7.

According to further preferred embodiments, the microorganism has a nucleotide sequence which contains a sequence region which has more than 99.80% sequence identity with the nucleotide sequence SEQ ID No. 7, and more preferably the nucleotide sequence contains a sequence region which has more than 99.90% sequence identity having the nucleotide sequence of SEQ ID NO: 7. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 7.

In preferred embodiments of the present invention, nucleotide mutations may be present at one or two positions or at three positions or at four positions or at five positions relative to the starting nucleotide sequence SEQ ID NO: 7. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text. The present invention also encompasses a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism having a nucleotide sequence containing a sequence region greater than 99.56 is present % Sequence identity with the nucleotide sequence of SEQ ID NO: 7, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 99.58% or greater than 99.60% or greater than 99 , 62% or more than 99.64% or more than 99.66% or more than 99.68% sequence identity with the nucleotide sequence SEQ ID NO: 7. Particularly preferably, the nucleotide sequence contains a sequence region which has more than 99.70% sequence identity with the nucleotide sequence SEQ ID No. 7.

According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range exceeding 99.80% sequence identity with the nucleotide sequence SEQ ID NO. 7 has. Most preferably, the nucleotide sequence contains a sequence region which has more than 99.90% sequence identity with the nucleotide sequence SEQ ID No. 7.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 7.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 7 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 7 in a method described above in connection with microorganisms of the species Methanothermobacter wolfeii for the fermentative production of biogas from biomass. The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO. 7 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. Preference is given to the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID No. 7 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in a method described above in connection with microorganisms of the species Methanothermobacter wolfeii fermentative production of biogas from biomass.

According to further preferred embodiments make said, characterized in terms of their nucleotide sequence microorganisms at least 10 ^'2%, preferably to at least 1% of the total present in the culture of microorganisms. Particularly preferably, the microorganisms make up at least 10%, particularly preferably at least 25%, of the total number of microorganisms present in the culture. Most preferably, the microorganisms described make up at least 50%, in particular at least 75%, of the total number of microorganisms present in the culture.

Also preferred are embodiments according to which the above-mentioned microorganisms make up at least 90% of the total number of microorganisms present in the culture. Particularly preferably it is a pure culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, which is a pure culture of a microorganism, as has been characterized above with respect to its nucleotide sequence.

Most preferably, in the cases described above, it is an immobilized culture of microorganisms.

In particular, the present invention includes the following aspects:

1. A process for the production of biogas from biomass in a fermentation reactor, characterized in that the biomass is added to a microorganism of the species Methanothermobacter wolfeii. 2. The method according to claim 1, characterized in that the microorganism of the species Methanothermobacter wolfeii is added in the form of a culture of microorganisms, wherein microorganisms of the species Methanothermobacter wolfeii account for at least 10 ^'4 % of the total number of microorganisms present in the culture.

3. The method according to claim 2, characterized in that constitute in the culture of microorganisms of the species Methanothermobacter wolfeii at least 10 ^'2 % of the total number of microorganisms present in the culture.

4. The method according to claim 3, characterized in that constitute in the culture of microorganisms of the species Methanothermobacter wolfeii at least 1% of the total number of microorganisms present in the culture.

5. The method according to claim 4, characterized in that constitute in the culture of microorganisms of the species Methanothermobacter wolfeii at least 10% of the total number of microorganisms present in the culture.

6. The method according to claim 5, characterized in that constitute in the culture of microorganisms of the species Methanothermobacter wolfeii at least 50% of the total number of microorganisms present in the culture.

7. The method according to claim 6, characterized in that constitute in the culture of microorganisms of the species Methanothermobacter wolfeii at least 75% of the total number of microorganisms present in the culture.

8. The method according to claim 7, characterized in that constitute in the culture of microorganisms of the species Methanothermobacter wolfeii at least 90% of the total number of microorganisms present in the culture.

9. The method according to at least one of claims 2 to 8, characterized in that a pure culture of the microorganism of the species Methanothermobacter wolfeii is added.

10. The method according to at least one of claims 2 to 9, characterized in that microorganisms of the species Methanothermobacter wolfeii the biomass are added as part of at least one immobilized culture of microorganisms. 1 1. Method according to at least one of claims 1 to 10, characterized in that timely to the addition of the microorganism of the species Methanothermobacter wolfeii additional biomass is added to the fermentation reactor.

12. The method according to at least one of claims 1 to 1 1, characterized in that the space load in the fermentation reactor is continuously increased by the continuous addition of biomass.

13. The method according to at least one of claims 1 to 12, characterized in that the production of biogas from biomass at a space load of ≥ 0.5 kg oTS / m ³ d, preferably ≥ 4.0 kg oTS / m ³ d, especially preferably ≥ 8.0 kg oTS / m ³ d.

14. The method according to at least one of claims 1 to 13, characterized in that the production of biogas from biomass takes place under constant mixing of the fermentation substrate.

15. The method according to at least one of claims 1 to 14, characterized in that the production of biogas from biomass at a temperature of 20 ^< € to 80 ⁰ C, preferably at a temperature of 40 ^ to 50 ⁰ C is performed.

16. The method according to at least one of claims 1 to 15, characterized in that the addition of fermentation substrate and the addition of the microorganism of the species Methanothermobacter wolfeii continuously.

17. The method according to at least one of claims 1 to 16, characterized in that the microorganism of the species Methanothermobacter wolfeii is added in an amount to the fermentation substrate that after addition of the proportion of the microorganism of the species Methanothermobacter wolfeii between 10 ^'8 % and 50% the total number in the

Fermentation substrate present microorganisms.

18. The method according to claim 17, characterized in that the microorganism of the species Methanothermobacter wolfeii is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanothermobacter wolfeii between 10 ^'6 % and 25% of the total in the Fermentation substrate present microorganisms. 19. The method according to claim 18, characterized in that the microorganism of the species Methanothermobacter wolfeii is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanothermobacter wolfeii between 10 ^"4 % and 10% of the total in Fermentation substrate present

Constitutes microorganisms.

20. The method according to claim 19, characterized in that the microorganism of the species Methanothermobacter wolfeii is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanothermobacter wolfeii between 10 ^'3 % and 1% of the total in Fermentation substrate present microorganisms.

21. Use of a microorganism of the species Methanothermobacter wolfeii for fermentative production of biogas from biomass.

22. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 99.56% sequence identity with the nucleotide sequence SEQ ID No. 7.

23. Microorganism according to claim 22, characterized in that the nucleotide sequence contains a sequence region which has more than 99.60% sequence identity with the nucleotide sequence SEQ ID No. 7.

24. Microorganism according to claim 23, characterized in that the nucleotide sequence contains a sequence region which has more than 99.70% sequence identity with the nucleotide sequence SEQ ID No. 7.

25. Microorganism according to claim 24, characterized in that the nucleotide sequence contains a sequence region which has more than 99.80% sequence identity with the nucleotide sequence SEQ ID No. 7.

26. Microorganism according to claim 25, characterized in that the nucleotide sequence contains a sequence region which has more than 99.90% sequence identity with the nucleotide sequence SEQ ID No. 7. 27. Microorganism according to claim 26, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 7.

28. Culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, characterized in that in the culture of microorganisms, a microorganism according to claims 22 to 27 is present, wherein the microorganism at least 10 ^'4 % of the total in the culture microorganisms present.

29. Culture of microorganisms according to claim 28, characterized in that the microorganism constitutes at least 10 ^'2 % of the total number of microorganisms present in the culture.

30. Culture of microorganisms according to claim 29, characterized in that the microorganism constitutes at least 1% of the total number of microorganisms present in the culture.

31. Culture of microorganisms according to claim 30, characterized in that the microorganism comprises at least 10% of the total number of existing in the culture

Constitutes microorganisms.

32. Culture of microorganisms according to claim 31, characterized in that the microorganism constitutes at least 50% of the total number of microorganisms present in the culture.

33. Culture of microorganisms according to claim 32, characterized in that the microorganism constitutes at least 90% of the total number of microorganisms present in the culture.

34. Culture of microorganisms according to claim 33, characterized in that it is a pure culture of a microorganism according to claims 22 to 27.

35. Culture of microorganisms according to at least one of claims 28 to 34, characterized in that it is an immobilized culture of microorganisms. 36. Use of a microorganism according to at least one of claims 22 to 27 for the fermentative production of biogas from biomass.

37. Use according to claim 36, characterized in that it is a method for the fermentative production of biogas from biomass according to at least one of

Claims 1 to 20 is.

38. Use of a culture of microorganisms according to at least one of claims 28 to 35 for the fermentative production of biogas from biomass.

39. Use according to claim 38, characterized in that it is a process for the fermentative production of biogas from biomass biomass according to at least one of claims 1 to 20.

In summary, a method for producing biogas from biomass in a fermentation reactor is described, wherein the biomass is added to a microorganism of the species Methanothermobacter wolfeii.

Methanobacterium oryzae

The present invention provides a method for producing biogas from biomass in a fermentation reactor. The biomass is added according to the invention a microorganism of the species Methanobacterium oryzae.

Surprisingly, it has been found that the addition of microorganisms of the species Methanobacterium oryzae to the fermentation substrate, the amount of biogas formed can be significantly increased. At constant space load, the addition of microorganisms of the species Methanobacterium oryzae causes a significant increase in the amount of biogas produced. Alternatively, the amount of methane produced can also be increased by increasing the volume load, with the addition of microorganisms of the species Methanobacterium oryzae preventing the fermentation process from becoming unstable under the altered conditions. The use of microorganisms of the species Methanobacterium oryzae therefore leads to an improvement in the efficiency and efficiency of biogas plants. The person skilled in the art knows which strains of microorganisms fall under the term "species Methanobacterium oryzae." Microorganisms of the species Methanobacterium oryzae have hitherto not been known in connection with the production of biogas by fermentation of organic substrates.

According to a preferred embodiment of the present invention, a microorganism of the species Methanobacterium oryzae is added in the form of a culture of microorganisms consisting predominantly of microorganisms of the species Methanobacterium oryzae. In fermentation substrates of biogas plants could microorganisms of the species Methanobacterium oryzae only in trace amounts of less than 10 ^'4% share of the total number of detected present microorganisms. Since the amount of microorganisms isolated from their natural occurrence is insufficient for the addition of the microorganisms, it is usually propagated in the form of a culture. In practice, it is found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out directly in the form of a culture of microorganisms.

The addition of the culture of Methanobacterium oryzae can be carried out in the form of a culture suspension or in the form of dry, freeze-dried or moist cell pellets.

Since the already mentioned various positive effects on the fermentation process are associated with the microorganism species Methanobacterium oryzae, this type of microorganism should be present in the added culture in a concentration exceeding the natural abundance. Of course, mixed cultures of any composition can be used for the addition. All that is required is that the species Methanobacterium oryzae is present in a concentration which is enriched in relation to the natural occurrence.

The determination of the proportions of different types of microorganisms in mixed cultures is not a problem for the expert. Thus, for example, with the help of fluorescence-labeled oligosensors specifically the proportion of microorganisms of the species Methanobacterium oryzae can be identified in a mixture. As already mentioned, microorganisms of the species Methanobacterium oryzae are preferably added to the fermentation substrate in the form of cultures of microorganisms, the cultures of microorganisms consisting predominantly of microorganisms of the species Methanobacterium oryzae. If, in addition to the determination of the number of microorganisms of the species Methanobacterium oryzae, the total number of microorganisms is also determined, the proportion of microorganisms of the species Methanobacterium oryzae in culture in percent. Microorganisms of the species Methanobacterium oryzae are in a mixed culture then the predominantly present type of microorganisms, if they have the highest percentage of the various types of microorganisms present in the mixed culture.

According to preferred embodiments of the present invention make microorganisms of the species Methanobacterium oryzae at least 10 ^'4%, especially at least ^{10' 2%,} more preferably at least 1% of the total number of existing in the added to the fermentation substrate culture microorganisms. According to further preferred embodiments, microorganisms of the species Methanobacterium oryzae account for at least 10%, in particular at least 50%, particularly preferably at least 90% of the total number of microorganisms present in the culture.

According to a very particularly preferred embodiment, a pure culture of a microorganism of the species Methanobacterium oryzae is added. Due to the specific metabolic processes and activities, the addition of a pure culture can especially contribute to an improved methane production.

According to another preferred embodiment of the present invention, a microorganism of the species Methanobacterium oryzae is added as part of at least one immobilized culture of microorganisms. Since the amount of microorganisms isolated from their natural occurrence is insufficient for the addition of the microorganisms, it is usually propagated in the form of a culture. In practice, it has been found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out in the form of an immobilized culture of microorganisms.

According to a preferred embodiment of the present invention, the microorganism of the species Methanobacterium oryzae is added in an amount to the fermentation substrate, so that after addition of the proportion of the microorganism of the species Methanobacterium oryzae between 10 ^'8 % and 50% of the total number of present in the fermentation substrate microorganisms accounts. In particular, depending on the fermenter size and thus as a function of the amount of fermentation substrate can be necessary to achieve the desired effect, an addition of very different amounts of microorganisms.

More preferably, a microorganism of the species Methanobacterium oryzae is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism the species Methanobacterium oryzae between ^{10 -6%} and 25% of the total number of microorganisms present in the fermentation substrate by weight. The microorganism of the species Methanobacterium is particularly preferred oryzae in an amount to the fermentation substrate is added that, after addition, the proportion of the microorganism of the species Methanobacterium oryzae between 10 ^"4% and 10% of the total number present in the fermentation substrate microorganisms accounts. Very particular preference is the microorganism of the species Methanobacterium oryzae added that, after addition, the proportion of the microorganism of the species Methanobacterium oryzae between ^{10 -3%} and 1% of the total number of microorganisms present in the fermentation substrate in a quantity makes to the fermentation substrate.

According to a particularly preferred embodiment of the present invention, the microorganism of the species Methanobacterium oryzae is a microorganism of the strain Bacterium clone HsA55fl. In all of the above-described embodiments, which deal with the addition of a microorganism of the species Methanobacterium oryzae, so preferably a microorganism of the strain Bacterium clone HsA55fl is added.

The present invention also encompasses the use of a microorganism of the species Methanobacterium oryzae for the fermentative production of biogas from biomass. Preferably, a microorganism of the strain Bacterium clone HsA55fl is used for the fermentative production of biogas from biomass.

The microorganisms Bacterium Methanobacterium oryzae SBG26 obtained from the fermentation substrate of a post-fermenter were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID NO: 23 comprises 1 146 nucleotides. The next related sequence identified was the uncultured bacterium clone HsA55fl. A comparison of the sequences revealed that a total of 36 exchanges of nucleotides or deletions are present. At a length of the particular nucleotide sequence of Methanobacterium oryzae SBG26 of 1 146 nucleotides and a length of the reference sequence of 1 125 nucleotides calculated using the FASTA algorithm a match of 96.80%.

The present invention also encompasses microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having more than 96.80% sequence identity with the nucleotide sequence SEQ ID NO: 23. More preferably, the nucleotide sequence contains a sequence range greater than 96.9% or greater than 97.0% or greater than 97.1% or greater than 97.2% or greater than 97.3% or greater than 97.4%. or more than 97.5% sequence identity with the nucleotide sequence SEQ ID NO: 23 and particularly preferably, the nucleotide sequence contains a sequence region having more than 98.0% sequence identity with the nucleotide sequence SEQ ID NO: 23.

According to further preferred embodiments, the microorganism has a nucleotide sequence containing a sequence region greater than 98.5% or more

99.0% sequence identity with the nucleotide sequence SEQ ID No. 23, and more preferably the nucleotide sequence contains a sequence region that exceeds 99.5%

Sequence identity with the nucleotide sequence SEQ ID NO. 23 has. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 23.

In preferred embodiments of the present invention, SEQ ID NO: 23 can be compared to the starting nucleotide sequence at one or two positions or at three positions or at four positions or at five positions or at six positions or at seven positions or at eight positions or at nine positions or ten positions or eleven positions or twelve positions or 13 positions or 14 positions or 15 positions or 16 positions or 17 positions or 18 positions or 19 positions or 20 positions or 21 positions or on 22 positions or 23 positions or 24 positions or 25 positions or 26 positions or 27 positions or 28 positions or 29 positions or 30 positions or 31 positions or 32 positions or 33 positions or 34 positions or at 35 positions or at 36 positions nucleotide mutations. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also encompasses a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism is present which has a nucleotide sequence containing a sequence region greater than 96.80 % Sequence identity with the nucleotide sequence SEQ ID NO: 23, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range exceeding 96.9% or more

97.0% or more than 97.1% or more than 97.2% or more than 97.3% or more 97.4% or more than 97.5% has sequence identity with the nucleotide sequence SEQ ID NO: 23. Most preferably, the nucleotide sequence contains a sequence region that has more than 97.6% sequence identity with the nucleotide sequence SEQ ID NO: 23.

According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 98.0% or greater 98.5% or greater has 99.0% sequence identity with the nucleotide sequence SEQ ID NO: 23. Most preferably, the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 23.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 23.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 23 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 23 in a method described above in connection with microorganisms of the species Methanobacterium oryzae for the fermentative production of biogas from biomass.

The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO. 23 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. It is preferable to use a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID No. 23 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in a method described above in connection with microorganisms of the species Methanobacterium oryzae for the fermentative production of biogas from biomass. According to further preferred embodiments make said, characterized in terms of their nucleotide sequence microorganisms at least 10 ^'2%, preferably to at least 1% of the total present in the culture of microorganisms. Particularly preferably, the microorganisms make up at least 10%, particularly preferably at least 25%, of the total number of microorganisms present in the culture. Most preferably, the microorganisms described make up at least 50%, in particular at least 75%, of the total number of microorganisms present in the culture.

Also preferred are embodiments according to which the above-mentioned microorganisms make up at least 90% of the total number of microorganisms present in the culture. Particularly preferably it is a pure culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, which is a pure culture of a microorganism, as has been characterized above with respect to its nucleotide sequence.

Most preferably, in the cases described above, it is an immobilized culture of microorganisms.

In particular, the present invention includes the following aspects:

1. A process for the production of biogas from biomass in a fermentation reactor, characterized in that the biomass is added to a microorganism of the species Methanobacterium oryzae.

2. The method according to claim 1, characterized in that the microorganism of the species Methanobacterium oryzae is added in the form of a culture of microorganisms, wherein microorganisms of the species Methanobacterium oryzae make up at least 10 ^'4 % of the total number of microorganisms present in the culture.

3. The method according to claim 2, characterized in that constitute in the culture of microorganisms of the species Methanobacterium oryzae at least 10 ^'2 % of the total number of microorganisms present in the culture.

4. The method according to claim 3, characterized in that constitute in the culture of microorganisms of the species Methanobacterium oryzae at least 1% of the total number of microorganisms present in the culture. 5. The method according to claim 4, characterized in that constitute in the culture of microorganisms of the species Methanobacterium oryzae at least 10% of the total number of microorganisms present in the culture.

6. The method according to claim 5, characterized in that make up in the culture of microorganisms of the species Methanobacterium oryzae at least 50% of the total number of microorganisms present in the culture.

7. The method according to claim 6, characterized in that constitute in the culture of microorganisms of the species Methanobacterium oryzae at least 75% of the total number of microorganisms present in the culture.

8. The method according to claim 7, characterized in that make up in the culture of microorganisms of the species Methanobacterium oryzae at least 90% of the total number of microorganisms present in the culture.

9. The method according to at least one of claims 2 to 8, characterized in that a pure culture of the microorganism of the species Methanobacterium oryzae is added.

10. The method according to at least one of claims 2 to 9, characterized in that microorganisms of the species Methanobacterium oryzae biomass are added as part of at least one immobilized culture of microorganisms.

1 1. Method according to at least one of claims 1 to 10, characterized in that in time for the addition of the microorganism of the species Methanobacterium oryzae additional biomass is added to the fermentation reactor.

12. The method according to at least one of claims 1 to 1 1, characterized in that the space load in the fermentation reactor by continuous addition of

Biomass is continuously increased.

13. The method according to at least one of claims 1 to 12, characterized in that the production of biogas from biomass at a space load of ≥ 0.5 kg oTS / m ³ d, preferably ≥ 4.0 kg oTS / m ³ d, especially preferably ≥ 8.0 kg oTS / m ³ d. 14. The method according to at least one of claims 1 to 13, characterized in that the production of biogas from biomass takes place under constant mixing of the fermentation substrate.

15. The method according to at least one of claims 1 to 14, characterized in that the production of biogas from biomass at a temperature of 20 ^ to 80 ⁰ C, preferably at a temperature of 40 ^< € to 50 ⁰ C is performed.

16. The method according to at least one of claims 1 to 15, characterized in that the addition of fermentation substrate and the addition of the microorganism of the type

Methanobacterium oryzae occur continuously.

17. The method according to at least one of claims 1 to 16, characterized in that the microorganism of the species Methanobacterium oryzae is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species

Methanobacterium oryzae accounts for between 10 ^-8 % and 50% of the total number of microorganisms present in the fermentation substrate.

18. The method according to claim 17, characterized in that the microorganism of the species Methanobacterium oryzae is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanobacterium oryzae between 10 ^"6 % and 25% of the total in Fermentation substrate present microorganisms.

19. The method according to claim 18, characterized in that the microorganism of the species Methanobacterium oryzae is added in an amount to the fermentation substrate that after addition of the proportion of the microorganism of the species Methanobacterium oryzae between 10 ^'4 % and 10% of the total in Fermentation substrate present microorganisms.

20. The method according to claim 19, characterized in that the microorganism of the species Methanobacterium oryzae is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanobacterium oryzae between 10 ^'3 % and 1% of the total in Fermentation substrate present microorganisms. 21. Process according to at least one of Claims 1 to 20, characterized in that the microorganism of the species Methanobacterium oryzae is a microorganism of the strain Bacterium clone HsA55fl.

22. Use of a microorganism of the species Methanobacterium oryzae for the fermentative production of biogas from biomass.

23. Use according to claim 22, characterized in that the microorganism of the species Methanobacterium oryzae is a microorganism of the strain Bacterium clone HsA55fl.

24. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 96.80% sequence identity with the nucleotide sequence SEQ ID No. 23.

25. Microorganism according to claim 24, characterized in that the nucleotide sequence contains a sequence region which has more than 97.5% sequence identity with the nucleotide sequence SEQ ID No. 23.

26. Microorganism according to claim 25, characterized in that the nucleotide sequence contains a sequence region which has more than 98.0% sequence identity with the nucleotide sequence SEQ ID No. 23.

27. Microorganism according to claim 26, characterized in that the nucleotide sequence contains a sequence region which has more than 99.0% sequence identity with the nucleotide sequence SEQ ID No. 23.

28. Microorganism according to claim 27, characterized in that the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 23.

29. Microorganism according to claim 28, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 23.

30. Culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, characterized in that in the culture of Microorganisms, a microorganism according to claims 24 to 29 is present, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

31. Culture of microorganisms according to claim 30, characterized in that the microorganism makes up at least ^{10 -2%} of the total number of microorganisms present in the culture.

32. Culture of microorganisms according to claim 31, characterized in that the microorganism at least 1% of the total number present in the culture

Constitutes microorganisms.

33. Culture of microorganisms according to claim 32, characterized in that the microorganism constitutes at least 10% of the total number of microorganisms present in the culture.

34. Culture of microorganisms according to claim 33, characterized in that the microorganism constitutes at least 50% of the total number of microorganisms present in the culture.

35. Culture of microorganisms according to claim 34, characterized in that the microorganism constitutes at least 90% of the total number of microorganisms present in the culture.

36. Culture of microorganisms according to claim 35, characterized in that it is a pure culture of a microorganism according to claims 24 to 29.

37. Culture of microorganisms according to at least one of claims 30 to 36, characterized in that it is an immobilized culture of microorganisms.

38. Use of a microorganism according to at least one of claims 24 to 29 for the fermentative production of biogas from biomass.

39. Use according to claim 38, characterized in that it is a process for the fermentative production of biogas from biomass according to at least one of claims 1 to 21. 40. Use of a culture of microorganisms according to at least one of claims 30 to 37 for the fermentative production of biogas from biomass.

41. Use according to claim 40, characterized in that it is a process for the fermentative production of biogas from biomass biomass according to at least one of claims 1 to 21.

In summary, a method for producing biogas from biomass in a fermentation reactor is described, wherein the biomass is added to a microorganism of the species Methanobacterium oryzae.

Archaeon clone C26A

The present invention provides a method for producing biogas from biomass in a fermentation reactor. The biomass is added according to the invention a microorganism of the strain Archaeon clone C26A.

Surprisingly, it has been shown that the addition of microorganisms of the strain Archaeon clone C26A to the fermentation substrate, the amount of biogas formed can be significantly increased. At constant space loading, the addition of microorganisms of the strain Archaeon clone C26A causes a significant increase in the amount of biogas produced. Alternatively, the amount of methane produced can also be increased by increasing the volume load, with the addition of Archaeon clone C26A microorganisms preventing the fermentation process from becoming unstable under the altered conditions. The use of microorganisms of the strain Archaeon clone C26A therefore leads to an improvement of efficiency and efficiency of biogas plants.

The person skilled in the art knows which microorganisms fall under the term "strain Archaeon clone C26A." In connection with the production of biogas by fermentation of organic substrates, microorganisms of the strain Archaeon clone C26A are hitherto unknown.

According to a preferred embodiment of the present invention, a microorganism of the strain Archaeon clone C26A is in the form of a culture of Microorganisms added, which consists predominantly of microorganisms of the strain Archaeon clone C26A. In fermentation substrates of biogas plants microorganisms of the genus archaeon C26a clone could be detected only in trace amounts of less than 10 ^'4% share of the total number of present microorganisms. Since the amount of microorganisms isolated from their natural occurrence is insufficient for the addition of the microorganisms, it is usually propagated in the form of a culture. In practice, it is found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out directly in the form of a culture of microorganisms.

The addition of the culture of Archaeon clone C26A can be carried out in the form of a culture suspension or in the form of dry, freeze-dried or moist cell pellets.

Since the already mentioned different positive effects on the fermentation process are connected with the microorganism strain Archaeon clone C26A, this strain of

Microorganisms in the added culture to be present in a concentration exceeding the natural occurrence. Of course, mixed cultures of any composition can be used for the addition. The only requirement is that the strain Archaeon clone C26A is present in a concentration that is enriched in relation to the natural occurrence.

The determination of the proportions of different types of microorganisms in mixed cultures is not a problem for the skilled worker. Thus, for example, with the help of fluorescence-labeled oligosensors specifically the proportion of microorganisms of the strain Archaeon clone C26A can be identified in a mixture. As already mentioned, microorganisms of the strain Archaeon clone C26A are preferably added to the fermentation substrate in the form of cultures of microorganisms, the cultures of microorganisms consisting predominantly of microorganisms of the strain Archaeon clone C26A. If, in addition to the determination of the number of microorganisms of the strain Archaeon clone C26A, the total number of microorganisms is also determined, the percentage of microorganisms of the strain Archaeon clone C26A in the culture can be stated as a percentage. Microorganisms of the strain Archaeon clone C26A are in a mixed culture then the predominant species of microorganisms, when they have the highest percentage of the various types of microorganisms present in the mixed culture. According to preferred embodiments of the present invention make microorganisms of the genus archaeon clone C26a at least 10 ^'4%, especially at least 10 ^{~ 2%,} more preferably at least 1% of the total number of the added to the fermentation substrate culture microorganisms present. According to further preferred embodiments, microorganisms of the strain Archaeon clone C26A account for at least 10%, in particular at least 50%, particularly preferably at least 90%, of the total number of microorganisms present in the culture.

According to a very particularly preferred embodiment, a pure culture of a microorganism of the strain Archaeon clone C26A is added. Due to the specific metabolic processes and activities, the addition of a pure culture can especially contribute to an improved methane production.

According to a further preferred embodiment of the present invention, a microorganism of the strain Archaeon clone C26A is added as a constituent of at least one immobilized culture of microorganisms. As for the addition of the

Microorganisms the amount of isolated from their natural occurrence

Microorganisms is insufficient, is usually made an increase in the form of a culture. In practice it has been found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily achieved in the form of an immobilized culture of

Microorganisms is made.

According to a preferred embodiment of the present invention, the microorganism of the strain Archaeon clone C26A is added in an amount to the fermentation substrate, so that after addition of the microorganism of the strain Archaeon clone C26A between 10 ^'8 % and 50% of the total in the fermentation substrate constitutes present microorganisms. In particular, depending on the fermenter size and thus as a function of the amount of fermentation substrate can be necessary to achieve the desired effect, an addition of very different amounts of microorganisms.

A microorganism of the strain archaeon clone C26a is particularly preferably added in an amount to the fermentation substrate that makes up after addition of the content of the microorganism of the strain archaeon clone C26a between ^{10 -6%} and 25% of the total number present in the fermentation substrate microorganisms. More preferably, the microorganism of the strain Archaeon clone C26A is added in an amount to the fermentation substrate such that, after addition, the proportion of the microorganism of the strain Archaeon clone C26a between 10 ^"4% and 10% of the total number present in the fermentation substrate microorganisms accounts. The microorganism of the genus archaeon clone C26a is very particularly preferably added in an amount to the fermentation substrate, that after addition the content of the microorganism of the strain archaeon clone C26a between 10 ^'3 % and 1% of the total number of microorganisms present in the fermentation substrate.

The present invention also encompasses the use of a microorganism of the strain Archaeon clone C26A for the fermentative production of biogas from biomass.

The obtained from the fermentation substrate of a Nachgärers methanogenic microorganisms of the strain Archaeon sp. SBG21 were subjected to sequence analysis. The determined 16S rRNA sequence SEQ ID No. 18 comprises 1 123 nucleotides. The next related sequence identified was the sequence of the uncultured Archaeon clone C26A. A comparison of the sequences revealed that there are a total of 57 exchanges of nucleotides or deletions. At a length of the particular nucleotide sequence of Archaeon sp. SBG21 of 1 123 nucleotides and a length of the reference sequence of 1 145 nucleotides is calculated using the FASTA algorithm, a 94.92% agreement.

The present invention also encompasses microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region that is greater than 94.92%.

Sequence identity with the nucleotide sequence SEQ ID NO. 18 has. More preferably, the nucleotide sequence contains a sequence region greater than 95.0% or greater than

95.1% or more than 95.2% or more than 95.3% or more than 95.4% or more than

95.5% or greater than 95.6% sequence identity with the nucleotide sequence SEQ ID NO: 18, and more preferably, the nucleotide sequence contains a sequence region having more than 96.0% sequence identity with the nucleotide sequence SEQ ID NO: 18.

According to further preferred embodiments, the microorganism has a nucleotide sequence which contains a sequence region which has more than 97.0% or more than 98.0% or more than 99.0% sequence identity with the nucleotide sequence SEQ ID No. 18 and is particularly preferred The nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 18. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 18.

In preferred embodiments of the present invention, as compared to the starting nucleotide sequence, SEQ ID NO: 18 may be attached at one or two positions three positions or four positions or five positions or six positions or seven positions or eight positions or nine positions or ten positions or eleven positions or twelve positions or 13 positions or 14 positions or 15 positions or at 16 positions or 17 positions or 18 positions or 19 positions or 20 positions or 21 positions or 22 positions or 23 positions or 24 positions or 25 positions or 26 positions or 27 positions or on 28 positions or 29 positions or 30 positions or 31 positions or 32 positions or 33 positions or 34 positions or 35 positions or 36 positions or 37 positions or 38 positions or 39 positions or 40 positions or at 41 positions or at 42 positions or at 43 positions or at 44 positions or at 45 positions or at 46 positions or at 47 posi or at 48 positions or 49 positions or 50 positions or 51 positions or 52 positions or 53 positions or 54 positions or 55 positions or 56 positions or 57 positions nucleotide mutations. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also encompasses a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism having a nucleotide sequence containing a sequence region greater than 94.92 is present having% sequence identity with the nucleotide sequence SEQ ID NO. 18, wherein the microorganism makes up at least 10 ^'4% of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 95.0% or greater than 95.1% or greater than 95 , 2% or more than 95.3% or more than 95.4% or more than 95.5% or more than 95.6% or more than 95.7% sequence identity with the nucleotide sequence SEQ ID NO: 18. Most preferably, the nucleotide sequence contains a sequence region having more than 96.0% sequence identity with the nucleotide sequence of SEQ ID NO: 18.

According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a Has sequence region having more than 97.0% or more than 98.0% or more than 99.0% sequence identity with the nucleotide sequence SEQ ID NO: 18. Most preferably, the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 18.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 18.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 18 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 18 in a method described above in connection with microorganisms of the strain Archaeon clone C26A for the fermentative production of biogas from biomass.

The present invention also includes the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms at least one microorganism as above with respect to its

Nucleotide sequence SEQ ID NO: 18 is defined and wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preference is given to the use of a culture of microorganisms for the fermentative production of biogas from biomass, the culture of microorganisms comprising at least one microorganism as described above with regard to its nucleotide sequence SEQ

ID NO. 18 has been defined, and wherein the microorganism is at least 10 ^'4% of the

Total number of microorganisms present in the culture, in one of the above - mentioned

In connection with microorganisms of the strain Archaeon clone C26A method described in more detail for the fermentative production of biogas from biomass.

According to further preferred embodiments make said, characterized in terms of their nucleotide sequence microorganisms at least 10 ^'2%, preferably to at least 1% of the total present in the culture of microorganisms. Particularly preferably, the microorganisms make up at least 10%, particularly preferably at least 25%, of the total number of microorganisms present in the culture. Most notably Preferably, the described microorganisms make up at least 50%, in particular at least 75%, of the total number of microorganisms present in the culture.

Also preferred are embodiments according to which the above-mentioned microorganisms make up at least 90% of the total number of microorganisms present in the culture. Particularly preferably it is a pure culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, which is a pure culture of a microorganism, as has been characterized above with respect to its nucleotide sequence.

Most preferably, in the cases described above, it is an immobilized culture of microorganisms.

In particular, the present invention includes the following aspects:

1. A method for producing biogas from biomass in a fermentation reactor, characterized in that the biomass is a microorganism of the strain

Archaeon clone C26A is added.

2. The method according to claim 1, characterized in that the microorganism of the strain Archaeon clone C26A is added in the form of a culture of microorganisms, wherein microorganisms of the strain Archaeon clone C26A account for at least 10 ^'4 % of the total number of microorganisms present in the culture.

3. The method according to claim 2, characterized in that constitute in the culture of microorganisms microorganisms of the strain Archaeon clone C26A at least 10 ^'2 % of the total number of microorganisms present in the culture.

4. The method according to claim 3, characterized in that constitute in the culture of microorganisms microorganisms of the strain Archaeon clone C26A at least 1% of the total number of microorganisms present in the culture.

5. The method according to claim 4, characterized in that constitute in the culture of microorganisms microorganisms of the strain Archaeon clone C26A at least 10% of the total number of microorganisms present in the culture. 6. The method according to claim 5, characterized in that in the culture of microorganisms microorganisms of the strain Archaeon clone C26A make up at least 50% of the total number of microorganisms present in the culture.

7. The method according to claim 6, characterized in that in the culture of microorganisms microorganisms of the strain Archaeon clone C26A make up at least 75% of the total number of microorganisms present in the culture.

8. The method according to claim 7, characterized in that constitute in the culture of microorganisms microorganisms of the strain Archaeon clone C26A at least 90% of the total number of microorganisms present in the culture.

9. The method according to at least one of claims 2 to 8, characterized in that a pure culture of the microorganism of the strain Archaeon clone C26A is added.

10. The method according to at least one of claims 2 to 9, characterized in that microorganisms of the strain Archaeon clone C26A biomass are added as part of at least one immobilized culture of microorganisms.

1 1. Method according to at least one of claims 1 to 10, characterized in that in time to the addition of the microorganism of the strain Archaeon clone C26A additional biomass is added to the fermentation reactor.

12. The method according to at least one of claims 1 to 1 1, characterized in that the space load in the fermentation reactor by continuous addition of

Biomass is continuously increased.

13. The method according to at least one of claims 1 to 12, characterized in that the production of biogas from biomass at a space load of ≥ 0.5 kg oTS / m ³ d, preferably ≥ 4.0 kg oTS / m ³ d, especially preferably ≥ 8.0 kg oTS / m ³ d.

14. The method according to at least one of claims 1 to 13, characterized in that the production of biogas from biomass takes place under constant mixing of the fermentation substrate. 15. The method according to at least one of claims 1 to 14, characterized in that the production of biogas from biomass at a temperature of 20 ^ to 80 ⁰ C, preferably at a temperature of 40 ^ to 50 ⁰ C is performed.

16. The method according to at least one of claims 1 to 15, characterized in that the addition of fermentation substrate and the addition of the microorganism of the strain Archaeon clone C26A carried out continuously.

17. The method according to at least one of claims 1 to 16, characterized in that the microorganism of the strain Archaeon clone C26A in an amount to the

Fermentation substrate is added, that after addition of the proportion of the microorganism of

Strain Archaeon clone C26A between 10 ^'8 % and 50% of the total in the

Fermentation substrate present microorganisms.

18. The method according to claim 17, characterized in that the microorganism of the strain Archaeon clone C26A is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the strain Archaeon clone C26A between 10 ^"6 % and 25% of the total in the fermentation substrate present microorganisms.

19. The method according to claim 18, characterized in that the microorganism of the strain Archaeon clone C26A is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the strain Archaeon clone C26A between 10 ^"4 % and 10% of the total in the fermentation substrate present microorganisms.

20. The method according to claim 19, characterized in that the microorganism of the strain Archaeon clone C26A is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the strain Archaeon clone C26A between 10 ^'3 % and 1% of the total present in the fermentation substrate

Constitutes microorganisms.

21. Use of a microorganism of the strain Archaeon clone C26A for fermentative production of biogas from biomass. 22. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 94.92% sequence identity with the nucleotide sequence SEQ ID No. 18.

23. Microorganism according to claim 22, characterized in that the nucleotide sequence contains a sequence region which has more than 96.0% sequence identity with the nucleotide sequence SEQ ID No. 18.

24. Microorganism according to claim 23, characterized in that the nucleotide sequence contains a sequence region which has more than 98.0% sequence identity with the nucleotide sequence SEQ ID No. 18.

25. Microorganism according to claim 24, characterized in that the nucleotide sequence contains a sequence region which has more than 99.0% sequence identity with the nucleotide sequence SEQ ID No. 18.

26. Microorganism according to claim 25, characterized in that the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 18.

27. Microorganism according to claim 26, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 18.

28. Culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, characterized in that in the culture of microorganisms, a microorganism according to claims 22 to 27 is present, wherein the microorganism at least 10 ^'4 % of the total in the culture microorganisms present.

29. Culture of microorganisms according to claim 28, characterized in that the microorganism constitutes at least 10 ^'2 % of the total number of microorganisms present in the culture.

30. Culture of microorganisms according to claim 29, characterized in that the microorganism constitutes at least 1% of the total number of microorganisms present in the culture. 31. Culture of microorganisms according to claim 30, characterized in that the microorganism constitutes at least 10% of the total number of microorganisms present in the culture.

32. Culture of microorganisms according to claim 31, characterized in that the microorganism constitutes at least 50% of the total number of microorganisms present in the culture.

33. Culture of microorganisms according to claim 32, characterized in that the microorganism constitutes at least 90% of the total number of microorganisms present in the culture.

34. Culture of microorganisms according to claim 33, characterized in that it is a pure culture of a microorganism according to claims 22 to 27.

35. Culture of microorganisms according to at least one of claims 28 to 34, characterized in that it is an immobilized culture of microorganisms.

36. Use of a microorganism according to at least one of claims 22 to 27 for the fermentative production of biogas from biomass.

37. Use according to claim 36, characterized in that it is a method for the fermentative production of biogas from biomass according to at least one of

Claims 1 to 20 is.

38. Use of a culture of microorganisms according to at least one of claims 28 to 35 for the fermentative production of biogas from biomass.

39. Use according to claim 38, characterized in that it is a process for the fermentative production of biogas from biomass biomass according to at least one of claims 1 to 20.

In summary, a process for the production of biogas from biomass in a fermentation reactor is described, wherein the biomass is added to a microorganism of the strain Archaeon clone C26A. Archaeon clone DF86

The present invention provides a method for producing biogas from biomass in a fermentation reactor. The biomass is added according to the invention a microorganism of the strain Archaeon clone DF86.

Surprisingly, it has been shown that the addition of microorganisms of the strain Archaeon clone DF86 to the fermentation substrate, the amount of biogas formed can be significantly increased. At constant space loading, the addition of microorganisms of the strain Archaeon clone DF86 causes a significant increase in the amount of biogas produced. Alternatively, the amount of methane produced can also be increased by increasing the volume load, with the addition of Archaeon clone DF86 microorganisms preventing the fermentation process from becoming unstable under the altered conditions. The use of microorganisms of the strain Archaeon clone DF86 therefore leads to an improvement of efficiency and efficiency of biogas plants.

It is known to those skilled in the art which microorganisms fall under the term "strain Archaeon clone DF8". In connection with the production of biogas by fermentation of organic substrates, microorganisms of the strain Archaeon clone DF86 are not yet known.

According to a preferred embodiment of the present invention, a microorganism of the strain Archaeon clone DF86 is added in the form of a culture of microorganisms consisting predominantly of microorganisms of the strain Archaeon clone DF86. In fermentation substrates of biogas plants microorganisms of the genus archaeon DF86 clone could be detected only in trace amounts of less than 10 ^'4% share of the total number of present microorganisms. Since the amount of microorganisms isolated from their natural occurrence is insufficient for the addition of the microorganisms, it is usually propagated in the form of a culture. In practice, it is found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out directly in the form of a culture of microorganisms. The addition of the culture of Archaeon clone DF86 can be carried out in the form of a culture suspension or in the form of dry, freeze-dried or moist cell pellets.

Since the already mentioned different positive effects on the fermentation process are connected with the microorganism strain Archaeon clone DF86, this strain of

Microorganisms in the added culture to be present in a concentration exceeding the natural occurrence. Of course, mixed cultures of any composition can be used for the addition. The only requirement is that the strain Archaeon clone DF86 is present in a concentration that is enriched in relation to the natural occurrence.

The determination of the proportions of different types of microorganisms in mixed cultures is not a problem for the person skilled in the art. For example, the proportion of microorganisms of the strain Archaeon clone DF86 in a mixture can be identified specifically with the aid of fluorescence-labeled oligopeptides. As already mentioned, microorganisms of the strain Archaeon clone DF86 are preferably added to the fermentation substrate in the form of cultures of microorganisms, the cultures of microorganisms predominantly consisting of microorganisms of the strain Archaeon clone DF86. If, in addition to the determination of the number of microorganisms of the strain Archaeon clone DF86, the total number of microorganisms is also determined, then the proportion of microorganisms of the strain Archaeon clone DF86 in the culture can be stated as a percentage. Microorganisms of the strain Archaeon clone DF86 are in a mixed culture then the predominant species of microorganisms, if they have the highest percentage of the different types of microorganisms present in the mixed culture.

According to preferred embodiments of the present invention make microorganisms of the genus archaeon clone DF86 at least 10 ^'4%, especially at least 10 ^"2%, more preferably at least 1% of the total number of the added to the fermentation substrate culture microorganisms present. According to make further preferred embodiments Microorganisms of the strain Archaeon clone DF86 at least 10%, in particular at least 50%, particularly preferably at least 90% of the total number of microorganisms present in the culture.

According to a very particularly preferred embodiment, a pure culture of a microorganism of the strain Archaeon clone DF86 is added. Due to the specific Metabolic processes and activities, the addition of a pure culture can contribute significantly to improved methane production.

According to another preferred embodiment of the present invention, a microorganism of the strain Archaeon clone DF86 is added as a component of at least one immobilized culture of microorganisms. As for the addition of the

Microorganisms the amount of isolated from their natural occurrence

Microorganisms is insufficient, is usually made an increase in the form of a culture. In practice it has been found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily achieved in the form of an immobilized culture of

Microorganisms is made.

According to a preferred embodiment of the present invention, the microorganism of the genus archaeon clone DF86 is added in an amount to the fermentation substrate so that after addition the content of the microorganism of the strain archaeon clone DF86 between ^{10 -8%} and 50% of the total in the fermentation substrate constitutes present microorganisms. In particular, depending on the fermenter size and thus as a function of the amount of fermentation substrate can be necessary to achieve the desired effect, an addition of very different amounts of microorganisms.

A microorganism of the strain archaeon clone DF86 is particularly preferably added in an amount to the fermentation substrate that makes up after addition of the content of the microorganism of the strain archaeon clone DF86 between ^{10 -6%} and 25% of the total number present in the fermentation substrate microorganisms. The microorganism of the genus archaeon clone DF86 is particularly preferably added in an amount to the fermentation substrate that makes up after addition of the content of the microorganism of the strain archaeon clone DF86 between ^{10 -4%} and 10% of the total number present in the fermentation substrate microorganisms. Most preferably, the microorganism of the strain Archaeon clone DF86 is added in an amount to the fermentation substrate such that after addition the proportion of the microorganism of the strain Archaeon clone DF86 is between 10 ^'3 % and 1% of the total number of microorganisms present in the fermentation substrate.

The present invention also encompasses the use of a microorganism of the strain Archaeon clone DF86 for the fermentative production of biogas from biomass. The methanogenic microorganisms Archaeon sp. SBG22 were subjected to sequence analysis. The determined 16S rRNA sequence SEQ ID No. 19 comprises 1 134 nucleotides. The next related sequence identified was the sequence of the non-cultured Archaeon clone DF86. A comparison of the sequences revealed that a total of 56 exchanges of nucleotides or deletions are present. At a length of the particular nucleotide sequence of Archaeon sp. SBG22 of 1 134 nucleotides and a length of the reference sequence of 1 106 nucleotides is calculated using the FASTA algorithm, a match of 95.06%.

The present invention also includes microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having more than 95.06% sequence identity with the nucleotide sequence of SEQ ID NO: 19. More preferably, the nucleotide sequence contains a sequence range greater than 95.1% or greater than 95.2% or greater than 95.3% or greater than 95.4% or greater than 95.5% or greater than 95.6%. or more than 95.7% sequence identity with the nucleotide sequence SEQ ID NO: 19, and more preferably, the nucleotide sequence contains a sequence region having more than 96.0% sequence identity with the nucleotide sequence SEQ ID NO: 19.

According to further preferred embodiments, the microorganism has a nucleotide sequence containing a sequence region greater than 97.0% or more

98.0% or more than 99.0% sequence identity with the nucleotide sequence SEQ ID NO: 19, and more preferably, the nucleotide sequence contains a sequence region having more than 99.5% sequence identity with the nucleotide sequence SEQ ID NO: 19. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 19.

In preferred embodiments of the present invention, SEQ ID NO: 19 can be compared to the starting nucleotide sequence at one or two positions or at three positions or at four positions or at five positions or at six positions or at seven positions or at eight positions or at nine positions or ten positions or eleven positions or twelve positions or 13 positions or 14 positions or 15 positions or 16 positions or 17 positions or 18 positions or 19 positions or 20 positions or 21 positions or on 22 positions or 23 positions or 24 positions or 25 positions or 26 positions or 27 positions or 28 positions or 29 positions or 30 positions or 31 positions or 32 positions or 33 positions or 34 positions or at 35 positions or 36 positions or 37 positions or 38 Positions or 39 positions or 40 positions or 41 positions or 42 positions or 43 positions or 44 positions or 45 positions or 46 positions or 47 positions or 48 positions or 49 positions or 50 positions or at 51 positions or at 52 positions or at 53 positions or at 54 positions or at 55 positions or at 56 positions nucleotide mutations. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also includes a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism is present, which contains a

Nucleotide sequence containing a sequence region exceeding 95.06%

Sequence identity with the nucleotide sequence SEQ ID NO. 19, wherein the

Microorganism accounts for at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 95.1% or greater than 95.2% or greater than 95 , 3% or more than 95.4% or more than 95.5% or more than 95.6% or more than 95.7% sequence identity with the nucleotide sequence SEQ ID NO: 19. More preferably, the nucleotide sequence contains a sequence region having more than 96.0% sequence identity with the nucleotide sequence SEQ ID NO: 19.

According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 97.0% or greater than 98.0% or greater than 99.0% sequence identity with the nucleotide sequence SEQ ID NO: 19. Most preferably, the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 19.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 19. The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 19 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 19 in a method described above in connection with microorganisms strain Archaeon clone DF86 for the fermentative production of biogas from biomass.

The present invention also includes the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms at least one microorganism as above with respect to its

Nucleotide sequence SEQ ID NO: 19 has been defined and wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preference is given to the use of a culture of microorganisms for the fermentative production of biogas from biomass, the culture of microorganisms comprising at least one microorganism as described above with regard to its nucleotide sequence SEQ

ID NO. 19 has been defined, and wherein the microorganism is at least 10 ^'4% of the

Total number of microorganisms present in the culture, in one of the above - mentioned

Correlation with microorganisms strain Archaeon clone DF86 described method for the fermentative production of biogas from biomass.

According to further preferred embodiments make said, characterized in terms of their nucleotide sequence microorganisms at least 10 ^'2%, preferably to at least 1% of the total present in the culture of microorganisms. Particularly preferably, the microorganisms make up at least 10%, particularly preferably at least 25%, of the total number of microorganisms present in the culture. Most preferably, the microorganisms described make up at least 50%, in particular at least 75%, of the total number of microorganisms present in the culture.

Also preferred are embodiments according to which the above-mentioned microorganisms make up at least 90% of the total number of microorganisms present in the culture. Particularly preferably it is a pure culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, which is a pure culture of a microorganism, as has been characterized above with respect to its nucleotide sequence. Most preferably, in the cases described above, it is an immobilized culture of microorganisms.

In particular, the present invention includes the following aspects:

1. A method for producing biogas from biomass in a fermentation reactor, characterized in that the biomass is a microorganism of the strain

Archaeon clone DF86 is added.

2. The method according to claim 1, characterized in that the microorganism of the strain Archaeon clone DF86 is added in the form of a culture of microorganisms, wherein microorganisms of the strain Archaeon clone DF86 make up at least 10 ^'4 % of the total number of microorganisms present in the culture.

3. The method according to claim 2, characterized in that constitute in the culture of microorganisms microorganisms of the strain Archaeon clone DF86 at least 10 ^'2 % of the total number of microorganisms present in the culture.

4. The method according to claim 3, characterized in that constitute in the culture of microorganisms microorganisms of the strain Archaeon clone DF86 at least 1% of the total number of microorganisms present in the culture.

5. The method according to claim 4, characterized in that constitute in the culture of microorganisms microorganisms of the strain Archaeon clone DF86 at least 10% of the total number of microorganisms present in the culture.

6. The method according to claim 5, characterized in that constitute in the culture of microorganisms microorganisms of the strain Archaeon clone DF86 at least 50% of the total number of microorganisms present in the culture.

7. The method according to claim 6, characterized in that constitute in the culture of microorganisms microorganisms of the strain Archaeon clone DF86 at least 75% of the total number of microorganisms present in the culture.

8. The method according to claim 7, characterized in that constitute in the culture of microorganisms microorganisms of the strain Archaeon clone DF86 at least 90% of the total number of microorganisms present in the culture. 9. The method according to at least one of claims 2 to 8, characterized in that a pure culture of the microorganism of the strain Archaeon clone DF86 is added.

10. The method according to at least one of claims 2 to 9, characterized in that microorganisms of the strain Archaeon clone DF86 biomass are added as part of at least one immobilized culture of microorganisms.

11. The method according to at least one of claims 1 to 10, characterized in that in time for the addition of the microorganism of the strain Archaeon clone DF86 additional

Biomass is added to the fermentation reactor.

12. The method according to at least one of claims 1 to 1 1, characterized in that the space load in the fermentation reactor is continuously increased by the continuous addition of biomass.

13. The method according to at least one of claims 1 to 12, characterized in that the production of biogas from biomass at a space load of ≥ 0.5 kg oTS / m ³ d, preferably ≥ 4.0 kg oTS / m ³ d, especially preferably ≥ 8.0 kg oTS / m ³ d.

14. The method according to at least one of claims 1 to 13, characterized in that the production of biogas from biomass takes place under constant mixing of the fermentation substrate.

15. The method according to at least one of claims 1 to 14, characterized in that the production of biogas from biomass at a temperature of 20 ^< € to 80 ⁰ C, preferably at a temperature of 40 ^ to 50 ⁰ C is performed.

16. The method according to at least one of claims 1 to 15, characterized in that the addition of fermentation substrate and the addition of the microorganism of the strain Archaeon clone DF86 done continuously.

17. The method according to at least one of claims 1 to 16, characterized in that the microorganism of the strain Archaeon clone DF86 in an amount to the

Fermentation substrate is added, that after addition of the proportion of the microorganism of Archaeon strain clone DF86 between ^{10 -8%} and 50% of the total constitutes at present in the fermentation substrate microorganisms.

18. The method according to claim 17, characterized in that the microorganism of the strain Archaeon clone DF86 is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the strain Archaeon clone DF86 between 10 ^"6 % and 25% of the total present in the fermentation substrate

Constitutes microorganisms.

19. The method according to claim 18, characterized in that the microorganism of the strain Archaeon clone DF86 is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the strain Archaeon clone DF86 between 10 ^'4 % and 10% of the total in the fermentation substrate present microorganisms.

20. The method according to claim 19, characterized in that the microorganism of the strain Archaeon clone DF86 is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the strain Archaeon clone DF86 between 10 ^'3 % and 1% of the total in the fermentation substrate present microorganisms.

21. Use of a microorganism of the strain Archaeon clone DF86 for the fermentative production of biogas from biomass.

22. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 95.06% sequence identity with the nucleotide sequence SEQ ID No. 19.

23. Microorganism according to claim 22, characterized in that the nucleotide sequence contains a sequence region which has more than 96.0% sequence identity with the nucleotide sequence SEQ ID No. 19.

24. Microorganism according to claim 23, characterized in that the nucleotide sequence contains a sequence region which has more than 98.0% sequence identity with the nucleotide sequence SEQ ID No. 19. 25. Microorganism according to claim 24, characterized in that the nucleotide sequence contains a sequence region which has more than 99.0% sequence identity with the nucleotide sequence SEQ ID No. 19.

26. Microorganism according to claim 25, characterized in that the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 19.

27. Microorganism according to claim 26, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID NO.

19 corresponds.

28. Culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, characterized in that in the culture of microorganisms, a microorganism according to claims 22 to 27 is present, wherein the microorganism at least 10 ^"4 % of the total in the culture microorganisms present.

29. Culture of microorganisms according to claim 28, characterized in that the microorganism at least 10 ^"2 % of the total number of existing in the culture

Constitutes microorganisms.

30. Culture of microorganisms according to claim 29, characterized in that the microorganism constitutes at least 1% of the total number of microorganisms present in the culture.

31. Culture of microorganisms according to claim 30, characterized in that the microorganism constitutes at least 10% of the total number of microorganisms present in the culture.

32. Culture of microorganisms according to claim 31, characterized in that the microorganism constitutes at least 50% of the total number of microorganisms present in the culture.

33. Culture of microorganisms according to claim 32, characterized in that the microorganism constitutes at least 90% of the total number of microorganisms present in the culture. 34. Culture of microorganisms according to claim 33, characterized in that it is a pure culture of a microorganism according to claims 22 to 27.

35. Culture of microorganisms according to at least one of claims 28 to 34, characterized in that it is an immobilized culture of microorganisms.

36. Use of a microorganism according to at least one of claims 22 to 27 for the fermentative production of biogas from biomass.

37. Use according to claim 36, characterized in that it is a process for the fermentative production of biogas from biomass according to at least one of claims 1 to 20.

38. Use of a culture of microorganisms according to at least one of claims 28 to 35 for the fermentative production of biogas from biomass.

39. Use according to claim 38, characterized in that it is a process for the fermentative production of biogas from biomass biomass according to at least one of claims 1 to 20.

In summary, a method for producing biogas from biomass in a fermentation reactor is described, wherein the biomass is added to a microorganism of the strain Archaeon clone DF86.

Methanobacterium subterraneum

The present invention provides a method for producing biogas from biomass in a fermentation reactor. The biomass is added according to the invention a microorganism of the species Methanobacterium subterraneum.

Surprisingly, it has been shown that the addition of microorganisms of the species Methanobacterium subterraneum to the fermentation substrate, the amount of biogas formed can be significantly increased. At constant space load causes the addition of

Microorganisms of the species Methanobacterium subterraneum a significant increase in Amount of biogas produced. Alternatively, the amount of methane produced can also be increased by increasing the volume load, with the addition of microorganisms of the species Methanobacterium subterraneum preventing the fermentation process from becoming unstable under the altered conditions. The use of microorganisms of the species Methanobacterium subterraneum therefore leads to an improvement in efficiency and efficiency of biogas plants.

The person skilled in the art knows which strains of microorganisms fall under the term "species Methanobacterium subterraneum." In connection with the production of biogas by fermentation of organic substrates, microorganisms of the species Methanobacterium subterraneum are hitherto unknown.

According to a preferred embodiment of the present invention, a microorganism of the species Methanobacterium subterraneum is added in the form of a culture of microorganisms consisting predominantly of microorganisms of the species Methanobacterium subterraneum. In fermentation substrates of biogas plants microorganisms of the species Methanobacterium could subterraneum only in trace amounts of less than 10 ^"4% share of the total number detected by present microorganisms. Since the amount for the addition of the microorganism at isolated from their natural occurrence microorganisms is insufficient, is In practice, it is found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out directly in the form of a culture of microorganisms.

The addition of the culture of Methanobacterium subterraneum can be carried out in the form of a culture suspension or in the form of dry, freeze-dried or moist cell pellets.

Since the already mentioned different positive effects on the fermentation process are associated with the microorganism species Methanobacterium subterraneum, this type of

Microorganisms in the added culture to be present in a concentration exceeding the natural occurrence. Of course, mixed cultures of any composition can be used for the addition. The only requirement is that the species Methanobacterium subterraneum is present in a concentration enriched with respect to the natural occurrence. The determination of the proportions of different types of microorganisms in mixed cultures is not a problem for the expert. Thus, for example, with the help of fluorescently labeled oligosensors specifically the proportion of microorganisms of the species Methanobacterium subterraneum can be identified in a mixture. As already mentioned, microorganisms of the species Methanobacterium subterraneum are preferably added to the fermentation substrate in the form of cultures of microorganisms, the cultures of microorganisms consisting predominantly of microorganisms of the species Methanobacterium subterraneum. If, in addition to the determination of the number of microorganisms of the species Methanobacterium subterraneum, the total number of microorganisms is also determined, the percentage of microorganisms of the species Methanobacterium subterraneum in the culture can be stated as a percentage. Microorganisms of the species Methanobacterium subterraneum are in a mixed culture then the predominantly present type of microorganisms, if they have the highest percentage of the various species of microorganisms present in the mixed culture.

According to preferred embodiments of the present invention make microorganisms of the species Methanobacterium subterraneum at least 10 ^'4%, especially at least 10 ^"2%, more preferably at least 1% of the total number of the added to the fermentation substrate culture microorganisms present. According to further preferred embodiments make microorganisms of the species Methanobacterium subterraneum at least 10%, in particular at least 50%, particularly preferably at least 90% of the total number of microorganisms present in the culture.

According to a very particularly preferred embodiment, a pure culture of a microorganism of the species Methanobacterium subterraneum is added. Due to the specific metabolic processes and activities, the addition of a pure culture can especially contribute to an improved methane production.

According to a further preferred embodiment of the present invention, a microorganism of the species Methanobacterium subterraneum is added as a constituent of at least one immobilized culture of microorganisms. As for the addition of the

Microorganisms the amount of isolated from their natural occurrence

Microorganisms is insufficient, is usually made an increase in the form of a culture. In practice it has been found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily achieved in the form of an immobilized culture of

Microorganisms is made. According to a preferred embodiment of the present invention, the microorganism of the species Methanobacterium subterraneum is added in an amount to the fermentation substrate, so that after addition of the proportion of microorganism of the species Methanobacterium subterraneum between 10 ^'8 % and 50% of the total number of present in the fermentation substrate microorganisms accounts. In particular, depending on the fermenter size and thus as a function of the amount of fermentation substrate can be necessary to achieve the desired effect, an addition of very different amounts of microorganisms.

A microorganism of the species Methanobacterium subterraneum is particularly preferably added in an amount to the fermentation substrate that constitutes subterraneum after addition of the content of the microorganism of the species Methanobacterium between ^{10 -6%} and 25% of the total number present in the fermentation substrate microorganisms. The microorganism of the species Methanobacterium subterraneum is particularly preferably added in an amount to the fermentation substrate that constitutes subterraneum after addition of the content of the microorganism of the species Methanobacterium between ^{10 -4%} and 10% of the total number present in the fermentation substrate microorganisms. The microorganism of the species Methanobacterium is very particularly preferably subterraneum added in an amount to the fermentation substrate that constitutes subterraneum after addition of the content of the microorganism of the species Methanobacterium between ^{10 -3%} and 1% of the total number present in the fermentation substrate microorganisms.

According to a particularly preferred embodiment of the present invention, the microorganism of the species Methanobacterium subterraneum is a microorganism of the strain Methanobacterium sp., Clone SMS-sludge-11. In all embodiments described above, which deal with the addition of a microorganism of the species Methanobacterium subterraneum, so preferably a microorganism of the strain Methanobacterium sp. Clone SMS-sludge-11 added.

According to another particularly preferred embodiment of the present invention, the microorganism of the species Methanobacterium subterraneum is a microorganism of the strain Bacterium clone HsA55fl. In all of the above-described embodiments, which deal with the addition of a microorganism of the species Methanobacterium subterraneum, so preferably a microorganism of the strain Bacterium clone HsA55fl is added. The present invention also includes the use of a microorganism of the species Methanobacterium subterraneum for the fermentative production of biogas from biomass. Preference is given to the fermentative production of biogas from biomass, a microorganism of the strain Methanobacterium sp. Clone SMS-sludge-11 used. Also preferred for the fermentative production of biogas from biomass is a microorganism of the strain Bacterium clone HsA55fl used.

The microorganisms Methanobacterium subterraneum SBG35 obtained from the fermentation substrate of a post-fermenter were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID NO: 32 comprises 801 nucleotides. As the next related 16S rRNA sequence, the sequence of uncultivated Methanobacterium sp. Clone SMS sludge-11 identified. A comparison of the sequences revealed that a total of 23 exchanges of nucleotides or deletions are present. With a length of the specific nucleotide sequence of 801 nucleotides of Methanobacterium subterraneum SBG35 and a length of the reference sequence of 804 nucleotides, a 97.13% agreement is calculated using the FASTA algorithm.

The present invention also encompasses microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having more than 97.13% sequence identity with the nucleotide sequence of SEQ ID NO: 32. More preferably, the nucleotide sequence contains a sequence range greater than 97.2% or greater than 97.3% or greater than 97.4% or greater than 97.5% or greater than 97.6% or greater than 97.7%. or more than 97.8% or more than 97.9% sequence identity with the nucleotide sequence SEQ ID NO: 32, and more preferably the nucleotide sequence contains a sequence region having more than 98.0% sequence identity with the nucleotide sequence SEQ ID NO: 32 ,

According to further preferred embodiments, the microorganism has a nucleotide sequence containing a sequence region having more than 98.5% sequence identity with the nucleotide sequence SEQ ID NO: 32 and more preferably the nucleotide sequence contains a sequence region having greater than 99.0% sequence identity having the nucleotide sequence of SEQ ID NO: 32. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 32.

In preferred embodiments of the present invention, SEQ ID NO: 32 may be at one or two positions or at the other than the starting nucleotide sequence three positions or four positions or five positions or six positions or seven positions or eight positions or nine positions or ten positions or eleven positions or twelve positions or 13 positions or 14 positions or 15 positions or at 16 positions or 17 positions or 18 positions or 19 positions or 20 positions or 21 positions or 22 positions or 23 positions nucleotide mutations. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also includes a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism is present, which contains a

Nucleotide sequence containing a sequence region exceeding 97.13%

Sequence identity with the nucleotide sequence SEQ ID NO. 32, wherein the

Microorganism accounts for at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 97.2% or greater than 97.3% or greater than 97 , 4% or more than 97.5% or more than 97.6% or more than 97.7% or more than 97.8% or more than 97.9% sequence identity with the nucleotide sequence SEQ ID NO: 32. More preferably, the nucleotide sequence contains a sequence region having more than 98.0% sequence identity with the nucleotide sequence SEQ ID NO: 32.

According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range of more than 98.5% sequence identity with the nucleotide sequence SEQ ID NO. 32 has. Most preferably, the nucleotide sequence contains a sequence region having more than 99.0% sequence identity with the nucleotide sequence SEQ ID NO: 32.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 32. The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 32 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 32 in a method described above in connection with microorganisms of the species Methanobacterium subterraneum for the fermentative production of biogas from biomass.

The present invention also encompasses the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 32 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture. It is preferable to use a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms comprises at least one microorganism as defined above with respect to its nucleotide sequence SEQ ID No. 32 and wherein the microorganism is at least 10 ^{' 4} % of the total number of microorganisms present in the culture, in a method described above in connection with microorganisms of the species Methanobacterium subterraneum for the fermentative production of biogas from biomass.

The microorganisms Methanobacterium subterraneum SBG34 obtained from the fermentation substrate of a post-fermenter were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID NO: 31 comprises 825 nucleotides. The next relative was identified as Bacterium clone HsA55fl. A comparison of the sequences revealed that there are a total of 3 exchanges of nucleotides or deletions. At a length of the specific nucleotide sequence of 825 nucleotides of Methanobacterium subterraneum SBG34 and a length of the reference sequence of 825 nucleotides, a 99.64% match is calculated using the FASTA algorithm.

The present invention also includes microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having greater than 99.64% sequence identity with the nucleotide sequence of SEQ ID NO: 31. More preferably, the nucleotide sequence contains a sequence range of greater than 99.66% or greater than 99.68% or greater than 99.70% or greater than 99.72% or greater than 99.74% or greater than 99.76%. or more than 99.78% or more than 99.80% or more than 99.82% or more than 99.84% has sequence identity with the nucleotide sequence SEQ ID NO: 31, and more preferably, the nucleotide sequence contains a sequence region having more than 99.86% sequence identity with the nucleotide sequence SEQ ID NO: 31.

According to further preferred embodiments, the microorganism has a nucleotide sequence containing a sequence region having more than 99.90% sequence identity with the nucleotide sequence SEQ ID NO: 31, and more preferably the nucleotide sequence contains a sequence region having more than 99.95% sequence identity having the nucleotide sequence of SEQ ID NO: 31. According to a very particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 31.

In preferred embodiments of the present invention, nucleotide mutations may be present at one or two positions or at three positions relative to the starting nucleotide sequence of SEQ ID NO: 31. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also encompasses a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, in which culture of microorganisms a microorganism is present which has a nucleotide sequence containing a sequence region greater than 99.64 % Sequence identity with the nucleotide sequence SEQ ID NO: 31, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 99.66% or greater than 99.68% or greater than 99 , 70% or more than 99.72% or more than 99.740% or more than 99.76% or more than 99.78% or more than 99.80% or more than 99.82% or more than 99.84% Sequence identity with the nucleotide sequence SEQ ID NO. 31 has. More preferably, the nucleotide sequence contains a sequence region that has more than 99.86% sequence identity with the nucleotide sequence SEQ ID NO: 31.

According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a Has sequence region having more than 99.90% sequence identity with the nucleotide sequence SEQ ID NO: 31. Most preferably, the nucleotide sequence contains a sequence region which has more than 99.95% sequence identity with the nucleotide sequence SEQ ID No. 31.

According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 31.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 31 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 31 in a method described above in connection with microorganisms of the species Methanobacterium subterraneum for the fermentative production of biogas from biomass.

The present invention also includes the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms at least one microorganism as above with respect to its

Nucleotide sequence SEQ ID NO: 31 is defined and wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preference is given to the use of a culture of microorganisms for the fermentative production of biogas from biomass, the culture of microorganisms comprising at least one microorganism as described above with regard to its nucleotide sequence SEQ

ID No. 31 is defined and wherein the microorganism is at least 10 ^'4 % of the

Total number of microorganisms present in the culture, in one of the above - mentioned

In connection with microorganisms of the species Methanobacterium subterraneum described in more detail methods for the fermentative production of biogas from biomass.

According to further preferred embodiments make said, characterized in terms of their nucleotide sequence microorganisms at least 10 ^'2%, preferably to at least 1% of the total present in the culture of microorganisms. Particularly preferably, the microorganisms make up at least 10%, particularly preferably at least 25%, of the total number of microorganisms present in the culture. Most notably Preferably, the described microorganisms make up at least 50%, in particular at least 75%, of the total number of microorganisms present in the culture.

Also preferred are embodiments according to which the above-mentioned microorganisms make up at least 90% of the total number of microorganisms present in the culture. Particularly preferably it is a pure culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, which is a pure culture of a microorganism, as has been characterized above with respect to its nucleotide sequence.

Most preferably, in the cases described above, it is an immobilized culture of microorganisms.

In particular, the present invention includes the following aspects:

1. A process for the production of biogas from biomass in a fermentation reactor, characterized in that the biomass is added to a microorganism of the species Methanobacterium subterraneum.

2. The method according to claim 1, characterized in that the microorganism of the species Methanobacterium subterraneum is added in the form of a culture of microorganisms, wherein microorganisms of the species Methanobacterium subterraneum make up at least 10 ^'4 % of the total number of microorganisms present in the culture.

3. The method according to claim 2, characterized in that make up in the culture of microorganisms of the species Methanobacterium subterraneum at least 10 ^'2 % of the total number of microorganisms present in the culture.

4. The method according to claim 3, characterized in that in the culture of microorganisms of the species Methanobacterium subterraneum at least 1% of

Total number of microorganisms present in the culture.

5. The method according to claim 4, characterized in that make up in the culture of microorganisms of the species Methanobacterium subterraneum at least 10% of the total number of microorganisms present in the culture. 6. The method according to claim 5, characterized in that make up in the culture of microorganisms of the species Methanobacterium subterraneum at least 50% of the total number of microorganisms present in the culture.

7. The method according to claim 6, characterized in that make up in the culture of microorganisms of the species Methanobacterium subterraneum at least 75% of the total number of microorganisms present in the culture.

8. The method according to claim 7, characterized in that in the culture of microorganisms of the species Methanobacterium subterraneum at least 90% of

Total number of microorganisms present in the culture.

9. The method according to at least one of claims 2 to 8, characterized in that a pure culture of the microorganism of the species Methanobacterium subterraneum is added.

10. The method according to at least one of claims 2 to 9, characterized in that microorganisms of the species Methanobacterium subterraneum the biomass are added as part of at least one immobilized culture of microorganisms.

1 1. Method according to at least one of claims 1 to 10, characterized in that in time for the addition of the microorganism of the species Methanobacterium subterraneum additional biomass is added to the fermentation reactor.

12. The method according to at least one of claims 1 to 1 1, characterized in that the space load in the fermentation reactor is continuously increased by the continuous addition of biomass.

13. The method according to at least one of claims 1 to 12, characterized in that the production of biogas from biomass at a space load of ≥ 0.5 kg oTS / m ³ d, preferably ≥ 4.0 kg oTS / m ³ d, especially preferably ≥ 8.0 kg oTS / m ³ d.

14. The method according to at least one of claims 1 to 13, characterized in that the production of biogas from biomass under constant mixing of

Fermentation substrate takes place. 15. The method according to at least one of claims 1 to 14, characterized in that the production of biogas from biomass at a temperature of 20 ^ to 80 ⁰ C, preferably at a temperature of 40 ^ to 50 ⁰ C is performed.

16. The method according to at least one of claims 1 to 15, characterized in that the addition of fermentation substrate and the addition of the microorganism of the species Methanobacterium subterraneum occur continuously.

17. The method according to at least one of claims 1 to 16, characterized in that the microorganism of the species Methanobacterium subterraneum in an amount to the

Fermentation substrate is added that after addition of the proportion of the microorganism of the species

Methanobacterium subterraneum between 10 ^'8 % and 50% of the total in the

Fermentation substrate present microorganisms.

18. The method according to claim 17, characterized in that the microorganism of the species Methanobacterium subterraneum is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanobacterium subterraneum between 10 ^"6 % and 25% of the total in Fermentation substrate present microorganisms.

19. The method according to claim 18, characterized in that the microorganism of the species Methanobacterium subterraneum is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanobacterium subterraneum between 10 ^"4 % and 10% of the total in Fermentation substrate present microorganisms.

20. The method according to claim 19, characterized in that the microorganism of the species Methanobacterium subterraneum is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanobacterium subterraneum between 10 ^'3 % and 1% of the total in Fermentation substrate present microorganisms.

21. Process according to at least one of Claims 1 to 20, characterized in that the microorganism of the species Methanobacterium subterraneum is a microorganism of the strain Methanobacterium sp. Clone SMS-sludge-11 trades. 22. The method according to at least one of claims 1 to 20, characterized in that it is the microorganism of the species Methanobacterium subterraneum to a microorganism of the strain Bacteήum clone HsA55fl.

23. Use of a microorganism of the species Methanobacterium subterraneum for the fermentative production of biogas from biomass.

24. Use according to claim 23, characterized in that it is a microorganism of the strain Methanobacterium sp. Clone SMS-sludge-11 trades.

25. Use according to claim 24, characterized in that it is a microorganism of the strain Bacterium clone HsA55fl in the microorganism of the species Methanobacterium subterraneum.

26. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 97.13% sequence identity with the nucleotide sequence SEQ ID No. 32.

27. Microorganism according to claim 26, characterized in that the nucleotide sequence contains a sequence region which has more than 98% sequence identity with the nucleotide sequence SEQ ID No. 32.

28. Microorganism according to claim 27, characterized in that the nucleotide sequence contains a sequence region which has more than 98.5% sequence identity with the nucleotide sequence SEQ ID No. 32.

29. Microorganism according to claim 28, characterized in that the nucleotide sequence contains a sequence region which has more than 99% sequence identity with the nucleotide sequence SEQ ID No. 32.

Microorganism according to claim 29, characterized in that the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 32. 31. Microorganism according to claim 30, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 32.

32. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 99.64% sequence identity with the nucleotide sequence SEQ ID No. 31.

33. The microorganism according to claim 32, characterized in that the nucleotide sequence contains a sequence region which has more than 99.7% sequence identity with the nucleotide sequence SEQ ID No. 31.

34. The microorganism according to claim 33, characterized in that the nucleotide sequence contains a sequence region which has more than 99.8% sequence identity with the nucleotide sequence SEQ ID No. 31.

35. The microorganism according to claim 34, characterized in that the nucleotide sequence contains a sequence region which has more than 99.9% sequence identity with the nucleotide sequence SEQ ID No. 31.

36. The microorganism according to claim 35, characterized in that the nucleotide sequence contains a sequence region which has more than 99.95% sequence identity with the nucleotide sequence SEQ ID No. 31.

37. Microorganism according to claim 36, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 31.

38. Culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, characterized in that in the culture of

Microorganisms, a microorganism according to claims 26 to 37 is present, wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

39. Culture of microorganisms according to claim 38, characterized in that the microorganism constitutes at least 10 ^'2 % of the total number of microorganisms present in the culture. 40. Culture of microorganisms according to claim 39, characterized in that the microorganism constitutes at least 1% of the total number of microorganisms present in the culture.

41. Culture of microorganisms according to claim 40, characterized in that the microorganism constitutes at least 10% of the total number of microorganisms present in the culture.

42. Culture of microorganisms according to claim 41, characterized in that the microorganism constitutes at least 50% of the total number of microorganisms present in the culture.

43. Culture of microorganisms according to claim 42, characterized in that the microorganism at least 90% of the total number present in the culture

Constitutes microorganisms.

44. Culture of microorganisms according to claim 43, characterized in that it is a pure culture of a microorganism according to claims 26 to 37.

45. Culture of microorganisms according to at least one of claims 38 to 44, characterized in that it is an immobilized culture of microorganisms.

46. Use of a microorganism according to at least one of claims 26 to 37 for the fermentative production of biogas from biomass.

47. Use according to claim 46, characterized in that it is a process for the fermentative production of biogas from biomass according to at least one of claims 1 to 22.

48. Use of a culture of microorganisms according to at least one of claims 38 to 45 for the fermentative production of biogas from biomass.

49. Use according to claim 48, characterized in that it is a method for the fermentative production of biogas from biomass biomass according to at least one of claims 1 to 22. In summary, a method for producing biogas from biomass in a fermentation reactor is described, wherein the biomass is added to a microorganism of the species Methanobacterium subterraneum.

Methanobacterium aarhusense

The present invention provides a method for producing biogas from biomass in a fermentation reactor. The biomass is added according to the invention a microorganism of the species Methanobacterium aarhusense.

Surprisingly, it has been shown that the amount of biogas formed can be significantly increased by the addition of microorganisms of the species Methanobacterium aarhusense to the fermentation substrate. At constant space load, the addition of microorganisms of the species Methanobacterium aarhusense causes a significant increase in the amount of biogas produced. Alternatively, the amount of methane formed can also be increased by increasing the space load, with the addition of microorganisms of the species Methanobacterium aarhusense preventing the fermentation process from becoming unstable under the altered conditions. The use of microorganisms of the species Methanobacterium aarhusense therefore leads to an improvement in the efficiency and efficiency of biogas plants.

The person skilled in the art knows which strains of microorganisms fall under the term "species Methanobacterium aarhusense." Microorganisms of the species Methanobacterium aarhusense have hitherto not been known in connection with the production of biogas by fermentation of organic substrates.

According to a preferred embodiment of the present invention, a microorganism of the species Methanobacterium aarhusense in the form of a culture of

Microorganisms added, mainly from microorganisms of the species

Methanobacterium aarhusense exists. In fermentation substrates of biogas plants could

Microorganisms of the species Methanobacterium aarhusense only in traces of less than 10 ^"

⁴ % proportion of the total number of microorganisms present are detected. As for the addition of microorganisms, the amount of their natural

Occurrence of isolated microorganisms is insufficient, is usually made an increase in the form of a culture. In practice it turns out that the addition of the Microorganisms to the fermentation substrate of a fermenter is most easily made directly in the form of a culture of microorganisms.

The addition of the culture of Methanobacterium aarhusense can be carried out in the form of a culture suspension or in the form of dry, freeze-dried or moist cell pellets.

Since the already mentioned various positive effects on the fermentation process are associated with the microorganism species Methanobacterium aarhusense, this type of microorganism should be present in the added culture in a concentration exceeding the natural abundance. Of course, mixed cultures of any composition can be used for the addition. All that is required is that the species Methanobacterium aarhusense is present in a concentration which is enriched in relation to the natural occurrence.

The determination of the proportions of different types of microorganisms in mixed cultures is not a problem for the expert. Thus, for example, with the help of fluorescence-labeled oligosensors specifically the proportion of microorganisms of the species Methanobacterium aarhusense be identified in a mixture. As already mentioned, microorganisms of the species Methanobacterium aarhusense are preferably added to the fermentation substrate in the form of cultures of microorganisms, the cultures of microorganisms consisting predominantly of microorganisms of the species Methanobacterium aarhusense. If, in addition to the determination of the number of microorganisms of the species Methanobacterium aarhusense, the total number of microorganisms is also determined, the percentage of microorganisms of the species Methanobacterium aarhusense in the culture can be stated as a percentage. Microorganisms of the species Methanobacterium aarhusense are in a mixed culture then the predominant species of microorganisms, if they have the highest percentage of the various species of microorganisms present in the mixed culture.

According to preferred embodiments of the present invention make microorganisms of the species Methanobacterium aarhusense at least 10 ^'4%, especially at least 10 ^"2%, more preferably at least 1% of the total number of the added to the fermentation substrate culture microorganisms present. According to further preferred embodiments make microorganisms of the species Methanobacterium aarhusense at least 10%, in particular at least 50%, particularly preferably at least 90% of the total number of microorganisms present in the culture. According to a very particularly preferred embodiment, a pure culture of a microorganism of the species Methanobacterium aarhusense is added. Due to the specific metabolic processes and activities, the addition of a pure culture can especially contribute to an improved methane production.

According to another preferred embodiment of the present invention, a microorganism of the species Methanobacterium aarhusense is added as a constituent of at least one immobilized culture of microorganisms. Since the amount of microorganisms isolated from their natural occurrence is insufficient for the addition of the microorganisms, it is usually propagated in the form of a culture. In practice, it has been found that the addition of the microorganisms to the fermentation substrate of a fermenter is most easily carried out in the form of an immobilized culture of microorganisms.

According to a preferred embodiment of the present invention, the microorganism of the species Methanobacterium aarhusense is added in an amount to the fermentation substrate, so that after addition of the proportion of the microorganism of the species Methanobacterium aarhusense between 10 ^'8 % and 50% of the total number of present in the fermentation substrate microorganisms accounts. In particular, depending on the fermenter size and thus as a function of the amount of fermentation substrate can be necessary to achieve the desired effect, an addition of very different amounts of microorganisms.

A microorganism of the species Methanobacterium is particularly preferred aarhusense in an amount added to the fermentation substrate, that after addition the content of the microorganism of the species Methanobacterium aarhusense between ^{10 -6%} and 25% of the total number of microorganisms present in the fermentation substrate by weight. More preferably, the microorganism of the species Methanobacterium aarhusense is added to the fermentation substrate in an amount such that, after addition, the proportion of the microorganism of the species Methanobacterium aarhusense is between 10 ^'4 % and 10% of the total number of microorganisms present in the fermentation substrate. Most preferably, the microorganism of the species Methanobacterium aarhusense is added to the fermentation substrate in an amount such that, after addition, the proportion of the microorganism of the species Methanobacterium aarhusense is between 10 ^-3 % and 1% of the total number of microorganisms present in the fermentation substrate. According to a particularly preferred embodiment of the present invention, the microorganism of the species Methanobacterium aarhusense is a microorganism of the strain Bacterium clone HsA55fl. In all of the above-described embodiments, which deal with the addition of a microorganism of the species Methanobacterium aarhusense, so preferably a microorganism of the strain Bacterium clone HsA55fl is added.

The present invention also encompasses the use of a microorganism of the species Methanobacterium aarhusense for the fermentative production of biogas from biomass. Preferably, a microorganism of the strain Bacterium clone HsA55fl is used for the fermentative production of biogas from biomass.

The microorganisms Methanobacterium aarhusense SBG36 obtained from the fermentation substrate of a post-fermenter were subjected to a sequence analysis. The determined 16S rRNA sequence SEQ ID NO: 33 comprises 910 nucleotides. The next related sequence identified was the uncultured bacterium clone HsA55fl. A comparison of the sequences revealed that a total of 22 exchanges of nucleotides or deletions are present. At a length of the specific nucleotide sequence of 910 nucleotides of Methanobacterium aarhusense SBG36 and a length of the reference sequence of 916 nucleotides, a 97.58% match is calculated using the FASTA algorithm.

The present invention also includes microorganisms having a nucleic acid having a nucleotide sequence containing a sequence region having more than 97.58% sequence identity with the nucleotide sequence of SEQ ID NO: 33. More preferably, the nucleotide sequence contains a sequence range greater than 97.6% or greater than 97.7% or greater than 97.8% or greater than 97.9% or greater than 98.0% or greater than 98.1%. or more than 98.2% sequence identity with the nucleotide sequence SEQ ID NO: 33, and more preferably, the nucleotide sequence contains a sequence region having more than 98.3% sequence identity with the nucleotide sequence SEQ ID NO: 33.

According to further preferred embodiments, the microorganism has a nucleotide sequence which contains a sequence region which has more than 98.5% or more than 99.0% sequence identity with the nucleotide sequence SEQ ID No. 33, and more preferably the nucleotide sequence contains a sequence region which greater than 99.5% sequence identity with the nucleotide sequence SEQ ID NO: 33. According to a whole In a particularly preferred embodiment, the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 33.

In preferred embodiments of the present invention, SEQ ID NO: 33 may be compared to the starting nucleotide sequence at one or two positions or at three positions or at four positions or at five positions or at six positions or at seven positions or at eight positions or at nine positions or at ten

Positions or eleven positions or twelve positions or 13 positions or 14

Positions or 15 positions or 16 positions or 17 positions or 18 positions or 19 positions or 20 positions or 21 positions or 22

Positions nucleotide mutations present. The meaning of the term "nucleotide mutation" is explained in the "Definitions" section of the present text.

The present invention also includes a culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, wherein in the culture of microorganisms a microorganism is present, which contains a

Having a nucleotide sequence containing a sequence range greater than 97.58%

Sequence identity with the nucleotide sequence SEQ ID NO. 33, wherein the

Microorganism accounts for at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preferably, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 97.6% or greater than 97.7% or greater than 97 , 8% or more than 97.9% or more than 98.0% or more than 98.1% or more than 98.2% sequence identity with the nucleotide sequence SEQ ID NO: 33. Particularly preferably, the nucleotide sequence contains a sequence region which has more than 98.3% sequence identity with the nucleotide sequence SEQ ID No. 33.

According to further preferred embodiments, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence with a sequence range greater than 98.4% or greater 98.5% or greater has 99.0% sequence identity with the nucleotide sequence SEQ ID NO: 33. Most preferably, the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID NO: 33. According to a very particularly preferred embodiment, in the culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, a microorganism is present which has a nucleotide sequence which contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 33.

The present invention also encompasses the use of a microorganism as defined above with respect to its nucleotide sequence SEQ ID NO: 33 for the fermentative production of biogas from biomass. Preference is given to the use of a microorganism as defined above with regard to its nucleotide sequence SEQ ID No. 33 in a method described above in connection with microorganisms of the species Methanobacterium aarhusense for the fermentative production of biogas from biomass.

The present invention also includes the use of a culture of microorganisms for the fermentative production of biogas from biomass, wherein the culture of microorganisms at least one microorganism as above with respect to its

Nucleotide sequence SEQ ID NO: 33 is defined and wherein the microorganism constitutes at least 10 ^'4 % of the total number of microorganisms present in the culture.

Preference is given to the use of a culture of microorganisms for the fermentative production of biogas from biomass, the culture of microorganisms comprising at least one microorganism as described above with regard to its nucleotide sequence SEQ

ID NO. 33 has been defined, and wherein the microorganism is at least 10 ^'4% of the

Total number of microorganisms present in the culture, in one of the above - mentioned

In connection with microorganisms of the species Methanobacterium aarhusense described method for the fermentative production of biogas from biomass.

According to further preferred embodiments make said, characterized in terms of their nucleotide sequence microorganisms at least 10 ^'2%, preferably to at least 1% of the total present in the culture of microorganisms. Particularly preferably, the microorganisms make up at least 10%, particularly preferably at least 25%, of the total number of microorganisms present in the culture. Most preferably, the microorganisms described make up at least 50%, in particular at least 75%, of the total number of microorganisms present in the culture.

Also preferred are embodiments according to which the above-mentioned microorganisms make up at least 90% of the total number of microorganisms present in the culture. Particularly preferred is a pure culture of Microorganisms suitable for use in a process for the fermentative production of biogas from biomass, which is a pure culture of a microorganism as characterized above with respect to its nucleotide sequence.

Most preferably, in the cases described above, it is an immobilized culture of microorganisms.

In particular, the present invention includes the following aspects:

1. A process for the production of biogas from biomass in a fermentation reactor, characterized in that the biomass is added to a microorganism of the species Methanobacterium aarhusense.

2. The method according to claim 1, characterized in that the microorganism of the species Methanobacterium aarhusense is added in the form of a culture of microorganisms, wherein microorganisms of the species Methanobacterium aarhusense make up at least 10 ^'4 % of the total number of microorganisms present in the culture.

3. The method according to claim 2, characterized in that in the culture of microorganisms of the species Methanobacterium aarhusense at least 10 ^'2 % of

Total number of microorganisms present in the culture.

4. The method according to claim 3, characterized in that constitute in the culture of microorganisms of the species Methanobacterium aarhusense at least 1% of the total number of microorganisms present in the culture.

5. The method according to claim 4, characterized in that constitute in the culture of microorganisms of the species Methanobacterium aarhusense at least 10% of the total number of microorganisms present in the culture.

6. The method according to claim 5, characterized in that make up in the culture of microorganisms of the species Methanobacterium aarhusense at least 50% of the total number of microorganisms present in the culture.

7. The method according to claim 6, characterized in that make up in the culture of microorganisms of the species Methanobacterium aarhusense at least 75% of the total number of microorganisms present in the culture. 8. The method according to claim 7, characterized in that make up in the culture of microorganisms of the species Methanobacterium aarhusense at least 90% of the total number of microorganisms present in the culture.

9. The method according to at least one of claims 2 to 8, characterized in that a pure culture of the microorganism of the species Methanobacterium aarhusense is added.

10. The method according to at least one of claims 2 to 9, characterized in that microorganisms of the species Methanobacterium aarhusense the biomass are added as part of at least one immobilized culture of microorganisms.

1 1. Method according to at least one of claims 1 to 10, characterized in that in time for the addition of the microorganism of the species Methanobacterium aarhusense additional biomass is added to the fermentation reactor.

12. The method according to at least one of claims 1 to 1 1, characterized in that the space load in the fermentation reactor is continuously increased by the continuous addition of biomass.

13. The method according to at least one of claims 1 to 12, characterized in that the production of biogas from biomass at a space load of ≥ 0.5 kg oTS / m ³ d, preferably ≥ 4.0 kg oTS / m ³ d, especially preferably ≥ 8.0 kg oTS / m ³ d.

14. The method according to at least one of claims 1 to 13, characterized in that the production of biogas from biomass takes place under constant mixing of the fermentation substrate.

15. The method according to at least one of claims 1 to 14, characterized in that the production of biogas from biomass at a temperature of 20 ^ to 80 ⁰ C, preferably at a temperature of 40 ^< € to 50 ⁰ C is performed.

16. The method according to at least one of claims 1 to 15, characterized in that the addition of fermentation substrate and the addition of the microorganism of the species Methanobacterium aarhusense carried out continuously. 17. The method according to at least one of claims 1 to 16, characterized in that the microorganism of the species Methanobacterium aarhusense is added in an amount to the fermentation substrate that after addition of the proportion of the microorganism of the species Methanobacterium aarhusense between 10 ^"8 % and 50% the total number in the

Fermentation substrate present microorganisms.

18. The method according to claim 17, characterized in that the microorganism of the species Methanobacterium aarhusense is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanobacterium aarhusense between 10 ^'6 % and 25% of the total in Fermentation substrate present microorganisms.

19. The method according to claim 18, characterized in that the microorganism of the species Methanobacterium aarhusense is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanobacterium aarhusense between 10 ^'4 % and 10% of the total in Fermentation substrate present microorganisms.

20. The method according to claim 19, characterized in that the microorganism of the species Methanobacterium aarhusense is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanobacterium aarhusense between 10 ^'3 % and 1% of the total in Fermentation substrate present microorganisms.

21. Process according to at least one of Claims 1 to 20, characterized in that the microorganism of the species Methanobacterium aarhusense is a microorganism of the strain Bacterium clone HsA55fl.

22. Use of a microorganism of the species Methanobacterium aarhusense for the fermentative production of biogas from biomass.

23. Use according to claim 22, characterized in that the microorganism of the species Methanobacterium aarhusense is a microorganism of the strain Bacterium clone HsA55fl. 24. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 97.58% sequence identity with the nucleotide sequence SEQ ID No. 33.

25. Microorganism according to claim 24, characterized in that the nucleotide sequence contains a sequence region which has more than 97.8% sequence identity with the nucleotide sequence SEQ ID No. 33.

26. Microorganism according to claim 25, characterized in that the nucleotide sequence contains a sequence region which has more than 98.0% sequence identity with the nucleotide sequence SEQ ID No. 33.

27. The microorganism according to claim 26, characterized in that the nucleotide sequence contains a sequence region which has more than 99.0% sequence identity with the nucleotide sequence SEQ ID No. 33.

28. A microorganism according to claim 27, characterized in that the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 33.

29. Microorganism according to claim 28, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 33.

30. Culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, characterized in that in the culture of microorganisms, a microorganism according to claims 24 to 29 is present, wherein the microorganism at least 10 ^'4 % of the total in the culture microorganisms present.

31. Culture of microorganisms according to claim 30, characterized in that the microorganism makes up at least ^{10 -2%} of the total number of microorganisms present in the culture.

32. Culture of microorganisms according to claim 31, characterized in that the microorganism constitutes at least 1% of the total number of microorganisms present in the culture. 33. Culture of microorganisms according to claim 32, characterized in that the microorganism constitutes at least 10% of the total number of microorganisms present in the culture.

34. Culture of microorganisms according to claim 33, characterized in that the microorganism constitutes at least 50% of the total number of microorganisms present in the culture.

35. Culture of microorganisms according to claim 34, characterized in that the microorganism constitutes at least 90% of the total number of microorganisms present in the culture.

36. Culture of microorganisms according to claim 35, characterized in that it is a pure culture of a microorganism according to claims 24 to 29.

37. Culture of microorganisms according to at least one of claims 30 to 36, characterized in that it is an immobilized culture of microorganisms.

38. Use of a microorganism according to at least one of claims 24 to 29 for the fermentative production of biogas from biomass.

39. Use according to claim 38, characterized in that it is a process for the fermentative production of biogas from biomass according to at least one of

Claims 1 to 21 is.

40. Use of a culture of microorganisms according to at least one of claims 30 to 37 for the fermentative production of biogas from biomass.

41. Use according to claim 40, characterized in that it is a process for the fermentative production of biogas from biomass biomass according to at least one of claims 1 to 21.

In summary, a process for the production of biogas from biomass in a fermentation reactor is described, wherein the biomass is added to a microorganism of the species Methanobacterium aarhusense. Brief description of the figures

To illustrate the invention and to illustrate its advantages, embodiments are given below. The exemplary embodiments will be explained in more detail in connection with FIGS. 1 to 3. It goes without saying that the statements made in connection with the exemplary embodiments are not intended to limit the invention. Show it:

Fig. 1 Results of a fermentation with the addition of microorganisms of the species Methanobacterium formicicum: Plotted is the gas yield, expressed in standard liters per day (Nl / d) and the space load, expressed in kg oTS / m ³ d of the fermenter against time, indicated in operating days;

Fig. 2A, measurement results of a further fermentation with the addition of microorganisms Fig. 2 B of the species Methanobacterium formicicum: Fig. 2A. Plotted is the gas yield, expressed in standard liters per day (Nl / d) and the volume load, expressed in kg oTS / m ³ d of the fermenter against time, indicated in operating days. Fig. 2B. Plotted are the pH and the measured Säuekonzentrationen of acetic acid, propionic acid and acetic acid equivalent, expressed in mg / l fermenter content against time, given in operating days;

Fig. 3A, measurement results of a control fermentation without addition of microorganisms: Fig. 3B Fig. 3A. Plotted is the gas yield, expressed in standard liters per day (Nl / d) and the volume load, expressed in kg oTS / m ³ d of the fermenter against time, indicated in operating days. Fig. 3B. Plotted are the pH and the measured Säuekonzentrationen of acetic acid, propionic acid and acetic acid equivalent, expressed in mg / l fermenter content against time, indicated in operating days. Ways to carry out the invention

The following examples illustrate the invention and are not to be construed as limiting. Unless otherwise indicated, standard molecular biology methods were used, such as e.g. by Sambrook et al., 1989, Molecular cloning: A Laboratory Manual 2nd Edition, Col. Spring Harbor Laboratory Press, Col. Spring Harbor, New York.

Methanoculleus bourgensis SBG7

DNA isolation

From the fermenter of a biogas plant substrate samples were taken and in a

Transferred Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol ^{I" 1} NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). After lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml) the cells were counted after addition of protease (20 ul of a 1% (w / v) proteinase K solution) and SDS ( 100 ul of a 10% solution) for 45 min at 65 ⁰ C incubated. The DNA was then extracted with one volume of phenol, one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1, v / v / v), and one volume of chloroform. From the aqueous supernatant (3.0 M, pH 5.2) the DNA was precipitated with 0.8 volume of isopropanol for 30 min. At -20 ⁰ C after the addition of 1/10 volume of sodium acetate solution. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

nucleotide sequence determination

To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers were used with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT (indicated in the 5 ^{^} → 3 direction, Y is C or T, H is C, T or A). The pieces of DNA obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR. The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones. From the respective clones, the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence using BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained the organism Methanoculleus sp. Dm2 can be assigned as nearest relatives.

Isolation and accumulation of microorganisms

Microorganisms Methanoculleus bourgensis SBG7 were isolated under anaerobic conditions with the aid of a suitable selection medium (medium 287 (DSMZ)) from the supernatant of material from a post-fermenter. Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 287 (DSMZ)), the bacteria Methanoculleus bourgensis SßG7 could be produced.

Fermentation with addition of microorganisms of the species Methanoculleus bourgensis

SBG7

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire generated Biogas in standard liters (gas volume at 273.15 K and 1013 mbar) determined per day (Nl / d). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). In each case, the cell mass from 1 l of a preculture of Methanoculleus bourgensis SBG7, which had been incubated for 5 days at a temperature of 40 ° C. in medium 287 (DSMZ), was added.

A comparison of the amount of biogas produced showed that with the addition of microorganisms Methanoculleus bourgensis SBG7 a significantly increased amount, but at least one by 5 % higher amount of biogas produced from a given amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanoculleus bourgensis SBG7 was used. Likewise, however, mixed cultures with a proportion of Methanoculleus bourgensis SBG7 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanoculleus bourgensis leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanoculleus bourgensis SBG12

DNA isolation

From the fermenter of a biogas plant substrate samples were taken and in a

Transferred Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol ^{I" 1} NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). After lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml) the cells were counted after addition of protease (20 ul of a 1% (w / v) proteinase K solution) and SDS ( 100 ul of a 10% solution) for 45 min at 65 ⁰ C incubated. The DNA was then extracted with one volume of phenol, one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1, v / v / v), and one volume of chloroform. From the aqueous supernatant (3.0 M, pH 5.2) the DNA was precipitated with 0.8 volume of isopropanol for 30 min. At -20 ⁰ C after the addition of 1/10 volume of sodium acetate solution. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

nucleotide sequence determination

To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this, the primer with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT (indicated in 5 ^' → 3 ^" direction, Y is C or T, H is C, T or A).) The DNA pieces obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-Drive), transformed into E. coli (according to QIAGEN PCR-Cloning-Handbook) and examined by colony-PCR The obtained colony PCR products were The corresponding plasmid DNA was isolated from the respective clones and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence using BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained assigned to the organism Archaeon clone GZK7 as the nearest relative can be.

Isolation and accumulation of microorganisms

Microorganisms Methanoculleus bourgensis SBG12 were isolated from the supernatant of material from a post-fermenter under anaerobic conditions using a suitable selection medium (Medium 287 (DSMZ)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 287 (DSMZ)), the bacteria Methanoculleus bourgensis SBG12 could be propagated.

Fermentation with addition of microorganisms of the species Methanoculleus bourgensis

SBG 12

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire generated Biogas in standard liters (gas volume at 273.15 K and 1013 mbar) determined per day (NUd). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). In each case, the cell mass was added from 1 l of a preculture of Methanoculleus bourgensis SBG12 which had been incubated for 5 days at a temperature of 4O ⁰ C in medium 287 (DSMZ).

A comparison of the amount of biogas produced showed that the addition of microorganisms Methanoculleus bourgensis SBG12 a significantly increased amount, but at least a 5% higher amount of biogas produced from a predetermined amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanoculleus bourgensis SBG 12 was used. However, mixed cultures with a proportion of Methanoculleus bourgensis SBG 12 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanoculleus bourgensis leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanoculleus bourgensis SBG13

DNA isolation

From the fermenter of a biogas plant substrate samples were taken and in a

Transferred Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol ^{I" 1} NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). After lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml) the cells were counted after addition of protease (20 ul of a 1% (w / v) proteinase K solution) and SDS ( 100 ul of a 10% solution) for 45 min at 65 ⁰ C incubated. The DNA was then extracted with one volume of phenol, one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1, v / v / v), and one volume of chloroform. From the aqueous supernatant, after addition of 1/10 volume of a sodium acetate solution (3.0 M, pH 5.2), the DNA was treated with 0.8 volume of isopropanol for 30 min. At -20 ⁰ C like. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

Nucleotide sequence determination To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT were used (indicated in 5 ^' → 3 ^" direction, Y is C or T, H is C, T or A) and the DNA pieces obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-Drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones from which the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence by means of BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained from the organism Archaeon clone 5.5ft C121A next Relatives can be assigned.

Isolation and accumulation of microorganisms

Microorganisms Methanoculleus bourgensis SBG13 were isolated under anaerobic conditions from the supernatant of material from a post-fermenter using a suitable selection medium (Medium 287 (DSMZ)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 287 (DSMZ)), the bacteria Methanoculleus bourgensis SBG13 could be propagated.

Fermentation with the addition of microorganisms of the species Methanoculleus bourgensis SBG13 During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a Period of several Months of the time course of the total biogas produced in standard liters (gas volume at 273.15 K and 1013 mbar) per day (Nl / d) determined. The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). The cell mass from 1 l of a preculture of Methanoculleus bourgensis SBG 13, which had been incubated for 5 days at a temperature of 4O ⁰ C in medium 287 (DSMZ) was added to each.

A comparison of the amount of biogas produced showed that the addition of microorganisms Methanoculleus bourgensis SBG13 a significantly increased amount, but at least a 5% higher amount of biogas produced from a predetermined amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanoculleus bourgensis SBG 13 was used. Likewise, mixed cultures with a proportion of Methanoculleus bourgensis SBG 13 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanoculleus bourgensis leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanoculleus bourgensis SBG14

DNA isolation

From the fermenter of a biogas plant substrate samples were taken and in a

Transferred Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol l ^-1 NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). after lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml), the cells (20 ul of a 1% after the addition of protease strength (w / v) proteinase K solution) and SDS (100 μl of a 10% solution) for 45 min at 65 ^° C. The DNA was then extracted with one volume of phenol, with one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1, (v / v / v)) and one volume of chloroform. From the aqueous supernatant (3.0 M, pH 5.2) the DNA was precipitated with 0.8 volume of isopropanol for 30 min. At -20 ⁰ C after the addition of 1/10 volume of sodium acetate solution. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

nucleotide sequence determination

To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers were used with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT (indicated in the 5 ^{^} → 3 direction, Y is C or T, H is C, T or A). The pieces of DNA obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR. The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones. From the respective clones, the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence by means of BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained from the organism Archaeon clone 5.5ft C121A next Relatives can be assigned.

Isolation and accumulation of microorganisms

Microorganisms Methanoculleus bourgensis SBG14 were isolated from the supernatant of material from a post-fermenter under anaerobic conditions using a suitable selection medium (Medium 287 (DSMZ)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 287 (DSMZ)), the bacteria Methanoculleus bourgensis SBG14 could be propagated. Fermentation with addition of microorganisms of the species Methanoculleus bourgensis SBG14

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire generated Biogas in standard liters (gas volume at 273.15 K and 1013 mbar) determined per day (NUd). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). The cell mass from 1 l of a preculture of Methanoculleus bourgensis SBG 14, which had been incubated for 5 days at a temperature of 4O ⁰ C in medium 287 (DSMZ) was added to each.

A comparison of the amount of biogas produced showed that the addition of microorganisms Methanoculleus bourgensis SBG14 a significantly increased amount, but at least a 5% higher amount of biogas produced from a predetermined amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanoculleus bourgensis SBG14 was used. Likewise, mixed cultures with a proportion of Methanoculleus bourgensis SBG14 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanoculleus bourgensis leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanoculleus bourgensis SBG15

DNA isolation

Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1300 μl saline extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol l ^-1 NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). After lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml) the cells were counted after addition of protease (20 ul of a 1% (w / v) proteinase K solution) and SDS ( 100 ul of a 10% solution) for 45 min at 65 ⁰ C incubated. The DNA was then extracted with one volume of phenol, one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1, v / v / v), and one volume of chloroform. From the aqueous supernatant (3.0 M, pH 5.2) the DNA was precipitated with 0.8 volume of isopropanol for 30 min. At -20 ⁰ C after the addition of 1/10 volume of sodium acetate solution. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

nucleotide sequence determination

To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT were used (indicated in 5 ^' → 3 ^" direction, Y is C or T, H is C, T or A) and the DNA pieces obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-Drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones from which the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence by means of BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained the next organism Archaeon clone 5.5ft C124A Relatives can be assigned.

Isolation and accumulation of microorganisms

Microorganisms Methanoculleus bourgensis SBG15 were isolated from the supernatant under anaerobic conditions using a suitable selection medium (Medium 287 (DSMZ)) isolated from material from a post-fermenter. Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 287 (DSMZ)), the bacteria Archaeon clone 5.5ft C124A could be propagated.

Fermentation with addition of microorganisms of the species Methanoculleus bourgensis SBG15

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire generated Biogas in standard liters (gas volume at 273.15 K and 1013 mbar) determined per day (Nl / d). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). The cell mass from 1 l of a preculture of Methanoculleus bourgensis SBG 15, which had been incubated for 5 days at a temperature of 4O ⁰ C in medium 287 (DSMZ) was added to each.

A comparison of the amount of biogas produced showed that the addition of microorganisms Methanoculleus bourgensis SSG75 a significantly increased amount, but at least a 5% higher amount of biogas produced from a predetermined amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanoculleus bourgensis SBG 15 was used. However, mixed cultures with a proportion of Methanoculleus bourgensis SBG 15 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanoculleus bourgensis leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanoculleus bourgensis SBG31

DNA isolation Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol ^{I" 1} NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). After lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml) the cells were counted after addition of protease (20 ul of a 1% (w / v) proteinase K solution) and SDS ( 100 ul of a 10% solution) for 45 min at 65 ⁰ C incubated. The DNA was then extracted with one volume of phenol, one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1, v / v / v), and one volume of chloroform. From the aqueous supernatant (3.0 M, pH 5.2) the DNA was precipitated with 0.8 volume of isopropanol for 30 min. At -20 ⁰ C after the addition of 1/10 volume of sodium acetate solution. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

nucleotide sequence determination

To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers were used with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT (indicated in the 5 ^{^} → 3 direction, Y is C or T, H is C, T or A). The pieces of DNA obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR. The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones. From the respective clones, the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence using BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained can be assigned to the organism Archaeon clone 5.5ft C141A as the nearest relative.

Isolation and Enrichment of Microorganisms Microorganisms Methanoculleus bourgensis SBG31 were isolated from the supernatant of material from a post-fermenter under anaerobic conditions with the aid of a suitable selection medium (Medium 287 (DSMZ)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 287 (DSMZ)), the bacteria Methanoculleus bourgensis SBG31 could be propagated.

Fermentation with addition of microorganisms of the species Methanoculleus bourgensis SBG 15

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire generated Biogas in standard liters (gas volume at 273.15 K and 1013 mbar) determined per day (Nl / d). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). The cell mass from 1 l of a preculture of Methanoculleus bourgensis SBG31 which had been incubated for 5 days at a temperature of 4O ⁰ C in medium 287 (DSMZ) was added to each.

A comparison of the amount of biogas produced showed that the addition of microorganisms Methanoculleus bourgensis SBG31 a significantly increased amount, but at least a 5% higher amount of biogas produced from a given amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanoculleus bourgensis SBG31 was used. However, mixed cultures with a proportion of Methanoculleus bourgensis SBG31 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanoculleus bourgensis leads to a significant improvement in the efficiency and efficiency of biogas plants. Methanoculleus bourgensis SBG30

DNA isolation

Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol ^{I" 1} NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). After lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml) the cells were counted after addition of protease (20 ul of a 1% (w / v) proteinase K solution) and SDS ( 100 ul of a 10% solution) for 45 min at 65 ⁰ C incubated. The DNA was then extracted with one volume of phenol, one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1, v / v / v), and one volume of chloroform. From the aqueous supernatant (3.0 M, pH 5.2) the DNA was precipitated with 0.8 volume of isopropanol for 30 min. At -20 ⁰ C after the addition of 1/10 volume of sodium acetate solution. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

nucleotide sequence determination

To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. The primers were used with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT (indicated in the 5 ^' → 3 direction, Y is C or T, H is C, T or A). The pieces of DNA obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR. The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones. From the respective clones, the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence by means of BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained assigned to the organism Archaeon clone A52 as the nearest relative can be.

Isolation and Enrichment of Microorganisms Microorganisms Methanoculleus bourgensis SBG30 were isolated from the supernatant of material from a post-fermenter under anaerobic conditions using a suitable selection medium (Medium 287 (DSMZ)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 287 (DSMZ)), the bacteria Methanoculleus bourgensis SBG30 could be increased.

Fermentation with addition of microorganisms of the species Methanoculleus bourgensis

SBG30

During a fermentation process in a test fermenter with a volume of

150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average volume load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of all biogas produced in standard liters (gas volume at 273.15 K and 1013 mbar) per day (NUd). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). The cell mass from 1 l of a preculture of Methanoculleus bourgensis SBG30 which had been incubated for 5 days at a temperature of 4O ⁰ C in medium 287 (DSMZ) was added to each.

A comparison of the amount of biogas produced showed that the addition of microorganisms Methanoculleus bourgensis SBG30 a significantly increased amount, but at least a 5% higher amount of biogas produced from a predetermined amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanoculleus bourgensis SBG30 was used. Likewise, however, mixed cultures with a share of

Methanoculleus bourgensis SBG30 can be used. In particular, mixed cultures of two or three types of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides are added.

The use of microorganisms of the species Methanoculleus bourgensis leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanoculleus bourgensis SBG23

DNA isolation

Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol l ^-1 NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). after lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml), the cells (20 ul of a 1% after the addition of protease strength (w / v) proteinase K solution) and SDS (100 μl of a 10% solution) for 45 min at 65 ^° C. The DNA was then mixed with one volume of phenol, with one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1). (v / v / v)) and extracted with one volume of chloroform From the aqueous supernatant, after addition of 1/10 volume of a sodium acetate solution (3.0 M, pH 5.2), the DNA was mixed with 0.8 volume of isopropanol 30 min. at -20 ⁰ C precipitated. The DNA was collected by centrifugation, washed with 70% ethanol twice and dried at room temperature in air.

nucleotide sequence determination

To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT were used (indicated in 5 ^' → 3 ^" direction, Y is C or T, H is C, T or A) and the DNA pieces obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-Drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones The corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

Sequence analysis Following sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed using the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence using BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained assigned to the organism Archaeon clone MCSArc_B6 as the nearest relative can be.

Isolation and accumulation of microorganisms

Microorganisms Methanoculleus bourgensis SBG23 were isolated from the supernatant of material from a post-fermenter under anaerobic conditions using a suitable selection medium (Medium 287 (DSMZ)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 287 (DSMZ)), the bacteria Methanoculleus bourgensis SBG23 could be propagated.

Fermentation with addition of microorganisms of the species Methanoculleus bourgensis SBG23

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire generated Biogas in standard liters (gas volume at 273.15 K and 1013 mbar) determined per day (NUd). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). The cell mass from 1 l of a preculture of Methanoculleus bourgensis SBG23 which had been incubated for 5 days at a temperature of 4O ⁰ C in medium 287 (DSMZ) was added to each.

A comparison of the amount of biogas produced showed that the addition of microorganisms Methanoculleus bourgensis SBG23 a significantly increased amount, but at least a 5% higher amount of biogas produced from a predetermined amount of organic dry matter is generated as without the addition of microorganisms. In the described embodiment, a pure culture of Methanoculleus bourgensis SBG23 was used. However, mixed cultures with a proportion of Methanoculleus bourgensis SBG23 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanoculleus bourgensis leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanoculleus bourgensis SBG28

DNA isolation Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol l ^-1 NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). after lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml), the cells (20 ul of a 1% after the addition of protease strength (w / v) proteinase K solution) and SDS (100 μl of a 10% solution) for 45 min at 65 ^° C. The DNA was then mixed with one volume of phenol, with one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1). (v / v / v)) and extracted with one volume of chloroform From the aqueous supernatant, after addition of 1/10 volume of a sodium acetate solution (3.0 M, pH 5.2), the DNA was mixed with 0.8 volume of isopropanol 30 min. at -20 ⁰ C precipitated. The DNA was collected by centrifugation, washed with 70% ethanol twice and dried at room temperature in air.

nucleotide sequence determination

To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT were used (indicated in 5 ^' → 3 ^" direction, Y is C or T, H is C, T or A) and the DNA pieces obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-drive), transformed into E. coli (according to QIAGEN PCR-Cloning-Handbook) and examined by colony PCR. The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones. From the respective clones, the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence using BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained the organism Euryarchaeote clone BSA 1A-03 as can be assigned to next relatives.

Isolation and accumulation of microorganisms

Microorganisms Methanoculleus bourgensis SBG28 were isolated from the supernatant of material from a post-fermenter under anaerobic conditions using a suitable selection medium (Medium 287 (DSMZ)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 287 (DSMZ)), the bacteria Methanoculleus bourgensis SBG28 could be propagated.

Fermentation with addition of microorganisms of the species Methanoculleus bourgensis SBG28

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire generated Biogas in standard liters (gas volume at 273.15 K and 1013 mbar) determined per day (Nl / d). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). The cell mass from 1 l of a preculture of Methanoculleus bourgensis SBG28 which had been incubated for 5 days at a temperature of 4O ⁰ C in medium 287 (DSMZ) was added to each. A comparison of the amount of biogas produced showed that with the addition of microorganisms Methanoculleus bourgensis SBG28 a significantly increased amount, but at least a 5% higher amount of biogas produced from a predetermined amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanoculleus bourgensis SBG28 was used. However, mixed cultures with a proportion of Methanoculleus bourgensis SBG28 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanoculleus bourgensis leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanoculleus bourgensis SBG29

DNA isolation Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol ^{I" 1} NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). After lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml) the cells were counted after addition of protease (20 ul of a 1% (w / v) proteinase K solution) and SDS ( 100 ul of a 10% solution) for 45 min at 65 ⁰ C incubated. The DNA was then extracted with one volume of phenol, one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1, v / v / v), and one volume of chloroform. From the aqueous supernatant (3.0 M, pH 5.2) the DNA was precipitated with 0.8 volume of isopropanol for 30 min. At -20 ⁰ C after the addition of 1/10 volume of sodium acetate solution. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

nucleotide sequence determination To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT were used (indicated in 5 ^' → 3 ^" direction, Y is C or T, H is C, T or A) and the DNA pieces obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-Drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones from which the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence by means of BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained the next organism Euryarchaeote clone BSA2A-02 Relatives can be assigned.

Isolation and accumulation of microorganisms

Microorganisms Methanoculleus bourgensis SBG29 were isolated under anaerobic conditions from the supernatant of material from a post-fermenter using a suitable selection medium (Medium 287 (DSMZ)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 287 (DSMZ)), the bacteria Methanoculleus bourgensis SBG29 could be propagated.

Fermentation with the addition of microorganisms of the species Methanoculleus bourgensis SBG29

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire generated Biogas in standard liters (gas volume at 273.15 K and 1013 mbar) determined per day (NUd). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once under multiple Addition (eg 2x weekly). The cell mass from 1 l of a preculture of Methanoculleus bourgensis SBG29 which had been incubated for 5 days at a temperature of 4O ⁰ C in medium 287 (DSMZ) was added to each.

A comparison of the amount of biogas produced showed that the addition of microorganisms Methanoculleus bourgensis SBG29 a significantly increased amount, but at least a 5% higher amount of biogas produced from a given amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanoculleus bourgensis SBG29 was used. However, mixed cultures with a proportion of Methanoculleus bourgensis SBG29 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanoculleus bourgensis leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanoculleus bourgensis SBG32

DNA isolation

Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol l ^-1 NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). after lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml), the cells (20 ul of a 1% after the addition of protease strength (w / v) proteinase K solution) and SDS (100 μl of a 10% solution) for 45 min at 65 ^° C. The DNA was then mixed with one volume of phenol, with one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1). (v / v / v)) and extracted with one volume of chloroform From the aqueous supernatant, after addition of 1/10 volume of a sodium acetate solution (3.0 M, pH 5.2), the DNA was mixed with 0.8 volume of isopropanol 30 minutes at -20 ⁰ C like. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

Nucleotide sequence determination To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT were used (indicated in 5 ^' → 3 ^" direction, Y is C or T, H is C, T or A) and the DNA pieces obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-Drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones from which the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence using BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained the next organism Euryarchaeote clone MAA-04 Relatives can be assigned.

Isolation and accumulation of microorganisms

Microorganisms Methanoculleus bourgensis SBG32 were isolated under anaerobic conditions from the supernatant of material from a post-fermenter using a suitable selection medium (medium 287 (DSMZ)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 287 (DSMZ)), the bacteria Methanoculleus bourgensis SBG32 could be propagated.

Fermentation with the addition of microorganisms of the species Methanoculleus bourgensis SBG32 During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a Period of several Months of the time course of the total biogas produced in standard liters (gas volume at 273.15 K and 1013 mbar) per day (Nl / d) determined. The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). The cell mass from 1 l of a preculture of Methanoculleus bourgensis SBG32 which had been incubated for 5 days at a temperature of 4O ⁰ C in medium 287 (DSMZ) was added to each.

A comparison of the amount of biogas produced showed that the addition of microorganisms Methanoculleus bourgensis SBG32 a significantly increased amount, but at least a 5% higher amount of biogas produced from a given amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanoculleus bourgensis SBG32 was used. Likewise, mixed cultures with a proportion of Methanoculleus bourgensis SBG32 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanoculleus bourgensis leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanoculleus chikugoensis SBG5

DNA isolation

From the fermenter of a biogas plant substrate samples were taken and in a

Transferred Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol l ^-1 NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). after lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml), the cells (20 ul of a 1% after the addition of protease strength (w / v) proteinase K solution) and SDS (100 μl of a 10% solution) for 45 min at 65 ^° C. The DNA was then extracted with one volume of phenol, with one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1, (v / v / v)) and one volume of chloroform. From the aqueous supernatant (3.0 M, pH 5.2) the DNA was precipitated with 0.8 volume of isopropanol for 30 min. At -20 ⁰ C after the addition of 1/10 volume of sodium acetate solution. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

nucleotide sequence determination

To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers were used with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT (indicated in the 5 ^{^} → 3 direction, Y is C or T, H is C, T or A). The pieces of DNA obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR. The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones. From the respective clones, the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence using BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained assigned to the organism Methanoculleus chikugoensis MG62 as the nearest relative can be.

Isolation and accumulation of microorganisms

Microorganisms of methanoculleus chikugoensis SBG5 were isolated under anaerobic conditions from the supernatant of material from a post-fermenter using a suitable selection medium (Medium 141 (DSMZ)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 141 (DSMZ)), the bacteria Methanoculleus chikugoensis SBG5 could be propagated. Fermentation with the addition of microorganisms of the species Methanoculleus chikugoensis SBG5

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire generated Biogas in standard liters (gas volume at 273.15 K and 1013 mbar) determined per day (NUd). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). The cell mass from 1 l of a preculture of Methanoculleus chikugoensis SBG5 which had been incubated for 5 days at a temperature of 4O ⁰ C in medium 141 (DSMZ) was added to each.

A comparison of the amount of biogas produced showed that with the addition of microorganisms Methanoculleus chikugoensis SBG5 a significantly increased amount, but at least a 5% higher amount of biogas produced from a given amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanoculleus chikugoensis SBG5 was used. However, mixed cultures with a proportion of methanoculleus chikugoensis SBG5 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanoculleus chikugoensis leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanoculleus marisnigri SBG6

DNA isolation

Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1300 μl saline extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol I ^"1 NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). After lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml) the cells were counted after addition of protease (20 ul of a 1% (w / v) proteinase K solution) and SDS (100 .mu.l of a 10% solution) was incubated for 45 min at 65 ^° C. The DNA was then extracted with one volume of phenol, with one volume of phenol: chloroform: isoamylalcohol (25: 24: 1, v / v / v) and one volume of chloroform aqueous supernatant was added after addition of 1/10 volume of a sodium acetate solution (3.0 M, pH 5.2) the DNA with 0.8 volume of isopropanol for 30 min at -20 ⁰ C. The DNA was removed by centrifugation, washed twice with Washed 70% ethanol and dried at room temperature in air.

nucleotide sequence determination

To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT were used (indicated in 5 ^' → 3 ^" direction, Y is C or T, H is C, T or A) and the DNA pieces obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-Drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones from which the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence using BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained assigned to the organism Methanoculleus marisnigri JR1 as the nearest relative can be.

Isolation and accumulation of microorganisms

Microorganisms Methanoculleus marisnigri SBG6 were prepared under anaerobic conditions with the aid of a suitable selection medium (Medium 532 (DSMZ)) from the supernatant of Material isolated from a post-fermenter. Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 532 (DSMZ)), the bacteria Methanoculleus marisnigή SBG6 could be propagated.

Fermentation with the addition of microorganisms of the species Methanoculleus marisnigή SBG6

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire generated Biogas in standard liters (gas volume at 273.15 K and 1013 mbar) determined per day (Nl / d). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). In each case, the cell mass from 1 l of a preculture of methanoculleus marisnigri SBG6, which had been incubated for 5 days at a temperature of 40 ° C. in medium 532 (DSMZ), was added.

A comparison of the amount of biogas produced showed that the addition of microorganisms Methanoculleus marisnigri SBG6 a significantly increased amount, but at least a 5% higher amount of biogas produced from a predetermined amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanoculleus marisnigri SBG6 was used. However, mixed cultures with a proportion of methanoculleus marisnigri SBG6 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanoculleus marisnigri leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanoculleus thermophilus SBG27

DNA isolation Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol ^{I" 1} NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). After lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml) the cells were counted after addition of protease (20 ul of a 1% (w / v) proteinase K solution) and SDS ( 100 ul of a 10% solution) for 45 min at 65 ⁰ C incubated. The DNA was then extracted with one volume of phenol, one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1, v / v / v), and one volume of chloroform. From the aqueous supernatant (3.0 M, pH 5.2) the DNA was precipitated with 0.8 volume of isopropanol for 30 min. At -20 ⁰ C after the addition of 1/10 volume of sodium acetate solution. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

nucleotide sequence determination

To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers were used with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT (indicated in the 5 ^{^} → 3 direction, Y is C or T, H is C, T or A). The pieces of DNA obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR. The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones. From the respective clones, the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence using BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained can be assigned to the organism Archaeon clone HDBW-WA02 as the closest relative.

Isolation and Enrichment of Microorganisms Microorganisms Methanoculleus thermophilus SBG27 were isolated from the supernatant of material from a post-fermenter under anaerobic conditions with the aid of a suitable selection medium (Medium 279 (DSMZ)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 279 (DSMZ)), the bacteria Methanoculleus thermophilus SBG27 could be propagated.

Fermentation with the addition of microorganisms of the species Methanoculleus thermophilus

SBG27

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire generated Biogas in standard liters (gas volume at 273.15 K and 1013 mbar) determined per day (NUd). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). The cell mass from 1 l of a preculture of Methanoculleus thermophilus SBG27 which had been incubated for 5 days at a temperature of 4O ⁰ C in medium 279 (DSMZ) was added to each.

A comparison of the amount of biogas produced showed that the addition of microorganisms Methanoculleus thermophilus SBG27 a significantly increased amount, but at least a 5% higher amount of biogas produced from a predetermined amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanoculleus thermophilus SBG27 was used. Likewise, however, mixed cultures with a proportion of Methanoculleus thermophilus SBG27 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added. The use of microorganisms of the species Methanoculleus thermophilus leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanosarcina acetivorans SBG 19

DNA isolation

From the fermenter of a biogas plant substrate samples were taken and in a

Transferred Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol ^{I" 1} NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). After lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml) the cells were counted after addition of protease (20 ul of a 1% (w / v) proteinase K solution) and SDS ( 100 ul of a 10% solution) for 45 min at 65 ⁰ C incubated. The DNA was then extracted with one volume of phenol, one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1, v / v / v), and one volume of chloroform. From the aqueous supernatant (3.0 M, pH 5.2) the DNA was precipitated with 0.8 volume of isopropanol for 30 min. At -20 ⁰ C after the addition of 1/10 volume of sodium acetate solution. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

nucleotide sequence determination

To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers were used with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT (indicated in the 5 ^{^} → 3 direction, Y is C or T, H is C, T or A). The pieces of DNA obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR. The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones. From the respective clones, the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977). sequence analysis

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence by means of BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained next to the organism Archaeon clone 5.5ft C85A next Relatives can be assigned.

Isolation and accumulation of microorganisms

Microorganisms Methanosarcina acetivorans SBG19 were isolated under anaerobic conditions from the supernatant of material from a post-fermenter using a suitable selection medium (Medium 304 (DSMZ)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable culture medium (Medium 304 (DSMZ)), the bacteria Methanosarcina acetivorans SBG19 could be propagated.

Fermentation with the addition of microorganisms of the species Methanosarcina acetivorans

SBG 19

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire generated Biogas in standard liters (gas volume at 273.15 K and 1013 mbar) determined per day (Nl / d). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). The cell mass from 1 l of a preculture of Methanosarcina acetivorans SBG19 which had been incubated for 5 days at a temperature of 4O ⁰ C in medium 304 (DSMZ) was added to each.

A comparison of the amount of biogas produced showed that the addition of microorganisms Methanosarcina acetivorans SBG19 a significantly increased amount, but at least a 5% higher amount of biogas produced from a predetermined amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanosarcina acetivorans SBG19 was used. However, mixed cultures with a proportion of Methanosarcina acetivorans SBG 19 can also be used. In particular, mixed cultures may be added from two or three types of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides.

The use of microorganisms of the species Methanosarcina acetivorans leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanobacterium formicicum

Lanqzeit fermentations for Bioqasproduktion on pilot scale with or without the addition of microorganisms of the species Methanobacterium formicicum

The fermentations for biogas production with the addition of microorganisms of the species Methanobacterium formicicum were carried out in technical fermenters with 5I content.

The bacterial cell counts were determined by counting bacterial cultures or their respective dilutions in a Thoma counting chamber. By means of a live-dead-staining (Baclight Bacterial Viability Kit, Molecular Probes / Netherlands) the total living bacteria concentration in the fermenter was determined.

The Technikumsfermenter were operated at 40 ⁰ C, contained a vertical agitator and a gas meter from Ritter. The fermenters were fed once a day with corn silage. The maximum level of 5I was regulated by an overflow. After several weeks of start-up time, a relatively constant biogas production at a volume load of about 2.5 kg oTS / m ³ d could be achieved at a level of about 10 Nl / d in this type of fermenter. In FIGS. 1 to 3, the gas yield to biogas is indicated in each case. The methane content of the biogas produced was on average at 50 to 52%.

In Figure 1, starting from this state at a constant space load (reference numeral 10) of about 2.5 kg oTS / m ³ d was added on the day of operation 44 a bacterial culture of Methanobacterium formicicum with a cell count of about 1, 2 x 10 ¹¹ cells (Reference 50). It was found that the added amount of methane bacteria of the species Methanobacterium formicicum corresponded to a very low concentration of 0.03% of the total bacterial count in the fermenter. Nevertheless, it could be observed after the addition of methane bacteria of the species Methanobacterium formicicum in this small amount that the average gas yield was about 10.4 Nl / d until about day 65, while the theoretically calculated gas yield was expected at about 9.3 Nl / d, which corresponded to an increase in gas yield of almost 12%. The broken line with reference numeral 20 represents the daily measured gas yield, the black line with reference numeral 30 a measured gas yield averaged over 6 days, the black line with reference numeral 40 the theoretically expected value for the gas yield. On day 66, methanobacterium formicicum methane bacteria were again added at a concentration equivalent to 0.03% of the total bacterial count in the fermenter (reference numeral 60). Thereupon there was another slight increase in the gas yield to values between 10.5 and 1 1, 0 Nl / d, which, however, leveled off again at about 10.5 Nl / d from about the 85th day of operation.

This experiment showed that the addition of bacteria of the species Methanobacterium formicicum can increase the gas yield in excess of the theoretically expected value even in relatively small amounts under moderate room loads.

In another long-term fermentation, an attempt was made to increase the volume of space continuously. This experiment is shown in FIGS. 2A and 2B. In FIG. 2A, the curve with the reference numeral 10 indicates the volume load, the broken line with reference numeral 20 the daily measured gas yield, the black line with reference 30 a measured gas yield averaged over 6 days, the black line with reference numeral 40 the theoretically expected value for the gas yield and the rhombus symbols with reference number 50 each indicate an addition of bacteria of the species Methanobacterium formicicum. From day 151 to day 267, the amount of bacteria from a 500 ml pre-culture was added about 2 to 3 times a week, each having a concentration in the range between 4x10 ⁸ cells / ml to 8x10 ⁸ cells / ml. Starting at a volume load of 2.5 kg oTS / m ³ d on operating day 100, the load on the room was increased to a value of 5 kg oTS / m ³ d until about day 130, but had to be restored to a value of approx 3.0 kg oTS / m ³ d because the fermenter became unstable. On the one hand, this was shown by the decreasing gas yield from day 130, but in particular by the increased production of free fatty acids, which are an indicator of the state of the fermentation process. During the experiment gas chromatographic concentrations of the volatile fatty acids acetic acid, propionic acid, butyric acid, valeric acid and the equivalent of acetic acid were measured. FIG. 2B shows the measured values (stated in mg of free fatty acids per I fermenter content) for the acetic acid equivalent (reference number 20), acetic acid (reference number 30) and propionic acid (reference number 40). The measured values are each by open circles marked, the lines represent only an interpolation. With reference numeral 10 in addition the measured pH values are shown. As a rule of thumb, the values for the free fatty acids should preferably not exceed a value of about 1000 mg / l in a stable fermentation process. However, since this value was clearly exceeded from the 125th day of operation, the load on the room was reduced. Beginning with a volume load of 3.5 kg oTS / m ³ d, 151 bacteria of the species Methanobacterium formicicum were repeatedly added from the day of operation, whereby the space load was continuously increased to values up to 7.0 kg oTS / m ³ d on the operating day 250 without that the free fatty acids would have risen to critical levels well above 1000 mg / l. However, as the generated biogas yield at the very high volume load of 7 kg oTS / m ³ d fell below the theoretically expected value, the space load was reduced slightly to 6 kg oTS / m ³ d and could be kept stable for 2 weeks. However, when no more bacteria were added from Day 268 onwards, the fermentation could not be maintained at this high level of room load and crashed from Day 280, reflecting the rapid drop in gas production.

Simultaneously with the long-term fermentation described in FIGS. 2A and 2B, a control experiment without the addition of bacteria was carried out in an identical fermenter. This is shown in FIGS. 3A and 3B. FIG. 3A in turn describes the results for biogas production. The curve with the reference numeral 10 indicates the space load, the broken line with reference numeral 20 the daily measured gas yield, the black line with reference numeral 30 each measured over 6 days measured gas yield, the black line with reference numeral 40 the theoretically expected value for the gas yield , Figure 3B shows the results for acid production during the fermentation process. Reference numeral 20 denotes the acetic acid equivalent, 30 the acetic acid, and 40 the propionic acid. The measured values are each marked by the open circles, the lines represent only an interpolation. Reference numeral 10 additionally shows the measured pH values. Starting with a volume load of 3.0 kg oTS / m ³ d on operating day 80, the volume load was increased to a value of 5.5 kg oTS / m ³ d by day 100, but with an increase in acid production and a decrease the gas yield was significantly below the theoretically expected value, so that the space load was slightly reduced and could be maintained at values between 4.5 kg oTS / m ³ d and 5.0 kg oTS / m ³ d until the operating day 220. From day 220 onwards, it was again attempted to increase the volume load without addition of bacteria to 7 kg oTS / m ³ d, which failed and in a fermenter crash with extreme increase of the free fatty acids and a decrease of the pH value to values up to 5 , 5 went along. The experiments shown in Figures 2A, 2B, 3A and 3B showed that when adding bacteria of the species Methanobacterium formicicum significant increases in space load are possible without causing over acidification of the fermenter content or a collapse of biogas production. An addition of bacteria of the species Methanobacterium formicicum thus caused a significant stabilization of the fermentation process, so that the biogas production can be made more efficient eg by increasing the space load.

The use of microorganisms of the species Methanobacterium formicicum leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanobacterium formicicum SBG4

DNA isolation

Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol l ^-1 NaCl, 30-40 mg of the acid washed PVPP contained) and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). after lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml), the cells (20 ul of a 1% after the addition of protease by weight (w / v) Proteinase K solution) and SDS (100 μl of a 10% solution) were incubated for 45 min at 65 ^° C. The DNA was then mixed with one volume of phenol, with one volume of phenol: chloroform: isoamylalcohol (25: 24: 1, ( v / v / v)) and extracted with one volume of chloroform From the aqueous supernatant, after addition of 1/10 volume of a sodium acetate solution (3.0 M, pH 5.2), the DNA was treated with 0.8 volume of isopropanol 30 min at -20 ⁰ C. precipitated. The DNA was collected by centrifugation, washed with 70% ethanol twice and dried at room temperature in air.

Nucleotide sequence determination To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT were used (indicated in 5 ^' → 3Εichtung; Y is C or T; H is C, T or A). The pieces of DNA obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR. The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones. From the respective clones, the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence using BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained the organism Methanobacterium sp. Clone SMS-sludge-11 can be assigned as nearest relative.

Isolation and Enrichment of the Microorganisms Microorganisms Methanobacterium formicicum SBG4 were isolated under anaerobic conditions from the supernatant of material from a post-fermenter using an appropriate selection medium (Medium 1 19 (DSMZ) suitable for Methanobacterium formicium (DSMZ strain: 1535)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable culture medium (Medium 1 19 (DSMZ)), the bacteria Methanobacterium formicicum SBG4 could be propagated.

Fermentation with addition of microorganisms of the species Methanobacterium formicicum

SBG4

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire generated Biogas in standard liters (gas volume at 273.15 K and 1013 mbar) determined per day (NUd). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). In each case, the cell mass was added from 1 l of a preculture Methanobacterium formicicum SBG4 of which had been incubated for 5 days at a temperature of 4O ⁰ C in Medium 1 19 (DSMZ).

A comparison of the amount of biogas produced showed that with the addition of microorganisms Methanobacterium formicicum SBG4 a significantly increased amount, but at least a 5% higher amount of biogas produced from a given amount of organic dry matter is generated than without the addition of microorganisms.

In the described embodiment, a pure culture of Methanobacterium formicicum SBG4 was used. Likewise, mixed cultures with a proportion of Methanobacterium formicicum SBG4 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanobacterium formicicum leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanobacterium formicicum SBG8

DNA isolation

From the fermenter of a biogas plant substrate samples were taken and in a

Transferred Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol ^{I" 1} NaCl, 30-40 mg of the acid washed PVPP contained) and then frozen three times (-80 ⁰ C) and Thawed (at 56 ⁰ C). After lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml) the cells were counted after addition of protease (20 ul of a 1% (w / v) proteinase K solution) and SDS ( 100 ul of a 10% solution) for 45 min at 65 ⁰ C incubated. The DNA was then extracted with one volume of phenol, one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1, v / v / v), and one volume of chloroform. From the aqueous supernatant, after addition of 1/10 volume of a sodium acetate solution (3.0 M, pH 5.2), the DNA was treated with 0.8 volume of isopropanol for 30 min. At -20 ⁰ C like. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

Nucleotide sequence determination To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT were used (indicated in 5 ^' → 3 ^" direction, Y is C or T, H is C, T or A) and the DNA pieces obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-Drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones from which the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence using BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained the organism Methanobacterium sp. 169 can be assigned as nearest relatives.

Isolation and accumulation of microorganisms

Microorganisms Methanobacterium formicicum SBG8 were isolated under anaerobic conditions with the aid of a suitable selection medium (Medium 1 19 (DSMZ) suitable for Methanobacterium formicium (DSMZ strain: 1535)) from the supernatant of material from a post-fermenter. Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable culture medium (Medium 1 19 (DSMZ)), the bacteria Methanobacterium formicicum SSGS could be propagated.

Fermentation with addition of microorganisms of the species Methanobacterium formicicum SBG8

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average Room load of 3 to 3.5 kg oTS / m ³ d) was determined over a period of several months, the time course of the total generated biogas in standard liters (gas volume at 273.15 K and 1013 mbar) per day (NUd). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). The cell mass from 1 l of a preculture of Methanobacterium formicicum SBG8 which had been incubated for 5 days at a temperature of 4O ⁰ C in Medium 1 19 (DSMZ) was added to each.

A comparison of the amount of biogas produced showed that the addition of microorganisms Methanobacterium formicicum SBG8 a significantly increased amount, but at least a 5% higher amount of biogas produced from a given amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanobacterium formicicum SBG8 was used. However, mixed cultures with a proportion of Methanobacterium formicicum SBG8 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanobacterium formicicum leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanobacterium formicicum SBG25

DNA isolation

Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol ^{I" 1} NaCl, 30-40 mg of the acid washed PVPP contained) and then frozen three times (-80 ⁰ C) and Thawed (at 56 ⁰ C). After lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml) the cells were counted after addition of protease (20 ul of a 1% (w / v) proteinase Solution) and SDS (100 ul of a 10% solution) for 45 min at 65 ⁰ C incubated. The DNA was then extracted with one volume of phenol, one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1, v / v / v), and one volume of chloroform. From the aqueous supernatant (3.0 M, pH 5.2) the DNA was precipitated with 0.8 volume of isopropanol for 30 min. At -20 ⁰ C after the addition of 1/10 volume of sodium acetate solution. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

Nucleotide sequence determination To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT were used (indicated in 5 ^' → 3 ^" direction, Y is C or T, H is C, T or A) and the DNA pieces obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-Drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones from which the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence by means of BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained the next Archaeon clone GC-4 Relatives can be assigned.

Isolation and accumulation of microorganisms

Microorganisms Methanobacterium formicicum SBG25 were isolated under anaerobic conditions with the aid of a suitable selection medium (Medium 1 19 (DSMZ) suitable for Methanobacterium formicium (DSMZ strain: 1535)) from the supernatant of material from a post-fermenter. Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable culture medium (Medium 1 19 (DSMZ)), the bacteria Methanobacterium formicicum SBG25 could be propagated. Fermentation with addition of microorganisms of the species Methanobacterium formicicum SBG25

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire generated Biogas in standard liters (gas volume at 273.15 K and 1013 mbar) determined per day (Nl / d). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). In each case, the cell mass was added from 1 l of a preculture of Methanobacterium formicicum SBG25, which had been incubated for 5 days at a temperature of 40 ^ in medium 1 19 (DSMZ).

A comparison of the amount of biogas produced showed that with the addition of microorganisms Methanobacterium formicicum SBG25 a significantly increased amount, but at least a 5% higher amount of biogas produced from a given amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanobacterium formicicum SBG25 was used. Likewise, mixed cultures with a proportion of Methanobacterium formicicum SBG25 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanobacterium formicicum leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanosarcina barkeri SBG 16

DNA isolation Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a bench centrifuge) a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench top centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol l ^-1 NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). after lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml), the cells (20 ul of a 1% after the addition of protease strength (w / v) proteinase K solution) and SDS (100 μl of a 10% solution) for 45 min at 65 ^° C. The DNA was then mixed with one volume of phenol, with one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1). (v / v / v)) and extracted with one volume of chloroform From the aqueous supernatant, after addition of 1/10 volume of a sodium acetate solution (3.0 M, pH 5.2), the DNA was mixed with 0.8 volume of isopropanol 30 min. at -20 ⁰ C precipitated. The DNA was collected by centrifugation, washed with 70% ethanol twice and dried at room temperature in air.

nucleotide sequence determination

To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT were used (indicated in 5 ^' → 3 ^" direction, Y is C or T, H is C, T or A) and the DNA pieces obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-Drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones from which the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence by means of BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained the next organism Archaeon clone 5.5ft C140A Relatives can be assigned.

Isolation and accumulation of microorganisms Microorganisms Methanosarcina barkeri SBG 16 were isolated under anaerobic conditions from the supernatant of material from a post-fermenter using a suitable selection medium (Medium 120 (DSMZ)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 120 (DSMZ)), the bacteria Methanosarcina barkeri SBG16 could be propagated.

Fermentation with the addition of microorganisms of the species Methanosarcina barkeri

SBG16

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire generated Biogas in standard liters (gas volume at 273.15 K and 1013 mbar) determined per day (Nl / d). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). The cell mass from 1 l of a preculture of Methanosarcina barkeri SBG16 which had been incubated for 5 days at a temperature of 4O ⁰ C in medium 120 (DSMZ) was added to each.

A comparison of the amount of biogas produced showed that the addition of microorganisms Methanosarcina barkeri SBG16 a significantly increased amount, but at least a 5% higher amount of biogas produced from a predetermined amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanosarcina barkeri SBG16 was used. However, mixed cultures with a proportion of Methanosarcina barkeri SBG 16 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanosarcina barkeri leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanosarcina barkeri SBG 17 DNA isolation

Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol l ^-1 NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). after lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml), the cells (20 ul of a 1% after the addition of protease strength (w / v) proteinase K solution) and SDS (100 μl of a 10% solution) for 45 min at 65 ^° C. The DNA was then mixed with one volume of phenol, with one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1). (v / v / v)) and extracted with one volume of chloroform From the aqueous supernatant, after addition of 1/10 volume of a sodium acetate solution (3.0 M, pH 5.2), the DNA was mixed with 0.8 volume of isopropanol 30 min. at -20 ⁰ C precipitated. The DNA was collected by centrifugation, washed with 70% ethanol twice and dried at room temperature in air.

Nucleotide sequence determination To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT were used (indicated in 5 ^' → 3 ^" direction, Y is C or T, H is C, T or A) and the DNA pieces obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-Drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones from which the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence using BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained can be assigned to the organism Archaeon clone 5.5ft C17A as the nearest relative.

Isolation and Enrichment of Microorganisms Microorganisms Methanosarcina barkeri SBG 17 were isolated under anaerobic conditions from the supernatant of material from a post-fermenter using a suitable selection medium (Medium 120 (DSMZ)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 120 (DSMZ)), the bacteria Methanosarcina barkeri SBG17 could be increased.

Fermentation with addition of microorganisms of the species Methanosarcina barkeri SBG 17

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire generated Biogas in standard liters (gas volume at 273.15 K and 1013 mbar) determined per day (Nl / d). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). The cell mass from 1 l of a preculture of Methanosarcina barkeri SBG17 which had been incubated for 5 days at a temperature of 4O ⁰ C in medium 120 (DSMZ) was added to each.

A comparison of the amount of biogas produced showed that the addition of microorganisms Methanosarcina barkeri SSG77 a significantly increased amount, but at least a 5% higher amount of generated biogas from a predetermined amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanosarcina barkeri SBG17 was used. Likewise, however, mixed cultures with a share of

Methanosarcina barkeri SBG 17 can be used. In particular, mixed cultures of two or three types of microorganisms selected from the group consisting of

Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides are added.

The use of microorganisms of the species Methanosarcina barkeri leads to a significant

Improvement of efficiency and efficiency of biogas plants. Methanosarcina barkeri SBG18

DNA isolation

Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol ^{I" 1} NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). After lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml) the cells were counted after addition of protease (20 ul of a 1% (w / v) proteinase K solution) and SDS ( 100 ul of a 10% solution) for 45 min at 65 ⁰ C incubated. The DNA was then extracted with one volume of phenol, one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1, v / v / v), and one volume of chloroform. From the aqueous supernatant (3.0 M, pH 5.2) the DNA was precipitated with 0.8 volume of isopropanol for 30 min. At -20 ⁰ C after the addition of 1/10 volume of sodium acetate solution. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

nucleotide sequence determination

To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. The primers were used with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT (indicated in the 5 ^' → 3 direction, Y is C or T, H is C, T or A). The pieces of DNA obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR. The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones. From the respective clones, the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence by means of BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained the next organism Archaeon clone 5.5ft C38A Relatives can be assigned.

Isolation and Enrichment of Microorganisms Microorganisms Methanosarcina barkeri SBG 18 were isolated under anaerobic conditions from the supernatant of material from a post-fermenter using a suitable selection medium (Medium 120 (DSMZ)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 120 (DSMZ)) the bacteria Methanosarcina barkeri SBG 18 could be increased.

Fermentation with the addition of microorganisms of the species Methanosarcina barkeri

SBG18

During a fermentation process in a test fermenter with a volume of

150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average volume load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of all biogas produced in standard liters (gas volume at 273.15 K and 1013 mbar) per day (NUd). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). The cell mass from 1 l of a preculture of Methanosarcina barkeri SBG18 which had been incubated for 5 days at a temperature of 4O ⁰ C in medium 120 (DSMZ) was added to each.

A comparison of the amount of biogas produced showed that the addition of microorganisms Methanosarcina barkeri SBG18 a significantly increased amount, but at least a 5% higher amount of biogas produced from a predetermined amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanosarcina barkeri SBG18 was used. Likewise, however, mixed cultures with a share of

Methanosarcina barkeri SBG 18 can be used. In particular, mixed cultures of two or three types of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides are added.

The use of microorganisms of the species Methanosarcina barkeri leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanosarcina barkeri SBG33

DNA isolation

Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol l ^-1 NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). after lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml), the cells (20 ul of a 1% after the addition of protease strength (w / v) proteinase K solution) and SDS (100 μl of a 10% solution) for 45 min at 65 ^° C. The DNA was then mixed with one volume of phenol, with one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1). (v / v / v)) and extracted with one volume of chloroform From the aqueous supernatant, after addition of 1/10 volume of a sodium acetate solution (3.0 M, pH 5.2), the DNA was mixed with 0.8 volume of isopropanol 30 min. at -20 ⁰ C precipitated. The DNA was collected by centrifugation, washed with 70% ethanol twice and dried at room temperature in air.

nucleotide sequence determination

To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT were used (indicated in 5 ^' → 3 ^" direction, Y is C or T, H is C, T or A) and the DNA pieces obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-Drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones The corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

Sequence analysis Following sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed using the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence using BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained to the organism Archaeon clone ATB KM1219 as the closest relative can be assigned.

Isolation and accumulation of microorganisms

Microorganisms Methanosarcina barkeri SBG33 were isolated under anaerobic conditions from the supernatant of material from a post-fermenter using a suitable selection medium (Medium 120 (DSMZ)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 120 (DSMZ)), the bacteria Methanosarcina barkeri SBG33 could be propagated.

Fermentation with the addition of microorganisms of the species Methanosarcina barkeri SBG33

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire generated Biogas in standard liters (gas volume at 273.15 K and 1013 mbar) determined per day (NUd). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). The cell mass from 1 l of a preculture of Methanosarcina barkeri SBG33 which had been incubated for 5 days at a temperature of 4O ⁰ C in medium 120 (DSMZ) was added to each.

A comparison of the amount of biogas produced showed that with the addition of microorganisms Methanosarcina barkeri SBG33 a significantly increased amount, but at least a 5% higher amount of biogas produced from a predetermined amount of organic dry matter is generated than without the addition of microorganisms. In the described embodiment, a pure culture of Methanosarcina barkeri SBG33 was used. However, mixed cultures with a proportion of Methanosarcina barkeri SBG33 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanosarcina barkeri leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanosarcina barkeri SBG20

DNA isolation Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol l ^-1 NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). after lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml), the cells (20 ul of a 1% after the addition of protease strength (w / v) proteinase K solution) and SDS (100 μl of a 10% solution) for 45 min at 65 ^° C. The DNA was then mixed with one volume of phenol, with one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1). (v / v / v)) and extracted with one volume of chloroform From the aqueous supernatant, after addition of 1/10 volume of a sodium acetate solution (3.0 M, pH 5.2), the DNA was mixed with 0.8 volume of isopropanol 30 min. at -20 ⁰ C precipitated. The DNA was collected by centrifugation, washed with 70% ethanol twice and dried at room temperature in air.

nucleotide sequence determination

To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT were used (indicated in 5 ^' → 3 ^" direction, Y is C or T, H is C, T or A) and the DNA pieces obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-drive), transformed into E. coli (according to QIAGEN PCR-Cloning-Handbook) and examined by colony PCR. The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones. From the respective clones, the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence by means of BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained next to the organism Archaeon clone ATB-KM1253 next Relatives can be assigned.

Isolation and accumulation of microorganisms

Microorganisms Methanosarcina barkeri SBG20 were isolated from the supernatant of material from a post-fermenter under anaerobic conditions using a suitable selection medium (Medium 120 (DSMZ)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 120 (DSMZ)), the bacteria Methanosarcina barkeri SBG20 could be propagated.

Fermentation with the addition of microorganisms of the species Methanosarcina barkeri SBG20

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire generated Biogas in standard liters (gas volume at 273.15 K and 1013 mbar) determined per day (Nl / d). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). The cell mass from 1 l of a preculture of Methanosarcina barkeri SBG20 which had been incubated for 5 days at a temperature of 4O ⁰ C in medium 120 (DSMZ) was added to each. A comparison of the amount of biogas produced showed that the addition of microorganisms Methanosarcina barkeri SBG20 a significantly increased amount, but at least a 5% higher amount of biogas produced from a predetermined amount of organic dry matter is generated than without the addition of microorganisms.

In the described embodiment, a pure culture of Methanosarcina barkeri SBG20 was used. However, mixed cultures with a proportion of Methanosarcina barkeri SBG20 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanosarcina barkeri leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanosarcina barkeri SBG24

DNA isolation Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol ^{I" 1} NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). After lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml) the cells were counted after addition of protease (20 ul of a 1% (w / v) proteinase K solution) and SDS ( 100 ul of a 10% solution) for 45 min at 65 ⁰ C incubated. The DNA was then extracted with one volume of phenol, one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1, v / v / v), and one volume of chloroform. From the aqueous supernatant (3.0 M, pH 5.2) the DNA was precipitated with 0.8 volume of isopropanol for 30 min. At -20 ⁰ C after the addition of 1/10 volume of sodium acetate solution. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

nucleotide sequence determination To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT were used (indicated in 5 ^' → 3 ^" direction, Y is C or T, H is C, T or A) and the DNA pieces obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-Drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones from which the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence using BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained to the organism Archaeon clone MH1492 3E as the closest relative can be assigned.

Isolation and accumulation of microorganisms

Microorganisms Methanosarcina barkeri SBG24 were isolated from the supernatant of material from a post-fermenter under anaerobic conditions using a suitable selection medium (Medium 120 (DSMZ)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 120 (DSMZ)) the bacteria Methanosarcina barkeri SBG24 could be propagated.

Fermentation with the addition of microorganisms of the species Methanosarcina barkeri SBG24

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire generated Biogas in standard liters (gas volume at 273.15 K and 1013 mbar) determined per day (NUd). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once under multiple Addition (eg 2x weekly). The cell mass from 1 l of a preculture of Methanosarcina barkeri SBG24 which had been incubated for 5 days at a temperature of 4O ⁰ C in medium 120 (DSMZ) was added to each.

A comparison of the amount of biogas produced showed that the addition of microorganisms Methanosarcina barkeri SBG24 a significantly increased amount, but at least a 5% higher amount of biogas produced from a predetermined amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanosarcina barkeri SBG24 was used. However, mixed cultures with a proportion of Methanosarcina barkeri SBG24 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanosarcina barkeri leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanosarcina mazei SBG9

DNA isolation

Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol l ^-1 NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). after lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml), the cells (20 ul of a 1% after the addition of protease strength (w / v) proteinase K solution) and SDS (100 μl of a 10% solution) for 45 min at 65 ^° C. The DNA was then mixed with one volume of phenol, with one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1). (v / v / v)) and extracted with one volume of chloroform From the aqueous supernatant, after addition of 1/10 volume of a sodium acetate solution (3.0 M, pH 5.2), the DNA was mixed with 0.8 volume of isopropanol 30 minutes at -20 ⁰ C like. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

Nucleotide sequence determination To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT were used (indicated in 5 ^' → 3 ^" direction, Y is C or T, H is C, T or A) and the DNA pieces obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-Drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones from which the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977)

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence using BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained assigned to the organism Methanosarcina mazei Goe1 as the nearest relative can be.

Isolation and accumulation of microorganisms

Microorganisms Methanosarcina mazei SBG9 were isolated under anaerobic conditions from the supernatant of material from a post-fermenter using a suitable selection medium (Medium 318 (DSMZ)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 318 (DSMZ)), the bacteria Methanosarcina mazei SBG9 could be propagated.

Fermentation with addition of microorganisms of the species Methanosarcina mazei SBG9 During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a Period of several months, the time course of the total biogas produced in standard liters (gas volume at 273.15 K and 1013 mbar) per day (NUd) determined. The fermentation process went under otherwise identical conditions, especially under the same load on the fermenter, once without the addition of microorganisms from and once with multiple addition (eg 2x weekly). In each case, the cell mass from 1 l of a preculture of Methanosarcina mazei SBG9, which had been incubated for 5 days at a temperature of 40 ° C. in medium 318 (DSMZ), was added.

A comparison of the amount of biogas produced showed that the addition of microorganisms Methanosarcina mazei SBG9 a significantly increased amount, but at least a 5% higher amount of biogas produced from a predetermined amount of organic dry matter is generated than without the addition of microorganisms.

In the described embodiment, a pure culture of Methanosarcina mazei SBG9 was used. However, mixed cultures with a proportion of Methanosarcina mazei SBG9 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanosarcina mazei leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanothermobacter wolfeii SBG10

DNA isolation

Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol l ^-1 NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). after lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml), the cells (20 ul of a 1% after the addition of protease strength (w / v) proteinase K solution) and SDS (100 μl of a 10% solution) for 45 min at 65 ^° C. The DNA was then mixed with one volume of phenol, with one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1). (v / v / v)) and with one volume of chloroform extracted. From the aqueous supernatant (3.0 M, pH 5.2) the DNA was precipitated with 0.8 volume of isopropanol for 30 min. At -20 ⁰ C after the addition of 1/10 volume of sodium acetate solution. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

nucleotide sequence determination

To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers were used with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT (indicated in the 5 ^{^} → 3 direction, Y is C or T, H is C, T or A). The pieces of DNA obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR. The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones. From the respective clones, the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

Sequence analysis Following sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed using the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence using BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained are assigned to the organism Methanothermobacter wolfeii as the nearest relative can.

Isolation and accumulation of the microorganisms Methanothermobacter wolfeii SBGW were isolated under anaerobic conditions with the aid of a suitable selection medium (Medium 1 19 (DSMZ) suitable for Methanobacterium formicium (DSMZ strain: 1535)) from the supernatant of material from a post-fermenter. Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable culture medium (Medium 1 19 (DSMZ)), the bacteria Methanothermobacter wolfeii SBG10 could be propagated.

Fermentation with addition of microorganisms of the species Methanothermobacter wolfeii SBG 10 During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire generated Biogas in standard liters (gas volume at 273.15 K and 1013 mbar) determined per day (NUd). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). The cell mass from 1 l of a preculture of Methanothermobacter wolfeii SBG10 which had been incubated for 5 days at a temperature of 4O ⁰ C in Medium 1 19 (DSMZ) was added to each.

A comparison of the amount of biogas produced showed that the addition of microorganisms Methanothermobacter wolfeii SBG 10 a significantly increased amount, but at least a 5% higher amount of biogas produced from a predetermined amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanothermobacter wolfeii SBG10 was used. Likewise, however, mixed cultures with a proportion of Methanothermobacter wolfeii SBG 10 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanothermobacter wolfeii leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanobacterium oryzae SBG26

DNA isolation

Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol ^{I" 1} NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). After lysis of the cells (30 min incubation at 37 ⁰ C after addition of 20 .mu.l of a lysozyme solution of 10 mg / ml), the cells after addition of protease (20 .mu.l of a 1% (w / v) ProteinaseK solution) and SDS (100 .mu.l of a 10 % solution) for 45 min at 65 ⁰ C incubated. The DNA was then extracted with one volume of phenol, one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1, v / v / v), and one volume of chloroform. From the aqueous supernatant (3.0 M, pH 5.2) the DNA was precipitated with 0.8 volume of isopropanol for 30 min. At -20 ⁰ C after the addition of 1/10 volume of sodium acetate solution. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

nucleotide sequence determination

To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. The primers were used with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT (indicated in the 5 ^' → 3 direction, Y is C or T, H is C, T or A). The pieces of DNA obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR. The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones. From the respective clones, the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

Sequence analysis Following sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed using the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence using BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained assigned to the organism Bacterium clone HsA55fl as the nearest relative can be.

Isolation and accumulation of microorganisms

Microorganisms Methanobacterium oryzae SBG26 were suitable under anaerobic conditions with the help of a suitable selection medium (Medium 1 19 (DSMZ)

Methanobacterium formicium (DSMZ strain: 1535)) from the supernatant of material isolated from a post-fermenter. A further selection of the liquid cultures was carried out with the aid of Dilution series. By selecting a suitable culture medium (Medium 1 19 (DSMZ)), the bacteria Methanobacterium oryzae SBG26 could be propagated.

Fermentation with addition of microorganisms of the species Methanobacterium oryzae SBG26

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire generated Biogas in standard liters (gas volume at 273.15 K and 1013 mbar) determined per day (NUd). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). The cell mass from 1 l of a preculture of Methanobacterium oryzae SBG26 which had been incubated for 5 days at a temperature of 4O ⁰ C in Medium 1 19 (DSMZ) was added to each.

A comparison of the amount of biogas produced showed that with the addition of microorganisms Methanobacterium oryzae SBG26 a significantly increased amount, but at least a 5% higher amount of biogas produced from a predetermined amount of organic dry matter is generated than without the addition of microorganisms.

In the described embodiment, a pure culture of Methanobacterium oryzae SBG26 was used. Likewise, however, mixed cultures with a proportion of Methanobacterium oryzae SBG26 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanobacterium oryzae leads to a significant improvement in the efficiency and efficiency of biogas plants.

Archaeon sp. SBG21

DNA isolation

Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After removal of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a bench top centrifuge) a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench top centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol ^{I" 1} NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). After lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml) the cells were counted after addition of protease (20 ul of a 1% (w / v) proteinase K solution) and SDS ( 100 ul of a 10% solution) for 45 min at 65 ⁰ C incubated. The DNA was then extracted with one volume of phenol, one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1, v / v / v), and one volume of chloroform. From the aqueous supernatant (3.0 M, pH 5.2) the DNA was precipitated with 0.8 volume of isopropanol for 30 min. At -20 ⁰ C after the addition of 1/10 volume of sodium acetate solution. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

nucleotide sequence determination

To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers were used with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT (indicated in the 5 ^{^} → 3 direction, Y is C or T, H is C, T or A). The pieces of DNA obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR. The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones. From the respective clones, the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

Sequence analysis Following sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed using the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence by means of BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained assigned to the organism Archaeon clone C26A as the closest relative can be. Isolation and accumulation of microorganisms

Microorganisms Archaeon sp. SBG21 were isolated under anaerobic conditions with the aid of a suitable selection medium (Medium 1 19 (DSMZ) suitable for Methanobacterium formicium (DSMZ strain: 1535)) from the supernatant of material from a post-fermenter. Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable culture medium (Medium 1 19 (DSMZ)), the bacteria Archaeon sp. SBG21 are propagated.

Fermentation with addition of microorganisms of the species Archaeon sp. SBG21 During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average volume load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire produced biogas in standard liters (gas volume at 273.15 K and 1013 mbar) per day (NUd) determined. The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). In each case, the cell mass was added from 1 l of a preculture of Archaeon sp. SBG21, which had been incubated for 5 days at a temperature of 40 ^ in medium 1 19 (DSMZ).

A comparison of the amount of biogas produced showed that when microorganisms were added, Archaeon sp. SBG21 a significantly increased amount, but at least a 5% higher amount of generated biogas from a predetermined amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Archaeon sp. SBG21 used. Likewise, however, mixed cultures with a proportion of Archaeon sp. SBG21 can be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Archaeon sp. SBG21 leads to a significant improvement in the efficiency and efficiency of biogas plants.

Archaeon sp. SBG22 DNA isolation

Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol ^{I" 1} NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). After lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml) the cells were counted after addition of protease (20 ul of a 1% (w / v) proteinase K solution) and SDS ( 100 ul of a 10% solution) for 45 min at 65 ⁰ C incubated. The DNA was then extracted with one volume of phenol, one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1, v / v / v), and one volume of chloroform. From the aqueous supernatant (3.0 M, pH 5.2) the DNA was precipitated with 0.8 volume of isopropanol for 30 min. At -20 ⁰ C after the addition of 1/10 volume of sodium acetate solution. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

nucleotide sequence determination

To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers were used with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT (indicated in the 5 ^{^} → 3 direction, Y is C or T, H is C, T or A). The pieces of DNA obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR. The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones. From the respective clones, the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA with the program package ARB (Ludwig et al., Nucleic Acids

Research. 2004, 32, 1363-1371) are analyzed phylogenetically. An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence by means of BLAST The program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained can be assigned to the organism Archaeon clone DF86 as the nearest relative.

Isolation and accumulation of microorganisms

Microorganisms Archaeon sp. SBG22 were isolated under anaerobic conditions from the supernatant of material from a post-fermenter using a suitable selection medium (Medium 1 19 (DSMZ) suitable for Methanobacterium formicium (DSMZ strain: 1535)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable culture medium (Medium 1 19 (DSMZ)), the bacteria Archaeon sp. SBG22 be propagated.

Fermentation with addition of microorganisms of the species Archaeon sp. SBG22 During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average volume load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire produced biogas in standard liters (gas volume at 273.15 K and 1013 mbar) per day (NUd) determined. The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). In each case, the cell mass was added from 1 l of a preculture of Archaeon sp. SBG22, which had been incubated for 5 days at a temperature of 40 ^ in medium 1 19 (DSMZ).

A comparison of the amount of biogas produced showed that when microorganisms were added, Archaeon sp. SBG22 a significantly increased amount, but at least a 5% higher amount of generated biogas from a predetermined amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Archaeon sp. SBG22 used. Likewise, however, mixed cultures with a proportion of Archaeon sp. SBG22 can be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Archaeon sp. SBG22 leads to a significant improvement in the efficiency and efficiency of biogas plants. Methanobacterium subterraneum SBG35

DNA isolation

Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol ^{I" 1} NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). After lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml) the cells were counted after addition of protease (20 ul of a 1% (w / v) proteinase K solution) and SDS ( 100 ul of a 10% solution) for 45 min at 65 ⁰ C incubated. The DNA was then extracted with one volume of phenol, one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1, v / v / v), and one volume of chloroform. From the aqueous supernatant (3.0 M, pH 5.2) the DNA was precipitated with 0.8 volume of isopropanol for 30 min. At -20 ⁰ C after the addition of 1/10 volume of sodium acetate solution. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

nucleotide sequence determination

To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. The primers were used with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT (indicated in the 5 ^' → 3 direction, Y is C or T, H is C, T or A). The pieces of DNA obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR. The resulting colony PCR products were subjected to restriction fragment length polymorphism analysis (RFLP) to select suitable clones. From the respective clones, the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence using BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained the organism Methanobacterium sp. Clone SMS-sludge-11 can be assigned as nearest relative.

Isolation and Enrichment of Microorganisms Microorganisms Methanobacterium subterraneum SBG35 were isolated under anaerobic conditions from the supernatant of material from a post-fermenter using a suitable selection medium (Medium 814 (DSMZ)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 814 (DSMZ)), the bacteria Methanobacterium subterraneum SBG35 could be propagated.

Fermentation with addition of microorganisms of the species Methanobacterium subterraneum SBG35

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire generated Biogas in standard liters (gas volume at 273.15 K and 1013 mbar) determined per day (Nl / d). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). The cell mass from 1 l of a preculture of methanobacterium subterraneum SBG35 that had been incubated for 5 days at a temperature of 40 ^<€ 814 in medium (DSMZ) was added to each.

A comparison of the amount of biogas produced showed that the addition of microorganisms Methanobacterium subterraneum SBG35 a significantly increased amount, but at least a 5% higher amount of biogas produced from a predetermined amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanobacterium subterraneum SBG35 was used. Likewise, mixed cultures with a proportion of Methanobacterium subterraneum SBG35 can also be used. In particular, you can Mixed cultures of two or three species of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanobacterium subterraneum leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanobacterium subterraneum SBG34

DNA isolation

Substrate samples were taken from the fermenter of a biogas plant and transferred to an Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol ^{I" 1} NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). After lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml) the cells were counted after addition of protease (20 ul of a 1% (w / v) proteinase K solution) and SDS ( 100 ul of a 10% solution) for 45 min at 65 ⁰ C incubated. The DNA was then extracted with one volume of phenol, one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1, v / v / v), and one volume of chloroform. From the aqueous supernatant (3.0 M, pH 5.2) the DNA was precipitated with 0.8 volume of isopropanol for 30 min. At -20 ⁰ C after the addition of 1/10 volume of sodium acetate solution. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

Nucleotide sequence determination To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this purpose, the primers were used with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT (indicated in the 5 ^{^} → 3 direction, Y is C or T, H is C, T or A). The pieces of DNA obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-drive), transformed into E. coli (according to QIAGEN PCR cloning Handbook) and examined by colony PCR. The resulting colony PCR products were subjected to restriction fragmentation. Length Polymorphism Analysis (RFLP) to select suitable clones. From the respective clones, the corresponding plasmid DNA was isolated and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence using BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained assigned to the organism Bacterium clone HsA55fl as the nearest relative can be.

Isolation and Enrichment of Microorganisms Microorganisms Methanobacterium subterraneum SBG34 were isolated under anaerobic conditions from the supernatant of material from a post-fermenter using a suitable selection medium (Medium 814 (DSMZ)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 814 (DSMZ)), the bacteria Methanobacterium subterraneum SBG34 could be propagated.

Fermentation with addition of microorganisms of the species Methanobacterium subterraneum SBG34

During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a period of several months, the time course of the entire generated Biogas in standard liters (gas volume at 273.15 K and 1013 mbar) determined per day (Nl / d). The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). In each case, the cell mass from 1 l of a preculture of Methanobacterium subterraneum SBG34, which had been incubated for 5 days at a temperature of 40 ° C. in medium 814 (DSMZ), was added.

A comparison of the amount of produced biogas showed that with the addition of microorganisms Methanobacterium subterraneum SBG34 a significantly increased amount, but at least one 5% higher amount of biogas produced from a given amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanobacterium subterraneum SBG34 was used. However, mixed cultures with a proportion of Methanobacterium subterraneum SBG34 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanobacterium subterraneum leads to a significant improvement in the efficiency and efficiency of biogas plants.

Methanobacterium aarhusense SBG36

DNA isolation

From the fermenter of a biogas plant substrate samples were taken and in a

Transferred Eppendorf reaction vessel. After separation of undigested plant material by centrifugation (30 s, 3000 rpm (revolutions per minute) in a table centrifuge), a cell pellet was recovered by centrifugation (30 min, 13000 rpm in a bench centrifuge). The cells were resuspended in 1,300 ul salinem extraction buffer (0.1 mol I ^"1 EDTA, 0.15 mol ^{I" 1} NaCl, 30-40 mg of the acid washed PVPP contained), and then frozen three times (-80 ⁰ C) and thawed (at 56 ⁰ C). After lysis of the cells (30 min incubation at 37 ⁰ C after the addition of 20 ul of a lysozyme solution of 10 mg / ml) the cells were counted after addition of protease (20 ul of a 1% (w / v) proteinase K solution) and SDS ( 100 ul of a 10% solution) for 45 min at 65 ⁰ C incubated. The DNA was then extracted with one volume of phenol, one volume of phenol: chloroform: isoamyl alcohol (25: 24: 1, v / v / v), and one volume of chloroform. From the aqueous supernatant (3.0 M, pH 5.2) the DNA was precipitated with 0.8 volume of isopropanol for 30 min. At -20 ⁰ C after the addition of 1/10 volume of sodium acetate solution. The DNA was collected by centrifugation, washed twice with 70% ethanol and dried in air at room temperature.

nucleotide sequence determination

To determine the 16S rRNA sequence, the gene for the 16S rRNA was amplified from the isolated DNA by PCR. For this, the primer with the sequences TCYGGTTGATCCTGCC and ACGG HTACCTTGTT ACG ACTT (indicated in 5 ^' → 3 ^" direction, Y is C or T, H is C, T or A).) The DNA pieces obtained as PCR products were then cloned into a cloning vector (ligation with the QIAGEN PCR cloning kit from QIAGEN / Hilden using the vector p-Drive), transformed into E. coli (according to QIAGEN PCR-Cloning-Handbook) and examined by colony-PCR The obtained colony PCR products were The corresponding plasmid DNA was isolated from the respective clones and sequenced by the chain termination method (Sanger et al., 1977).

sequence analysis

After sequence analysis of the colonies, the 16S rDNA or its transformation into the corresponding 16S rRNA could be phylogenetically analyzed with the program package ARB (Ludwig et al., Nucleic Acids Research., 2004, 32, 1363-1371). An analysis of the cloned 16S rDNA or the corresponding 16S rRNA sequence using BLAST program (basic local alignment search tool) of the database www.ncbi.nlm.nih.gov showed that a sequence obtained assigned to the organism Bacterium clone HsA55fl as the nearest relative can be.

Isolation and accumulation of microorganisms

Microorganisms Methanobacterium aarhusense SBG36 were isolated under anaerobic conditions from the supernatant of material from a post-fermenter using a suitable selection medium (Medium 141 (DSMZ)). Further selection of the liquid cultures was carried out with the aid of dilution series. By selecting a suitable nutrient medium (Medium 141 (DSMZ)), the bacteria Methanobacterium aarhusense SBG36 could be propagated.

Fermentation with addition of microorganisms of the species Methanobacterium aarhusense SBG36 During a fermentation process in a test fermenter with a volume of 150 l under realistic plant conditions (temperature ~ 40 ⁰ C, average space load of 3 to 3.5 kg oTS / m ³ d) was over a Period of several months, the time course of the total biogas produced in standard liters (gas volume at 273.15 K and 1013 mbar) per day (Nl / d) determined. The fermentation process was carried out under otherwise identical conditions, in particular under the same volume loading of the fermenter, once without the addition of microorganisms and once with multiple addition (eg twice a week). In each case, the cell mass was added from 1 l of a preculture of Methanobacterium aarhusense SBG36 which had been incubated for 5 days at a temperature of 4O ⁰ C in medium 141 (DSMZ).

A comparison of the amount of biogas produced showed that when adding microorganisms Methanobacterium aarhusense SBG36 a significantly increased amount, but at least a 5% higher amount of biogas produced from a predetermined amount of organic dry matter is generated as without the addition of microorganisms.

In the described embodiment, a pure culture of Methanobacterium aarhusense SBG36 was used. However, mixed cultures with a proportion of Methanobacterium aarhusense SBG36 can also be used. In particular, mixed cultures of two or three kinds of microorganisms selected from the group consisting of Paenibacillus macerans, Clostridium sartagoformum and Clostridium sporosphaeroides can be added.

The use of microorganisms of the species Methanobacterium aarhusense leads to a significant improvement in the efficiency and efficiency of biogas plants.

Claims

claims 1. A process for the production of biogas from biomass in a fermentation reactor, characterized in that the biomass is added to a microorganism of the species Methanoculleus bourgensis. 2. The method according to claim 1, characterized in that the microorganism of the species Methanoculleus bourgensis is added in the form of a culture of microorganisms, wherein microorganisms of the species Methanoculleus bourgensis make up at least 10 ^'4 % of the total number of microorganisms present in the culture. 3. The method according to claim 2, characterized in that in the culture of microorganisms microorganisms of the species Methanoculleus bourgensis make up at least 10 ^{" 2} % of the total number of microorganisms present in the culture. 4. The method according to claim 3, characterized in that in the culture of microorganisms microorganisms of the species Methanoculleus bourgensis make up at least 1% of the total number of microorganisms present in the culture. 5. The method according to claim 4, characterized in that in the culture of microorganisms microorganisms of the species Methanoculleus bourgensis make up at least 10% of the total number of microorganisms present in the culture. 6. The method according to claim 5, characterized in that in the culture of microorganisms microorganisms of the species Methanoculleus bourgensis make up at least 50% of the total number of microorganisms present in the culture. 7. The method according to claim 6, characterized in that in the culture of microorganisms microorganisms of the species Methanoculleus bourgensis make up at least 75% of the total number of microorganisms present in the culture. 8. The method according to claim 7, characterized in that in the culture of microorganisms microorganisms of the species Methanoculleus bourgensis make up at least 90% of the total number of microorganisms present in the culture. 9. The method according to at least one of claims 2 to 8, characterized in that a pure culture of the microorganism of the species Methanoculleus bourgensis is added. 10. The method according to at least one of claims 2 to 9, characterized in that microorganisms of the species Methanoculleus bourgensis the biomass are added as part of at least one immobilized culture of microorganisms. 1 1. Method according to at least one of claims 1 to 10, characterized in that in time for the addition of the microorganism of the species Methanoculleus bourgensis additional biomass is added to the fermentation reactor. 12. The method according to at least one of claims 1 to 1 1, characterized in that the space load in the fermentation reactor is continuously increased by the continuous addition of biomass. 13. The method according to at least one of claims 1 to 12, characterized in that the production of biogas from biomass at a space load of ≥ 0.5 kg oTS / m ³ d, preferably ≥ 4.0 kg oTS / m ³ d, especially preferably ≥ 8.0 kg oTS / m ³ d. 14. The method according to at least one of claims 1 to 13, characterized in that the production of biogas from biomass takes place under constant mixing of the fermentation substrate. 15. The method according to at least one of claims 1 to 14, characterized in that the production of biogas from biomass at a temperature of 2O ⁰ C to 80 ⁰ C, preferably at a temperature of 40 ⁰ C to 5O ⁰ C is performed. 16. The method according to at least one of claims 1 to 15, characterized in that the addition of fermentation substrate and the addition of the microorganism of the species Methanoculleus bourgensis carried out continuously. 17. The method according to at least one of claims 1 to 16, characterized in that the microorganism of the species Methanoculleus bourgensis in an amount to the Fermentation substrate is added that after addition of the proportion of the microorganism of the species Methanoculleus bourgensis constitutes at present in the fermentation substrate of microorganisms between ^{10 -8%} and 50% of the total. 18. The method according to claim 17, characterized in that the microorganism of the species Methanoculleus bourgensis is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanoculleus bourgensis between 10 ^"6 % and 25% of the total in Fermentation substrate present Constitutes microorganisms. 19. The method according to claim 18, characterized in that the microorganism of the species Methanoculleus bourgensis is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanoculleus bourgensis between 10 ^'4 % and 10% of the total in Fermentation substrate present microorganisms. 20. The method according to claim 19, characterized in that the microorganism of the species Methanoculleus bourgensis is added in an amount to the fermentation substrate, that after addition of the proportion of the microorganism of the species Methanoculleus bourgensis between 10 ^'3 % and 1% of the total in Fermentation substrate present microorganisms. 21. Process according to at least one of Claims 1 to 20, characterized in that the microorganism of the species Methanoculleus bourgensis is a microorganism of the strain Methanoculleus sp. dm2 acts. 22. The method according to at least one of claims 1 to 20, characterized in that it is a microorganism of the strain Archaeon clone GZK7 in the microorganism of the species Methanoculleus bourgensis. 23. The method according to at least one of claims 1 to 20, characterized in that it is a microorganism of the strain Archaeon clone 5.5ft C121A in the microorganism of the species Methanoculleus bourgensis. 24. The method according to at least one of claims 1 to 20, characterized in that it is in the microorganism of the species Methanoculleus bourgensis to a Microorganism of the strain Archaeon clone 5.5ft C124A acts. 25. The method according to at least one of claims 1 to 20, characterized in that it is the microorganism of the species Methanoculleus bourgensis to a microorganism of the strain Archaeon clone 5.5Ü C141A. 26. The method according to at least one of claims 1 to 20, characterized in that it is the microorganism of the species Methanoculleus bourgensis to a microorganism of the strain Archaeon clone A52. 27. The method according to at least one of claims 1 to 20, characterized in that it is in the microorganism of the species Methanoculleus bourgensis to a Microorganism of the strain Archaeon clone MCSArc_B6 acts. 28. The method according to at least one of claims 1 to 20, characterized in that it is the microorganism of the species Methanoculleus bourgensis a microorganism of the strain Euryarchaeote clone BSA1A-03. 29. The method according to at least one of claims 1 to 20, characterized in that it is the microorganism of the species Methanoculleus bourgensis to a microorganism of the strain Euryarchaeote clone BSA2A-02. 30. The method according to at least one of claims 1 to 20, characterized in that it is the microorganism of the species Methanoculleus bourgensis to a microorganism of the strain Euryarchaeote clone MAA04. 31. Use of a microorganism of the species Methanoculleus bourgensis for the fermentative production of biogas from biomass. 32. Use according to claim 31, characterized in that it is a microorganism of the strain Methanoculleus sp. dm2 acts. 33. Use according to claim 31, characterized in that the microorganism of the species Methanoculleus bourgensis is a microorganism of the strain Archaeon clone GZK7. 34. Use according to claim 31, characterized in that it is a microorganism of the strain Archaeon clone 5.5ft C121A in the microorganism of the species Methanoculleus bourgensis. 35. Use according to claim 31, characterized in that the microorganism of the species Methanoculleus bourgensis is a microorganism of the strain Archaeon clone 5.5ft C124A. 36. Use according to claim 31, characterized in that the microorganism of the species Methanoculleus bourgensis is a microorganism of the Tribe Archaeon clone 5.5ft C141A is traded. 37. Use according to claim 31, characterized in that the microorganism of the species Methanoculleus bourgensis is a microorganism of the strain Archaeon clone A52. 38. Use according to claim 31, characterized in that the microorganism of the species Methanoculleus bourgensis is a microorganism of the strain Archaeon clone MCSArc_B6. 39. Use according to claim 31, characterized in that it is the microorganism of the species Methanoculleus bourgensis a microorganism of the strain Euryarchaeote clone BSA1A-03. 40. Use according to claim 31, characterized in that it is the microorganism of the species Methanoculleus bourgensis a microorganism of the strain Euryarchaeote clone BSA2A-02. 41. Use according to claim 31, characterized in that the microorganism of the species Methanoculleus bourgensis is a microorganism of the Tribal Euryarchaeote clone MAA04 acts. 42. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 97.31% sequence identity with the nucleotide sequence SEQ ID No. 4. 43. The microorganism according to claim 42, characterized in that the nucleotide sequence contains a sequence region which has more than 98.0% sequence identity with the nucleotide sequence SEQ ID No. 4. 44. The microorganism according to claim 43, characterized in that the nucleotide sequence contains a sequence region which has more than 98.5% sequence identity with the nucleotide sequence SEQ ID No. 4. 45. The microorganism according to claim 44, characterized in that the nucleotide sequence contains a sequence region which has more than 99.0% sequence identity with the nucleotide sequence SEQ ID No. 4. 46. The microorganism according to claim 45, characterized in that the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 4. 47. Microorganism according to claim 46, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 4. 48. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 97.54% sequence identity with the nucleotide sequence SEQ ID No. 9. 49. The microorganism according to claim 48, characterized in that the nucleotide sequence contains a sequence region which has more than 98.0% sequence identity with the nucleotide sequence SEQ ID No. 9. 50. The microorganism according to claim 49, characterized in that the nucleotide sequence contains a sequence region which has more than 98.5% sequence identity with the nucleotide sequence SEQ ID No. 9. 51. Microorganism according to claim 50, characterized in that the nucleotide sequence contains a sequence region which has more than 99.0% sequence identity with the nucleotide sequence SEQ ID No. 9. 52. The microorganism according to claim 51, characterized in that the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 9. 53. Microorganism according to claim 52, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID NO. 9 corresponds. 54. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 98.04% sequence identity with the nucleotide sequence SEQ ID No. 10. 55. The microorganism according to claim 54, characterized in that the nucleotide sequence contains a sequence region which has more than 98.2% sequence identity with the nucleotide sequence SEQ ID No. 10. 56. The microorganism according to claim 55, characterized in that the nucleotide sequence contains a sequence region which has more than 98.5% sequence identity with the nucleotide sequence SEQ ID No. 10. 57. The microorganism according to claim 56, characterized in that the nucleotide sequence contains a sequence region which has more than 99.0% sequence identity with the nucleotide sequence SEQ ID No. 10. 58. The microorganism according to claim 57, characterized in that the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 10. 59. Microorganism according to claim 58, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID NO. 10 corresponds. 60. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 97.53% sequence identity with the nucleotide sequence SEQ ID No. 11. 61. Microorganism according to claim 60, characterized in that the nucleotide sequence contains a sequence region which has more than 98.0% sequence identity with the nucleotide sequence SEQ ID No. 11. 62. The microorganism according to claim 61, characterized in that the nucleotide sequence contains a sequence region which has more than 98.5% sequence identity with the nucleotide sequence SEQ ID No. 11. 63. The microorganism according to claim 62, characterized in that the nucleotide sequence contains a sequence region which has more than 99.0% sequence identity with the nucleotide sequence SEQ ID No. 11. 64. The microorganism according to claim 63, characterized in that the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 11. 65. Microorganism according to claim 64, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 11. 66. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 96.88% sequence identity with the nucleotide sequence SEQ ID No. 12. 67. The microorganism according to claim 66, characterized in that the nucleotide sequence contains a sequence region which has more than 97.5% sequence identity with the nucleotide sequence SEQ ID No. 12. 68. The microorganism according to claim 67, characterized in that the nucleotide sequence contains a sequence region which has more than 98.0% sequence identity with the nucleotide sequence SEQ ID No. 12. 69. The microorganism according to claim 68, characterized in that the nucleotide sequence contains a sequence region which has more than 99.0% sequence identity with the nucleotide sequence SEQ ID No. 12. 70. The microorganism according to claim 69, characterized in that the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 12. 71. Microorganism according to claim 70, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 12. 72. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 99.1% sequence identity with the nucleotide sequence SEQ ID No. 28. 73. The microorganism according to claim 72, characterized in that the nucleotide sequence contains a sequence region which has more than 99.3% sequence identity with the nucleotide sequence SEQ ID No. 28. 74. The microorganism according to claim 73, characterized in that the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 28. 75. The microorganism according to claim 74, characterized in that the nucleotide sequence contains a sequence region which has more than 99.7% sequence identity with the nucleotide sequence SEQ ID No. 28. 76. The microorganism according to claim 75, characterized in that the nucleotide sequence contains a sequence region which has more than 99.9% sequence identity with the nucleotide sequence SEQ ID No. 28. 77. Microorganism according to claim 76, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID NO. 28 corresponds. 78. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 95.82% sequence identity with the nucleotide sequence SEQ ID No. 27. 79. The microorganism according to claim 78, characterized in that the nucleotide sequence contains a sequence region which has more than 96.0% sequence identity with the nucleotide sequence SEQ ID No. 27. 80. The microorganism according to claim 79, characterized in that the nucleotide sequence contains a sequence region which has more than 97.0% sequence identity with the nucleotide sequence SEQ ID No. 27. 81. Microorganism according to claim 80, characterized in that the nucleotide sequence contains a sequence region which has more than 98.0% sequence identity with the nucleotide sequence SEQ ID No. 27. 82. The microorganism according to claim 81, characterized in that the nucleotide sequence contains a sequence region which has more than 99.0% sequence identity with the nucleotide sequence SEQ ID No. 27. 83. Microorganism according to claim 82, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 27. 84. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 95.88% sequence identity with the nucleotide sequence SEQ ID No. 20. 85. The microorganism according to claim 84, characterized in that the nucleotide sequence contains a sequence region which has more than 96.0% sequence identity with the nucleotide sequence SEQ ID No. 20. 86. The microorganism according to claim 85, characterized in that the nucleotide sequence contains a sequence region which has more than 97.0% sequence identity with the nucleotide sequence SEQ ID No. 20. 87. The microorganism according to claim 86, characterized in that the nucleotide sequence contains a sequence region which has more than 98.0% sequence identity with the nucleotide sequence SEQ ID No. 20. 88. The microorganism according to claim 87, characterized in that the nucleotide sequence contains a sequence region which has more than 99.0% sequence identity with the nucleotide sequence SEQ ID No. 20. 89. Microorganism according to claim 88, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 20. 90. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 99.63% sequence identity with the nucleotide sequence SEQ ID No. 25. 91. Microorganism according to claim 90, characterized in that the nucleotide sequence contains a sequence region which has more than 99.65% sequence identity with the nucleotide sequence SEQ ID No. 25. 92. Microorganism according to claim 91, characterized in that the nucleotide sequence contains a sequence region which has more than 99.7% sequence identity with the nucleotide sequence SEQ ID No. 25. 93. The microorganism according to claim 92, characterized in that the nucleotide sequence contains a sequence region which has more than 99.8% sequence identity with the nucleotide sequence SEQ ID No. 25. 94. The microorganism according to claim 93, characterized in that the nucleotide sequence contains a sequence region which has more than 99.9% sequence identity with the nucleotide sequence SEQ ID No. 25. 95. Microorganism according to claim 94, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID NO. 25 corresponds. 96. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 99.26% sequence identity with the nucleotide sequence SEQ ID No. 26. 97. The microorganism according to claim 96, characterized in that the nucleotide sequence contains a sequence region which has more than 99.3% sequence identity with the nucleotide sequence SEQ ID No. 26. 98. The microorganism according to claim 97, characterized in that the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 26. 99. Microorganism according to claim 98, characterized in that the nucleotide sequence contains a sequence region which has more than 99.7% sequence identity with the nucleotide sequence SEQ ID No. 26. 100. Microorganism according to claim 99, characterized in that the nucleotide sequence contains a sequence region which has more than 99.9% sequence identity with the nucleotide sequence SEQ ID No. 26. 101. Microorganism according to claim 100, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 26. 102. A microorganism comprising a nucleic acid having a nucleotide sequence, characterized in that the nucleotide sequence contains a sequence region which has more than 97.54% sequence identity with the nucleotide sequence SEQ ID No. 29. 103. Microorganism according to claim 102, characterized in that the nucleotide sequence contains a sequence region which has more than 98.0% sequence identity with the nucleotide sequence SEQ ID No. 29. 104. The microorganism according to claim 103, characterized in that the nucleotide sequence contains a sequence region which has more than 98.5% sequence identity with the nucleotide sequence SEQ ID No. 29. 105. The microorganism according to claim 104, characterized in that the nucleotide sequence contains a sequence region which has more than 99.0% sequence identity with the nucleotide sequence SEQ ID No. 29. 106. The microorganism according to claim 105, characterized in that the nucleotide sequence contains a sequence region which has more than 99.5% sequence identity with the nucleotide sequence SEQ ID No. 29. 107. Microorganism according to claim 106, characterized in that the nucleotide sequence contains a sequence region which corresponds to the nucleotide sequence SEQ ID No. 29. 108. Culture of microorganisms suitable for use in a process for the fermentative production of biogas from biomass, characterized in that in the culture of microorganisms, a microorganism according to claims 42 to 107, wherein the microorganism accounts for at least 10 ^{~ 4} % of the total number of microorganisms present in the culture. 109. Culture of microorganisms according to claim 108, characterized in that the microorganism constitutes at least 10 ^"2 % of the total number of microorganisms present in the culture. 1 10. Culture of microorganisms according to claim 109, characterized in that the microorganism at least 1% of the total number of existing in the culture Constitutes microorganisms. 1 1 1. Culture of microorganisms according to claim 1 10, characterized in that the microorganism constitutes at least 10% of the total number of microorganisms present in the culture. 1 12. Culture of microorganisms according to claim 1 1 1, characterized in that the microorganism constitutes at least 50% of the total number of microorganisms present in the culture. 1 13. Culture of microorganisms according to claim 1 12, characterized in that the microorganism constitutes at least 90% of the total number of microorganisms present in the culture. 1 14. Culture of microorganisms according to claim 1 13, characterized in that it is a pure culture of a microorganism according to claims 42 to 107. 1 15. Culture of microorganisms according to at least one of claims 108 to 1 14, characterized in that it is an immobilized culture of microorganisms. 1 16. Use of a microorganism according to at least one of claims 42 to 107 for the fermentative production of biogas from biomass. 1 17. Use according to claim 1 16, characterized in that it is a method for the fermentative production of biogas from biomass according to at least one of claims 1 to 30. 1 18. Use of a culture of microorganisms according to at least one of claims 108 to 1 15 for the fermentative production of biogas from biomass. 1 19. Use according to claim 1 18, characterized in that it is a method for the fermentative production of biogas from biomass biomass according to at least one of claims 1 to 30 is.