WO2015104031A1

WO2015104031A1 - Disruption of biomass by endogenous pressure generation from fermentation

Info

Publication number: WO2015104031A1
Application number: PCT/EP2014/000050
Authority: WO
Inventors: Wolfgang H. SCHWARZ; Vladimir V. ZVERLOV
Original assignee: Individual
Current assignee: Individual
Priority date: 2014-01-10
Filing date: 2014-01-10
Publication date: 2015-07-16
Anticipated expiration: 2016-07-10

Abstract

The present invention relates to a method of disrupting biomass by an abrupt pressure release. For disrupting biomass, in particular as pretreatment of biomass for improved degradability, in an environmentally friendly manner, without harsh chemical substances and with reduced energy input and costs, the inventive method performs the steps of fermenting the biomass with a microorganism or with a consortium of microorganisms producing gas in a vessel, said gas increasing the pressure in the vessel; and disrupting the biomass by abruptly releasing the increased pressure.

Description

Disruption of biomass by endogenous pressure generation from fermentation

The present invention relates to a method of disrupting biomass by an abrupt pressure release.

Biomass, such as plant biomass is a renewable source of carbon compounds for liquid and gaseous biofuels as well as for platform chemicals for the chemical industry. It can be sustainably produced, making it a valuable source of energy in a "carbon free" future, in which our energy and material needs do not add to the already overstressed C0₂ burden of the atmosphere. Many technologies for biomass utilization, fuel or chemical production and biotransformation are based on fermentation, utilizing the sugar content of biomass. These sugars are not only found in the soluble sugars of plant tissues, but also in the starch and the cellulose and hemicellulose of stalks and stems, as well as in residual materials from the food, forestry and agricultural industries. Sugars are an indispensable source for biological fermentation substrates, whose large-scale implementation can supplement the hitherto petrochemical-based production of gaseous and liquid fuels and bulk chemicals. While such processes form the basis for sustainable production pathways, biomass degradation is still too slow, too inefficient and therefore too expensive.

Efficiency and economy of biotechnology depend to a large degree on the availability of plant biomass, which can be greatly enhanced by increasing its degradability, i.e. through plant cell wall depolymerization and degradation. Degradation processes consist of two parts: substrate pretreatment and biochemical or thermochemical depolymerization. Among these technologies, degradation by exogenous enzymes (industrial saccharification) and also degradation by hydrolytic bacteria in integrated processes (such as consolidated bioprocessing) are hampered by the physical inaccessibility of the inner parts of the insoluble and partially crystalline cell wall material, by the heterogeneity of its composition (cellulose, hemicellulose, pectin and many others) and by its chemical derivatization (e.g. esterification). Various technical methods have been used to disrupt the biomass, i.e. disintegrate or destroy the compound cell wall material as a pretreatment of biomass for improving its degradability. The first step usually involves chopping the branches, leaves, stems etc. to a size of a few centimeters for improved storability, compressibility and transportability. For certain processes pretreatment begins with impregnation with acids or alkali. One widely used process is "cooking" with or without addition of chemicals or solvents, using usually pressure from 8 to 15 bar followed by instant expansion (steam explosion). This leads to disruption of the compound cell wall which makes the fibers and its constituent polymers more accessible to degradation by hydrolytic enzymes and also may dissolve a part of the polysaccharide material. The concept of what is now known as steam explosion was documented in US Patent No. 1 ,578,609 by Mason, in which he describes an invention for disintegrating lignocellulosic material by subjecting it to an aqueous gas mixture (hot steam at temperatures of about 180°C to 240°C) at pressures of at least 275 psi (about 19 bar) but preferably in the range of 400 to 600 psi (about 28 to 41 bar). The gas is then expanded via a small orifice in the pressure vessel, forcing the gas and materials out of the vessel and causing them to explode.

Subsequent advances and current State of the Art with regard to steam explosion techniques are summarized by Stelte, W. ('Steam explosion for Biomass Pre-treatment', Danish Technological Institute, Taastrup, Denmark, 2013) in a recent report. These include several mechanical advancements, such as variations between batch and continuous processes, as well as chemical changes due to pressure, temperature and chemical additives and the effects thereof on the lignocellulosic products.

WO 2009/068875 refers to a method for using pressurized steam to disrupt eukaryotic cells, and also mentions utilizing certain additives such as ammonia and sulfuric acid, among others, to facilitate the degradation of organic matter, in this case with an aim to sterilize the cell-containing substrate.

US 2009/221814 discusses a continuous steam explosion process in which hydrolysis occurs in the pressurized stage of the steam explosion process. The reactor receives flows of one or more of a mild acid, sulfur dioxide gas (S0₂), oxygen, compressed air, ammonia, water, water vapor, steam (for heating and maintaining temperature) and catalyzing agents, with the purpose of promoting hydrolysis and providing pressurization.

WO 2006/032282 relates to a hydrolysis process within a pressure vessel including the addition of chemicals (extreme pH values) and/or heat above 80 °C.

The impregnation of substrates with acids prior to and facilitating steam explosion has been documented by Zimbardi, et al. (Zimbardi, F., Viola, E., Nanna, F., Larocca, E., Cardinale, M., & Barisano, D. 'Acid impregnation and steam explosion of corn stover in batch processes', Industrial Crops and Products, Volume 26, Issue 2, 195-206, 2007), and Wang, et al. (Wang, L, Fan, X., Tang, P., & Yuan, Q., 'Xylitol fermentation using hemicellulose hydrolysate prepared by acid pre-impregnated steam explosion of corncob', Journal of Chemical Technology and Biotechnology, Volume 88, Issue 1 1 , pages 2067-2074, November 2013).

The use of pressurized carbon dioxide to rupture cells has been documented and is discussed by Zheng, et al. (Zheng, Y., Lin, H., & Tsao, G., Pretreatment for Cellulose Hydrolysis by Carbon Dioxide Explosion, West Lafayette: Purdue University, 1998). The use of C0₂ to disrupt cells as a mechanism for sterilization was also explored, among others by Nakamura, et al. ('Disruption of Microbial Cells by the Flash Discharge of High-Pressure Carbon Dioxide', Bioscience, Biotechnology, and Biochemistry, Volume 58, Issue 7, 1297-1301 , 1998). All of the above technologies used for pretreatment and disruption of biomass are hampered by high energy costs (for heating) or material costs (for acids and alkali) which constitute a considerable part of the operating costs for the production of biofuels or platform chemicals in second generation white biotechnology. Every reduction in operation costs is a step toward the mass-scale introduction of second generation fuels and base chemicals and the host of benefits it will provide the global economy.

The technical problem to be solved by the present invention is thus to provide a method of disrupting biomass, in particular as pre-treatment of biomass for improved degradability, that is environmentally friendly, avoids harsh chemical substances such as acids and alkali, while at the same time reducing the energy costs and thus reducing the operational costs. The method of disrupting biomass according to the present invention solves this technical problem by the steps of fermenting the biomass with a microorganism or with a consortium of microorganisms producing gas in a vessel, said gas increasing the pressure in the vessel; and disrupting the biomass by abruptly releasing the increased pressure.

The build-up of the gas that is endogenously produced by the microorganism (in the following, the expression "microorganism" also refers to a consortium of microorganisms, unless explicitly stated otherwise) during the fermentation causes the pressure in the vessel to rise, and leads to an even distribution of the gases in the biomass, such as for example plant tissue slurry among others, between the cell wall constituents. When the pressure is abruptly released, the dissolved gas rapidly expands and mechanically disrupts the cell walls and other biological material, making e.g. the plant cell wall components better accessible to enzymatic degradation by the activity of hydrolytic bacteria and enzymes. Particularly, the non-covalent association of cellulose and the hemicellulose-lignin layer in primary and secondary cell walls are disrupted thereby, making the cellulose much more readily accessible for depolymerizing enzymes. Unlike steam explosion processes needing hot steam of 180°C and more, no excessive energy input is required for disrupting the biomass according to the present invention because the gases are produced internally, principally without external supply of energy (except the low to moderate heat used to initially heat up the fermentation process and to keep the temperature at the appropriate value). This environmentally friendly and heat efficient way of disrupting biomass reduces the operating costs for biomass pre-treatment. The use of aggressive chemicals like acid or alkali is not necessary. Contrary thereto, in the prior art, several additives are used to facilitate hydrolysis of the substrate, and these substances are added at pressure and high temperatures. The prior art 5 does not mention the role of biological processes in contributing to the pressure build-up.

US patent application no. 2007/224669 A1 relates to a process whereby anaerobic digestion of biomass is used to generate methane gas inside a vessel. However, the pressure is mechanically down-regulated by releasing the gas, contrary to the inventive principle of using pressure generated from bacteria to cause mechanical damage to materials.

I0 The above-mentioned solution according to the present invention may be combined in any way with any one of the following advantageous embodiments of the present invention respectively and thus further improved.

According to a first embodiment, the microorganism or the consortium of microorganisms produces gas, preferably C0₂, during growth and fermentation on the biomass. This way of 15 internally producing gas by fermentation on the biomass reduces the necessity to add expensive substrates for growth of the microorganism, thereby further improving the economy of the inventive disruption method.

According to a further embodiment, the microorganism, or at least one or preferably all of the microorganisms in the consortium of microorganisms, comprises at least one of the following

!O characteristics: anaerobic or facultatively anaerobic; saccharolytic; mesophilic or thermophilic, preferably having a growth optimum between 20 and 85 °C, most preferably between 50 and 65 °C; hydrolytic; capability of producing organic acids or short-chain fatty acids, as well as optionally alcohols during the fermentation; growth at a pressure of 5 bar, preferably of 10 bar, more preferably of 15 bar and most preferably of 25 bar. For example, anaerobic hydrolytic

!5 bacteria produce sugars with their secreted hydrolytic enzymes and ferment them together with anaerobic and facultatively anaerobic fermentative saccharolytic bacteria to carbonic acids, alcohols and gases, such as C0₂ and H₂. These bacteria can be highly pressure resistant, growing at a pressure of 5 bar or even at 15 bar or 25 bar. In a closed vessel they can generate high pressures from the gasses that they produce. Pressure values beyond 15 bar have been

!O observed by the inventors in fermentation. The produced acids lower the pH which can shift the C0₂-hydrogencarbonate equilibrium towards C0₂ gas increasing the gas pressure further. By moderately increasing the fermentation temperature from ambient temperature up to 80°C, preferably to 37°C to 65°C, and most preferably around 60 °C, a number of positive effects conducive to the degradation process and handling of the biomass slurry occur: the solubility of C0₂ is decreased (only 4 g of C0₂ are soluble in 1 liter of water at 10 bar and 60 °C and hence the gas pressure is increased), the viscosity is decreased, and the degradation velocity is increased. The resulting high pressure can be used for abrupt expansion to efficiently disrupt the cell wall components mechanically. According to a further embodiment, the microorganism or the consortium of microorganisms, (i) is derived from a sample from a biofuel plant, an environmental sample, rotten biomass, compost, a digestate, a fermentative residue, a bioprocess liquid and/or a sewage plant; and/or (ii) comprises a member of the phyla Firmicutes, Fibrobacteres, Proteobacteria, Thermotogae and Bacteroidetes, preferably a member of the order Clostridiales, Thermobacterales, Halanaerobiales, Bacillales, or Thermotogae. The appropriate microorganism or consortium of microorganisms for fermenting the biomass, e.g. a specific culture of selected microorganism, can be individually chosen depending on the biomass to be disrupted, whereas the microorganism/consortium of microorganisms from group (i) above is a waste-/by-product that is cheap, abundant and easy to obtain. Members of (ii) above have shown to be efficient saccharolytic bacteria forming gas and acid during fermentation at moderate temperatures of between 20 to 85°C, preferably between 50 and 65°C, which is far below the energy required for heating a sample to above 180°C, as required for conventional steam explosion.

A "biofuel plant" is any facility producing liquid and/or gaseous fuel, such as for example hydrogen or methane or ethanol or another alcohol containing energy produced, from living organisms. The term "biomass" refers to any biological material derived from living or recently living organisms. It encompasses, among others, plants or plant-derived materials. An "environmental sample" is any sample taken from surroundings or an organism or population of organisms, such as soil or residues from food, forestry or agriculture for example. "Rotten biomass", also referred to as biodegraded biomass, is the residue from dissolution of material by bacteria or other biological means. A "digestate" is the material remaining after the anaerobic or facultatively anaerobic digestion of a biodegrable feedstock/sample. A "fermentative residue" is any substance resulting from a fermentation process, except for the fermentative product. A "fermentative residue" for example encompasses the filter cake of a fermentation product or the fermentative liquid after the product has been purified therefrom. A "bioprocess liquid" is any liquid resulting from a bioprocess, i.e. a process that uses complete living cells or their components to obtain a desired product, like for example remaining fermentation broth.

According to a further embodiment, the pressure is abruptly released after the pressure in the vessel is increased above 5 bar, preferably above 10 bar, and most preferably above 15 bar. These high pressures ensure that the endogenous gas produced upon fermentation by the microorganism/consortium of microorganisms dissolved in the biomass expands rapidly upon pressure release and thereby mechanically disrupts the biomass.

According to a further embodiment, the pressure is released from near the bottom of the vessel. This ensures that the vessel can be completely emptied without moving it (e.g. by tilting) since the release opening is at the bottom. Further, the slurry situated at the bottom of the vessel and not the gas phase is ejected with full pressure, using the gas phase above the slurry. In another embodiment, the produced gas may be harvested. The produced gas may be used for energy generation, biological and/or chemical conversion, like C0₂ or H₂ for example. If hydrogen gas is among the gases produced, this can be used for energy generation. Other gases, such as for example C0₂, may be used as an educt in a conversion reaction. A chemical conversion may also be referred to as chemical transformation of a substrate/educt to a product in a chemical process/chemical reaction. A biological conversion is similar thereto, with the exception that the transformation does not occur chemically, but rather in a biological reaction using living matter or components of living matter such as for example enzymes. According to a further embodiment, the disrupted biomass is, in a downstream process, (i) degraded enzymatically and/or chemically, preferably by enzymes produced during the fermentation, (ii) fed into a biofuel fermentation process, (iii) subject to biorefinery and/or (iv) subject to chemical conversion. The microorganisms used in the fermentation may also be partially disrupted by the pressure release. The enzymes produced by the microorganism, however, such as depolymerizing enzymes like ligninolytic, proteolytic, peptolytic, lipidolytic, amylolytic, pectinolytic, hemicellulolytic, glycolytic and cellulolytic or other depolymerizing enzymes are still active and may be used to enzymatically degrade the disrupted biomass. "Biorefinery" refers to the process occurring in the facility that integrates biomass conversion processes and equipment to produce fuels, power, heat and chemicals, like for example biodiesel, bioethanol or for example lactic acid, succinic acid, etc. An "upstream process" is any process occurring in a facility prior to the method of disrupting the biomass, whereas a downstream process is any process step occurring subsequent to, i.e. following, the disruption of the biomass.

In a further embodiment, the biomass is mechanically broken before fermenting it. The biomass may be mechanically broken by grinding and/or milling. This way, the biomass is pre-disrupted and thereby made more readily accessible for an even distribution of the gases produced by the microorganism during fermentation. The biomass may be mechanically broken into pieces of 1 cm to 20 cm, preferably into pieces of 2 cm to 10 cm, measured along the longest dimension of the broken biomass. According to a further embodiment, the biomass may be mashed with liquid producing a slurry, preferably a slurry having a dry mass content of 0.1 to 40 % by weight, most preferably of 1 to 30 % by weight, before fermenting it. A slurry having such dry mass content has been shown to be particularly suited as a biomass medium for the fermentation. The fermentation temperature may be from 15°C to 80°C, preferably 37°C to 65°C. The optimum temperature inter alia depends on the microorganism used, and could be around the optimum growth temperature.

According to a further embodiment, the liquid used for mashing and producing the slurry may be water, a watery process liquid or a fermentation digestate. The process liquid or digestate may be from an upstream and/or downstream process of the disrupted biomass in the whole process of pre-treatment and subsequent production of e.g. biofuels or bio-refinery products or platform chemicals.

The biomass may be inoculated with a process liquid or digestate comprising the microorganism or the consortium of microorganisms from a downstream process or with a fermentative residue from a previous fermentation comprising the microorganism or the consortium of microorganisms. This way, the inoculation may occur during the mashing, when using a process liquid or digestate. Further, the inoculum is already adapted to the fermentation of the biomass to be disrupted, when being derived from a downstream process.

According to a further embodiment, a buffering agent may be present in the fermentation. The buffering agent improves the fermentation process by keeping the pH in a range optimal for fermentation. The buffering agent may be a carbonate. Carbonates improve the production of C0₂ because acids produced upon fermentation lower the pH and shift the carbonate equilibrium to C0₂ gas, which further increases the amount of gas formed and thus the pressure inside the vessel.

According to a further embodiment, the fermentation may be performed in a batch or in a continuous process mode, or in a mode of removing a part of the disrupted biomass at intervals and replacing the removed part with new biomass.

The invention will be described in more detail by way of example hereinafter with reference to the accompanying drawings, which illustrate advantageous embodiments, as well as by reference to experiments. The described embodiments and experiments are only possible configurations in which the individual features may, however, as described above, be implemented independently of each other, or be omitted. Corresponding elements illustrated in the drawings are provided with the same reference signs. In the drawings:

Fig. 1 is a schematic representation of the inventive process;

Fig. 2 shows chaffed maize-hay silage used for the experiments;

Fig. 3 depicts an embodiment of a vessel, in which the inventive method is conducted;

Fig. 4 shows a glass bottle of similar geometry to the pressure vessel of Fig. 3 for the pressure- less control experiments;

Fig. 5 shows cellulolytic bacteria, species Clostridium thermocellum, free and attached to a plant fibre; and

Fig. 6 is a diagram showing the effect of expansion on the degree of degradation.

Fig. 1 is a schematic representation of one embodiment of the method according to the present invention. Fig. 1 shows the following:

1) the grinding/milling step,

2) the mashing step,

3) fermentation including the pressure measurement,

4) the fixed volume vessel under which self-pressurizing fermentation occurs,

5) the expansion and

6) the expansion vessel.

For degradation and gas build-up, the biomass material may, in the first step, be ground or milled, for example to a size from 1 cm to 20 cm. These biomass chips or fibres are then mixed, e.g. with water or watery process liquids, biogas fermenter digestate or any other suitable watery mixture which allows for bacterial growth, to a dry mass content of 0.1 to 40 %, preferably 1 to 30 % (w/v) in mashing step 2. This mixture (or slurry) is introduced into the reaction vessel V.

The vessel V comprises a manometer M at the headstock, as well as an expansion line with an expansion value EV at the bottom of the vessel. Through the expansion line, the disrupted biomaterial is released into an expansion vessel E.

The biomass slurry may be filled into the pressure resistant vessel V (containing a manometer device M) in the two following modes:

1. Batch process: the vessel V is filled shortly below the rim; after pressure has risen sufficiently, the vessel V expands its content through an opening or a EV valve for complete expansion. The opening may be placed at or near the lowest point of the vessel V.

Continuous process: a suitable mechanical device presses new slurry into the vessel V, whereas the pressure is regulated by letting small parts of the content repeatedly through a suitable device for rapid expansion. The geometry of the vessel V and the geometry of the inlet port, and/or a technical device ensure sufficient mixing and average residential time for the biomass.

The slurry or mash may be inoculated with suitable bacteria which are selected for optimal gas production from ground lignocellulosic biomass. The selection of optimal bacteria is performed dependent on the substrate to be used. Alternatively, liquid digestate from a biomass fermenter downstream of the production line may be used for inoculation. Alternatively, the vessel is run continuously and does not need inoculation. Alternatively, the vessel is not emptied completely nor cleaned to ensure inoculation with a remaining part of the hydrolytic and fermentative bacteria. Bacteria suitable for inoculation are, for example, saccharolytic bacteria which form gas and acids during fermentation, such as members of the Clostridium, Ruminococcus, Thermoanaerobacter genera, as well as other fermentative, anaerobic or facultatively anaerobic bacteria. Preferably, thermophilic bacteria with a growth optimum between 50 and 65 °C are used, such as C. thermocellum, C. stercorarium, C. cellulosi, Th. thermohydrolsulfuricum and other bacteria with a high potential to degrade the oligo- or polysaccharides in plant biomass and to metablise them to gases.

In one embodiment, mesophilic or thermophilic hydrolytic bacteria can be inoculated into a closed vessel with resistance to pressures over 25 bar. This vessel may be kept at the appropriate temperature for optimal growth of the bacteria (from 15 to 75 °C, preferably 37 to 65 °C). The bacteria begin to grow by metabolising the easily digestible sugars and polysaccharides of the biomass, degrading the polysaccharides and fermenting the resulting sugars to predominately organic acids, alcohols and the gasses C0₂ and H₂. Depolymerizing enzymes such as the ligninolytic, proteolytic, peptolytic, lipidolytic, amylolytic, pectinolytic, hemicellulolytic, glycolytic and cellulolytic or other depolymerizing enzymes may be produced by the microorganism and/or added to the fermentation medium.

If appropriate, the built-up gas may be released from the head space. It may be used for energy production or chemical conversion. The vessel may include a redundant safety device in case the pressure regulation mechanism is defective or the substrate outlet is blocked. When a sufficiently high pressure, e.g. at least 5 bar, or at least 15 bar, has built up to the gas production by the microorganism inside the vessel, the pressure is abruptly released via the expansion valve EV and the disrupted biomass is released into the expansion vessel E.

If the downstream process is a biogas process or another fermentation process requiring 5 specific anaerobic conditions, it can be ensured that the expansion occurs into a closed expansion vessel E to avoid contamination with oxygen and to use the fermentation gasses (especially the hydrogen gas) for the production of biogas.

Although the bacteria may also be partially disrupted by the abrupt expansion, the produced enzymes are still active. These enzymes - and/or externally produced enzymes or enzyme I0 producing microorganisms added in addition - can now more effectively degrade the plant biomass for the production of sugars. These can be used in situ, in a separate reaction or in a chain of reactions for fermentative or chemical processes. Alternatively the internal constituents of bacterial cells or other constituents of the biomass can be used for biotechnological or research purposes (biorefinery).

15 In the following, experiments conducted are described as exemplarily illustrating the present invention.

Methods

50 g of maize-hay silage (50 % wet w/w each, chaffed to 2 mm with a disk mill) (see Fig. 2) was incubated in a pressure vessel V equipped with an expansion valve EV and a manometer M.

»0 The structure of the silage is shown in Fig. 2. The added liquid volume was 450 ml of water (containing 50 mM MOPS buffer, pH 7.0) and 50 ml of a thermophilic anaerobic bacterial culture, added under semi-oxygen-free conditions (flushing with C0₂ gas). The bacterial culture was selected to degrade biomass silage efficiently under production of hydrolysis gas; it was derived from a biogas plant by repeated passaging with cellulose as carbon source and consisted of

!5 about 40% of Clostridium thermocellum.

The pressure vessel V was incubated at 60 °C and the pressure was read on the manometer M until the pressure reached at least 10 bar (Fig. 3). Under the laboratory conditions described above, a pressure above 10 bar was reached after 3 days.

The content was released after the time indicated by opening the 8 mm valve EV (head over) iO and expanding the pressurized content into a bucket. Samples of the solid material (fibers) were washed through a fine mesh with water, dried for weight determination at 80 °C overnight and incubated with commercial cellulase preparations used in industrial applications for degradation of plant biomass. Incubation conditions were as per indications by the manufacturer. Representative samples of the material before and after incubation were dried at 80 °C overnight and weighed. The difference between the two measurements (before and after enzyme treatment) was taken as the degree of degradation. Concurrent samples were prepared and incubated for control in an unpressurized bottle of similar geometry, containing a gas filter for permanent release of produced gases (Fig. 4). Control experiments without addition of bacteria were performed where appropriate. The experiment was repeated with different lengths of incubation under pressure, as well as different incubation times with various enzyme concentrations. All measurement points were performed in duplicate.

Results

After 3 days incubation at 60 °C, hydrolytic gas (mainly C0₂) was accumulated due to the metabolic activity of the bacteria and pressures of more than 10 bar were consistently achieved despite a headspace of 1/5^th volume. The quantity of gas produced depended on the volume, the substrate and the incubation conditions and, as such, significantly higher pressures (approaching 20 bar) may be achieved under conditions of different cultures and, particularly, larger vessel volumes, smaller headspace to total volume ratios, and substrates containing more accessible sugars (data not shown). The bacteria could grow and ferment, and the enzymes produced were active under the high pressures achieved. Fig. 5 shows cellulolytic bacteria, species Clostridium thermocellum, free and attached to a plant fiber. The enzymes can support the enzymatic after-treatment in a downstream process. The inactivation rate of living bacteria due to the fast expansion will depend dramatically on the expansion technology used (in particular on the geometry of the expansion opening and the built up pressure) and was not tested with the small equipment used in this experiment. A large proportion of the bacteria can be expected to survive the treatment and could be used as inoculation for a next fermentation round.

The 2 mm pieces of plant biomass are disrupted upon sudden pressure release by the fast expansion of the gas bubbles developing within the plant cells and between structural features of the plant cell walls, and in addition by the sheering forces during the fast movement through the narrow expansion valve. This disruption allows better access of the various enzymes to their specific target substrates. This leads thus to a higher velocity and efficiency of enzymatic degradation. The enzymatic degradation of the pressure-pretreated material was up to 33 % more efficient than with the non-pressurized pretreated material (see Fig. 6). Thus, the treated sample was degraded by far better, and more hardly degradable material has been hydrolyzed.

The conditions for enzymatic hydrolysis were the standard (optimal) conditions suggested by the manufacturer (fungal enzymes from Trichoderma reesei): 0.5 % treated substrate fibers (w/w dry), 90 mkg enzyme, and 20 h incubation at 50 °C. The enzymatic reaction was carried out in reaction buffer (0.1 M MES, pH 5.0, 10 mM CaCI₂). The pressure-treated substrate was degraded to 4.8 % in this measurement. Longer enzymatic incubation leads to a higher degree of degradation, but also to reduced increases in degradability by the enzyme (27 % for 40 h). Similar results were obtained with higher enzyme loads, albeit with higher substrate degradation degrees. The results indicate unequivocally a higher accessibility of the substrate fibers to the enzymes when plant biomass is disrupted by the pressure-expansion pretreatment.

The rate of degradation depends on the biomass load (w/v), the enzymatic treatment time and the enzyme load. These parameters must be adjusted to the technology and substrate used, to the bacteria in the inoculation, to the incubation temperature, and to other parameters of the process.

Claims

Method of disrupting biomass by the steps of:

fermenting the biomass with a microorganism or with a consortium of microorganisms producing gas in a vessel, said gas increasing the pressure in the vessel; and

disrupting the biomass by abruptly releasing the increased pressure.

Method of claim 1 , wherein the microorganism or the consortium of microorganisms produces gas, preferably C0₂, during growth and fermentation on biomass.

Method of claim 1 or 2, wherein the microorganism or at least one, preferably all of the microorganisms in the consortium of microorganisms comprises at least one of the following characteristics:

anaerobic or facultatively anaerobic,

saccharolytic,

mesophilic or thermophilic, preferably having a growth optimum between

20 and 85 °C, most preferably between 50 and 65 °C,

hydrolytic,

capability of producing organic acids or short-chain fatty acids, as well as optionally alcohols during the fermentation,

growth at a pressure of 5 bar, preferably of 10 bar, more preferably of 15 bar and most preferably of 25 bar.

Method of any one of claims 1 to 3, wherein the microorganism or the consortium of microorganisms:

(i) is derived from a sample from a biofuel plant, an environmental sample, rotten biomass, compost, a digestate, a fermentative residue, a bioprocess liquid and/or sewage plant; and/or

(ii) comprises a member of the phyla Firmicutes, Fibrobacteres, Proteobacteria, Thermotogae and Bacteroidetes, preferably a member of the order Clostridiales, Thermobacterales, Halanaerobiales, Bacillales, or Thermotogae.

5. Method of any one of claims 1 to 4, wherein the biomass is inoculated with a process liquid or digestate comprising the microorganism or the consortium of microorganisms from a downstream process or with a fermentative residue from a previous fermentation comprising the microorganism or the consortium of microorganisms.

5

6. Method of any one of claims 1 to 5, wherein the pressure is abruptly released after the pressure in the vessel increased above 5 bar, preferably above 10 bar, and most preferably above 15 bar.

10 7. Method of any one of claims 1 to 6, wherein the pressure is released from or near the bottom of the vessel.

8. Method of any one of claims 1 to 7, wherein the produced gas is harvested.

15 9. Method of claim 8, wherein the harvested gas is used for energy generation, biological and/or chemical conversion.

10. Method of any one of claims 1 to 9, wherein the disrupted biomass is, in a downstream process, (i) degraded enzymatically and/or chemically, preferably by enzymes produced 0 during the fermenting step, (ii) fed into a biofuel fermentation process, (iii) subject to biorefinery and/or (iv) subject to chemical conversion.

1 1. Method of any one of claims 1 to 10, wherein the biomass is mechanically broken before fermenting it, preferably by grinding and/or milling into pieces of 1 cm to 20 cm,

15 preferably into pieces of 2 cm to 10 cm.

12. Method of any one of claims 1 to 1 1 , wherein the biomass is mashed with liquid producing slurry, preferably a slurry having a dry mass content of 0.1 to 40 % (w/v) most preferably of 1 to 30 % (w/v), before fermenting it.

JO

13. Method of claim 12, wherein the liquid is water, a watery process liquid or a fermenter digestate, said process liquid or digestate preferably being from an upstream and/or downstream process of the disrupted biomass.

$5 14. Method of any one of claims 1 to 13, wherein a buffering agent is present in the fermentation, said buffering agent preferably being a carbonate. Method of any one of claims 1 to 14, wherein the fermentation is performed in a batch or in a continuous process mode, or in a mode of removing a part of the disrupted biomass at intervals and replacing the removed part with new biomass.