MXPA00006211A - Bacterial nitrite oxidizer and method of use thereof - Google Patents

Abstract

The present invention provides an isolated bacterial strain capable of oxidizing nitrite to nitrate and a method of use thereof for preventing or alleviating the accumulation of nitrite in an aqueous medium.

Description

BACTERIAL NITRITE OXIDIZER AND METHOD OF USE BACKGROUND OF THE INVENTION 1. Field of the invention The invention relates generally to a nitrite oxidant and specifically to a novel bacterium capable of oxidizing nitrite to nitrate. 2. Background information The oxidation of nitrate to nitrite by oxidative bacteria nitrite qui iolitoautotróficas (NOB) in fish culture systems, extended from the home aquarium to commercial aquaculture systems, is an important process. The accumulation of high concentrations of nitrite, toxic to fish and other aquatic organisms, is prevented by an active nitrite remover by nitrifying microorganisms. Nitrite is formed in aquarium systems of ammonium oxidation, the main nitrogenous waste of teleosts, by the autotrophic ammonium oxidizing bacterium (AOB). Thus, closed aquatic filtration systems usually provide a solid substrate, confined to a biological filter or biofilter, which promotes the growth of AOB and NOB. A variety of materials can form the substrate of a biofilter, ranging from gravel to specially designed molded plastics. The biofilters can be submerged in the flow path of the filtration system, or it can be located so that the water distills or leaks through a medium located in the atmosphere outside the aquarium, before flowing back into the tank.

Traditionally, it was considered that the bacteria responsible for the oxidation of ammonium and nitrite in the aquarium were Ni trosomas europaea and Ni trobacter winogradskyi, or their close relatives, respectively (Heaton, F. W. 1977. Aquacultural Enginbeering.

Wiley & Sons, Inc. New York; Wheaton, F. W., J.

Hochhei er, and G. E. Kaiser. 1991. Fixed film nitrification in filters for aquaculture, p. 272-303. In D.E. Bruñe and J. R. Tomasso (eds.) Aquaculture and Water Quality. The World Aquaculture Society, Baton Rouge, LA.). However, there are many indications that both Ni trochos europaea and Ni trobacter winogradskyi can not be predominant components of the aquarium freshwater nitrifier actively (Hovanec, TA and EF DeLong 1996. Comparative Analysis of nitrifying bacteria associated with freshwater and marine aquaria Appl. Environ Microbiol. 62: 2888-2896. These references, and all other references cited herein are hereby incorporated by reference.) In seawater aquarium, Nitrosomas europaea and close relatives make their appearance to encompass a significant proportion of the total eubacterial community, but Ni trobacter winogradskyi was above the detection limits in the study by Hovanec and Delong (1996). Chemolyteautotrophic nitrite oxidizing bacteria are phylogenetically diverse, occurring in many subdivisions of the Proteobacteria (Figure 1). The best-studied members of this group of organisms (for example, Nitrobacter winogradskyi and close relatives) belong to the subdivision pí of the Proteobacteria (Watson, SW And JB Waterbury 1971 Characteristics of two marine nitrite oxidizing bacteria, Ni trospina gracilis nov. Nov. Sp. And Ni Trococcus mobilis Nov. Gen. Nov. Sp.Arch. Mikrobiol. 77: 203-230.) Neither trospina gracilis and Ni trococcus mobilis, isolated first by Watson and Waterburry, were determined to be members of the subdivisions. and T of the Protobacteria, respectively (Teske, A., E. Alm, JM Regan, S. Toze, BE Rittmann, and DA Stahl, 1994. Evolutionary relationships among ammonia- and nitrite-oxidizing bacteria J. Bacteriol 176: 6623-6630. ) Another NOB, Nor marine trospira, is phylogenetically affiliated with non-oxidizing nitrite bacteria such as Leptospirillum ferroxidans (Enrich, SD, Behrens, E. Ledeva, W. Ludwig, and E. Bock, 1995. A new obligately chemolithoautotrophic, nitrite oxidizing bacterium, Ni trospira moscoviensis sp. nov., and its phylogenetic relationship, Arch. Microbiol. 164: 16-23.) Based on the phylogenetic analysis of 16S rRNA sequences, Erlich et al. proposed a new phylum within the domain of Bacteria for these organisms (Figural). A nitrite oxidizing bacterium discovered again in a freshwater environment (a worn steel tube in a heating system), Neither trospira moscoviensis, was recently found to be phylogenetically related to marine Nitrospira.

Whether in a pure culture or in biofilters, NOBs are slow-growing organisms with doubling times of 12 to 32 hours (Belser, LW and EL Schmidt, 1978. Diversity in the ammonia-oxidizing nitrifier population of a soil. Environ Microbiol 36: 584-588, Carlucci, AF and DH Strckland, 1968. The isolation, purifucation and some kinetic studies of marine nitrifying bacteria, Exp. Mar. Biol. Ecol. 2: 156-166.) Therefore , in the newly available aquarium, ammonium and nitrite can reach toxic concentrations for the fish before a sufficient biomass of AOB and NOB becomes established. To reduce the length of time for the establishment of NOB in biofilters, commercial preparations of these organisms, in various forms of preservation, are available to germinate in the aquarium environment. These preparations range from essentially pure cultures of Ni trobacter species, to mixed cultures of autologous organisms AOB NOB, to products which combine autotrophic nitrifying bacteria with several species of heterotrophic bacteria. Studies from the past have generally shown that these mixtures are ineffective, but specific reasons for their poor performance have not been elucidated (Bower, CE and T. Turner, 1981. Accelerated nitrification in new seawater culture systems: effectiveness of commercial additives and seed media Aquaculture, 24.1-9; Timmermans, JA and R. Gerard, 1990. Observations sur 1 'utilization in etangs de suspensions bacteriennes du coerce, Fr. Peche Piscic 316: 28-30.) Therefore, there is a need for a product containing a suitable bacterial culture to establish a sufficient biomass of nitrite-oxidizing bacteria in freshwater aquarium before the nitrite in the aquarium reaches toxic concentrations for the fish.

BRIEF DESCRIPTION OF THE INVENTION It is an object of the present invention to provide an isolated bacterial or bacterial strain capable of oxidizing nitrite to nitrate. In a particularly preferred embodiment, the r.AJDN16S of the bacterial or bacterial strain has the nucleotide sequence of SEQ ID NO: 1, or a variant thereof which is at least 95% similar to SEQ ID NO: 1.

The invention is also proposed to include nucleic acid sequences and bacteria with sequences which are at least 90% similar, preferably 95% similar, more preferably 99% similar to SEQ ID NO.

• NO: 1. For the purposes of this application, "95% similar" means that substitutions of single bases can occur up to 5% of the bases. By "99% similar" it is reported that substitutions of single bases can occur from above up to 1% of bases. In a preferred embodiment, the strain of bacteria or < bacterial will be in the form of a biologically pure culture of a bacterial strain capable of oxidizing nitrite to nitrate. It is another object of the present invention to provide a mixture which includes a culture of concentrated bacteria capable of oxidizing nitrite to nitrate.

It is furthermore an object of the present invention to provide a method for alleviating the accumulation of nitrite in a medium. The method comprises adding to the medium a bacterial or bacterial strain capable of oxidizing nitrate to nitrite. It is still another object of the present invention to provide a probe which can be used to detect and determine the amount of bacteria capable of oxidizing nitrite to nitrate.

Accordingly, an object of the present invention is to provide a biologically pure culture of a bacterial strain capable of oxidizing nitrite to nitrate, where the 16S rDNA of the bacterial strain has the nucleotide sequence of SEQ ID NO: 1.

Another objective of the present invention is to provide a mixture comprising a concentrate of bacteria capable of oxidizing nitrite to nitrate, wherein the 16S rDNA of the bacterium has a nucleotide sequence of SEQ ID NO: 1.

A further aspect of the present invention is a method of alleviating the accumulation of nitrite in a medium containing water. Such means include, but are not limited to, freshwater aquariums, saltwater aquariums, wastewater, marshes, and other environments in which it is desirable to reduce the nitrite load. The method comprises a step of placing within the medium a sufficient amount of bacteria capable of oxidizing nitrate to nitrite to lighten the accumulation of nitrite in the medium. In a preferred embodiment of the method, the 16S rDNA of the bacterium has a nucleotide sequence of SEQ ID NO: 1.

A further aspect of the present invention is a .DNA probe and a method for the detection and determination of the amount of bacteria capable of oxidizing nitrite to nitrate in a medium containing bacteria in the gravel.

The present invention provides a number of advantages. As we explain in more detail below, the bacterium of the present invention can effectively lighten or eliminate the accumulation of nitrite in a medium, particularly in freshwater aquariums. The DNA probes of the present invention and the methods of using the probes according to the present invention provide an effective tool for one to detect and measure the existence of a bacterial strain which is capable of oxidizing nitrite to nitrate in a given medium. .

The bacteria of the present invention are well suited for use in freshwater aquariums, saltwater aquariums and wastewater to lighten the accumulation of nitrite. These can also be used in a bioreparation process to reduce the level of contamination caused by nitrite.

In the use of the bacterium according to the present invention, the bacterium can be placed on substrates such as a rotating biological contactor (RBC), a biofilter unit, or other suitable substrate. The bacteria can be used in the form of powder, liquid or solid form (for example, dry cooling). The compositions may include preservatives, nutrients and the like, as desirable to stabilize the bacteria and promote growth in the medium. The compositions may also include other bacterial strains. The inclusion of such additional strains may be desirable, for example, for the removal of other contaminants or undesirable substances which may be found in the medium.

BRIEF DESCRIPTION OF THE DRAWINGS.

The foregoing and other features of this invention and the manner of obtaining it will become more apparent, and will be understood in the best way by reference to the following description, taken in conjunction with the accompanying drawings. These drawings represent a typical embodiment of the invention and therefore do not limit its scope. These serve to add specificity and detail, in which: Figure 1 shows the phylogenetic relationships of the autotrophic nitrite oxidizing bacteria (NOB) in the subdivision Protobacteria and the Ni trospira group. Clone 710-9 a rDNA clone that originates from aquariums with active nitrite-oxidizing bacterial populations, is more similar to the nitrite oxidizing bacteria of the Ni trospira group. The specificity of two nucleotide probes designed for Ni trospira spp. they are indicated by the framed sections.

Figures 2A and 2B show the results of the temperature dissociation experiments for the probes SG-Ntspa-0685-aA-22 and S-* - Ntspa-0454-aA-19 with 50% temperature dissociation marked by the vertical line. The r.RNA of Ni trospira marina (O), and RNA transcribed from Clone 710-9 (< •, o).

Figures 3A-3C show the specificity of the nucleotide probes targeting the nitrite oxidizing bacteria (NOB) of the Ni trospira group and the Clone 710-9 identified in this study. The order of the probe is Eubacterial probe SD-Bact-0338-aA-18 (A), NOB probe similar to Ni trospira SG-Ntspa-0685-aA-22 (B), and NOB probe similar to Ni trospira S - * - Ntspa-0454-aA-19 (C), with rRNA, transcribed RNA (trRNA) or rDNA amplified with PCR, in the following order: channel a-1, Comamonas testosteroni; channel a-2, Alcaligenes eutrophus; channel a-3, Alcaligenes faecalis; channel a-4, Comamonas acidovorans; channel a-5, Ni trobacter winogradskyi (rDNA); channel a-6 Ni trobacter agilis (rDNA); channel b-1, Clone 710-9 (rDNA); Clone b-2, Clone 710-9 (trRNA); channel b-3, Ni trospira marina (rDNA); channel b-4, Ni trospira marina (trARN); channel b-5, Ni trospira gracilis; channel b-6, Shewanella putrefaciens.

Figures 4A-4C show chemically ammonium (A), nitrite (B) and nitrate (C) for an aquarium set up for 138 days. The serrated saw model for ammonium is the result of increasing the frequency of dosing with ammonium chloride when nitrification was being established. Water was exchanged from sweet to sea water on day 80. mM Figure 5 shows the denaturing gradient in gel electrophoresis (DGGE) the time profile of the series of a biofilm of a freshwater aquarium during the establishment of nitrification. The aquarium was exchanged for sea water on day 80. Lines A, G and J contain two clones that include Clone 710-9, a putative nitrite oxidizing bacterium that shows close similarity to the Ni trospira group. The band corresponding to this organism appears first with significant intensity on Day 22. Lines B, C, D, E, and F are sampling data before crossing into seawater. Lines H and I are sampling data after exchanging seawater. The chemistry of water for Several forms of nitrogen in this aquarium are shown in Figure 4. Figures 6A and 6B show the values of inorganic nitrogen for a newly established freshwater aquarium dosed with ammonium chloride for 33 days. (A): values along ammonium (0) with additional data of ammonium chloride (A). (B): values of nitrite (E2) and nitrate (•) for the same aquarium. A DGGE profile of the nitrifying assembly associated with this aquarium is presented in Figure 7.

Figure 7A shows the denaturing gradient in gel electrophoresis (DGGE) of selected data during the first 18 days after the start-up of a freshwater aquarium during which the nitrification time is coming to be established. Clone 710-9, a putative nitrite-oxidizing bacterium similar to Nitrospira, beginning to appear around day 12 (Line G). Figure 7B shows the relative intensity of the band for Clone 710-9 in each sample data. The associated water chemistry data for this aquarium are presented in Figure 6.

Figures 8A-8C show the water chemistry data and the results of the nucleic acid hybridization probe for a freshwater aquarium during the first 57 days after starting it. The nitrite (A) and nitrate (B) are for two tanks: Tank 4 (which did not receive a commercial bacterial mixture and Tank 16 (0) which received weekly additions of a commercial bacterial mixture (CYCLE®, Rolf C, Hagen Corp., Mansfield, MA 02048, USA) for the first 4 weeks. (Nitrate data not obtained for days 18 and up to 40.) (C) percentage of hybridization related to that of a bacterial probe SD-Bact -0338-aA-18) for specific probes for nitrite oxidizing bacteria (NOB). Probes S-G-Ntspa-0685-a-A-22 and S - * - Ntspa-0454-a-A-19 directed to Nitrospira spp., While the probe S - * - Nbac-1017-a-A-20 is for the subdivision ¿x. protobacteriana of NOB.

Figure 9 is the nucleotide sequence of the 16S rDNA of a bacterial strain of the present invention (SEQ ID NO: 1).

DETAILED DESCRIPTION OF THE INVENTION.

The present invention is based on the surprising discovery of a novel bacterial strain which is responsible for the oxidation of nitrite in freshwater aquariums.

The present invention provides an isolated bacterial strain or a biologically pure culture of a bacterial strain capable of oxidizing nitrite to nitrate, wherein the 16S fDNA of the bacterial strain has a nucleotide sequence of SEQ ID NO: 1 as shown in Figure 9 For the purposes of the present invention, an isolated bacterial strain is one that has been subjected to some degree of purification from its natural environment. A culture of a bacterium is considered biologically pure if at least 20% of the bacteria are from a bacterial strain. However, it is preferable if the culture is at least 33% pure, more preferably at least 45% pure and more preferably at least 90% pure. • The bacterial strain of the present invention may also be combined with other species of bacteria, nutrients, and / or other components to provide a The composition for maintaining or purifying the medium containing water. It may be desirable, for example, to combine the bacteria of the present invention with bacteria capable of removing other contaminants or undesirable compounds from the medium containing water. Examples of such bacteria include ammonium oxidizing bacteria (chemolytotoxic bacteria that oxidize ammonium to nitrate), heterotrophic bacteria (which mineralize the organic material into ammonium), and other bacteria which will be known by those of skill in the state of the art. The bacteria • Ammonium oxidants, for example, are known from the beta subdivision of Protobacteria, for example species of the genus Ni trosomas and Ni trosospira. Nitrate reducing bacteria are known from the genus Azoarcus, Pseudomonas and Alcaligenes. The heterotrophic microorganisms are known from the genus Bacillus, Pseudomonas, and Al caligenes. Such are available from known sources (eg, American Type Culture Collection, 10801 University Blvd., Manassas VA 20110, USA) or could be isolated directly from aquarium biofilters.

For example, the bacterial strains of the present invention could be combined with ammonium oxidizing bacteria such that the ammonium present in the water system would be oxidized to nitrite and the nitrite oxidized to nitrate. Another example would be to combine the strain Bacterial of the present invention with an aerobic or anaerobic denitrifying bacterium. In this case, the ? Nitrate that is produced by the bacterial strain of the present invention would be reduced to dinitrogen or other nitrogen-based products. A third example would be combine the bacterial strain of the present invention with heterotrophic bacteria which mineralize organic matter into simpler inorganic substances . which, subsequently, can be used as substrates by ammonium and oxidizing bacteria, or the strain of the present invention.

Isolated bacterial strains of the present invention comprise bacteria that are similar to members of the Ni trospira group in that these can be detected with oligonucleotide probes having the sequence 5'- CACCGGGAATTCCGCTCCTC'3 '(SEQ ID NO: 2) or 5'- TCCATCTTCCCTCCCGAAAA-3' (SEQ ID NO: 3).

The present invention also provides a mixture comprising a concentrated bacterial strain capable of oxidizing nitrite to nitrate, wherein the 16S rDNA of the bacterium has a nucleotide sequence of SEQ ID NO: 1. According to the invention, the bacterial strain is considered to be it is concentrated if the bacterial strain occurs in a concentration which is higher than its concentration in nature. In general, the concentration of the bacterial strain will be at least 20% of the total cells in the sample as determined by standard techniques such as' molecular probes using fluorescent in situ hybridization techniques, which will be known to those enabled in the state of the technique, using appropriate controls and numbering methods. More preferably, the concentration of the bacterial strain would be 33% or greater of the total cells, even more preferably 45%, and more preferably 90% or greater of the total cells.

It is understood that the bacterial strain, and the mixture of the present invention may be in a powder, liquid, frozen form and a cold dried form. Such forms include, but are not limited to: a liquid form where the strain is a liquid solution containing inorganic salts or organic compounds such that the viability of the cells is not destroyed during the course of storage; a frozen form, where the strain in a liquid mixture as above, optionally includes cryoprotective compounds to prevent cell lysis, is frozen and stored at a temperature at or below 32 F; a powder form, which has been produced by drying to freezing or other means, where the dehydrated form of the strain or mixture can be stored at normal room temperature without loss of viability.

Obtaining a proper form of the bacterial strain and the mixture of the present invention is well within the skill in the state of the art in view of the present disclosure. It is also understood that the bacterial strain and the mixture of the present invention can be used alone, or in combination with other components. Examples of such components include, but are not limited to, ammonium oxidizing bacteria, heterotrophic ammonium oxidizing bacteria, heterotrophic nitrite oxidizing bacteria, and the like. All forms of the biologically pure bacterial strain can also contain nutrients, amino acids, vitamins and other compounds which serve to preserve and promote the growth of the bacterial strain. The bacterial strain and the mixtures and compositions of the present invention can be used in freshwater aquariums, saltwater aquariums and wastewater to lighten nitrite accumulation. These can be used in a bioreparation process to reduce the level of contamination caused by a nitrite. A biomedical process, also called bioacretation, includes, but is not limited to, the supplemental addition of microorganisms to a system (for example, a site where biological or chemical contamination has occurred) for the purposes of promotion or biological establishment and / or chemical processes that result in the change of one or more forms of chemical compounds present in the original system.

Accordingly, an aspect of the present invention provides a method of alleviating the accumulation of nitrite in a medium. The method includes a step of placing within a medium a sufficient amount of a bacterial strain capable of oxidizing nitrite nitrate to lighten the accumulation of nitrite in the medium, where the r.RNA 16S of the strain of the bacterium has a sequence of nucleotide of SEQ ID NO: 1. The amount of the bacterial strain is sufficient if the added bacteria can lighten or prevent the accumulation of nitrite in the medium. In general, the addition of the bacterial strain of the invention to a freshwater or saltwater aquarium is expected to minimize the nitrite concentration by at least 30% above the level that would be reached in the absence of the bacterial strain.

It will be appreciated that the current levels carried out in a given environment will be a function of the size and contents of the system, for example the number of fish, plants, etc. contained in the system. In a fresh 37-liter aquarium with 10 adult fish, the nitrite concentration can reach 8 mg / l or higher without the addition of the bacterial strain although the maximum level can be reduced to around 3 mg / l by addition of the bacterial strain. In general, it would not be expected that the maximum concentration of nitrite exceeds 5 mg / l if the bacterial strain of the invention is added to such a system. When the system reaches a stable system, the nitrite levels recede below 0.25 mg / l, a process that occurs more rapidly when the bacterial strain of the invention is present.

In one embodiment of the present invention the bacterial strain of the present invention is placed directly into a medium such as, but not limited to, freshwater aquariums, saltwater aquariums, and wastewater. Preferably, the bacterium can first be grown in a rotating biological contactor and then placed in a medium. In a different embodiment, the bacterium of the present invention can be placed in a biofilter unit contained in a medium.

As used herein, the term "aquarium" is proposed to mean a container which may be made of, in combination or in its entirety, but not limited to, glass, plastic, or wood containing water and in which are placed live aquatic organisms, (such as fish, plants, bacteria and invertebrates) and the contents of these. An aquarium can be for the purposes of displaying aquatic organisms, for its short and long term hold, for scientific study, for transportation or other purposes. A freshwater aquarium is usually an aquarium in which the liquid medium has a salinity of less than 15 parts per hundred. A saltwater aquarium is usually an aquarium in which the liquid medium has a salinity of more than 15 parts per thousand. The term "aquarium water" is used to refer to the medium that is contained within the aquarium, and its associated filter systems, in which aquatic organisms reside. Aquarium water can contain an extensive range of inorganic or organic chemicals and, therefore, can have a wide range of concentrations of such parameters as salts, pH, total dissolved solids, and temperature to name a few.

As used herein, "waste water" generally refers to a liquid medium that is the product of an industrial or human process. This may need to be treated by one or more filtration methods to make it less harmful to the environment such that it is subject to standard downloads as determined by a government agency. A waste water can also be recycled so that it is not discharged into the • ambient.

As used herein, "biofilter" generally refers to a type of filter whose purpose is to promote the growth of microorganisms or provide a substrate for the fixation and growth of microorganisms. A biofilter can be part of an aquarium filtration system to a wastewater filtration system. As used herein, the term "rotating biological contactor" generally refers to a type of Biofilter that rotates in water or medium. This can be completely or partially submerged in the water or in the middle. Persons skilled in the art will recognize rotating biological contactors as personified in US Pat. Nos. 2,085,217; 2,172,067; 5,423,978; 8,419,831; 5,679,253; • 5,779,885 and all continuations, improvements and foreign counterparts, being the same commonly agreed upon by the assignment of the present invention and each one respectively incorporated expressly herein by reference.

As used herein, "beard filter" refers to an irregularly configured natural or synthetic multi-braid material which can serve as a biofilter, a mechanical filter or a combination thereof.

As used here, "aquarium gravel" refers to a substrate commonly placed inside, at the bottom of an aquarium. This may be composed of regular or irregular shaped rock pieces, coral, plastic, or other material. This can serve as a biofilter, a mechanical filter, for decorative purposes to a combination of these.

As used herein, the term "sponge filter" refers to a natural or synthetic material which when used in an aquarium or as part of an aquarium filtration system can serve as a mechanical filter or a biofilter or both. .

As used herein, "plastic filter medium" refers to a man-made material that serves as a biofilter or a mechanical filter or both. This can be molded from plastic or molded injected.

The present invention provides a nucleotide probe for detecting and measuring the amount of bacteria of the present invention which are present in a medium. The probe has a nucleotide sequence of 5'-CACCGGGAATTCCGCGCTCCTC'3 '(SEQ ID NO: 2) or a 5'-nucleotide sequence -TCCATCTTCCCTCCCGAAAA-3' (SEQ ID NO: 3). The nucleotide probes of the present invention can be synthesized by methods that are known in the state of the art. The nucleotide probes of the present invention can be labeled by any of • the markers that are detectable. Examples of suitable labels include, but are not limited to, radioactive labels, fluorescent labels and the like. Suitable marking materials are commercially available and would be known by those of ordinary skill in the state of the art. The methods of labeling an oligonucleotide or • a polynucleotide are also known to those of ordinary skill in the state of the art. (See, for example, Sambrook, J., E. F. Fitsch, and T. Maniatis.

Molecular Cloning-A Laboratory Manual, 2a. Edition, 1989, Cold Spring Harbor Press.) The nucleotide probes of the present invention can hybridize with 16S rDNA of the bacterial strain of the present invention. Accordingly, the nucleotide probes of the present invention are well used for use in a method for the detection and determination of the amount of bacteria of the present invention. In one aspect of the present invention, there is provided a method for the detection and determination of the amount of bacteria capable of oxidizing nitrate to nitrite in a medium, wherein the 16S rDNA of the bacteria has a nucleotide sequence of SEQ ID NO: 1. The method includes the stages of: (a) providing a detectably labeled probe of the present invention; (b) total isolation of the DNA from a medium; (c) exposure of the isolated total DNA to a probe detectably labeled under conditions under which the probe hybridizes only the nucleic acid of the bacteria, where the 16S rDNA of the bacteria has a nucleotide sequence of SEQ ID NO: 1; (d) detection and measurement of the hybridized probe for detection and measurement of the amount of bacteria.

The medium can be aquarium water, where DNA is isolated from there. The medium may also contain a material selected from a group consisting of aquarium gravel, sponge filter material, beard filter, plastic filter media, but is not considered to be limited thereto. Accordingly, the .DNA can be isolated from the above and other sources where you can expect to find such bacteria.

The detection method of the present invention provides an effective tool for one to monitor and detect the occurrence of bacteria capable of oxidizing nitrite to nitrate in a medium. The method also provides a tool for one to review commercial additives to determine the effectiveness of the additives, particularly in freshwater aquariums, by measuring the occurrence or amount of the bacteria of the present invention.

The examples of the embodiments of the present invention are stated in detail below.

MATERIALS AND METHODS.

Sampling and extraction of nucleic acid. For the rRNA extractions of the aquarium gravel, individual samples of gravel (10 g) were placed in a polypropylene tube covered with 2.5 ml of low pH buffer (50 mM sodium acetate, 10 M disodium EDTA) and processed as previously described (Hovanec, T. A. and E.F. DeLong, 1996. Comparative analysis of nitrifying bacteria associated with freshwater and marine aquaria, Api Environ Microbiol. 62: 2888-2895.). For DNA extraction, the gravel samples were resuspended in a cell lysis buffer (40 mM EDTA, 50 mM Tris-HCl, 0.75 M sucrose) and processed as previously described. Samples were stored at minus 20 ° C until extraction.

The DNA was quantified by fluorometry and binding of Hoechst 33258 type dye (DynaQuant 200, Hoefer Pharmacia Biotech Inc., San Francisco, California). The rRNA was quantified by measurement of absorbance at 260 nm (Perkin Elmer, Lambda 3B, The Perkin-Elmer Coporation, 761 Main Avenue, Norwalk, CT 06859), assuming that unit 1 A260 corresponds to 40: g / ml RNA .

Clones of rRNA gene clones amplified by PCR. The clone libraries were derived from • nucleic acid extracts from aquarium samples. The bacterial ribosomal RNA gene fragments were amplified with the primers S-D-Bact-0011-a-S-17 (8f; GTT TGA TCC TGG CTC AG) (SEQ ID NO: 4) and 1492r (eubacterial; GGT TAC CTT GTT ACG ACT T) (SEQ ID NO: 5) or S - * - UniV-0519-aA-18 (519r; GWA TTA CCG CGG CKG CTG) (SEQ ID NO: 6). The PCR conditions, cycle parameters, and reaction components were like previously described (DeLong, E. F. 1992. Archaea in • coastal marine environments. Proc. Nati Acad. Sci. USA 89: 5685-5689.) The PCR products were evaluated by agarose gel electrophoresis. The PCR fragments were cloned with a TA cloning kit (Invitrogen, Carlsbad, Calif.), As described in the manufacturers' instructions.

Analysis and profiling DGGE. For DGGE analysis, the rRNA fragments were amplified using the 358f (eubacterial; CCT AGG GGC AGG AGT) (SEQ ID NO: 7) with a 40 bp GC fastener at the 5 'end as described by Murray et al. (Murray, AL, JT Hollibaugh, and C. Orrego, 1996. Phylogenetic compositions of bacterioplankton from two California estuaries compared by denaturing gradient gel electrophoresis of 16S rDNA fragments, Appl. Environ Microbiol. 62: 2615-2620), and the initiator reverse S - * - Univ-0519-aA-18 (519r; GWA TTA CCG CGG CKG CTG) (SEQ ID NO: 6). The PCR was performed on a Stratagene Robocycler Gradient 96 (La Jolla, Calif.) Using the manufacturer's reagents. The PCR conditions included a warm start (80 ° C) and a landing procedure. The initial denaturation at 94 ° C for 3 minutes was followed by a denaturation at 94 ° C for one minute a tempering landing from 65 ° C to 55 ° C for 1 minute 29 seconds (the tempering time during landing increased by 1.4 see per cycle), and the extension of the initiator at 72 ° C to 56 see (the extension time was increased by 1.4 seconds per cycle). The final temperature series of the previous thermal cycle was repeated for 20 total cycles, followed by a final extension at 72 ° C for 5 minutes. The amplifications were examined by agarose gel electrophoresis.

The DGGE was performed with a Bio-Rad D-GENE System (BioRad Laboratories, Hercules, Calif.). All gels were 8.5% Acrylamide / Bis using Bio-Rad reagents (from GENE Electrophoresis Reagent Kit, Bio-Rad Laboratories Hercules, Calif.). The gel gradients were emptied using Bio-Rad reagents (from GENE Electrophoresis Reagent Kit, Bio-Rad Laboratories Hercules, Calif.) With a denaturation gradient of 20 to 60% (where 100% denaturant is a mixture of 40 % deionized formamide and 7 M urea) and the Bio-Rad gradient release system (model 475, Bio-Rad Laboratories Hercules, CA, USA). All gels were run at 200 volts for 6 hours. The gels were visualized in one of two ways. For visualization and recovery of the discrete DNA bands, the gels were first maintained for 10 minutes in 250 ml of buffer 1 x TAE in which 100: 1 ethidium bromide (1 mg / ml) was added, then washed for 10 minutes. minutes in shock absorber 1 x TAE. For documentation purposes some gels were placed in Vistra Green (diluted 1: 10,000) (Molecular Dinamics, Sunnyvale, Calif.) For 20 minutes, followed by a 20 minute wash in 1 x T.AE buffer and then scanned using a Fluorlmager SI (Molecular Dynamics, Sunnyvale, Calif.).

The individual bands were extracted from DGGE gels using scalpels sterilized in alcohol. The extraction of the DNA from the gel followed the methods of Ferris et al. (Ferris, MJ, G. Muyzer, and DM Ward 1996. Denaturing gradient gel electrophoresis profiles of 16S rRA-defined population inhabitig a hot spring microbial mat community, Appl. Environ Microbiol. 62: 340-346.) The extracted bands were placed in a sterile 2 ml screw cap tube with 500: 1 sterile deionized water. The tubes were filled in half with glass beads (Cat. No. 11079-101 BioSpec Products Inc., Bartlesville, Okla.) And placed in a mechanical bead beater (Mini-beadbeater-8, BioSpec Products Inc., Bartlesville , Okla.) For three minutes in the middle higher. The processed .DNA remained in the tubes at 4 ° C throughout the night. After storage overnight, the tubes were centrifuged at 3200 x g for 8 minutes at 4 ° C to concentrate the gel fragments. The supernatant was transferred to a clean eppendorf tube.

To review the extraction efficiency, the supernatant was reamplified with DGGE primers and reanalyzed by DGGE. An extraction was considered acceptable if it produced a single band in the DGGE analysis which co-migrated with the origin band of DGGE in the sample of the mixed population.

Development of the oligonucleotide probe and hybridization procedures. Two oligonucleotide probes were designed which hybridize specifically with the sequence of the rRNA gene isolated in these studies of the biofilters of Ni trospira marina, Nistrospira moscoviensis, and similar to Ni trospira. A probe (S-G-Ntspa-0685-a-A-22) targets the bacteria similar to Ni trospira derived from the biofilters, and both Ni trospira marina and Ni trospira moscoviensis. The second probe (S - * - Ntspa-0454-a-A-19) targets the Nitrospira-like bacterium derived from the bisfilter and its close cultured relative, Ni trospira moscoviensis (Figure 1). The sets of probes were initially projected using BLAST (Altschul, S. F., W. Gish, W.

Miller, E. W. Myers, and D. J. Lipman. 1990. Basic local alignment toool. J. Mol. Biol 215: 403-410) and CHECK PROBÉ (Maidak, BL, N. Larse, MJ McCaughey, R. Overbeek, GJ Olsen, K. Fogel, J. Blandy, and CR Woese, 1994-The ribosomal datbase, Nucleic Acid Res. 22: 3485-3487.) probes were synthesized by Operon Tech, Inc. (Alameda, Calif.). The nucleotide sequences and positions of the probes are shown in Table 1. • or TABLE 1. Nucleotide sequences and positions of the oligonucleotide probe for oxidized bacteria wave "Position Base sequence Td Wash temperature Group ob (nucleotides) 15 (5 'to 3') (° C) ° (° C) G-Ntspa-0685-aA-22 664-685 CAC CGG GAATTC CGC GCTCCTC 63.0 60.0 Nitrospi mos * -Ntspa-0454-aA-19 435-454 TCCACTTTCCCTCCCGAAAA 58.5 56.0 N. mari Clo Nitrospir moscov Clone 7 Names of the tests designated using the standard proposed by Alm et al. 1996 (Alm, E. W., D.B.Oerther, KLarsen, D.A. Stahl, and L. Ras tábase, Appl. Environ Microbiol. 62: 3557-3559). Escherichia coli umeration. Dissociation temperature (TD) is defined as the temperature at which 50% of the bound probe is released from the homologous hybrid.

Since the rRNA of the bacterium similar to Ni trospira derived from a biofilter is not available, a 16S rRNA transcribed in vi tro was used as tempered for the determinations of the dissociation temperature (Td) and as a control in the hybridization experiments. . The 16S RARN transcribed in vi tro was synthesized as described by Polz and Cavanaugh (Polz, MF and CM Cavanaugh, 1997. A simple etod for quantification of uncultured microorganisms in the environment based on in vitro transcription of 16S rRNA. Microbiol 63: 1028-1033.) The dissociation temperature (Td) of the oligonucleotide probes was determined by measuring the amount of probe eluded over a series of increasing wash temperatures (Raskin, L., JM Stromley, BE Ritt ann, and DA Stahl. Group-specific 16S rRNA hybridization probes to describe natural co-munities of ethanogens, Appl. Environ Microbiol 60: 1232-1240.) For these probes, 200 ng of annealing was immobilized on a nylon membrane (Hybond-N, A ersham ) and hybridized overnight 45 C with a labeled probe 32r After hybridization, the membrane was washed at room temperature in IX SET (150 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 7.8) and 1% SDS for 30 minutes in a stirring table. The individual filter bands were then placed in a 0.5 ml eppendorf tube containing 500 μl 1XSET / 1% SDS preheated to the temperature of the initial probe. The eppendorf tubes were placed in a thermal cycler (Perkin Elmer) and incubated for 30 minutes. The membrane was transferred to a new eppendorf tube containing 1XSET / 1% SDS, and the temperature increased, and held at elevated temperature for 30 minutes. After each wash, the wash buffer was transferred to a scintillation flask containing 3 ml of the scintillation cocktail (Liquiscint, National Diagnostics, Atlanta, Geog.) Mixed, and the radioactivity quantified by liquid scintillation counting. Each profile was carried out in duplicate. The rRNA of the aquarium was transferred by slot and quantified using nucleic acid probes developed in this and in a previous study (Hovanec, TA and EF DeLong, 1996. Co-operative analysis of nitrifying bacteria associated with freshwater and marine aquaria. Microbiol., 62: 2888-2896.) Under previously described conditions (Hovanec, TA and EF DeLong, 1996. Comparative analysis of vitrifying bacteria associated with freshwater and marine aquaria, Appl. Environ Microbiol. 62: 2888-2896.) The methods to determine the relative amount of the specific RARN hybridization signal from each probe were the same as previously described (Hovanec, TA and EF DeLong, 1996. Comparative analysis of nitrifying bacteria associated with freshwater and marine aquaria "Appl. Environ. Microbiol. 62: 2888-2896.) Sequenced The sequencing of rDNA SSU excised from DGGE clones or gels was carried out using directly Secuenase 2.0 (United States Biochemical, Cleveland, Ohio).

Experimental Aquarium Systems. Three sets of experiments were put into operation in an aquarium to a) study the establishment of nitrifying bacteria and b) determine the effect of a bacterial additive. New aquariums, filtering systems, and save it were used for each probe. Samples of the aquarium water for the three probes were analyzed for ammonium (gas diffusion membrane method), nitrite (azo staining method) and nitrate (cadmium-azo reduction staining method) by flow injection analysis as previously described (Hovanec, TA and EF DeLong, 1996. Comparative analysis of nitrifying bacteria associated with freshwater and marine aquaria, Appl. Environ Microbiol. 62: 2888-2896.) Probe of Bacterial Additive. Six glass aquariums were established with a filtering system under airborne gravel (Model KF720, Neptuno Products, Moorpark, Calif.) In a controlled temperature laboratory (average air temperature 26.0 ° C ± 1.5 ° C). The aquarium was covered with glass covers but it was not illuminated except with the ceiling lights of the room which were in a light cycle: darkness 5 of 14hr: 10hr. 6.8 kg of natural aquarium gravel (Kaytee Products, Irwindale, Calif.) Were placed on top of the filter plate. 30 1 of tap water from the city, which passed through activated carbon, were added to each aquarium. The filtered air was provided to each aquarium of a common air source. 6 fish (Danio aequipinnatus) were placed in each aquarium and fed 0.5 g fish food (Aquarian, Kal Kan Foods, Vernon, Calif.) Daily during two feeding periods. Three of the aquaria (the treatment group) were each dosed with 8 ml of bacterial additive (Cycle®, Rolf C. Hagen Inc., Mansfield, Mass.) In the. first day and once every 7 days later for an additional 3 weeks. The other three aquaria were the control group and not received no additives. • Two samples of 10 g of gravel were collected from each aquarium on a weekly basis and the nucleic acids extracted and analyzed as described above. 25 Time of the NOB Appearance. Three glass aquariums were established as described above. 34 1 of tap water from the city, which passed through activated carbon, were added to each aquarium which contained 4.53 kg of gravel. Initially, 0.71 mmol of ammonium chloride sterilized by filtration (0.2 μm) were added to each tank followed by a dosage • additional 5.0 mmoles of NH4C1 on the fourth day. On days 10, 15, 18, 23 and 30, more ammonium additions of 8.9 mmoles were made to each aquarium. During the probe a total of 50.4 mmoles of ammonium were added to each aquarium. Water samples were collected daily.

Two samples of 10 g of gravel were collected from each aquarium daily for 33 days. To a sample, 2 ml of lysis buffer was added and the sample was frozen (-20 ° C) until the rDNA was extracted by previously described methods. The rRNA was subjected to DGGE after crossing the PCR with the primers and conditions stated above. The other sample was preserved with 2 ml of cushion with bed pulse. 20 • Time series. Three aquariums were placed as described previously with 4.53 kg of gravel and filled with 30 1 of city water which had passed through activated carbon. The probe was carried out during 25 138 days during which the aquarium was dosed individually with 8.9 mmoles of ammonium (as ammonium chloride) sterilized by filtering (0.2 μl) on the first and second day of the probe. From days 12 to 78 of the probe, more additions of 8.9 mmole of ammonium were made on average every 3 days. A total of 246 mmoles of ammonium were added to each tank during the probe. The water was sampled three times a week for chemical analysis. The aquariums were set up for 80 days with fresh water at which time they were changed to seawater (32 ppt) by drying and filling with water mixed with artificial marine salts (Marineland Commercial Aquariums, Moorpark, Calif.). After the change the aquariums were put into operation for an additional period of 57 days.

Nucleotide sequences and access numbers to the repository. The nucleotide sequence has been deposited in the GenBank database under accession number AF035813 for Clone 710-9. Clone 710-9 has been deposited at the American Type Culture Collection, 108001 University Blvd., Manassas, VA 20110-2209, USA, as the access number in The formula of the mineral medium used to grow the bacterial strain of the present invention: in an embodiment of the present invention, one can isolate and grow the strain of the bacteria of the present invention in an isolated medium disclosed by Ehrich et. al .. (S. Ehrich, D. Behrens, E. Lebedeva, W. Ludwig, and E. Bock, A new obligatory chemolithoatotrophic, nitrite-oxidizing bacterium, Ni trospira moscoviensis sp. nov. and its phylogenetic relationship .Arch Microbial (1995) 164: 16-23. An example of such isolated media has the following formula. 7.25 mmol of sodium nitrite, 0.07 mmol of calcium carbonate, 8.56 mmol of sodium chloride, 0.25 mmol of magnesium chloride, 0.86 mmol of potassium phosphate, 10 0.15: mol of magnesium sulfate, 0.79: mol of boric acid , 0.15: mol of zinc sulfate, • 0.03: mol of molybdic acid, 0.10: mol of cupric sulfate, and 3.50: mol of ferrous sulfate, in one liter of distilled H20. Autoclave pH 8.6 with pH paper Methods for the isolation and growth of the strain • Bacterial of the present invention: Three samples of an aquarium were obtained as follows: 1. 50 ml of aquarium water 2. 5 grams of aquarium gravel 3. One debarking of a BioWheel® filter from Marineland Aquariu Products (a rotating biological contactor.) Each sample was placed in 75 ml of culture medium • ore, (see above), with a concentration of nitrite at 7 M. These were incubated in the dark at 27 ° C in culture flasks with foam stoppers in tissue culture flasks. 10 Nitrite consumption was frequently measured by the azo dye method when the nitrite concentration • descended samples were observed under the microscope, many types of cells were observed, in particular canes, cocci and spherical packages, indicating that the crop was not pure.

To obtain a biologically pure culture, serial decuples of a culture were made known that contains the white organism. Generally, six to nine dilutions are made so that the original culture is diluted one million to one billion times. The growth of the nitrite oxidizing bacteria is then monitored in the cultures of the dilutions. In the case of the present organism, growth and cell division is achieved by measuring the nitrite concentration in the cultures, the concentration of nitrite in the cultures containing the nitrite oxidizing bacteria will decrease over time. Sometimes, a dilution will not show growth (the nitrite concentration will not decrease). This means that it was very diluted (there are no cells present). After a certain amount of time the more dilute sample that exhibited growth of the nitrite oxidizing bacteria is checked for its purity.

This is done by adding a small amount of the division to an organic culture medium to check for the presence of heterotrophic bacteria as described in Watson and Waterbury 1971 (Watson, SW and JB Waterbury, 1971. Characteristics of two marine nitrite oxidizing bacteria Ni trospina gracilis Nov. Gen. Nov. Sp. and Ní Troccoc? s Mobilis Nov. Gen. Nov. Arch. Mikrobiol. 77: 203-230. If there is a positive signal so that the culture is not yet pure (the solution will become cloudy), after the dilution of the culture is subjected to other series of dilutions and the process repeated until a pure culture is obtained. Once the pure culture is obtained, a relatively long number of cells can be grown. These cells can be preserved in the form of freezing, dry freezing or in liquid suspension. The cells for freezing are concentrated from a culture flask by centrifugation, quickly frozen in a flask placed in a mixture of dry ice and acetone and stored in the laboratory refrigerator at -80 ° C. The dry freezing of the microorganisms is completed with bench-top units available from a number of manufacturers, as will be familiar to those qualified in the state of the art.

In this way, a heterotrophic culture medium can be used to test whether the culture of nitrite oxidizing bacteria is pure. Only heterotrophic bacteria will grow in this medium (they will not grow in this nitrifying autotrophic). Thus, if a culture sample of nitrite oxidizing bacteria is grown in this medium and it becomes turbid after a day or two, that culture was not pure. This is one of the means which can be used to test the purity of the isolated bacterial cells; other means will be known to those of skill in the state of the art.

RESULTS Isolation of putative nitrite oxidizing bacteria. Two ways were taken to identify nitrite oxidizing bacteria in the aquarium samples. The first way was to develop clone libraries of gravel samples from an aquarium several times during the vitrification establishment. Samples were taken 17 days and 31 days after the aquarium was established and ammonium additions began. A third library was constructed of DNA extracted from the material of a biofilter constructed of thermoplastic material (Model CBW-1, Aquaria INC., Moorpark, Calif.). This filter had been set for 109 days in a system with daily doses of ammonium chloride.

The second route used to monitor and identify the vitrification of microorganisms was gradient denaturation in gel electrophoresis (DGGE). DNA extracted from aquarium gravel samples taken during the establishment of nitrification was subjected to DGGE to produce a discrete band model. The band models were compared with each other, and the band models produced by a mixture of known nitrifiers. The single bands were cut from the gels and sequenced.

The sequences of the clone and DGGE libraries were compared to bacterial sequences found in public databases (BLAST (Altschul, SF, W. Gish, W. Miller, EW Myers, and DJ Lip an. 1990. Basic local alignment seach tool J. Mol. Biol. 215: 403-410) and RDP (Maidak, BL, N. Larsen, MJ McCaughey, R. Overbeek, GJ Olsen, K. Fogel, J. Blandy, and CR Woese, 1994. the ribosomal datábase project Nucleic Acids Res. 22 3485-3487.) Some sequences, which showed close similarity to known nitrite oxidizing organisms, were more completely sequenced.

Identification of putative NOB similar to Ni trospira. Five samples of nitrite oxidizing bacteria were projected or by development of clone library or DGGE. A total of 96 clones or cut bands were partially sequenced. Of these, 11 were highly similar to the members of the Ni trospira group but none was similar to Ni trobacter spp. . The partial sequences were more highly similar to Ni trospira marina and Ni trospira moscoviensis (data not shown). The 16S rDNA of a representative clone which contains the rDNA similar to Nitrospira was completely sequenced, and a phylogenetic tree was inferred. The phylogenetic analysis indicated a high similarity between this cloned rDNA (710-9) and members of the Ni trospira group, Ni trospira moscoviensis and Ni trospira marina (Figure 1). The rDNA contained in clone 710-9 was 96.1% similar to Ni trospira moscoviensis and 87.4% similar to Ni trospira marina (Table 2). • f o < ? ! -3 fu ¡O ro pu H H- K) O * C / l ¡a (A u> g £ fl) o LQ C 0) ro a? c H- 3 O H- < T > H (0 H- * < rt re d 0 a H- CD Cii ro tu tu tu o DH DH DH DH DH DH DH DH DH DH DH DH DH DH DH Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos Pos -9, numbering from E. coli S? O Pl rt w H P- OW?? I-1 Ti & > l- '. HO 0). Ro Specificity of oligonucleotide probe.

Oligonucleotide probe sequences. The position (numbering of E. coli), dissociation temperature Tds, wash temperature, and target groups for the probes are shown in Table 1. For the probe S - * - Ntspa-0454-aA-19, the Td was 58.5 ° C, while the Td was 63 ° C for the SG-Ntspa-0685-aA-22 probe (Figure 2).

The channel transfer experiments confirmed that the S-G-Ntspa-0685-a-A-22 probe was specific to the known NOB of the Ni trospira group, as well as the clone 710-9 (Figure 3). As predicted, the probe S - * - Ntspa-0454-a-A-19 hybridized to clone 710-9, but not to Ni trospira marina. In addition, the experiments showed that neither the probe hybridized with nitrite oxidizing bacteria which are members of the subdivisions. or & of the Protobacteria (Figure 3).

Detection of NOB in aquariums. Table 3 summarizes the sounding results of many aquarium biofilms with the NOB probes. The S-G-Ntspa-0685-a-A-22 probe produced a positive signal with all the freshwater and saltwater aquariums tested. The probe S - * - Ntspa-0454-a-A-19 detected bacteria similar to Ni trospira in all freshwater aquariums but not in all saltwater aquariums (Table 3). There were no cases of positive detection by a test which targets species of Nitrobacter to Proteobacteria.

• • • Table 3. Results of rRNA probing extracted from aquarium biofilms with nucleic acid tests for nitrite oxidizing bacteria Time Series: The values of ammonium, nitrite and nitrate for a representative aquarium test dosed with ammonium chloride for 138 days are shown in Figure 4. The data show the desired model for the establishment of nitrification in aquariums.

Initially the ammonium was increased after it decreased to undetectable levels around day 12 (The dentate model seen from the ammonium values is the result of the increase in the frequency of the ammonium additions.) By day 12, the nitrite was increased, reaching its maximum value on day 22. By day 38 the nitrite was essentially zero the nitrate was strongly increased (Figure 4) .The change from fresh water to sea water on day 80 resulted in an immediate increase in ammonium and, subsequently, in the nitrite It took about 20 days for the oxidation of the ammonium to be reestablished The re-establishment of the nitrite oxidation took approximately 40 days.

A DGGE profile for selected days above the first 101 days for this same aquarium shows that the rDNA sequence similar to Ni trospira appeared weakly on day 15, corresponds to the beginning of nutrient oxidation (Figure 5). Around day 22 the band corresponding to the rRNA sequence similar to Nitrospira increased in relative intensity, and kept the intensity above the next two sampling data. After switching to seawater, the relative intensity of the band similar to Nitrospira decreased. The general band model also changed qualitatively between the sampling data of freshwater and seawater. The pattern of bands around day 87 (seven days after the change) appeared closer to the model for day 57 (fresh water), than the model for day 101 (sea water).

Bacterial appearance time similar to Ni trospira. The daily concentrations of ammonium, nitrite and nitrate above the first 33 days after the disposal of a new aquarium 27 are presented in Figure 6. The trends were as expected with the ammonium peak around day 12. The values of nitrite increased starting on day 12 and peaking at day 21, and decreasing below detection limits around day 26. Nitrate increased steadily from around day 15 onwards. The DGGE showed that the band corresponding to Clone 710-9, the putative NOB, appeared first on day 12 with the relative intensity of the band 710-9 increasing daily based on relative fluorescence units of the rDNA amplifications (Figure 7) .

Commercial Additive The addition of a commercial bacterial mixture (C? CLE®) which contains Ni trobacter sp. , but not Ni trospira sp. , did not result in the detection of Ni trobacter species by oligonucleotide probe hybridization experiments. However, a band that co-migrated with a control derived from AD? of pure? itrobacter could be detected in the original commercial mixture by DGGE analysis. The rAR? similar to? itrospira was easily detected in the aquarium. The specific tests for the ?rospira group indicated that the tank that received the additive had a significantly greater percentage of the rAR? of? yyrospira species (Figure 8). By day 16, approximately 5% of the AR? eubacterial hybridized with the group-specific general test? itrospira, compared to only 0.33% of the rAR? eubacterial in the tancjue which did not receive an additive (Figure 8). By day 50, the values were 3.39 and 1.52 for the additive and non-additive aquariums, respectively (Figure 8). From these results it can be concluded that the commercial mixture does not promote the growth of N. winogradskyi, which contains this, but instead promotes slightly the growth of bacteria similar to Ni trospira because it has some type of fertilization effect.

The nitrite concentrations in the two aquaria decreased as did the relative percentage of rAR? similar to? itrospira increased. By day 22, the nitrite had reached a maximum in the tank that received the additive. The nitrite concentrations reached the maximum in the aquarium not added around day 32. By day 38, nitrite levels in both aquariums were essentially below their detection limits, and nitrate levels were equivalent in the treated aquariums and not treated (Figure 8).

The results of the DGGE analysis, rRNA probing, and sequencing strongly indicated that a bacteria similar to Ni trospira is responsible for the oxidation of nitrite in freshwater aquariums. The combined use of molecular phylogenetic techniques and water monitoring chemically suggested a correspondence between the changes in the microbial community of the biofilm which coincide with the initiation of the oxidation of ammonium and nitrite. The onset of nitrite oxidation coincided with the appearance of the Ni-like nitrite-like putative nitrite-like bacteria. The results provide support for the conclusion of an earlier study (Hovanec and DeLong 1996), which suggested that the nitrite oxidizing bacteria of Protobacterial K subdivision (Ni trobacter types) were not major components of the bacterial populations of oxidative nitrite in freshwater aquariums.

The results regarding the beneficial effects of the addition of a bacterial additive containing species of Ni trobacter were equivocal. While nitrite levels dropped earlier in treated aquaria and in untreated aquaria, there was no evidence that Ni trobacter species were actively growing in these aquariums. The fact that bacteria similar to Ni trospira were easily detected and their establishment coincided with the oxidation of nitrite supports the conclusion that organisms similar to Ni trospira and not the species of Ni trobacter, are the major oxidants of nitrite in the environment of freshwater aquarium. It is possible that the addition of bacterial mixtures could provide vitamins and other nutrients which generally stimulate the growth of nitrifying assemblies, promoting their growth and development, and indirectly stimulating the oxidation of nitrite.

The foregoing is proposed to illustrate, but not limit the scope of the invention. Surely, those of ordinary skill in the state of the art can promptly prevent and produce additional modalities, based on the teachings contained herein, without undue experimentation.

The present invention can be embodied in other specific forms without departing from its essential characteristics. The modality described was considered in everything that it regards as illustrative and not as restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalence of the claims are to be adopted within their scope.

It is noted in relation to this date, that the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, the contents of the following are declared as property:

Claims (29)

.CLAIMS

1. An isolated bacterial strain capable of oxidizing nitrite to nitrate, characterized in that it comprises a nucleotide sequence which is at least 95% similar to that which is declared in SEQ ID NO: 1.

2. The bacterial strain of claim 1 characterized in that it comprises the nucleotide sequence which is identical to SEQ ID NO: 1.

3. A biologically pure culture of a bacterial strain capable of oxidizing nitrite to nitrate, characterized in that the 16S rDNA of the bacterial strain has the nucleotide sequence declared in SEQ ID NO: 1.

4. The culture of claim 3, characterized in that said strain is Clone 710-9.

5. A composition comprising an isolated bacterial strain capable of oxidizing nitrite to nitrate, characterized in that said bacterial strain comprises the nucleotide sequence declared in SEQ ID NO: 1.

6. The composition of claim 5 characterized in that it is in a frozen form.

7. The composition of claim 5, characterized in that it is in a dry form of freezing.

8. The . composition of claim 5 > characterized 5 by is in the form of a powder. •

9. A composition comprising a concentrated bacterial strain capable of oxidizing nitrite to nitrate, characterized in that the 16S rDNA of the bacterial strain 10 has the oligonucleotide sequence declared in SEQ ID NO 1

10. The composition of claim 9, characterized in that said bacterial strain has a sequence of 15 rDNA 16S which is at least 95% similar to SEQ ID NO: 1.

11. The composition of claim 9, characterized in that it additionally comprises ammonium oxidizing microorganisms, in a mineral salt solution.

12. The composition of claim 9, characterized in that it additionally comprises nitrate reducing microorganisms, in a mineral salt solution.

13. The composition of claim 9, characterized in that it additionally comprises heterotrophic microorganisms, in a mineral salt and organic based solutions.

14. The composition of claim 13, characterized in that it additionally comprises microorganisms • Ammonium oxidants and nitrate reducing microorganisms.

15. A "nucleotide sequence" characterized in that it comprises the sequence declared in SEQ ID NO: 1, or a variant thereof which is at least 95% similar.

• 16. A method for lightening or preventing the accumulation of nitrite in a medium, characterized in that 15 comprises a step of placing within the medium a sufficient amount of a bacterial strain capable of oxidizing nitrite to nitrate to alleviate the accumulation of nitrite in the media, wherein said bacterium comprises the nucleotide sequence of SEQ ID NO: 1, or 20 a variant of this which is at least 95% similar.

17. The method of claim 16, characterized in that the nitrite is reduced by at least 30% above the levels which would be present on the other hand.

18. The method of claim 16, characterized in that the medium is an aquarium.

19. The method of claim 18, characterized in that the aquarium is a freshwater aquarium.

20. The method of claim 18, characterized in that the aquarium is an aquarium of seawater.

21. The method of claim 17, characterized in that the medium comprises waste water.

22. A process of biorepair, characterized in that it comprises the method of claim 15.

23. The method of claim 16, characterized in that the bacterial strain is placed within a medium by means of a rotating biological contactor.

24. The method of claim 16, characterized in that the bacterial strain is placed within a medium by means of a biofilter.

25. An oligosaccharide probe selected from the group consisting of 5 '-CACCGGGAATTCCGCGCTCCTC-3' (SEQ ID NO: 2) and 5f-TCCATCTTCCCTCCCGAAAA-3 '(SEQ ID NO: 3).

26. A method for the detection and determination of the amount of bacteria capable of oxidizing nitrite to nitrate in a medium, characterized in that the 16S rDNA of the bacterium has a nucleotide sequence of SEQ ID NO: 1, said method comprises the steps of: a ) providing a detectably labeled probe according to claim 20; b) total isolation of the DNA from the medium; c) exposure of the total of isolated DNA to the probe detectably labeled under conditions which hybridize to the probe for only the nucleic acid of the bacterium, where the 16S rDNA of the bacterium has a nucleotide sequence of SEQ ID NO: 1; d) detection and measurement of the hybridized probe for detection and measurement of the amount of the bacterium, where the 16S rDNA of the bacterium has a nucleotide sequence of SEQ ID NO: 1.

27. The method of claim 26, characterized in that the medium is aquarium water.

28. The method of claim 27, characterized in that the medium includes a material selected from a group consisting of aquarium gravel, sponge filters, beard filter, and plastic filter media.

29. The method of claim 28, characterized in that the total DNA is isolated from the material selected from a group consisting of aquarium gravel, sponge filters, beard filter, plastic filter media.