CN1807594A

CN1807594A - Separation and authentication method for denitrifying bacteria with anaerobic ammoxidation activity

Info

Publication number: CN1807594A
Application number: CNA200610049500XA
Authority: CN
Inventors: 郑平; 胡宝兰
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2006-02-14
Filing date: 2006-02-14
Publication date: 2006-07-26

Abstract

The invention discloses a separation and authentication method for denitrifying bacteria with anaerobic ammoxidation activity, wherein a novel anaerobic ammonoxidation bacterium segregation and evaluation program is employed, wherein denitrifying bacteria is isolated first, then the capability of ammonia and nitrite simultaneous transformation under anaerobic condition is used as the criterion for confirmation of anaerobic ammonoxidation bacterium, finally the classification status of the bacterium is identified.

Description

Separation and identification method of denitrifying bacteria with anaerobic ammonium oxidation activity

Technical Field

The invention relates to a method for separating and identifying denitrifying bacteria with anaerobic ammonium oxidation activity.

Background

Anammox (Anammox), a biological reaction in which ammonia is used as an electron donor and nitrite is used as an electron acceptor and nitrogen gas (A) is the reaction product, was first reported in 1990 )^[1]. In the field of biological denitrification of wastewater, the method breaks through the classic nitrification-denitrification theory, and people develop a brand-new biological denitrification process based on the classic nitrification-denitrification theory, such as a shortcut nitrification (SHARON) -anaerobic ammonia oxidation process^[2]OLAND process^[3]And CANON process^[4]And the like. The microorganisms involved in the biological denitrification processes are autotrophic bacteria, no additional organic carbon source is needed, the operation cost can be saved by more than 40%, and an ideal path is provided for solving the worldwide problem of treating high-concentration nitrogen-containing wastewater (particularly low-carbon-nitrogen-ratio wastewater, such as sludge press filtrate, coking wastewater and other chemical wastewater, monosodium glutamate wastewater and other food processing wastewater). In the field of microorganisms, Anammox breaks through the concept that oxygen is required to participate in ammonia oxidation for a long time, and discloses a class of unprecedented microorganisms, namely anaerobic ammonia oxidizing bacteria, which enrich microbiological content.

In natural ecosystems, ammonia oxidation to nitrite or reduction of nitrate to nitrite, with coexistence of ammonia and nitrite, often occurs due to insufficient oxygen supply or limited electron donors (sulfides or organic matter). It has been demonstrated that anammox activity is detectable in marine sediments, deep ocean anoxic zones, lakesediments, waterlogged soils, and many sewage treatment plants^[2-7]. The natural habitat sample or the artificial habitat sample containing ammonia and nitrite (or nitrate) is a good separation source of the anaerobic ammonia oxidizing bacteria.

The growth of the anaerobic ammonium oxidation bacteria is slow, and the multiplication time is as long as 11d^[11]In addition, the cell density required by the bacteria to show anammox activity is higher, and only when the cell density is higher than 10¹⁰Since anammox activity can be detected only at one mL, it is often difficult to detect anammox activity by a classical bacterial isolation technique based on a single cell (or a single colony) and a conventional bacterial function identification method based on the Koch criterion. After repeated attempts fail, the first anammox strain was successfully isolated in 1999 using Percoll density gradient centrifugation, and was identified as a Phycomycetes Brocadia anammoxans based on 16S rDNA by comparison with nucleic acid sequences in GenBank database^[12]. To date, five anammox bacteria have been reported in the literature, and they are classified into Candidatus Brocadia and Candidatus Kuenenia^[13]And Candidatus Scalindua^[14，15]Three areThe genus of Phycomycetes. The main defect of adopting Percoll density gradient centrifugation technology is that the separated thallus contains a small amount of mixed bacteria, and when the separated thallus is used as a strain to carry out microbiological research, the obtained result is often questioned; the growth conditions of the separated strains are difficult to determine and are not easy to expand in large quantityAnd (5) increasing.

From an electron donor perspective, Anammox is an oxidation reaction of ammonia, similar to nitration; from an electron acceptor perspective, however, Anammox is a nitrite reduction reaction, similar to denitrification. Studies have shown that successful start-up of an Anammox reactor can be achieved by inoculating sludge using denitrification, and then do some denitrifying bacteria have Anammox activity? If a positive answer can be obtained, the population range of the Anammox bacteria can be enlarged, the microbiological knowledge is further enriched, and fast-growing strains can be screened from the Anammox bacteria, so that conditions are created for the industrial application of the Anammox process.

Reference documents:

[1]Van de Graaf AA，Mulder A，De Bruijn P，et al.Anaerobic oxidation of ammoniais a biologically mediated process.Applied and Environmental Microbiology，1995，61(4)：1246-1251

[2]Fux C，Boehler M，Huber P，et al.Biological treatment of ammonium-richwastewater by partial nitritation and subsequent anaerobic ammonium oxidation(anammox)in a pilot plant.Journal of Biotechnology，2002，99：295-306

[3]Kuai L and Verstraete W () Ammonia removal by the oxygen-limited autotrophicnitrification-denitrification system.Applied and Environmental Microbiology，1998，64(11)：4500-4506.

[4]Sliekers AO，Derwort N，Gomez JLC，et al.(2002)Completely autotrophicnitrogen removal over nitrite in one single reactor.Water Research，36：2475-2482.

[5]Schmid M，Twachtmann U，Klein M，et al.Molecular evidence for genus leveldiversity of bacteria capable of catalyzing anaerobic ammonium oxidation.SystAppl microbial，2000，23：93-106

[6]Gable J E.Anaerobic ammonium oxidation during soil aquifer treatmentD).Arizona state university，2002.

[7]Thamdrup B and Dalsgaard T.Production of N₂through anaerobic ammoniaoxidation coupled to nitrate reduction in marine sediments.Applied andEnvironmental Microbiology，2002，68(3)：1312-1318

[8]Toh SK and Ashbolt NJ.Adaptation of anaerobic ammonium-oxidizingconsortium to synthetic coke-ovens wastewater.Appl Microbiol Biotechnol，2002，59：344-352

[9]Trimmer M，Nicholls JC，Deflandre B.Anaerobic ammonium oxidation measuredin sediments along the Thames Estuary，United Kingdom.Appl.Environ.Microbiol.2003，69：6447-6454.

[10]Devol A H.Solution to a marine mystery[J].Nature，2003，422(6932)：575～576.

[11]Strous M，Heijnen J J，Kuenen J G，et al.The sequencing batch reactor as apowerful tool for the study of slowly growing anaerobic ammonium-oxidizingmicroorganisms [J].Appl.Microbiol.Biotechnol.，1998，50(27)：589-596.

[12]Strous M，Fuerst JA，Kramer EHM，et al.Missing lithotroph identified as newplanctomycete.Nature 1999，400：446-449

[13]Egli K，Fanger U.，Alvarez Pjj，et al.Enrichment and characterization of ananammox bacterium from a rotating biological contactor treatingammonium-rich leachate.Arch Microbiol 2001，175：198-207.

[14]Kuypers M M M，Sliekers A O，Lavik G，et al.Anaerobic ammonium oxidationby anammox bacteria in the black sea [J].Nature，2003，422(6932)：608～611

[15]Schmid，M.，Walsh，K.，Webb，R.，et al.Candidatus ″Scalindua brodae″，sp.nov.，Candidatus ″Scalindua wagnei″，sp.nov.，two new species of anaerobicammonium oxidizing bacteria.Systematic and Applied Microbiology 2003，26，529-538

[16]dongxu pearl, zeisingling, et al.

[17]Holt JG.，Krieg NR.，Sneath PHA.et al.Bergey′s manual of determinativebacteriology(9th edition )Williams&Wilkins，1994：427-455.

[18]Staley JT，Bryant MP，Pfennig N and Holt JG.Bergey’s manual of systematicbacteriology(Vol.3).Williams&Wilkins，1989，p.1807-1835

Disclosure of Invention

The invention provides a method for separating and identifying denitrifying bacteria with anaerobic ammonium oxidation activity.

The method for separating denitrifying bacteria with anaerobic ammonium oxidation activity comprises the following steps:

1. selecting natural habitat and artificial habitat samples with ammonia and nitrate or nitrite as separation sources of denitrifying bacteria with anaerobic ammonium oxidation activity, wherein the natural habitat samples mainly comprise waterlogged soil, marsh land sludge, lake bottom sludge and marine sediments, and the artificial habitat samples mainly comprise aeration tank sludge of an urban sewage treatment plant, anaerobic digestion tank sludge of the urban sewage treatment plant and activated sludge in various biological wastewater denitrification treatment systems.

2. Taking a basic inorganic salt liquid culture medium containing 0.1-1 mg/L yeast extract or glucose as an enrichment matrix, and culturing an anaerobic ammonia oxidation biomembrane, floc or granular sludge in a continuous culture or batch culture mode;

3. taking anaerobic ammonia oxidation biomembranes, flocs or granular sludge, crushing the anaerobic ammonia oxidation biomembranes, the flocs or the granular sludge by ultrasonic waves to prepare primary bacterial suspension, and diluting the primary bacterial suspension step by step to prepare serial bacterial suspensions.

4. Separating denitrifying bacteria with denitrifying culture medium by anaerobic plate streaking method, and anaerobically culturing at 28-30 deg.C. The single colony growing on the plate is picked into liquid culture medium and anaerobically cultured at 28-30 deg.c. After culturing, the single colony is separated by adopting an anaerobic plate marking method. This was repeated several times. When the bacterial cell morphology observed under the microscope is consistent, the target strain is considered to be purified.

The identification method of denitrifying bacteria with anaerobic ammonium oxidation activity comprises the following steps:

amplifying a pure culture of denitrifying bacteria in a liquid denitrifying culture medium, centrifuging at 5000rpm to obtain thallus cells, preparing a thallus suspension by using a basic inorganic salt culture medium, adding ammonia and nitrite, and judging whether the strain has anammox capability, namely whether the strain is anammox bacteria or not by simultaneously converting the ammonia and the nitrite under the anaerobic condition;

2. determining the classification status of the pure strain according to the morphological characteristics, physiological and biochemical characteristics, carbon source utilization characteristics and molecular biological characteristics of the pure strain, wherein the morphological characteristics comprise observation of cultured colonies, observation of cultured thalli and observation of thalli cell structures; the physiological and biochemical characteristics are identified by a Vitek GN system; the carbon source utilization characteristics are identified by adopting a Biolog microplate system; the molecular biological characteristics comprise the determination of G + C% (mol/mol) content and 16SrDNA sequence, the determination of G + C% (mol/mol) adopts a thermal denaturation method, the determination of the 16S rDNA sequence comprises the steps of extracting strain genome DNA, amplifying 16S rDNA by PCR, purifying 16S rDNA, determining the 16S rDNA sequence, comparing the determined 16S rDNA sequence with the 16S rDNA sequence existing in GenBank (www.ncbi.com), and determining the classification status of the strain.

Detailed Description

According to the characteristic that the anaerobic ammonia oxidation reaction is similar to the denitrification reaction, denitrifying bacteria are separated firstly, and then the anaerobic ammonia oxidation activity of the pure culture strain is verified. The method is a novel anaerobic ammonium oxidation bacteria separation method, and practice proves that the method is effective.

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.

Example 1

1. in the embodiment, activated sludge in an aeration tank of an urban sewage treatment plant is selected as a separation source of denitrifying bacteria with anaerobic ammonia oxidation activity.

2. To add NaNO₂And (NH)₄)₂SO₄And a small amount of yeast extract as enrichment substrate, and culturing anaerobic ammonium oxidation floc in a continuous culture mode.

3. Taking anaerobic ammonia oxidation floc, carrying out mild crushing by using ultrasonic waves to prepare primary bacterial suspension, and then diluting step by step to prepare serial bacterial suspension.

5. The method comprises the steps of amplifying denitrifying bacteria by using a liquid denitrifying culture medium, centrifuging to obtain thallus cells, preparing thallus suspension with a certain concentration by using a basic inorganic salt culture medium, adding ammonia and nitrite, and judging whether the strain has anammox activity or not by using the capability of simultaneously converting the ammonia and the nitrite under an anaerobic condition.

6. The classification status of the strains is determined according to morphological characteristics, physiological and biochemical characteristics, carbon source utilization characteristics and molecular biological characteristics of pure culture, and is specifically referred to in the literatures [17 and 18].

1) The morphological characteristics include observation of cultured colonies, observation of cultured cells and observation of cell structure of cells. The morphology of the bacterial cells was observed by staining according to the method described in the literature [16]and by using a Leica research microscope (DMLB + QCOLite) in Germany. Observing the bacterial cell morphology by an electron microscope, centrifugally collecting bacterial cells in a culture solution, suspending the bacterial cells by using normal saline, then negatively staining the bacterial cells by using 2% phosphotungstic acid, preparing a hanging drop sample, and observing the hanging drop sample by adopting a JEM-1200EX transmission electron microscope. And (4) observing the bacterial cell structure by using an electron microscope, and observing by using a JEM-1200EX Transmission Electron Microscope (TEM) after ultrathin slicing.

2) The physiological and biochemical characteristics were identified using the Vitek GN system.

3) Carbon source utilization characteristics were characterized using a Biolog microplate system.

4) Molecular biological characteristics include the determination of G + C% (mol/mol) content and 16S rDNA sequence.

A) G + C% (mol/mol) was determined by thermal denaturation.

B) The steps for determining the 16S rDNA sequence are as follows:

a) and extracting and purifying the genome DNA of the separated bacteria by using a bacterial DNA extraction kit.

b) Amplification of 16S rDNA Using a pair of PCR Universal primers^[16]. Forward primer BSF 8/20: 5'-AGAGTTTGAT CCTGG CTCAG-3' (Escherichia coli corresponding to position 8-27); reverse primer BSR 1495/20: 5'-ACGGC TACCT TGTTA CGACT-3' (Escherichia coli corresponds to position 1495-. The PCR reaction was carried out in a 100. mu.L reaction system: DNA template (1.0. mu. mol/L) 5. mu.L, dNTP mix (final concentration 100. mu. mol) 2. mu.L, Taq DNA polymerase (2.5U) 1. mu.L, forward primer Pf (50pmol) 2. mu.L, reverse primer Pf (50pmol) 2. mu.L, buffer L0. mu.L, water 78. mu.L. And (3) PCR reaction conditions: 94 ℃ for 1 min; 52 ℃, 1min, 72 ℃, 3min, 30 cycles.

c) The 16S rDNA was purified and the sequence of the 16S rDNA was determined by Biotech Ltd.

d) The determined 16S rDNA sequence was aligned with the existing sequences in GenBank (www.ncbi.com) to determine the taxonomic status of the isolates.

Example 2

1. in the embodiment, waterlogged soil is selected as a separation source of denitrifying bacteria with anaerobic ammonium oxidation activity.

A) G + C% (mol/mol) was determined by thermal denaturation.

B) The steps for determining the 16S rDNA sequence are as follows:

b) Amplification of 16S rDNA Using a pair of PCR Universal primers^[16]. Forward primer BSF 8/20: 5'-AGAGTTTGAT CCTGG CTCAG-3' (Escherichia coli corresponding to position 8-27); reverse primer BSRl 495/20: 5'-ACGGC TACCT TGTTA CGACT-3' (Escherichia coli is in the corresponding position1495-1514). The PCR reaction was carried out in a 100. mu.L reaction system: DNA template (1.0. mu. mol/L) 5. mu.L, dNTP mix (final concentration 100. mu. mol) 2. mu.L, Taq DNA polymerase (2.5U) 1. mu.L, forward primer Pf (50pmol) 2. mu.L, reverse primer Pf (50pmol) 2. mu.L, buffer 10. mu.L, water 78. mu.L. And (3) PCR reaction conditions: 94 ℃ for 1 min; 52 ℃, 1min, 72 ℃, 3min, 30 cycles.

Compared with example 1, the present example can separate denitrifying bacteria having anammox activity using a different separation source.

Example 3

2. To add NaNO₂And (NH)₄)₂SO₄And a small amount of glucose in a basic inorganic salt culture medium as an enrichment matrix, and culturing the anaerobic ammonia oxidation floc in a continuous culture mode.

A) G + C% (mol/mol) was determined by thermal denaturation.

B) The steps for determining the 16S rDNA sequence are as follows:

b) Amplification of 16S rDNA Using a pair of PCR Universal primers^[16]. Forward primer BSF 8/20: 5'-AGAGTTTGAT CCTGG CTCAG-3' (Escherichia coli corresponding to position 8-27); reverse primer BSR1495/20: 5'-ACGGC TACCT TGTTA CGACT-3' (Escherichia coli corresponds to position 1495-. The PCR reaction was carried out in a 100. mu.L reaction system: DNA template (1.0. mu. mol/L) 5. mu.L, dNTP mix (final concentration 100. mu. mol) 2. mu.L, Taq DNA polymerase (2.5U) 1. mu.L, forward primer Pf (50pmol) 2. mu.L, reverse primer Pf (50pmol) 2. mu.L, buffer 10. mu.L, water 78. mu.L. And (3) PCR reaction conditions: 94 ℃ for 1 min; 52 ℃, 1min, 72 ℃, 3min, 30 cycles.

In this example, as compared with example 1, denitrifying bacteria having anammox activity were isolated by replacing yeast extract with glucose.

Example 4

3. Taking anaerobic ammonia oxidation floc, crushing the floc by using a glass homogenizer to prepare primary bacterial suspension, and then diluting the primary bacterial suspension step by step to prepare serial bacterial suspensions.

A) G + C% (mol/mol) was determined by thermal denaturation.

B) The steps for determining the 16S rDNA sequence are as follows:

b) Amplification of 16S rDNA Using a pair of PCR Universal primers^[16]. Forward primer BSF 8/20: 5'-AGAGTTTGAT CCTGG CTCAG-3' (Escherichia coli corresponding to position 8-27); reverse primer BSR 1495/20: 5'-ACGGC TACCT TGTTA CGACT-3' (Escherichia coli corresponds to position 1495-. The PCR reaction was carried out in a 100. mu.L reaction system: DNA template (1.0. mu. mol/L) 5. mu.L, dNTP mix (final concentration 100. mu. mol) 2. mu.L, Taq DNA polymerase (2.5U) 1. mu.L, forward primer Pf (50pmol) 2. mu.L, reverse primer Pf (50pmol) 2. mu.L, buffer 10. mu.L, water 78. mu.L. And (3) PCR reaction conditions:94 ℃ for 1 min; 52 ℃, 1min, 72 ℃, 3min, 30 cycles.

Compared with example 1, the present example can also separate denitrifying bacteria with anammox activity by a different disruption method.

Example 5 comparative experiment^[12]

The anaerobic ammonium oxidation bacteria density gradient centrifugal separation method comprises the following steps:

1. active sludge in the biological denitrification fluidized bed reactor is selected as a separation source of anaerobic ammonium oxidation bacteria.

2. The anaerobic ammonia oxidation floc is cultured by taking a basic inorganic salt culture medium as an enrichment matrix and adopting a batch culture mode.

3. Taking anaerobic ammonia oxidation floc, carrying out mild disruption by ultrasonic waves, and centrifuging to remove cell debris to prepare cell suspension.

4. The cell suspension was mixed with Percoll solution and subjected to density gradient centrifugation. After centrifugation, a band of red cells appeared and was in a specific position in the centrifuge tube. The red cell zone is the anammox bacteria.

5. The red cell band in the centrifugal tube is carefully sucked out by a sterile pipette, and the purity of the separated strain can reach that only one polluted cell is contained in every 200-800 cells.

6. Making the isolated strain to have high cell density (higher than 10)¹⁰one/mL) of the strain, adding a liquid inorganic salt culture medium containing ammonia and nitrite, detecting the disappearance of the ammonia and the nitrite, and if the ammonia and the nitrite disappear simultaneously, determining that the strain is anaerobic ammonia oxidizing bacteria.

7. Extracting and measuring the 16S rDNA sequence of the separated strain, and determining the classification status of the strain by comparing with GenBank.

Comparing the two methods described above, the advantages of the invention (examples 1, 2, 3, 4) are: 1) can obtain a pure culture (without pollution cells) of the anaerobic ammonium oxidation bacteria, and provides pure species for microbiological research and industrial application; 2) the denitrifying bacteria with anaerobic ammonium oxidation activity grow faster, the physiological and biochemical characteristics of the separated strains are easy to be ascertained, and the strain preservation and large-scale amplification are facilitated; 3) the separation and identification procedures are simple, the result is credible, and the popularization and the application are easy.

Claims

1. A method for separatingdenitrifying bacteria having anaerobic ammonium oxidation activity, which comprises the steps of:

1) selecting natural habitat and artificial habitat samples with ammonia and nitrate or nitrite as separation sources of denitrifying bacteria with anaerobic ammonium oxidation activity, wherein the natural habitat samples mainly comprise waterlogged soil, marsh land sludge, lake bottom sludge and marine sediments, and the artificial habitat samples mainly comprise aeration tank sludge of an urban sewage treatment plant, anaerobic digestion tank sludge of the urban sewage treatment plant and activated sludge in various biological wastewater denitrification treatment systems.

2) Taking a basic inorganic salt liquid culture medium containing 0.1-1 mg/L yeast extract or glucose as an enrichment matrix, and culturing an anaerobic ammonia oxidation biomembrane, floc or granular sludge in a continuous culture or batch culture mode;

3) taking anaerobic ammonia oxidation biomembranes, flocs or granular sludge, crushing the anaerobic ammonia oxidation biomembranes, the flocs or the granular sludge by ultrasonic waves to prepare primary bacterial suspension, and diluting the primary bacterial suspension step by step to prepare serial bacterial suspensions.

4) Separating denitrifying bacteria with denitrifying culture medium by anaerobic plate streaking method, and anaerobically culturing at 28-30 deg.C. Picking single colony growing on the plate into liquid culture medium, anaerobic culturing at 28-30 deg.C, separating single colony by anaerobic plate streaking, and repeating the above steps for several times. When the bacterial cell morphology observed under the microscope is consistent, the target strain is considered to be purified.

2. The method for separating denitrifying bacteria having anaerobic ammonium oxidation activity according to claim 1The culture medium is characterized in that the formula of the liquid basic inorganic salt culture medium is as follows: KHCO₃500mg/L，KH₂PO₄27.2mg/L，MgSO₄·7H₂O 300mg/L，CaCl₂136mg/L, 1mL/L of trace element I and 1mL/L of trace element II; the formula of the trace element I is as follows: EDTA 5000mg/L, FeSO₄5000 mg/L; the formula of the trace element II comprises: : EDTA 15000mg/L, ZnSO₄·7H₂O 430mg/L，CoCl₂·6H₂O 240mg/L，MnCl₂·4H₂O 990mg/L，CuSO₄·5H₂O 250mg/L，Na₂MoO₄·2H₂O 220mg/L，NiCl₂·6H₂O 190mg/L，Na₂SeO₄·10H₂O 210mg/L，H₃BO₄14 mg/L; the formula of the yeast extract or the glucose stock solution is as follows: yeast extract or glucose 100 mg/L.

3. The method for separating denitrifying bacteria with anaerobic ammonium oxidation activity according to claim 1, wherein the formula of the liquid denitrifying medium is as follows: KH (Perkin Elmer)₂PO₄30mg/L，KHCO₃500mg/L，MgSO₄200mg/L，FeCl₃100mg/L，CaCl₂30mg/L，CH₃COONa 860mg/L，(NH₄)₂SO₄660mg/L，NaNO₃850 mg/L. The formula of the solid denitrification culture medium is as follows: agar of 20g/L is added on the basis of the formula of the liquid culture medium.

4. A method for identifying denitrifying bacteria having anaerobic ammonium oxidation activity, which comprises the steps of:

1) amplifying a pure culture of denitrifying bacteria in a liquid denitrifying culture medium, centrifuging at 5000rpm to obtain thallus cells, preparing a thallus suspension by using a basic inorganic salt culture medium, adding ammonia and nitrite, and judging whether the strain has anammox capability, namely anammox bacteria, according to the capability of simultaneously converting ammonia and nitrite under anaerobic conditions;

2) determining the classification status of the pure strain according to the morphological characteristics, physiological and biochemical characteristics, carbon source utilization characteristics and molecular biological characteristics of the pure strain, wherein the morphological characteristics comprise observation of cultured colonies, observation of cultured thalli and observation of thalli cell structures; the physiological and biochemical characteristics are identified by a Vitek GN system; the carbon source utilization characteristics are identified by adopting a Biolog microplate system; the molecular biological characteristics comprise the determination of G + C% (mol/mol) content and 16SrDNA sequence, the determination of G + C% (mol/mol) adopts a thermal denaturation method, the determination of the 16S rDNA sequence comprises the steps of extracting strain genome DNA, amplifying 16S rDNA by PCR, purifying 16S rDNA, determining the 16S rDNA sequence, comparing the determined 16S rDNA sequence with the 16S rDNA sequence existing in GenBank (www.ncbi.com), and determining the classification status of the strain.