CN1807593A - Separation and authentication method for Planctomyces with anaerobic ammoxidation activity - Google Patents
Separation and authentication method for Planctomyces with anaerobic ammoxidation activity Download PDFInfo
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Abstract
This invention discloses a method for separating and attesting of anaerobic ammoxidation active planctomycete, comprising these steps: Firstly separate planctomycete; affirm the bacterium is anaerobic ammoxidation bacterium according to its ability to transform ammonia and oxonitrite simultaly under anaerobic condition; finally attest classification status of the bacterium. Add minim yeast cream or glucose in basis abio-salt culture medium, which is suitable for planctomycete growing. At the same time, adopt a new attest procedure for anaerobic ammoxidation bacterium: firstly purify strain and affirm anaerobic ammoxidation active; then attest its bacterial. The advantanges of this method contains: 1) pure culture of anaerobic ammoxidation bacterium can be achieved, according can supply bacterial for microbiological studies and industrialization appliance; 2) physiology and biochemistry characteristics of the separated strain can be proved up easily, and this is convenient for the conservation and large expandation of the strain; 3) the procedure of separation and attesting is simple and the result is believable.
Description
Technical Field
The invention relates to a method for separating and identifying a planctomycete with anaerobic ammonia 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, it breaks through the classic nitrification-denitrification theory,based on the above, people have developed new biological denitrification processes, such as short-cut nitrification (SHARON) -anaerobic ammonia oxidation process, OLAND process, CANON process, etc. 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 can be detected in marine sediments, deep-water anoxic zones, lake sediments, 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 anammox bacteria reported in the literature so far have five species, which are respectively assigned to three genera of Phycomycetes[2，8，9]. The five anaerobic ammonium oxidation bacteria are obtained by separation by adopting a density gradient centrifugation method[10]No report of successfully obtaining the anammox bacteria by adopting a classical microorganism separation method exists. In other words, until now no pure culture of anammox bacteria has been obtained.
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]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
[3]Gable J E.Anaerobic ammonium oxidation during soil aquifer treatment(D).Arizona state university，2002.
[4]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
[5]Toh SK and Ashbolt NJ.Adaptation of anaerobic ammonium-oxidizingconsortium to synthetic coke-ovens wastewater.Appl Microbiol Biotechnol，2002，59：344-352
[6]Trimmer M，Nicholls JC，Deflandre B.Anaerobic ammonium oxidation measuredin sediments along the Thames Estuary，United Kingdom.Appl.Environ.Microbiol.2003，69：6447-6454.
[7]Devol A H.Solution to a marine mystery [J].Nature，2003，422(6932)：575～576.
[8]Strous M，Fuerst JA，Kramer EHM，et al.Missing lithotroph identified as newplanctomycete.Nature 1999，400：446-449
[9]Schmid，M.，Walsh，K.，Webb，R.，et al.andidatus ″Scalindua brodae″，sp.nov.，Candidatus ″Scalindua wagnei″，sp.nov.，two new species of anaerobicammonium oxidizing bacteria.Systematic and Applied Microbiology 2003，26，529-538
[10]Strous M，Kuenen JG，Fuerst JA，et al.The anammox--a new experimentalmanifesto for microbiological eco-physiology.Antonie van Leeuvenoek，2002，81：693-702.
[11]dongxu pearl, zeisingling, et al.
[12]Holt JG.，Krieg NR.，Sneath PHA.et al.Bergey′s manual of determinativebacteriology(9th edition )Williams&Wilkins，1994：427-455.
[13]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 a planctomycete with anaerobic ammonia oxidation activity.
The separation steps of the mycoides with anaerobic ammonia oxidation activity are as follows:
1) selecting natural habitat and artificial habitat samples with ammonia and nitrate or nitrite as separation sources of the hymenium with anaerobic ammonia 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 manner;
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) mixing the series of bacterial suspensions in a melted solid culture medium, carrying out anaerobic rolling tube method culture at 28-30 ℃, picking single bacterial colonies growing on an anaerobic rolling tube into a liquid culture medium by using a capillary tube, carrying out anaerobic culture at 28-30 ℃, separating the single bacterial colonies by adopting the anaerobic rolling tube method after culture, repeating the steps for a plurality of times, and considering that the target bacterial strain is purified when the bacterial cells observed under a microscope have consistent shapes.
The formula of the liquid culture medium is as follows: KHCO3500mg/L，KH2PO427.2mg/L，MgSO47H2O 300mg/L，CaCl2136mg/L，NaNO2140mg/L，NH4)2SO4140mg/L, 1mL/L of trace element I, 1mL/L of trace element II and 1mL/L of yeast extract or glucose stock solution; wherein the formula of the trace element I is as follows: EDTA 5000mg/L, FeSO45000 mg/L; the formula of the trace element II comprises: EDTA15000mg/L, ZnSO47H2O 430mg/L，CoCl26H2O 240mg/L，MnCl24H2O 990mg/L，CuSO45H2O 250mg/L，Na2MoO42H2O 220mg/L，NiCl26H2O 190mg/L，Na2SeO410H2O 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.
The solid medium was prepared by adding agar to the above liquid medium at a concentration of 20 g/L.
The identification method of the phytophthora with anaerobic ammonia oxidation activity comprises the following steps:
1) amplifying a pure culture of the planctomycete in a liquid 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 is an anammox bacterium or not according to the capability of simultaneously converting the ammonia and the nitrite under an anaerobic condition;
2) the classification status of the strain is determined by the thallus morphology and the 16S rDNA sequence, the thallus morphology comprises the observation of a culture colony, the observation of a culture thallus and the observation of a thallus cell structure, the determination step of the 16S rDNA sequence comprises the steps of extracting the genome DNA of the strain, amplifying the 16S rDNA by PCR, purifying the 16S rDNA, determining the 16SrDNA sequence, and comparing the determined 16S rDNA sequence with the 16SrDNA sequence existing in GenBank (www.ncbi.com) to determine the classification status of the strain.
The invention has the advantages that: 1) can obtain a pure culture of the anaerobic ammonium oxidation bacteria, and provides strains for microbiological research and industrial application; 2) the physiological and biochemical characteristics of the separated strain are known, so that the strain is convenient to preserve and massively amplify; 3) the identification procedure is simple, and the result is credible.
Detailed Description
According to the present invention, based on the current situation that five kinds of separated anammox bacteria are the hymexazol bacteria, a pure culture of the hymexazol bacteria is successfully obtained by adopting a culture medium which is independently developed and suitable for the growth of the hymexazol bacteria, and the anammox activity of the pure culture strain is confirmed.
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto.
Example 1
The steps for separating and identifying the anammox bacteria are as follows:
1. in the embodiment, activated sludge in an aeration tank of an urban sewage treatment plant is selected as a separation source of the hymexazol with anaerobic ammonia oxidation activity.
2. And (3) adopting a continuous culture mode, and taking a basic inorganic salt liquid culture medium added with trace yeast extract as an enrichment matrix to culture the anaerobic ammonia oxidation flocs.
3. Taking anaerobic ammonia oxidation floc, crushing the floc by ultrasonic waves to prepare bacterial suspension, and then diluting the bacterial suspension step by step to prepare the bacterial suspension.
4. Mixing the series of bacterial suspensions in melted solid culture medium, performing anaerobic rolling tube, and performing anaerobic culture at 28-30 deg.C. After culturing for 7-14 days, picking the single colony growing on the anaerobic roller into a liquid culture medium by using a capillary tube, and continuing anaerobic culture for 7-14 days at 28-30 ℃. After culturing, separating single bacterial colony by anaerobic tube rolling 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.
5. Amplifying a pure culture by using a liquid culture medium, centrifuging to obtain thallus cells, preparing thallus suspension 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 the anaerobic condition.
6. The classification status of the strains is determined according to morphological characteristics and molecular biological characteristics of the pure culture, and is specifically referred to documents [12 and 13].
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 reference [11]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 molecular biological characteristics are mainly 16S rDNA sequences, and the determination steps 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[11]. 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.
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
The steps for separating and identifying the anammox bacteria are as follows:
1. in the embodiment, activated sludge in an aeration tank of an urban sewage treatment plant is selected as a separation source of the hymexazol with anaerobic ammonia oxidation activity.
2. And (3) adopting a continuous culture mode, and taking a basic inorganic salt liquid culture medium added with trace glucose as an enrichment matrix to culture the anaerobic ammonia oxidation flocs.
3. Taking anaerobic ammonia oxidation floc, crushing the floc by ultrasonic waves to prepare bacterial suspension, and then diluting the bacterial suspension step by step to prepare the bacterial suspension.
4. Mixing the series of bacterial suspensions in melted solid culture medium, performing anaerobic rolling tube, and performing anaerobic culture at 28-30 deg.C. After culturing for 7-14 days, picking the single colony growing on the anaerobic roller into a liquid culture medium by using a capillary tube, and continuing anaerobic culture for 7-14 days at 28-30 ℃. After culturing, separating single bacterial colony by anaerobic tube rolling 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.
5. Amplifying a pure culture by using a liquid culture medium, centrifuging to obtain thallus cells, preparing thallus suspension 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 the anaerobic condition.
6. The classification status of the strains is determined according to morphological characteristics and molecular biological characteristics of the pure culture, and is specifically referred to documents [12 and 13].
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 reference [11]and by using a Leica research microscope (DMLB + QCOLite) in Germany. Observing the bacterial cell morphology by anelectron 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 molecular biological characteristics are mainly 16S rDNA sequences, and the determination steps 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[11]. 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.
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.
In this example, the yeast extract was changed to glucose in comparison with example 1, and the isolated mycosphaerella fungi having anammox activity wasalso obtained.
Example 3
The steps for separating and identifying the anammox bacteria are as follows:
1. in the example, waterlogged soil is selected as a separation source of the mycoderm fungi with anaerobic ammonia oxidation activity.
2. And (3) adopting a continuous culture mode, and taking a basic inorganic salt liquid culture medium added with trace yeast extract as an enrichment matrix to culture the anaerobic ammonia oxidation flocs.
3. Taking anaerobic ammonia oxidation floc, crushing the floc by using a glass homogenizer to prepare bacterial suspension, and then diluting the bacterial suspension step by step in series to prepare the bacterial suspension.
4. Mixing the series of bacterial suspensions in melted solid culture medium, performing anaerobic rolling tube, and performing anaerobic culture at 28-30 deg.C. After culturing for 7-14 days, picking the single colony growing on the anaerobic roller into a liquid culture medium by using a capillary tube, and continuing anaerobic culture for 7-14 days at 28-30 ℃. After culturing, separating single bacterial colony by anaerobic tube rolling 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.
5. Amplifying a pure culture by using a liquid culture medium, centrifuging to obtain thallus cells, preparing thallus suspension 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 the anaerobic condition.
6. The classification status of the strains is determined according to morphological characteristics and molecular biological characteristics of the pure culture, and is specifically referred to documents [12 and 13].
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 reference [11]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 molecular biological characteristics are mainly 16S rDNA sequences, and the determination steps 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[11]. 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.
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.
Compared with the example 1, the method can also separate the phytophthora parasitica with anammox activity by adopting different separation sources and different anaerobic ammonia oxidation floc crushing modes.
Example 4
The steps for separating and identifying the anammox bacteria are as follows:
1. in the embodiment, activated sludge in an aeration tank of an urban sewage treatment plant is selected as a separation source of the hymexazol with anaerobic ammonia oxidation activity.
2. And (3) adopting a continuous culture mode, and taking a basic inorganic salt liquid culture medium as an enrichment matrix to culture the anaerobic ammonia oxidation flocs.
3. Taking anaerobic ammonia oxidation floc, crushing the floc by using a glass homogenizer to prepare bacterial suspension, and then diluting the bacterial suspension step by step in series to prepare the bacterial suspension.
4. Mixing the series of bacterial suspensions in melted solid culture medium, performing anaerobic rolling tube, and performing anaerobic culture at 28-30 deg.C. After culturing for 7-14 days, picking the single colony growing on the anaerobic roller into a liquid culture medium by using a capillary tube, and continuing anaerobic culture for 7-14 days at 28-30 ℃. After culturing, separating single bacterial colony by anaerobic tube rolling 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.
5. Amplifying a pure culture by using a liquid culture medium, centrifuging to obtain thallus cells, preparing thallus suspension 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 the anaerobic condition.
6. The classification status of the strains is determined according to morphological characteristics and molecular biological characteristics of the pure culture, and is specifically referred to documents [12 and 13].
In contrast to example 1, this example uses a different enrichment and isolation medium, i.e.no yeast extract or glucose was added to the minimal medium. In this example, the culture mixture of anammox bacteria was enriched, but a pure culture of planctomycete having anammox activity could not be isolated.
Example 5 comparative experiment[10]
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, crushing by ultrasonic waves, centrifuging to remove cell debris, and preparing 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 above examples, the enrichment and separation of anammox bacteria are not affected by using different sources of separation and disruption, but only the mixed culture of anammox bacteria can be enriched and the pure culture of the anammox bacteria can not be obtained if yeast extract or glucose is not added to the culture medium.
The advantages of the invention (examples 1, 2, 3) compared to the comparative experiment (example 5) 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 physiological and biochemical characteristics of the separated strain are easy to be ascertained, so that the strain preservation and the large-scale amplification are convenient; 3) the separation and identification procedures are simple, and the result is credible.
Claims (4)
1. A method for separating a fungus having anaerobic ammonia oxidation activity, comprising the steps of:
1) selecting natural habitat and artificial habitat samples with ammonia and nitrate or ammonia and nitrite as separation sources of the pumice fungi with anaerobic ammonia oxidation activity, wherein the natural habitat samples mainly comprise waterlogged soil, marsh land sludge, lake sediment 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) mixing the series of bacterial suspensions in a melted solid culture medium, carrying out anaerobic rolling tube method culture at 28-30 ℃, picking single bacterial colonies growing on an anaerobic rolling tube into a liquid culture medium by using a capillary tube, carrying out anaerobic culture at 28-30 ℃, separating the single bacterial colonies by adopting the anaerobic rolling tube method after culture, repeating the steps for a plurality of times, and considering that the target bacterial strain is purified when the bacterial cells observed under a microscope have consistent shapes.
2. The method for isolating Phycomycetes having anammox activity according to claim 1, wherein the liquid medium comprises: KHCO3500mg/L，KH2PO427.2mg/L，MgSO47H2O 300mg/L，CaCl2136mg/L，NaNO2140mg/L，(NH4)2SO4140mg/L, 1mL/L of trace element I, 1mL/L of trace element II and 1mL/L of yeast extract or glucose stock solution; wherein the formula of the trace element I is as follows: EDTA 5000mg/L, FeSO45000 mg/L; trace element II formulaThe method comprises the following steps: EDTA15000mg/L, ZnSO47H2O 430mg/L，CoCl26H2O 240mg/L，MnCl24H2O990mg/L，CuSO45H2O 250mg/L，Na2MoO42H2O 220mg/L，NiCl26H2O 190mg/L，Na2SeO410H2O 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 a planctomycete having anammox activity according to claim 1 or 2, wherein the solid medium is agar added in an amount of 20g/L to the liquid medium.
4. A method for identifying a planctomycete having anammox activity, the method comprising the steps of:
1) amplifying a pure culture of the planctomycete in a liquid 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 is an anammox bacterium or not according to the capability of simultaneously converting the ammonia and the nitrite under an anaerobic condition;
2) the classification status of the strain is determined by the thallus morphology and the 16S rDNA sequence, the thallus morphology comprises the observation of a culture colony, the observation of a culture thallus and the observation of a thallus cell structure, the determination step of the 16S rDNA sequence comprises the steps of extracting the genome DNA of the strain, amplifying the 16S rDNA by PCR, purifying the 16S rDNA, determining the 16SrDNA sequence, and comparing the determined 16S rDNA sequence with the 16SrDNA sequence existing in GenBank (www.ncbi.com) to determine the classification status of the strain.
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2006
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101402990B (en) * | 2008-07-18 | 2011-04-20 | 哈尔滨工业大学 | Culture medium for sifting anaerobic denitrifying bacteria and method for sifting anaerobic denitrifying bacteria |
| CN101886056A (en) * | 2010-07-02 | 2010-11-17 | 北京桑德环保集团有限公司 | Method for separating anaerobic ammonia oxidizing bacteria |
| CN102329874A (en) * | 2011-10-10 | 2012-01-25 | 北京工业大学 | Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method of ammonia-oxidizing bacteria in active sludge |
| CN102329874B (en) * | 2011-10-10 | 2013-11-06 | 北京工业大学 | Real-time fluorescent quantitative PCR (polymerase chain reaction) detection method of ammonia-oxidizing bacteria in active sludge |
| CN103614284A (en) * | 2013-12-05 | 2014-03-05 | 哈尔滨工业大学 | Enrichment device of free-state anaerobic ammonium oxidation bacteria and using method thereof |
| CN103614284B (en) * | 2013-12-05 | 2015-06-17 | 哈尔滨工业大学 | Enrichment device of free-state anaerobic ammonium oxidation bacteria and using method thereof |
| CN110055343A (en) * | 2019-04-30 | 2019-07-26 | 清华大学 | A kind of specific primer polluted for monitoring the quick-acting phosphorus loads of deposit |
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| CN100400650C (en) | 2008-07-09 |
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