CN113249254A - Pseudomonas nitroreducens strain and application thereof - Google Patents
Pseudomonas nitroreducens strain and application thereof Download PDFInfo
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- 241000204735 Pseudomonas nitroreducens Species 0.000 title claims abstract description 53
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 claims abstract description 19
- 241000589516 Pseudomonas Species 0.000 claims abstract description 15
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000002351 wastewater Substances 0.000 claims abstract description 13
- 238000004321 preservation Methods 0.000 claims abstract description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 38
- 229910052757 nitrogen Inorganic materials 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 229910002651 NO3 Inorganic materials 0.000 claims description 11
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 9
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 2
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 2
- 230000003321 amplification Effects 0.000 claims description 2
- 238000009360 aquaculture Methods 0.000 claims description 2
- 244000144974 aquaculture Species 0.000 claims description 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 2
- 238000000746 purification Methods 0.000 claims description 2
- 238000000926 separation method Methods 0.000 claims description 2
- 238000011081 inoculation Methods 0.000 claims 1
- 230000001788 irregular Effects 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 9
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 6
- 229910052799 carbon Inorganic materials 0.000 abstract description 6
- 230000008569 process Effects 0.000 abstract description 5
- 238000012423 maintenance Methods 0.000 abstract description 2
- 239000001963 growth medium Substances 0.000 description 11
- 239000002609 medium Substances 0.000 description 10
- MMDJDBSEMBIJBB-UHFFFAOYSA-N [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] Chemical compound [O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O.[NH6+3] MMDJDBSEMBIJBB-UHFFFAOYSA-N 0.000 description 9
- 239000007788 liquid Substances 0.000 description 7
- 108020004465 16S ribosomal RNA Proteins 0.000 description 5
- 241000894006 Bacteria Species 0.000 description 5
- IPQVRLSXWJPESU-UHFFFAOYSA-N [N].ON=O Chemical compound [N].ON=O IPQVRLSXWJPESU-UHFFFAOYSA-N 0.000 description 5
- 229910052564 epsomite Inorganic materials 0.000 description 5
- 239000001509 sodium citrate Substances 0.000 description 5
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 5
- 244000005700 microbiome Species 0.000 description 4
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 4
- 238000002798 spectrophotometry method Methods 0.000 description 4
- 239000007836 KH2PO4 Substances 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- ZPLCXHWYPWVJDL-UHFFFAOYSA-N 4-[(4-hydroxyphenyl)methyl]-1,3-oxazolidin-2-one Chemical compound C1=CC(O)=CC=C1CC1NC(=O)OC1 ZPLCXHWYPWVJDL-UHFFFAOYSA-N 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- JVMRPSJZNHXORP-UHFFFAOYSA-N ON=O.ON=O.ON=O.N Chemical compound ON=O.ON=O.ON=O.N JVMRPSJZNHXORP-UHFFFAOYSA-N 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 239000013067 intermediate product Substances 0.000 description 2
- 229910052603 melanterite Inorganic materials 0.000 description 2
- 230000001546 nitrifying effect Effects 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000012266 salt solution Substances 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- ZCUQOPGIJRGJDA-UHFFFAOYSA-N 1-naphthalen-1-ylethane-1,2-diamine Chemical compound C1=CC=C2C(C(N)CN)=CC=CC2=C1 ZCUQOPGIJRGJDA-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- 241000589774 Pseudomonas sp. Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 229910052927 chalcanthite Inorganic materials 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 150000002826 nitrites Chemical class 0.000 description 1
- GQPLMRYTRLFLPF-UHFFFAOYSA-N nitrous oxide Inorganic materials [O-][N+]#N GQPLMRYTRLFLPF-UHFFFAOYSA-N 0.000 description 1
- 238000013081 phylogenetic analysis Methods 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 235000010333 potassium nitrate Nutrition 0.000 description 1
- 239000004323 potassium nitrate Substances 0.000 description 1
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 238000011218 seed culture Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229940074404 sodium succinate Drugs 0.000 description 1
- ZDQYSKICYIVCPN-UHFFFAOYSA-L sodium succinate (anhydrous) Chemical compound [Na+].[Na+].[O-]C(=O)CCC([O-])=O ZDQYSKICYIVCPN-UHFFFAOYSA-L 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 238000003911 water pollution Methods 0.000 description 1
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- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/30—Aerobic and anaerobic processes
- C02F3/302—Nitrification and denitrification treatment
- C02F3/303—Nitrification and denitrification treatment characterised by the nitrification
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- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/30—Aerobic and anaerobic processes
- C02F3/302—Nitrification and denitrification treatment
- C02F3/305—Nitrification and denitrification treatment characterised by the denitrification
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- C02F2101/10—Inorganic compounds
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Abstract
A nitroreduction Pseudomonas strain and application thereof are disclosed, wherein the strain is nitroreduction Pseudomonas (Pseudomonas nitroreducens) pf2 which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2021274. The strain can synchronously carry out nitrification and denitrification by taking organic carbon as a carbon source under aerobic conditions, and is suitable for removing NH from organic wastewater containing ammonia nitrogen or nitrate4 +The removal rate of-N can reach 92.7 percent, and NO3 ‑The N removal rate can reach 100 percent; and greatly simplifying the treatment process and improving the treatment effectAnd the operation and maintenance cost is reduced.
Description
Technical Field
The invention belongs to the technical field of environmental protection, and particularly relates to a pseudomonas nitroreducens strain with heterotrophic nitrification and aerobic denitrification functions and application thereof.
Background
Nitrogen pollution of water bodies becomes the most serious environmental problem in the world, and biological denitrification is the most economical and effective disposal method in water pollution treatment. The traditional biological denitrification mainly comprises nitrification and denitrification. The biological nitrification and denitrification processes are interdependent, but because of the great difference in physiology and biochemistry between nitrifying and denitrifying microorganisms, there are different tolerances and manifestations to Dissolved Oxygen (DO), temperature, pH, growth rate, Chemical Oxygen Demand (COD), and nitrates and nitrites. This makes it difficult to balance and coordinate the conventional nitrification and denitrification processes, resulting in a very complicated denitrification process and low efficiency. In recent years, many microorganisms having heterotrophic nitrification-aerobic denitrification (HN-AD) functions have been screened and applied to biological denitrification systems. HN-AD bacteria have higher cell growth rate than the traditional nitrifying microorganisms, and can utilize an organic substrate as a carbon source and simultaneously treat nitrogen pollutants with different forms, such as ammonia Nitrogen (NH)4 +-N), nitrate Nitrogen (NO)3 --N) and nitrous Nitrogen (NO)2 --N) into a nitrogen-containing gas.
Disclosure of Invention
The invention provides a nitroreduction pseudomonas strain for enriching a denitrification strain resource library, wherein the nitroreduction pseudomonas strain is a heterotrophic nitrification-aerobic denitrification strain, can enable nitrification and denitrification to be carried out simultaneously, and has great application value in the aspect of sewage treatment.
The technical scheme of the invention is as follows:
a nitroreduction Pseudomonas strain is a nitroreduction Pseudomonas (Pseudomonas nitroreducens) pf2, which is preserved in China Center for Type Culture Collection (CCTCC) at 3 months and 25 days 2021, and the preservation number is CCTCC NO: m2021274; the address is as follows: wuhan university school of eight-channel 299 # in Wuchang area of Wuhan city, Hubei province.
In a further embodiment, the effective sequence length of the 16SrDNA of Pseudomonas nitroreducens (Pseudomonas nitroreducens) pf2 is 1498bp, and the sequence is shown as SEQ ID NO: 1 is shown.
In a further scheme, the Pseudomonas nitroreducens (Pseudomonas nitroreducens) pf2 is obtained by enrichment culture, separation and purification of bottom mud of an aquaculture pond.
In a further scheme, the Pseudomonas nitroreducens (Pseudomonas nitroreducens) pf2 is a gram-negative bacillus, is in a short rod shape, has neat edges and has milky colony.
In a further embodiment, the Pseudomonas nitroreducens pf2 is grown using ammonium sulfate or nitrate as the sole nitrogen source.
Another object of the invention is to provide an application of the Pseudomonas nitroreducens strain, wherein pf2 is used for nitrogen removal of nitrogen-containing water.
In a further scheme, the nitrogen-containing water body refers to organic wastewater containing ammonia nitrogen and nitrate.
In a further scheme, the Pseudomonas nitroreducens (Pseudomonas nitroreducens) pf2 is inoculated in a nitrogen-containing water body for aerobic continuous culture after being subjected to expanded culture.
In a further scheme, the temperature of the aerobic continuous culture is 30-35 ℃; the inoculum size of the Pseudomonas nitroreducens pf2 was 1% (volume by volume).
The effective sequence length of the 16SrDNA of the nitroreduction Pseudomonas (Pseudomonas nitroreducens) pf2 is 1498bp, and the sequence is shown as SEQ ID NO: 1 is shown. The strain was identified to belong to the genus Pseudomonas (Pseudomonas sp.) by BLAST sequence homology alignment and was designated as Pseudomonas nitroreducens (pf 2).
The technical scheme of the invention has the following advantages:
1. the Pseudomonas nitroreducens pf2 is a heterotrophic nitrification-aerobic denitrification strain, namely, the Pseudomonas nitroreducens (Pseudomonas nitroreducens) can synchronously carry out nitrification and denitrification by taking organic carbon as a carbon source under aerobic conditions, and is suitable for removing organic wastewater containing ammonia nitrogen or nitrate.
2. Compared with the traditional denitrification technology, the invention adopts Pseudomonas nitroreducens (pf 2) for denitrification of the nitrogen-containing water body, thereby greatly simplifying the treatment process, improving the treatment efficiency and reducing the operation and maintenance cost.
3. The Pseudomonas nitroreducens (Pseudomonas nitroreducens) pf2 has a remarkable ammonia nitrogen removal effect. The nitroreduction pseudomonas pf2 is added into ammonia nitrogen wastewater with the initial concentration of 175mg/L, the temperature is 30 ℃, the rotating speed is 220rpm, the TN removal rate can reach 100 percent, and the NH content is4 +The removal rate of-N can reach 92.7%, the accumulation of intermediate products is less, and the ammonia nitrogen removal effect is good.
4. The strain pf2 has aerobic denitrification capability: in nitrate wastewater with the initial concentration of 100mg/L, the temperature is 30 ℃, the rotating speed is 220rpm, the TN removal rate can reach 95.5 percent, and NO is3 -The removal rate of-N can reach 100 percent, and the aerobic denitrification capability is obvious.
Drawings
The biological denitrification of Pseudomonas nitroreducens pf2 will be illustrated by the drawings in the present application.
FIG. 1 is a 16S rDNA phylogenetic clade of Pseudomonas nitroreducens pf 2;
FIG. 2 shows the growth characteristics of said Pseudomonas nitroreducens with ammoniacal nitrogen and nitrate nitrogen as the sole nitrogen source;
FIG. 3 shows the denitrification capacity of Pseudomonas nitroreducens pf2 in ammonia nitrogen wastewater with an initial concentration of 175 mg/L;
FIG. 4 is a denitrification performance analysis of Pseudomonas nitroreducens pf2 using nitrate as a nitrogen source.
Detailed Description
The present invention will be described in further detail with reference to the following examples and the accompanying drawings.
The methods for measuring total nitrogen, ammonia nitrogen, nitrate nitrogen and nitrite nitrogen referred to in the following examples are as follows:
the total nitrogen is measured by alkaline potassium persulfate digestion ultraviolet spectrophotometry (GB 11894-89);
ammonia nitrogen is measured by adopting a method for measuring the ammonia nitrogen in water by adopting a nano reagent spectrophotometry (HJ 535-2009);
nitrate Nitrogen (NO)3 --N) respectively adopting a method (HZ-HJ-SZ-0138) for measuring nitrate nitrogen by adopting ultraviolet spectrophotometry water quality;
nitrous acid Nitrogen (NO)2 -The measurement of-N) was carried out by using naphthyl ethylenediamine hydrochloride spectrophotometry.
Example 1:
a nitroreduction Pseudomonas strain is a nitroreduction Pseudomonas (Pseudomonas nitroreducens) pf2, which is preserved in China Center for Type Culture Collection (CCTCC) at 3 months and 25 days 2021, with the preservation numbers as follows: CCTCC NO: m2021274; the address is as follows: wuhan university school of eight-channel 299 # in Wuchang area of Wuhan city, Hubei province.
1. Enrichment and isolation of strains
1.1 evenly mixing and standing part of bottom mud collected from the culture pond by using normal saline, and taking supernatant as a sample dilution stock solution. Adding the diluted stock solution into a heterotrophic nitrification enrichment medium according to the volume ratio of 10 percent, and performing shake culture at the temperature of 30 ℃ and the rpm/min for 3 days.
Wherein, the heterotrophic nitrification enrichment culture medium: will be (NH)4)2SO40.472g, 4.052g of sodium succinate and 50ml of Vickers salt solution, adding water for dissolution, supplementing distilled water to 1L, and adjusting the pH to 7.0. Wherein the solute in the Vickers salt solution (g/L) is composed of the following raw materials: k2HPO4 5.0g，FeSO4·7H2O 0.05g，NaCl 2.5g，MgSO4·7H2O 2.5g，MnSO4·4H2O 0.05g。
1.2 inoculating the domesticated microorganisms into an aerobic denitrification culture medium for further screening, wherein the aerobic denitrification culture medium takes potassium nitrate as a unique nitrogen source.
Aerobic denitrification culture medium: 0.36g/L KNO3，10.55g/L Na2HPO4·12H2O，1.5g/L KH2PO4， 0.1g/L MgSO4·7H2O, 4.0g/L sodium citrate and 0.2 percent (volume ratio) of trace element solution. Wherein the trace element solution: 50g/L EDTA-Na2, 2.2g/L ZnSO4，5.5g/L CaCl2，5.06g/L MnCl2·4H2O， 5.0g/L FeSO4·7H2O，1.57g/L CuSO4·5H2O，1.61g/L CoCl2·6H2O。
1.3 taking 1mL of sample which is subjected to primary screening by the aerobic denitrification culture medium, uniformly coating the sample on a solid GN chromogenic culture medium, and then placing the sample in a constant temperature incubator at 30 ℃ for culture to obtain bacterial colonies.
1.4 to further verify that the single colony picked has the property of changing the GN color medium to blue, the single colony which is blue in the solid GN color medium was picked and inoculated into 3mL of sterilized liquid GN color medium, shaking cultured at 30 ℃ and 150r/min, the color change was observed, and a strain which can change the liquid GN color medium from green to blue was selected as a rescreened strain.
Wherein the liquid GN chromogenic medium consists of: 1.0g/L KNO38.5g/L sodium citrate, 1.0g/L L-asparagine, 1.0g/L KH2PO4，1.0g/L MgSO4·7H2O，0.2g/L CaCl2·6H2O，0.05g/L FeCl3·6H2O, 0.1% (by volume) bromothymol blue (BTB) solution, and the pH was adjusted to 7.0.
Adding agar 2% to the liquid chromogenic medium to prepare solid chromogenic medium, and sterilizing at 121 deg.C for 20 min.
2. Molecular biological identification
Selecting single colony of one of the rescreened bacteria for 16S rRNA gene amplification, and performing PC by using common primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5 '-TACGGYTACCTTGTTACGACTT-3') for 16S rRNA geneAnd (3) amplifying R. Wherein the 50 mu L PCR reaction system comprises: 2 XTaq PCR Master Mix 25. mu.L, 27F (10. mu.M) 1. mu.L, 1492R (10. mu.M) 1. mu.L, DNA template as single colony picked, ddH2O24. mu.L. Setting a PCR program: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 45s, and extension at 72 ℃ for 40s, and amplification for 30 cycles; final extension at 72 ℃ for 10 min. The 16S rDNA Sequence obtained by sequencing was submitted to Genbank database (https:// blast.ncbi.nlm.nih.gov/blast.cgi) for Sequence alignment, the database used was 16S ribosome RNAAdatabes, and the most similar species to the target strain was determined according to the highest Sequence coverage (Sequence coverage) and similarity (Identity).
The effective sequence length of the sequenced 16SrDNA is 1498bp, and the sequence is shown as SEQ ID NO: 1 is shown. The genetic relationship between the strain and other strains is shown in figure 1, phylogenetic analysis confirms that the 16S rDNA sequence of the bacterium has more than 99 percent of homology with nitroreduction Pseudomonas (Pseudomonas nitroreducens) published in NCBI database, so that the bacterium is confirmed to be nitroreduction Pseudomonas and named as nitroreduction Pseudomonas pf2, the strain is sent to China center for type culture collection for preservation, and the preservation numbers are as follows: CCTCC NO: m2021274; the preservation date is as follows: 3 months and 25 days 2021; the address is as follows: wuhan university school of eight-channel 299 # in Wuchang area of Wuhan city, Hubei province.
Example 2, the removal capacity of pseudomonas nitroreducens pf2 on ammonia nitrogen and total nitrogen in wastewater.
By NH4 +N is nitrogen source, sodium citrate is carbon source, NH is in waste water4 +The growth and ammonia nitrogen and total nitrogen removal capacity of strain pf2 were examined at an N concentration of 175 mg/L.
Selecting the bacterial colony of the re-screened bacteria separated in the example 1 to a heterotrophic nitrification ammonia nitrogen culture medium (NM) and culturing for 18h at the speed of 220rpm of a 30 ℃ gas bath shaking table, and specifically comprising the following steps: taking liquid strains (2ml) according to the volume ratio of 1% of the culture medium, and inoculating the liquid strains into 200ml NM (heterotrophic nitrification ammonia nitrogen culture medium). The formula of NM culture medium is: (NH)4)2SO40.945g/L, 16.34g/L sodium citrate and MgSO4·7H2O 1g/L，KH2PO4 0.25g/L，Na2HPO4 0.3g/L。
As can be seen from FIG. 2, the strain was grown in log phase for 8h, in plateau phase for 24h, and the OD600 of the strain was raised to 1.909 for 32 h. As can be seen from FIG. 3, the removal rate of the bacterial strain to ammonia nitrogen reaches 91.1%, the removal rate to total nitrogen reaches 100%, and nitrate nitrogen and nitrite nitrogen as intermediate products accumulate less. The strain pf2 is shown to have better treatment effect on high-concentration ammonia nitrogen wastewater.
Example 3 removal of nitrate Nitrogen from wastewater by Pseudomonas nitroreducens pf2
The strain pf2 takes nitrate as a nitrogen source, and the aerobic denitrification capability of the strain is examined by detecting the degradation condition of the nitrogen source in the wastewater. Initial TN concentration of the nitrate wastewater is 147mg/L, and initial NO3 -The liquid seed culture (2ml) of the rescreened strain isolated in example 1 was inoculated into 200ml of a nitrate medium at an N concentration of 99mg/L at a medium volume ratio of 1%, and cultured at 30 ℃ and a rotation speed of 220 rpm. The formula of the culture medium is as follows: KNO30.722 g/L, sodium citrate 5g/L, MgSO4·7H2O 1g/L，KH2PO4 0.25g/L，Na2HPO40.3 g/L. As can be seen from FIG. 2, the strain pf2 was cultured for 16h, and the OD600 reached up to 1.338. As shown in FIG. 4, the removal rate of nitrate nitrogen by the strain at this time reached 100%, and the total nitrogen removal rate was 95.5%. The experimental result shows that the strain pf2 can perform aerobic denitrification by taking nitrate nitrogen as a substrate under aerobic conditions.
The above-described embodiments are merely preferred embodiments of the present invention, and the embodiments of the present invention are not limited to the above-described embodiments, and it should be understood that many other modifications and embodiments can be devised by those skilled in the art, which will fall within the spirit and scope of the principles of this disclosure.
Sequence listing
<110> aquatic research institute of agricultural science institute of Anhui province
<120> Pseudomonas nitroreducens strain and application thereof
<141> 2021-03-24
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1498
<212> DNA
<213> Pseudomonas nitroreducens (Pseudomonas nitroreducens) pf2
<400> 1
agagtttgat cctggcttag attgaacgct ggcggcaggc ctaacacatg caagtcgagc 60
ggatgagtgg agcttgctcc atgattcagc ggcggacggg tgagtaatgc ctaggaatct 120
gcctggtagt gggggacaac gtttcgaaag gaacgctaat accgcatacg tcctacggga 180
gaaagcaggg gaccttcggg ccttgcgcta tcagatgagc ctaggtcgga ttagctagtt 240
ggtggggtaa aggcctacca aggcgacgat ccgtaactgg tctgagagga tgatcagtca 300
cactggaact gagacacggt ccagactcct acgggaggca gcagtgggga atattggaca 360
atgggcgaaa gcctgatcca gccatgccgc gtgtgtgaag aaggtcttcg gattgtaaag 420
cactttaagt tgggaggaag ggcagtaagt taataccttg ctgttttgac gttaccaaca 480
gaataagcac cggctaactt cgtgccagca gccgcggtaa tacgaagggt gcaagcgtta 540
atcggaatta ctgggcgtaa agcgcgcgta ggtggtttgg taagatggat gtgaaatccc 600
cgggctcaac ctgggaactg catccataac tgcctgacta gagtacggta gagggtggtg 660
gaatttcctg tgtagcggtg aaatgcgtag atataggaag gaacaccagt ggcgaaggcg 720
accacctgga ctgatactga cactgaggtg cgaaagcgtg gggagcaaac aggattagat 780
accctggtag tccacgccgt aaacgatgtc gactagccgt tgggatcctt gagatcttag 840
tggcgcagct aacgcgataa gtcgaccgcc tggggagtac ggccgcaagg ttaaaactca 900
aatgaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg 960
aagaacctta cctggccttg acatgtccgg aaccttgcag agatgcgagg gtgccttcgg 1020
gaatcggaac acaggtgctg catggctgtc gtcagctcgt gtcgtgagat gttgggttaa 1080
gtcccgtaac gagcgcaacc cttgtcctta gttaccagca cgttatggtg ggcactctaa 1140
ggagactgcc ggtgacaaac cggaggaagg tggggatgac gtcaagtcat catggccctt 1200
acggccaggg ctacacacgt gctacaatgg tcggtacaga gggttgccaa gccgcgaggt 1260
ggagctaatc ccataaaacc gatcgtagtc cggatcgcag tctgcaactc gactgcgtga 1320
agtcggaatc gctagtaatc gtgaatcaga atgtcacggt gaatacgttc ccgggccttg 1380
tacacaccgc ccgtcacacc atgggagtgg gttgctccag aagtagctag tctaaccgca 1440
agggggacgg ttaccacgga gtgattcatg actggggtga agtcgtaaca aggtagcc 1498
Claims (9)
1. A nitroreduction pseudomonas strain is characterized in that: the strain is Pseudomonas nitroreducens (Pseudomonas nitroreducens) pf2, which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2021274.
2. The pseudomonas nitroreducens strain of claim 1, wherein: the effective sequence length of the 16SrDNA of the Pseudomonas nitroreducens (Pseudomonas nitroreducens) pf2 is 1498bp, and the sequence is shown as SEQ ID NO: 1 is shown.
3. The pseudomonas nitroreducens strain of claim 1, wherein: the Pseudomonas nitroreducens (Pseudomonas nitroreducens) pf2 is obtained by enrichment culture, separation and purification of bottom mud of an aquaculture pond.
4. The pseudomonas nitroreducens strain of claim 1, wherein: the nitropseudomonas nitroreducens (pf 2) is a gram-negative bacillus, is in a short rod shape, has irregular edges and is milky white in colony.
5. The pseudomonas nitroreducens strain of claim 1, wherein: the Pseudomonas nitroreducens pf2 was grown using ammonium sulfate or nitrate as the sole nitrogen source.
6. Use of a strain of pseudomonas nitroreducens according to any one of claims 1 to 5, characterized in that: the Pseudomonas nitroreducens (Pseudomonas nitroreducens) pf2 is used for denitrification of nitrogen-containing water bodies.
7. Use according to claim 6, characterized in that: the nitrogen-containing water body refers to organic wastewater containing ammonia nitrogen or nitrate.
8. Use according to claim 6, characterized in that: the Pseudomonas nitroreducens (Pseudomonas nitroreducens) pf2 is inoculated in a nitrogen-containing water body for aerobic continuous culture after amplification culture.
9. Use according to claim 8, characterized in that: the temperature of the aerobic continuous culture is 30-35 ℃; the inoculation amount of the Pseudomonas nitroreducens pf2 is 1 percent of the volume of the nitrogen-containing water body.
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| CN115948288B (en) * | 2022-12-06 | 2023-09-26 | 浙江大学 | Aerobic high-efficiency denitrification compound flora and application thereof |
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