CN117844669A - Microbacterium north and its culture and application - Google Patents
Microbacterium north and its culture and application Download PDFInfo
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- 241001467578 Microbacterium Species 0.000 title claims abstract description 32
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 52
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims abstract description 36
- 229910052698 phosphorus Inorganic materials 0.000 claims abstract description 36
- 239000011574 phosphorus Substances 0.000 claims abstract description 36
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 26
- 238000009629 microbiological culture Methods 0.000 claims abstract description 5
- 239000010865 sewage Substances 0.000 claims description 25
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 claims description 23
- 239000002351 wastewater Substances 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 22
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 14
- 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 claims description 12
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 11
- 230000008569 process Effects 0.000 claims description 11
- 239000001632 sodium acetate Substances 0.000 claims description 11
- 235000017281 sodium acetate Nutrition 0.000 claims description 11
- 229910019142 PO4 Inorganic materials 0.000 claims description 10
- 239000010452 phosphate Substances 0.000 claims description 10
- 239000001963 growth medium Substances 0.000 claims description 8
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 claims description 7
- 238000012258 culturing Methods 0.000 claims description 7
- 238000004321 preservation Methods 0.000 claims description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 6
- 229910052799 carbon Inorganic materials 0.000 claims description 6
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 5
- 229910052760 oxygen Inorganic materials 0.000 claims description 5
- 239000001301 oxygen Substances 0.000 claims description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 4
- 238000009395 breeding Methods 0.000 claims description 4
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 claims description 4
- 238000009360 aquaculture Methods 0.000 claims description 3
- 244000144974 aquaculture Species 0.000 claims description 3
- 230000001488 breeding effect Effects 0.000 claims description 3
- 239000000149 chemical water pollutant Substances 0.000 claims description 3
- 244000144972 livestock Species 0.000 claims description 3
- 244000144977 poultry Species 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-M dihydrogenphosphate Chemical compound OP(O)([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-M 0.000 claims description 2
- 125000005341 metaphosphate group Chemical group 0.000 claims description 2
- 230000000877 morphologic effect Effects 0.000 claims description 2
- 235000011056 potassium acetate Nutrition 0.000 claims description 2
- 229940074404 sodium succinate Drugs 0.000 claims description 2
- ZDQYSKICYIVCPN-UHFFFAOYSA-L sodium succinate (anhydrous) Chemical compound [Na+].[Na+].[O-]C(=O)CCC([O-])=O ZDQYSKICYIVCPN-UHFFFAOYSA-L 0.000 claims description 2
- 238000012136 culture method Methods 0.000 claims 4
- 244000005700 microbiome Species 0.000 abstract description 6
- 230000001360 synchronised effect Effects 0.000 abstract description 5
- 241000894006 Bacteria Species 0.000 description 17
- 230000001580 bacterial effect Effects 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 108020004465 16S ribosomal RNA Proteins 0.000 description 6
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 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 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 230000001546 nitrifying effect Effects 0.000 description 4
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 239000010802 sludge Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 238000002798 spectrophotometry method Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 235000019270 ammonium chloride Nutrition 0.000 description 3
- 230000001651 autotrophic effect Effects 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 229910002651 NO3 Inorganic materials 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 2
- 235000019797 dipotassium phosphate Nutrition 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 235000019319 peptone Nutrition 0.000 description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
- 229920013639 polyalphaolefin Polymers 0.000 description 2
- 239000004323 potassium nitrate Substances 0.000 description 2
- 235000010333 potassium nitrate Nutrition 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000002864 sequence alignment Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000219104 Cucurbitaceae Species 0.000 description 1
- 241000500375 Microbacterium sp. Species 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 229920000388 Polyphosphate Polymers 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 241000625324 Pseudomonas guariconensis Species 0.000 description 1
- HDELZEHCSGXWFK-UHFFFAOYSA-N [N].OC1=CC=CC=C1 Chemical compound [N].OC1=CC=CC=C1 HDELZEHCSGXWFK-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000011609 ammonium molybdate Substances 0.000 description 1
- 235000018660 ammonium molybdate Nutrition 0.000 description 1
- APUPEJJSWDHEBO-UHFFFAOYSA-P ammonium molybdate Chemical compound [NH4+].[NH4+].[O-][Mo]([O-])(=O)=O APUPEJJSWDHEBO-UHFFFAOYSA-P 0.000 description 1
- 229940010552 ammonium molybdate Drugs 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000015784 hyperosmotic salinity response Effects 0.000 description 1
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000005360 mashing Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000001205 polyphosphate Substances 0.000 description 1
- 235000011176 polyphosphates Nutrition 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 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
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
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- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
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- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a strain of Microbacterium northwestMicrobacterium kitamiense) PR with heterotrophic nitrification-aerobic denitrification and dephosphorization properties has been deposited in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on the day 27 of 2022, with the deposit numberCGMCC No.25182. The strain can simultaneously perform heterotrophic nitrification and aerobic denitrification and dephosphorization, realizes synchronous removal of nitrogen and phosphorus under aerobic conditions, and has good application prospects.
Description
Technical Field
The invention belongs to the technical field of environmental microorganism and water pollution treatment, and particularly relates to a strain of Microbacterium northwest, and culture and application thereof.
Background
Denitrification and phosphorus accumulation are two very important reaction processes in the biological denitrification and phosphorus removal process of sewage, and denitrifying bacteria (denitrifiers) and dephosphorizing bacteria (polyphosphate accumulating organisms, PAOs) are also gradually known and understood as denitrification and phosphorus removal technologies are continuously and deeply researched in the microorganism field. In the traditional biological denitrification and dephosphorization process, ammonia nitrogen in sewage is firstly oxidized into nitrite nitrogen or nitrate nitrogen under the aerobic condition by autotrophic nitrification, and then reduced into nitrogen by heterotrophic denitrification under the anaerobic condition; under the action of heterotrophic phosphorus accumulating bacteria (PAOs), orthophosphate in the sewage is absorbed in the cells in excess under the aerobic condition and stored in the form of phosphorus accumulating particles, and the orthophosphate is released by the phosphorus accumulating particles under the anaerobic condition, so that the phosphorus in the system is removed by a sludge discharge mode. The difference of biological denitrification and dephosphorization mechanisms makes the two processes difficult to unify, and the competition of the dephosphorization bacteria and the denitrifying bacteria for the carbon source always exists; the nitrifying bacteria, the denitrifying bacteria and the dephosphorizing bacteria have different bacterial ages, and various bacterial groups are mutually restricted by being mixed together, so that the optimal running condition of the system is difficult to achieve. Therefore, the traditional removal process requires the staged treatment of anaerobic phosphorus release, anoxic denitrification and aerobic nitrification, which not only increases the complexity of equipment, but also increases the occupied area and the operation cost.
In recent years, the discovery of synchronous heterotrophic nitrification aerobic denitrification dephosphorization bacteria (SNDPR) enables the assumption that the strains can convert ammonia nitrogen into nitrogen under aerobic conditions and absorb excessive orthophosphate into cells, so that the denitrification dephosphorization process can be completed in one reactor, the competition problem of different functional strains for substrates is avoided, the number of the reactors can be reduced, the denitrification process is simplified, and the running cost can be reduced.
CN106635855A discloses a Microbacterium northwest strain which is Microbacterium northwest strainMicrobacterium kitamiense)FSTB-4 has been deposited in China general microbiological culture Collection center, china Committee for culture Collection of microorganisms, with a accession number of CGMCC No.10939, 1, 6, 2015. The strain can be applied to the efficient removal of COD in the salt-containing wastewater with the salt content of 1.0-5.0 wt%. Can rapidly degrade and remove COD in the high-salt-content wastewater, and has the characteristics of strong salt tolerance, good treatment effect and the like. However, this strain cannot denitrify ammonia nitrogen as a substrate.
CN110407338A discloses a pseudomonas strain Pseudomonas guariconensis PP-d4 of the cucurbitaceae family of low temperature denitrification dephosphorization bacteria, which has the deposit number: cctccc NO: m2018653. The strain PP-d4 has strong low-temperature denitrification capability and wide ammonia nitrogen concentration tolerance, and can resist NH with low, medium and high concentrations at low temperature of 5 DEG C 4 + The degradation rate of the N can reach 88.0% -74.5%, 94.6% -90.6% and 81.7% -42.7% respectively. The strain PP-d4 not only can utilize ammonia nitrogen, nitrite or nitrate as a unique nitrogen source for growth and metabolism, but also can achieve the aim of synchronous denitrification and dephosphorization, and NO in an ADM-P culture medium 3 - -N and TP degradation rates of 85.9% and 77.2%, respectively; in NADM-P medium, NO 2 - The degradation rates of N and TP were 74.6% and 56.6%, respectively. The strain PP-d4 can be applied to various water treatment processes such as livestock and poultry breeding wastewater, domestic sewage, industrial organic wastewater, aquaculture tail water, landfill leachate and the like and river course restoration, strengthens the denitrification and dephosphorization effects of a water treatment system, and has higher application value in the water treatment process.
However, the number of SNDPR bacteria separated at present is less, so that more phosphorus removal bacteria with heterotrophic nitrification and aerobic denitrification capability are bred, which is beneficial to expanding the application field of the microorganisms and is more suitable for industrial application.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a Microbacterium northwest strain and a culture application thereof. The strain can simultaneously perform heterotrophic nitrification and aerobic denitrification and dephosphorization, realizes synchronous removal of nitrogen and phosphorus under aerobic conditions, and has good application prospects.
The invention provides a strain of Microbacterium northwestMicrobacterium kitamiense) PR (PR) withHas heterotrophic nitrification-aerobic denitrification and dephosphorization properties, and is preserved in China general microbiological culture Collection center (CGMCC) No.25182 in the year 2022, month 06 and day 27.
The invention provides a Microbacterium northwest strainMicrobacterium kitamiense) The main morphological features of PR are: culturing in TSA culture medium at 30deg.C for 3d, and culturing to obtain yellow colony, round colony, wet surface, opacity, and regular edge.
The invention provides a Microbacterium northwest strainMicrobacterium kitamiense) PR cells are short rods under a microscope, 0.4-0.6μm×0.7-1.6 μm, are arranged singly or in pairs, and are gram-positive.
The invention provides a Microbacterium northwest strainMicrobacterium kitamiense) The result of the sequencing analysis of the PR 16S rDNA gene is shown in a sequence table. According to sequence alignment, microbacterium North seesMicrobacterium kitamiense) PR is different from published 16S rDNA data of Microbacterium northwest.
The second aspect of the invention provides a Microbacterium northwest strainMicrobacterium kitamiense) PR is cultured in a medium containing ammonia nitrogen and phosphate with acetate as carbon source. Wherein the phosphate is at least one of orthophosphate, metaphosphate, hydrogen phosphate, dihydrogen phosphate, polymerized phosphate and the like; the acetate is at least one of sodium acetate, potassium acetate and the like. The culture conditions are as follows: COD/TN is 15:1-30:1, the culture temperature is 20-35 ℃, the pH value is 6-9, and the rotating speed is 50-300rpm.
In a third aspect, the invention provides a Microbacterium northwest strainMicrobacterium kitamiense) The PR is applied to ammonia nitrogen removal and phosphorus removal of sewage. The strain can simultaneously perform heterotrophic nitrification and aerobic denitrification and dephosphorization, namely can be used for treating wastewater or sewage containing ammonia nitrogen and phosphate, and the concentration of the ammonia nitrogen in the water body is not higher than 300mg/L, preferably 100-250mg/L, and the total phosphorus concentration is not higher than 30mg/L, preferably 5-20mg/L.
A fourth aspect of the present invention provides Microbacterium North-visible bacteriaMicrobacterium kitamiense) PR is applied to denitrification and dephosphorization of sewage. The strainCan be used for aerobic denitrification nitrogen and phosphorus removal, namely can be used for treating wastewater or sewage containing nitrate nitrogen/nitrite nitrogen and phosphate, wherein the concentration of the nitrate nitrogen/nitrite nitrogen in the water body is not higher than 150mg/L, preferably 50-150mg/L, and the total phosphorus concentration is not higher than 30mg/L, preferably 5-20mg/L.
In the application of the invention, the treatment conditions are as follows: the COD/TN ratio is 15:1-30:1, the concentration of dissolved oxygen is 0.5-2.5mg/L, the treatment temperature is 20-35 ℃, and the pH is 6-9.
In the application of the invention, at least one of sodium acetate as a carbon source, sodium succinate, methanol and the like is added in the treatment process, and sodium acetate is preferred. The addition amount is determined according to the COD/TN ratio of 15:1-30:1.
In the application of the invention, the wastewater or the sewage is at least one of municipal sewage, livestock and poultry breeding wastewater, domestic sewage, industrial organic wastewater, aquaculture tail water, landfill leachate and other wastewater or sewage, wherein the total nitrogen concentration is not higher than 500mg/, and the total phosphorus concentration is not higher than 30mg/L.
Compared with the prior art, the invention has the following beneficial effects:
(1) The breeding method of the invention shows that the Bacillus northlasii is a speciesMicrobacterium kitamiense) PR can simultaneously perform heterotrophic nitrification and aerobic denitrification and dephosphorization, and can realize synchronous removal of nitrogen and phosphorus under aerobic conditions, so that the process flow is simplified, and the equipment and investment cost is saved.
(2) Microbacterium North HeiMicrobacterium kitamiense) PR can be applied to the treatment of wastewater or sewage containing ammonia nitrogen and phosphorus for heterotrophic nitrification and aerobic denitrification and dephosphorization; can also be applied to the wastewater or sewage treatment containing nitrate nitrogen/nitrite nitrogen and phosphorus to carry out denitrification and dephosphorization. In addition, denitrification and dephosphorization are synchronously carried out, so that the problem of competition of denitrifying bacteria and phosphorus accumulating bacteria on a carbon source is solved.
(3) Compared with autotrophic nitrifying bacteria, the heterotrophic nitrifying bacteria have the advantages of high growth rate and high cell yield, and can effectively solve the problems of slow proliferation of the autotrophic nitrifying bacteria, long hydraulic retention time of the system and the like.
Description of biological Material preservation
The invention provides a Microbacterium northwest strainMicrobacterium kitamiense) PR, deposited in China general microbiological culture Collection center (China Committee for culture Collection); preservation number: CGMCC No. 25182; preservation date: 2022, 06, 27; preservation address: the institute of microorganisms of national academy of sciences of China, no.1, no. 3, north Chen West Lu, the Korean region of Beijing.
Drawings
FIG. 1 is a photograph of colony morphology of the strain of the present invention on a plate medium.
FIG. 2 is a photograph of a cell of a strain of the present invention at 1000 Xmagnification under a microscope.
Detailed Description
The isolation and selection of the strains of the invention and their use in cultivation are described in further detail below in connection with the examples. The embodiments and specific operation procedures are given on the premise of the technical scheme of the invention, but the protection scope of the invention is not limited to the following embodiments.
The experimental methods in the following examples, unless otherwise specified, are all conventional in the art. The experimental materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. In the invention, the percentage content is weight percentage content.
In the invention, the ammonia nitrogen concentration is measured by GB7478-87 determination of ammonium in water-distillation and titration method; the nitrate nitrogen concentration is measured by using GB7480-1987 method for measuring Water quality-nitrate nitrogen-phenol disulfonic acid spectrophotometry; the nitrite nitrogen concentration is measured by using a water quality-determination of nitrite nitrogen-spectrophotometry of GB 7493-87; the total nitrogen concentration adopts GB 11894-89 water quality-total nitrogen determination-alkaline potassium persulfate digestion ultraviolet spectrophotometry, and the total phosphorus concentration adopts GB11893-89 ammonium molybdate spectrophotometry.
EXAMPLE 1 isolation screening of Strain PR
The strain is obtained by separating and screening the bacterial strain from the active sludge bacterial colony by adopting a dilution plate separation method, and the active sludge is obtained from a sewage treatment plant of an enterprise in Erdos city of inner Mongolia in 10 months in 2018.
(1) Dilution plate separation method: pouring out the supernatant after solid-liquid separation of the sample, taking 10g of sludge, adding deionized water, mashing for 30min on a magnetic stirrer, sucking out 10mL of supernatant by a pipette after the muddy water mixture is kept stand for 2h, placing the supernatant into a triangular flask containing 100mL of enrichment medium and sealing the bottle by gauze, placing the bottle into a constant-temperature shake culture device, and shake culturing at 30 ℃ for 150 r/min. After 24h of culture, sampling, coating, diluting and coating the culture medium into YG (YG) under aseptic condition, culturing for 48h to obtain a plurality of single colonies, picking single colonies, culturing on a shake flask, observing with a microscope to determine whether the single colonies are pure strains, and repeating the steps until the single colonies are determined to be pure strains. And (3) obtaining 3 pure strains through repeated purification and culture.
(2) And (3) inoculating the pure strain screened in the step (1) into a screening culture medium, performing shake culture at 30 ℃ for 24 hours at 150r/min, centrifuging for 5 minutes at 8000r/min, taking supernatant to measure total phosphorus, total nitrogen and ammonia nitrogen content, repeating the test for multiple times, and finally obtaining the strain PR with high denitrification and dephosphorization efficiency.
The enrichment medium is: glucose 0.1g, peptone 0.1g, yeast extract 0.01g, sodium chloride 0.05g, dipotassium phosphate 0.1g, potassium dihydrogen phosphate 0.1g, magnesium sulfate 0.01g, calcium chloride 0.005g, ammonium chloride 0.5-0.6 g, potassium nitrate 0.2-0.6 g, water 1L, pH7.0-7.2.
YG medium: 1g of yeast extract powder, 1g of glucose, 0.3g of dipotassium hydrogen phosphate, 0.25g of potassium dihydrogen phosphate, 1L of water and pH7.0-7.2.
Screening the culture medium: 4.5g of sodium acetate, 0.1g of peptone, 0.01g of yeast extract powder, 0.05g of sodium chloride, 0.1g of dipotassium hydrogen phosphate, 0.1g of monopotassium hydrogen phosphate, 0.01g of magnesium sulfate, 0.005g of calcium chloride, 0.5-0.6 g of ammonium chloride, 0.2-0.6 g of potassium nitrate, 8.5g of PIPE buffer solution and 1L of water, wherein the pH value is 7.0-7.2.
Example 2 identification of strains
The obtained pure strain PR is sent to China center for type culture collection (CICC) for strain identification. The physiological and biochemical characteristics are shown in table 1. The sequencing analysis result of the 16S rDNA gene is shown in a sequence table, and the sequence of the 16S rDNA sequence is compared with a professional database (Update 2021.04.21) taking NCBI database as a base source (as shown in figure 1) to obtain the sequence table, and the sequence table is compared with the Microbacterium northern Europe (Microbacterium sp.) of the genus MicrobacteriumMicrobacterium kitamiense) Homology is closest. Adopting MEGA software, adopting an orthographic ligation method to display a phylogenetic tree (shown in figure 2) of the PR and the 16S rDNA sequences of related species, carrying out repeated similarity calculation for 1000 times, wherein in the figure, a node of the phylogenetic tree only displays a numerical value with a Bootstrap value greater than 50%, and the superscript T represents a model strain. The strain is identified as Microbacterium northwestMicrobacterium kitamiense）。
TABLE 1 physiological and Biochemical characteristics
* In the table: "+", positive; "+ w ", weak positive; "-", negative.
FIG. 1 sequence alignment
FIG. 2 phylogenetic tree of "PR" and related species 16S rDNA sequences
EXAMPLE 3 cultivation of Microbacterium North Americi PR
Preparing a culture solution: preparing a culture solution from ammonium chloride, potassium hydrogen phosphate, sodium acetate and water, wherein the concentration of ammonia nitrogen in the culture solution is 200mg/L, the total phosphorus is 20mg/L, and the COD is 3500mg/L. Packaging into triangular bottles, and sterilizing.
The pure strain is activated and cultured to OD 660 Over 0.7, the activation culture adopts LB culture medium, then is inoculated into shake flask culture solution according to the volume ratio of 1%, and shake-cultured for 30h at 30 ℃ and pH of 7-7.5 at 150r/min, and OD 660 Reaching more than 0.7.
Through sampling detection, the total nitrogen concentration is 52mg/L, the ammonia nitrogen concentration is 5.6mg/L, and the total phosphorus content is 0.8mg/L, COD and 446mg/L.
EXAMPLE 4 application of Microbacterium North-see PR
The wastewater A of the sewage treatment field of a certain enterprise comprises the following components: the total nitrogen concentration is 155mg/L, the ammonia nitrogen concentration is 135mg/L, the total phosphorus concentration is 18mg/L, and the COD is 2006mg/L.
Inoculating the fresh bacterial liquid prepared in the example 3 into the wastewater A for treatment, wherein the inoculation volume ratio is 5%, adding sodium acetate to ensure that the COD/TN is 20:1, and the treatment conditions are as follows: the dissolved oxygen concentration is 1.0-2.5mg/L, the culture temperature is 25 ℃, the pH value is 7.0-7.5, and the water index is detected after 48h of culture.
The detection result is as follows: the total nitrogen concentration is 35mg/L, the ammonia nitrogen concentration is 0.5mg/L, the total phosphorus concentration is 0.6 mg/L, and the COD is 415mg/L.
EXAMPLE 5 application of Microbacterium North-see PR
The wastewater B of the sewage treatment plant of a certain enterprise comprises the following components: the total nitrogen concentration is 145mg/L, the nitrate nitrogen concentration is 128mg/L, the total phosphorus concentration is 22mg/L, and the COD is 2105mg/L.
Inoculating the fresh bacterial liquid prepared in the example 3 into the wastewater B for treatment, wherein the inoculation volume ratio is 5%, adding sodium acetate to ensure that the COD/TN is 25:1, and the treatment conditions are as follows: the dissolved oxygen concentration is 1.0-2.5mg/L, the culture temperature is 30 ℃, the pH value is 7.5-8.0, and the water index is detected after 48h of culture.
The detection result is as follows: the total nitrogen concentration is 49mg/L, the nitrate nitrogen concentration is 15mg/L, and the total phosphorus concentration is 0.5mg/L, COD and 96mg/L.
EXAMPLE 6 application of Microbacterium North-see PR
The composition of the wastewater C of the sewage treatment plant of a certain enterprise is as follows: the total nitrogen concentration is 161mg/L, the ammonia nitrogen concentration is 45mg/L, the nitrate nitrogen concentration is 38mg/L, the nitrite nitrogen concentration is 48mg/L, the total phosphorus concentration is 20mg/L, and the COD is 1998mg/L.
Inoculating the fresh bacterial liquid prepared in the embodiment 3 into the wastewater C for treatment, wherein the inoculation volume ratio is 5%, adding sodium acetate to ensure that the COD/TN is 15:1, and the treatment conditions are as follows: the dissolved oxygen concentration is 1.0-1.5mg/L, the culture temperature is 20 ℃, the pH value is 7.0-7.5, and the water index is detected after 48h of culture.
The detection result is as follows: the total nitrogen concentration is 37mg/L, the ammonia nitrogen concentration is 1.1mg/L, the nitrate nitrogen concentration is 11.5mg/L, the nitrite nitrogen concentration is 13.2mg/L, the total phosphorus concentration is 0.5mg/L, and the COD is 364mg/L.
Comparative example 1
The difference from example 4 is that Microbacterium northwest disclosed in CN106635855A is adoptedMicrobacterium kitamiense) FSTB-4 with a preservation number of CGMCC No.10939. The detection result is as follows: the total nitrogen concentration is 135mg/L, the ammonia nitrogen concentration is 121mg/L, the total phosphorus concentration is 11.5mg/L, and the COD is 209mg/L.
Comparative example 2
The difference from example 5 is that Microbacterium northwest disclosed in CN106635855A is adoptedMicrobacterium kitamiense) FSTB-4 with a preservation number of CGMCC No.10939. The detection result is as follows: the total nitrogen concentration is 115mg/L, the nitrate nitrogen concentration is 88mg/L, the total phosphorus concentration is 13.1mg/L, and the COD is 188mg/L.
Claims (13)
1. A strain of Microbacterium northsonii is Microbacterium northsoniiMicrobacterium kitamiense) PR with heterotrophic nitrification-aerobic denitrification and dephosphorization performance is preserved in China general microbiological culture collection center (CGMCC) with the preservation number of 25182 in the year 2022, month 06 and day 27.
2. A strain according to claim 1, characterized in that: the North Microbacterium is described asMicrobacterium kitamiense) The main morphological features of PR are: culturing in TSA culture medium at 30deg.C for 3d, and culturing to obtain yellow colony, round colony, wet surface, opacity and regular edge; under microscope, the thallus is in short rod shape, 0.4-0.6μm×0.7-1.6μm, single or paired arrangement, gram positive.
3. Microbacterium northwest (Fr.) karstMicrobacterium kitamiense) A PR culture method is characterized in that: in a culture medium containing ammonia nitrogen and phosphate, acetate is used as a carbon source for culture.
4. A culture method according to claim 3, wherein: the phosphate is at least one of orthophosphate, metaphosphate, hydrogen phosphate, dihydrogen phosphate and polymerized phosphate.
5. A culture method according to claim 3, wherein: the acetate is at least one of sodium acetate and potassium acetate.
6. A culture method according to claim 3, wherein: the culture conditions are as follows: COD/TN is 15:1-30:1, the culture temperature is 20-35 ℃, the pH value is 6-9, and the rotating speed is 50-300rpm.
7. Microbacterium northwest (Fr.) karstMicrobacterium kitamiense) The PR is applied to ammonia nitrogen removal and phosphorus removal of sewage.
8. The use according to claim 7, characterized in that: the strain can simultaneously carry out heterotrophic nitrification and aerobic denitrification and dephosphorization, namely is used for treating wastewater or sewage containing ammonia nitrogen and phosphate, the concentration of the ammonia nitrogen in the water body is not higher than 300mg/L, preferably 100-250mg/L, and the total phosphorus concentration is not higher than 30mg/L, preferably 5-20mg/L.
9. Microbacterium northwest (Fr.) karstMicrobacterium kitamiense) The PR is applied to aerobic denitrification and dephosphorization of sewage.
10. The use according to claim 9, characterized in that: the strain can perform aerobic denitrification and dephosphorization, namely is used for treating wastewater or sewage containing nitrate nitrogen/nitrite nitrogen and phosphate, the concentration of the nitrate nitrogen/nitrite nitrogen in a water body is not higher than 150mg/L, preferably 50-150mg/L, and the total phosphorus concentration is not higher than 30mg/L, preferably 5-20mg/L.
11. Use according to any one of claims 7-10, characterized in that: the treatment conditions are as follows: the COD/TN ratio is 15:1-30:1, the concentration of dissolved oxygen is 0.5-2.5mg/L, the treatment temperature is 20-35 ℃, and the pH is 6-9.
12. Use according to any one of claims 7-10, characterized in that: in the treatment process, at least one of sodium acetate as a carbon source, sodium succinate and methanol is added, preferably sodium acetate; the addition amount is determined according to the COD/TN ratio of 15:1-30:1.
13. Use according to any one of claims 7-10, characterized in that: the wastewater or sewage is at least one of municipal sewage, livestock and poultry breeding wastewater, domestic sewage, industrial organic wastewater, aquaculture tail water, landfill leachate and the like, wherein the total nitrogen concentration is not higher than 500mg/, and the total phosphorus concentration is not higher than 30mg/L.
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