CN116891809B - Pseudomonas asiatica and microbial agent and application thereof - Google Patents
Pseudomonas asiatica and microbial agent and application thereof Download PDFInfo
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- CN116891809B CN116891809B CN202211556971.5A CN202211556971A CN116891809B CN 116891809 B CN116891809 B CN 116891809B CN 202211556971 A CN202211556971 A CN 202211556971A CN 116891809 B CN116891809 B CN 116891809B
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- Prior art keywords
- pseudomonas
- asiatica
- culture
- phosphorus
- strain
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- 241000589516 Pseudomonas Species 0.000 title claims abstract description 34
- 230000000813 microbial effect Effects 0.000 title claims abstract description 10
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- 229910052698 phosphorus Inorganic materials 0.000 claims abstract description 62
- 239000011574 phosphorus Substances 0.000 claims abstract description 62
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 30
- 239000002351 wastewater Substances 0.000 claims abstract description 28
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 15
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 8
- 238000004321 preservation Methods 0.000 claims abstract description 8
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- 125000003729 nucleotide group Chemical group 0.000 claims description 10
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 9
- 235000013365 dairy product Nutrition 0.000 claims description 5
- BHEPBYXIRTUNPN-UHFFFAOYSA-N hydridophosphorus(.) (triplet) Chemical compound [PH] BHEPBYXIRTUNPN-UHFFFAOYSA-N 0.000 claims description 5
- 239000010840 domestic wastewater Substances 0.000 claims description 3
- 239000003337 fertilizer Substances 0.000 claims description 2
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- XKLJHFLUAHKGGU-UHFFFAOYSA-N nitrous amide Chemical compound ON=N XKLJHFLUAHKGGU-UHFFFAOYSA-N 0.000 claims description 2
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- 239000010908 plant waste Substances 0.000 claims 1
- 229910052799 carbon Inorganic materials 0.000 abstract description 20
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- QRXMUCSWCMTJGU-UHFFFAOYSA-N 5-bromo-4-chloro-3-indolyl phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP(O)(=O)O)=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
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- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- BDKLKNJTMLIAFE-UHFFFAOYSA-N 2-(3-fluorophenyl)-1,3-oxazole-4-carbaldehyde Chemical compound FC1=CC=CC(C=2OC=C(C=O)N=2)=C1 BDKLKNJTMLIAFE-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 101100409047 Chlorobaculum tepidum (strain ATCC 49652 / DSM 12025 / NBRC 103806 / TLS) ppk2 gene Proteins 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 238000000116 DAPI staining Methods 0.000 description 1
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- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
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- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
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- 241000631636 Ishige Species 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 101710129824 Polyphosphate kinase 1 Proteins 0.000 description 1
- 101710129823 Polyphosphate kinase 2 Proteins 0.000 description 1
- 229920000037 Polyproline Polymers 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
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- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- GFEWRKPOYXKQKX-UHFFFAOYSA-I [C+4].C(C)(=O)[O-].[Na+].C(C)(=O)[O-].C(C)(=O)[O-].C(C)(=O)[O-].C(C)(=O)[O-] Chemical compound [C+4].C(C)(=O)[O-].[Na+].C(C)(=O)[O-].C(C)(=O)[O-].C(C)(=O)[O-].C(C)(=O)[O-] GFEWRKPOYXKQKX-UHFFFAOYSA-I 0.000 description 1
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- YYRMJZQKEFZXMX-UHFFFAOYSA-N calcium;phosphoric acid Chemical compound [Ca+2].OP(O)(O)=O.OP(O)(O)=O YYRMJZQKEFZXMX-UHFFFAOYSA-N 0.000 description 1
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- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- FFUAGWLWBBFQJT-UHFFFAOYSA-N hexamethyldisilazane Chemical compound C[Si](C)(C)N[Si](C)(C)C FFUAGWLWBBFQJT-UHFFFAOYSA-N 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
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- MEFBJEMVZONFCJ-UHFFFAOYSA-N molybdate Chemical compound [O-][Mo]([O-])(=O)=O MEFBJEMVZONFCJ-UHFFFAOYSA-N 0.000 description 1
- 239000010813 municipal solid waste Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229910052762 osmium Inorganic materials 0.000 description 1
- SYQBFIAQOQZEGI-UHFFFAOYSA-N osmium atom Chemical compound [Os] SYQBFIAQOQZEGI-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- -1 phosphate anhydride Chemical class 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920013639 polyalphaolefin Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 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
- 239000000843 powder Substances 0.000 description 1
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- 239000011734 sodium Substances 0.000 description 1
- 229940087562 sodium acetate trihydrate Drugs 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000004544 sputter deposition Methods 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000002426 superphosphate Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000007671 third-generation sequencing Methods 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1229—Phosphotransferases with a phosphate group as acceptor (2.7.4)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/04—Phosphotransferases with a phosphate group as acceptor (2.7.4)
- C12Y207/04001—Polyphosphate kinase (2.7.4.1)
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/105—Phosphorus compounds
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/38—Pseudomonas
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Molecular Biology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Hydrology & Water Resources (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
The invention relates to the technical field of microorganisms, in particular to Pseudomonas asiatica, a microbial agent and application thereof. The preservation number of the strain is CCTCC NO: M2022729. The strain has obvious effect of removing phosphorus and nitrogen in wastewater, has especially obvious effect of removing phosphorus which can reach 100%, and can be used for ecological reconstruction or in-situ restoration of natural water. And has wide adaptability of carbon source concentration, and can be used for treating low-carbon source wastewater.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to Pseudomonas asiatica, a microbial agent and application thereof.
Background
Along with human activities, eutrophication of water bodies has become a global problem, and many studies find that one of the main causes of eutrophication is the discharge of phosphorus-rich domestic wastewater, agricultural wastewater, industrial wastewater into water bodies. Therefore, the removal of accumulated phosphorus from water is a key for preventing and controlling eutrophication of water, chemical phosphorus removal and biological phosphorus removal are mainly used in sewage treatment at present, and an enhanced biological phosphorus removal method (EBPR) utilizes phosphorus removal bacteria (PAOs) to hydrolyze intracellular polyphosphate (poly-P) under anaerobic conditions to release phosphorus, and gathers excessive phosphorus from sewage to intracellular accumulated poly-P under aerobic conditions, and finally, phosphorus-rich sludge containing phosphorus removal bacteria is discharged to remove phosphorus from sewage. Among them, the enhanced biological phosphorus removal system is widely used because of its low cost and high efficiency. Phosphorus removal bacteria are a class of bacteria that are super-absorbent for phosphorus, not a single microorganism, but rather a diverse group of microorganisms that are capable of providing phosphorus in the form of polyphosphate particles within cells. Poly-P formation plays a critical role in the "super-phosphate" process of phosphate-removing microorganisms, and scientists have studied to find that Poly-P exists in many organisms and that Poly-P can be used as a phosphate reserve for organisms to provide phosphate anhydride linkages. The formation of poly-P was found to be closely related to polyphosphate kinase (PPK 1), and the transfer of the phosphate residue at the ATP terminus to the poly-P chain was reversible under the action of PPK 1. In 2002, ishige K et al found another polyphosphate kinase (PPK 2) from Pseudomonas aeruginosa, which catalyzes both the transfer of the phosphate group on GTP to poly-P and the production of GTP-producing enzyme PPK2 from poly-P, however, the nitrogen and phosphorus removal effect was not ideal.
Disclosure of Invention
In view of the above, the invention provides a Pseudomonas asiatica, a microbial agent and application thereof, wherein the strain has remarkable removal effect on phosphorus and nitrogen in wastewater, has wide adaptability of carbon source concentration, and can be used for treating low-carbon source wastewater.
According to the invention, sludge samples of sewage treatment plants in Zhejiang, jiangsu, shandong and other places are randomly collected, 85 primary screening strains are obtained in total, the dephosphorization capability of 41 strains reaches more than 20%, wherein the highest dephosphorization rate and the highest denitrification rate of Pseudomonas asiatica ZM on artificial wastewater reach 100% and 75.9%, after the whole genome of the strain is sequenced, 1 ppk1 gene and 1 ppk2 gene are identified, and effective strain resources and gene resources are provided for the rapid and efficient treatment of eutrophic water bodies.
The invention provides Pseudomonas asiatica (Pseudomonas asiatica) ZM16 with a preservation number of CCTCC NO: M2022729.
ZM16 strain was isolated and identified as follows:
(1) Culture media A-E were prepared separately:
the culture medium A is a strain separation, purification and preservation culture medium, and comprises the following components: each liter of culture medium A contains 10g of peptone, 5g of yeast extract, 2g of sodium acetate, 19g of agar and the balance of water, and the pH is regulated to 7.0-7.2.
Medium B was low P (1.8 mg/L) medium consisting of: each liter of medium B contains CH 3 COONa 3.42g，K 2 HPO 4 10 mg，(NH4) 2 SO 4 1.18 g，NH 4 Cl 0.955g，CaCl 2 60mg，MgSO 4 82 mg, HEPES 7g, trace elements 2mL, and the balance of water, and adjusting the pH to 7.0-7.2.
Medium C was a P-rich (19.9 mg/L) medium consisting of: each liter of medium C contains CH 3 COONa 3.42g，MgSO 4 82 mg，FeSO 4 3.7 mg，CaCl 2 60 mg，(NH4) 2 SO 4 1.18g，NH 4 Cl 0.955g, yeast extract 0.1g, KNO 3 1.81 g(N 250mg/L)、NaNO 2 1.22g(N 250mg/L)、KH 2 PO 4 56 mg，K 2 HPO 4 40mg, HEPES 7g, trace elements 2mL, and the balance of water, and adjusting the pH to 7.0-7.2.
The culture medium D is a phosphorus-rich nitrate culture medium, and comprises the following components: each liter of medium D contains CH 3 COONa 3.42g，MgSO 4 82 mg，FeSO 4 3.7 mg，CaCl 2 60 mg，NH 4 Cl 191.1mg，KNO 3 1.81 g(N 250mg/L)，(NH4) 2 SO 4 235 mg, yeast extract 0.1g, KH 2 PO 4 56mg，K 2 HPO 4 40mg, HEPES 7g, trace elements 2mL, and the balance of water, and adjusting the pH to 7.0-7.2.
The culture medium E is a phosphorus-rich nitrite culture medium, and comprises the following components: each liter of medium E contains CH 3 COONa 3.42g，MgSO 4 82 mg，FeSO 4 3.7 mg，CaCl 2 60 mg，NH 4 Cl 191.1mg，NaNO 2 1.22 g(N 250mg/L)，(NH4) 2 SO 4 235 mg, yeast extract 0.1g, KH 2 PO 4 56 mg，K 2 HPO 4 40mg, HEPES 7g, trace elements 2mL, and the balance of water, and adjusting the pH to 7.0-7.2.
The trace elements comprise the following components: the trace elements per liter contain FeSO 4 ·7H 2 O 100mg，H 3 BO 3 20mg，CuSO 4 ·5H 2 O20 mg，AlK(SO 4 ) 2 ·12H 2 O 15mg,KI 100mg，MnSO 4 ·7H 2 O11mg，60mg，ZnSO 4 ·7H 2 O 100mg，CoCl 2 ·6H 2 O 100mg，Na 2 MoO 4 50 mg, 10g of ethylenediamine tetraacetic acid and the balance of water.
(2) Enrichment culture
Taking 20g to 100mL of activated sludge of sewage treatment pools of different treatment plants, carrying out shaking culture for 12h at 30 ℃, transferring 10mL of mixed liquor after shaking culture into 100mL of culture medium B with phosphorus concentration of 5mg/L, carrying out enrichment culture for 3d at 30 ℃, transferring into the culture medium C, carrying out one round of culture for 3 days, transferring 4 rounds of culture, and carrying out enrichment culture for 24d to obtain enrichment culture liquid.
(3) Separation and purification
Diluting the enriched culture solution to 10 -6 ～10 -3 Sucking 500 μl of the diluent into a separation and purification plate, mixing with the culture medium A at 40-42 ℃, solidifying, inverting, and culturing at 30 ℃ to obtain bacterial colonies; picking single bacterial colony on the flat plate, carrying out repeated streak purification on the flat plate, and observing to show that the bacteria are free of impurities, thus obtaining purified bacterial strain; colonies growing rapidly were picked up and inoculated in 100. Mu.L of BCIP developer-coated medium for cultivation, and blue-green colonies growing rapidly were used as candidate strains.
(4) Screening of phosphorus removal bacteria
Inoculating the purified strain into a conical flask filled with 100mL of culture medium B, performing shaking culture at 130r/min and 30 ℃ for 24 hours to obtain a preculture solution, transferring 10% of the preculture solution into the conical flask filled with 100mL of culture medium C, and performing shaking culture at 130r/min and 30 ℃ for 2 days; wherein, anaerobic culture is carried out for 24 hours and then aerobic culture is carried out for 24 hours; and (3) centrifuging 8000g of culture solution for 10min after the culture is finished, taking supernatant, measuring the phosphorus content in the liquid by using a molybdenum-antimony spectrophotometry, and examining the total phosphorus removal rate of the strain, thereby screening out a phosphorus-removing bacterium ZM16 with higher phosphorus removal effect, wherein the nucleotide sequence of the 16S rRNA gene is shown as SEQ ID NO. 1.
The ZM16 strain is separated from sewage treatment pool sludge of a Tianzi mountain refuse treatment plant in Hangzhou, zhejiang, and presents light yellow circular colonies on an LB culture medium and an inorganic salt sodium acetate carbon source culture medium, and has round luster and neat edges. After gram staining, DAPI staining and Nile red staining, the bacteria were identified as gram negative bacteria, with green colored metachromatic particles (polyP) and red colored lipid Particles (PHB) in the cells. The scanning electron microscope observed the thallus in rod shape, without special cell structure such as flagellum, pili, micro capsule, etc., and the physiological and biochemical characteristics are shown in table 1.
Table 1: physiological and biochemical identification of ZM16 strain
According to morphological characteristics and physiological and biochemical characteristics of ZM16, the 16S rRNA sequence of the strain ZM16 is compared and identified, and the similarity between the strain ZM16 and the 16S rRNA sequence of Pseudomonas asiatica RYU (T) (GeneBank: MH 517510.1) is highest, and the 16S rRNA sequence reaches 99.44%, and finally the strain is classified into Pseudomonas, named Pseudomonas asiatica ZM, and is preserved in China center for type culture collection, and the preservation number is CCTCC NO: m2022729, the preservation date is 2022, 5 and 26.
The invention further analyzes the dephosphorization mechanism of the strain Pseudomonas asiatica ZM, uses a second generation and third generation sequencing technology to combine to complete genome scanning sequencing of the high-efficiency dephosphorization strain Pseudomonas asiatica ZM, uses Unicycler splicing software to splice a plurality of Kmer parameters on an optimized sequence, obtains an optimal assembly result, and then uses Pilon software to carry out local hole filling and base correction on the assembly result. And comprehensively evaluating the assembly results of a plurality of Kmers according to the total length of the spliced sequences, the number of the scafold, the scafold N50 and other technical indexes, and finally obtaining the genome with the size of 5543632bp and the GC content of 58.12% by means of ZM16 genome sequencing analysis.
Bacterial gene prediction was performed using Glimmer 3.02 software, the protein sequences of the predicted genes were blasted against NR, GENES, STRING and GO databases, respectively (BLAST 2.2.28 +), strain ZM16 was identified to contain 1 ppk1 gene and 1 ppk2 gene, designated ppk1-ZM16 (as shown in SEQ NO. 2) and ppk2-ZM16 (as shown in SEQ NO. 3), and Open Reading Frames (ORFs) were 2241bp and 981bp in size, encoding 747 amino acids (as shown in SEQ NO. 4) and 327 amino acids (as shown in SEQ NO. 5), respectively.
The polyphosphate kinase genes ppk1-zm16 and ppk2-zm16 are characterized by the following sequence amplification methods:
the complete gene fragments ppk1-zm16 and ppk2-zm16 are obtained by using the genome of the strain Pseudomonas asiatica ZM as a template and utilizing a PCR method.
The primers used for the PCR are shown in Table 2:
table 2: tables of information on the sequences of the primers for the ppk1 and ppk2 genes
Experiments show that the nitrogen and phosphorus removal rates of ZM16 strain on agricultural sewage and raw water of a dairy plant are 100 percent.
The invention also provides application of the Pseudomonas asiatica in wastewater dephosphorization and nitrogen removal.
Wherein the wastewater comprises at least one of agricultural wastewater, rural domestic wastewater, dairy wastewater, chemical fertilizer plant wastewater and kitchen waste water. The phosphorus content in the wastewater is less than or equal to 40mg/L, and the nitrosamine content in the wastewater is less than or equal to 100mg/L.
The invention also provides a microbial agent for removing phosphorus and nitrogen, which comprises a Pseudomonas asiatica ZM16 strain.
The invention also provides a method for treating wastewater, comprising applying the Pseudomonas Asiatica ZM16 strain or the microbial agent to wastewater containing nitrogen and/or phosphorus.
In the method for treating wastewater, the OD value of the Pseudomonas asiatica to the Pseudomonas asiatica is more than or equal to 0.1.
The invention provides a polyphosphate kinase gene, the nucleotide sequence of which is any one of 1) to 3):
1) SEQ ID NO:2 or SEQ ID NO:3, a nucleotide sequence shown in figure 3;
2) A nucleotide sequence which is a substitution, deletion or addition of one or more nucleotides in the nucleotide sequence shown in 1) and which encodes the same protein as the nucleotide sequence shown in 1) or which encodes a protein functionally identical or similar to the protein;
3) A nucleotide sequence which is at least 90% homologous to the nucleotide sequence shown in 1) or 2) and which encodes a protein functionally identical or similar.
The invention also provides a protein coded by the polyphosphate kinase gene, and the amino acid sequence of the protein is as follows:
i) SEQ ID NO:4 or SEQ ID NO:5, and a polypeptide sequence shown in the figure;
II), an amino acid sequence which is functionally identical or similar to the protein of the amino acid sequence shown in I) and in which one or more amino acids are substituted, deleted or added in the amino acid sequence shown in I);
III) an amino acid sequence which is at least 90% homologous to the amino acid sequence indicated under I) or II) and which has the same or similar function to the protein.
The Asian pseudomonas Pseudomonas asiatica ZM provided by the invention has remarkable removal effect on phosphorus and nitrogen in wastewater, and the phosphorus removal rate reaches 100%, so that the Asian pseudomonas Pseudomonas asiatica ZM can be used for natural water ecological reconstruction or in-situ restoration; and has wide adaptability of carbon source concentration, and can be used for treating low-carbon source wastewater.
Drawings
FIG. 1 is a graph of Pseudomonas asiatica ZM bacterial stain; 1-a PHB staining pattern; 1-b, a staining chart of DAPI polyphosphate particles;
FIG. 2 is a scanning electron microscope image of Pseudomonas asiatica ZM cells, 2-a is the observation result of strain ZM16 under a 5,000-fold scanning electron microscope, and 2-b is the observation result of strain ZM16 under a 30,000-fold scanning electron microscope;
FIG. 3 is a agarose gel electrophoresis of the dephosphorizing gene; 3-a is agarose gel electrophoresis diagram of polyphosphate kinase 1 gene, ppk1-zm16 gene PCR product; 3-b is agarose gel electrophoresis diagram of polyphosphate kinase 2 gene, ppk2-zm16 gene PCR product;
FIG. 4 is a graph of phosphorous removal performance for different carbon to phosphorous ratios;
FIG. 5 is a graph of phosphorous removal performance for different carbon sources;
FIG. 6 is a graph of phosphorous removal performance at different pH's;
FIG. 7 is a graph of optimal dephosphorization and denitrification performance, 7-a being the dephosphorization result and 7-b being the denitrification result;
FIG. 8 is a plot of phosphorus removal window performance;
FIG. 9 is a graph showing the performance of dephosphorization and denitrification of artificially synthesized wastewater, wherein 9-a is the dephosphorization result and 9-b is the denitrification result;
FIG. 10 is a graph of wastewater dephosphorization and denitrification performance of a sewage treatment plant; 10-a is the phosphorus removal result, 10-b is the effect of removing paranitronitrogen, and 10-c is the effect of removing paranitronitrogen.
Description of biological preservation
Pseudomonas asiatica ZM16, 5.26 days 2022, is preserved in China center for type culture Collection, with the addresses of China, the university of Wuhan and Wuhan, and the preservation number of the Chinese and Wuhan is CCTCCNO: M2022729.
Detailed Description
The examples provided below are presented in more detail to facilitate a further understanding of the present invention. These examples are provided only for better understanding of the present invention and are not intended to limit the scope or practice of the present invention, and the embodiments of the present invention are not limited to the following.
Enzymes, kits and other reagents used in the following examples were purchased from the biotechnology company of nanking and nuozhen.
The primers used in the following examples were synthesized by Beijing qing Biotech Co.
Example 1: isolation and identification of dephosphorization bacteria Pseudomonas asiaticaZM16
1. Sample processing
Under the aseptic condition, 20g of activated sludge of a sewage treatment pool of a Tianzi mountain garbage treatment plant in Zhejiang Hangzhou is taken to be sterilized 100mL of sterile water containing glass beads, the shaking culture is carried out for 12h at 30 ℃,10 mL of mixed liquor after the shaking culture is transferred to 100mL of culture medium B with the phosphorus concentration of 5mg/L, enrichment culture is carried out for 3d at 30 ℃, and the mixed liquor is transferred to 100mL of culture medium C with the phosphorus concentration of 49.5mg/L for 3 days to be one round, and the total transfer is carried out for 4 rounds under the aseptic condition, and the enrichment culture is carried out for 24d, thus obtaining the enrichment culture liquid.
2. Separation and purification
Diluting the enriched culture solution to 10 under aseptic condition -6 ～10 -3 Sucking 500 μl of the diluent into a separation and purification plate, mixing with 20mL of culture medium A at 40-42 ℃, solidifying, inverting, and culturing at 30 ℃ to obtain bacterial colonies; picking single colony on the flat plate, and carrying out repeated zigzag streak purification on the flat plate until observation shows that the bacteria are free, thus obtaining purified strain; colonies growing rapidly were picked up and inoculated in 100. Mu.L of BCIP developer-coated medium B for cultivation, and blue-green colonies growing rapidly were used as candidate strains. The strains screened above are identified by combining morphological and physiological characteristics and biochemical characteristics of bacteria and 16S rRNA sequences.
3. Screening of phosphorus removal bacteria
Inoculating the purified strain into a conical flask filled with 100mL of culture medium B, carrying out shake culture at 130r/min and 30 ℃ under anaerobic condition for 24 hours to obtain a preculture solution, transferring 10% of the preculture solution into a conical flask filled with 100mL of culture medium C, and carrying out shake culture at 130r/min and 30 ℃ for 2 days; wherein, anaerobic culture is carried out for 24 hours and then aerobic culture is carried out for 24 hours; and (3) centrifuging 8000g of culture solution for 10min after the culture is finished, taking supernatant, measuring the phosphorus content in the liquid by using a molybdenum-antimony anti-spectrophotometry at 710nm, and examining the total phosphorus removal rate of the strain, so that the screened phosphorus removal bacteria with higher phosphorus removal effect are obtained.
4. Dyeing identification
1) PHB identification after anaerobic culture of thalli
Taking a proper amount of bacterial liquid in the late phase of anaerobic culture to logarithmic phase, centrifuging for 5min at 8000g, removing supernatant, washing the precipitated bacterial body twice with 0.1M PBS buffer solution, and re-suspending cells to OD 600 =0.8, 1mL of bacterial solution was mixed with 15 μl of nile red dye (mass concentration 0.1mg/mL acetone solution) and homogenized at room temperatureThe color is 5min, the excitation wavelength is 480nm, the absorption wavelength is 575nm, and the intracellular red is PHB when observed under a fluorescence microscope. The results are shown in FIG. 1-B.
2) Identification of poly-P after aerobic culture of thalli
Taking a proper amount of bacterial liquid which is cultivated to the late stage of the logarithmic phase, centrifuging for 5min at 8000g, removing the supernatant, washing the precipitated bacterial body twice with 0.1M PBS buffer solution, and re-suspending the cells to OD 600 1mL of bacterial solution is added with 100 mu L of DAPI dye solution (the mass concentration is 1mg/mL solution), the bacterial solution is dyed for 10min at room temperature in a dark place, the excitation wavelength is 480nm, the absorption wavelength is 575nm, and the intracellular green fluorescence is poly-P particles when observed under a fluorescence microscope. The results are shown in FIG. 1-C.
5. Characterization of morphological features
1) Scanning electron microscope
Picking single colony in 5mL LB culture medium with an inoculating loop, culturing at 30 ℃ for 12h at 130rpm, centrifuging at 4 ℃ for 5min at 6000 Xg, collecting 500 mu L of bacterial liquid, washing bacterial cells 3 times with precooled 0.1MPBS, suspending bacterial cells in 500 mu L of precooled 2.5% glutaraldehyde fixing liquid, and fixing at 4 ℃ in a dark place for 24h; washing three times with 0.1M PBS; 400. Mu.L of 1% osmium acid was added and the mixture was fixed for 2 hours; eluting with 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% ethanol for 15min, and dehydrating with anhydrous ethanol for 15min (repeating twice); in a ventilated kitchen, the samples were immersed in Hexamethyldisilazane (HMDS) for 15min, and repeated twice; placing the sample into a critical drier at room temperature for CO 2 Replacing and drying for 60min, cutting a cover glass by scissors, and attaching a proper sample to the copper plate; plating metal platinum film in a sputtering instrument with the thickness of gold plating being about 20-30nm; the sample was observed under a scanning electron microscope at a suitable magnification and photographed, with the results shown in fig. 2.
2) Genomic DNA extraction
Extraction was performed using a microbial genomic DNA flash extraction kit and detected by 1.0% agarose electrophoresis, the results of which are shown in FIG. 3-A.
3) PCR amplification and identification of 16S rRNA
The bacterial 16S rRNA sequence general primer F27/R1492 is selected for PCR amplification, and the F27 primer sequence is as follows: the primer sequence of the 5-AGAGTTTGATCCTGGCTCAG-3' and the R1492 is as follows: 5'-GGTTACCTTGTTACGACTT-3';
the PCR reaction system comprises: PCR mix buffer 25. Mu.L, 3'primer 1. Mu.L, 5' primer 01. Mu.L, DNA template 2. Mu.L, ddH 2 O21. Mu.L, total volume 50. Mu.L.
The PCR amplification conditions were: pre-denaturation at 94℃for 5min;30 cycles: denaturation at 94℃for 1min, annealing at 50℃for 1min, and extension at 72℃for 2min; finally, the mixture is incubated at 72 ℃ for 10min and preserved at 4 ℃.
mu.L of DNA product was taken and detected by electrophoresis on 1.0% agarose gel.
The BLAST software is used for comparing with the registered 16S rRNA sequence in GenBank, the homology of the strain ZM16 and Pseudomonas asiatica is 99.6%, the ANI value is 98.72%, and the strain belongs to the genus Pseudomonas.
Example 2 optimal Nitrogen and phosphorus removal conditions for Strain ZM16 of the present invention
1. The method for measuring total phosphorus by using the molybdenum-antimony anti-spectrophotometry comprises the following steps:
a standard use solution of phosphorus was formulated. And respectively taking 0, 0.2, 0.4, 0.8, 1.2, 1.6, 2.0, 3.0, 4.0 and 5.0mL of phosphorus standard solution into a test tube with a cover, adding distilled water until the total volume is 10mL, adding 1.6mL of potassium persulfate, uniformly mixing, and digesting for 30min at 120 ℃. After cooling, adding 0.4mL of ascorbic acid solution, and uniformly mixing; after 30s, 0.8mL of molybdate mixed solution is added, and the mixture is uniformly mixed and kept stand at room temperature for 15min. At a wavelength of 700nm, absorbance was measured with reference to the blank No.1, and a standard curve was drawn.
2. Determination of optimal carbon-phosphorus ratio
The strain ZM16 preserved by the invention is inoculated into a culture medium C with different carbon-nitrogen-phosphorus ratios after enrichment culture, and the initial OD 600 =0.1, c: n: p is 25:10: 1. 50:10: 1. 100:10: 1. 200:10:1 and 300:10:1, wherein the N sources are each formed of NH 4 CL-N：KNO 3 -N：NaNO 2 -n=1: 2:2, composing; shake culturing at 30deg.C for 48 hr, anaerobic culturing for 24 hr, aerobic culturing for 24 hr, and detecting OD of culture medium 600 The values and the phosphorus removal rate, and the detection results are shown in FIG. 5. As can be seen from the results of FIG. 1, the strain of the present invention has a wide adaptability of carbon source concentration, and the ratio of C: N: P50: 10:1 to 300:10:1 has better dephosphorization effect and can be used for wastewater treatment under the condition of low carbon source.
3. Determination of optimal carbon source
The strain ZM16 preserved by the invention is inoculated into phosphorus-rich culture medium C with different carbon sources after enrichment culture, and the initial OD 600 =0.1, shake culture at 30 ℃ for 48h, wherein anaerobic culture is performed for 24h, then aerobic culture is performed for 24h, and OD of the culture medium is detected after 48h 600 The values and the phosphorus removal rate, and the detection results are shown in FIG. 6. The phosphorus-rich culture medium with different carbon sources refers to a culture medium C, wherein glucose with equal carbon content is used for replacing sodium acetate in the culture medium C to obtain a culture medium C1, fructose with equal carbon content is used for replacing sodium acetate in the culture medium C to obtain a culture medium C2, sucrose with equal carbon content is used for replacing sodium acetate in the culture medium C to obtain a culture medium C3, and lactose with equal carbon content is used for replacing sodium acetate in the culture medium C to obtain a culture medium C4; and replacing sodium acetate in the culture medium C with starch with equal carbon content to obtain the culture medium C5. As can be seen from the results of FIG. 6, the strain of the present invention can well utilize various carbon sources, and the most suitable carbon source is sodium acetate.
4. Determination of the optimum pH
The strain ZM16 preserved by the invention is inoculated into culture medium C with different pH values after enrichment culture, and the initial OD 600 =0.1, shake culture at 20 ℃ for 48h, wherein anaerobic culture is performed for 24h, then aerobic culture is performed for 24h, and OD of medium C is detected after 48h 600 The values and the phosphorus removal rate, and the detection results are shown in FIG. 7. The above-mentioned culture media with different pH values respectively refer to: a culture medium C; medium C6: the components are the same as the culture medium C, and the pH value is 5.0; medium C7: the components are the same as the culture medium C, and the pH value is 6.0; medium C8: the components are the same as the culture medium C, and the pH value is 8.0; medium C9: the components are the same as the culture medium C, and the pH value is 9.0; medium C10: the components are the same as the culture medium C, and the pH value is 10.0; as is clear from the results of FIG. 7, the strain of the present invention has a strong dephosphorization activity in the pH range of 7 to 9.
5. Determination of phosphorus removal Capacity under optimal conditions
The strain ZM16 preserved by the invention is inoculated in a culture medium C after enrichment culture, and the initial OD 600 Shake culture at 20deg.C for 48 hr, anaerobic culture for 24 hr, aerobic culture for 24 hr, and whole cultureThe real-time OD value and the dephosphorization rate, the denitrification rate and the denitrification rate in the process are shown in figure 7, the dephosphorization rate reaches 100% after 48 hours, and the denitrification rate reach 85.6% and 92.3% respectively.
Example 3: determination of phosphorus removal range of phosphorus removal bacteria
The strain preserved by the invention is inoculated in the optimal condition culture medium C after enrichment culture, and the initial OD 600 =0.1, ph=7.5, keeping the C content unchanged, adjusting C: N to 10:1, wherein the N sources are each formed of NH 4 Cl-N：KNO 3 -N：NaNO 2 -n=1: 2:2, and setting the P content to be 1mg/L, 2mg/L, 4mg/L, 8mg/L, 16mg/L, 24mg/L, 32mg/L and 40mg/L respectively; shake culturing at 30deg.C for 48 hr, anaerobic culturing for 24 hr, aerobic culturing for 24 hr, and detecting OD of culture medium 600 The values and the phosphorus removal rate, and the detection results are shown in FIG. 8. As can be seen from FIG. 8, the strain of the invention can remove P of 28.16mg/L at most in 48 hours, and has 100% dephosphorization rate on water bodies with P content of 20mg/L or less.
Example 4: determination of phosphorus removal rate of artificially synthesized wastewater
1. The formula of the synthetic wastewater culture medium is as follows:
890mg of sodium acetate trihydrate, 10mg of yeast powder, 100g of peptone and K 2 HPO 4 56.25mg、KH 2 PO 4 44 mg、KNO 3 288 mg、NaNO 2 197 mg、CaCl 2 28 mg、NaCl 50mg、NaHCO 3 75 mg、MgSO 4 75 mg、ddH 2 O1L. The P content is 20mg/L.
2. Dephosphorization rate of the strain: the strain preserved by the invention is inoculated in a culture medium C, is subjected to anaerobic culture for 12 hours after enrichment culture for 12 hours, is inoculated in 200ml of a synthetic culture medium after co-culture for 24 hours, and has initial OD 600 After 24h of aerobic culture, OD of the culture medium was detected =1 600 The values and the amounts of nitrogen and phosphorus removed are shown in FIG. 9. The phosphorus removal amount of ZM16 was calculated to be 20mg/L (100%), and the amounts of nitrate and nitrite removed were found to be 33.3mg/L (83.5%) and 27.3mg/L (68.3%), respectively, on average.
Example 5: sewage nitrogen and phosphorus removal for sewage treatment plant
Taking agricultural sewage raw water and effluent from Zhejiang Jinhua and dairy sewage, and respectively measuring the pH, total phosphorus, nitrate nitrogen and nitrite nitrogen content as follows:
TABLE 3 Table 3
Inoculating ZM16 strain into conical bottle containing 100mL of agricultural sewage water, agricultural sewage water and dairy sewage water, and initial OD 600 =1, cultured aerobically at 130r/min, at 30 ℃ for 6h; after the culture is completed, 8000g of culture solution is taken and centrifuged for 10min, supernatant is taken, the total phosphorus, nitrate nitrogen and nitrite nitrogen concentration of the supernatant is measured, the removal rate of the strain on the total phosphorus, nitrate nitrogen and nitrite nitrogen is examined, and the detection result is shown in figure 10. As a result, the nitrogen and phosphorus removal rates of ZM16 were 100%.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (8)
1. Pseudomonas asiaticaPseudomonas asiatica) Characterized in that the preservation number is CCTCC NO: M2022729。
2. Pseudomonas asiatica according to claim 1，The nucleotide sequence of the 16S rRNA gene is shown as SEQ ID NO. 1.
3. Use of pseudomonas asiatica according to claim 1 or 2 for the dephosphorization and nitrogen removal of waste water.
4. The use according to claim 3, wherein the waste water comprises at least one of agricultural waste water, rural domestic waste water, dairy waste water, fertilizer plant waste water, kitchen waste water.
5. The method according to claim 3, wherein the wastewater contains less than or equal to 40mg/L of phosphorus and less than or equal to 100mg/L of nitrosamine.
6. A microbial agent for phosphorous and nitrogen removal comprising the pseudomonas asiatica of claim 1 or 2.
7. A method of treating wastewater comprising applying the pseudomonas asiatica of claim 1 or 2 or the microbial agent of claim 6 to wastewater containing phosphorus and/or nitrogen.
8. The method of claim 7, wherein the OD value of the pseudomonas asiatica to pseudomonas asiatica is greater than or equal to 0.1.
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| CN106244502A (en) * | 2016-09-27 | 2016-12-21 | 湖北大学 | One strain efficient dephosphorization and the pseudomonas of degraded lecithin |
| CN110217895A (en) * | 2019-05-31 | 2019-09-10 | 中国科学院微生物研究所 | A kind of complex micro organism fungicide and its application for water environment treatment |
| CN113999836A (en) * | 2021-11-03 | 2022-02-01 | 河北天泓环保科技有限公司 | Compound microbial agent for sewage treatment, preparation method and application of compound microbial agent in river treatment and black and odorous water treatment |
| CN115369063A (en) * | 2022-09-29 | 2022-11-22 | 武汉益多康生物技术有限公司 | Microbial agent for water environment treatment and preparation method thereof |
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| CN106244502A (en) * | 2016-09-27 | 2016-12-21 | 湖北大学 | One strain efficient dephosphorization and the pseudomonas of degraded lecithin |
| CN110217895A (en) * | 2019-05-31 | 2019-09-10 | 中国科学院微生物研究所 | A kind of complex micro organism fungicide and its application for water environment treatment |
| CN113999836A (en) * | 2021-11-03 | 2022-02-01 | 河北天泓环保科技有限公司 | Compound microbial agent for sewage treatment, preparation method and application of compound microbial agent in river treatment and black and odorous water treatment |
| CN115369063A (en) * | 2022-09-29 | 2022-11-22 | 武汉益多康生物技术有限公司 | Microbial agent for water environment treatment and preparation method thereof |
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| "Genomic Analysis of Pseudomonas asiatica JP233: An Efficient Phosphate-Solubilizing Bacterium";Linlin Wang 等;《Genes》;第13卷(第2290期);全文 * |
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