CN114480159B - Synchronous heterotrophic nitrification aerobic denitrification dephosphorization bacterium and application thereof - Google Patents
Synchronous heterotrophic nitrification aerobic denitrification dephosphorization bacterium and application thereof Download PDFInfo
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- 230000001360 synchronised effect Effects 0.000 title claims abstract description 22
- 241000894006 Bacteria Species 0.000 title abstract description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 62
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims abstract description 53
- 229910052698 phosphorus Inorganic materials 0.000 claims abstract description 52
- 239000011574 phosphorus Substances 0.000 claims abstract description 52
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 41
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 claims abstract description 24
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims abstract description 24
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 16
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 16
- JVMRPSJZNHXORP-UHFFFAOYSA-N ON=O.ON=O.ON=O.N Chemical compound ON=O.ON=O.ON=O.N JVMRPSJZNHXORP-UHFFFAOYSA-N 0.000 claims abstract description 12
- 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 abstract description 11
- 239000002351 wastewater Substances 0.000 claims abstract description 8
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- 238000000034 method Methods 0.000 claims description 35
- 239000001509 sodium citrate Substances 0.000 claims description 17
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- 229910019142 PO4 Inorganic materials 0.000 claims description 13
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- 239000001963 growth medium Substances 0.000 description 13
- 230000001580 bacterial effect Effects 0.000 description 10
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- 210000004027 cell Anatomy 0.000 description 7
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 7
- 235000019341 magnesium sulphate Nutrition 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- 239000000725 suspension Substances 0.000 description 7
- 239000011790 ferrous sulphate Substances 0.000 description 6
- 235000003891 ferrous sulphate Nutrition 0.000 description 6
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 6
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 5
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 5
- 235000011130 ammonium sulphate Nutrition 0.000 description 5
- XQGPKZUNMMFTAL-UHFFFAOYSA-L dipotassium;hydrogen phosphate;trihydrate Chemical compound O.O.O.[K+].[K+].OP([O-])([O-])=O XQGPKZUNMMFTAL-UHFFFAOYSA-L 0.000 description 5
- 238000011081 inoculation Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
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- 239000002609 medium Substances 0.000 description 5
- 230000001546 nitrifying effect Effects 0.000 description 5
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- 230000005526 G1 to G0 transition Effects 0.000 description 4
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- 238000012258 culturing Methods 0.000 description 4
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 4
- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Chemical compound [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
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- 229920000388 Polyphosphate Polymers 0.000 description 2
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- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 239000013067 intermediate product Substances 0.000 description 2
- 239000001205 polyphosphate Substances 0.000 description 2
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- 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
- 229910021586 Nickel(II) chloride Inorganic materials 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
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- 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
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- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 1
- 230000019771 cognition Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 150000005690 diesters Chemical class 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 235000019797 dipotassium phosphate Nutrition 0.000 description 1
- 239000010840 domestic wastewater Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
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- 238000005259 measurement Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- QMMRZOWCJAIUJA-UHFFFAOYSA-L nickel dichloride Chemical compound Cl[Ni]Cl QMMRZOWCJAIUJA-UHFFFAOYSA-L 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
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- 229920013639 polyalphaolefin Polymers 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
- 238000002360 preparation method Methods 0.000 description 1
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- 238000000926 separation method Methods 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
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- 239000011684 sodium molybdate Substances 0.000 description 1
- 235000015393 sodium molybdate Nutrition 0.000 description 1
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 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
- 230000006641 stabilisation Effects 0.000 description 1
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- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
Classifications
-
- 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
- C02F3/02—Aerobic processes
-
- 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
- C02F3/348—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the way or the form in which the microorganisms are added or dosed
-
- 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
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic 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
- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2305/00—Use of specific compounds during water treatment
- C02F2305/06—Nutrients for stimulating the growth of microorganisms
-
- 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
Abstract
A synchronous heterotrophic nitrification aerobic denitrification dephosphorization bacterium and application thereof belong to the technical field of microorganisms. The strain is preserved in China general microbiological culture Collection center (GGMGG) with the preservation number of GGMGG No.23440 at 9 and 17 of 2021. Pseudomonas aeruginosaSNDP-01 is a strain which plays the functions of heterotrophic nitrification, aerobic denitrification and aerobic excess phosphorus absorption simultaneously, inorganic nitrogen (ammonia nitrogen, nitrite nitrogen and nitrate nitrogen) in water is converted into nitrogen in one step by utilizing an organic carbon source under the conditions that the temperature is 30-35 ℃, the pH value is=7.5-8.5, the C/N (mass ratio) =10-15, the P/N (mass ratio) =0.1-0.2 and the rotating speed is=120-160 rpm, and meanwhile, excess orthophosphate is absorbed and stored in extracellular secretion (EPS) in a phosphorus single fat form, so that the synchronous denitrification and dephosphorization of high-nitrogen and phosphorus wastewater are realized, and meanwhile, the unique phosphorus gathering mode is also beneficial to the resource recovery of phosphorus elements in the wastewater. The strain can play an important role in the fields of living sewage treatment, water body restoration, river treatment, resource recovery and the like.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and relates to pseudomonas aeruginosa (Pseudomonas aeruginosa) SNDP-01 and application thereof. The strain has the functions of synchronous heterotrophic nitrification and aerobic denitrification and excessive phosphorus absorption, and can be inoculated into domestic sewage to realize synchronous denitrification and dephosphorization of the wastewater in one reactor.
Background
With the progress of society and the development of economy, the excessive discharge of the domestic sewage and the industrial wastewater rich in nitrogen and phosphorus gradually prominences the eutrophication problem of various water bodies in China, and not only causes damage to the ecological system of the water bodies, but also brings great threat to the health of human beings. Therefore, limiting the concentration of nitrogen and phosphorus in the effluent is critical to a sewage treatment plant. The sewage treatment method is mainly divided into a chemical method, a physical method and a biological method, wherein the biological treatment method is widely applied to the treatment of domestic sewage due to the characteristics of high efficiency, low cost, environmental friendliness and the like.
In the traditional biological denitrification and dephosphorization process, ammonia nitrogen in sewage is firstly subjected to autotrophic nitrification under an aerobic condition to be oxidized into nitrite nitrogen or nitrate nitrogen, and then subjected to heterotrophic denitrification under an anaerobic condition to be reduced into nitrogen; 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. Therefore, the traditional removal process requires 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 addition, competition of heterotrophic denitrifying bacteria and phosphorus accumulating bacteria for carbon sources is also unfavorable for constructing a stable and sustainable denitrification and dephosphorization biological system. Thus, the best solution is to combine these separate processes to achieve simultaneous nitrogen and phosphorus removal in a single reactor.
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.
However, the number of SNDPR bacteria separated at present is small, most of the strains have the problem of anaerobic phosphorus release, and secondary pollution of water is easily caused. Therefore, the discovery and separation of more phosphorus removal bacteria with heterotrophic nitrification and aerobic denitrification capability is beneficial to expanding the application field of the microorganisms and promoting the cognition of the microorganisms in nature.
The invention screens out a dephosphorization bacterium with heterotrophic nitrification and aerobic denitrification function from the activated sludge in the secondary sedimentation tank of the sewage treatment plant. Under strict aerobic conditions, the strain can realize high-efficiency synchronous denitrification and dephosphorization functions, and the removed phosphorus is stored in extracellular secretion (EPS) in the form of phosphorus monoester, so that the release of anaerobic phosphate is not existed, and the recovery of phosphorus resources is facilitated.
Disclosure of Invention
The invention provides a phosphorus removal bacterium with heterotrophic nitrification and aerobic denitrification capability, namely pseudomonas aeruginosa (Pseudomonas aeruginosa) SNDP-01, which breaks through the limitation of the traditional biological nitrogen and phosphorus removal theory and can realize synchronous nitrogen and phosphorus removal in one reactor.
The invention provides application of pseudomonas aeruginosa (Pseudomonas aeruginosa) SNDP-01 in the treatment of real domestic sewage, and the pseudomonas aeruginosa can be inoculated into high-nitrogen and phosphorus wastewater, and can synchronously denitrify and dephosphorize in an aerobic single-stage reactor, thereby overcoming the technical bottlenecks that the nitrification process, the denitrification process and the phosphorus absorption/release process of the existing biological denitrification and dephosphorization process need to be carried out in sections, and having wide application prospect and good economic and social benefits.
Compared with the traditional biological denitrification and dephosphorization process, the application of the invention is characterized in that the nitrification process is not the traditional autotrophic condition but the heterotrophic condition, the denitrification process is not the traditional anaerobic condition but the aerobic condition, and the dephosphorization process is not performed under the anaerobic/aerobic alternating condition by utilizing the phosphorus accumulating bacteria, but is performed under the complete aerobic condition by utilizing the aerobic phosphorus uptake bacteria. The denitrification and dephosphorization process is completed by one functional strain under one aerobic heterotrophic state, complex condition transformation is not needed, and the problem of mutual competition of different functional bacterial groups in the traditional denitrification and dephosphorization process is avoided.
The pseudomonas aeruginosa (Pseudomonas aeruginosa) SNDP-01 provided by the invention is preserved in China general microbiological culture Collection center (GGMGG), and the preservation address is: no. 3 of North Chen Xili No. 1, no.23440 of North Chaoyang district of Beijing city, and the preservation date is 2021, 9 months and 17 days. The length of the 16S rDNA nucleotide sequence is 1421bp.
The pseudomonas aeruginosa (Pseudomonas aeruginosa) SNDP-01 provided by the invention grows on a basic solid culture medium, and the preparation method of the basic solid culture medium comprises the following steps: 3.5833g of sodium citrate, 0.472g of ammonium sulfate, 0.075g of dipotassium hydrogen phosphate, 0.05g of magnesium sulfate, 0.12g of sodium chloride, 1ml of trace elements and 15-20g of agar are weighed, the medicines are dissolved in 1L of deionized water, and the deionized water is poured into a culture dish after being sterilized at 121 ℃ for 20min to prepare a solid culture medium.
After the pseudomonas aeruginosa (Pseudomonas aeruginosa) SNDP-01 provided by the invention is inoculated to a solid basic culture medium and cultured for 48 hours, white colonies with smooth and moist surfaces, slightly convex middle parts and irregular edges are presented, and a water-soluble pigment which enables the culture medium to be blue-green can be secreted. Gram staining was negative. The scanning electron microscope result shows that the size of the strain is (1.5-5) mu m x (0.5-1) mu m, and the strain is in a rod shape or a short rod shape.
The pseudomonas aeruginosa (Pseudomonas aeruginosa) SNDP-01 provided by the invention can convert ammonia nitrogen into nitrite nitrogen or nitrate nitrogen under an aerobic condition by taking an organic matter as an electron donor, so as to realize a heterotrophic nitrification process; the organic matters are used as electron donors under the aerobic condition, so that nitrite nitrogen or nitrate nitrogen is reduced into nitrogen, and the aerobic denitrification process is realized; meanwhile, the synchronous nitrification and denitrification process can be realized by taking ammonia nitrogen, nitrite nitrogen, ammonia nitrogen and nitrate nitrogen as mixed nitrogen sources under the aerobic condition.
The pseudomonas aeruginosa (Pseudomonas aeruginosa) SNDP-01 provided by the invention can transport orthophosphate into cells while performing aerobic denitrification, and finally the orthophosphate is mainly stored in EPS in the form of phosphoric monoester, so that a direct aerobic phosphorus absorption process without taking anaerobic phosphorus release as a premise is realized.
The pseudomonas aeruginosa (Pseudomonas aeruginosa) SNDP-01 provided by the invention has the best conditions of playing an excellent synchronous denitrification and dephosphorization role that: the carbon source is sodium citrate, the C/N is 10-15, the P/N is 0.1-0.2, the culture temperature is 30-35 ℃, the pH is 7.5-8.5, and the rotating speed is 120-160rpm.
The invention provides pseudomonas aeruginosa (Pseudomonas aeruginosa) SNDP-01 in the aerobic denitrification process is free of intermediate (NO) 2 - And NO 3 - ) Has good application prospect.
The invention provides a pseudomonas aeruginosa (Pseudomonas aeruginosa) SNDP-01 which has the following aerobic phosphate utilization paths: part of orthophosphate is used for intracellular metabolism and cell membrane synthesis, and the other part of orthophosphate is stored in EPS after forming phosphate monoester, and the process of anaerobic phosphate release does not exist, so that the resource recovery of phosphorus element is facilitated.
Drawings
FIG. 1 shows the growth and denitrification and dephosphorization performance of Pseudomonas aeruginosa (Pseudomonas aeruginosa) SNDP-01 under various culture conditions.
FIG. 2 shows the NH content of Pseudomonas aeruginosa (Pseudomonas aeruginosa) SNDP-01 4 + The growth and denitrification and dephosphorization characteristics of the synchronous heterotrophic nitrification and dephosphorization process are carried out for the sole nitrogen source.
FIG. 3 shows the SNDP-01 of Pseudomonas aeruginosa (Pseudomonas aeruginosa) with NO respectively 2 - And NO 3 - The growth and denitrification and dephosphorization characteristics of the synchronous aerobic denitrification and dephosphorization process are carried out for the sole nitrogen source.
FIG. 4 shows the SNDP-01 of Pseudomonas aeruginosa (Pseudomonas aeruginosa) with NH respectively 4 + And NO 2 - 、NH 4 + And NO 3 - The growth and denitrification dephosphorization characteristics of the synchronous nitrification and denitrification dephosphorization process are carried out for the mixed nitrogen source.
FIG. 5 shows denitrification and dephosphorization properties of Pseudomonas aeruginosa (Pseudomonas aeruginosa) SNDP-01 for treating real life wastewater.
FIG. 6 shows phosphorus distribution and presence of phosphorus in EPS during heterotrophic nitrification and phosphorus removal by Pseudomonas aeruginosa (Pseudomonas aeruginosa) SNDP-01.
Fig. 7 shows the presence of phosphorus on EPS.
Detailed Description
The invention will be further elaborated in connection with the drawings and the specific embodiments described below, which are intended to illustrate the invention only and not to limit the scope of the invention. The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are commercially available.
The media used in the examples are as follows:
basal medium: ammonium sulfate 0.472g/L, dipotassium phosphate trihydrate 0.075g/L, magnesium sulfate 0.050g/L, sodium chloride 0.120g/L, ferrous sulfate 0.010g/L, and trace elements 1ml/L.
Nitrifying culture medium: 3.583g/L of sodium citrate, 0.472g/L of ammonium sulfate, 0.075g/L of dipotassium phosphate, 0.050g/L of magnesium sulfate, 0.120g/L of sodium chloride, 0.010g/L of ferrous sulfate and 1ml/L of trace elements.
Denitrification medium I: 3.583g/L sodium citrate, 0.493g/L sodium nitrite, 0.075g/L dipotassium phosphate trihydrate, 0.050g/L magnesium sulfate, 0.120g/L sodium chloride, 0.010g/L ferrous sulfate, and 1ml/L trace elements.
Denitrification medium II: 3.583g/L sodium citrate, 0.607g/L sodium nitrate, 0.075g/L dipotassium phosphate trihydrate, 0.050g/L magnesium sulfate, 0.120g/L sodium chloride, 0.010g/L ferrous sulfate, and 1ml/L trace elements.
Mixed nitrogen source medium i: 3.583g/L of sodium citrate, 0.236g/L of ammonium sulfate, 0.246g/L of sodium nitrite, 0.075g/L of dipotassium phosphate trihydrate, 0.050g/L of magnesium sulfate, 0.120g/L of sodium chloride, 0.010g/L of ferrous sulfate and 1ml/L of trace elements.
Mixed nitrogen source medium ii: 3.583g/L of sodium citrate, 0.236g/L of ammonium sulfate, 0.304g/L of sodium nitrate, 0.075g/L of dipotassium phosphate trihydrate, 0.050g/L of magnesium sulfate, 0.120g/L of sodium chloride, 0.010g/L of ferrous sulfate and 1ml/L of trace elements.
Trace elements: 1g/L of zinc sulfate, 0.3g/L of manganese chloride, 3g/L of boric acid, 2g/L of cobalt chloride, 0.1g/L of copper chloride, 0.2g/L of nickel chloride and 0.3g/L of sodium molybdate.
The domestic sewage used in the invention is obtained from the effluent of a septic tank, and the basic components comprise GOD 170-210mg/L and NH 4 + -N 62-75mg/L,NO 2 - -N 0.01-0.12mg/L,NO 3 - -N 0.2-1.2mg/L,PO 4 3- -P 5.6-6.8mg/L。
Example 1
Optimization of the optimal synchronous denitrification and dephosphorization conditions of pseudomonas aeruginosa Pseudomonas aeruginosa SNDP-01.
The strain preserved in glycerol at-20deg.C (preserved in China general microbiological culture Collection center, accession No. 23440) was inoculated into 100ml of sterilized basal medium, placed in a shaker at 30deg.C at 120rpm for shaking culture for 18-20 hours to grow the cells to the late logarithmic phase, and the bacterial suspension was used for inoculation (hereinafter the same). 3.5833g of sodium citrate, 3.4167g of sodium acetate, 2.375g of sucrose, 2.5g of glucose and 3.375 g of sodium succinate are respectively added into 5 conical flasks containing 300ml of liquid basal medium as carbon sources, then 3ml of bacterial suspension are respectively inoculated into the conical flasks, the flasks are placed into an air bath shaker at 35 ℃ and 120rpm for culture, sampling is carried out at the 0 th and 24 th hours and NH is measured after centrifugation at 8000rpm for 10min 4 + -N、PO 4 3- Concentration and OD of P 600 Values. As shown in FIG. 1a, the ammonia nitrogen and phosphate removal rate of the strain is highest when the carbon source is sodium citrate, so that the carbon source condition of the strain exerting the optimal denitrification and dephosphorization capability is sodium citrate.
And in the same way, sodium citrate is used as a carbon source, the C/N (mass ratio, the same applies below) of the basic culture medium is adjusted to be 2.5, 5, 10, 15 and 20 respectively, and the culture and test strip conditions are the same as above. As a result, as shown in FIG. 1b, the ammonia nitrogen and phosphate removal rate of the strain was highest when C/N=10-15, and C/N=10-15 was taken as the optimal C/N condition.
Similarly, the P/N (mass ratio, the same applies hereinafter) of the basal medium was adjusted to 0.1, 0.2, 0.4, 0.6 and 0.8 under the conditions of optimal carbon source and C/N ratio, respectively, and the culture and test conditions were the same as above. As shown in FIG. 1c, the ammonia nitrogen and phosphate removal rate of the strain is highest when P/N=0.1-0.2, and the optimal P/N ratio condition is taken as P/N=0.1-0.2.
Similarly, under the condition that the optimal carbon source is sodium citrate and C/N/P=100/10/1 (mass ratio, the same applies below), the culture temperatures are respectively adjusted to 20, 25, 30, 35 and 40 ℃, and the test strips are the same as above. As shown in FIG. 1d, the ammonia nitrogen and phosphate removal rate of the strain is highest when the temperature is 30-35 ℃, and the optimal temperature condition is 30-35 ℃.
Similarly, the pH was adjusted to 5.5, 6.5, 7.5, 8.5 and 9.5 at a temperature of 30-35℃with sodium citrate as the optimal carbon source, C/N/P=100/10/1, and the culture and measurement conditions were as above. As a result, as shown in fig. 1e, ammonia nitrogen and phosphate removal rates were highest when ph=7.5 to 8.5, so ph=7.5 to 8.5 was taken as the optimal pH condition.
Similarly, under the conditions that the optimal carbon source is sodium citrate, the C/N/P=100/10/1, the temperature is 30 ℃, the pH is 7.5-8.5, the rotating speeds are respectively adjusted to 40, 80, 120, 160 and 200rpm, and the test strip conditions are the same as above. As a result, as shown in FIG. 1f, the ammonia nitrogen and phosphate removal rates were highest when the rotational speed was 120-160rpm, and thus the rotational speed was 120-160rpm as the optimum rotational speed condition.
In summary, pseudomonas aeruginosa (Pseudomonas aeruginosa) SNDP-01 exerts optimal synchronous denitrification and dephosphorization conditions as follows: the carbon source is sodium citrate, the C/N is 10-15, the P/N is 0.1-0.2, the temperature is 30-35 ℃, the pH is 7.5-8.5, and the rotating speed is 120-160rpm.
Example 2
Pseudomonas aeruginosa Pseudomonas aeruginosa SNDP-01 takes ammonia nitrogen as the only nitrogen source to perform synchronous nitrification and dephosphorization, and has the characteristics of growth and pollutant removal.
3ml of the bacterial suspension was inoculated into an Erlenmeyer flask containing 300ml of liquid nitrifying medium at 30℃and 120rpm. NH was measured every 4 hours 4 + -N、NO 2 - -N and NO 3 - -N、PO 4 3- Concentration and OD of P 600 Values.
The results are shown in FIG. 2. The strain rapidly enters the log phase after inoculation, enters a short stationary phase at 24h and rapidly decays. The strain breaks down NH while growing 4 + -N and absorb PO 4 3- P, max NH 4 + The N removal rate was 6.62mg N/(L/h), maximum PO 4 3- The P removal rate was 1.03mg P/(L/h). At 20h, NH 4 + The N removal rate reaches the maximum value of 82%, 24h, PO 4 3- The removal rate of P reaches a maximum of 97%. In addition, NH 4 + Almost no degradation of NNO 2 - -N and NO 3 - -N accumulation. As the strain decays, part of the intracellular nitrogen and phosphorus is eluted causing the ammonia nitrogen and phosphorus concentration to start to rise at 24 h.
Example 3
Pseudomonas aeruginosa Pseudomonas aeruginosa SNDP-01 takes nitrite nitrogen and nitrate nitrogen as the only nitrogen source to perform synchronous denitrification dephosphorization.
Inoculating 3ml of bacterial suspension into conical flask containing 300ml of liquid denitrification culture medium I and denitrification culture medium II, culturing in air bath shaking table (30deg.C, 120 rpm) for 32 hr, and measuring NH every 4 hr 4 + -N、NO 2 - -N and NO 3 - -N、PO 4 3- Concentration and OD of P 600 Values.
During denitrification dephosphorization with high concentration of nitrite nitrogen (100 mg/L) as substrate (FIG. 3 a), the strain enters the logarithmic phase at 8h after inoculation and enters the stationary phase at 24 h. Part of NO 2 - N is assimilated by the strain for growth and reproduction, another part of NO 2 - N is removed by aerobic denitrification with maximum NO 2 - The N removal rate was 12.83mg N/(L/h). Meanwhile, PO in the denitrification process 4 3- -rapid decrease in P concentration, maximum PO 4 3- The P removal rate was 0.64mg P/(L/h). At 28h, NO 2 - The removal rate of N reaches 100%, PO 4 3- The removal rate of P reaches a maximum of 91%. In addition, NO 2 - Almost no NH during degradation of N 4 + Accumulation of-N, although there is a small amount of NO at the early stage 3 - N is produced but is completely degraded after 28 h. As the strain decays, part of the intracellular material dissolves out resulting in a slight increase in phosphorus content at 32 h.
During denitrification dephosphorization with high concentration of nitrate nitrogen (100 mg/L) as substrate (FIG. 3 b), the strain enters the logarithmic phase at 8h after inoculation and enters the stationary phase at 24 h. The strain can take NO 3 - N is the sole nitrogen source for growth and reproduction, and PO is absorbed simultaneously 4 3- -P. Of these, the mostLarge NO 3 - The N removal rate was 10.43mg N/(L/h), maximum PO 4 3- The P removal rate was 0.86mg P/(L/h). At 24h, NO 3 - -the N removal rate reaches a maximum of 99%; 28h, PO 4 3- The removal rate of P reaches a maximum of 87%. In addition, NO 3 - Almost no NH during degradation of N 4 + Accumulation of-N, NO 2 - -N drops rapidly to 0 after a slight increase. As the strain decays, part of the intracellular material dissolves out resulting in a slight increase in ammonia nitrogen and phosphorus concentrations.
Example 4
Pseudomonas aeruginosa Pseudomonas aeruginosa SNDP-01 utilizes the characteristics of growth, denitrification and dephosphorization when the mixed nitrogen source is used for synchronous nitrification and denitrification.
Inoculating 3ml of bacterial suspension into conical flask containing 300ml of liquid nitrification and denitrification culture medium I and nitrification and denitrification culture medium II, culturing in air bath shaking table (30 deg.C, 120 rpm) for 32 hr, and measuring NH every 4 hr 4 + -N、NO 2 - -N and NO 3 - -N、PO 4 3- Concentration and OD of P 600 Values.
In the process of nitrifying, denitrifying and dephosphorizing by taking ammonia nitrogen and nitrite nitrogen as mixed nitrogen sources (figure 4 a), 8h after bacterial strain inoculation enters a logarithmic phase, 24h enters a stabilization phase, and the bacterial strain decomposes an inorganic nitrogen source substrate and absorbs phosphate while growing. Wherein NH is 4 + -N、NO 2 - -N、PO 4 3- The maximum removal rate of P was 5.34mg N/(L/h), 5.74mg N/(L/h), 0.82mg P/(L/h), respectively. At 20h, NH 4 + -the removal rate of N reaches a maximum of 96%; at 28h, PO 4 3- -the removal rate of P reaches a maximum of 99%; 32h, NO 2 - The removal rate of N reaches 100 percent. In the process of nitrifying and denitrifying with ammonia nitrogen and nitrate nitrogen as mixed nitrogen sources (figure 4 b), the growth performance and pollutant removal performance of the strain are similar to those of the process with ammonia nitrogen and nitrite nitrogen as substrates. Wherein NH is 4 + -N、NO 3 - -N、PO 4 3- The maximum removal rate of P was 3.90mg N/(L/h), 3.58mg N/(L/h), 1.15mg P/(L/h), respectively. At 16h, NH 4 + -the removal rate of N reaches a maximum of 96%; at 32h, NO 3 - -N and PO 4 3- The removal rate of P reaches a maximum of 99% and 96%, respectively. In addition, little intermediate product is accumulated in the synchronous nitrification and denitrification process. As the strain decays, part of the intracellular material dissolves out, resulting in a slight increase in nitrogen and phosphorus content at 28 h.
Example 5
The pollutant removal performance of the pseudomonas aeruginosa Pseudomonas aeruginosa SNDP-01 in real life wastewater.
Inoculating 3ml of bacterial suspension into conical flask containing 300ml of domestic wastewater, culturing in air bath shaking table (30deg.C, 120 rpm) for 32 hr, and measuring NH every 4 hr 4 + -N、NO 2 - -N and NO 3 - -N、PO 4 3- -concentration of P.
The results are shown in FIG. 5. After pseudomonas aeruginosa Pseudomonas aeruginosa SNDP-01 is inoculated to domestic sewage, the concentration of ammonia nitrogen and orthophosphate is rapidly reduced. Wherein NH is 4 + -N and PO 4 3- The maximum removal rate of P was 6.20mg N/(L/h) and 1.19mg P/(L/h), respectively. At 16h, NH 4 + -N and PO 4 3- The removal rate of P reaches a maximum of 99% and 93%. Little intermediate product accumulation is observed in the denitrification process. As the strain decays, from 16h, part of the intracellular material dissolves out resulting in an increase in phosphorus content.
Example 6
P. aeruginosa (Pseudomonas aeruginosa) SNDP-01 distribution of phosphorus during heterotrophic nitrification and phosphorus removal and the existence form of phosphorus in EPS.
Inoculating 3ml of bacterial suspension into a conical flask containing 300ml of liquid nitrifying culture medium, placing the conical flask in an air bath shaking table (30 ℃ C., 120 rpm) for culturing for 32 hours, respectively measuring total phosphorus content in solution, intracellular, EPS and cell membrane in 8 hours, 16 hours and 24 hours, and measuring phosphorus forms existing in the intracellular and EPS in 24 hours.
The results are shown in FIG. 6. Under aerobic conditions, with the growth of pseudomonas aeruginosa (Pseudomonas aeruginosa) SNDP-01, phosphate in the culture medium is continuously absorbed by the strain into cells to perform cellular metabolism and synthesize cell membranes, and after the strain grows to a stationary phase, phosphorus is mainly distributed on EPS and cell membranes of the strain. As also shown in fig. 7, phosphorus on EPS exists mainly in the form of phosphoric monoester, while small amounts of phosphorus in cells exist mainly in the form of orthophosphate, phosphoric monoester, and phosphoric diester, which is significantly different from the conventional polyphosphate accumulating bacteria in which phosphorus is stored in cells in the form of polyphosphate particles, which is advantageous for recovery of phosphorus element.
Sequence listing
<110> university of Beijing industry
<120> a synchronous heterotrophic nitrification aerobic denitrification dephosphorization bacterium and application thereof
<141> 2021-11-23
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ccagactcct acgggaggca gcagtgggga atattggaca atgggcgaaa gcctgatcca 360
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agcgcgcgta ggtggttcag caagttggat gtgaaatccc cgggctcaac ctgggaactg 600
catccaaaac tactgagcta gagtacggta gagggtggtg gaatttcctg tgtagcggtg 660
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cactgaggtg cgaaagcgtg gggagcaaac aggattagat accctggtag tccacgccgt 780
aaacgatgtc gactagccgt tgggatcctt gagatcttag tggcgcagct aacgcgataa 840
gtcgaccgcc tggggagtac ggccgcaagg ttaaaactca aatgaattga cgggggcccg 900
cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg aagaacctta cctggccttg 960
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Claims (3)
1. A synchronous heterotrophic nitrification aerobic denitrification dephosphorization bacterium-pseudomonas aeruginosa (Pseudomonas aeruginosa) SNDP-01 is characterized in that: the strain is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 2021, 9 months and 17 days, the preservation number is CGMCC No.23440, and the length of a 16S rDNA gene sequence is 1421bp.
2. A method of using pseudomonas aeruginosa (Pseudomonas aeruginosa) SNDP-01 according to claim 1, characterized by: the organic carbon source which can be utilized is sodium citrate, and the inorganic nitrogen source which can be utilized is ammonia nitrogen, nitrite nitrogen and nitrate nitrogen; the inorganic phosphorus source that can be utilized is orthophosphate.
3. A method of using pseudomonas aeruginosa (Pseudomonas aeruginosa) SNDP-01 according to claim 1, characterized by: the conditions of ammonia nitrogen and phosphate removal performance are:
inoculating the strain into synthetic wastewater with ammonia nitrogen, nitrite nitrogen or nitrate nitrogen as a unique nitrogen source, and removing carbon, nitrogen and phosphorus in one step under the optimal culture condition;
or inoculating the strain into synthetic wastewater with ammonia nitrogen, nitrite nitrogen, ammonia nitrogen and nitrate nitrogen as mixed nitrogen sources, and removing carbon, nitrogen and phosphorus in one step under the optimal culture condition; the culture conditions are as follows: the carbon source is sodium citrate, the mass ratio of C/N is 10-15, the mass ratio of P/N is 0.1-0.2, the culture temperature is 30-35 ℃, the pH is 7.5-8.5, and the rotating speed is 120-160rpm.
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