Microbial composite flora with nitrogen removal capacity as well as construction method and application of microbial composite flora

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to a microbial composite flora with nitrogen removal capacity as well as a construction method and application of the microbial composite flora. The microbial composite flora with the nitrogen removal capability comprises mixed leissella luricola, leissella luricola, bacillus mucilaginosus, selaginella hydrocarbonoclada and rosinia aggresticus A S419 (Roseium aggresticus A S419), and the microbial composite flora with the nitrogen removal capability is prepared from mixed leissella luricola. The ratio of the viable counts of the mixed leptosticia parviflora to the viable counts of the leptosticia parviflora to the viable counts of the bacillus mucilaginosus to the viable counts of the septosticia parviflora to the viable counts of the septosticia parviflora to the viable counts of the septosticia parviflora to the viable counts of the septosticia parviflora to the viable counts of the septosticia parviflora to the Research shows that the constructed microbial composite flora with the nitrogen removal capacity is a flora capable of efficiently removing nitrate nitrogen and nitrite nitrogen in high-salinity water in mariculture and the like, and a simple and effective operation method is provided for nitrogen removal of high-salinity wastewater.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and in particular relates to a microbial composite flora with nitrogen removal capability, a construction method and an application thereof. Background Art

[0002] High-salinity wastewater refers to wastewater with a total salt content of at least 3.5%wt, and its sources include a variety of industrial and human activities, such as industrial production, mining, smelting, agricultural drainage and urban sewage treatment. High-salinity wastewater generated by industries such as marine aquaculture, petroleum and pharmaceuticals is also characterized by high concentrations of nitrogen-containing pollutants. Nitrates and nitrites present in high-salinity wastewater pose a threat to ecosystems and human health and may cause a variety of problems. Therefore, removing inorganic pollutants such as nitrates and nitrites from high-salinity wastewater is very important for human health and the environment.

[0003] The mainstream methods for treating nitrogen-containing wastewater include physical and chemical methods and biological methods. Among them, physical and chemical methods consume a lot of energy, are costly, and have low nitrogen removal efficiency. Biological methods are environmentally friendly, low-cost, and have high nitrogen removal efficiency. Therefore, biological methods are more effective in treating such wastewater. Commonly used microorganisms in biological methods include denitrifying strains and denitrifying synthetic bacteria.

[0004] At present, there have been many reports on biological denitrification strains and denitrifying synthetic bacteria for high-salt nitrogen-containing wastewater. The main genera of existing denitrifying strains are: Vibrio, Marinobacter, Acinetobacter, Halomonas, Flavosporium, Pseudomonas, Halophilic Vibrio, Klebsiella, Arthrobacter, and Alkanophage. The bacterial genus combinations that appear in the denitrifying synthetic bacteria are: Alcaligenes, Bacillus and Halomonas; Chabel, Halomonas and Vibrio; Vibrio and Pseudomonas; Cladosporium, Halomonas and Marinobacter; Hydrogen-eating water-producing bacteria and Cladosporium; Halomonas and Bacillus; Marinobacter, Azovibrio, Paracoccus, Bacillus and Pseudomonas. Among them, the Latin name of the genus Vibrio is Vibrio ; The Latin name of the genus Marinobacter is Marinobacter ; The Latin name for the genus Acinetobacter is Acinetobacter; The Latin name of the genus Halomonas is Halomonas ; The Latin name of the genus Flavoxella is Lihuangia ; The Latin name for Pseudomonas is Pseudomonas ; The Latin name of the genus Halovibrio is Halovibrio ; The Latin name for Klebsiella is Klebsiella ; The Latin name for the genus Arthrobacter is Arthrobacter ; The Latin name of the genus Alkanophaga is Alcanivorax ; The Latin name for the genus Alcaligenes is Alcaligenes ; The Latin name for Bacillus is Bacillus ; The Latin name of the genus Halomonas is Halomonas; The Latin name for the genus Chabelella is Zobellella ; The Latin name of the genus Halomonas is Halomona ; The Latin name for Vibrio is Vibrio ; The Latin name for the genus Vibrio is Vibrio ; The Latin name for Pseudomonas is Pseudomonas ; The Latin name for the genus Bacillus is Virgibacillus ; The Latin name of the genus Halomonas is Halmonas ; and the Latin name for the genus Marinobacter Marinobacter ; The Latin name of the genus Hydrogen-eating Water-producing Bacteria is Hydrogenophaga The Latin name for the genus Bacillus is Virgibacillu ; The Latin name of the genus Halomonas is Halmonas ; The Latin name for Bacillus is Bacillus ; The Latin name of the genus Marinobacter is Marinobacter ; The Latin name of the genus Azoarium is Azoarcus ; The Latin name for Paracoccus is Paracoccus ; The Latin name for the genus Bacillus is Bacillus ; and the Latin name for Pseudomonas Pseudomonas .

[0005] About half of the strains in the above synthetic bacterial consortium come from freshwater and need sufficient carbon source to efficiently remove nitrate and nitrite. Therefore, this type of microorganism used to treat high-salinity wastewater has low nitrogen removal efficiency when treating high-salinity wastewater. Summary of the invention

[0006] In order to solve the problem of low nitrogen removal efficiency of microorganisms in high-salt wastewater in the prior art, the present invention proposes a microbial composite flora with nitrogen removal ability and a construction method and application thereof. To achieve the above purpose, the present invention adopts the following technical solution.

[0007] The present invention provides a microbial composite flora with nitrogen removal ability, including mixed water reisingella, reisingella, sticking bacillus, hydrocarbon-removing sea rod-shaped bacteria, agglomerative rose bacteria_AS419 ( Roseibium aggregatum _S419).

[0008] The ratio of the live bacterial counts of the mixed water Riesingerella, the Riesingerella, the sticking Bacillus, the hydrocarbon-removing sea rod-shaped bacteria, and the agglomerated rose bacteria_AS419 is 65~75:17~28:4~5:0.01~0.015:0.8~1.0.

[0009] The components of the above-mentioned microbial composite flora with nitrogen removal ability provided by the present invention include the mixed water Leisingerella, Leisingerella, the sticking bacillus, the hydrocarbon removal sea rod-shaped bacteria, and the agglomerated rose bacteria-AS419, all of which are from biofilms with strong stress resistance in the ocean, and can stably exist and efficiently remove nitrogen under high salinity conditions. The microbial composite flora with nitrogen removal ability provided by the present invention solves the problem of low nitrogen removal efficiency of existing microorganisms in high-salinity wastewater. Among them, the "high salinity" condition refers to the total salt content in water of 3.5%wt and 7%wt.

[0010] Among them, the Latin name of the mixed water Singh's bacteria is Leisingera aquaemixtae ; The Latin name of Reisingerella is Leisingera sp; the Latin name for Bacillus sp Tenacibaculum sp; the Latin name of the hydrocarbon-removing bacillus Marinobacter hydrocarbonoclasticus ; The Latin name of rose fungus A S419 is Roseibium aggregatum _S419.

[0011] The mixed Singella spp. is a mixed bacteria of mixed Singella spp. M597, mixed Singella spp. M602, mixed Singella spp. M583 and mixed Singella spp. S166.

[0012] The ratio of the live bacteria counts of the mixed Singerella mite M597, the mixed Singerella mite M602, the mixed Singerella mite M583 and the mixed Singerella mite S166 is 1:1:1:1.

[0013] The classification name of the mixed water Singella M597 is Leisingera aquaemixtae M597, which was deposited in the China Center for Type Culture Collection on September 2, 2024, with the deposit number CCTCC M 20241898; the classification name of the mixed water mine Singhia M602 is Leisingera aquaemixtae M602, which was deposited in the China Center for Type Culture Collection on September 2, 2024, with the deposit number CCTCC M 20241899; the classification name of the mixed water mine Singhia M583 is Leisingera aquaemixtae M583, which was deposited in the China Center for Type Culture Collection on September 2, 2024, with the deposit number CCTCC M 20241897; the classification name of the mixed water mine Singerella S166 is Leisingera aquaemixtae S166 was deposited in the China Center for Type Culture Collection on September 2, 2024, with the deposit number CCTCCM 20241900.

[0014] The classification name of the agglomerative rose bacteria_AS419 isRoseibium aggregatum _S419, was deposited in the China Center for Type Culture Collection on July 8, 2024, with the deposit number CCTCC M 20241515.

[0015] The described Ratsinger's bacteria is Ratsinger's bacteria M658; the classification name of the described Ratsinger's bacteria M658 is Leisingera sp. M658, which was deposited in the China Center for Type Culture Collection on July 8, 2024, with the deposit number CCTCC M 20241512.

[0016] The adhesin Bacillus is a mixed bacteria of the adhesin Bacillus S095, the adhesin Bacillus S105 and the adhesin Bacillus Z003; the ratio of the live bacteria counts of the adhesin Bacillus S095, the adhesin Bacillus S105 and the adhesin Bacillus Z003 is 1:1:1.

[0017] The classification name of the sticky bacillus S095 is Tenacibaculum sp. S095, which was deposited in the China Center for Type Culture Collection on September 2, 2024, with the deposit number CCTCC M 20241896; the classification name of the sticky bacillus S105 is Tenacibaculum sp. S105, which was deposited in the China Center for Type Culture Collection on July 8, 2024, with the deposit number CCTCC M 20241513; the classification name of the sticky bacillus Z003 is Tenacibaculum sp. Z003, which was deposited in the China Center for Type Culture Collection on September 2, 2024, with the deposit number CCTCC M 20241901.

[0018] The hydrocarbon-removing marine bacilli are hydrocarbon-removing marine bacilli S418, and the classification name of the hydrocarbon-removing marine bacilli S418 is Marinobacter hydrocarbonoclasticus S418 was deposited in the China Center for Type Culture Collection on July 8, 2024, with the deposit number CCTCC M 20241511.

[0019] The present invention also provides a method for constructing the composite microbial flora with nitrogen removal ability, comprising the following steps: The microbial composite flora with nitrogen removal ability is obtained by mixing the live bacteria counts of the mixed water Bacillus, the Bacillus, the sticking Bacillus, the hydrocarbon-removing sea rod-shaped bacteria, and the agglomerated rose bacteria_AS419 in a ratio of 65~75:17~28:4~5:0.01~0.015:0.8~1.0.

[0020] The mixed water mine Singella is a mixed bacteria of the mixed water mine Singella M597, the mixed water mine Singella M602, the mixed water mine Singella M583 and the mixed water mine Singella S166. The ratio of the number of live bacteria of the mixed water mine Singella M597, the mixed water mine Singella M602, the mixed water mine Singella M583 and the mixed water mine Singella S166 is 1:1:1:1.

[0021] The described bacteria is bacteria M658.

[0022] The adhesin Bacillus is a mixed bacteria of the adhesin Bacillus S095, the adhesin Bacillus S105 and the adhesin Bacillus Z003; the ratio of the live bacteria counts of the adhesin Bacillus S095, the adhesin Bacillus S105 and the adhesin Bacillus Z003 is 1:1:1.

[0023] The hydrocarbon-removing marine bacillus is hydrocarbon-removing marine bacillus S418.

[0024] The agglomerative rose-colored bacteria_A is agglomerative rose-colored bacteria_A S419.

[0025] Based on microbial genome analysis and screening, the present invention constructed a NO3 - The microbial complex with nitrogen removal ability composed of complete pathway bacteria can quickly remove NO3 under high salt concentration. - -N and NO2 - -N. Among them, NO3 - -N is nitrate nitrogen; NO2 - -N is nitrite nitrogen.

[0026] In addition, currently members of the genus Reisingerella are usually halophilic or salt-tolerant, and can tolerate a salinity of up to 18%wt. Only a few literature reports have reported that members of this genus have denitrification capabilities, and members of this genus do not appear in any patents. Existing articles have reported that members of the genus Adhesibacter have denitrification capabilities and the ability to adapt to different marine environments, including low-nutrient and high-salinity conditions. The relevant patents of the genus Adhesibacter only involve fish disease vaccines. Only a few literature reports have reported that Roseobacter agglomerans have denitrification functions, and there is only one patent for its fermentation to produce polyhydroxyalkanoates. Therefore, the microbial composite flora with efficient nitrogen removal capabilities constructed by the above-mentioned 10 strains of the present invention is a new microbial synthetic flora.

[0027] The present invention also provides the use of the microbial composite flora with nitrogen removal ability in treating high-salt culture medium and marine aquaculture wastewater.

[0028] Preferably, the application is nitrogen removal.

[0029] Preferably, the nitrogen removal refers to removing nitrate nitrogen and nitrite nitrogen from the high-salt culture medium and the marine aquaculture wastewater.

[0030] Preferably, the marine aquaculture wastewater is marine white shrimp aquaculture wastewater.

[0031] Preferably, the temperature of the high-salt culture medium and the marine aquaculture wastewater are both 23°C~28°C, and the pH is 7~8; the total salt content in the high-salt culture medium is 3.5%wt and / or 7%wt; the total salt content in the marine aquaculture wastewater is 3.5%wt.

[0032] Preferably, the microbial composite flora with nitrogen removal ability is inoculated into the high-salt culture medium and the marine aquaculture wastewater for 3h to 48h, respectively, to remove nitrate nitrogen and nitrite nitrogen in the high-salt culture medium and the marine aquaculture wastewater. The denitrifying strains in the microbial composite flora with nitrogen removal ability described in the present invention all come from marine biofilms with strong stress resistance. Most of the aerobic denitrifying bacteria currently used in factories come from fresh water, so the number of strain types used in previously reported high-salt wastewater can be enriched to a certain extent. After the microbial composite flora with nitrogen removal ability of the present invention is inoculated into the marine white shrimp aquaculture wastewater, all nitrate nitrogen in the wastewater can be removed without adding additional carbon sources, and the decrease in cell density is relatively small. This reflects the effectiveness of the microbial composite flora with nitrogen removal ability in the present invention on NO3 - -N and NO2 - -The actual removal efficiency of N.

[0033] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a microbial composite community with nitrogen removal ability and its construction method and application. The present invention screened and synthesized a microbial composite community derived from marine biofilm, which has strong stress resistance and NO3 - The microbial complex with complete reduction pathway and most of the strain members are not found in denitrification, this microbial complex has high efficiency in removing NO3 under high salt static conditions - -N and NO2 - -N capacity, thus, can provide a simple new method for removing nitrogen from high-salinity wastewater, where the total salt content in the high-salinity wastewater is 3.5%wt.

[0034] 2. The present invention selects NO3 - The 10 bacterial strains that restored the complete pathway were mixed evenly at equal cell density to form a denitrification synthetic bacterial community, that is, a microbial composite community with nitrogen removal ability. The synthesis standard of the microbial composite community is OD 600 =0.2, the standard for detecting denitrification rate is OD600 =0.8; Determination of NO3 in bacterial solution by vanadium chloride reduction method - -N concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The figure is a schematic diagram showing the locations of the key denitrification genes of the 10 bacterial strains in the present invention on their genomes.

[0036] Figure 2 OD is the absorbance of the modified 2216E liquid culture medium of two salinities in the present invention 540 With NO3 - -N concentration linear curve; where, Figure 2 Figure A shows the absorbance OD in 3.5%wt salinity-modified 2216E liquid culture medium 540 With NO3 - -Linear curve of N concentration; Figure 2 Figure B shows the absorbance OD in 7% wt salinity-modified 2216E liquid culture medium 540 With NO3 - -Linear curve of N concentration.

[0037] Figure 3 The nitrogen removal efficiency diagram of the microbial composite flora with nitrogen removal ability in the present invention and the commercially available bacterial agent at 3.5%wt and 7%wt salinity; wherein, Figure 3 Figure A shows the NO3 in the culture medium - -N concentration; Figure 3 Figure B shows the NO2 in the culture medium - -N concentration; Figure 3 Panel C shows the cell density in the culture medium.

[0038] Figure 4 The nitrogen removal efficiency diagram of the microbial composite flora with nitrogen removal ability in the present invention at 3.5%wt and 7%wt salinity; wherein, Figure 4 Figure A shows the NO3 in the culture medium - -N concentration; Figure 4 Figure B shows the NO2 in the culture medium - -N concentration; Figure 4 Panel C shows the cell density in the culture medium.

[0039] Figure 5 The figure is a nitrogen removal efficiency diagram of the composite microbial flora with nitrogen removal ability in the present invention and the commercially available bacterial agent in marine white shrimp aquaculture wastewater; wherein, Figure 5 Figure A shows NO3 in wastewater - -N concentration; Figure 5 Figure B shows NO2 in wastewater - -N concentration; Figure 5Figure C shows the cell density in wastewater. DETAILED DESCRIPTION

[0040] The following will be combined with the drawings and specific embodiments of the present invention to clearly and completely describe the technical solutions in the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] The methods described in the embodiments of the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used can be obtained from commercial sources unless otherwise specified.

[0042] The culture medium, reagents and experimental animals used in the following examples are as follows: (1) Culture medium All culture media used in the present invention were purchased from Haibo Biotechnology.

[0043] Marine broth 2216E agar medium formula: peptone 5.0g, yeast extract powder 1.0g, ferric citrate 0.1g, sodium chloride 19.45g, magnesium chloride 5.98g, sodium sulfate 3.24g, calcium chloride 1.8g, potassium chloride 0.55g, sodium carbonate 0.16g, potassium bromide 0.08g, strontium chloride 0.034g, boric acid 0.022g, sodium silicate 0.004g, sodium fluoride 0.0024g, ammonium nitrate 0.0016g, disodium hydrogen phosphate 0.008g, agar 15g, the solvent is ultrapure water, add ultrapure water to make up to 1L.

[0044] Marine broth 2216E liquid culture medium formula: peptone 5.0g, yeast extract powder 1.0g, ferric citrate 0.1g, sodium chloride 19.45g, magnesium chloride 5.98g, sodium sulfate 3.24g, calcium chloride 1.8g, potassium chloride 0.55g, sodium carbonate 0.16g, potassium bromide 0.08g, strontium chloride 0.034g, boric acid 0.022g, sodium silicate 0.004g, sodium fluoride 0.0024g, ammonium nitrate 0.0016g, disodium hydrogen phosphate 0.008g, the solvent is ultrapure water, add ultrapure water to make up to 1L.

[0045] Formula of 3.5%wt salinity improved 2216E liquid medium: 0.5g peptone and 0.1g yeast extract powder, 0.1g ferric citrate, 19.45g sodium chloride, 5.9g magnesium chloride, 3.24g magnesium sulfate, 3.24g sodium sulfate, 1.8g calcium chloride, 0.55g potassium chloride, 0.16g sodium carbonate, 0.08g potassium bromide, 0.034g strontium chloride, 0.022g boric acid, 0.004g sodium silicate, 0.0024g sodium fluoride, 0.0016g ammonium nitrate, 0.008g disodium hydrogen phosphate, the solvent is ultrapure water, add ultrapure water to 1L. Note: In order to avoid the influence of minerals and salinity, the concentration of inorganic nutrients in the mother liquor is the same as that of Marine broth 2216E liquid medium. In addition: if 35g / L sea salt is added to the medium, the salinity is adjusted to 7%wt.

[0046] (2) Reagents and materials The DNA extraction kit was purchased from Nanjing Vinozan Biotechnology Co., Ltd., FastPure Bacteria DNA Isolation Mini Kit, Cat: DC103-1.

[0047] Ultra-high-speed PCR premix was purchased from Shanghai Yisheng Biotechnology Co., Ltd. 2×Hieff ® Ultra-RapidHotStart PCR Master Mix, Cat: 10157ES03.

[0048] NO2 - -N content determination kit was purchased from Shanghai Runyu Biotechnology Co., Ltd., CAS: RY-0430W.

[0049] (3) Equipment Vertical full temperature shaking incubator: Zhichu ZQLY-180.

[0050] Benchtop microcentrifuge: BECKMAN Microfuge 16 Centrifuge.

[0051] Constant temperature mixer: Yisheng ES-TM100.

[0052] Gradient PCR amplifier: Biometra TOne, Germany.

[0053] Micro vortex mixer: BioMixer 4K.

[0054] Constant temperature water bath: Digital constant temperature water bath HHS-11-2.

[0055] Agarose gel electrophoresis system: Bio-Rad DYCP-31DN, USA.

[0056] Nanodrop micro-volume spectrophotometer: Tiangen OSE-260-03.

[0057] Microplate reader: Thermo Scientific MULTISKAN FC.

[0058] (4) Experimental animals The marine whiteleg shrimp aquaculture wastewater was collected from a marine whiteleg shrimp aquaculture farm in Haiyang City, Yantai City, Shandong Province.

[0059] Example 1 1. Sample collection and processing 1.1. Sample collection and pretreatment: Rocks located at a depth of 1.5 m in the subtidal zone were collected in the intertidal zone of Maidao, Qingdao, Shandong Province, China: 36°03′13.3″, 120°25′31.5″. Natural marine biofilm samples were then scraped from the rock surface with a sterile cotton swab, and the collected samples were immediately transferred to the Marine Biofilm Laboratory in the Teaching Building, Area X, Fushan Campus, Ocean University of China.

[0060] Under sterile conditions, take 10 mL of seawater to thoroughly rinse the cotton swab to obtain the sample stock solution, take 1 mL of the sample stock solution and dilute it in 9 mL of sterilized seawater to obtain 10 -1 Repeat this step to obtain 3 dilutions of different concentrations, i.e. 10 -2 Concentration of dilution, 10 -3 Concentration of dilution and 10 -4 Concentration of dilution.

[0061] The seawater formula is as follows: dissolve 35g of sea salt in 1L of ultrapure water, sterilize at 121°C for 15min, filter with a 0.22μm filter membrane after cooling, and place in a 4°C refrigerator for later use.

[0062] 1.2. Sample culture and separation and purification: Take 200 μL of bacterial suspension from each gradient dilution and spread it on Marine broth 2216E agar medium, then put the solid plate into a 25°C constant temperature biochemical incubator and invert it for culture. After 3 days of culture, use an inoculation loop to select bacterial colonies of different colors and shapes, and subculture 8 times to obtain a pure culture solid plate.

[0063] All single colonies with different shapes and colors were inoculated into 10 mL centrifuge tubes containing 4 mL Marine broth 2216E liquid culture medium, and the centrifuge tubes were placed in a vertical full-temperature shaking incubator. After shaking and culturing at 180 rpm × 25 ° C until the logarithmic growth phase, 1.0 mL of the bacterial solution was mixed with 50% glycerol by volume at a volume ratio of 1:1 to obtain glycerol-frozen bacterial solution. The glycerol-frozen solution was stored in a -80 ° C refrigerator.

[0064] 2. DNA Extraction and Genome Sequencing 2.1. Preliminary identification of species: Take 1 mL of the remaining bacterial solution and place it in a 1.5 mL centrifuge tube and centrifuge it at 6,000 rpm for 5 min using a desktop microcentrifuge, then discard the supernatant. Add 200 μL of ddH2O to the centrifuge tube to resuspend the bacteria, place it in a constant temperature mixer, and heat it at 100°C for 10 min to release bacterial DNA.

[0065] The target fragment was amplified using a gradient PCR amplifier, ultra-high-speed PCR premix, and 16S-rRNA gene sequence universal primers 27F and 1492R.

[0066] The PCR reaction system was as follows: 1 μL of template DNA, 10 μM forward primer: 1 μL, 10 μM reverse primer: 1 μL, 10 μL Taq enzyme premix containing dye, and sterile ddH2O added to a total volume of 25 μL.

[0067] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min before entering the cycle, denaturation at 95°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 60 s, and after 35 cycles, extension at 72°C for 10 min.

[0068] The nucleotide sequence of primer 27F is shown in SEQ ID NO. 1: 5'-AGAGTTTGATCCTGGCTCAG-3'; the nucleotide sequence of primer 1492R is shown in SEQ ID NO. 2: 5'-GGTTACCTTGTTACGACTT-3'.

[0069] 2.5 μL of PCR amplification product was taken for 2% agarose gel electrophoresis detection. The qualified PCR products were sent to Shanghai Sangon Biotechnology Co., Ltd. for Sanger sequencing.

[0070] Based on the Sanger sequencing information, the bacterial species were preliminarily annotated using the NCBI database in the United States. Then, the cd-hit software version 4.8.1 was used to remove duplicates based on the 16s sequence information of all single colonies, and the sequence similarity parameter threshold was set to 0.9999, and finally the species information and sample number information corresponding to all non-redundant sequences were obtained.

[0071] Among them, the full name of NCBI is: National Center for Biotechnology Information.

[0072] 2.2. Genome extraction and sequencing: Following the culture procedure in step 1.2, all non-redundant bacteria were cultured to the logarithmic phase and then centrifuged to collect approximately 1.0×10 9 The DNA extraction and purification of all strains were carried out according to the processing steps of Gram-positive bacteria samples in the FastPure Bacteria DNA Isolation Mini Kit kit instructions.

[0073] The DNA extraction process is as follows: (1) Add 180 μL of homemade Lysozyme solution to the centrifuge tube containing the bacteria, mix the centrifuge tube using a micro vortex mixer, and then immediately place it in a 37°C constant temperature water bath and incubate for 1 h.

[0074] (2) Add 20 μL of Proteinase K to the centrifuge tube and vortex to mix.

[0075] (3) Add 250 μL of Buffer GB and vortex to mix.

[0076] (4) Place the centrifuge tube in a constant temperature mixer at 70°C and incubate for 10 min to obtain a mixed solution.

[0077] The DNA purification process is as follows: (1) Add 180 μL of anhydrous ethanol to the mixture and vortex to mix. Centrifuge briefly to collect the liquid on the tube cap and inner wall.

[0078] (2) Transfer the mixture to the adsorption column in the collection tube, centrifuge at 12,000 rpm for 1 min and discard the filtrate.

[0079] (3) Add 500 μL of Buffer PB, 600 μL of Buffer PW, and 600 μL of Buffer PW to the column in sequence. Centrifuge at 12,000 rpm for 1 min after each addition and discard the filtrate.

[0080] (4) Place the adsorption column back into the collection tube and centrifuge at 12,000 rpm for 2 min. Open the cover of the adsorption column and place it on a clean bench for 5 min to allow the residual ethanol to evaporate completely.

[0081] (5) Transfer the adsorption column to a new 1.5 mL centrifuge tube, add 50 μL of ddH2O to the center of the adsorption column membrane, place it on a clean bench for 5 minutes, and then centrifuge it at 12,000 rpm for 1 minute to obtain the purified DNA.

[0082] (6) Remove and discard the adsorption column, mark the cap of the 1.5 mL centrifuge tube, and store it in a -20°C refrigerator.

[0083] The integrity, concentration and purity of all non-redundant bacterial DNA were tested by 2% agarose gel electrophoresis and Nanodrop micro-spectrophotometer. The qualified DNA samples were sent to Novogene China and sequenced based on the Illumina NovaSeq PE 150 platform, with a sequencing volume of 2Gb per bacterial sample. Finally, the bacterial genome data of all non-redundant marine rock biofilms were obtained.

[0084] Lysozyme is lysozyme. Lysozyme solution formula: accurately weigh 2g of Lysozyme dry powder, dissolve it with 1×TE buffer at pH=8 and make up to volume in a 100mL volumetric flask to obtain a 20mg / mL Lysozyme solution.

[0085] 2.3. Genome analysis: All non-redundant bacterial genomes were denovo assembled using SPAdes software version 3.13.0 on a local Linux system. The K-mer parameters for the assembly were set to "21, 33, 55, 77, 99, 101", and the "--careful" and "--cov-cutoff auto" parameters were added to reduce errors in the assembly process, remove regions with low or abnormally high coverage, and improve the accuracy of the assembly results. The quality and completeness of the bacterial genomes were evaluated using the checkm lineage_wf workflow in CheckM software version 1.1.2, and strains with higher genomic data quality were selected from the evaluation results. The ORFs of the bacterial genome were predicted using Prodigal software version 2.60, and the "-c" parameter was selected to output complete ORFs with stop codons. Gene function annotations were performed on the ORFs of the bacterial genome based on the KEGG database and BLAST command on the local Linux system, and the E-value threshold was set to 10 -7 To retain the alignment results with high credibility and sequence similarity.

[0086] Among them, high quality of genomic data means that the completeness is greater than 95% and the contamination is less than 5%.

[0087] The full name of ORFs is: open reading frames, and its Chinese name is open reading frames.

[0088] The full name of KEGG is: Kyoto Encyclopedia of Genes and Genomes, and its Chinese name is Kyoto Encyclopedia of Genes and Genomes.

[0089] The full name of BLAST is: Basic Local Alignment Search Tool.

[0090] Screen out strains containing key denitrification genes in their genomes, including: narG , narH , narI / V or napA , napB , nirK or nirS , norB or norC as well as nosZ .

[0091] Based on the functional analysis of genomic data, 10 strains with denitrification potential were screened, such as Figure 1 The 10 strains are: mixed water minesingella M597, mixed water minesingella M602, mixed water minesingella M583, mixed water minesingella S166, minesingella M658, agglomerative rose bacteria_A S419, hydrocarbon removal sea rod-shaped bacteria S418, sticking bacteria S095, sticking bacteria S105 and sticking bacteria Z003.

[0092] Among them, the classification name of mixed water Singella M597 is Leisingera aquaemixtae M597, which was deposited in the China Center for Type Culture Collection on September 2, 2024, with the deposit number CCTCC M 20241898; the classification name of the mixed water mine Singhia M602 is Leisingera aquaemixtae M602, which was deposited in the China Center for Type Culture Collection on September 2, 2024, with the deposit number CCTCC M 20241899; the classification name of the mixed water mine Singhia M583 is Leisingera aquaemixtae M583, which was deposited in the China Center for Type Culture Collection on September 2, 2024, with the deposit number CCTCC M 20241897; the classification name of the mixed water mine Singella S166 is Leisingera aquaemixtaeS166, which was deposited in the China Center for Type Culture Collection on September 2, 2024, with the deposit number CCTCCM 20241900; the classification name of Ratsingerella M658 is Leisingera sp. M658, which was deposited in the China Center for Type Culture Collection on July 8, 2024, with the deposit number CCTCC M 20241512; the classification name of Bacillus viscidus S095 is Tenacibaculum sp. S095, which was deposited in the China Center for Type Culture Collection on September 2, 2024, with the deposit number CCTCC M 20241896; the classification name of Bacillus fasciatus S105 is Tenacibaculum sp. S105, which was deposited in the China Center for Type Culture Collection on July 8, 2024, with the deposit number CCTCC M 20241513; the classification name of Bacillus adhering to Z003 is Tenacibaculum sp. Z003, which was deposited in the China Center for Type Culture Collection on September 2, 2024, with the deposit number CCTCC M 20241901; the classification name of the hydrocarbon-removing bacillus S418 is Marinobacter hydrocarbonoclasticus S418, which was deposited in the China Center for Type Culture Collection on July 8, 2024, with the deposit number CCTCC M 20241511; the classification name of R. agglomerans_AS419 is Roseibium aggregatum_ AS419 was deposited in the China Center for Type Culture Collection on July 8, 2024, with the deposit number CCTCC M 20241515.

[0093] 3. Nitrogen removal rate of synthetic bacterial flora under high salinity 3.1. Synthesis of denitrifying bacteria: The above 10 strains were inoculated into 15 mL centrifuge tubes containing 5 mL Marine broth 2216E liquid medium at a v / v ratio of 1%, and cultured at 180 rpm × 25 °C until the logarithmic phase, when the absorbance value was about 0.8 at 600 nm using a microplate reader, i.e., OD 600 =0.8. The OD of each bacterium was adjusted using Marine broth 2216E liquid medium. 600 All were adjusted to 0.2.

[0094] The bacterial suspensions of the 10 bacterial strains obtained above were uniformly mixed at a cell density ratio of 1:1, and after adding 50% glycerol in an amount equal to the total bacterial suspension, the mixture was dispensed into 2 mL cryovials to obtain a synthetic bacterial colony, and the cryovials containing the synthetic bacterial colony were stored in a -80°C refrigerator.

[0095] The synthetic bacterial community is a microbial composite community with the ability to remove nitrogen, also known as a denitrifying bacterial community or a microbial composite community. The microbial composite community with the ability to remove nitrogen is referred to as a synthetic bacterial community below.

[0096] The synthetic bacterial community includes mixed water Leisingella, Leisingella, Adhesive Bacillus, hydrocarbon removal sea rod-shaped bacteria, and agglomerative rose-colored bacteria_A S419. The ratio of the live bacteria counts of mixed water Leisingella, Leisingella, Adhesive Bacillus, hydrocarbon removal sea rod-shaped bacteria, and agglomerative rose-colored bacteria_A S419 is 70:22.5:4.5:0.0125:0.9.

[0097] The mixed Singella spp. is a mixed bacteria of mixed Singella spp. M597, mixed Singella spp. M602, mixed Singella spp. M583 and mixed Singella spp. S166. The ratio of the number of live bacteria of mixed Singella spp. M597, mixed Singella spp. M602, mixed Singella spp. M583 and mixed Singella spp. S166 is 1:1:1:1.

[0098] The bacterium Ratsinger was Ratsinger M658.

[0099] The adhesin bacteria are a mixture of adhesin bacteria S095, adhesin bacteria S105 and adhesin bacteria Z003. The ratio of the live counts of adhesin bacteria S095, adhesin bacteria S105 and adhesin bacteria Z003 is 1:1:1.

[0100] The hydrocarbon-removing marine bacillus is hydrocarbon-removing marine bacillus S418.

[0101] 3.2、Cultivation of synthetic flora: The cryopreserved synthetic bacterial flora was inoculated into a 15 mL centrifuge tube containing 5 mL of Marine broth 2216E liquid culture medium at a v / v ratio of 1:5, and activated by shaking at 180 rpm × 25 ° C for 18 hours. The activated bacterial liquid was inoculated into a 500 mL conical flask containing 200 mL of Marine broth 2216E liquid culture medium at a v / v ratio of 1%, and cultured by shaking at 180 rpm × 25 ° C for 24 hours.

[0102] 3.3 NO3 - -N and NO2 - -N detection: NO3 - -N is quantitatively detected by vanadium chloride reduction method, NO2 - -N is quantitatively detected using the Griess reagent method. - -N is nitrate nitrogen; NO2 - -N is nitrite nitrogen, and vanadium chloride is VCl3.

[0103] 3.3.1. Required reagents and formulas: (1) KNO3 stock solution: Accurately weigh 10.1 g of KNO3 that has been dried at 110°C for 2 h, dissolve it in ultrapure water in a 100 mL volumetric flask to obtain NO3 - -N KNO3 stock solution with a concentration of 1M.

[0104] (2) KNO3 working solution: Accurately pipette 1.2 mL of KNO3 stock solution and dilute to 100 mL volumetric flask with ultrapure water to obtain NO3 - -N concentration is 60mM KNO3 working solution.

[0105] (3) VCl3 reducing solution: Accurately weigh 800 mg of VCl3 and dissolve it completely in 20 mL of ultrapure water. Then add 8.4 mL of 37% wt concentrated hydrochloric acid and dilute to a 100 mL volumetric flask with ultrapure water to obtain a saturated VCl3 reducing solution.

[0106] Note: If there are suspended particles in the saturated VCl3 reduction solution, filtration is required.

[0107] (4) N-1-naphthylethylenediamine dihydrochloride solution: Accurately weigh 100 mg of NEDD, dissolve it in ultrapure water and dilute it to a 100 mL volumetric flask to obtain a 1 g / L NEDD solution. The English abbreviation of N-1-naphthylethylenediamine dihydrochloride is NEDD.

[0108] (5) Sulfonamide solution: Accurately weigh 2g of Sulfanilamide, and after it is completely dissolved in 20mL of ultrapure water, add 5mL of 37%wt concentrated hydrochloric acid, and dilute to a 100mL volumetric flask with ultrapure water to obtain a 20g / L Sulfanilamide solution. The English name of sulfonamide is Sulfanilamide.

[0109] (6) NO3 - -N detection reagent: Take 5 parts of saturated VCl3 reduction solution, 1 part of 1g / L NEDD solution and 1 part of 20g / L Sulfanilamide solution respectively, vortex and mix to get NO3 - -N detection reagent.

[0110] (7) Aminosulfonic acid solution: Accurately weigh 583 mg of SA, dissolve it in ultrapure water and dilute it to a 100 mL volumetric flask to obtain a 60 mM SA solution. The English abbreviation of aminosulfonic acid is SA.

[0111] Note: All the above medicines should be stored in a 4℃ refrigerator away from light and have a shelf life of 1 month.

[0112] 3.3.2. Absorbance OD of bacterial solution 540With NO3 - -Linear curve of N concentration: Select a strain of marine biofilm-derived bacteria that does not have denitrification ability Psychrobacter nivimaris H023, determination of different NO3 contents under two salinity conditions - -N concentration of the culture medium at 540nm absorbance, that is, OD 540 , draw OD 540 With NO3 - -N concentration linear curve. By measuring the OD of the synthetic bacterial community in the two salinity culture media in the denitrification experiment 540 , thus according to OD 540 With NO3 - -N concentrations, estimating the residual NO3 in the synthetic bacterial culture medium - -N concentration, calculate the NO3 of the synthetic bacterial community - -N removal efficiency.

[0113] The specific steps are as follows: Follow the relevant steps in step 3.2 to Psychrobacter nivimaris H023 was activated and expanded to obtain the strain Psychrobacter nivimaris The bacterial suspension of H023 was divided into two parts and placed in 50 mL sterile centrifuge tubes. The bacterial precipitate was collected by centrifugation at 4,500 rpm for 8 min. The bacterial suspension was resuspended in 3.5% wt salinity and 7% wt salinity modified 2216E liquid medium respectively. The OD value of the resuspended bacterial suspension was calculated. 600 Controlled at 0.8.

[0114] Take out 10 1mL bacterial liquids from the two resuspended bacterial liquids and put them into new 1.5mL sterile centrifuge tubes. Mark each group of centrifuge tubes in the order of 0-9, and aspirate 0, 10μL, 20μL, 30μL, 40μL, 50μL, 60μL, 70μL, 80μL, and 90μL bacterial liquids from the centrifuge tubes and discard them, and then add 60mM KNO3 working solution to each centrifuge tube to make the volume of each tube 1mL. Then use 3.5%wt salinity and 7%wt salinity to improve 2216E liquid culture medium, dilute the above two groups of bacterial liquids by 20 times, so that the NO3 in the two groups of bacterial liquids is 0. - The -N concentration was adjusted to 0, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, and 90 μM, respectively.

[0115] Centrifuge at 12,000 rpm for 2 min to precipitate most of the bacteria, then pipette 180 μL of the supernatant into a 96-well polypropylene microplate. Add 30 μL of SA solution to the wells and react for 15 min to remove NO2 in the sample.- -N. Then place the 96-well polypropylene microplate in an ELISA reader and shake for 30 seconds to remove bubbles generated during the reaction. Take out the 96-well polypropylene microplate and add 120 μL NO3 - -N detection reagent, pipette and mix well, place it in a 25℃ biochemical incubator and incubate for 60 minutes, then immediately use an ELISA reader to detect the sample absorbance at a wavelength of 540nm, and use the obtained data to plot the OD 540 With NO3 - -Linear curve of N concentration.

[0116] 3.5%wt salinity improved NO3 in 2216E liquid culture medium - -N concentration calculation formula: △A=A 测量值 -A 空白 ; NO3 - -N (μM) = (△A-0.0035)÷0.005×B; Wherein, B: dilution factor, undiluted is 1; 10μM, 20μM, 30μM, 40μM, 50μM, 60μM, 70μM, 80μM, 90μM concentration of NO3 - -N of A 测量值 They are 0.166, 0.204, 0.250, 0.301, 0.361, 0.414, 0.461, 0.508 and 0.559 respectively; 空白 It is 0.112.

[0117] OD in 3.5%wt salinity-modified 2216E liquid medium 540 With NO3 - -N concentration linear curve, see Figure 2 Figure A in .

[0118] NO3 in 2216E liquid culture medium with 7%wt salinity improvement - -N concentration calculation formula: △A=A measured value-A blank; NO3 - -N (μM) = (△A-0.0005)÷0.0048×B; Wherein, B: dilution factor, undiluted is 1; 10μM, 20μM, 30μM, 40μM, 50μM, 60μM, 70μM, 80μM, 90μM concentration of NO3 - -N of A 测量值 They are 0.230, 0.283, 0.320, 0.370, 0.427, 0.476, 0.517, 0.568 and 0.617 respectively; A 空白It is 0.182.

[0119] Absorbance OD in 7%wt salinity improved 2216E liquid culture medium 540 With NO3 - -N concentration linear curve, see Figure 2 Figure B in .

[0120] 3.3.3 NO2 in culture medium - -N content determination method: According to NO2 - -N content determination kit instructions. Among them, NO2 - The -N content determination kit was purchased from Shanghai Runyu Biotechnology Co., Ltd., namely, the nitrite content determination kit RY-0430W in water and soil.

[0121] Two tubes of standard solution were prepared using 3.5%wt salinity modified 2216E liquid medium and 7%wt salinity modified 2216E liquid medium, respectively. The specific steps are as follows: 2mL of 3.5% salinity modified 2216E liquid medium and 7%wt salinity modified 2216E liquid medium were used to dissolve the standard to obtain mother solution 1, then 100μL of mother solution 1 was put into a centrifuge tube containing 900μL of 3.5% salinity modified 2216E liquid medium and 7%wt salinity modified 2216E liquid medium to obtain mother solution 2, and the ten-fold dilution method was used continuously to finally dilute the mother solution 1 5000 times to obtain 2ml NO2 - -N final concentration of 23.5 μM standard solution. The standard solution is a standard solution prepared from 3.5% salinity improved 2216E liquid culture medium and 7%wt salinity improved 2216E liquid culture medium.

[0122] Take 150 μL of 3.5% salinity modified 2216E liquid medium and 7% wt salinity modified 2216E liquid medium, 3.5% salinity modified 2216E liquid medium and 7% wt salinity modified 2216E liquid medium respectively, and put 50 μL of reagent 1 and reagent 2 into the microwells of the 96-well polypropylene microplate in turn, and mix them by pipetting. Place the 96-well polypropylene microplate in a 25°C biochemical incubator for 15 minutes, and then immediately place it in an ELISA reader to measure OD 540 The detected value is taken as NO2 - -N content determination kit provides the calculation formula constant, which is used for the subsequent NO2 - -Calculation of N content.

[0123] NO2 - -N content determination kit solution calculation formula: NO2 - -N=△A÷(A 标准 -A 空白 )×C 标 ×D; △A=A 测量值 -A 空白 ; Among them, NO2 - -N: NO2 in the culture medium tested - -N concentration, in μM; C 标 : Standard concentration, in NO2 - -N meter, C 标 23.5μM; D: dilution factor, D=1 when undiluted; A 空白 is 0μM NO2 - -N medium absorbance value; A 标准 is 23.5μM NO2 - -N absorbance, A 测量值 It is the value measured by diluting the test medium to an appropriate multiple, and △A is the actual absorbance value minus 0μM NO2 - -N medium absorbance value obtained.

[0124] 3.5%wt salinity improved A in 2216E liquid culture medium 标准 is 0.587; A 空白 is 0.05; when calculating the NO2 in 3.5%wt salinity solution - -N concentration, it is necessary to 标准 =0.587 and A 空白 =0.05 is substituted into the above formula to calculate the NO2 in the culture medium tested. - -N concentration.

[0125] 7%wt salinity improved 2216E liquid culture medium 标准 is 0.603; A 空白 is 0.05. When calculating the NO2 in 7%wt salinity solution - -N concentration, it is necessary to 标准 =0.603 and A 空白 =0.05 is substituted into the above formula to calculate the NO2 in the culture medium tested. - -N concentration.

[0126] 3.4. Determination of nitrogen removal efficiency and cell density of synthetic bacterial flora: The activated bacterial suspension of the synthetic bacterial colony was divided into two portions, each placed in a 50 mL sterile centrifuge tube, and centrifuged at 4,500 rpm for 8 min to collect the bacterial precipitate. The above two portions of bacterial cells were resuspended in modified 2216E medium containing 3.5% wt salinity and 7% wt salinity, respectively, so that the OD of the synthetic bacterial colony was 600 About 0.8. Add KNO3 stock solution to the above resuspended bacterial solution and add NO3 - The final concentration of -N was adjusted to 1.6mM. Three experimental replicates were set for each group, and the denitrifying bacteria agent purchased from Weifang Zhonghong Environmental Protection Technology Co., Ltd. was used as a positive control. The above bacterial solution was placed in a 25°C biochemical incubator for static culture. Every 12 hours, 300μL of the test bacterial solution was placed in a 1.5mL centrifuge tube and centrifuged at 12,000rpm for 2min to precipitate most of the bacteria. Then, the NO3 in the bacterial solution was measured and calculated. - -N and NO2 - -N content. According to the above method: the synthetic bacterial flora was inoculated into 3.5%wt salinity improved 2216E medium and 7%wt salinity improved 2216E medium respectively, and the above detection steps were repeated every 3 hours; the synthetic bacterial flora and commercially available bacterial agents were inoculated into the wastewater of marine white shrimp farming, and the above detection steps were repeated every 12 hours.

[0127] 3.4.1. NO3 in bacterial solution - -N content determination: Take 180 μL of the supernatant after centrifugation and put it into a 96-well polypropylene microplate. Add 30 μL of SA solution to the wells and react for 15 minutes to remove NO2 in the sample. - -N, place the 96-well polypropylene microplate in an ELISA reader and shake for 30 seconds to remove bubbles generated during the reaction, and add 120 μL NO3 - -N detection reagent, place the 96-well polypropylene microplate in a 25℃ biochemical incubator for 60 minutes, and then immediately place it in an ELISA reader to measure the OD of the bacterial solution 540 , according to the OD determined above 540 With NO3 - -N concentration linear curve, calculation of NO3 in synthetic bacterial flora and commercial bacterial agent solution - -N content, results see Figure 3 , Figure 4 and Figure 5 Figure A in .

[0128] 3.4.2 NO2 in bacterial solution - -N content determination: Take 20 μL of the supernatant after centrifugation and add it to a 1.5 mL centrifuge tube containing 980 μL of ddH2O and mix by pipetting. Take 150 μL of the dilution from the centrifuge tube and place it in a 96-well polypropylene microplate. - -N content determination kit instructions, add 50 μL of reagent 1 and reagent 2 to the microwells containing diluent and mix them by pipetting. Place the 96-well polypropylene microplate in a 25℃ biochemical incubator for 15 minutes, and then immediately place it in an ELISA reader to measure the OD of the bacterial solution. 540 According to the formula provided by the kit, the NO2 in the synthetic bacterial flora and commercially available bacterial agent solution was calculated. - -N content, results see Figure 3 , Figure 4 and Figure 5 Figure B in .

[0129] Among them, NO2 - The -N content determination kit was purchased from Shanghai Runyu Biotechnology Co., Ltd., namely, the nitrite content determination kit RY-0430W in water and soil.

[0130] 3.4.3. Synthetic bacterial colony cell density detection: Take 200 μL of bacterial solution and put it into a 96-well polypropylene microplate. Immediately place it in an ELISA reader to measure and record the OD value of the bacterial solution. 600 , the results are shown in Figure 3 , Figure 4 and Figure 5 Figure C in .

[0131] Under the two salinity conditions, the synthetic bacteria were able to convert NO3 - -N was completely removed, and NO2 was present at both 0 and 12h - -N accumulation, while NO2 - -N accumulation was significantly less than that under 3.5%wt salinity. - The -N removal efficiency and average removal rate were 100% and 0.15 mM / h, respectively. The bacterial density of the synthetic bacterial community increased slowly within 24 h, namely: OD 600 The values ​​of the commercially available bacterial agents increased from 0.802 and 0.803 to 0.916 and 0.938 respectively. However, under the two salinity conditions, the commercially available bacterial agents failed to convert NO3 - -N is completely removed, and all have NO2 - -N accumulation, NO2 - The maximum accumulation of -N is 0.28mM at 3.5%wt salinity in 24h. The commercially available bacterial agent has a NO3 --N removal efficiency and average removal rate were 3% and 13%, 0.006 and 0.02 mM / h respectively. The bacterial density of the commercially available bacterial agent continued to decrease to almost the same value within 24 hours, namely: OD 600 The cell density decreased from 0.801 and 0.800 to 0.181 and 0.164 respectively under the two salinity conditions. The cell density under the 3.5%wt salinity condition decreased faster.

[0132] In order to further determine the nitrogen removal efficiency of the synthetic bacterial community, the NO3 - -N and NO2 - -N content and cell density, the results are as follows Figure 4 shown.

[0133] Under the two salinity conditions, the synthetic bacteria were able to convert NO3 - -N is completely removed and NO2 appears - -N accumulation, except for 0h and 12h, at other time points, the NO2 of the synthetic bacteria under 3.5%wt salinity conditions - -N accumulation was slightly lower than that of the synthetic bacteria under 7%wt salinity conditions. - -N accumulation, the maximum accumulation was 1.77 and 1.85 mM at 3 h, respectively. Under the two salinity conditions, the synthetic bacteria accumulated NO3 - The -N removal efficiency and average removal rate were 95.2% and 95.3%, 0.584mM / h and 0.585mM / h, respectively. The bacterial density increased slowly and the overall trend was almost the same, namely: OD 600 They increased from 0.804 and 0.805 to 0.916 and 0.918 respectively.

[0134] The above experimental results show that the synthetic bacteria can convert NO3 in the culture medium into - -N is completely removed. NO3 of synthetic bacteria under 7%wt salinity conditions - -N removal efficiency did not decrease. During this period, salinity, NO3 - -N and NO2 - The presence of -N did not affect cell growth.

[0135] In the wastewater of Penaeus vannamei aquaculture, the synthetic bacteria can convert NO3 - -N was completely removed, and NO2 was present at 0, 12h and 24h. - -N accumulation, the maximum accumulation was 0.56mM at 12h. NO3 -The -N removal efficiency and average removal rate were 86.6% and 0.1 mM / h, respectively, and the bacterial density decreased slowly, namely: OD 600 The commercially available microbial agents can also reduce NO3 - -N is completely removed, and NO2 is present within 48 hours - -N accumulation, the maximum accumulation is 0.76mM at 24h. The commercially available bacterial agent has NO3 - The -N removal efficiency and average removal rate were 55% and 0.066 mM / h, respectively, and the bacterial density continued to decrease, namely: OD 600 They decreased from 0.807 to 0.350 respectively.

[0136] The above experimental results show that the synthetic bacterial flora can also convert NO3 into - -N is completely removed, and NO3 - -N, salinity and lack of carbon source have little effect on its cell density.

[0137] The present invention screened and synthesized marine biofilm-derived, stress-resistant, NO3 - The complete pathway of reduction and most of the strain members are not found in the denitrifying microbial complex. This microbial complex has high salt tolerance and can effectively remove NO3 under high salt static conditions. - -N and NO2 - -N. Therefore, it can be used to treat high-salinity nitrogen-containing wastewater, providing a simple new method for wastewater nitrogen removal.

[0138] The microbial composite flora with nitrogen removal ability provided by the present invention can stably exist and efficiently remove nitrogen under high salinity conditions, thus solving the problem of low nitrogen removal efficiency of existing microorganisms when treating high-salinity wastewater.

[0139] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0140] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. A microbial composite flora having nitrogen removal capability, characterized in that: Including mixed water Leisinger's bacteria, Leisinger's bacteria, sticky bacteria, hydrocarbon-removing sea rod-shaped bacteria, and agglomerated rose bacteria_A S419 ( Roseibium aggregatum _A S419); the deposit number of the agglomerated rose bacteria_A S419 is CCTCC M 20241515; The ratio of the live bacterial counts of the mixed water Riesingella, the Riesingella, the sticking bacteria, the hydrocarbon-removing sea rod-shaped bacteria, and the agglomerated rose bacteria_AS419 is 65-75:17-28:4-5:0.01-0.015:0.8-1.0; The mixed water mine Singh's bacteria is mixed water mine Singh's bacteria M597 ( Leisingera aquaemixtae M597), mixed water minesingella M602 ( Leisingera aquaemixtae M602), mixed water singella M583 ( Leisingera aquaemixtae M583) and mixed Singella leucogeneticum S166 ( Leisingera aquaemixtae S166); the deposit number of the mixed water Singella leuprorein M597 is CCTCC M 20241898; the deposit number of the mixed water Singella leuprorein M602 is CCTCC M 20241899; the deposit number of the mixed water Singella leuprorein M583 is CCTCC M 20241897; the deposit number of the mixed water Singella leuprorein S166 is CCTCC M 20241900; The ratio of the viable bacteria counts of the mixed Singella pyrenoidosa M597, the mixed Singella pyrenoidosa M602, the mixed Singella pyrenoidosa M583 and the mixed Singella pyrenoidosa S166 is 1:1:1:1; The described bacteria is bacteria M658 ( Leisingera sp. M658); the sp. relzingerella M658 was deposited in China Center for Type Culture Collection on July 8, 2024, with the deposit number of CCTCC M 20241512; The sticking bacillus is sticking bacillus S095 ( Tenacibaculum sp. S095), the sticky Bacillus S105 ( Tenacibaculum sp. S105) and the adhensinic bacteria Z003 ( Tenacibaculum sp. Z003); the ratio of the number of live bacteria of the adhesin Bacillus S095, the adhesin Bacillus S105 and the adhesin Bacillus Z003 is 1:1:1; The deposit number of the Bacillus adhesus S095 is CCTCC M 20241896; the deposit number of the Bacillus adhesus S105 is CCTCC M 20241513; the deposit number of the Bacillus adhesus Z003 is CCTCC M 20241901; The hydrocarbon-removing marine bacillus is the hydrocarbon-removing marine bacillus S418 ( Marinobacter hydrocarbonoclasticus S418); the deposit number of the hydrocarbon-removing marine bacillus S418 is CCTCC M 20241511; The agglomerated rose bacteria_A S419, the mixed water mine Singh's bacteria M597, the mixed water mine Singh's bacteria M602, the mixed water mine Singh's bacteria M583, the mixed water mine Singh's bacteria S166, the sticking bacteria S095, the sticking bacteria S105, the sticking bacteria Z003, and the hydrocarbon-removing sea rod-shaped bacteria S418 are all preserved in the China Center for Type Culture Collection.

2. The method for constructing a composite microbial flora having nitrogen removal ability according to claim 1, characterized in that: The steps include: The mixed water reisinger's bacteria, the reisinger's bacteria, the sticking bacillus, the hydrocarbon-removing sea rod-shaped bacteria, and the agglomerated rose bacteria_AS419 are mixed according to the ratio of the number of live bacteria of 65~75:17~28:4~5:0.01~0.015:0.8~1.0 to obtain the microbial composite flora with nitrogen removal ability.

3. Use of the microbial complex with nitrogen removal ability as claimed in claim 1 in treating high-salt culture medium and marine aquaculture wastewater.

4. The use of the composite microbial flora with nitrogen removal ability according to claim 3 in treating high-salt culture medium and marine aquaculture wastewater, characterized in that: The application described is nitrogen removal.

5. The use of the composite microbial flora with nitrogen removal ability according to claim 4 in treating high-salt culture medium and marine aquaculture wastewater, characterized in that: The nitrogen removal refers to the removal of nitrate nitrogen and nitrite nitrogen in the high-salt culture medium and the marine aquaculture wastewater.

6. The use of the composite microbial flora with nitrogen removal ability according to claim 3 in treating high-salt culture medium and marine aquaculture wastewater, characterized in that: The marine aquaculture wastewater is marine white shrimp aquaculture wastewater.

7. The use of the composite microbial flora with nitrogen removal ability according to claim 3 in treating high-salt culture medium and marine aquaculture wastewater, characterized in that: The temperature of the high-salt culture medium and the marine aquaculture wastewater are both 23° C. to 28° C., and the pH is both 7 to 8; The total salt content in the high-salt culture medium is 3.5%wt and / or 7%wt; the total salt content in the marine aquaculture wastewater is 3.5%wt.

8. The use of the composite microbial flora with nitrogen removal ability according to claim 3 in treating high-salt culture medium and marine aquaculture wastewater, characterized in that: The microbial composite flora with nitrogen removal ability is inoculated into the high-salt culture medium and the marine aquaculture wastewater for 3 hours to 48 hours respectively, so as to remove nitrate nitrogen and nitrite nitrogen in the high-salt culture medium and the marine aquaculture wastewater.

Images