Denitrifying bacteria, microbial inoculum and application of denitrifying bacteria and microbial inoculum

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to denitrifying bacteria, a microbial agent and application of the denitrifying bacteria and the microbial agent. According to the invention, a denitrifying bacterium, namely Shania sp. Pe10, which is derived from a plastic marine organism envelope, is strong in stress resistance and has a NO3 <-> reduction complete pathway is screened, the bacterium is preserved in China Center for Type Culture Collection on February 24, 2025, the preservation number is CCTCC NO: M 2025284, and the strain has the capability of efficiently removing NO3 <->-N and NO2 <->-N under the conditions of standing, low carbon and high salt.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to a denitrifying bacterium, a bacterial agent and applications thereof. Background Art

[0002] Biological methods in sewage treatment generally use anaerobic denitrifying bacteria, autotrophic denitrifying bacteria, activated sludge method, aerobic denitrifying bacteria, denitrifying bacteria, etc.

[0003] Anaerobic denitrification agents have high requirements for actual sewage treatment equipment, which increases hidden costs. During sewage treatment, autotrophic nitrifying bacteria and anaerobic denitrifying bacteria are carried out separately under different conditions, resulting in a long denitrification process. Although autotrophic denitrifying bacteria do not need to add additional carbon sources when removing nitrogen, they have problems such as slow growth, low denitrification efficiency, harsh living conditions, and weak resistance to shock loads. The activated sludge method has a better nitrogen removal effect, but it has problems such as large operating equipment footprint and high operating costs, and secondary pollution caused by poorly treated sludge products. , most activated sludges have a relatively ideal nitrogen removal effect only under low salinity conditions, and only a very small number of activated sludges can remove nitrogen with less than ideal efficiency under higher salinity; aerobic denitrifying bacteria usually need higher C / N conditions to stably and efficiently remove nitrogen, and excessive addition of carbon source may cause secondary pollution of water bodies; most denitrifying strains come from freshwater environments, and often have problems such as low tolerance to salinity and poor growth activity. Therefore, in actual processes, the nitrogen removal efficiency of such bacteria in high-salinity wastewater is extremely low, so it is necessary to provide a salt-tolerant denitrifying bacteria. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a denitrifying bacteria, a bacterial agent and applications thereof.

[0005] A denitrifying bacterium, wherein the denitrifying bacterium is Shen's bacteria ( Shimia sp.) pe10, which was deposited in the China Center for Type Culture Collection on February 24, 2025, with the deposit number: CCTCC NO: M 2025284.

[0006] A bacterial agent comprises the denitrifying bacteria.

[0007] Application of the denitrifying bacteria or the bacterial agent in sewage treatment.

[0008] Preferably, the sewage treatment refers to sewage denitrification.

[0009] Preferably, the sewage is saline sewage.

[0010] Preferably, the salt content in the saline wastewater is greater than 2% by mass.

[0011] Preferably, the application comprises the following steps: After the denitrifying bacteria are activated, the bacterial precipitate is collected by centrifugation; The bacterial precipitate is added to the sewage to be treated and treated for at least 6 hours to perform sewage denitrification. The mass volume ratio of the bacterial precipitate to the sewage to be treated is 30g~100g:1L.

[0012] Preferably, the salt content in the sewage to be treated is 3% to 7% by mass.

[0013] Preferably, the method for activating the denitrifying bacteria is to inoculate the strain into 2216E liquid culture medium at a volume ratio of 1:4-6 and activate for 15h-22h.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The present invention screens out plastic marine biofilms that are highly resistant to stress and have NO3 - The denitrifying bacteria that restore the complete pathway are Shen's bacteria ( Shimia sp.) pe10, which was deposited in the China Center for Type Culture Collection on February 24, 2025, with the deposit number: CCTCC NO: M 2025284. This strain has a high efficiency in removing NO3 under static low-carbon and high-salt conditions. - -N (nitrate nitrogen) and NO2 - -N (nitrite nitrogen) capacity, thus providing a new green and low-cost approach for treating nitrogen-containing wastewater with actual high salinity and low nutrients.

[0015] The denitrifying bacteria provided by the present invention can denitrify 20 mg / L NO3 in the culture medium within 24 hours under the salinity conditions of 3.5% wt and 7% wt. - -N is completely removed, and this strain can efficiently remove NO3 in low carbon source concentration and high salt culture medium under static conditions - -N and NO2 - -N. is a beneficial bacteria in the intestinal tract of many marine organisms, has no potential harm to the environment and aquaculture systems, and provides a new feasible solution for rapid biological nitrogen removal from high-salinity wastewater. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the data analysis and experimental operation process of the present invention.

[0017] Figure 2 For the strain of the present invention Shimia Schematic diagram of the location of key denitrification genes in the genome of sp.

[0018] Figure 3 For the denitrifying strain of the present invention Shimiasp. nitrogen removal efficiency diagram 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 NO2 in the culture medium - -N concentration; Figure 3 Panel C shows the cell density in the culture medium.

[0019] Figure 4 This is a characterization diagram of the denitrification ability of the strain 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 - -The color development corresponding to the concentration of N; Figure 4 Figure B shows NO2 in the culture medium - -N concentration corresponding to the color development. DETAILED DESCRIPTION

[0020] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0021] Culture medium and reagents used in the present invention 1. Culture medium All culture media used in the present invention were purchased from Haibo Biotechnology.

[0022] Marine broth 2216E agar medium formula: peptone 5.0 g, yeast extract powder 1.0 g, ferric citrate 0.1 g, sodium chloride 19.45 g, magnesium chloride 5.98 g, sodium sulfate 3.24 g, calcium chloride 1.8 g, potassium chloride 0.55 g, sodium carbonate 0.16 g, potassium bromide 0.08 g, strontium chloride 0.034 g, boric acid 0.022 g, sodium silicate 0.004 g, sodium fluoride 0.0024 g, ammonium nitrate 0.0016 g, disodium hydrogen phosphate 0.008 g, agar 15 g. The solvent is ultrapure water, and the volume is adjusted to 1 L with ultrapure water.

[0023] Marine broth 2216E liquid culture medium formula: peptone 5.0 g, yeast extract powder 1.0 g, ferric citrate 0.1 g, sodium chloride 19.45 g, magnesium chloride 5.98 g, sodium sulfate 3.24 g, calcium chloride 1.8 g, potassium chloride 0.55 g, sodium carbonate 0.16 g, potassium bromide 0.08 g, strontium chloride 0.034 g, boric acid 0.022 g, sodium silicate 0.004 g, sodium fluoride 0.0024 g, ammonium nitrate 0.0016 g, disodium hydrogen phosphate 0.008 g. The solvent is ultrapure water, and the volume is adjusted to 1 L with ultrapure water.

[0024] 3.5% wt salinity modified 2216E liquid medium recipe: 0.5g peptone, 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. Ultrapure water was used as the solvent and the volume was adjusted to 1L. Note: To minimize the effects of minerals and salinity, the inorganic nutrient concentrations in the mother liquor were the same as those in Marine broth 2216E liquid medium. In addition, 35g / L sea salt was added to this medium to adjust the salinity to 7% wt.

[0025] 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.

[0026] Ultra-high-speed PCR premix was purchased from Shanghai Yisheng Biotechnology Co., Ltd. 2×Hieff ® Ultra-RapidHotStart PCR Master Mix, Cat: 10157ES03.

[0027] NO2 - -N content determination kit was purchased from Shanghai Runyu Biotechnology Co., Ltd., CAS: RY-0430W.

[0028] 3. Equipment Vertical full-temperature oscillating incubator: Zhichu ZQLY-180.

[0029] Benchtop microcentrifuge: BECKMAN Microfuge 16 Centrifuge.

[0030] Constant temperature mixer: Yisheng ES-TM100.

[0031] Gradient PCR amplifier: Biometra TOne, Germany.

[0032] Micro vortex mixer: Sangon Mixer 4K.

[0033] Constant temperature water bath: digital display constant temperature water bath HHS-11-2.

[0034] Agarose gel electrophoresis system: Bio-Rad DYCP-31DN, USA.

[0035] Nanodrop micro-volume spectrophotometer: Tiangen OSE-260-03.

[0036] Microplate reader: Thermo Scientific MULTISKAN FC.

[0037] Example 1 1. Sample Sampling and Pretreatment 1. Sample collection Nylon mesh bags containing four types of plastic were placed 2 meters underwater in the subtidal zone of Dingjiazui, Huangdao District, Qingdao, Shandong Province, China (120°8'19''E, 35°54'55''N). Three replicates of each type were collected, and the sampling locations were marked with suspended buoys. Biofilm samples were then collected from the plastic surfaces using sterile cotton swabs at 10 and 20 days post-sampling. The collected samples were immediately transferred to the Marine Biofilm Laboratory in the Teaching Building, Area X, Fushan Campus, Ocean University of China.

[0038] Under sterile conditions, 10 mL of Marine broth 2216E liquid culture medium was taken to thoroughly rinse the cotton swab to obtain the sample stock solution. 1 mL of the sample stock solution was diluted in 9 mL of culture medium to obtain 10 1 Repeat this step to obtain 4 dilutions of different concentrations, i.e. 10 2 Concentration of dilution, 10 3 Concentration of dilution, 10 4 Concentration of dilution, and 10 5 Concentration of dilution.

[0039] 2. Separation and purification Spread 200 μL of bacterial suspension from each dilution onto a solid plate containing Marine Broth 2216E agar. Place the plate in a 25°C incubator and invert it for incubation. After 3 days of incubation, use an inoculating loop to select bacterial colonies of varying color and shape. Subculture the plate eight times to obtain a pure culture solid plate.

[0040] Inoculate all selected single colonies of varying morphology and color into 10 mL centrifuge tubes containing 4 mL of Marine broth 2216E liquid culture medium. Place the centrifuge tubes in a vertical shaking incubator and culture at 180 rpm at 25°C until the logarithmic growth phase. Prepare a culture suspension. Mix 1.0 mL of the suspension with 50% glycerol at a volume ratio of 1:1 to prepare a glycerol-frozen suspension. Store the suspension in a -80°C freezer.

[0041] 2. DNA Extraction and Genome Sequencing 1. Preliminary species identification Take 1 mL of the above bacterial solution and place it in a 1.5 mL centrifuge tube. Centrifuge it at 6000 rpm for 5 min in a desktop microcentrifuge. Discard the supernatant and add ddH2O to the bacteria at the bottom of the centrifuge tube to resuspend them to obtain ddH2O-suspended bacteria. Take 200 μL of ddH2O-suspended bacteria, place it in a constant temperature mixer, and heat it at 100°C for 10 min to release bacterial DNA.

[0042] 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.

[0043] The PCR reaction system was as follows: 1 μL template DNA, 10 μM forward primer: 1 μL, 10 μM reverse primer: 1 μL, 10 μL Taq enzyme premix containing dye, and sterile ddH2O was added to a total volume of 25 μL.

[0044] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min followed by cycling, 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.

[0045] 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'.

[0046] 2.5 μL of PCR amplification product was subjected to 2% agarose gel electrophoresis. Qualified PCR products were sent to Shanghai Sangon Biotechnology Co., Ltd. for Sanger sequencing.

[0047] Based on Sanger sequencing data, bacterial species were preliminarily annotated using the NCBI database. Duplicates were then removed from the 16S sequences of all individual colonies using cd-hit software, version 4.8.1, with a sequence similarity threshold of 0.9999. Ultimately, species information and sample IDs were obtained for all non-redundant sequences.

[0048] Among them, the full name of NCBI is: National Center for Biotechnology Information.

[0049] 2. Genome sequencing According to the culture steps in the above separation and purification steps, all non-redundant bacteria were cultured to the logarithmic phase and then centrifuged to collect about 1.0×10 9 DNA extraction and purification of all strains were performed according to the procedures for processing Gram-positive bacteria samples in the FastPure Bacteria DNA Isolation Mini Kit instructions.

[0050] 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 with a micro-vortex mixer, and then immediately place it in a 37°C constant temperature water bath and incubate for 1 hour.

[0051] (2) Add 20 μL of Proteinase K to the centrifuge tube and vortex to mix.

[0052] (3) Add 250 μL of Buffer GB and vortex to mix.

[0053] (4) Place the centrifuge tube in a constant temperature mixer at 70°C and incubate for 10 minutes to obtain a mixed solution.

[0054] 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.

[0055] (2) Transfer the mixture to the adsorption column in the collection tube, centrifuge at 12,000 rpm for 1 min and discard the filtrate.

[0056] (3) Add 500 μL of Buffer PB, 600 μL of Buffer PW, and 600 μL of Buffer PW to the adsorption column in sequence. Centrifuge at 12,000 rpm for 1 min after each addition and discard the filtrate.

[0057] (4) Place the adsorption column back into the collection tube and centrifuge at 12,000 rpm for 2 min. Open the cap of the adsorption column and place it on a clean bench for 5 min to allow the residual ethanol to evaporate completely.

[0058] (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.

[0059] (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.

[0060] The integrity, concentration, and purity of all non-redundant bacterial DNA were determined using 2% agarose gel electrophoresis and a Nanodrop microspectrophotometer. Qualified DNA samples were sent to Novogene China for sequencing on the Illumina NovaSeq PE 150 platform, with a sequencing capacity of 2 Gb per bacterial sample. Ultimately, genomic data for all non-redundant bacterial species from marine rock biofilms were obtained.

[0061] Lysozyme is lysozyme. Lysozyme solution formula: Accurately weigh 2 g of Lysozyme dry powder, dissolve it in 1× TE buffer (pH 8), and dilute to volume in a 100 mL volumetric flask to obtain a 20 mg / mL Lysozyme solution.

[0062] 3. Genomic 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 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 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 screened from the evaluation results. The ORFs of bacterial genomes were predicted using Prodigal software version 2.60, and the "-c" parameter was selected to output complete ORFs with stop codons. Gene function annotation of ORFs of bacterial genomes was performed 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.

[0063] Among them, high-quality genomic data refers to a completeness greater than 95% and a contamination degree less than 5%.

[0064] The full name of ORFs is: open reading frames, and its Chinese name is open reading frames.

[0065] The full name of KEGG is: Kyoto Encyclopedia of Genes and Genomes, and its Chinese name is Kyoto Encyclopedia of Genes and Genomes.

[0066] The full name of BLAST is: Basic Local Alignment Search Tool.

[0067] 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 .

[0068] Based on the functional analysis of genomic data, a bacterial strain with denitrification potential was screened out, such as Figure 2 This strain is: Shen's pe10. The classification name of Shen's pe10 is Shimia sp. pe10 was deposited in the China Center for Type Culture Collection on February 24, 2025, with the deposit number: CCTCC NO: M 2025284.

[0069] 3. Activation culture of denitrifying bacteria, nitrogen removal efficiency and cell density determination 1. Activation culture The cryopreserved strain was inoculated into a 15-mL centrifuge tube containing 5 mL of Marine broth 2216E liquid medium at a v / v ratio of 1:5 and activated by shaking at 180 rpm at 25°C for 18 hours. The activated bacterial suspension was inoculated into a 500-mL conical flask containing 200 mL of Marine broth 2216E liquid medium at a volume fraction of 1% and cultured at 180 rpm at 25°C for 24 hours.

[0070] 2. Determination of nitrogen removal efficiency and cell density The activated bacterial solution was divided into two parts, each of which was placed in a 50 mL sterile centrifuge tube and centrifuged at 4500 rpm for 8 min to collect the bacterial precipitate. The above two bacterial precipitates were resuspended in 3.5% wt salinity modified 2216E liquid medium and 7% wt salinity modified 2216E medium respectively, so that the OD of the synthetic bacterial colony was 600 ≈0.8, and two resuspended bacterial solutions were obtained, of which the mass of bacterial pellet was 50 g, and the volume of 3.5%wt salinity modified 2216E liquid medium and 7%wt salinity modified 2216E liquid medium were both 1 L. Potassium nitrate (KNO3) mother solution was added to the resuspended bacterial solution to reduce the NO3 in the resuspended bacterial solution. - The final concentration of -N was adjusted to 20 mg / L, and three experimental replicates were set for each group. The resuspended bacterial solution was placed in a 25°C biochemical incubator for static culture to obtain the test bacterial solution. At 6 hours and 24 hours, 300 μL of the test bacterial solution was placed in a 1.5 mL centrifuge tube and centrifuged at 12000 rpm for 2 minutes to precipitate most of the bacteria. The NO3 in the test bacterial solution was then measured and calculated. - -N and NO2 - -N content.

[0071] Among them, vanadium chloride reduction method is used to reduce NO3 - -N quantitative detection, using Griess reagent for NO2 - Quantitative detection of -N.

[0072] The results and color development are shown in Figure 3 and Figure 4 Take 200 μL of the bacterial solution to be tested and place it in a 96-well polypropylene microplate. Immediately place it in a microplate reader to measure and record the OD value of the bacterial solution. 600 , the results are shown in Figure 3 Figure C. Under 3.5% wt salinity, the strain converted NO3 to - -N was completely removed and NO2 did not appear in the culture medium - -N accumulation, bacterial density slightly increased, namely: OD 600 They increased from about 0.8 to about 0.82. Under 7% wt salinity conditions, the NO3 in the culture medium at 6 h - -N residual concentration was 0.63 mg / L, and NO2 did not appear in the culture medium. - -N accumulation, and there was almost no significant increase in bacterial density, that is: OD 600 The strains increased from about 0.8 to about 0.803 under the two salinity conditions within 6 hours. - The -N removal efficiency and average removal rate were 100% and 97%, respectively, and 3.33 mg / L·h -1 and 3.22 mg / L·h-1 .

[0073] The above experimental results show that the synthetic bacteria can convert NO3 in the culture medium into - -N is completely removed and no NO2 appears - -N accumulation. NO3 of synthetic bacterial consortium under 7%wt salinity conditions - -N removal efficiency decreased slightly, and during denitrification, salinity, NO3 - -N and NO2 - The presence of -N did not affect cell growth.

[0074] It should be noted that when the claims of the present invention involve numerical ranges, 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 preferred embodiments.

[0075] Although the 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 have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0076] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. A denitrifying bacterium, characterized in that The denitrifying bacteria is Shen's bacteria ( Shimia sp.) pe10, which was deposited in the China Center for Type Culture Collection on February 24, 2025, with the deposit number: CCTCC NO: M 2025284.

2. A bacterial agent, characterized in that The invention comprises the denitrifying bacteria according to claim 1.

3. Use of the denitrifying bacteria according to claim 1 or the bacterial agent according to claim 2 in sewage treatment.

4. The use according to claim 3, characterized in that The sewage treatment refers to sewage denitrification.

5. The use according to claim 4, characterized in that The sewage is saline sewage.

6. The use according to claim 5, wherein the salt content in the saline wastewater is greater than 2% by mass.

7. The use according to claim 3, characterized in that The application comprises the following steps: After the denitrifying bacteria are activated, the bacterial precipitate is collected by centrifugation; The bacterial precipitate is added to the sewage to be treated and treated for at least 6 hours to perform sewage denitrification. The mass volume ratio of the bacterial precipitate to the sewage to be treated is 30g~100g:1L.

8. The use according to claim 7, characterized in that The salt content in the sewage to be treated is 3% to 7% by mass.

9. The use according to claim 6, characterized in that The denitrifying bacteria activation method comprises inoculating the strain into 2216E liquid culture medium at a volume ratio of 1:4-6 and activating the culture medium for 15h-22h.

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