Nitrate breathing bacterium and application thereof

Abstract

The invention discloses nitrate breathing bacteria and application thereof, and belongs to the field of energy conservation and environmental protection. The nitrate respiration bacterium disclosed by the invention is Shigella flexneri CCTCC 77, and is preserved in the China Center for Type Culture Collection on June 6, 2024, and the preservation number is CCTCC NO: M 20241159. The nitrate breathing bacteria are separated from the pig farm wastewater treatment process; the strain can optimize, regulate and control the denitrification treatment process of culture high-nitrogen wastewater in situ, improve the conversion efficiency of nitrite and nitrous oxide, and reduce the addition amount of an exogenous carbon source, the output of sludge and the aeration energy consumption in the wastewater treatment process.

Description

Technical Field

[0001] The invention relates to the field of energy conservation and environmental protection, and in particular to nitrate-respiring bacteria and applications thereof. Background Art

[0002] Piggery wastewater is a typical high NH4 + -N and low carbon-nitrogen ratio (C / N) wastewater have a relatively concentrated discharge time, a high pollution load, are difficult to quickly control and utilize, and the sludge produced is difficult to degrade and increases the risk of secondary pollution. Improperly treated high-nitrogen wastewater can easily lead to eutrophication problems in water bodies.

[0003] Traditional biological denitrification includes nitrification and denitrification. Denitrification is an important part of the wastewater denitrification process. It uses denitrifying bacteria to convert nitrate (NO3 - ) into nitrogen (N2). Short-range denitrification is the first step of the denitrification reaction, which converts nitrate (NO3 - ) is reduced to nitrite (NO2 - ), which can be used as a reaction matrix for anaerobic ammonium oxidation (Anammox). Short-range denitrification and anaerobic ammonium oxidation can be combined into a new biological denitrification process. Compared with traditional denitrification processes, new denitrification processes have the advantages of saving electron donors (replacing carbon sources with ammonia nitrogen) and avoiding greenhouse gas (N2O) emissions.

[0004] Nitrate respiration refers to the process by which denitrifying bacteria utilize nitrate (NO3 - ) as an electron acceptor to complete respiration to obtain energy, mainly generating nitrite (NO2 - At present, the research on denitrifying bacteria is mainly based on Pseudomonas ( Pseudomonas ), Alcaligenes spp. Alcaligenes ), Paracoccus ( Paracoccus ) and Bacillus ( Bacillus Most denitrifying bacteria are isolated from natural environments for wastewater treatment, which may have certain inadaptability. There are relatively few strains and application studies based on in situ domestication processes. Summary of the invention

[0005] The purpose of the present invention is to provide a nitrate-respiring bacteria and an application thereof. The nitrate-respiring bacteria of the present invention are separated from a pig farm wastewater treatment process; the strain can in situ optimize and regulate the denitrification treatment process of aquaculture high-nitrogen wastewater, improve the conversion efficiency of nitrite and nitrous oxide, and reduce the amount of exogenous carbon source added, sludge generation and aeration energy consumption in the wastewater treatment process.

[0006] The present invention first provides a Shigella flexneri ( Shigella flexneri), which was deposited in the China Center for Type Culture Collection (CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on June 6, 2024, with the deposit number of CCTCC NO: M 20241159.

[0007] The above-mentioned Shigella flexneri ( Shigella flexneri ) were characterized by nitrate reduction and utilization, mannitol fermentation, catalase and indole production.

[0008] Furthermore, the present invention also provides a bacterial agent, which contains the above-mentioned Shigella flexneri ( Shigella flexneri ).

[0009] The active ingredient of the bacterial agent is the Shigella flexneri ( Shigella flexneri ).

[0010] Finally, the present invention provides the above-mentioned Shigella flexneri ( Shigella flexneri ) in short-range denitrification.

[0011] In the above application, in the short-range denitrification, the temperature is 30°C±1°C; and the carbon-nitrogen mass ratio is 3:1.

[0012] The present invention also provides the above-mentioned Shigella flexneri ( Shigella flexneri ) in the conversion of nitrate nitrogen into nitrite nitrogen.

[0013] Shigella flexneri Shigella flexneri CCTCC77 is able to carry out nitrate respiration, converting nitrate nitrogen into nitrite nitrogen.

[0014] The strain of the invention is obtained by domestication and separation from pig farm wastewater. The strain is a Gram-negative bacterium in a rod or rod shape, with a bacterial body length of 2.0-2.5 μm and a width of 1-1.2 μm, without capsules and spores, and can be attached and arranged for growth. After being cultured on a nutrient broth agar medium for one day, the colonies are round, translucent, slightly light yellow, with a colony diameter of 1-1.5 mm, and a growth temperature range of 7-42°C. The strain of the invention shows positive indicators of nitrate reduction and utilization, mannitol fermentation, catalase, and indole production.

[0015] Instructions for the Deposit of Biological Materials Classification and nomenclature of biological materials: Shigella flexneri ( Shigella flexneri ) Strain number of biological material: CCTCC77 Name of the depository of biological materials: China Center for Type Culture Collection Abbreviation of the depository of biological materials: CCTCC Address of the depository of biological materials: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072 Deposit date of biological material: June 6, 2024 The registration number of the Collection Center of Biological Materials: CCTCC NO: M 20241159 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Shigella flexneri ( Shigella flexneri Transmission electron microscope photo of CCTCC77); Figure 2 Shigella flexneri ( Shigella flexneri CCTCC77) growth curve; Figure 3 Shigella flexneri ( Shigella flexneri Denitrification efficiency diagram of CCTCC77). DETAILED DESCRIPTION

[0016] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0017] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0018] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0019] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0020] The composition and concentration of the nutrient broth liquid culture medium used in the following examples are as follows (g / L): beef extract 5, peptone 10, NaNO2 1; the solvent is water.

[0021] The composition and concentration of nutrient broth solid culture medium are as follows (g / L): beef extract 5, peptone 10, NaNO2 1; the solvent is water; add 2% agar of the prepared culture medium volume.

[0022] Example 1, Shigella flexneri Shigella flexneri Isolation and identification of CCTCC77 1. Isolation of strains 1. Sludge sample collection Activated sludge from pig farm wastewater treatment at Muyuan No. 2 Farm in Guangfu Town, Nanchang County, Nanchang City, Jiangxi Province was collected (collection time: July 2023, collector: Nie Hanbing, contact information: 18811371960).

[0023] 2. Isolation and purification of strains The collected activated sludge from pig farm wastewater was put into a sequencing batch reactor (SBR) for 17 days. The reactor had a volume of 10L, a built-in agitator, and 5 equally spaced outlets on the wall for sampling, drainage, and water inlet. The composition of the inlet substances was as follows (g / L): sodium nitrite 0.66, ammonium chloride 0.384, sodium acetate 0.166, sodium bicarbonate 1.04, potassium dihydrogen phosphate 0.072, calcium chloride 0.3, and magnesium chloride 0.1647. The inlet and outlet were connected to the outlet of the SBR reactor through a peristaltic pump, and the temperature of the SBR reactor was controlled at 30℃±(1-2)℃ through a circulating pump connected to a water bath. Subsequently, the water sample in the reactor (which can be regarded as a bacterial suspension) was diluted and inoculated into a denitrification solid culture medium and cultured in a constant temperature incubator at 37°C for 2 to 3 days. Typical colonies with different morphologies and clear colony characteristics were picked out and enriched with nutrient broth liquid culture medium for 1 to 2 days, and then spread on the denitrification solid culture medium. The above enrichment and purification steps were repeated 2 to 3 times until a single colony culture was finally formed. The strain was named CCTCC77.

[0024] Pick out the purified single colony and inoculate it into nutrient broth liquid culture medium or nutrient broth solid culture medium for later use.

[0025] The components and concentrations of the denitrification solid culture medium used above are as follows (g / L): ammonium chloride 0.08, potassium nitrate 0.6, sodium acetate 1, potassium hydrogen phosphate trihydrate 0.2, calcium chloride dihydrate 0.1, magnesium sulfate heptahydrate 0.04, iron sulfate dihydrate 0.02, and trace element solutions A and B are added 1mL / L each. Among them, the components and concentrations of trace element A are as follows (g / L): disodium ethylenediaminetetraacetate 6.39, iron sulfate heptahydrate 5. The components and concentrations of trace element B are as follows (g / L): disodium ethylenediaminetetraacetate 19.11, boric acid 0.014, zinc sulfate heptahydrate 0.43, cobalt dichloride hexahydrate 0.24, manganese chloride tetrahydrate 0.99, copper sulfate pentahydrate 0.25, nickel chloride hexahydrate 0.19, sodium molybdate 0.22; the solvent is water.

[0026] 2. Shigella flexneri Shigella flexneri Identification of CCTCC77 (1) Morphological identification Select bacterial strains, streak on nutrient broth solid culture medium, seal it and place it in a 37°C constant temperature incubator for 1 day to observe the colony morphology.

[0027] Morphological identification results: The colonies are round, translucent, white with a slight yellowish tint, and the diameter of the colonies is 1-1.5 mm. The bacteria are observed under a transmission electron microscope at a magnification of 15,000 times. The length of the bacteria is 2.0-2.5 μm, the width is 1-1.2 μm, and they are rod-shaped or rod-shaped. They have no capsule or spores and can grow in an attached arrangement. Figure 1 .

[0028] (2) Physiological and biochemical identification According to the eighth edition of Bergey's Manual of Bacterial Identification, the identification result was Shigella flexneri ( Shigella flexneri ), Gram-negative bacteria, with a growth temperature range of 7-42°C, and an optimum growth temperature of 25-37°C. The bacteria do not grow at 4°C but can grow at 42°C. The strain does not require organic growth factors. The strain was tested for physiological and biochemical indicators of nitrate reduction and utilization, mannitol fermentation, catalase, and indole production, and the results are shown in Table 1.

[0029] Table 1 Shigella flexneri ( Shigella flexneri )

[0030] (3) Molecular identification The genomic DNA of the strain was extracted and PCR amplified. The amplified product was recovered using the AxyPrep DNA gel recovery kit, and then DNA sequencing was performed using the sequencer ABI3730-XL. The sequencing result is shown in sequence 1 of length 1411. The spliced ​​sequence file was compared with the data in the NCBI 16S database using the NCBIBlast program. The bacteria belonged to Shigella flexneri. The strain was deposited in the China Center for Type Culture Collection CCTCC on June 6, 2024, with the deposit number: CCTCCNO: M 20241159. The strain was classified and named Shigella flexneri ( Shigella flexneri ). Sequence 1 is as follows: PCR amplification reaction system construction process: 1) Primer design sequence: Primer 1 27F: 5′- AGAGTTTGATCCTGGCTCAG-3′ Primer 2 1492R: 5′- GGTTACCTTGTTACGACTT-3′; 2) Establishment of PCR amplification system (50 μL): Genomic DNA (20 ng / μL) 1.0 μL 10×Buffer (containing 2.5 mM Mg 2+ )5.0μL Taq polymerase (5u / μL) 1.0μL dNTP (10mM) 1.0μL 27F Primer 1 (10 μM) 1.5 μL 1492R Primer 2 (10 μM) 1.5 μL ddH2O39.0 μL Total volume 50.0 μL; 3) PCR amplification reaction program setting: Pre-denaturation at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min 30 s, 35 cycles, and final extension at 72°C for 7 min.

[0031] Example 2, Shigella flexneri ( Shigella flexneri Verification of denitrification function of CCTCC77) 1. Shigella flexneri ( Shigella flexneri CCTCC77) growth curve assay The frozen strain was activated, dissolved in nutrient broth liquid medium, and then streaked and inoculated on nutrient broth solid medium, cultured at 37°C for 24 hours. After the colonies grew on the plate, typical colonies were picked and inoculated in nutrient broth liquid medium at 37°C for 18 hours as the first-level seed liquid. The first-level seed liquid was divided into 10% of the original volume and inoculated into sterile PP tubes. The culture liquid was taken every 1 hour, and the OD600 value (Optical Density) of the bacterial liquid was measured at a wavelength of 600nm by photoelectric turbidimetry to obtain Shigella flexneri ( Shigella flexneri CCTCC77) growth curve Figure 2 As shown. Figure 2 As can be seen in the figure, Shigella flexneri ( Shigella flexneriThe incubation period of the secondary seed liquid growth curve of CCTCC77 is relatively short, about 2 hours, which may be because the strain is more active and the culture conditions before and after inoculation are similar, so it can adapt to the new environment in a short time after inoculation. Shigella flexneri The logarithmic growth phase of CCTCC77 is about 2-4 hours, the stable phase is 4-7 hours, and it enters the decline phase after 7 hours.

[0032] 2. Shigella flexneri ( Shigella flexneri CCTCC77) Denitrification efficiency determination Under the condition of C / N=3 (mass ratio), the Shigella flexneri ( Shigella flexneri CCTCC77). Pick a single colony purified with denitrification solid culture medium and add it to 1L nutrient broth liquid culture medium to make a bacterial solution, which is stored in a constant temperature incubator at 30°C for 24-48h for use. Add 100mL of reaction solution with a C / N mass ratio of 3 (nitrate nitrogen concentration of 108mg / L) prepared with analytically pure potassium nitrate and sodium acetate to a 100mL screw-capped reagent bottle, and then inoculate the above 1L bacterial solution after centrifugation (about 63.8mg) into a screw-capped bottle and culture at 30°C for 10h. Before starting, blow nitrogen into the reactor to make it anaerobic. After the reaction starts, take 5mL of water sample every 2 hours, filter it with a 0.22μm filter head, and detect the concentrations of nitrate nitrogen and nitrite nitrogen. Figure 3 The results show that the denitrification effect of the strain under this condition is that with the passage of time, the concentration of nitrate nitrogen is constantly decreasing, while the concentration of nitrite nitrogen is gradually increasing. The nitrate nitrogen removal rate of the reaction system is constantly increasing, and the removal rate of 10h is 37.48%. The nitrite nitrogen conversion rate reaches 99.5% in 2h, and the highest nitrite nitrogen conversion rate is 100% in 6h, indicating that the removed nitrate nitrogen is almost completely converted into nitrite nitrogen. The above shows that Shigella flexneri ( Shigella flexneri .CCTCC77) can carry out nitrate respiration, effectively removing nitrate nitrogen and converting it into nitrite nitrogen, with a conversion rate of 88.5%~100%.

Claims

1. Shigella flexneri ( Shigella flexneri ), its deposit number is CCTCC NO: M 20241159.

2. The Shigella flexneri according to claim 1 ( Shigella flexneri ), characterized in that: The Shigella flexneri ( Shigella flexneri ) were characterized by nitrate reduction and utilization, mannitol fermentation, catalase and indole production.

3. A bacterial agent comprising the Shigella flexneri according to claim 1 ( Shigella flexneri ).

4. The Shigella flexneri according to claim 1 ( Shigella flexneri ) in short-range denitrification.

5. The use according to claim 1, characterized in that: In the short-range denitrification, the temperature is 30°C±1°C; and the carbon-nitrogen mass ratio is 3:

1.

6. The Shigella flexneri according to claim 1 ( Shigella flexneri ) in the conversion of nitrate nitrogen into nitrite nitrogen.

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