A nitrate-respiring bacterium and its application

Abstract

The present invention discloses a nitrate-respiring bacterium and its application, belonging to the field of energy conservation and environmental protection. The nitrate-respiring bacterium of the present invention is Shigella flexneri Shigella flexneri CCTCC77, which was deposited at the China Center for Type Culture Collection on June 6, 2024, with the deposit number CCTCC NO: M 20241159. The nitrate-respiring bacterium of the present invention was isolated from the process of pig farm wastewater treatment; this strain can in-situ optimize and regulate the denitrification treatment process of high-nitrogen wastewater in aquaculture, improve the conversion efficiency of nitrite and nitrous oxide, and reduce the dosage of exogenous carbon source, sludge production and aeration energy consumption during the wastewater treatment process.

Description

Technical Field

[0001] The present 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 relatively concentrated drainage time, 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, which uses denitrifying bacteria to convert nitrate (NO3 - ) is decomposed into nitrogen (N2). Short-cut denitrification is the first step of the denitrification reaction, which converts nitrate (NO3 - ) is reduced to nitrite (NO2 - ), which can serve as a reaction substrate for anaerobic ammonium oxidation (Anammox). Short-cut denitrification combined with anaerobic ammonium oxidation can form a novel biological denitrification process. Compared with traditional denitrification processes, this new process offers advantages such as conserving 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 the genus Pseudomonas ( Pseudomonas ), Alcaligenes spp. ( Alcaligenes ), Paracoccus ( Paracoccus ) and Bacillus spp. ( Bacillus Most denitrifying bacteria isolated from natural environments for wastewater treatment may have some degree of inadaptability, and research on bacteria and their applications based on in situ acclimation processes is relatively limited. Summary of the Invention

[0005] The purpose of the present invention is to provide a nitrate-respiring bacterium and its application. The nitrate-respiring bacterium of the present invention is isolated from the 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 at the China Center for Type Culture Collection (abbreviated as CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on June 6, 2024, with the deposit number CCTCC NO: M 20241159.

[0007] The above-mentioned Shigella flexneri ( Shigella flexneri ) has the physiological and biochemical characteristics of nitrate reduction utilization, mannitol fermentation, catalase and indole production being positive.

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

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

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

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

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

[0013] The Shigella flexneri Shigella flexneri CCTCC77 can carry out nitrate respiration and convert nitrate nitrogen to nitrite nitrogen.

[0014] The strain of the present invention was domesticated and isolated from pig farm wastewater. It is a Gram-negative bacterium, rod-shaped or bacillus-shaped, with a cell length of 2.0 - 2.5 μm and a width of 1 - 1.2 μm, without a capsule and spores, and can grow by adhering and arranging; after culturing on nutrient broth agar medium for 1 day, the colonies are round, semi-transparent, slightly light yellow, with a colony diameter of 1 - 1.5 mm, and the growth temperature range is 7 - 42°C; the strain of the present invention shows positive indicators of nitrate reduction utilization, mannitol fermentation, catalase, and indole production.

[0015] Description of the deposit of biological material

[0016] Classification and naming of biological material: Shigella flexneri ( Shigella flexneri )

[0017] Strain number of biological material: CCTCC77

[0018] Name of the depositary institution of biological material: China Center for Type Culture Collection

[0019] Abbreviation of the depositary institution for biological materials: CCTCC

[0020] Address of the depositary institution for biological materials: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postcode: 430072

[0021] Deposit date of biological materials: June 6, 2024

[0022] Registration number of the biological materials in the depositary center: CCTCC NO: M 20241159 Description of the drawings

[0023] Figure 1 It is a transmission electron microscope photograph of Shigella flexneri ( Shigella flexneri CCTCC77);

[0024] Figure 2 It is a growth curve of Shigella flexneri ( Shigella flexneri CCTCC77);

[0025] Figure 3 It is a denitrification efficiency diagram of Shigella flexneri ( Shigella flexneri CCTCC77). Specific implementation manners

[0026] The present invention will be further described in detail below in conjunction with specific implementation manners. The examples given are only for clarifying the present invention, rather than limiting the scope of the present invention.

[0027] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0028] In the following examples, quantitative tests are all set with three repeated experiments, and the results are averaged.

[0029] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0030] The composition and concentration of the nutrient broth liquid medium used in the following examples are as follows (g / L): beef extract 5, peptone 10, NaNO2 1; the solvent is water.

[0031] The composition and concentration of the nutrient broth solid medium are as follows (g / L): beef extract 5, peptone 10, NaNO2 1; the solvent is water; 2% agar of the volume of the prepared medium is added.

[0032] Example 1, Shigella flexneri Shigella flexneri Isolation and identification of CCTCC77

[0033] I. Isolation of strains

[0034] 1. Collection of sludge samples

[0035] Collect the activated sludge from the wastewater treatment of the second pig farm in Guangfu Town, Nanchang County, Nanchang City, Jiangxi Province (Collection time: July 2023, Collector: Nie Hanbing, Contact number: 18811371960).

[0036] 2. Isolation and purification of strains

[0037] Put the collected activated sludge from the wastewater treatment of the pig farm into a Sequencing Batch Reactor (SBR) and run it for 17 days. The volume of this reactor is 10L, equipped with a stirrer, and there are 5 equally spaced water outlets on the reactor wall for sampling, draining, feeding, etc. The composition of the influent substances is 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, magnesium chloride 0.1647. The influent and effluent are connected to the outlet of the SBR reactor through a peristaltic pump, and the temperature of the SBR reactor is controlled at 30℃±(1 - 2)℃ by connecting to a water bath through a circulation pump. Subsequently, take the water sample in the reactor (which can be regarded as a bacterial suspension), dilute and inoculate it on the denitrifying solid medium, and culture it in a constant temperature incubator at 37℃ for 2 - 3 days. Select typical colonies with different morphologies and clear colony characteristics and enrich them in the nutrient broth liquid medium for 1 - 2 days, then spread them on the denitrifying solid medium, and repeat the above enrichment and purification steps 2 - 3 times until a single colony culture is finally formed; name this strain CCTCC77.

[0038] Pick the purified single colony and inoculate it into the nutrient broth liquid medium or nutrient broth solid medium for standby.

[0039] The composition and concentration of the above-mentioned denitrifying solid medium are as follows (g / L): ammonium chloride 0.08, potassium nitrate 0.6, sodium acetate 1, dipotassium hydrogen phosphate trihydrate 0.2, calcium chloride dihydrate 0.1, magnesium sulfate heptahydrate 0.04, ferric sulfate dihydrate 0.02, add 1 mL / L of trace element solution A and B respectively. Among them, the composition and concentration of trace element A are as follows (g / L): disodium ethylenediaminetetraacetate 6.39, ferric sulfate heptahydrate 5. The composition and concentration 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.

[0040] II. Shigella flexneri Shigella flexneri Identification of CCTCC77

[0041] (1) Morphological identification

[0042] Select a bacterial strain, streak it on a nutrient broth solid medium, seal it well and incubate it in a constant temperature incubator at 37°C for 1 day, and observe the colony morphology.

[0043] Results of morphological identification: The colonies are round, semi-transparent, white with a slightly yellowish tint, and the colony diameter is 1 - 1.5 mm. The bacterial cells were observed under a transmission electron microscope magnified 15,000 times. The cell length is 2.0 - 2.5 μm, the width is 1 - 1.2 μm, rod-shaped or bacillus-shaped, without a capsule and spores, and can grow attached and arranged. Figure 1 。

[0044] (2) Physiological and biochemical identification

[0045] Referring to the eighth edition of Bergey's Manual of Determinative Bacteriology, the identification result is Shigella flexneri ( Shigella flexneri ), a Gram-negative bacterium, with a growth temperature range of 7 - 42°C and an optimum growth temperature of 25 - 37°C. This bacterium does not grow at 4°C but can grow at 42°C. This strain does not require organic growth factors. Physiological and biochemical index tests such as nitrate reduction utilization, mannitol fermentation, catalase, and indole production were carried out on the strain, and the results are shown in Table 1.

[0046] Table 1 Physiological and biochemical indexes of Shigella flexneri ( Shigella flexneri )

[0047]

[0048] (3) Molecular identification

[0049] Extract the genomic DNA of the strain, perform PCR amplification, recover the amplified product using the AxyPrep DNA Gel Extraction Kit, and then use the sequencer ABI3730-XL for DNA sequencing. The sequencing result is shown as Sequence 1 with a length of 1411. Use the NCBI Blast program to compare the spliced sequence file with the data in the NCBI 16S database. This bacterium belongs to Shigella flexneri. This strain was deposited in the China Center for Type Culture Collection (CCTCC) on June 6, 2024, with the deposit number: CCTCC NO: M 20241159, and the taxonomic name of this strain is Shigella flexneri ( Shigella flexneri ). Sequence 1 is as follows:

[0050]

[0051] Construction process of PCR amplification reaction system:

[0052] 1) Primer design sequence:

[0053] Primer 1 27F: 5′- AGAGTTTGATCCTGGCTCAG-3′

[0054] Primer 2 1492R: 5′- GGTTACCTTGTTACGACTT-3′;

[0055] 2) Establishment of PCR amplification system (50 μL):

[0056] Genomic DNA (20 ng / μL) 1.0 μL

[0057] 10× Buffer (containing 2.5 mM Mg 2+ ) 5.0 μL

[0058] Taq polymerase (5 u / μL) 1.0 μL

[0059] dNTP (10 mM) 1.0 μL

[0060] Primer 1 27F (10 μM) 1.5 μL

[0061] Primer 2 1492R (10 μM) 1.5 μL

[0062] ddH2O 39.0 μL

[0063] Total volume 50.0 μL;

[0064] 3) Setting of PCR amplification reaction program:

[0065] 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, number of cycles is 35 times, and final extension at 72°C for 7 min.

[0066] Example 2, Verification of denitrification function of Shigella flexneri ( Shigella flexneri CCTCC77)

[0067] 1. Determination of growth curve of Shigella flexneri ( Shigella flexneri CCTCC77)

[0068] Activate the cryopreserved strain, dissolve it with nutrient broth liquid medium, then streak and inoculate it on the nutrient broth solid medium, and culture it at 37°C for 24 h. After colonies grow on the plate, pick typical colonies and inoculate them into the nutrient broth liquid medium, and culture at 37°C for 18 h to obtain the primary seed solution. Aliquot and inoculate the primary seed solution into sterile pp tubes at 10% of the original volume. Take the culture solution every 1 h, and use the photoelectric turbidimetry method to measure the OD600 value (Optical Density) of the bacterial solution at a wavelength of 600 nm to obtain the growth curve of Shigella flexneri ( Shigella flexneri CCTCC 77) as shown in Figure 2 . It can be seen from Figure 2 that the growth curve of the secondary seed solution of Shigella flexneri ( Shigella flexneri CCTCC 77) has a relatively short latency period, about 2 h, probably because the activity of this strain is strong, and the culture conditions before and after inoculation are similar, so it can adapt to the new environment in a short time after inoculation. The logarithmic growth phase of Shigella flexneri ( Shigella flexneri CCTCC 77) is about 2 - 4 h, the stationary phase is 4 - 7 h, and the decline phase begins after 7 h.

[0069] 2. Denitrification efficiency determination of Shigella flexneri ( Shigella flexneri CCTCC 77)

[0070] Under the condition of C / N = 3 (mass ratio), explore the denitrification effect of Shigella flexneri ( Shigella flexneri CCTCC 77). Pick a single colony purified by the denitrification solid medium and transfer it to 1 L of nutrient broth liquid medium to make a bacterial solution, and store it in a constant temperature incubator at 30°C for 24 - 48 h for use. Add 100 mL of a reaction solution with a C / N mass ratio of 3 (nitrate nitrogen concentration of 108 mg / L) prepared with analytical pure potassium nitrate and sodium acetate to a 100 mL screw-cap reagent bottle, and then inoculate the cells (about 63.8 mg) after centrifuging the above 1 L of bacterial solution into the screw-cap bottle and culture at 30°C for 10 h. Before starting, blow nitrogen into the reactor to make it in an anaerobic state. After the reaction starts, take 5 mL of water samples every 2 hours, filter them with a 0.22 μm filter head, and detect the nitrate nitrogen and nitrite nitrogen concentrations. Figure 3 shows the denitrification effect of this strain under this condition. The results show that with the passage of time, the nitrate nitrogen concentration is continuously decreasing, and the nitrite nitrogen concentration is gradually increasing. The nitrate nitrogen removal rate of the reaction system is continuously increasing, and the removal rate at 10 h is 37.48%. The nitrite nitrogen conversion rate reaches 99.5% at 2 h and has the highest nitrite nitrogen conversion rate of 100% at 6 h, indicating that the removed nitrate nitrogen is almost completely converted into nitrite nitrogen. From the above, it shows that Shigella flexneri ( Shigella flexneri .CCTCC 77) can carry out nitrate respiration, effectively remove nitrate nitrogen and convert it into nitrite nitrogen, with a conversion rate of 88.5% - 100%.

Claims

1. Shigella flexneri ( Shigella flexneri ), with the preservation number of CCTCC NO: M 20241159.

2. A bacterial agent containing the Shigella flexneri described in claim 1 ( Shigella flexneri ).

3. Use of Shigella flexneri according to claim 1 in Shigella flexneri short-term denitrification.

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

1.

5. Use of Shigella flexneri as claimed in claim 1 ( Shigella flexneri ) in the conversion of nitrate nitrogen to nitrite nitrogen.

Images