Application of a salt-tolerant heterotrophic nitrification-aerobic denitrification bacterium

Abstract

The invention discloses a salt-tolerant heterotrophic nitrification-aerobic denitrification bacterium and its application. The salt-tolerant heterotrophic nitrification-aerobic denitrification bacterium Halomonas elongate 5300 has a preservation number of CGMCC No. 25768. The strain has a wide tolerance range for salt concentration and can grow at a salt concentration of 0% to 26%. When the salt concentration is 4% to 16%, it can efficiently remove NO in water. 2 ‑ ‑N and NH 4 + ‑N, where NO 2 ‑ The removal efficiency of ‑N reaches 100.00%, NH4 + -N exceeds 78.51%. Strain 5300 can effectively solve the problem of high salt inhibiting the activity of functional microorganisms in nitrogen-contaminated water bodies in the previous biological denitrification process, and is suitable for denitrification of nitrogen-contaminated water bodies under high salt and aerobic conditions.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering and environmental engineering, and relates to a salt-tolerant heterotrophic nitrification-aerobic denitrification bacterial strain and application thereof, which is used for biological denitrification of high-salt nitrogen-containing polluted water bodies. Background Art

[0002] With the development of economy, industrial fields such as petrochemical industry, coal chemical industry, fertilizer industry, plastic industry, pharmaceutical industry and dye industry all produce extremely difficult to treat high-salinity wastewater with salinity of about 10%-25%. In addition, coastal cities mostly use seawater for production and life, which has led to a significant increase in the discharge of high-salinity wastewater. High-salinity wastewater is not only rich in organic matter that is difficult to degrade, but also has an increased nitrogen content. When it enters the ecological environment, it will lead to eutrophication of lakes, rivers and other environments, and produce stress and toxic effects on aquatic organisms, thereby endangering human health. Therefore, it is urgent to strengthen the nitrogen treatment of high-salinity wastewater.

[0003] At present, biological methods are the most economical and efficient wastewater treatment, which is green and has no secondary hazards. However, high salt will inhibit the biochemical reaction process of microorganisms and significantly reduce the efficiency of wastewater treatment. So far, most of the heterotrophic nitrification-aerobic denitrification microorganisms that have been discovered cannot stably perform the nitrogen removal function when the salinity exceeds 2%. Under high salt conditions, microbial growth will be inhibited to varying degrees, and the denitrification efficiency will drop sharply. Therefore, screening heterotrophic nitrification-aerobic denitrification strains that can efficiently perform biological denitrification in a high-salt environment has important application value for the denitrification treatment of high-salt nitrogen-containing water bodies. In addition, there are studies on the salt-tolerant heterotrophic nitrification and aerobic denitrification functions of the genus Halomonas, but there is no research on the function of Halomonaselongate as a heterotrophic nitrification-aerobic denitrification bacteria.

[0004] Dong Lingxi. Isolation and denitrification characteristics of a heterotrophic nitrification-aerobic denitrification bacterium Halomonas piezotolerans HN2 [D]. Zhejiang Ocean University, 2022. The salinity tolerance is only 3.0%; Su Zhaopeng, Li Yun, Pan Luqing, etc. Denitrification performance and quantitative detection of a new heterotrophic nitrification-aerobic denitrification bacterium GJWA3 [J]. Journal of Ocean University of China (Natural Science Edition), 2021, 51(10) It was verified that Halomonas meridiana strain SCSIO 43005 showed excellent NH+4-N and NO-2-N removal capabilities under the conditions of pH 7.0-8.5, temperature 25-35℃, C / N 10-20, and salinity 24-40. Ma Hongjing, Liu Ying, Mu Xinting, et al. Isolation, identification and denitrification performance of salt-tolerant and efficient aerobic denitrifying bacteria Halomonas sp.HRL-11[J]. Journal of Dalian Ocean University, 2022, 37(02) It was verified that a strain of Halomonas zhaodongensis could denitrify nitrate nitrogen (NO) under high salinity (31) conditions. - The optimal conditions for the reduction of NO were as follows: shaking speed 150 r / min (initial DO concentration 7.48 mg / L), C / N 10, and temperature 30 °C. Under these conditions, strain HRL-11 was - 3-N was the only nitrogen source. After 48 hours of reaction, its OD 600 nm The value is 1.4, for NO3 - The removal efficiencies of -N, TOC and TN were 91.5%, 69.4% and 65.6%, respectively.

[0005] Chinese patent CN201210458579 discloses a salt pan Halomonas BMEN3 (Halomonas campisalis.BMEN3) with heterotrophic nitrification and aerobic denitrification functions. It still has heterotrophic nitrification and aerobic denitrification capabilities under high salt and high alkaline conditions, but it is mainly used for the treatment of high-salt and nitrogen-containing wastewater and flue gas. It can remove 100 mg / L NH4 in 12 hours at a pH of 9-10 and a salinity of only 4%. + -N and 140mg / L NO3-N.

[0006] Chinese patent CN201410138167.4 discloses a marine halomonas strain HGMN521 with aerobic denitrification ability and its application, which is mainly used to treat seawater contaminated by inorganic nitrogen.

[0007] CN201710421768 Halomonasalkaliphila X3 was screened from the eutrophic sediment environment of the cage fish farming area in Xiangshan Port. -1 In the test solution, after 24 hours, the removal rates of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen were 98.29%, 99.07% and 96.48% respectively, and it can grow under the condition of only 1%-8% NaCl.

[0008] Halomonas lactosivorans CYQ1-6-1 of Chinese patent CN201510315663 has strong tolerance to high concentrations of nitrite and high denitrification efficiency, and can denitrify 1000 mg / L NO2 within 72 hours. - -N is completely removed, however, the present application discovered a new bacterial species, Halomonas elongate, which has not been reported in previous studies to have the function of heterotrophic nitrification-aerobic denitrification bacteria. Summary of the invention

[0009] The purpose of the present invention is to provide a salt-tolerant heterotrophic nitrification-aerobic denitrification bacterium that can efficiently remove nitrogen under high-salt conditions, so as to enrich the current microbial resource library for high-salt nitrogen-containing wastewater treatment.

[0010] The technical solution of the present invention is as follows:

[0011] The application of a salt-tolerant heterotrophic nitrification-aerobic denitrification bacterium, the strain is 5300, which is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration, the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is September 21, 2022, the classification name is Halomonas elongate, the deposit number is CGMCC No.25768, and it is used for biological denitrification of high-salt nitrogen-containing polluted water bodies.

[0012] The heterotrophic nitrification-aerobic denitrification bacteria provided by the present invention are salt-tolerant bacteria, can grow in a salt environment of 0% to 26%, have a good growth state, and have a wider growth range than common salt-tolerant bacteria.

[0013] The salt-tolerant heterotrophic nitrification-aerobic denitrification bacteria of the present invention have biological characteristics: the colony is light yellow and round, and the surface is moist and easy to pick up. The salt-tolerant heterotrophic nitrification-aerobic denitrification bacteria are inoculated into nitrogen-containing high-salt artificial wastewater, and the salt concentration is added to 0% to 24%, the nitrogen source concentration is 20 to 200 mg / L, the inoculation amount is 1% to 5%, different carbon sources (sucrose, glucose, sodium pyruvate, trehalose, inosine, sodium acetate, stachyose), C / N is 1 to 20, pH is 5 to 9, 28 to 40°C, and 0 to 220 r / min to carry out heterotrophic nitrification-aerobic denitrification.

[0014] The suitable salt concentration for denitrification of the strain is 4% to 16%, with NO2 - -N and NH4 + -N is the indicator of the appropriate nitrogen source concentration is 20 ~ 40mg / L and 20 ~ 80mg / L, respectively, with NO2 - -N and NH4 + -N is the index, the suitable inoculation amount is 2% to 4% and 1% to 2% respectively, the suitable carbon source is sodium pyruvate, C / N is 15, pH is 7.0, the culture temperature is 37℃ to 40℃, and the rotation speed is 165 to 220r / min.

[0015] The present invention has the following beneficial effects:

[0016] The heterotrophic nitrification-aerobic denitrification bacteria of the present invention are salt-tolerant bacteria, which can adapt to the growth environment with a salt concentration of 0-26%, and provide bacterial species resources for denitrification treatment of high-salinity wastewater.

[0017] The salt-tolerant heterotrophic nitrification-aerobic denitrification bacteria of the present invention can efficiently carry out heterotrophic nitrification and aerobic denitrification reactions when the salt concentration is 4% to 16%. - -N removal rate is 100%, NH4 + The removal rate of -N is above 78.51%. It can grow under the condition of 4% to 16% salt concentration, using sodium pyruvate as carbon source and NO2 - -N and NH4 + -N is the indicator of the appropriate nitrogen source concentration of 20 ~ 40mg / L and 20 ~ 80mg / L, NO2 - -N and NH4 + -N as the index, the appropriate inoculation amount was 2% to 4% and 1% to 2%, C / N was 15, pH was 7.0 and the rotation speed was 165 to 220 r / min. After 7 days of aerobic culture under the above conditions, the strain was sensitive to NO2 - -N and NH4 + -N has a good denitrification effect, which is of great practical significance for achieving heterotrophic nitrification-aerobic denitrification in a high-salt environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The phylogenetic tree of the 16S rRNA gene of the salt-tolerant heterotrophic nitrification-aerobic denitrifying bacteria 5300 of the present invention.

[0019] Figure 2 Schematic diagram of the effect of salt concentration on the denitrification of the salt-tolerant heterotrophic nitrification-aerobic denitrifying bacteria 5300 of the present invention.

[0020] Figure 3 Schematic diagram of the effect of nitrogen source concentration on the denitrification of the salt-tolerant heterotrophic nitrification-aerobic denitrifying bacteria 5300 of the present invention.

[0021] Figure 4 Schematic diagram of the effect of inoculum dosage on the denitrification of the salt-tolerant heterotrophic nitrification-aerobic denitrifying bacteria 5300 of the present invention.

[0022] Figure 5 Schematic diagram of the effect of different carbon sources on the denitrification of the salt-tolerant heterotrophic nitrification-aerobic denitrification bacteria 5300 of the present invention, wherein 1: sucrose; 2: glucose; 3: sodium pyruvate; 4: trehalose; 5: inosine; 6: sodium acetate; 7: stachyose.

[0023] Figure 6 Schematic diagram of the effect of carbon-nitrogen ratio on the denitrification of the salt-tolerant heterotrophic nitrification-aerobic denitrifying bacteria 5300 of the present invention.

[0024] Figure 7 Schematic diagram of the effect of pH on the denitrification of the salt-tolerant heterotrophic nitrification-aerobic denitrification bacteria 5300 of the present invention.

[0025] Figure 8 Schematic diagram of the effect of temperature on the denitrification of the salt-tolerant heterotrophic nitrification-aerobic denitrification bacteria 5300 of the present invention.

[0026] Fig. 9 Schematic diagram of the effect of rotation speed on the denitrification of the salt-tolerant heterotrophic nitrification-aerobic denitrification bacteria 5300 of the present invention. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with specific implementations, but the protection scope of the present invention is not limited thereto:

[0028] Embodiment 1:

[0029] Screening and identification of a salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacterium.

[0030] Samples were collected from the Qijiaojing Salt Lake in Xinjiang. 5 g of mixed soil sample was added to a conical flask filled with sterile water. After shaking on a shaker for 0.5 h, 1 mL of soil bacterial suspension was drawn into a test tube filled with sterile water and diluted to a bacterial concentration of 10 -1 , 10 -2100 μL of the dilution was taken from each sample and spread on the seawater 2216E medium. The sample was inverted and cultured at 37°C for 1 month. The colonies with different morphological characteristics were streaked and separated for more than 3 times. The colonies were determined to be single colonies after the growth characteristics of the colonies were kept consistent. The single colonies were cultured on the slant medium and stored in a 4°C refrigerator.

[0031] 2216E medium (g / L): peptone 5, yeast extract 1, ferric phosphate 0.1, agar 20, NaCl 80.0, trace element solution 2mL, pH 7.2–7.5. The above trace element solution (g / L): EDTA 50, ZnSO4 2.2, CaCl2 5.5, MnCl2·4H2O2.06, FeSO4·7H2O 5.0, (NH4)6Mo7O 24 ·4H2O 1.1, CuSO4·5H2O 1.6, CoCl2·6H2O 1.61.

[0032] The purified strains were inoculated into BTB medium for initial screening, inverted and cultured at 37°C for 3 days, and the strain that turned blue the fastest and most obviously on the medium was named 5300, stored in 20% glycerol and placed in a -80°C refrigerator for long-term storage.

[0033] BTB medium components (g / L): C6H5Na3O7·2H2O 5.70, KNO3 2.0, K2HPO4 1.0, MgSO4·7H2O0.2, NaCl 80.0, agar 20, 1% bromothymol blue ethanol solution 1 mL, water 1000 mL, pH 7.0.

[0034] The extraction of strain genomic DNA was carried out according to the instructions of the TaKaRa kit (model 9769S). The extracted genomic DNA was used as a template to amplify the bacterial 16S rRNA gene using universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-TACGGTTACCTTGTTACGACTT-3′). The PCR product was sent to Shanghai Biotechnology Co., Ltd. for sequencing after being tested by 1% agarose gel electrophoresis.

[0035] The length of the sequence of strain 5300 was 1372 bp. The strain sequence was uploaded to the NCBI database and compared with the known bacterial 16SrRNA gene sequence results. The results showed that strain 5300 was Halomonas elongata. The strain phylogenetic tree was constructed using MEGA7.0. The results are as follows Figure 1 .

[0036] Embodiment 2:

[0037] The growth of strain 5300 is suitable for a range of salt concentrations.

[0038] The strain 5300 was activated in advance, and a 2216E culture medium with a salt concentration gradient of 0%, 2%, 4%, 6%, 8%, 10%, 12%, 14%, 16%, 18%, 20%, 22%, 24%, 25% and 26% was prepared. The strain was inoculated on culture medium plates with different salt concentrations, and cultured upside down at 37°C for 7 days to observe the growth of the strain under different salt concentrations.

[0039] The results showed that strain 5300 could grow normally at salt concentrations of 0% to 26%, and the optimal growth range was also 0% to 26%. The bacteria had a wide range of salt tolerance and strong adaptability.

[0040] Table 1: Growth of strain 5300 at different salt concentrations

[0041]

[0042] Note: “+++” indicates good growth

[0043] Embodiment 3:

[0044] Suitable salt concentration for denitrification by strain 5300.

[0045] Nitrification medium (g / L): (NH4)2SO4 0.0896, CH3COONa 2.0, K2HPO4 0.5, MgSO4·7H2O0.41, pH=7.0-7.2, and the rest is water.

[0046] Denitrification medium 1 and 2: Replace (NH4)2SO4 0.0896 in the nitrification medium with NaNO2 0.1, and the rest of the formula remains unchanged.

[0047] Treatment method: After the strain is inoculated into liquid nitrification medium, denitrification medium 1 and 2 at 1%, culture at 37℃, 165r / min for 7 days, take samples regularly, and measure NO2--N and NH4 in the liquid + -N removal efficiency.

[0048] The salt concentrations of nitrification medium, denitrification medium 1 and 2 were set to 0, 4, 8, 12, 16, 20 and 24%, and 1% bacterial suspension was inoculated. The other culture conditions remained unchanged and the culture was carried out for 7 days to determine the denitrification capacity.

[0049] The results are as follows Figure 2 As shown in the figure, when the salt concentration is 4% to 16%, the strain can efficiently carry out heterotrophic nitrification and aerobic denitrification reactions, with a NO2--N removal rate of 100% and NH4 + The removal rate of -N is above 78.51%.

[0050] Embodiment 4:

[0051] Effects of different growth conditions on the efficient heterotrophic nitrification-aerobic denitrification characteristics of strain 5300.

[0052] The strain 5300 was activated in advance, and different gradients of nitrogen source concentration (20, 40, 80, 120, 160 and 200 mg / L), inoculation amount (1, 2, 3, 4 and 5%), carbon source (sucrose, glucose, sodium pyruvate, trehalose, inosine, sodium acetate and stachyose), carbon-nitrogen ratio (1, 5, 10, 15 and 20), pH (5, 6, 7, 8 and 9), culture temperature (28, 37 and 40°C) and rotation speed (0, 85, 165 and 220 r / min) were set in sequence. The experimental operation was consistent with Example 3, and samples were taken regularly to determine the denitrification efficiency.

[0053] The results show that ( Figure 3 to Figure 9 ), the strain is sensitive to NO2--N and NH4 + -N optimal removal rate reached 100.00% and 86.63%. - -N and NH4 + -N is the indicator of the appropriate nitrogen source concentration is 20 ~ 40mg / L and 20 ~ 80mg / L, respectively, with NO2 - -N and NH4 + The suitable inoculation amounts with -N as the indicator are 2% to 4% and 1% to 2% respectively, the suitable carbon source is sodium pyruvate, the suitable C / N is 15, the suitable pH is 7, the suitable culture temperature is 37°C to 40°C, and the suitable rotation speed is 165 to 220 r / min.

Claims

1. Use of a salt-tolerant heterotrophic nitrification-aerobic denitrification bacterium, characterized by: The salt-tolerant heterotrophic nitrification-aerobic denitrification bacteria is Halomonas elongate 5300, with a preservation number of CGMCC No. 25768. The bacterium is used for biological denitrification of high-salt nitrogen-containing polluted water bodies, wherein the high salt concentration is 4% to 16%.

2. The use of a salt-tolerant heterotrophic nitrification-aerobic denitrification bacterium according to claim 1, characterized in that: The application includes inoculating Halomonas elongate 5300 into high-salinity nitrogen-contaminated water bodies. The bacteria can remove NO2 in water bodies when the salt concentration is 4% to 16%. - -N and NH4 + -N.

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