Method for efficiently purifying high-salt nitrogen-containing water body

Abstract

The invention relates to a method for efficiently purifying a high-salt nitrogen-containing water body. Mainly relates to a salt-tolerant heterotrophic nitrification-aerobic denitrification bacterium Halomonas ramblica 5505 with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.30683. The strain is mainly used for removing ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in water through assimilation, and the assimilation rates respectively reach 62.68%, 51.08% and 52.66%; meanwhile, part of inorganic nitrogen can be dissimilated into nitrogen-containing gas, and the final nitrogen removal efficiency reaches 100%, 100% and 74.4% respectively. The removal rate of the strain on ammonia nitrogen and nitrate nitrogen within 12 hours in simulated nitrogen-containing wastewater reaches 90% or above, and accumulated nitrite nitrogen can be completely removed by increasing the carbon-nitrogen ratio. The strain can effectively solve the problem of inhibition of high-salt nitrogen-containing wastewater on biological nitrogen removal function microorganisms, the nitrogen removal process is green and efficient, energy consumption is low, and the strain can be applied to ecological management and restoration of high-salt nitrogen polluted water.

Description

Technical Field

[0001] The invention belongs to the field of bioengineering and environmental engineering, and relates to the application of a salt-tolerant heterotrophic nitrification-aerobic denitrification bacterium, and specifically to the denitrification of high-salt nitrogen-containing wastewater by the salt-tolerant heterotrophic nitrification-aerobic denitrification bacteria. Background Art

[0002] Human activities have input high loads of nitrogen into the ecosystem, causing nitrogen imbalance in the environmental system, which has led to many environmental problems such as eutrophication of water bodies, algae blooms, and reduced biodiversity, which seriously threaten human survival and health. How to remove nitrogen has become the number one problem in water pollution control.

[0003] In addition to the increasingly serious nitrogen pollution problem in water bodies, the salinity of sewage has also increased significantly. Coastal areas use seawater for production and life, and the salinity of marine aquaculture wastewater and offshore urban domestic sewage is generally around 3%. More wastewater from the chemical industry, such as the petrochemical industry, coal chemical industry, fertilizer industry, plastic industry, pharmaceutical industry, dye industry, and rubber industry, has a salinity of about 10%-25%, which is currently one of the most difficult wastewaters to treat.

[0004] Biological denitrification technology has gradually become the mainstream technology for treating nitrogen-containing wastewater because of its cleanliness, high efficiency and low energy consumption. At present, most heterotrophic nitrification-aerobic denitrification microorganisms cannot stably perform denitrification functions in an environment with a salt concentration of more than 2%. In a high-salt environment, the microbial osmotic pressure is unbalanced, the biological enzyme activity is inhibited, and the microbial growth is hindered, which limits the application of heterotrophic nitrification-aerobic denitrification microorganisms in treating high-salt wastewater.

[0005] Meng Xin et al., Research on the treatment of high-salinity wastewater by salt-tolerant bacteria Halomonas alimentaria NY [J]. Chemical Engineering Management, 2023(23): 61-64+86. The optimal growth temperature and salinity are 30°C and 4%, respectively. When the initial nitrogen concentration is 100-250 mg / L, the bacteria can convert nitrate into nitrite within 24 hours and has no nitrite removal ability. Shang Jia. Identification and salt tolerance mechanism analysis of four new species of Halomonas [D]. Inner Mongolia University, 2023. The optimal salt concentration of the four new species of Halomonas is no more than 5%. Song Kunnan, Heterotrophic nitrification-aerobic denitrification mechanism of Halomonas CD01 under high-salinity conditions [D]. Chongqing University, 2022.NH 4 + -N removal rate is only 76.0+6.5%, NO 2 --N removal efficiency was only 78.7±2.9%, NO 3--N removal rate is only 76.0+3.7%. Tang Jing et al., Isolation and identification of a salt-tolerant aerobic denitrifying bacterium and its denitrification characteristics [D]. Journal of Environmental Engineering, 2014.12.05, the strain can only grow and denitrify well in a culture medium with a salinity of 3%-7%, and the optimal salinity is 3% (in terms of NaCl). The strain can use NO 3 --N denitrification, NO was denitrified in a denitrification medium with a salinity of 3% within 24 hours. 3 --N removal rate is only 92.6%, and it takes 36 hours to completely remove it. Zhang Peiyu et al., Isolation and identification of a mildly halophilic denitrifying bacterium and preliminary study on its denitrification characteristics [D]. Microbiology Bulletin, 2009.04.20 Halomonas sp. This strain can only grow in a culture medium with a salinity of 10%, with an optimal salinity of 2% to 7%, an optimal pH of 7.5 to 8.5, and the best carbon source is sucrose. The denitrification efficiency is more than 80% in the temperature range of 25℃ to 30℃.

[0006] Chinese patent CN202410252922 A salt-tolerant aerobic denitrifying bacterium DWK9 and its application, although its salt tolerance range is 0% to 35%, the actual salinity range is 3 to 14%. The nitrification ability is also lower than the strain of this application. Chinese patent CN202210719750 A moderately halophilic bacterium with high-salt wastewater assimilation and denitrification function and its application, moderately halophilic, with a salinity of only 20-60g / L NaCl.

[0007] Therefore, screening heterotrophic nitrification and aerobic denitrification strains that can efficiently and stably remove nitrogen in a high-salt environment has important application value for treating high-salt nitrogen-containing water bodies. At present, there have been studies on the salt-tolerant heterotrophic nitrification-aerobic denitrification function of Halomonas genus, but there is no research on the related function of Halomonas ramblicola. Summary of the invention

[0008] The invention provides a method for efficiently purifying a high-salt nitrogen-containing water body. The salt-tolerant heterotrophic nitrification-aerobic denitrification bacteria are Halomonas ramblicola 5505, which are deposited in the General Microbiological Center of China National Committee for Microbiological Culture Collection, with a deposit address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, a deposit number of CGMCC No. 30683, and a deposit date of 2024-05-17.

[0009] The salt-tolerant heterotrophic nitrification-aerobic denitrification bacteria of the present invention provide denitrification efficiency for high-salt nitrogen-containing wastewater, with a growth salt concentration range of 0%-25%, an optimum salt concentration range of 3%-8%, and optimum denitrification conditions: ammonia nitrogen, nitrate nitrogen and nitrite nitrogen concentrations are all 100 mg / L, C / N is 8-16, the carbon source is sodium citrate and sodium pyruvate, pH is 7-8, the rotation speed is 200 r / min, and the temperature is 30-37°C.

[0010] The salt-tolerant heterotrophic nitrification-aerobic denitrification bacteria of the present invention have biological characteristics and physiological and biochemical indicators: the colony is milky white, with round protrusions, regular edges, moist and smooth surface, and opaque. It is identified as a Gram-negative bacterium, has nitrate reduction ability, can hydrolyze starch and Tween-40 and Tween-60; can utilize gluconate, malic acid and a small amount of citric acid; catalase reaction is positive, and oxidase result is weakly positive. The results of the antimicrobial drug experiment show that the strain is insensitive to penicillin and erythromycin, and is sensitive to piperacillin, imipenem, ceftriaxone ciprofloxacin, chloramphenicol, tetracycline, rifampicin and other drugs.

[0011] The beneficial effects of the present invention are:

[0012] 1. The bacteria can grow normally in the salt concentration range of 0%-25%, and the optimal growth salt concentration range is 3%-8%. The optimal nitrogen source, carbon source, carbon-nitrogen ratio, rotation speed, temperature, and pH for nitrogen removal of the strain were screened, and the relationship between nitrogen removal effect and growth amount was explored. Under alkaline conditions of pH 7-8, it can achieve efficient removal of inorganic nitrogen in water, with ammonia nitrogen and nitrate nitrogen removal rates of 100%, and nitrite nitrogen removal rate of 74.4%.

[0013] 2. Under mixed nitrogen source conditions, the strain preferentially utilizes ammonia nitrogen, and its heterotrophic nitrification ability is superior to its aerobic denitrification ability, which is beneficial to the reuse of wastewater after nitrogen removal. The strain mainly removes ammonia nitrogen, nitrate nitrogen and nitrite nitrogen in water by assimilation. The nitrogen removal mechanism of the strain was studied, and the assimilation rates reached 62.68%, 51.08% and 52.66% respectively; at the same time, it can dissimilate part of inorganic nitrogen into nitrogen-containing gas. The removal rate of soluble total nitrogen is as high as 87.89%.

[0014] 3. The strain can remove more than 90% of ammonia nitrogen and nitrate nitrogen in simulated nitrogen-containing wastewater within 12 hours. Improving the carbon-nitrogen ratio can completely remove the accumulated nitrite nitrogen. The strain can effectively solve the problem of high-salt nitrogen-containing wastewater inhibiting biological denitrification microorganisms. Its nitrogen removal process is green and efficient, with low energy consumption, and can be applied to the ecological management and restoration of high-salt nitrogen-polluted water bodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Phylogenetic tree of strain 5505 constructed based on bacterial 16S rRNA gene sequence homology.

[0016] Figure 2 Effects of strain 5505 on the removal efficiency and growth of ammonia nitrogen, nitrate nitrogen and nitrite under different conditions:

[0017] abc: effects of salt concentration on the removal effect and growth of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen by strain 5505; def: effects of nitrogen source concentration on the removal effect and growth of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen by strain 5505; ghi: effects of carbon source type on the removal effect and growth of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen by strain 5505; jkl: effects of carbon-nitrogen ratio on the removal effect and growth of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen by strain 5505; mno: effects of rotation speed on the removal effect and growth of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen by strain 5505; pqr: effects of pH on the removal effect and growth of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen by strain 5505; stu: effects of temperature on the removal effect and growth of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen by strain 5505.

[0018] Figure 3 Effects of different nitrogen source combinations on the denitrification performance of the strain. af corresponds to NO 3 --NNH 4 + -N, NO 2 --N, NO 3 --N and NO 2 --N, NO 3 --N and NH 4 + -N, NO 2 --N and NH 4 + -N,NO 3 --N and NO 2 --N.

[0019] Figure 4 Effect of simulated nitrogen-containing wastewater on the deammonification performance of strains.

[0020] Figure 5 Effect of compound nitrogen sources on strain growth density. DETAILED DESCRIPTION

[0021] Example 1: Screening and identification of a salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacterium 5505

[0022] Screening of bacterial strains and self-built halophilic bacterial strain library in the laboratory

[0023] Use 2216E medium for activation and purification of strains. Inoculate the purified strains in BTB medium for initial screening and incubate at 37°C for 3 days. The strain that turns blue fastest and most obviously on the medium is named 5505, stored in 20% glycerol, and placed in a -80°C refrigerator for long-term storage.

[0024] 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, ZnSO 4 2.2, CaCl 2 5.5, MnCl 2 '4H 2 O2.06, FeSO 4 7H 2 O5.0, (NH 4 ) 6 Mo 7 O 24 ·4H 2 O1.1, CuSO 4 ·5H 2 O1.6, CoCl 2 6H 2 O1.61.

[0025] BTB medium composition (g / L): C 6 H 5 Na 3 O 7 ·2H 2 O 5.70, KNO 3 2.0,K 2 HPO 4 1.0, MgSO 4 '7H 2 O0.2, NaCl 80.0, agar 20, 1% bromothymol blue ethanol solution 1mL, water 1000mL, pH 7.0.

[0026] The bacterial genomic DNA was extracted using the Takara kit, and the extracted gene DNA was used as a template for PCR amplification using 16S rDNA amplification primers: 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGTTACCTTGTTACGACTT-3'), and the PCR product was sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing. The sequencing results were submitted to the NCBI database for homology comparison, and the related sequences with higher homology were selected to construct a phylogenetic tree using the Neighbor-joining method of MEGA 11 software.

[0027] Example 2: Analysis of salt tolerance of strain 5505

[0028] Select R2A basal culture medium to prepare solid culture media with salt concentration gradients of 0%, 1%, 3%, 5%, 7%, 9%, 10%, 12%, 15%, 18%, 20%, 22%, and 25%, and observe the growth on solid culture media with different salt concentrations.

[0029] Table 1 Growth of strain 5505 on medium with different salt concentrations

[0030]

[0031] -: no growth; +: some growth; ++: a small amount of growth; +++: a lot of growth

[0032] Liquid culture media with salt concentrations of 0%, 3%, 5%, 8%, 10%, 15%, and 20% were prepared respectively, and the activated bacterial suspension was inoculated into the liquid culture medium at 2% (v / v), and placed in a constant temperature shaker at 37°C and 180r / min. Samples were taken at regular intervals of 2h to determine the OD600 value of the bacterial liquid, and a strain growth curve was established.

[0033] R2A basic medium (g / L): 0.25% tryptone, 0.5% acid hydrolyzed casein, 0.5% yeast extract powder, 0.5% soluble starch, 0.5% K 2 HPO 4 0.3,MgSO 4 7H 2 O0.1, sodium pyruvate 0.3, peptone 0.25, glucose 0.5, NaCl 50.0, agar 20.0, pH adjusted to 7.0-7.2.

[0034] Example 3: Physiological and biochemical characteristics of strain 5505

[0035] With reference to the Manual of Common Bacterial Identification, the strains were subjected to the following tests: enzymatic assay (oxidase, urease, catalase); hydrolysis test (cellulose, starch, chitin, Tween basal medium added with 1%); acid production test (Mérieux API 20NE); and 11 conventional antibiotic resistance tests using antimicrobial drug sensitivity papers.

[0036] The results showed that strain 5505 was a Gram-negative bacterium with nitrate reduction ability, able to hydrolyze starch, Tween-40 and Tween-60, able to utilize gluconate, malic acid and a small amount of citric acid, positive for catalase reaction and weakly positive for oxidase. The results of antimicrobial drug experiments showed that the strain was insensitive to penicillin and erythromycin, but sensitive to piperacillin, imipenem, ceftriaxone, ciprofloxacin, chloramphenicol, tetracycline, rifampicin and other drugs.

[0037] Embodiment 4:

[0038] Verification of efficient denitrification conditions

[0039] Denitrification liquid medium (g / L): MgSO 4 7H 2 O0.10,K 2 HPO 4 ·3H 2 O6.00, KH 2 PO 4 1.50, NaCl 50.00, trace elements 2.00 mL, pH 7.0-7.2, carbon source and nitrogen source are added according to experimental requirements.

[0040] Treatment method: The initial conditions of the experiment were based on sodium acetate as the carbon source, NO 3 --N, NO 2 --N, NH 4 + -N mass concentration was 100 mg / L, C / N (mass ratio) was 16:1, pH was 7.0, 2 groups of blank controls and 3 groups of parallel experiments were set up, and the inoculation amount was 2% (v / v). The single-factor control variable method was used to explore the effects of different environmental factors on the aerobic denitrification performance of the strain. The strains were cultured under different salt concentrations (0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15% and 20%), nitrogen source content (100, 200, 300, 400, 500 and 1000 mg / L), C / N (mass ratio) (2:1, 4:1, 8:1, 16:1, 32:1), carbon source type (sodium acetate, sodium citrate, sodium pyruvate, glucose, glycerol), pH (6, 7, 8), speed (0, 120, 165, 200 r / min), temperature (20℃, 30℃, 37℃). The above experiments were carried out in sequence to determine the optimal nitrogen removal conditions. The culture was shaken at 37℃ and 165 r / min for 72 h. The bacterial solution was centrifuged at 4000 r / min for 5 min every 24 h, and the supernatant was taken to determine the NO in the solution. 3 -N, NO 2 -N and NH4 + -N concentration was used to investigate the effects of different factors on the nitrogen removal ability of the target strain.

[0041] The results showed that strain 5505 can achieve efficient removal of three substrates, ammonia nitrogen, nitrate nitrogen and nitrite nitrogen, under high salt conditions, with the best nitrogen removal efficiencies reaching 100%, 100% and 74.4% respectively. The optimum denitrification salt concentration range is 3%-8%, the nitrogen source concentration is 100mg / L, the suitable carbon sources are sodium citrate and sodium pyruvate, the suitable carbon-nitrogen ratio is 8-16, the suitable speed is 200r / min, the suitable pH is 7-8, and the suitable temperature is 30℃-37℃.

[0042] Table 2 Effect of nitrogen concentration on denitrification efficiency of strain 5505

[0043]

[0044]

[0045] Embodiment 5:

[0046] Verification of denitrification effect of strain 5505 under optimal culture conditions

[0047] According to the single factor experimental results, NO 3 --N, NO 2 --N, NH 4 + -N denitrification culture medium was placed in the optimal culture conditions for cultivation, samples were taken to determine the residual nitrogen concentration in the culture medium, and the nitrogen removal efficiency was calculated.

[0048] The results showed that under the optimal culture conditions, the denitrification efficiency of strain 5505 for ammonia nitrogen and nitrate nitrogen could reach 100%, and the denitrification efficiency for nitrite nitrogen was 74%.

[0049] Table 3 Denitrification efficiency of strain 5505 under optimal denitrification conditions

[0050]

[0051] Embodiment 6:

[0052] Verification of denitrification ability of strains under compound nitrogen source conditions

[0053] Using different nitrogen source combinations (NO 3 --N and NO 2 --N; NO 3 --N and NH 4 + -N;NO 2 --N and NH 4 + -N; and NH 4 + -N and NO 3 --N and NO 2--N) mode, the initial nitrogen concentration of the nitrogen source was maintained at 100 mg / L, and the other conditions were maintained at the optimal experimental conditions. The nitrogen concentration in the culture medium was measured every 24 hours to determine the effect of different types of nitrogen source combinations on the denitrification efficiency of the strain.

[0054] The results showed that strain 5505 preferentially used ammonia nitrogen under the conditions of multiple nitrogen sources and completed the removal of ammonia nitrogen within 12 hours; when ammonia nitrogen was present in the composite nitrogen source, the removal efficiency of nitrate nitrogen and nitrite nitrogen by the strain was improved. Compared with the single nitrogen source condition, ammonia nitrogen could significantly (P<0.05) increase the cell density of the strain in the composite nitrogen source nitrogen removal. Adding a carbon source to the culture medium could remove the accumulated nitrite.

[0055] Example 7: Verification of denitrification ability of strains under simulated wastewater conditions

[0056] Configure the initial nitrogen concentration to 100 mg / L containing NO 3 --N, NO 2 --N, NH 4 + -N simulated nitrogen-containing wastewater, and samples were taken every 24 hours to determine the remaining nitrogen concentration in the culture medium. Effect of simulated wastewater on the denitrification efficiency of the strain.

[0057] The results showed that strain 5505 3 --N, NO 2 --N, NH 4 + -N in wastewater, the removal rate of ammonia nitrogen and nitrate nitrogen can reach more than 90% within 12 hours, nitrite nitrogen accumulates within 12 hours, and ammonia nitrogen and nitrate nitrogen can be completely removed after 24 hours. Increasing the carbon-nitrogen ratio or adding carbon source to the culture medium can completely remove the accumulated nitrite.

[0058] Embodiment 8:

[0059] Strain 5505 was inoculated into NO 3 --N, NO 2 --N and NH 4 + -N denitrification liquid medium, respectively, to determine NO 3 --N, NO 2 --N and NH 4 + The nitrogen balance relationship was established by comparing the removal rate of -N and the dissolved total nitrogen (DTN) and total nitrogen (TN) in the bacterial solution.

[0060] The results showed that strain 5505 had a preference for ammonia nitrogen, as its heterotrophic nitrification ability was better than its aerobic denitrification ability. 5505 mainly removed inorganic nitrogen in water through assimilation, with an ammonia nitrogen assimilation rate of 62.68%, a nitrate nitrogen assimilation rate of 51.08%, and a nitrite nitrogen assimilation rate of 52.66%. The nitrogen assimilation ability was better than the dissimilation ability.

[0061] Table 4 Nitrogen balance analysis during denitrification

[0062]

[0063] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for efficiently purifying high-salinity nitrogen-containing water, characterized in that: Salt-tolerant heterotrophic nitrification-aerobic denitrification bacteria are used. The salt-tolerant heterotrophic nitrification-aerobic denitrification bacteria are Halomonas ramblicola5505, which are deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No.30683, and the deposit date is 2024-05-17. The optimal salt concentration, nitrogen source, carbon source, carbon-nitrogen ratio, rotation speed, temperature, and pH are screened.

2. A method for efficiently purifying high-salinity nitrogen-containing water according to claim 1, characterized in that: The method treats ammonia nitrogen, nitrate nitrogen or nitrite nitrogen in sewage with a salt concentration of 0%-25%.

3. A method for efficiently purifying high-salinity nitrogen-containing water according to claim 1, characterized in that: The optimum salt concentration range is 3%-8%.

4. A method for efficiently purifying high-salinity nitrogen-containing water according to claim 1, characterized in that: The optimum denitrification conditions include ammonia nitrogen, nitrate nitrogen and nitrite nitrogen concentrations of 100 mg / L.

5. The method for efficiently purifying high-salinity nitrogen-containing water according to claim 1, characterized in that: The C / N is 8-16.

6. The method for efficiently purifying high-salinity nitrogen-containing water according to claim 1, characterized in that: The carbon sources are sodium citrate and sodium pyruvate.

7. The method for efficiently purifying high-salinity nitrogen-containing water according to claim 1, characterized in that: The reaction pH is 7-8.

8. The method for efficiently purifying high-salinity nitrogen-containing water according to claim 1, characterized in that: The rotating speed is 200r / min.

9. The method for efficiently purifying high-salinity nitrogen-containing water according to claim 1, characterized in that: The temperature is 30-37°C.

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