Application of a halomonas sp. strain with high-efficiency salt-tolerant denitrification

Abstract

This invention relates to the application of a highly efficient halophilic denitrifying bacterium of the genus *Halomonas*. The strain is *Halomonas ramblicola* 5505, a salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacterium, with accession number CGMCC No. 30683. The strain primarily removes ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen from water through assimilation, achieving assimilation rates of 62.68%, 51.08%, and 52.66%, respectively; it can also dissimilate some inorganic nitrogen into nitrogen-containing gases. In simulated nitrogen-containing wastewater, the strain achieved a removal rate of over 90% for ammonia nitrogen and nitrate nitrogen within 12 hours, and increasing the carbon-to-nitrogen ratio completely removed accumulated nitrite nitrogen. This strain effectively solves the problem of inhibition of biological denitrification microorganisms by high-salt nitrogen-containing wastewater. Its nitrogen removal process is green, efficient, and energy-saving, and can be applied to the ecological treatment and restoration of water bodies polluted by high salt and nitrogen.

Description

Technical Field

[0001] This invention belongs to the fields of bioengineering and environmental engineering, and relates to the application of a highly efficient halophilic denitrifying bacterium of the genus Halomonas, specifically to the denitrification of high-salt nitrogen-containing wastewater by salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacteria. Background Technology

[0002] Human activities have introduced high levels of nitrogen into ecosystems, causing nitrogen imbalances in the environment and leading to numerous environmental problems, such as eutrophication, algal blooms, and reduced biodiversity. These issues seriously threaten human survival and health, making nitrogen removal the number one challenge in water pollution control.

[0003] In addition to the increasingly serious problem of nitrogen pollution in water bodies, the salinity of wastewater has also increased significantly. Coastal areas use seawater for production and daily life, and the salinity of mariculture wastewater and domestic sewage from near-shore cities is generally around 3%. More wastewater comes from chemical industries, such as petrochemical, coal chemical, fertilizer, plastics, pharmaceutical, dye, and rubber industries, with salinity reaching around 10%-25%, making it one of the most difficult wastewaters to treat.

[0004] Biological denitrification technology has gradually become the mainstream technology for treating nitrogen-containing wastewater due to its cleanliness, high efficiency, and low energy consumption. Currently, most heterotrophic nitrifying-aerobic denitrifying microorganisms cannot stably perform their denitrification function in environments with a salt concentration exceeding 2%. Under high-salt conditions, the osmotic pressure of microorganisms is imbalanced, the activity of biological enzymes is inhibited, and the growth of microorganisms is hindered, limiting the application of heterotrophic nitrifying-aerobic denitrifying microorganisms in the treatment of high-salt wastewater.

[0005] Meng Xin et al. Study on the treatment of high-salt wastewater by halophilic bacterium Halomonas alimentaria NY [J]. Chemical Industry Management, 2023(23): 61-64+86. The optimal growth temperature and salinity are 30℃ and 4%, respectively. When the initial nitrogen concentration is 100-250 mg / L, this bacterium can convert nitrate to nitrite within 24 h, but 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 does not exceed 5%. Song Kunnan. Heterotrophic nitrification-aerobic denitrification mechanism of Halomonas CD01 under high salt conditions [D]. Chongqing University, 2022. NH4 + -N removal rate was only 76.0±6.5%, NO2 - -N removal rate was only 78.7±2.9%, NO3 --N removal rate was 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 medium with a salinity of 3%-7%, and the optimal salinity is 3% (calculated as NaCl). This strain can utilize NO3- - -N undergoes denitrification, and within 24 hours in a denitrification medium with a salinity of 3%, NO3- is reduced. - The removal rate of -N was only 92.6%, and it took 36 hours to remove it almost completely. Zhang Peiyu et al., Isolation, Identification and Preliminary Study on Denitrification Characteristics of a Mildly Halophilic Denitrifying Bacterium [D]. Bulletin of Microbiology, 2009.04.20. Halomonas (Halomonas sp.). This strain can only grow in 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 being sucrose. The nitrogen removal efficiency reaches more than 80% in the temperature range of 25℃ to 30℃.

[0006] Chinese patent CN202410252922 describes a salt-tolerant aerobic denitrifying bacterium, DWK9, and its application. While its salt tolerance range is 0%–35%, its actual salinity range is 3–14. Its nitrification capacity is also lower than that of the strain described in this application. Chinese patent CN202210719750 describes a moderately halophilic bacterium with assimilation and denitrification functions in high-salt wastewater and its application. This bacterium is moderately halophilic, with a salinity of only 20-60 g / L NaCl.

[0007] Therefore, screening for heterotrophic nitrification-aerobic denitrification strains capable of efficient and stable nitrogen removal under high-salt environments is of significant application value for treating high-salt nitrogen-containing water bodies. Currently, there are reports on the salt-tolerant heterotrophic nitrification-aerobic denitrification functions of the *Halomonas* genus, but no studies have been conducted on the related functions of *Halomonas ramblicola*. Summary of the Invention

[0008] This invention provides the application of a highly efficient halophilic denitrifying bacterium of the genus Halomonas. The halophilic heterotrophic nitrifying-aerobic denitrifying bacterium is Halomonas ramblicola 5505, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 30683 and deposit date of 2024-05-17.

[0009] This invention provides denitrification efficiency for high-salt nitrogen-containing wastewater using salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacteria. The growth salt concentration range is 0%-25%, with an optimal salt concentration range of 3%-8%. The optimal denitrification conditions are: ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen concentrations are all 100 mg / L, C / N ratio is 8-16, carbon sources are sodium citrate and sodium pyruvate, pH is 7-8, rotation speed is 200 r / min, and temperature is 30-37℃.

[0010] This invention relates to salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacteria, characterized by the following biological features and physiological and biochemical indicators: colonies are milky white, round and raised, with regular edges, a moist and smooth surface, and are opaque. It has been identified as a Gram-negative bacterium with nitrate-reducing ability, capable of hydrolyzing starch and Tween-40 and Tween-60; it can utilize gluconate, malic acid, and a small amount of citric acid; it is positive for catalase and weakly positive for oxidase. Antimicrobial drug experiments show that the strain is not sensitive to penicillin and erythromycin, but sensitive to piperacillin, imipenem, ceftriaxone, ciprofloxacin, chloramphenicol, tetracycline, rifampin, and other drugs.

[0011] The beneficial effects of this invention are:

[0012] 1. This bacterium can grow normally in a salt concentration range of 0%-25%, with an optimal salt concentration range of 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 efficiency and growth rate was investigated. Under slightly alkaline conditions (pH 7-8), it can achieve highly efficient removal of inorganic nitrogen in water, with ammonia and nitrate nitrogen removal rates of 100% and nitrite nitrogen removal rates of 74.4%.

[0013] 2. Under mixed nitrogen source conditions, this strain preferentially utilizes ammonia nitrogen, exhibiting superior heterotrophic nitrification compared to aerobic denitrification, which facilitates the reuse of wastewater after nitrogen removal for aquatic aquaculture. The strain primarily removes ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen from water through assimilation. The nitrogen removal mechanism was investigated, with assimilation rates reaching 62.68%, 51.08%, and 52.66%, respectively. Simultaneously, it can dissimilate some inorganic nitrogen into nitrogen-containing gases. The removal rate of soluble total nitrogen is as high as 87.89%.

[0014] 3. This strain achieved a removal rate of over 90% for ammonia nitrogen and nitrate nitrogen in simulated nitrogen-containing wastewater within 12 hours. Increasing the carbon-to-nitrogen ratio can completely remove accumulated nitrite nitrogen. This strain can effectively solve the problem of inhibition of biological denitrification microorganisms by high-salt nitrogen-containing wastewater. Its nitrogen removal process is green, efficient, and energy-saving, and can be applied to the ecological treatment and restoration of high-salt nitrogen-polluted water bodies. Attached Figure Description

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

[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: Effect of salt concentration on the removal efficiency and growth rate of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen by strain 5505; d ef: Effect of nitrogen source concentration on the removal efficiency and growth rate of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen by strain 5505; ghi: Effect of carbon source type on the removal efficiency and growth rate of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen by strain 5505; j k1: Effect of carbon-to-nitrogen ratio on the removal efficiency and growth rate of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen by strain 5505; m n0: Effect of rotation speed on the removal efficiency and growth rate of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen by strain 5505; pq r: Effect of pH on the removal efficiency and growth rate of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen by strain 5505; stu: Effect of temperature on the removal efficiency and growth rate of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen by strain 5505.

[0018] Figure 3 The effect of different nitrogen source combinations on the nitrogen removal performance of the strain. af correspond to NO3-, respectively. - -NNH4 + -N, NO2 - -N, NO3 - -N and NO2 - -N, NO3 - -N and NH4 + -N, NO2 - -N and NH4 + -N, NO3 - -N and NO2 - N.

[0019] Figure 4 The effect of simulated nitrogen-containing wastewater on the ammonia removal performance of the bacterial strain.

[0020] Figure 5 The effect of compound nitrogen sources on the growth density of bacterial strains. Detailed Implementation

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

[0022] Strain screening was conducted using a self-built halophilic bacteria strain bank in the laboratory.

[0023] 2216E medium was used for the activation and purification of the strain. The purified strain was inoculated into BTB medium for initial screening, inverted and cultured at 37℃ for 3 days. The strain that turned blue fastest and most obviously on the medium was named 5505, stored in 20% glycerol, and stored in a -80℃ refrigerator for long-term preservation.

[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 trace element solution (g / L) is: EDTA 50, ZnSO4 2.2, CaCl2 5.5, MnCl2·4H2O 2.06, FeSO4·7H2O 5.0, (NH4)6Mo 7O 24 ·4H2O1.1, CuSO4·5H2O1.6, CoCl2·6H2O1.61.

[0025] BTB medium composition (g / L): C6H5Na3O7·2H2O 5.70, KNO3 2.0, K2HPO4 1.0, MgSO4·7H2O 0.2, NaCl 80.0, agar 20, 1% bromothymol blue ethanol solution 1 mL, water 1000 mL, pH 7.0.

[0026] Bacterial genomic DNA was extracted using a Takara kit. Using the extracted genomic DNA as a template, PCR amplification was performed using 16S rDNA amplification primers: 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGTTACCTTGTTACGACTT-3'). The PCR products were sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequencing results were submitted to the NCBI database for homology comparison. Phylogenetic trees were constructed using the Neighbor-J oining method with high homology sequences selected through MEGA 11 software.

[0027] Example 2: Salt tolerance analysis of strain 5505

[0028] R2A basal medium was used 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 the growth on solid culture media with different salt concentrations was observed.

[0029] Table 1. Growth of strain 5505 on culture media with different salt concentrations.

[0030]

[0031] -: No growth; +: Growth; ++: Slight growth; +++: Significant growth

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

[0033] R2A basal medium (g / L): tryptone 0.25, acid-hydrolyzed casein 0.5, yeast extract 0.5, soluble starch 0.5, K2HPO4 0.3, MgSO4·7H2O 0.1, 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: Examination of the physiological and biochemical characteristics of strain 5505

[0035] Referring to the "Handbook for Identification of Common Bacteria", the following tests were performed on the strain: enzymatic assays (oxidase, urease, catalase); hydrolysis experiments (cellulose, starch, chitin, and Tween basal medium with 1% added); acid production experiments (bioMérieux API 20NE); and resistance tests to 11 common antibiotics were conducted using antimicrobial susceptibility testing discs.

[0036] The results showed that strain 5505 is a Gram-negative bacterium with nitrate-reducing ability, capable of hydrolyzing starch and Tween-40 and Tween-60; it can utilize gluconate, malic acid, and can utilize a small amount of citric acid; it is positive for catalase and weakly positive for oxidase. Antimicrobial drug tests showed that the strain was not sensitive to penicillin and erythromycin, but sensitive to piperacillin, imipenem, ceftriaxone, ciprofloxacin, chloramphenicol, tetracycline, rifampin, and other drugs.

[0037] Example 4:

[0038] Verification of efficient nitrogen removal conditions

[0039] Denitrification liquid culture medium (g / L): MgSO4·7H2O 0.10, K2HPO4·3H2O 6.00, KH2PO4 1.50, NaCl 50.00, trace elements 2.00mL, pH 7.0-7.2, carbon source and nitrogen source added according to experimental requirements.

[0040] Treatment method: The initial experimental conditions used sodium acetate as the basic carbon source, and NO3... - -N, NO2 - -N, NH4 +The concentrations of nitrogen (N) were 100 mg / L, the C / N ratio was 16:1, the pH was 7.0, two blank control groups and three parallel experiments were set up, and the inoculum size was 2% (v / v). The effects of different environmental factors on the denitrification performance of the strain were investigated using a single-factor controlled variable method. The strain was subjected to experiments in sequence at 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, and 32:1), carbon source type (sodium acetate, sodium citrate, sodium pyruvate, glucose, and glycerol), pH (6, 7, and 8), rotation speed (0, 120, 165, and 200 r / min), and temperature (20℃, 30℃, and 37℃) to determine the optimal nitrogen removal conditions. The strain was cultured at 37℃ and 165 r / min with shaking for 72 h. Every 24 h, the bacterial culture was centrifuged at 4000 r / min for 5 min, and the supernatant was used to determine the NO3 content in the solution. - -N, NO2 - -N and NH4 + -N concentration was used to investigate the effects of different factors on the nitrogen removal capacity of the target strain.

[0041] The results demonstrated that strain 5505 could achieve highly efficient removal of three substrates—ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen—under high-salt conditions, with optimal nitrogen removal efficiencies of 100%, 100%, and 74.4%, respectively. The optimal denitrification salt concentration range was 3%–8%, the nitrogen source concentration was 100 mg / L, the suitable carbon sources were sodium citrate and sodium pyruvate, the suitable carbon-to-nitrogen ratio was 8–16, the suitable rotation speed was 200 r / min, the suitable pH was 7–8, and the suitable temperature was 30℃–37℃.

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

[0043]

[0044] Example 5:

[0045] Verification of denitrification effect of strain 5505 under optimal culture conditions

[0046] Based on the results of the single-factor experiment, NO3 was configured respectively. - -N, NO2 - -N, NH4 + -N denitrification medium was cultured under optimal conditions, and samples were taken to determine the residual nitrogen concentration in the medium and the nitrogen removal efficiency was calculated.

[0047] The results showed that strain 5505 could achieve a 100% denitrification efficiency for ammonia nitrogen and nitrate nitrogen and a 74% denitrification efficiency for nitrite nitrogen under optimal culture conditions.

[0048] Table 3. Nitrogen removal efficiency of strain 5505 under optimal nitrogen removal conditions.

[0049]

[0050] Example 6:

[0051] Verification of strain denitrification ability under combined nitrogen source conditions

[0052] Using different nitrogen source combinations (NO3) - -N and NO2 - -N, NO3 - -N and NH4 + -N, NO2 - -N and NH4 + -N and NH4 + -N and NO3 - -N and NO2 - Using the (-N) method, the initial nitrogen concentration of the nitrogen source was maintained at 100 mg / L, and all other conditions were kept at the optimal experimental conditions. Samples were taken every 24 hours to measure the nitrogen concentration in the culture medium. The effect of different combinations of nitrogen sources on the denitrification efficiency of the strains was determined.

[0053] The results showed that strain 5505 preferentially utilized ammonia nitrogen under multi-nitrogen source conditions, completing the removal of ammonia nitrogen within 12 hours. The presence of ammonia nitrogen in the combined nitrogen source improved the strain's removal efficiency for nitrate and nitrite nitrogen. Compared to single nitrogen source conditions, ammonia nitrogen significantly (P<0.05) increased the cell density of the strain during nitrogen removal using a combined nitrogen source. Adding a carbon source to the culture medium could remove accumulated nitrite.

[0054] Example 7:

[0055] Verification of strain denitrification ability under simulated wastewater conditions

[0056] Configure an initial nitrogen concentration of 100 mg / L containing NO3. - -N, NO2 - -N, NH4 + Simulated nitrogen-containing wastewater (-N) was used, and samples were taken every 24 hours to determine the residual nitrogen concentration in the culture medium. The effect of simulated wastewater on the denitrification efficiency of the strain was investigated.

[0057] The results showed that strain 5505 thrives in NO3-rich environments. - -N, NO2 - -N, NH4 + In wastewater containing -N, the removal rate of ammonia nitrogen and nitrate nitrogen can reach over 90% within 12 hours. Nitrite nitrogen accumulates within 12 hours, but ammonia nitrogen and nitrate nitrogen can be completely removed after 24 hours. Increasing the carbon-nitrogen ratio or adding a carbon source to the culture medium can completely remove accumulated nitrite.

[0058] Example 8:

[0059] Strain 5505 was inoculated into NO3. - -N, NO2 - -N, NH4 + -N denitrification liquid culture medium, NO3 was measured separately. - -N, NO2 - -N and NH4 + The nitrogen balance relationship was established by the removal rate of -N and the dissolved total nitrogen (DTN) and total nitrogen (TN) in the bacterial culture.

[0060] The results showed that strain 5505 had a preference for ammonia nitrogen utilization, exhibiting superior heterotrophic nitrification capacity compared to aerobic denitrification capacity. The removal of inorganic nitrogen from water by strain 5505 was mainly achieved 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%. Nitrogen assimilation capacity was superior to dissimilation capacity.

[0061] Table 4. Nitrogen balance analysis during the denitrification process.

[0062]

[0063] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a halomonas halodurans bacterium in the treatment of ammonia, nitrate or nitrite nitrogen, characterized in that: The salt-tolerant heterotrophic nitrification-aerobic denitrification bacterium Halomonas ramblicola 5505 is preserved in the China General Microbiological Culture Collection Center, located at No. 1, Xibei Road, Beichen, Chaoyang District, Beijing, with a preservation number of CGMCC No. 30683.

2. The use of a halomonas denitrifying bacterium according to claim 1 for the treatment of ammonia, nitrate or nitrite nitrogen, characterized in that: The optimal salt concentration range is 3%-8%.

3. The application of a halomonas denitrifying bacteria according to claim 1 in the treatment of ammonia, nitrate or nitrite nitrogen, characterized in that: The optimal denitrification conditions include an ammonia nitrogen, nitric acid nitrogen and nitrous acid nitrogen concentration of 100 mg / L.

4. Use of a Halomonas denitrifying bacterium according to claim 1 for the treatment of ammonia, nitrate or nitrite nitrogen, characterized in that: The C / N is 8-16.

5. Use of a Halomonas denitrifying bacterium according to claim 1 for the treatment of ammonia, nitrate or nitrite nitrogen, characterized in that: The carbon source is sodium citrate and sodium pyruvate.

6. Use of a Halomonas denitrifying bacterium according to claim 1 for the treatment of ammonia, nitrate or nitrite nitrogen, characterized in that: The reaction pH is 7-8.

7. Use of a Halomonas denitrifying bacterium according to claim 1 for the treatment of ammonia, nitrate or nitrite nitrogen, characterized in that: The rotation speed is 200 r / min.

8. Use of a Halomonas denitrifying bacterium according to claim 1 for the treatment of ammonia, nitrate or nitrite nitrogen, characterized in that: The temperature is 30-37°C.

Images