A highly salt-tolerant Acinetobacter baumannii strain for heterotrophic nitrification and aerobic denitrification in wastewater, its inoculant, and its applications.

Abstract

This invention relates to the fields of environmental microbiology and wastewater treatment technology, specifically to a highly salt-tolerant Acinetobacter baumannii strain for heterotrophic nitrification and aerobic denitrification of wastewater, a microbial agent, and its application. The strain is Acinetobacter baumannii. Acinetobacter baumannii M4, deposited on May 12, 2025, at the Guangdong Provincial Microbial Culture Collection Center, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number GDMCC No: 66302. This invention utilizes the *Acinetobacter baumannii* strain M4, domesticated using a multi-round salt-beating method. It can grow in high-salt-alkali environments and exhibits both heterotrophic nitrification and aerobic denitrification capabilities. It can be widely applied to the biological denitrification and phosphorus removal treatment of high-salt-alkali nitrogen and phosphorus wastewater, including wastewater from aquaculture, pickling, and industrial processes.

Description

Technical Field

[0001] This invention relates to the fields of environmental microbiology and wastewater treatment, specifically to a highly salt-tolerant Acinetobacter baumannii strain for heterotrophic nitrification and aerobic denitrification of wastewater, a microbial agent, and its application in the biological denitrification and phosphorus removal treatment of high-salt, alkaline, nitrogen- and phosphorus-containing wastewater. Background Technology

[0002] In existing technologies, the biological treatment of high-salinity, high-alkalinity wastewater containing ammonia nitrogen is a recognized technical challenge in the industry. These wastewaters come from a wide range of sources, including the pickling food industry, seafood processing wastewater, and chemical or brackish water aquaculture tailwater. They present the problem of synergistic pollution from high salinity, high alkalinity, and ammonia nitrogen, as well as the strong inhibitory effect of salinity and alkalinity on biological treatment. When salinity > 3% (30 g / L), the activity of conventional biological denitrification microorganisms (such as common nitrifying bacteria) is significantly inhibited, and cells face the risk of plasmolysis. For wastewater with high salinity, high alkalinity, and high ammonia nitrogen, such as wastewater from pickling food processing (pickled vegetables, kimchi, salted seafood), deep processing of seafood, pesticide / pharmaceutical chemical mother liquor, and landfill leachate, MVR evaporation, halophilic bacterial biological treatment, or anaerobic ammonia oxidation are often required. Anammox It is processed using special techniques such as […].

[0003] In existing reports, the standard strains of Acinetobacter baumannii are tolerant to a salt concentration of less than 1.5% and an alkalinity of 7.0-7.5, with a significant decrease in growth rate after deviating from these values. For example, a strain of Acinetobacter baumannii disclosed in CN106987547A... Acinetobacter baumannii AL-6 can only treat freshwater wastewater, has a salinity tolerance of less than 1.5%, and a pH range of 5-9. It cannot treat wastewater with high alkalinity or high salinity. CN119709485A discloses Acinetobacter baumannii N-1, which has a pH range of 7-9 and does not address salt tolerance. CN105586294A discloses Acinetobacter baumannii WZUF26, which has a temperature range of 15-30℃ and does not address salt or alkali tolerance.

[0004] The existing Acinetobacter baumannii strains disclosed above all suffer from insufficient salt and alkali tolerance, failing to meet the treatment requirements of high-salt and high-alkali wastewater. Therefore, there is an urgent need to develop an Acinetobacter baumannii strain capable of simultaneously tolerating high-salt (≥3% NaCl) and high-alkali (pH≥9) environments while maintaining highly efficient nitrogen and phosphorus removal performance to meet industry demands. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to provide a domesticated strain of Acinetobacter baumannii, highly salt- and alkali-tolerant, for heterotrophic nitrification and aerobic denitrification of wastewater, along with a microbial agent, enabling it to efficiently degrade NH4 under high salinity and alkalinity conditions.+ -N, NO3 - -N, NO2 - -N and PO4 3- -P, this strain has good environmental adaptability and high safety, and can be applied to the treatment of various high salinity and alkalinity wastewater, significantly expanding the application field of Acinetobacter baumannii strains.

[0006] This invention provides the following technical solutions: A strain of *Amoebae baumannii* capable of heterotrophic nitrification and aerobic denitrification in high-salinity wastewater, characterized in that it is a strain of *Amoebae baumannii*. Acinetobacter baumannii M4 was deposited on May 12, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number GDMCC No: 66302.

[0007] The Acinetobacter baumannii M4 strain was originally collected from aquaculture rafts in Huidong County, Huizhou City, Guangdong Province. After multiple isolation and screening, it was selected as an optimal strain and then subjected to multiple rounds of salt shock acclimatization to develop high salt tolerance. This strain can survive in water environments with a salinity of no more than 10%, grow in environments with a salinity of no more than 7%, and achieve an average removal rate of over 40% of ammonia nitrogen in wastewater in environments with a salinity of 3-6%.

[0008] The multi-round salt shock acclimatization includes the following steps: S1: Activation of strains and preparation of seed culture: The target strains with good screening performance were streaked in three zones on LB plates and incubated upside down in a 30℃ constant temperature biochemical incubator for 24-48 hours; plump single colonies were picked and inoculated into 250mL Erlenmeyer flasks containing 50mL LB liquid medium and cultured in a shaker at 30℃ and 160rpm for 12-16 hours until the logarithmic growth phase, which is used as the 0th generation seed culture for later use; S2: Basic Salt Tolerance Pre-test and Acclimation Pressure Confirmation: The seed culture prepared in S1 was inoculated at a rate of 1% into basic salt culture media with salinity gradients of 0%, 1.5%, 3%, and 7% NaCl, with two replicates for each gradient. After incubation at 30℃ and 160rpm for 24-48 hours, the OD600 value of each bottle was measured to assess the inherent salt tolerance of the strain. The 3% NaCl concentration was determined as the starting pressure point for the first salt shock acclimation, and 20mM betaine was used as the fixed concentration for subsequent acclimation. S3: Initial Salt Shock Acclimation and Betaine-Assisted Adaptation: A basal salt acclimation medium containing 3% NaCl and 20 mmol / L anhydrous betaine was prepared. To protect the activity of betaine, betaine and carbon source were separately prepared, filtered, and sterilized before being aseptically added to the sterilized basal salt medium. The S1 seed culture was centrifuged and the bacterial cells were washed once with sterile physiological saline. The bacterial cells were inoculated into the above 3% NaCl acclimation medium at a 5% inoculum size. The culture was carried out at 30℃ and 160 rpm for 48-72 hours with shaking. During this stage, growth was slow. At the end of the culture, a detectable increase in OD600 was observed, indicating that the strain had begun to adapt with the assistance of betaine. S4: Gradient Salt Shock Acclimation: Take the bacterial culture from the end of S3, centrifuge and wash the cells, then transfer it at a 5% inoculum to a fresh acclimation medium with the same composition for continuous subculturing; monitor OD600 daily until the strain can enter a stable logarithmic growth phase within 24-48 hours at this salinity; when the strain grows stably at 3% salinity, centrifuge and wash the bacterial culture, then inoculate it into a fresh acclimation medium with a salinity increased to 4%; repeat the above "adaptation-increase" process, gradually increasing the salinity of the medium according to the gradient of 4%→7%; after each increase, subculture continuously and stably at this salinity for at least 2-3 times; when the strain can reach a maximum OD600 value of 0.6 or above after two consecutive generations of culture in an acclimation medium with a 7% NaCl concentration, the first generation of acclimation is completed, and a mixed first-generation salt-tolerant bacterial group is obtained; S5: Denitrification performance verification and screening of dominant bacterial groups: The first-generation salt-tolerant bacterial culture obtained in S4 was centrifuged and washed to remove betaine; 1% inoculum was added to basal salt media without betaine and with salinities of 0%, 1.5%, 3%, 4%, and 7% NaCl, respectively; after 48 hours of cultivation, the ammonia nitrogen removal rate was measured; cultures with ammonia nitrogen removal rates still higher than 90% at 3% and 4% salinity, and significant removal capacity at 7% high salinity, were screened; these dominant cultures were mixed and the bacterial cells were collected by centrifugation; the above dominant bacterial groups were re-inoculated into an acclimatization medium of 7% NaCl and 20mM betaine for 2-3 generations of rejuvenation culture to obtain the second-generation salt-tolerant bacterial group; S6: Single-strain isolation and purification: Pick several rapidly growing single colonies from the plate and inoculate them into liquid medium containing 7% NaCl for verification. Compare the growth rate and ammonia nitrogen removal efficiency of each strain. Screen out the single colony culture that grows the fastest and has the highest ammonia nitrogen removal rate at 7% salinity. After multiple streak purifications, obtain the third-generation salt-tolerant denitrification pure strain. A microbial inoculant containing the aforementioned Acinetobacter baumannii strain. Acinetobacter baumannii M4 is the active ingredient.

[0009] Application of the Alkali Acinetobacter baumannii strain M4, or the microbial agent, in the biological treatment of high-salt, alkaline, nitrogen- and phosphorus-containing wastewater.

[0010] The carbon source of the high-salt, alkaline, nitrogen- and phosphorus-containing wastewater is at least one of sodium citrate, sodium succinate, sodium oxalate, and sodium acetate; the carbon-to-nitrogen ratio is 0-20, the phosphorus-to-nitrogen ratio is 0-1.0; the pH value is 6-12, the temperature is 25℃-40℃, and the salinity is 0%-7.0%.

[0011] The beneficial effects of the present invention include at least the following: (1) The Acinetobacter baumannii M4 strain domesticated by the present invention has significantly enhanced salt tolerance. In the prior art, the salt tolerance of the standard strain of Acinetobacter baumannii is less than 1.5%, while the M4 strain of the present invention, through multiple rounds of salt shock domestication combined with betaine-assisted domestication, enables the strain to grow normally in a high salinity environment of 3-7%, and can tolerate a maximum salinity of 10%, breaking through the technical bottleneck of the salt tolerance of Acinetobacter baumannii and filling the technical gap in the field of high salinity (≥3%) wastewater treatment.

[0012] (2) The Acinetobacter baumannii M4 strain of the present invention has significantly enhanced alkali resistance. The pH range of Acinetobacter baumannii in the prior art is usually 7-9, while the pH range of the M4 strain of the present invention is 6-12. It can still maintain a high denitrification and phosphorus removal efficiency at pH 11, which significantly expands the application range of Acinetobacter baumannii in the treatment of highly alkaline water.

[0013] (3) The Acinetobacter baumannii M4 strain of the present invention has high phosphorus-nitrogen ratio tolerance. Most strains in the prior art cannot efficiently remove nitrogen and phosphorus under high phosphorus-nitrogen ratio conditions, while the M4 strain of the present invention can still have strong nitrogen and phosphorus removal capabilities under phosphorus-nitrogen ratio conditions of 1.0. For water bodies with high phosphorus-nitrogen ratio, this strain can efficiently remove nitrogen and phosphorus, comprehensively improve the water environment quality, reduce eutrophication of water bodies, and promote the sustainable use of water resources.

[0014] (4) The Acinetobacter baumannii M4 strain of the present invention is applied to the field of nitrogen and phosphorus wastewater treatment. Under completely aerobic conditions, this strain can utilize NH4+ and NH4+ respectively. + -N, NO3 — N and NO2 - -N was used as the sole inorganic nitrogen source for aerobic nitrification and denitrification; its degradation efficiencies reached a maximum of 97.03%, 95.64%, and 99.91%, respectively, corresponding to PO4. 3- -P removal efficiency is 100%.

[0015] (5) The Acinetobacter baumannii M4 strain domesticated in this invention has both heterotrophic nitrification and aerobic denitrification functions; it can utilize a variety of organic carbon sources while having strong tolerance to high concentrations of organic carbon, and has good organic carbon removal capacity in water bodies. This strain is particularly suitable for the treatment of nitrogen-containing wastewater with high C / N ratios.

[0016] (6) The Acinetobacter baumannii M4 of the present invention can better utilize organic substrates, grow rapidly, easily reach a high biomass concentration, and achieve organic carbon removal while maintaining a high denitrification rate.

[0017] (7) The Acinetobacter baumannii M4 of the present invention can tolerate a phosphorus-nitrogen ratio range of 0 to 1.0, and the denitrification and phosphorus removal effect within this phosphorus-nitrogen ratio range is basically unaffected. This bacterium can not only be applied to the phosphorus-nitrogen ratio range of conventional aquaculture water, but also to the denitrification and phosphorus removal of water with a high phosphorus-nitrogen ratio, including aquaculture, pickling and industrial wastewater, etc., which significantly breaks through the limitation of microbial treatment of phosphorus-containing wastewater in water with a high phosphorus-nitrogen ratio.

[0018] (8) The Acinetobacter baumannii M4 strain of the present invention can overcome the incompatibility problem of nitrification and denitrification caused by different oxygen demands, making it possible for nitrification and denitrification to occur simultaneously in the same aerobic reactor. Applying this strain to the microbial denitrification process of various high-salt and alkaline wastewater can help reduce the equipment footprint and construction costs, improve treatment efficiency, and significantly reduce the periodic water changes in aquaculture, thus having good economic and environmental benefits and broad application prospects. Attached Figure Description

[0019] Figure 1 This is a colony morphology diagram of Acinetobacter baumannii M4 of the present invention on a nutrient agar plate; Figure 2 This is a Gram staining image of Acinetobacter baumannii M4 according to the present invention; Figure 3 The scanning electron microscope (SEM) image of Acinetobacter baumannii M4 for the present invention includes AC, wherein A is the SEM image of M4, and B and C are the length and width of M4; Figure 4 This is a graph showing the time-survival rate of zebrafish in the Acinetobacter baumannii M41 bacterial culture of the present invention; Figure 5 This is a comparative diagram showing the growth and denitrification efficiency of Acinetobacter baumannii M4 under different organic carbon sources and different inorganic nitrogen sources, including AC, where A is the sole nitrogen source of NH4. + -N,B's only nitrogen source is NO3. - -N,C's only nitrogen source is NO2. - -N; Figure 6This is a comparative diagram showing the growth and denitrification efficiency of Acinetobacter baumannii M4 under different C / N ratios and different inorganic nitrogen sources, including AC, where A has NH4 as the sole nitrogen source. + -N,B's only nitrogen source is NO3. - -N,C's only nitrogen source is NO2. - -N; Figure 7 This is a comparative diagram showing the growth and denitrification efficiency of Acinetobacter baumannii M4 under different P / N ratios and different inorganic nitrogen sources, including AC, where A has NH4 as the sole nitrogen source. + -N,B's only nitrogen source is NO3. - -N,C's only nitrogen source is NO2. - -N; Figure 8 This is a comparative graph showing the growth and denitrification efficiency of Acinetobacter baumannii M4 under different pH and inorganic nitrogen source conditions, including AC, where A has NH4 as the sole nitrogen source. + -N,B's only nitrogen source is NO3. - -N,C's only nitrogen source is NO2. - -N; Figure 9 This is a comparative graph showing the growth and denitrification efficiency of Acinetobacter baumannii M4 under different salinity and inorganic nitrogen source conditions, including AC, where A has NH4 as the sole nitrogen source. + -N,B's only nitrogen source is NO3. - -N,C's only nitrogen source is NO2. - -N; Figure 10 This is a comparative graph showing the growth and denitrification efficiency of Acinetobacter baumannii M4 under different temperatures (°C) and different inorganic nitrogen sources, including AC, where A has NH4 as the sole nitrogen source. + -N,B's only nitrogen source is NO3. - -N,C's only nitrogen source is NO2. - -N; Figure 11 This diagram illustrates the antibacterial effect of commonly used clinical antibiotics on Acinetobacter baumannii M4 as described in this invention. Detailed Implementation

[0020] To more clearly illustrate the present invention and to gain a clearer understanding of its technical features, objectives, and beneficial effects, the technical solution of the present invention will now be described in detail with reference to the accompanying drawings.

[0021] In the experiment of this invention, NH4 + NO3 - NO2 - The determination and analysis methods for the three nitrogen elements are all based on national standards, including: NH4 + The determination and analysis were performed according to the "Water Quality - Determination of Ammonia Nitrogen - Nessler's Reagent Spectrophotometric Method" (GB HJ535-2009). NO3 - The determination and analysis were performed in accordance with the "Water Quality - Determination of Nitrate Nitrogen - Ultraviolet Spectrophotometry" (GB HJ / T346-2007). NO2 - The determination and analysis were performed according to the "Water Quality - Determination of Nitrite Nitrogen - Spectrophotometric Method" (GB 7493-87). PO4 3- -P was obtained using the ammonium molybdate spectrophotometric method.

[0022] The strain Acinetobacter baumannii provided by this invention ( Acinetobacter baumannii M4 was deposited on May 12, 2025, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number 66302. The deposit conclusion is viable. See also Figure 1 and Figure 2 The Acinetobacter baumannii M4 strain is Gram-negative, slightly raised, with a smooth surface and neat edges. It is easily picked up with an inoculation loop, and is round and pale yellow with a transparent ring around the colony. It is 3-5 mm in diameter and has no special odor.

[0023] Example 1

[0024] This invention relates to the collection, enrichment, isolation, and screening of the original strain of Acinetobacter baumannii M4. 1. Sample collection The original wild strain of Acinetobacter baumannii M4 of the present invention was screened from aquaculture rafts in Huidong County, Huizhou City, Guangdong Province. The samples were collected according to the "mixed sample collection method" in the "Technical Specification for Soil Environmental Monitoring" (HJ / T 166-2004), using the plum blossom sampling method to collect surface, middle and deep water and bottom sediment from the aquaculture pond in sterile sampling bags, and transported and stored at 4°C for later use.

[0025] 2. Preparation of culture medium and solution (1) Salt solution (g / L): NaCl 2.5g, MgSO4·7H2O 2.5g, MnSO4·4H2O 0.05g, FeSO4·7H2O 0.05g; adjust pH to 6, store in a sealed container at 4℃ away from light; (2) Trace element solution (g / L): MgSO4·7H2O 50.0g, CaCl2·2H2O 5.5g, CuSO4·5H2O 1.57g, ZnSO4·7H2O 2.2g, FeSO4·7H2O 5.0g, MnCl2·4H2O, CoCl2·6H2O 1.60g, Na2EDTA 50.0g; (3) Enrichment medium (g / L): sodium succinate 5.62g, KH2PO4 0.087g, (NH4)2 SO4 0.472g, NaNO3 0.24g, NaNO2 0.165g, salt solution 50mL; (4) BTB medium (g / L): 5.66g anhydrous sodium citrate, 1.5g KH2PO4, 0.2g MgSO4·7H2O, 7.9g Na2HPO4, 0.842g NaNO3, 0.362g NaNO2, 0.192g NH4Cl, 25g agar, 1mL 1% BTB ethanol solution, 2mL trace element solution, adjust pH to 7.0-7.5; (5) Single nitrogen source fermentation medium (DMⅠ) (g / L): 5.62g sodium succinate, 0.131g KH2PO4, 0.472g (NH4)2 SO4, 2 mL trace element solution, pH 7.0; (6) Single nitrogen source fermentation medium (DMⅡ) (g / L): 5.62g sodium succinate, 0.131g KH2PO4, 0.607g NaNO3, 2 mL trace element solution, pH 7.0; (7) Single nitrogen source fermentation medium (DMⅢ) (g / L): 5.62g sodium succinate, 0.131g KH2PO4, 0.4928g NaNO2, 2 mL trace element solution, pH 7.0.

[0026] The basic culture medium used in this experiment was sterilized by autoclaving at 121℃ for 20 minutes.

[0027] 2. Enrichment, isolation, and screening of heterotrophic nitrification-aerobic denitrification strains (1) Sample pretreatment: Weigh 10g of sediment sample into an Erlenmeyer flask containing 90mL of sterile physiological saline and a small amount of sterile glass beads. This is a 10-fold diluted suspension. Plug the flask with a cotton plug (this process is performed in a laminar flow hood). Shake at 160r / min for 1h to ensure the sediment sample is evenly dispersed and the microorganisms are fully suspended in the physiological saline. (The same applies to water samples). (2) Enrichment culture: Take 22.2 mL of the above mixture (10 -1The sediment was incubated in 200 mL of enrichment medium at 30°C and 160 rpm for 24 h; 10 mL of water sample was incubated in 90 mL of enrichment medium at 30°C and 160 rpm for 24 h. This step mainly increases the quantity of the target strain to facilitate subsequent isolation and purification. (3) Plate coating of enriched sample solution: Take 1 mL each of the water sample and mud sample enriched and cultured above, add them to 9 mL of sterile physiological saline and dilute to 10. -2 Take 55 test tubes, each containing 9 mL of sterile water, and label them "Dilution Factor + Water Sample / Sludge Sample". Use a pipette to draw 1 mL of the suspension from each test tube and add it to the tube labeled "Sludge Sample-10". -2 The sample was placed in a test tube and gently rinsed several times with hot air to obtain 10. -2 The mud sample was diluted sequentially to 10 using the same method. -10 The mud sample was diluted. The water sample was diluted using the same method. The process was repeated to dilute both the water and sediment samples to a concentration of 10. -10 Take 10 respectively -7 ~10 -10 Water samples and sediment samples (3 replicates per gradient, 1 blank control) were prepared. 100 μL of the diluted solution was placed on a pre-prepared BTB plate and spread evenly in one direction using a flaming spreader. The plates were then incubated upside down in a 30℃ biochemical incubator for 2 days. (4) Isolation and purification: Observe the plates at different dilutions, select plates with colony counts of 30-300, and discard plates that do not meet the requirements. Use an inoculation loop to pick single colonies of different shapes and textures, and use a plate to streak the plates in a zigzag pattern into four zones. Use an inoculation loop to pick colonies in zone one and streak several times, then streak the plates in zones two, three, and four in sequence. After each zone is streaked, sterilize the inoculation loop and cool it before streaking the next zone (repeat 3-4 times). After drying at room temperature, invert the plates and incubate them in a 30℃ constant temperature biochemical incubator for 2-3 days. Pick single colonies from zone three or four and stain them with ammonium oxalate crystal violet, then test for purity under a microscope. If they are not pure, continue to repeat the streaking process or increase the dilution factor for enrichment culture until purified single colonies are obtained. Finally, observe and photograph the plates using a 100× oil immersion microscope. (5) Spot inoculation screening: Use an inoculation needle to pick up the purified strain and spot inoculate it into BTB denitrification identification medium for 2-3 days. Select strains with high denitrification ability based on colony growth and the size of the blue halo around the colony in BTB medium. Generally speaking, the larger the blue halo, the higher the denitrification ability. After inoculation on slant and constant temperature incubation at 30℃ for 2-3 days, store the test tubes at 4℃. (6) Expanding the culture of the strain: Prepare a nutrient broth medium and autoclave it at 121℃ for 20 min; inoculate the strain into the sterilized nutrient broth medium and incubate it at 30℃ and 160 rpm for 16 h to allow the strain to grow to the logarithmic growth phase. (7) Re-screening of nitrification and denitrification performance: Prepare and sterilize the re-screening medium, add the inoculum to the re-screening medium, three replicates per group, and incubate at 30℃ and 160rpm; take appropriate samples at 0h, 12h, 24h, 36h and 48h respectively, and measure OD. 600 After centrifugation at 8000 rpm and 4℃ for 5 min, the NH4 content of the supernatant was measured. + -N, NO3 - -N, NO2 - -N and PO4 3- -P content.

[0028] Example 2 This embodiment uses the salt shock acclimatization method to acclimate the re-screened strains to salt and alkali tolerance, which includes the following steps: S1: Strain activation and seed culture preparation: The target strains with good screening performance were streaked in three zones on LB plates and incubated upside down in a 30℃ constant temperature biochemical incubator for 24-48 hours; plump single colonies were picked and inoculated into 250mL Erlenmeyer flasks containing 50mL LB liquid medium and cultured in a shaker at 30℃ and 160rpm for 12-16 hours until the logarithmic growth phase, which is used as the 0th generation seed culture for later use.

[0029] S2: Preliminary Salt Tolerance Test and Acclimation Pressure Confirmation: Seed culture prepared in S1 was inoculated at a rate of 1% into basal salt media with salinity gradients of 0%, 1.5%, 3%, and 7% NaCl, with two replicates for each gradient. After incubation at 30℃ and 160 rpm for 24-48 hours, the OD600 value of each bottle was measured to assess the strain's inherent salt tolerance. It was expected that the strain would grow better at salinities from 0% to 1.5%, and its growth would be significantly inhibited at 3% salinity. Therefore, the 3% NaCl concentration was determined as the initial pressure point for the first salt shock acclimation.

[0030] In step S2, the optimal auxiliary concentration of betaine was determined using the following steps: the seed culture prepared in step S1 was taken, centrifuged, and the bacterial cells were washed once with sterile physiological saline; 5% inoculum was added to basal salt acclimatization media containing 3% NaCl and betaine concentration gradients of 20mM, 30mM, 40mM, and 50mM, with two replicates for each concentration; the culture was incubated at 30℃ and 160rpm for 48-72 hours with shaking; after the culture was completed, the OD600 value of each bottle was measured to compare the promoting effects of different betaine concentrations on the initial adaptation and growth of the strain at 3% salinity; after comprehensive comparison, the betaine concentration of 20mM with the best growth performance of the strain (highest OD600) was selected as the fixed concentration for subsequent acclimatization.

[0031] S3: Initial Salt Shock Acclimation and Betaine-Assisted Adaptation: Prepare a basal salt acclimation medium containing 3% NaCl and 20 mM anhydrous betaine. To protect betaine activity, separately prepare and sterilize betaine and carbon source solutions, then aseptically add them to the sterilized basal salt medium. Centrifuge the seed culture from step S1 and wash the bacterial cells once with sterile physiological saline. Inoculate the bacterial cells into the above 3% NaCl acclimation medium at a 5% inoculum size. Incubate at 30°C and 160 rpm for 48-72 hours with shaking. Growth is slow during this stage; the goal is to achieve a detectable increase in OD600 at the end of the culture, indicating that the strain has begun to adapt with the assistance of betaine.

[0032] S4: Gradient Salt Shock Acclimation: After the S3 culture, the bacterial culture was centrifuged and washed, then transferred at a 5% inoculum to a fresh acclimation medium with the same composition for continuous subculturing. OD600 was monitored daily until the strain could enter a stable logarithmic growth phase within 24-48 hours at that salinity. Once the strain was stable at 3% salinity, the culture was centrifuged and washed, then inoculated into a fresh acclimation medium with a salinity increased to 4%. The above "adaptation-increase" process was repeated, gradually increasing the medium salinity from 4% to 7%. After each increase, the strain was continuously and stably subcultured at that salinity for at least 2-3 times. When the strain reached a maximum OD600 value of 0.6 or higher after two consecutive generations in an acclimation medium with a 7% NaCl concentration, the first generation of acclimation was completed, yielding a mixed first-generation salt-tolerant bacterial population.

[0033] S5: Denitrification Performance Verification and Screening for Rejuvenation of Dominant Bacterial Species: The first-generation salt-tolerant bacterial culture obtained in S4 was centrifuged and washed to remove betaine. It was then inoculated at a 1% inoculum into basal salt media containing no betaine and with salinities of 0%, 1.5%, 3%, 4%, and 7% NaCl, respectively. After 48 hours of cultivation, the ammonia nitrogen removal rate was measured. Cultures exhibiting ammonia nitrogen removal rates exceeding 90% at 3% and 4% salinity, and significant removal capacity at 7% high salinity, were prioritized for screening. These dominant cultures were mixed and centrifuged to collect the bacterial cells. The aforementioned dominant bacterial groups were re-inoculated into an acclimatization medium containing 7% NaCl + 20mM betaine for 2-3 generations of rejuvenation culture to obtain the second-generation salt-tolerant bacterial species.

[0034] S6: Single-strain isolation and purification: Multiple rapidly growing single colonies were picked from the plates and inoculated into liquid medium containing 7% NaCl for verification. The growth rate and ammonia nitrogen removal efficiency of each strain were compared. The single colony culture with the fastest growth and highest ammonia nitrogen removal rate at 7% salinity was screened out. After repeated streak purification, a third-generation salt-tolerant denitrification pure strain was obtained.

[0035] S7: Comprehensive Performance Evaluation and Strain Preservation: For the finally obtained third-generation salt-tolerant denitrifying pure strains, their growth curves and ammonia nitrogen removal efficiency were systematically measured under a NaCl gradient from 0% to 7% to comprehensively evaluate their adaptability and denitrification function under different salinity environments. The bacterial culture in the logarithmic growth phase was mixed with an equal volume of sterile glycerol, thoroughly mixed, and then dispensed into sterile strain preservation tubes and immediately placed in an ultra-low temperature freezer at -80℃ for long-term preservation.

[0036] The salt shock acclimatization method used in this embodiment directly exposes the strain to a high-salt environment, activating its stress response mechanism, promoting the accumulation of compatible solutes and the activity of ion pumps, thereby rapidly obtaining strains with high salt tolerance. This method incorporates the auxiliary role of betaine as a compatible solute precursor, significantly improving acclimatization efficiency and effect. Combined with metabolic engineering techniques, by adding compatible solute precursors or optimizing nutrient conditions, the salt tolerance and denitrification performance of this strain were further improved. Salt-beat acclimatization mechanisms include: (1) Activation of osmotic stress response: When a strain is suddenly exposed to a high-salt environment (such as 3% NaCl), the extracellular osmotic pressure increases sharply, causing the cell to lose water and triggering the osmotic stress response of the cell. This "salt shock" can quickly activate the strain's physiological defense mechanism. (2) Mechanism of compatible solute accumulation: With the assistance of betaine, the strain accumulates compatible solutes through the following pathways: A. Active uptake of exogenous betaine: The strain takes up betaine from the culture medium through high-affinity betaine transport proteins (such as ProU and ProP systems); B. Synthesis of endogenous compatible solutes: Activates the biosynthetic pathways of compatible solutes such as betaine, proline, and trehalose; C. Intracellular accumulation of compatible solutes: Maintains the osmotic pressure balance inside and outside the cell, and protects enzyme activity and protein structure.

[0037] (3) Adaptive changes in cell membrane: Long-term salt shock acclimatization induces changes in cell membrane composition: increasing the proportion of branched-chain fatty acids and improving cell membrane fluidity; adjusting the composition of phospholipid head groups and enhancing membrane stability; changing the expression profile of membrane proteins and enhancing ion-selective permeability.

[0038] The auxiliary mechanisms of action of betaine include: (1) Maintenance of osmotic pressure balance: Betaine accumulates in high concentrations in cells (up to 20-30% of the dry weight of cells), and maintains the osmotic pressure balance of cells through the "compatible solute" mechanism without interfering with normal cell metabolism.

[0039] (2) Protein protection: Betaine protects protein structure and function in the following ways: - Preferred exclusion mechanism: Betaine is excluded from the protein hydration layer, stabilizing the protein's native conformation. - Regulation of hydrophobic interactions: Maintaining the stability of the protein's hydrophobic core - Enzyme activity protection: Maintaining the activity of denitrification-related enzymes (such as nitrification reductase and nitrosification reductase) under high-salt conditions. (3) Molecular chaperone function: Betaine helps proteins fold correctly and reduces protein aggregation and denaturation induced by high salt.

[0040] 3. The genetic adaptation mechanisms of gradient domestication include: (1) Epigenetic regulation: Salt shock acclimatization induces changes in DNA methylation patterns and histone modifications, which regulate the expression of salt tolerance-related genes.

[0041] (2) Gene mutation and selection: The selective pressure exerted by the high-salt environment promotes the accumulation of favorable mutations: Mutations in transport protein genes enhance ion transport efficiency; mutations in compatibility solute synthesis genes improve synthesis capacity; mutations in regulatory genes enhance stress response efficiency. (3) Horizontal gene transfer: During the domestication of mixed bacterial communities, salt-tolerant genes may be transferred horizontally between bacterial communities through mobile genetic elements such as plasmids and transposons, which can accelerate the improvement of the overall salt tolerance of the bacterial community.

[0042] 4. Molecular basis of the effect Through multiple rounds of salt shock acclimatization, the strain acquired the following adaptive changes at the molecular level: (1) At the genomic level: increased copy number or upregulated expression of salt tolerance-related genes (such as the bet, pro, and tre gene clusters); enhanced expression of ion transporter genes (such as nhaA, nhaB, and chaA); constitutive expression of heat shock protein genes (such as groEL and dnaK); (2) Transcriptome level: Enhanced activity of stress response-related transcription factors (such as σS and σE); remodeling of non-coding RNA regulatory networks; and adjustment of the activity of global regulatory factors (such as CRP and FNR). (3) Proteome level: The abundance of compatible solute synthases (such as ProA, ProB, Trehalose synthases) increased; the expression of ion transport proteins (such as Na+ / H+ antitransporters) increased; the activity and stability of denitrification enzyme systems (such as Nar, Nir, Nor, Nos) were enhanced. (4) Metabolomics level: The concentrations of betaine, proline and trehalose in cells were significantly increased; the ATP / ADP ratio was optimized and the energy supply was sufficient; the redox balance was maintained (NADH / NAD+ ratio was stable).

[0043] Compared with traditional gradual acclimatization methods, the salt-impact acclimatization method of this invention has the following technical advantages: (1) Short acclimatization period: By directly exposing the strain to a high-salt environment, the strain's stress mechanism is quickly activated, shortening the acclimatization period by more than 50%.

[0044] (2) Stable domestication effect: Multiple rounds of salt shock combined with betaine assistance result in high genetic stability of the high salt tolerance trait, which is not easily degenerated.

[0045] (3) Denitrification performance maintenance: Denitrification performance screening is carried out simultaneously during the domestication process to ensure that the obtained high salt-tolerant strains maintain excellent denitrification ability.

[0046] (4) Wide range of applications: This method is not only applicable to Acinetobacter baumannii, but can also be extended to the salt tolerance enhancement of other microbial strains with denitrification function.

[0047] (5) High cost-effectiveness: Betaine is a food additive with low cost, simple domestication process and easy industrial scale-up.

[0048] This embodiment, through multiple rounds of salt shock acclimatization, screened out strains that can still maintain efficient nitrogen and phosphorus removal functions in high-salt environments. Their salt tolerance and nitrogen and phosphorus removal capabilities have been significantly improved. They can grow normally and perform nitrogen and phosphorus removal functions in extreme environments with salinity of 7% and pH of 12, breaking through the limitations of existing technologies and providing an effective technical solution for the biological treatment of high-salt and high-alkali wastewater.

[0049] Example 3 This example demonstrates the identification of strain M4.

[0050] (1) Morphological identification: After the above screening, isolation, screening and salt shock acclimatization, a heterotrophic nitrifying-aerobic denitrifying strain M4 was obtained. The strain was Gram-negative, slightly raised, smooth, with neat edges, and easily picked up with an inoculation loop. It was round and pale yellow, with a transparent ring around the colony, 3-5 mm in diameter, and had no special odor. Under scanning electron microscopy, the bacteria were short rod-shaped, without flagella, and about 0.753±0.0025 μm in length.

[0051] (2) Molecular biological identification: DNA from strain M4 was extracted using Takara Lysis Buffer for Microorganism to Direct PCR lyase. Its 16S rDNA was amplified using this template with a pair of universal primers: upstream primer (27F): 5'-AGAGTTTGATCCTGGCTCAG-3'; downstream primer (1492R): 5'-GGCTACCTTGTTACGACTT-3'. The universal primers were synthesized by Shanghai Bioengineering Co., Ltd. PCR reaction system (25 μL): 2× Unique TM 12.5 μL Taq Master Mix (With Dye), 1 μL each of upstream and downstream primers, 1 μL DNA template, and 9.5 μL ddH2O were used. The PCR program was as follows: ① 94℃, 5 min; ② 94℃ pre-denaturation, 1 min; ③ 55℃ annealing, 1 min; ④ 72℃ extension, 1.5 min; ⑤ 72℃, 10 min; cycles ②-④ were repeated 30 times. Results were analyzed by 1% agarose gel electrophoresis. Sequencing of the PCR-amplified 16S rDNA product was performed by Shanghai Bioengineering Co., Ltd.

[0052] Table 1 Physiological and biochemical characteristics of strain M4

[0053] Note: "+" indicates a positive result; "-" indicates a negative result. Based on its 16S rDNA, bacterial morphology, colony morphology, and physiological and biochemical identification items, strain M4 was identified as Acinetobacter baumannii ( ). Acinetobacter baumannii ).

[0054] Example 4 This embodiment evaluates the environmental safety of strain M4.

[0055] (1) Fish toxicity test: Healthy zebrafish with a body length of 3±1cm were selected. Danio rerioThey were temporarily kept in a large, continuously aerated water body for 30 days, during which time they were fed normally and had their water changed regularly; after their condition stabilized, they were randomly assigned to 15L glass tanks, and experimental groups with added bacterial solution were set up ( Acinetobacter baumannii M4) and a control group (CRT) with an equal volume of sterile water were used. Each experimental group consisted of 30 zebrafish, with 3 replicates per group. The overnight culture was centrifuged at 8000 rpm for 5 minutes, the supernatant was discarded, and the culture was resuspended in sterile PBS buffer. This process was repeated 1-2 times, followed by resuscitation with sterile water. OD was calculated based on the standard curve. 600 The relationship between bacterial concentration and the experimental water concentration was adjusted to approximately 1×10⁻⁶. 6 CFU / mL, while the blank control group was given an equal amount of sterile water; during the experiment, the subjects were fed normally and the experimental water was completely changed every three days. After the water was changed, the above method was repeated to add bacterial solution and sterile water respectively. The survival rate of zebrafish in each group was recorded. The experiment lasted for 14 days. See appendix Figure 4 Under normal conditions, after 14 days of rearing, the survival rate of zebrafish in the control group was 100%, while the concentration of test bacteria in the water of the experimental group was 10. 6 CFU / mL, higher than the pathogenic dose of common pathogens (10 4 The survival rate of zebrafish in the experimental group was 100%, with no significant difference compared to the control group (p>0.05); it was preliminarily determined that strain M4 has high aquatic organism safety.

[0056] (2) Commonly used antibiotic resistance tests: The experimental methods, procedures, and evaluation criteria for antibiotic resistance tests (antibiotic disc susceptibility testing) are based on the "M100 Standard for the Implementation of Antimicrobial Susceptibility Testing (13th Edition)" and the "M02-A10 Standard for the Implementation of Antimicrobial Susceptibility Testing Disc Method (Vol. 32 No. 1)"; the specific steps are as follows: a) Prepare MHA (Guangdong Huankai Biotechnology Co., Ltd.) plates and correct the pH to 7.2~7.4; b) Prepare discs with the corresponding drug content using sterile blank drug sensitivity test discs (Hangzhou Microbial Reagent Co., Ltd.); c) Inoculate the strain into nutrient broth medium and incubate at 30℃ and 180r / min until the logarithmic phase. Dilute to the specified concentration before the experiment. d) Use a disposable sterile cotton swab to dip into the diluted bacterial solution, squeeze out the water from the centrifuge tube wall, and then spread it evenly on an MHA plate. Let it dry at room temperature for 5 minutes. e) Using sterile forceps, place the paper containing the antibiotic in the center of the MHA plate. Set up 3 parallel replicates for each experiment. f) Invert the plate within 15 minutes and incubate at 30℃ for 18 hours; g) Measure the diameter of the inhibition zone using an IP54 metal-cased digital vernier caliper (Yongkang Jingsida Trading Co., Ltd.); Observe the size of the inhibition zone, see Appendix Figure 11 (AQ were neomycin, cefotaxime, streptomycin, tetracycline, metronidazole, penicillin, cefazolin, norfloxacin, erythromycin, oxacillin, polymyxin B, imipenem, amoxicillin, ciprofloxacin, cefoxitin, aztreonam, and sulfamethoxazole, respectively.) The results showed that strain M4 was sensitive to neomycin, cefotaxime, streptomycin, tetracycline, cefazolin, norfloxacin, erythromycin, imipenem, amoxicillin, ciprofloxacin, cefoxitin, and sulfamethoxazole, but resistant to metronidazole, oxacillin, polymyxin B, and aztreonam.

[0057] Therefore, strain M4 is sensitive to a variety of antibiotics, providing a reference for the administration of antibiotics in aquaculture water in future practical applications, providing a means of killing the strain, and improving the biosafety of the strain.

[0058] Table 2. M4 Antibiotic Resistance Test

[0059] Example 5

[0060] In this embodiment, Acinetobacter baumannii M4 was applied to wastewater treatment, and its optimal growth and denitrification conditions were tested.

[0061] (1) Testing the effects of different organic carbon sources on the growth and denitrification performance of Acinetobacter baumannii M4 Four carbon sources—sodium oxalate, sodium succinate, sodium acetate, and sodium citrate—were selected, with a fixed C / N ratio of 10, and the culture conditions maintained at 30℃, 160 rpm, and pH 7.0. Using DM fermentation medium as a base, the amounts of sodium acetate, sodium succinate, sodium oxalate, and sodium citrate added per liter of medium were 5.708 g, 5.62 g, 5.583 g, and 4.08 g, respectively; the amounts of (NH4)2SO4, NaNO3, and NaNO2 added per liter of medium as single inorganic nitrogen sources were 0.472 g, 0.607 g, and 0.493 g, respectively. Candidate strains were inoculated into nutrient broth and cultured at 30℃ and 160 rpm for 16 h. Then, 1% of the inoculum was added to the aforementioned denitrification media containing different organic carbon sources. The OD values ​​of the culture were measured at 0 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h. 600 Centrifuge at 8000 r / min, 4℃ for 3 min, then take the supernatant and determine the NH4 content. + -N, NO3 - -N, NO2 - -N three nitrogen elements and PO4 3--P content. The experiment included three technically replicated experimental groups and a blank control group, with the control group receiving an equal inoculum volume of physiological saline. The effects of four different organic carbon sources—sodium oxalate, sodium succinate, sodium acetate, and sodium citrate—on the growth and nitrogen and phosphorus removal efficiency of M4 cells were analyzed.

[0062] (2) Testing the effects of different C / N ratios on the growth and denitrification performance of M4 Sodium succinate was selected as the carbon source for the denitrification medium. Conditions were fixed at 30℃, 160 rpm, P / N = 0.3, and pH = 7.0, with C / N gradients of 0, 5, 10, 15, and 20. Sodium succinate was added to different gradient media at concentrations of 0 g / L, 2.812 g / L, 5.625 g / L, 8.438 g / L, and 11.25 g / L, respectively. The amounts of (NH4)2SO4, NaNO3, and NaNO2 as single inorganic nitrogen sources were added per liter of medium at 0.472 g, 0.607 g, and 0.493 g, respectively. Candidate strains were inoculated into nutrient broth and cultured at 30℃ and 160 rpm for 16 h. Then, 1% of the inoculum was added to the aforementioned media. The OD values ​​of the culture medium were measured at 0 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h. 600， After centrifugation at 8000 r / min and 4℃ for 3 min, the supernatant was collected and the NH4 content was determined. + -N, NO3 - -N, NO2 - -N three nitrogen elements and PO4 3- -P content. The experiment included three technically replicated experimental groups and a blank control group, with the control group receiving an equal inoculum volume of physiological saline. The effects of five different C / N ratios (0, 5, 10, 15, and 20) on the growth and nitrogen and phosphorus removal efficiency of M4 cells were analyzed.

[0063] (3) Test the effect of different P / N ratios on the growth and denitrification performance of M4 Sodium succinate was selected as the carbon source for the denitrification medium. Conditions were fixed at 30℃, 160 r / min, C / N = 10, and pH = 7.0. P / N gradients of 0, 0.1, 0.3, 0.4, 0.8, and 1.0 were established. The amount of KH₂PO₄ added to the medium for each gradient was 0 g / L, 0.0439 g / L, 0.1312 g / L, 0.1756 g / L, 0.3512 g / L, and 0.439 g / L, respectively. The amounts of (NH₄)₂SO₄, NaNO₃, and NaNO₂ added per liter of medium as single inorganic nitrogen sources were 0.472 g, 0.607 g, and 0.493 g, respectively. Candidate strains were inoculated into nutrient broth medium and cultured at 30℃ and 160 r / min for 16 h. Then, 1% of the inoculum was added to the above medium. The OD values ​​of the culture medium were measured at 0 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h. 600 Centrifuge at 8000 r / min, 4℃ for 3 min, then take the supernatant and determine NH4. + -N, NO3 - -N, NO2 - -N three nitrogen elements and PO4 3- -P content. The experiment included three technically replicated experimental groups and a blank control group, with the control group receiving an equal inoculum volume of physiological saline. The effects of six different P / N ratios (0, 0.1, 0.3, 0.4, 0.8, and 1.0) on the growth and nitrogen and phosphorus removal efficiency of M4 cells were analyzed.

[0064] (4) Test the effect of different pH values ​​on the growth and denitrification performance of M4 Under fixed conditions of C / N=10, P / N=0.3, 30℃, 160r / min, and sodium succinate as the sole organic carbon source, pH gradients of 6, 7, 8, 9, 10, 11, and 12 were established. The amounts of (NH4)2SO4, NaNO3, and NaNO2 added per liter of culture medium as sole inorganic nitrogen sources were 0.472 g, 0.607 g, and 0.493 g, respectively. Candidate strains were inoculated into nutrient broth and cultured at 30℃ and 160r / min for 16 h. Then, 1% of the inoculum was added to the aforementioned culture medium. The OD values ​​of the culture medium were measured at 0 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h. 600 Centrifuge at 8000 r / min, 4℃ for 3 min, then take the supernatant and determine NH4. + -N, NO3 - -N, NO2 - -N three nitrogen elements and PO4 3--P content. The experiment included three technically replicated experimental groups and a blank control group, with the control group receiving an equal inoculum volume of physiological saline. The effects of seven different pH values ​​(6, 7, 8, 9, 10, 11, and 12) on the growth and nitrogen and phosphorus removal efficiency of M4 cells were analyzed.

[0065] (5) Test the effect of different temperatures on the growth and denitrification performance of M4 Under fixed conditions of C / N = 10, pH = 7.0, 160 r / min, and sodium succinate as the sole organic carbon source, temperature gradients of 25℃, 30℃, 35℃, and 40℃ were established. The amounts of (NH4)2SO4, NaNO3, and NaNO2 added per liter of culture medium as sole inorganic nitrogen sources were 0.472 g, 0.607 g, and 0.493 g, respectively. Candidate strains were inoculated into nutrient broth and cultured at 30℃ and 160 r / min for 16 h. Then, 1% of the inoculum was added to the aforementioned culture medium, and the OD values ​​of the culture medium were measured at 0 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h. 600 Centrifuge at 8000 r / min, 4℃ for 3 min, then take the supernatant and determine NH4. + -N, NO3 - -N, NO2 - -N three nitrogen elements and PO4 3- -P content. The experiment included three technically replicated experimental groups and a blank control group, with the control group receiving an equal inoculum volume of physiological saline. The effects of four different temperatures (25℃, 30℃, 35℃, and 40℃) on the growth and nitrogen and phosphorus removal efficiency of M4 cells were analyzed.

[0066] (6) Test the effect of different salinities on the growth and denitrification performance of M4 With fixed conditions including C / N=10, P / N=0.3, pH=7.0, 160 r / min, and sodium succinate as the sole organic carbon source, salinity gradients were set at 0%, 1.5%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, and 10.0%. Salinity was controlled by adding NaCl at amounts of 0 g, 1.5 g, 2.0 g, 3.0 g, 4.0 g, 5.0 g, 6.0 g, 7.0 g, and 10.0 g per liter. The amounts of (NH4)2SO4, NaNO3, and NaNO2 as sole inorganic nitrogen sources were added at 0.472 g, 0.607 g, and 0.493 g per liter of culture medium, respectively. Candidate strains were inoculated into nutrient broth medium and cultured at 30℃ and 160 r / min for 16 h. Then, 1% of the inoculum was added to the above medium. The OD values ​​of the culture medium were measured at 0 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h. 600 Centrifuge at 8000 r / min, 4℃ for 3 min, then take the supernatant and determine NH4.+ -N, NO3 - -N, NO2 - -N three nitrogen elements and PO4 3- -P content. The experiment included three technically replicated experimental groups and a blank control group, with the control group receiving an equal inoculum volume of physiological saline. The effects of nine different salinities (0%, 1.5%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, and 10.0%) on the growth and nitrogen and phosphorus removal efficiency of M4 cells were analyzed.

[0067] Based on the test results of this embodiment, Figures 5-10 It can be seen that strain M4 can grow using various organic carbon sources such as sodium citrate and sodium succinate. Growth and nitrogen and phosphorus removal are best when using sodium succinate. It can grow under conditions of C / N ratio of 0–20, P / N ratio of 0–1, pH of 6–11, salinity of 0%–7%, and temperature of 25℃–40℃. At a C / N ratio of 0, M4 growth is poor, indicating that it requires a carbon source as a basis for energy and structure during growth and metabolism. Insufficient carbon source leads to limited energy metabolism, thus affecting growth. When the P / N ratio is 0.1, the nitrogen removal effect is significantly worse than when the P / N ratio is 0.3, indicating that phosphorus is an essential element for M4 growth and metabolism. When the salinity increases from 0 to 1.5%, the growth of the strain improves accordingly, indicating that the bacterium is suitable for low-salt conditions. Furthermore, it continues to grow well under high-salt conditions when the salinity increases from 3% to 7%, demonstrating tolerance to high salinity. However, growth significantly decreases at extremely high salinity of 10%. When NH4+ is present... + When nitrogen (N) is the sole nitrogen source, the bacteria can grow normally at temperatures ranging from 25℃ to 40℃, maintaining a removal rate of over 95% under these conditions, indicating a preference for ammoniacal nitrogen. The optimal denitrification conditions were: sodium succinate as the carbon source, C / N = 10, P / N = 0.3, pH = 7, and T = 30℃. The denitrification efficiencies of M4 were 97.03%, 95.64%, and 99.91%, respectively, with corresponding phosphorus removal rates of 100%. Under the optimal denitrification and phosphorus removal conditions, there was no statistically significant difference in nitrogen and phosphorus removal rates as the pH increased from 7 to 11, indicating good tolerance to alkaline environments.

[0068] The M4 strain domesticated in this invention provides a new solution for the treatment of high-salinity and alkaline wastewater. This strain has no adverse effects on aquaculture organisms, exhibits high biocompatibility with aquatic organisms, and is suitable for application in most aquaculture water bodies. It simultaneously possesses heterotrophic nitrification, aerobic denitrification, and phosphorus removal functions; it can utilize multiple organic carbon sources while exhibiting strong tolerance to high concentrations of organic carbon and extremely alkaline environments, demonstrating good organic carbon removal capabilities. This strain can also be applied to the treatment of high-salinity and alkaline food processing wastewater or aquaculture tailwater projects. Such wastewater contains large amounts of sugars and other organic matter, which M4 can quickly adapt to and utilize, converting them into energy for its own growth. M4 is comprehensive and unique, possessing not only heterotrophic nitrification, aerobic denitrification capabilities, but also efficient phosphorus removal. M4 exhibits extremely high tolerance to high concentrations of organic carbon and extremely alkaline environments. This strain is particularly suitable for the treatment of nitrogen-containing wastewater with high C / N ratios and high salinity and alkalinity; under fully aerobic conditions, this strain can utilize NH4+. + -N, NO3 - -N and NO2 - -N is used as the sole inorganic nitrogen source for aerobic nitrification and denitrification. This strain can overcome the incompatibility problem of nitrification and denitrification caused by different oxygen demands, making it possible for nitrification and denitrification to be carried out simultaneously in the same aerobic reactor. It has good economic and environmental benefits and broad application prospects.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention; other technical solutions obtained by those skilled in the art without creative effort within the scope of the present invention are all within the scope of protection of the present invention.

Claims

1. A highly salt-tolerant Acinetobacter baumannii strain capable of heterotrophic nitrification and aerobic denitrification in wastewater, characterized in that, It is a strain of Acinetobacter baumannii. Acinetobacter baumannii M4 was deposited on May 12, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou, Guangdong Province, with accession number GDMCC No: 66302.

2. The Acinetobacter baumannii strain according to claim 1, characterized in that, The Acinetobacter baumannii M4 strain was originally collected from aquaculture rafts in Huidong County, Huizhou City, Guangdong Province. After multiple separation and screening, an optimal strain was obtained. Then, after multiple rounds of salt shock acclimatization, the strain was acclimatized to enhance its high salt and alkali tolerance. This strain can survive in a water environment with a salinity of 10% and grow in an environment with a salinity of 7%. Furthermore, it can achieve an average removal rate of over 40% of ammonia nitrogen in wastewater in an environment with a salinity of 3-6%.

3. The Acinetobacter baumannii strain according to claim 2, characterized in that, The multi-round salt shock acclimatization includes the following steps: S1: Strain activation and seed culture preparation: The target strains with good screening performance were streaked in three zones on LB plates and incubated upside down in a 30℃ constant temperature biochemical incubator for 24-48 hours; plump single colonies were picked and inoculated into 250mL Erlenmeyer flasks containing 50mL LB liquid medium and cultured in a shaker at 30℃ and 160rpm for 12-16 hours until the logarithmic growth phase, which is used as the 0th generation seed culture for later use; S2: Basic Salt Tolerance Pre-test and Acclimation Pressure Confirmation: The seed culture prepared in S1 was inoculated at a rate of 1% into basic salt culture media with salinity gradients of 0%, 1.5%, 3%, and 7% NaCl, with two replicates for each gradient. After incubation at 30℃ and 160rpm for 24-48 hours, the OD600 value of each bottle was measured to assess the inherent salt tolerance of the strain. The 3% NaCl concentration was determined as the starting pressure point for the first salt shock acclimation, and 20 mmol / L betaine was used as the fixed concentration for subsequent acclimation. S3: Initial Salt Shock Acclimation and Betaine-Assisted Adaptation: A basal salt acclimation medium containing 3% NaCl and 20mM anhydrous betaine was prepared. To protect the activity of betaine, betaine and carbon source were separately prepared, filtered, and sterilized before being aseptically added to the sterilized basal salt medium. The S1 seed culture was centrifuged and the cells were washed once with sterile physiological saline. The cells were inoculated into the above 3% NaCl acclimation medium at a 5% inoculum size. The culture was carried out at 30℃ and 160rpm for 48-72 hours with shaking. Growth was slow during this stage, and a detectable increase in OD600 was observed at the end of the culture, indicating that the strain had begun to adapt with the assistance of betaine. S4: Gradient Salt Shock Acclimation: Take the bacterial culture from the end of S3 culture, centrifuge and wash the cells, then transfer it at a 5% inoculum to a fresh acclimation medium with the same composition for continuous subculturing; monitor OD600 daily until the strain can enter a stable logarithmic growth phase within 24-48 hours at this salinity; when the strain grows stably at 3% salinity, centrifuge and wash the bacterial culture, then inoculate it into a fresh acclimation medium with a salinity increased to 4%; repeat the above "adaptation-increase" process, gradually increasing the salinity of the medium according to a gradient of 4%→7%; after each increase, subculture continuously and stably at this salinity for at least 2-3 times; When a strain can reach a maximum OD600 value of 0.6 or above after two consecutive generations of cultivation in an acclimatization medium with a concentration of 7% NaCl, then one generation of acclimatization is completed, and a mixed first-generation salt-tolerant bacterial group is obtained. S5: Denitrification performance verification and screening of dominant bacterial groups: The first-generation salt-tolerant bacterial culture obtained in S4 was centrifuged and washed to remove betaine; 1% inoculum was added to basal salt media without betaine and with salinities of 0%, 1.5%, 3%, 4%, and 7% NaCl, respectively; after 48 hours of cultivation, the ammonia nitrogen removal rate was measured; cultures with ammonia nitrogen removal rates still higher than 90% at 3% and 4% salinity, and significant removal capacity at 7% high salinity, were screened; these dominant cultures were mixed and the bacterial cells were collected by centrifugation; the above dominant bacterial groups were re-inoculated into an acclimatization medium of 7% NaCl and 20mM betaine for 2-3 generations of rejuvenation culture to obtain the second-generation salt-tolerant bacterial group; S6: Single-strain isolation and purification: Select several rapidly growing single colonies from the plate and inoculate them into liquid medium containing 7% NaCl for verification. Compare the growth rate and ammonia nitrogen removal efficiency of each strain. Select the single colony culture that grows the fastest and has the highest ammonia nitrogen removal rate at 7% salinity. After multiple streak purifications, obtain the third-generation salt-tolerant denitrification pure strain.

4. A microbial inoculant, characterized in that, It uses the Acinetobacter baumannii strain as described in claim 1. Acinetobacter baumannii M4 is the active ingredient.

5. The application of the Acinetobacter baumannii strain according to claim 1, or the microbial agent according to claim 4, in the biological treatment of high-salt, alkaline, nitrogen- and phosphorus-containing wastewater.

6. The application according to claim 5, characterized in that, The carbon source of the high-salt, alkaline, nitrogen- and phosphorus-containing wastewater is at least one of sodium citrate, sodium succinate, sodium oxalate, and sodium acetate.

7. The application according to claim 5, characterized in that, The carbon-to-nitrogen ratio of the high-salt, alkaline, nitrogen- and phosphorus-containing wastewater is 0-20, and the phosphorus-to-nitrogen ratio is 0-1.

0.

8. The application according to claim 5, characterized in that, The pH value of the high-salt, alkaline, nitrogen- and phosphorus-containing wastewater is 6-12.

9. The application according to claim 5, characterized in that, The temperature of the high-salt, alkaline, nitrogen- and phosphorus-containing wastewater is 25℃-40℃.

10. The application according to claim 5, characterized in that, The salinity of the high-salinity, alkaline, nitrogen- and phosphorus-containing wastewater is 0%-7.0%.

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