Heterotrophic nitrification and aerobic denitrification nitrogen and phosphorus removal strain with salt-tolerant characteristic, microbial inoculum and application

Abstract

The invention relates to the field of environmental microorganisms, and discloses a heterotrophic nitrification aerobic denitrification nitrogen and phosphorus removal strain with a salt-tolerant characteristic, a microbial inoculum and application, the strain is Acinetobacter haemolticus GE10 which is preserved in the Guangdong Microbiological Culture Collection Center on May 12, 2025, the preservation address is the fifth floor, No. 59 building, No. 100 Courtyard, Xianlie Middle Road, Guangzhou City, Guangdong Province, and the preservation number is CGMCC NO.987. The invention further discloses a preparation method of the heterotrophic nitrification aerobic denitrification nitrogen and phosphorus removal strain with the salt-tolerant characteristic. The preservation number of the strain is GDMCC NO. 66303. The acinetobacter hemolyticus strain GE10 domesticated by the invention has the functions of salt resistance, heterotrophic nitrification and aerobic denitrification at the same time, has better water organic carbon removal capacity, and is particularly suitable for treatment of high-salinity nitrogen-containing sewage.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology, specifically to a heterotrophic nitrifying aerobic denitrifying nitrogen and phosphorus removal strain, inoculant, and its application, which are salt-tolerant. Background Technology

[0002] Aquaculture is increasingly recognized as a viable solution to meet the growing demand for freshwater and seafood, especially considering the limitations of wild fisheries production. Aquaculture has become a potentially key contributor to global food production, particularly in Asia, where it accounts for over 90% of the world's total aquaculture output. Aquaculture not only supports rural economies but also reduces pressure on wild fish populations, thus protecting ecosystems to some extent. However, the development of aquaculture has not been without its benefits. Due to the disproportionate area of ​​aquaculture compared to the yield of aquatic products, high-density and highly intensive farming models have emerged. This model results in the discharge of large amounts of nitrogen-containing wastewater, and high concentrations of organic pollution severely impact the environment, consequently affecting the health and survival of plants and animals. Direct discharge of untreated wastewater leads to pollution and eutrophication of aquaculture water bodies. Statistics show that pollutant emissions from aquatic animal and plant farming environments account for more than 5% of my country's total water pollution emissions. Therefore, large-scale, efficient, and low-cost treatment of aquaculture wastewater is essential.

[0003] Such a severe situation necessitates efficient solutions. One of the most common methods is the biological treatment of wastewater using denitrifying bacteria. However, traditional wastewater treatment processes involve independent stages of autotrophic nitrification and denitrification, inevitably increasing operating costs. Furthermore, the slow growth rate of autotrophic nitrifying bacteria makes them susceptible to fluctuations in wastewater composition, thus limiting the efficiency and applicability of traditional biological nitrogen removal methods in practical applications. Traditional phosphorus removal relies on polyphosphate-accumulating bacteria accumulating and releasing phosphorus under aerobic-anoxic conditions. This requires specific environmental conditions and contradicts the traditional denitrification pathway. In previous nitrogen removal processes, nitrogen is converted to N2 under anaerobic conditions, while phosphate removal occurs under aerobic conditions. Simultaneously achieving denitrification and phosphorus removal presents significant challenges in practical operation.

[0004] In existing technologies, some heterotrophic nitrifying and aerobic denitrifying (HNAD) bacteria have shown potential value in wastewater treatment due to their ability to simultaneously perform nitrification and denitrification under aerobic conditions and their faster growth rate. However, the efficiency of such bacterial treatment processes decreases significantly under harsh environments. In particular, the denitrification capacity of HNAD bacteria is inhibited under high osmotic conditions, leading to excessive accumulation of nitrite and nitrous oxide, and even strain dehydration and death. For example, the heterotrophic nitrifying-aerobic denitrifying and polyphosphate-accumulating Acinetobacter WZUF26 disclosed in CN105586294A is difficult to meet the industrial demand for large-scale, efficient, and low-cost treatment of marine aquaculture wastewater. Summary of the Invention

[0005] To address the problems existing in the prior art, the purpose of this invention is to provide a heterotrophic nitrifying aerobic denitrifying nitrogen and phosphorus removal strain with salt tolerance and its application. Original strains were collected from mariculture farms, and through cultivation, screening, and domestication, a heterotrophic nitrifying aerobic denitrifying nitrogen and phosphorus removal strain with salt tolerance was obtained. Furthermore, the environmental adaptability and safety of this strain were improved to meet the industry's demand for large-scale, efficient, and low-cost treatment of mariculture wastewater.

[0006] The objective of this invention is achieved through the following technical solution: A heterotrophic nitrifying aerobic denitrifying and phosphorus-removing bacterial strain with salt tolerance, this strain is Acinetobacter hemolyticus. Acinetobacter haemolyticus GE10 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.66303.

[0007] Acinetobacter hemolyticus Acinetobacter haemolyticus GE10 is a dominant salt-tolerant strain of Acinetobacter hemolyticus (a marine functionally specialized strain) obtained from water and mud samples collected, screened, domesticated, and isolated from marine aquaculture farms.

[0008] A microbial agent, which is the aforementioned Acinetobacter hemolyticus. Acinetobacter haemolyticus GE10 is the active ingredient.

[0009] The application of the salt-tolerant heterotrophic nitrifying aerobic denitrifying nitrogen and phosphorus removal strains, or the microbial agents, in the biological nitrogen and phosphorus removal treatment of saline, nitrogenous, and phosphorus-containing wastewater.

[0010] The beneficial effects of this invention are as follows: (1) The hemolytic amoebae strain GE10 of the present invention is applied to the field of nitrogen and phosphorus wastewater treatment. Under completely aerobic conditions, this strain can utilize NH4+. + NO3 -and NO2 - As the sole inorganic nitrogen source, it is used for aerobic nitrification and denitrification nitrogen removal; its degradation efficiency reaches a maximum of 99.99%, 99.95%, and 99.88%, respectively.

[0011] (2) The hemolytic amoebae strain GE10 of the present invention has heterotrophic nitrification and aerobic denitrification functions; it can utilize a variety of organic carbon sources while having a strong tolerance to high concentrations of organic carbon, and has a good ability to remove organic carbon from water.

[0012] (3) The hemolytic immobile bacillus strain GE10 of the present invention can better utilize organic substrates, grow rapidly, and easily reach a high biomass concentration in a short period of time, achieving the removal of organic carbon while maintaining a high denitrification rate.

[0013] (4) The strain GE10 screened and domesticated in this invention not only performs well in the HNAD process under high salinity, but also effectively removes phosphate. It exhibits good nitrogen and phosphorus removal performance in a salinity range of 0% to 8%, with the best performance in the 0% to 5% salinity range. Tests show that this GE10 strain can efficiently degrade NH4 under 8% salinity conditions. + -N, NO3 - -N, NO2 - -N and PO4 3- -P is a marine functionalized strain of Acinetobacter hemolyticus with heterotrophic nitrification and aerobic denitrification functions. This strain can be used not only in conventional aquaculture waters, but also for denitrification and phosphorus removal in saline waters. It can be widely used in the treatment of tailwater from marine aquaculture farms and has broad application prospects.

[0014] (5) The hemolytic amoebae strain GE10 of the present invention can overcome the incompatibility problem of nitrification and denitrification caused by different oxygen requirements in saline wastewater, so that nitrification and denitrification can be carried out simultaneously in the same aerobic reactor. Applying this strain to the microbial denitrification process of seawater aquaculture water is beneficial to reducing the equipment footprint and construction cost, improving treatment efficiency, and also greatly reducing the periodic water exchange in the aquaculture process. It has good economic and environmental benefits and broad application prospects. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the colony morphology of strain GE10 on solid culture medium in an embodiment of the present invention; Figure 2 This is a schematic diagram illustrating the Gram staining experiment results in an embodiment of the present invention; Figure 3 This is a schematic diagram illustrating the effect of different organic carbon sources on ammonia and phosphorus removal efficiency in embodiments of the present invention; NH4 + -N,PO4 2--P removal rate and OD600 value (a); NO2 - -N,PO4 2- -P removal rate and OD600 value (b); NO3 - -N PO4 2- -P removal rate and OD600 value (c); Figure 4 This is a schematic diagram illustrating the effect of different C / N ratios on nitrogen and phosphorus removal efficiency in this embodiment of the invention; NH4 + -N,PO4 2- -P removal rate and OD600 value (a); NO2 - -N,PO4 2- -P removal rate and OD600 value (b); NO3 - -N PO4 2- -P removal rate and OD600 value (c); Figure 5 This is a schematic diagram illustrating the effect of different P / N ratios on nitrogen and phosphorus removal efficiency in an embodiment of the present invention; NH4 + -N,PO4 2- -P removal rate and OD600 value (a); NO2 - -N,PO4 2- -P removal rate and OD600 value (b); NO3 - -N PO4 2- -P removal rate and OD600 value (c); Figure 6 This is a schematic diagram illustrating the effect of different temperatures on nitrogen and phosphorus removal efficiency in an embodiment of the present invention; NH4 + -N,PO4 2- -P removal rate and OD600 value (a); NO2 - -N,PO4 2- -P removal rate and OD600 value (b); NO3 - -N PO4 2- -P removal rate and OD600 value (c); Figure 7 This is a schematic diagram illustrating the effect of different pH values ​​on nitrogen and phosphorus removal efficiency in an embodiment of the present invention; NH4 + -N,PO4 2- -P removal rate and OD600 value (a); NO2 - -N,PO4 2- -P removal rate and OD600 value (b); NO3 - -N PO4 2- -P removal rate and OD600 value (c); Figure 8This is a schematic diagram illustrating the effect of different salinities on nitrogen and phosphorus removal efficiency in an embodiment of the present invention; NH4 + -N,PO4 2- -P removal rate and OD600 value (a); NO2 - -N,PO4 2- -P removal rate and OD600 value (b); NO3 - -N PO4 2- -P removal rate and OD600 value (c). Detailed Implementation

[0016] 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 below, but this should not be construed as limiting the scope of the present invention.

[0017] Unless otherwise specified, the raw materials, reagents or devices used in the following examples are available from conventional commercial sources or can be obtained by existing known methods.

[0018] The present invention will be further described below with reference to the accompanying drawings and several embodiments.

[0019] 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.

[0020] Basic Implementation See appendix Figure 1 and Figure 2 The present invention provides a bacterium with both heterotrophic nitrification and aerobic denitrification functions, the strain being Acinetobacter hemolyticus. Acinetobacter haemolyticusGE10, the heterotrophic nitrifying and aerobic denitrifying bacteria Acinetobacter hemolyticus GE10, is Gram-negative, its colonies are white and opaque on nutrient agar, with raised, round, smooth and moist surfaces, intact edges, and the bacteria are coccoid in shape, without spores or flagella.

[0021] This invention provides a salt-tolerant heterotrophic nitrifying aerobic denitrifying nitrogen and phosphorus removal strain, which is Acinetobacter hemolyticus. Acinetobacter haemolyticus GE10 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. 66303.

[0022] The Acinetobacter haemolyticus GE10 is a dominant salt-tolerant strain of Acinetobacter haemolyticus (marine functionally specialized strain) obtained from water and mud samples of marine aquaculture farms through collection, screening, domestication, and isolation. Its domestication process includes the following steps: S1: Initial bacterial strains were obtained by screening and separating water and mud samples from oyster farms in Zhangpu County, Zhangzhou City, Fujian Province. Sample collection was carried out according to the "mixed sample collection method" in the "Technical Specification for Soil Environmental Monitoring" (HJ / T 166-2004). The plum blossom sampling method was used to collect surface, middle and deep water and bottom mud from the aquaculture pond in sterile sampling bags. Initial bacterial groups were obtained by screening and separated, and transported and stored at 4℃ for later use. S2: Prepare an enrichment medium with a salinity of 1.5%, inoculate the obtained initial strain into the medium, and after cultivation, enrichment, isolation and screening, obtain the salt-tolerant dominant bacterial group, thus obtaining the first generation of dominant salt-tolerant bacterial group; Then, an enrichment medium with a salinity of 2% or 3% was prepared, and the first-generation dominant salt-tolerant bacteria were inoculated into the medium for culture. After isolation and screening, the domesticated second-generation salt-tolerant dominant bacteria were obtained. S3: Repeat step S2. Based on the second-generation dominant salt-tolerant bacteria, increase the salinity of the enrichment medium by 1.5-2%, then cultivate and domesticate three times. After isolation and screening, the third-generation salt-tolerant dominant bacteria are obtained. This process is repeated, increasing the salinity of the enrichment medium by 1.5-2% for each generation until the salinity is increased to 8%. After cultivation, enrichment, isolation and screening, more salt-tolerant dominant bacteria are obtained. After screening, the multi-generation domesticated dominant salt-tolerant bacteria are finally obtained. S4: A strain with the best growth was selected from multiple generations of superior salt-tolerant strains and named Acinetobacter hemolyticus GE10.

[0023] A microbial agent that contains the aforementioned Acinetobacter hemolyticus. Acinetobacter haemolyticus GE10 is the active ingredient.

[0024] The application of the salt-tolerant heterotrophic nitrifying aerobic denitrifying nitrogen and phosphorus removal strains, or the microbial agents, in the treatment of saline wastewater, wherein the wastewater is saline and contains nitrogen and phosphorus, with a salinity of 0-8; a C / N ratio of 0-15; a P / N (phosphorus / nitrogen) ratio of 0-1; a pH value of 5-8; a temperature of 25℃-40℃; and a carbon source of either sodium citrate or sodium succinate.

[0025] Example 1 This embodiment provides a salt-tolerant heterotrophic nitrifying aerobic denitrifying nitrogen and phosphorus removal strain GE10 and its application. Based on the basic embodiment, it also includes the following technical solutions: 1. Sample collection The Acinetobacter hemolyticus GE10 of this invention was isolated from water and mud samples from an oyster farm in Zhangpu County, Zhangzhou City, Fujian 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 mud from the aquaculture pond in sterile sampling bags, and transported and stored at 4°C for later use.

[0026] 2. Preparation of culture medium and solution (1) Salt solution (g / L): NaCl 2.5g, MgSO4·7H2O 2.5g, FeSO4·7H2O 0.05g, MnSO4·4H2O 0.05g; (2) Trace element solution (g / L): MgSO4·7H2O 50.0g, CaCl2 5.5g, CuSO4·5H2O 1.57g, ZnSO4·7H2O 2.2g, FeSO4 5.0g, MnCl2·4H2O 5.06g, CoCl2·6H2O 1.60g, Na2EDTA 50.0g; (3) Enrichment medium (g / L): 5.62g sodium succinate, 0.087g KH2PO4, 0.24g NaNO3, 0.165g NaNO2, 0.472g (NH4)2SO4, 50mL salt solution; Furthermore, the salinity of the prepared enrichment culture media was set to 2%, 4%, 6%, and 8%, respectively. (4) BTB medium (g / L): 6.45 g sodium citrate dihydrate, 1 mL 1% BTB ethanol solution, 1.5 g KH2PO4, 0.01 g MgSO4·7H2O, 7.9 g Na2HPO4, 0.8415 g NaNO3, 0.192 g NH4Cl, 0.362 g NaNO2, 2 mL trace element solution, 20 g agar, pH 7.0~7.5; (5) Single nitrogen source fermentation medium (DMⅠ) (g / L): 5.62g sodium succinate, 0.087g KH2PO4, 0.472g (NH4)2SO4, 2 mL trace element solution, pH 7.0; (6) Single nitrogen source fermentation medium (DMⅡ) (g / L): 5.62g sodium succinate, 0.087g 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, 0.087g KH2PO4, 0.4928g NaNO2, 2 mL trace element solution, pH 7.0.

[0027] The basic culture medium used in the experiment was sterilized by autoclaving at 121°C for 20 minutes.

[0028] Example 2 This embodiment provides a salt-tolerant heterotrophic nitrifying aerobic denitrifying nitrogen and phosphorus removal strain GE10 and its application. Based on the basic embodiment and Example 1, its enrichment, isolation and screening further include the following technical solutions: (1) Sample pretreatment: Prepare LB nutrient broth medium and autoclave at 121℃ for 20 min. Take 10g of pond bottom mud and transfer it to a 300mL Erlenmeyer flask containing 90ml of LB nutrient broth medium in a clean bench. Shake at 160r / min and incubate at 30℃ for 12-16h to fully activate the microorganisms in the mud sample. The same procedure applies to water samples. (2) Enrichment culture: Take 10 mL of the above mixed liquid and add it to a 300 mL Erlenmeyer flask containing 100 mL of enrichment medium (different salinities). Incubate each generation of strains at 30 °C and 160 r / min for 2-3 days in a shaker. For water samples, take 10 mL and inoculate it into a 300 mL Erlenmeyer flask containing 90 mL of enrichment medium. Incubate at 30 °C and 160 r / min for 2-3 days in a shaker. (3) Sample dilution and coating: Take the enriched water sample and mud sample stock solution from (2) respectively, and connect them to test tubes containing sterile physiological saline in a clean bench. The dilution gradient is 10. -4 10-5 10 -6 10 -7 And mix gently by pipetting or shaking. Take 10 ml of each. -1 ~10 -4 100µL~200µL of stock solution of sediment and water sample concentration gradients were directly spread on BTB plate medium. Three parallel groups and one blank control group were set up for each gradient. The plates were incubated in an incubator at 30℃ for 2~3 days. (4) Isolation and purification: Pick colonies of different morphologies using an inoculation loop. Use the streak plate method to isolate and purify the colonies by streaking them onto BTB solid agar plates. After streaking, place the plates upright in a clean bench with a small opening at room temperature for 5 minutes, then invert them and incubate at 30°C for 2-3 days. Repeat this step, picking single colonies and streaking 3-4 times. After observing colonies without abnormal morphology, pick single colonies, stain them with crystal violet, and examine them under a microscope (100x oil immersion) for purity. (5) Spot inoculation screening: Use an inoculation needle to pick up the purified strain and spot inoculate it in BTB denitrification identification medium for 2-3 days; select strains with high denitrification ability based on the growth of the colony and the size of the blue halo in the BTB medium around the colony (generally speaking, the larger the blue halo, the higher the denitrification ability); inoculate it in LB agar medium and incubate at 30℃ for 2-3 days, and then store the test tubes that form new colonies at 4℃. (6) Re-screening of nitrification and denitrification performance: The cultured colonies were divided into multiple portions, and the strains obtained in (5) above were inoculated into nutrient broth using an inoculation loop. The broth was then incubated at 30°C and 160 r / min for 1 day on a shaker, and the OD was measured. 600 Then, 1% of the inoculum was inoculated into the enrichment medium and cultured at 30°C with shaking at 160 rpm. The OD values ​​of the culture medium were measured at 0 h, 12 h, 24 h, 36 h, and 48 h. 600 Centrifuge at 12000 rpm for 5 min at 4℃, then collect the supernatant and determine the NH4 content. + -N, NO3 - -N, NO2 - -N and PO4 3- -P content; NH4 content was selected. + -N, NO3 - -N, NO2 - -N and PO4 3 The group of colonies that best degraded -P was selected as candidate colonies for further screening. (7) Salt tolerance acclimatization: The candidate colonies were acclimatized for multiple generations by gradually increasing different salinities (2% per generation). The conditions were fixed, such as C / N=10, P / N=0.2, pH=7.0, 160r / min, and sodium succinate as the single organic carbon source. The salinity gradient was set to 0%, 1.5%, 3%, 5%, 8% or 0%, 2%, 4%, 6%, 8%. The salinity gradient was achieved by adding NaCl. In this embodiment, the candidate colonies were inoculated into nutrient broth medium with salinity gradients of 0%, 1.5%, 3%, 5%, 8% in sequence. Each time, the culture was carried out at 30℃ and 160r / min for 1 day to obtain the final surviving colonies. The last generation of the strain with the highest survival ability was selected based on the growth of the colonies and the size of the blue halo in the BTB medium around the colonies. It was named GE10. (8) Using an inoculation loop, take the strain GE10 obtained in (7) above and inoculate it into nutrient broth. Incubate on a shaker at 30°C and 160 r / min for 1 day, and measure its OD. 600 Then, 1% of the culture medium was inoculated into the enrichment medium and cultured at 30°C with shaking at 160 rpm. The OD values ​​of the culture medium were measured at 0 h, 12 h, 24 h, 36 h, and 48 h. 600 Centrifuge at 12000 rpm for 5 min at 4℃, then collect the supernatant and determine the NH4 content. + -N, NO3 - -N, NO2 - -N and PO4 3- The content of -P was used to verify its performance.

[0029] Example 3 This embodiment provides a salt-tolerant heterotrophic nitrifying aerobic denitrifying nitrogen and phosphorus removal strain GE10 and its application. Based on Examples 1 and 2, the obtained strain GE10 was identified, including the following: (1) Morphological identification: After the above screening and isolation, a heterotrophic nitrification-aerobic denitrification strain GE10 was obtained. This strain is Gram-negative, coccobacilli-shaped, and without flagella. The colonies on nutrient agar are white and opaque, with raised, round, smooth and moist surfaces and intact edges.

[0030] (2) Molecular biological identification: DNA from strain GE10 was extracted using the Bacterial DNA Mini-Extraction Kit B from MyBio Biotechnology Co., Ltd. 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× UniqueTM 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; ②~④ cycles 30 times. Sequencing of the PCR products was performed by Shanghai Bioengineering Co., Ltd. The main physiological and biochemical characteristics of strain GE10 are shown in Table 1 below.

[0031] Table 1

[0032] Note: "+" indicates a positive result; "-" indicates a negative result.

[0033] After the above screening steps, a salt-tolerant heterotrophic nitrifying-aerobic denitrifying strain, GE10, was obtained. Based on its 16S rDNA, bacterial morphology, colony morphology, and physiological and biochemical characteristics, strain A4 was identified as Acinetobacter haemolyticus.

[0034] Example 4 This embodiment provides a salt-tolerant heterotrophic nitrifying aerobic denitrifying nitrogen and phosphorus removal strain GE10 and its application, which further validates the performance of GE10, including the following: Validating the optimal growth and denitrification conditions for hemolytic Acinetobacter hemolyticus strain GE10: (1) Effects of different organic carbon sources on the growth and denitrification performance of Acinetobacter hemolyticus GE10 Four carbon sources—oxaloacetic acid, 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 the four organic carbon sources added per liter of medium were 5.00 g, 5.62 g, 2.50 g, and 4.08 g, respectively; the amounts of (NH4)2SO4, NaNO3, and NaNO2, used 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 rpm for 1 day. 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, 12 h, 24 h, 36 h, and 48 h. 600 Centrifuge at 8000 r / min, 4℃, for 5-10 min, then collect 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—sucrose, sodium succinate, glucose, and sodium citrate—on the growth and nitrogen and phosphorus removal efficiency of GE10 were analyzed.

[0035] (2) Effects of different C / N ratios on the growth and denitrification performance of GE10 Sodium succinate was selected as the carbon source for the denitrification medium. Conditions were fixed at 30℃, 160 rpm, P / N = 0.2, and pH = 7.0, with C / N gradients of 0, 2, 5, 10, and 15. The amount of sodium succinate added to the medium for each gradient was 0 g / L, 1.125 g / L, 2.812 g / L, 5.62 g / L, and 8.44 g / L, respectively. The amounts of (NH4)2SO4, NaNO3, and NaNO2 as single inorganic nitrogen sources were 0.472 g, 0.607 g, and 0.493 g per liter of medium, respectively. Candidate strains were inoculated into nutrient broth and cultured at 30℃ and 160 rpm for 1 day. Then, 1% of the inoculum was added to the aforementioned medium. The OD values ​​of the culture medium were measured at 0 h, 12 h, 24 h, 36 h, and 48 h. 600 Centrifuge at 8000 r / min, 4℃ for 5-10 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 five different C / N ratios (0, 2, 5, 10, and 15) on the growth and nitrogen and phosphorus removal efficiency of GE10 were analyzed.

[0036] (3) Effects of different P / N ratios on the growth and denitrification performance of GE10 Sodium succinate was selected as the carbon source for the denitrification medium. Conditions were fixed at 30℃, 160 rpm, C / N = 10, and pH = 7.0. P / N gradients of 0, 0.1, 0.2, 0.5, and 1 were established. The amount of KH₂PO₄ added to the medium for each gradient was 0.0000 g / L, 0.0430 g / L, 0.0870 g / L, 0.2193 g / L, and 0.4387 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 rpm for 1 day. Then, 1% of the inoculum was added to the aforementioned medium. The OD values ​​of the culture medium were measured at 0 h, 12 h, 24 h, 36 h, and 48 h. 600 Centrifuge at 8000 r / min, 4℃ for 5-10 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 five different P / N ratios (0, 0.1, 0.2, 0.5, and 1) on the growth and nitrogen and phosphorus removal efficiency of GE10 were analyzed.

[0037] (4) Effects of different pH values ​​on the growth and denitrification performance of GE10 Under fixed conditions of C / N=10, P / N=0.2, 30℃, 160r / min, and sodium succinate as the sole organic carbon source, pH gradients of 5, 6, 7, and 8 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 medium and cultured at 30℃ and 160r / min for 1 day. Then, 1% of the inoculum was added to the aforementioned culture medium. The OD values ​​of the culture medium were measured at 0 h, 12 h, 24 h, 36 h, and 48 h. 600 Centrifuge at 8000 r / min, 4℃ for 5-10 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 five different pH values ​​(5, 6, 7, and 8) on the growth and nitrogen and phosphorus removal efficiency of GE10 were analyzed.

[0038] (5) Effects of different temperatures on the growth and denitrification performance of GE10 Under fixed conditions of C / N = 10, pH = 7.0, 160 r / min, and sodium citrate 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 medium and cultured at 30℃ and 160 r / min for 1 day. Then, 1% of the inoculum was added to the aforementioned culture medium. The OD values ​​of the culture medium were measured at 0 h, 12 h, 24 h, 36 h, and 48 h. 600 Centrifuge at 8000 r / min, 4℃ for 5-10 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 temperatures (25℃, 30℃, 35℃, and 40℃) on the growth and nitrogen and phosphorus removal efficiency of GE10 were analyzed.

[0039] (6) Effects of different salinities on the growth and denitrification performance of GE10 Under fixed conditions of C / N=10, P / N=0.2, pH=7.0, 160 r / min, and sodium succinate as the sole organic carbon source, salinity gradients of 0%, 1.5%, 3%, 5%, 8%, and 10% were established. Salinity was controlled by adding NaCl at amounts of 0 g, 1.5%, 3 g, 5 g, 8 g, and 10 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 and cultured at 30°C and 160 r / min for 1 day. Then, 1% of the inoculum was added to the aforementioned culture medium. The OD values ​​of the culture medium were measured at 0 h, 12 h, 24 h, 36 h, and 48 h. 600 Centrifuge at 8000 r / min, 4℃ for 5-10 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 six different salinities (0%, 1.5%, 3%, 5%, 8%, and 10%) on the growth and nitrogen and phosphorus removal efficiency of GE10 were analyzed.

[0040] pass Figure 7 and Figure 4 Other test results show that GE10 can be grown 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 0–15, P / N ratio of 0–1, pH of 6–8, and temperature of 25℃–40℃. Even at a C / N ratio of 0, GE10 can still grow, indicating that it can metabolize energy through phosphorus accumulation and release. When the P / N ratio is 0, the nitrogen removal effect is significantly less than when the P / N ratio is 0.1, indicating that phosphorus is an essential element for GE10 growth and metabolism. The optimal denitrification conditions are sodium succinate as the carbon source, C / N=10, P / N=0.2, pH=7, and T=30℃. The nitrogen removal efficiencies of GE10 are 99.65%, 99.98%, and 99.89%, respectively, with corresponding phosphorus removal rates of 98.86%, 95.55%, and 97.49%. Under optimal nitrogen and phosphorus removal conditions, there was no statistically significant difference in nitrogen and phosphorus removal rates when the salinity increased from 0% to 3%. It also maintained good nitrogen and phosphorus removal rates even when the salinity reached 5%. It could survive when the salinity reached 8% but could not survive when the salinity reached 10%, indicating that it has good tolerance to salinity.

[0041] In summary, the GE10 strain is derived from marine aquaculture farms, is salinity tolerant, and possesses highly efficient nitrogen and phosphorus removal (NH4+) removal capabilities. + -N / NO3 - -N / NO2 - -N / PO4 3- Key characteristics include -P), heterotrophic nitrification-aerobic denitrification (HN-AD) function, and its similarity to the hemolytic Acinetobacter model strain (such as the standard strain CIP 64.3). T ATCC 17906 TThe differences between the GE10 strain and other strains (all isolated from soil / freshwater environments, lacking efficient nitrogen removal and salt tolerance) are mainly reflected in three dimensions: ecological adaptation, functional phenotype, and genetic basis. The phylogenetic tree exhibits characteristics of "species clustering uniformity + functional adaptation differentiation." Therefore, strain GE10 belongs to the genus *Anoxybacillus*, which is mostly isolated from high-temperature environments. Its 16S rRNA sequence and phylogenetic position should be significantly different from those of the genus *Acinetobacter*. Taxonomically, *Anoxybacillus* and *Acinetobacter* belong to different groups. It is speculated that GE10 will cluster with other thermophilic bacteria on the phylogenetic tree, while remaining far from the *Acinetobacter* branch to which JS-1 belongs, reflecting the significant differentiation resulting from adaptation to different environmental pressures during their evolutionary process.

[0042] The essential difference between GE10 and the model strain (such as ATCC 17906) is that the model strain is a "conservative representative" of Acinetobacter hemolyticus (adapted to the normal environment and with basic functions), while GE10 is an "ecologically specialized representative" (adapted to the high salinity and high nitrogen and phosphorus environment of seawater and possessing HN-AD + polyphosphate function). The core difference stems from gene variation driven by environmental selection pressure and the acquisition of exogenous functional genes.

[0043] From a phylogenetic perspective, GE10, based on its core genes (16S rRNA / gyrB / rpoB), belongs to the Acinetobacter haemolyticus family, but has formed an independent subclade due to ecological adaptation, reflecting "species unity + ecological differentiation." Based on its functional genes (amoA / ppk), GE10's functional genes originate from horizontal transfer from marine bacteria and are completely differentiated from the model strain, reflecting "evolutionary origin of functional specialization." In terms of application scenarios, GE10's unique characteristics make it a dedicated strain for "nitrogen and phosphorus removal from marine aquaculture tailwater" (solving the problems of traditional model strains' inability to tolerate salt and remove nitrogen). The GE10 strain is a specialized strain with high application potential, acclimatized to the environment. Its difference from the Acinetobacter haemolyticus model strain is not only "different origin" but also a fundamental differentiation in "ecological function" and "genetic characteristics."

[0044] Testing has shown that the GE10 strain of this invention, when applied to the treatment of saline nitrogen and phosphorus aquaculture wastewater, has no adverse effects on aquatic organisms and exhibits high biocompatibility. It 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 salinity, demonstrating good organic carbon removal capacity. This strain is particularly suitable for treating nitrogenous wastewater with a high C / N ratio; under fully aerobic conditions, this strain can utilize NH4+... + NO3 - and NO2- This strain can be used as the sole inorganic nitrogen source for aerobic nitrification and denitrification. It can overcome the incompatibility between nitrification and denitrification caused by different oxygen demands, making it possible for nitrification and denitrification to occur simultaneously in the same aerobic reactor. It has good economic and environmental benefits and broad application prospects.

[0045] 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. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A heterotrophic nitrifying aerobic denitrifying nitrogen and phosphorus removal strain with salt tolerance, characterized in that, This strain is Acinetobacter hemolyticus. Acinetobacter haemolyticus GE10 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. 66303.

2. The heterotrophic nitrifying aerobic denitrifying nitrogen and phosphorus removal strain with salt tolerance according to claim 1, characterized in that, Acinetobacter hemolyticus Acinetobacter haemolyticus GE10 is a dominant salt-tolerant strain of Acinetobacter hemolyticus obtained from water and mud samples collected, screened, domesticated, and isolated from marine aquaculture farms.

3. A microbial inoculant, characterized in that, It is the hemolytic Acinetobacter as described in claim 1 Acinetobacter haemolyticus GE10 is the active ingredient.

4. The application of the salt-tolerant heterotrophic nitrifying aerobic denitrifying nitrogen and phosphorus removal strain according to claim 1 or 2, or the microbial agent according to claim 3, in the treatment of saline wastewater.

5. The application according to claim 4, characterized in that, The carbon source of the wastewater is at least one of sodium citrate and sodium succinate.

6. The application according to claim 4, characterized in that, The C / N ratio of the wastewater is 0~15.

7. The application according to claim 4, characterized in that, The P / N ratio of the wastewater is 0~1.

8. The application according to claim 4, characterized in that, The pH value of the wastewater is 5-8.

9. The application according to claim 4, characterized in that, The temperature of the wastewater is 25℃~40℃.

10. The application according to claim 4, characterized in that, The salinity of the wastewater is 0-8%.

Images