Culture and domestication method of salt-tolerant heterotrophic nitrification and denitrification microbial agent

Abstract

The invention discloses a culture and domestication method of a salt-tolerant heterotrophic nitrification and denitrification microbial agent, and belongs to the technical field of environmental microorganisms. Comprising the following steps: inoculating a heterotrophic nitrification culture medium with a halomonas seed solution, and culturing until a strain reaches a stable growth period to finish domestication. The Halomonas sp. SOB56 strain provided by the invention can be used for effectively removing nitrogen-containing substances in high-salinity wastewater such as mariculture wastewater and the like, and can be adapted to high-salinity wastewater with different salinity through domestication. The removal rates of the strain on ammonia nitrogen and nitrate nitrogen are 99.6% and 94.22% respectively within 36 hours when the strain enters a logarithmic phase under the condition that the temperature is 30 DEG C and the salinity is 5%. The biological nitrogen removal method belongs to the technical field of novel biological nitrogen removal, nitrate nitrogen and nitrite nitrogen are not generated in the ammonia nitrogen removal process, and the biological nitrogen removal method has a good application prospect in actual application of high-salinity wastewater such as mariculture wastewater.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology, and in particular to a method for cultivating and acclimatizing salt-tolerant heterotrophic nitrifying and denitrifying microbial agents. Background Technology

[0002] With the rapid development of mariculture, the discharge of high-salinity nitrogenous wastewater has increased significantly. Excessive nitrogen discharge leads to eutrophication, harming the environment and human health. High salt concentrations severely impact the survival of aquatic organisms, water potability, and agricultural production, causing soil salinization at the discharge sites. Microbial denitrification is widely used in wastewater treatment due to its high efficiency, low cost, and environmental friendliness. However, high salinity causes cell dehydration, reduces cell activation capacity, and decreases enzyme activity, leading to poorer biological treatment effects and posing a significant challenge to microbial wastewater treatment systems. Traditional denitrification processes generally use aerobic bacteria, but nitrification and denitrification need to be carried out in two steps, which increases treatment time and energy consumption. Halomonas bacteria, a genus with salt tolerance and nitrification / denitrification capabilities, greatly enhances its value in treating mariculture wastewater and provides theoretical support for the research and development of biological denitrification technology for high-salinity wastewater. Summary of the Invention

[0003] The purpose of this invention is to provide a method for cultivating and acclimatizing salt-tolerant heterotrophic nitrifying and denitrifying microbial agents, thereby solving the problems existing in the prior art. To enrich the heterotrophic nitrifying-aerobic denitrifying strain resource library, this invention provides a strain of *Halomonas*. Vreelandella titanicae strain SOB56 is a salt-tolerant heterotrophic nitrifying-aerobic denitrifying bacterium that enables efficient denitrification of marine aquaculture wastewater and has great value in wastewater treatment.

[0004] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of the present invention is a method for cultivating and acclimatizing salt-tolerant heterotrophic nitrifying and denitrifying microbial agents, comprising the following steps: inoculating the seed liquid of Halomonas bacteria into a heterotrophic nitrification medium and culturing until the strain reaches a stable growth period, thus completing the acclimatization.

[0005] The second technical solution of the present invention is the application of the halomonas bacteria obtained by the cultivation and domestication method in the treatment of marine aquaculture wastewater.

[0006] The third technical solution of the present invention is a method for treating marine aquaculture wastewater, which uses Haloxylon ammodendron obtained by the aforementioned cultivation and domestication method to treat marine aquaculture wastewater and reduce its ammonia nitrogen and nitrate nitrogen content.

[0007] Based on the above technical solution, the present invention has the following technical effects: The Halomonas bacterium provided by this inventionVreelandella titanicae Strain SOB56 can effectively remove nitrogenous substances from high-salinity wastewater such as mariculture wastewater, and can be acclimatized to high-salinity wastewater of varying salinity. At a salinity of 5%, this strain achieved removal rates of 99.6% for ammonia nitrogen and 94.22% for nitrate nitrogen within 36 hours of entering the logarithmic growth phase. This biological denitrification method belongs to the field of novel biological denitrification technology. It removes ammonia nitrogen without generating nitrate or nitrite nitrogen, and shows great promise for practical applications in treating high-salinity wastewater such as mariculture wastewater. Attached Figure Description

[0008] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0009] Figure 1 Halomonas Vreelandella titanicae Growth morphology of strain SOB56 on solid culture medium.

[0010] Figure 2 Halomonas Vreelandella titanicae Scanning electron microscope image of strain SOB56.

[0011] Figure 3 To assess the heterotrophic nitrification growth and nitrogen removal efficiency of heterotrophic nitrifying-aerobic denitrifying bacteria.

[0012] Figure 4 To improve the aerobic denitrification growth and nitrogen removal efficiency of heterotrophic nitrifying-aerobic denitrifying bacteria.

[0013] Figure 5 The nitrogen removal efficiency of mariculture wastewater is given, where 'a' represents the actual nitrogen concentration change in mariculture wastewater, and 'b' represents the nitrogen content of *Halomonas* bacteria. Vreelandella titanicae Changes in nitrogen concentration in marine aquaculture wastewater when treated with strain SOB56. Detailed Implementation

[0014] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0015] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0016] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0017] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0018] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0019] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0020] This invention provides a method for cultivating and acclimatizing salt-tolerant heterotrophic nitrifying and denitrifying microbial agents, comprising the following steps: inoculating Halomonas seed liquid into a heterotrophic nitrification medium and culturing until the strain reaches a stable growth period, thus completing the acclimatization.

[0021] In some specific implementations, the *Haloxymonas* is *Haloxymonas*. Vreelandella titanicae strain SOB56 was deposited on December 19, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC 67508.

[0022] In some specific embodiments, the heterotrophic nitrification medium consists of: (NH4)2SO4 0.4717 g·L⁻¹ -1Sodium acetate 3.417 g·L -1 5 mL of vitamin solution and 5 mL of trace element solution, pH 7.1±0.2, sterilized by moist heat at 121 ℃ for 30 min.

[0023] In some specific implementations, the culture conditions include: culture in an air-cooled shaker at 30 °C and 160 rpm.

[0024] This invention also provides the application of the Halomonas bacteria obtained by the culture and domestication method in the treatment of marine aquaculture wastewater.

[0025] In some specific implementation schemes, Halomonas bacteria are used to treat marine aquaculture wastewater to reduce its ammonia nitrogen and nitrate nitrogen content.

[0026] This invention also provides a method for treating marine aquaculture wastewater, which uses Haloxylon ammodendron obtained through the cultivation and domestication method to treat the wastewater and reduce its ammonia nitrogen and nitrate nitrogen content.

[0027] The present invention contains halomonas. Vreelandella titanicae Strain SOB56 is a heterotrophic nitrifying-aerobic denitrifying bacterium. This strain uses sodium acetate as a carbon source to aerobically degrade ammonia nitrogen and nitrate nitrogen in water. During nitrification, no nitrite or nitrate nitrogen is produced, thus avoiding secondary pollution of the water body. Therefore, this invention seeks to protect *Halomonas*. Vreelandella titanicae Application of strain SOB56 in wastewater denitrification. This invention further protects *Halomonas*. Vreelandella titanicae Application of strain SOB56 in the simultaneous removal of ammonia nitrogen and COD from wastewater. Furthermore, this strain can be gradually adapted to denitrification of high-salinity wastewater with higher salinity through acclimatization, while maintaining a high denitrification rate. Therefore, this invention seeks to protect *Halomonas*. Vreelandella titanicae Domestication method of strain SOB56.

[0028] Upon testing, the *Haloxymonas* strain of this invention... Vreelandella titanicae strain SOB56 is a highly efficient heterotrophic nitrifying-aerobic denitrifying bacterium that can easily adapt to high-salinity wastewater of varying salinity through acclimatization. It is a Gram-negative bacterium with a cell size of (0.37 ± 0.12) × (2.76 ± 1.22) µm. It can utilize both ammonia nitrogen and nitrate nitrogen as its sole nitrogen sources.

[0029] The culture medium formulations used in the examples are as follows: LB medium (enrichment medium): 10 g / L peptone -1 5 g·L yeast extract -1 NaCl 50 g·L -1pH 7.1±0.2, sterilized by moist heat at 121 ℃ for 30 min.

[0030] LB solid medium: 10 g / L peptone -1 5 g·L yeast extract -1 NaCl 50 g·L -1 2 g agar, pH 7.1±0.2, sterilized by moist heat at 121 ℃ for 30 min.

[0031] Heterotrophic nitrification medium: (NH4)2SO4 0.4717 g·L -1 Sodium acetate 3.417 g·L -1 5 mL of vitamin solution and 5 mL of trace element solution, pH 7.1±0.2, sterilized by moist heat at 121 ℃ for 30 min.

[0032] Aerobic denitrification medium: NaNO3 0.607 g·L -1 Sodium acetate 3.417 g·L -1 5 mL of vitamin solution and 5 mL of trace element solution, pH 7.1±0.2, sterilized by moist heat at 121 ℃ for 30 min.

[0033] General fermentation medium: 4 g·L corn steep liquor powder -1 2 g·L soybean meal -1 3 g·L of cell wall broken yeast powder -1 3 g·L of beef extract -1 Fish meal peptone 2 g·L -1 5 g·L glucose -1 Magnesium sulfate 0.1 g·L -1 Potassium dihydrogen phosphate 0.08 g·L -1 Manganese sulfate 0.02 g·L -1 Sodium chloride 0.1 g·L -1 Tween 80 0.01 g·L -1 .

[0034] Halomonas culture medium: 15.0 g·L⁻¹ tryptone -1 5.0 g·L soybean peptone -1 Sodium chloride 40.0 g·L -1 .

[0035] Denitrification medium: KNO3 0.7214 mg·L -1 Sodium acetate 1.4643 mg·L -1 MgSO4 0.05 mg·L -1 0.2 mg·L⁻¹ CaCO₃ -1KH2PO4 0.022 mg·L -1 1 mL of trace elements.

[0036] Vitamin solution: Folic acid 2 mg / L -1 Biotin 2 mg·L -1 Thiamine hydrochloride 5 mg·L -1 Vitamin B 12 0.1 mg·L -1 Vitamin B6 10 mg·L -1 Riboflavin 5 mg·L -1 nicotinic acid 5 mg·L -1 D-Calcium Pantothenate 5 mg·L -1 para-aminobenzoic acid 5 mg·L -1 .

[0037] Trace element solution: 1.5 g·L⁻¹ nitric acid triacetic acid -1 MnSO4·H2O 0.5 g·L -1 FeSO4·H2O 0.1g·L -1 CuSO4·7H2O 0.01 g·L -1 MgSO4·7H2O 3.0 g·L -1 NaCl 1.0 g·L -1 , CoCl2·6H2O, CaCl20.076 g·L -1 ZnSO4·7H2O 0.1 g·L -1 , KAl(SO4)2·12 H2O 0.02 g·L -1 HBO 30.01g·L -1 .

[0038] Example 1 Strain screening and identification: High-salt activated sludge from seawater was homogenized and inoculated at a 5% inoculum into 100 mL of general fermentation medium, Haloxylon ammodendron medium, and LB medium and denitrification medium with different salinity gradients (0%, 3%, 4%, 5%). The cultures were then aerobically cultured overnight at 32°C and 180 rpm for strain screening. A medium with good denitrification was selected, and the pre-cultured bacterial solution was filtered through a 40 μm cell strainer (BKMAM Cell Strainer 40 μm) to remove large solid particles. The solution was then diluted to the appropriate concentration with sterile PBS buffer and placed in 5 mL sorting tubes (BD FALCON). Sorting was performed using a flow cytometer (BDFACSAria Fusion SORP), and single bacteria that met the sorting criteria were aliquoted at 10*10 onto corresponding pre-cultured solid medium plates. The plates were inverted and placed in a 32°C constant temperature biochemical incubator to observe growth until single colonies appeared. Single colonies were identified using the universal forward primer 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and the reverse primer 1492R: 5'-GGTTACCTTGTTACGACTT-3'. Based on 16S rRNA alignment, strain SOB56 was identified as belonging to the genus *Halomonas*.

[0039] Halomonas Vreelandella titanicae SOB56, this strain was deposited on December 19, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC 67508.

[0040] Bacterial culture activation and preparation: Halomonas Vreelandella titanicae The seed culture of strain SOB56 was inoculated into LB solid medium and incubated at 30°C and 160 rpm for 24 h. After incubation, a single colony was picked and activated in 100 mL of LB medium for 12 h. Then, 10 mL of the activated colony was inoculated into fresh LB medium and incubated at 30°C and 160 rpm for 24 h. The process was repeated two to three times depending on the growth.

[0041] Take bacterial culture in the logarithmic growth phase, centrifuge, wash and resuspend two to three times with PBS to prepare inoculum (D). 600 =2.100±0.200), with an inoculum size of 2.5% (volume ratio) in each culture medium.

[0042] Example 2 strains Vreelandella titanicae Degradation performance of strain SOB56 for ammonia nitrogen and test methods (1) Determination of ammonia nitrogen degradation capacity: Halomonas bacteria Vreelandella titanicaestrain SOB56 was inoculated into heterotrophic nitrification medium, and samples were taken and centrifuged at regular intervals. The absorbance at 420 nm was measured using a UV spectrophotometer, and the corresponding ammonia nitrogen concentration was calculated by referring to the standard curve.

[0043] (2) Ammonia nitrogen utilization characteristics: The inoculum was inoculated into a 250 mL Erlenmeyer flask containing 200 mL of the above heterotrophic nitrification medium, and after thorough mixing, it was cultured in an air-cooled shaker at 30 ℃ and 160 rpm. NH4+ in the supernatant was measured at regular intervals. + –N, NH2OH–N, NO3 — -N、NO2 — –N, TN and OD 600 Value. See results. Figure 3 ,according to Figure 3 The results show Vreelandella titanicae strain SOB56 entered the logarithmic phase after a lag phase of 42 hours, and then entered the stationary phase after another 42 hours of logarithmic phase. NH4 + –N removal rate reached its maximum at 60-72 h, with only 0.276 mg / L removed during the entire culture process. -1 NO3 — N, 0.023 mg L -1 NO2 — N, 0.051 mg L -1 The increase in NH2OH–N suggests that no nitrate nitrogen, nitrite nitrogen, or hydroxylamine was produced during the nitrification process of the strain.

[0044] (3) According to Figure 3 As a result, when ammonia nitrogen was used as the sole nitrogen source, the ammonia nitrogen degradation rate was 15.16% after 60 h, 57.53% after 72 h, 91.87% after 84 h, and 99.6% after 96 h. This is consistent with the OD... 600 The change corresponds to the entry into the logarithmic growth phase at 42 hours and the entry into the stationary phase at 96 hours.

[0045] Example 3 strains Vreelandella titanicae Degradation performance of strain SOB56 for nitrate nitrogen and test methods (1) Determination of nitrate nitrogen degradation capacity: Halomonas bacteria were tested. Vreelandella titanicae strain SOB56 was inoculated into aerobic denitrification medium, and samples were taken and centrifuged at regular intervals. The absorbance values ​​at 220 nm and 275 nm were measured using a UV spectrophotometer. The corresponding nitrate nitrogen concentration was calculated by referring to the standard curve. The absorbance value at 540 nm was measured using a UV spectrophotometer. The corresponding nitrite nitrogen concentration was calculated by referring to the standard curve.

[0046] (2) Utilization characteristics of nitrate nitrogen: Inoculate the inoculum into a 250 mL Erlenmeyer flask containing 200 mL of the above aerobic denitrification medium, shake thoroughly, and then incubate in an air-cooled shaker at 30 ℃ and 160 rpm. Take samples at regular intervals to determine the NH4+ content in the supernatant. + –N, NH2OH–N, NO3 — -N、NO2 — –N, TN and OD 600 Value. See results. Figure 4 ,according to Figure 4 The results show Vreelandella titanicae strain SOB56 entered the logarithmic phase after a lag phase of 42 hours, and then entered the stationary phase after another 42 hours of logarithmic phase. — The removal rate of nitrogen reaches its maximum at 60-72 h. A small amount of ammonia nitrogen accumulates during the entire process, while the accumulation of nitrite nitrogen is relatively small.

[0047] (3) According to Figure 4 As a result, when the strain used nitrate nitrogen as the sole nitrogen source, nitrate nitrogen degradation reached 25.67% after 42 h, 81.08% after 60 h, and 94.22% after 96 h. This is consistent with OD... 600 The change corresponds to the entry into the logarithmic growth phase at 42 hours and the entry into the stationary phase at 96 hours.

[0048] Example 4 strains Vreelandella titanicae Domestication of strain SOB56 The inoculum was inoculated into a heterotrophic nitrification medium (salinity 10%: NaCl 100 g·L⁻¹). -1 Salinity 15%: NaCl 150g·L -1 After thorough mixing, the mixture was incubated in an air-controlled shaker at 30 ℃ and 160 rpm. After two and three weeks of incubation, the strain reached a stable growth phase, with an ammonia nitrogen removal rate of 99%.

[0049] If the inorganic salt medium (heterotrophic nitrification medium and aerobic denitrification medium) is replaced with LB medium, the time it takes for the strain to reach the logarithmic phase will be shortened. LB medium is richer in nutrients than inorganic salt medium, so it will grow faster.

[0050] A method for denitrifying high-salinity wastewater includes the following steps: introducing *Haloxylon ammodendron*... Vreelandella titanicae strain SOB56 was inoculated into the wastewater at an inoculum rate of 2.5% (volume ratio) and the wastewater was treated at 30 ℃ and 160 rpm for 72 h.

[0051] A comparison was made between actual wastewater inoculated with the strain and actual wastewater without the strain. It was found that after 72 hours, the ammonia nitrogen in the wastewater inoculated with the strain was almost completely degraded, with a degradation rate approaching 100%, while the ammonia nitrogen in the wastewater without strain SOB56 showed almost no change. This indicates that the strain can effectively degrade nitrogen in actual wastewater. Furthermore, the total nitrogen content in the treated wastewater was less than 1 mg·L⁻¹. -1 This meets the effluent discharge requirements for marine aquaculture wastewater.

[0052] Before the experiment, relevant parameters of the actual seawater were measured, and the results showed that, with Cl... -1 When measuring salinity, the actual salinity of mariculture wastewater is 2.5%, and the total nitrogen concentration is 5.599 mg·L⁻¹. -1 The ammonia nitrogen concentration was 4.769 mg·L⁻¹. -1 The hydroxylamine concentration was 0.094 mg·L⁻¹. -1 The nitrate nitrogen concentration was 0.732 mg·L⁻¹. -1 The concentration of nitrite nitrogen was 0.004 mg·L⁻¹. -1 The total nitrogen concentration after degradation was 0.805 mg·L⁻¹. -1 The degradation rate reached 85.6%.

[0053] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for cultivating and acclimatizing salt-tolerant heterotrophic nitrifying and denitrifying microbial agents, characterized in that, Includes the following steps: The acclimatization process is completed when the *Haloxymonas* seed culture is inoculated into a heterotrophic nitrification medium and cultured until the strain reaches a stable growth period.

2. The cultivation and domestication method according to claim 1, characterized in that, The halometa is halometa. Vreelandella titanicae strain SOB56 was deposited on December 19, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC67508.

3. The cultivation and domestication method according to claim 1, characterized in that, The heterotrophic nitrification medium consists of: (NH4)2SO4 0.4717 g·L⁻¹ -1 Sodium acetate 3.417 g·L -1 5 mL of vitamin solution and 5 mL of trace element solution, pH 7.1±0.2, sterilized by moist heat at 121 ℃ for 30 min.

4. The cultivation and domestication method according to claim 1, characterized in that, The cultivation conditions include: cultivation in an air-controlled shaker at 30 ℃ and 160 rpm.

5. The application of the halomonas bacteria obtained by the cultivation and domestication method according to any one of claims 1-3 in the treatment of marine aquaculture wastewater.

6. The application according to claim 4, characterized in that, Halomonas bacteria were used to treat marine aquaculture wastewater to reduce its ammonia nitrogen and nitrate nitrogen content.

7. A method for treating marine aquaculture wastewater, characterized in that, The halometabolites obtained by the cultivation and domestication method according to any one of claims 1-3 are used to treat marine aquaculture wastewater to reduce its ammonia nitrogen and nitrate nitrogen content.

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