A strain of alkaliphilic halomonas with salt-tolerant aerobic denitrification characteristics and its application

Abstract

The present invention discloses a strain of alkaliphilic Halomonas bacteria with salt-tolerant aerobic denitrification properties and its application, relating to the field of environmental protection technology. The alkaliphilic Halomonas bacteria is designated HYJ1 and deposited with CGMCC No. 27111. The strain HYJ1 is most suitable for a carbon source of sodium acetate, with an optimal C / N ratio of 4-8 and an optimal pH of 9.0. Denitrification efficiency is highest at a salinity of 2%, and the optimal oxygen volume ratio range is 10% to 30%.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental protection, and more particularly to a strain of alkaliphilic halomonas with salt-tolerant aerobic denitrification characteristics and its application. Background Art

[0002] As the world's population increases, marine aquaculture is becoming increasingly common around the world. Marine aquaculture is mainly concentrated in China's eastern coastal provinces, such as Liaoning, Zhejiang, and Shandong. Pollutants in marine aquaculture tailwater mainly come from feed, animal feces, fish mucus, and chemicals, mainly from excessive feed. Some of the nitrogen and phosphorus in synthetic feed will be absorbed by fish, and the rest will enter the water body and its sediments. Therefore, marine aquaculture tailwater mainly contains NH4 + -N and NO2 - -N, NO3 - -N, phosphates, organic matter, etc. Compared to municipal sewage, marine aquaculture wastewater has the characteristics of high salinity, large volume, low pollutant concentration, and low temperature. If untreated marine aquaculture wastewater is discharged directly into the sea, it will not only pollute the receiving water bodies and affect the health of aquatic life, but also pose an increasing threat to the marine ecosystem. Therefore, denitrification treatment of marine aquaculture wastewater is particularly important.

[0003] Biological methods are a well-established wastewater treatment technology favored for their wide adaptability. However, traditional biological denitrification processes are often ineffective in treating saline wastewater. This is primarily due to the high osmotic pressure generated by the high salinity environment, which causes dehydration and plasmolysis of microbial cells, inhibiting enzyme activity and growth, and ultimately leading to their death. Identifying salt-tolerant and halophilic bacteria capable of treating saline wastewater and studying their optimal environmental conditions for pollutant degradation will be crucial for improving the efficiency of marine aquaculture tailwater treatment and developing novel treatment processes.

[0004] Therefore, providing an aerobic denitrifying bacterial strain with high denitrification performance and adaptability to the high-salinity environment of seawater is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides a strain of alkaliphilic halomonas with salt-tolerant aerobic denitrification characteristics and its application.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A strain of alkaliphilic halomonas having salt-tolerant aerobic denitrification properties, the alkaliphilic halomonas being named HYJ1, belonging to the alkaliphilic halomonas, with a Latin name: Halomonas alkaliphila, deposited in the General Microbiology Center of China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The registration number is CGMCC No. 27111, and the preservation date is April 13, 2023.

[0008] Furthermore, the liquid salt-tolerant denitrification culture medium for culturing the alkaliphilic halomonas is composed of: 1.71g sodium acetate, 0.6gNaNO3, 1.6gK2HPO4, 30g NaCl, 0.02gCaCl2, 0.1gMgSO4•7H2O, 0.005gFeSO4•7H2O, 0.1gMgSO4•7H2O, and 0.1mL trace metal mother liquor.

[0009] Furthermore, the trace metal mother liquor comprises: MgSO4•H2O 0.0344 g / L, H3BO3 0.05 g / L, ZnCl2 0.07 g / L, Na2MoO4•2H2O 0.0726 g / L, CaCl2•2H2O 0.02 g / L, NiCl2•6H2O 0.024 g / L, CoCl2•6H2O 0.08 g / L, and FeSO47H2O 1.0 g / L.

[0010] Application of a salt-tolerant aerobic denitrifying alkaliphilic halomonas strain in wastewater purification.

[0011] It can be seen from the above technical solution that compared with the prior art, the beneficial effects of the present invention are:

[0012] The present invention successfully isolated a salt-tolerant aerobic denitrifying strain HYJ1 with high denitrification performance from the biofilm of a marine aquaculture bioreactor in Hainan. The bacterium was identified as an alkaliphilic Halomonas by 16S rDNA sequence analysis. Simultaneously, the effects of various factors on its denitrification performance were explored under different carbon sources, salinity, carbon-nitrogen ratio, pH, and oxygen demand (DO) conditions. The most suitable carbon source for strain HYJ1 was sodium acetate, with an optimal C / N ratio of 4-8, an optimal pH of 9.0, and the highest denitrification efficiency at a salinity of 2%, with an optimal oxygen volume ratio range of 10% to 30%. The discovery of this strain's salt-tolerant aerobic denitrification properties provides a theoretical basis for achieving efficient denitrification treatment of marine aquaculture wastewater and simultaneous nitrification and denitrification processes in sewage treatment plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0014] Figure 1 is the phylogenetic tree of strain HYJ1;

[0015] Figure 2 The effect of conventional carbon sources on aerobic denitrification of the strain;

[0016] Figure 3 The effect of different carbon-nitrogen ratios on the aerobic denitrification effect of the strain;

[0017] Figure 4 The effect of different salinity on aerobic denitrification of the strain;

[0018] Figure 5 The effect of different pH on the aerobic denitrification effect of the strain;

[0019] Figure 6 This is the effect of different dissolved oxygen on the aerobic denitrification effect of the strain. DETAILED DESCRIPTION

[0020] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0021] Experimental Materials:

[0022] 1 L of enrichment medium (liquid salt-tolerant denitrification medium) contains: 1.71 g sodium acetate, 0.6 g NaNO3, 1.6 g K2HPO4, 30 g NaCl, 0.02 g CaCl2, 0.1 g MgSO4•7H2O, 0.005 g FeSO4•7H2O, 0.1 g MgSO4•7H2O, and 0.1 mL trace metal stock solution.

[0023] The trace metal mother solution contains: MgSO4•H2O 0.0344 g / L, H3BO3 0.05 g / L, ZnCl2 0.07 g / L, Na2MoO4•2H2O 0.0726 g / L, CaCl2•2H2O 0.02 g / L, NiCl2•6H2O 0.024 g / L, CoCl2•6H2O 0.08g / L, and FeSO47H2O 1.0 g / L.

[0024] Agar plate culture medium was prepared by adding 1 mL of BTB (dissolved in 1% anhydrous alcohol) and 20 g of agar to the enriched medium, with the remaining ingredients remaining unchanged. All culture media were sterilized at 120°C for 90 minutes to ensure the absence of microbial contamination and cooled to room temperature before use.

[0025] Example 1: Screening, identification and preservation of salt-tolerant aerobic denitrifying bacteria

[0026] (1) Enrichment culture: Biofilm fillers were collected from the marine aquaculture tailwater treatment bioreactor of Charoen Pokphand Aquatic Products (Oriental) Co., Ltd. 20 mL of PBS buffer solution was added to two of the fillers. The fillers were squeezed by hand until the color of the fillers became lighter and closer to the original color. At this point, most of the aerobic denitrifying bacteria attached to the surface of the biofilm fillers had entered the PBS buffer solution.

[0027] Prepare a 100mL serum bottle, add 50mL of the prepared enrichment medium, securely stopper the bottle, and sterilize it in an autoclave. After sterilization, remove the bottle and place the culture medium in a UV sterilizer for 30 minutes. Allow the solution to cool to room temperature. Add 5mL of the PBS eluate to the serum bottle, securely stopper the bottle, and incubate it in a shaker set at 30°C and 130 rpm for approximately 32 hours, until the culture medium in the serum bottle becomes turbid and cannot be seen through.

[0028] The enrichment operation was repeated twice to obtain a bacterial suspension after thrice-enrichment and acclimation.

[0029] (2) Separation and purification: Prepare 5 sterilized test tubes, add 9 mL of sterilized clean and sterile ultrapure water to each test tube, and mark them as test tube No. 1, test tube No. 2, test tube No. 3, test tube No. 4, and test tube No. 5 respectively.

[0030] Add 1 mL of the bacterial suspension, enriched and acclimated three times, to test tube 1. Shake thoroughly, then pipette 1 mL of liquid from test tube 1 and add it to test tube 2. Repeat this process to obtain five different concentrations of bacterial suspension. Then, pipette 50 μL of the bacterial suspension from each test tube and place it onto a sterilized, cooled agar plate. Use a spreader that has been cooled and burned by an alcohol burner to evenly distribute the bacterial suspension. Prepare two replicates for each gradient and label them. Place the culture plates upside down in a constant temperature incubator at 30°C for three days.

[0031] When colonies were observed growing in the culture medium, individual colonies were selected, streaked on plates, and isolated again to obtain pure bacteria. Denitrification experiments were conducted using different pure bacteria at an initial nitrate-nitrogen concentration of 100 mg / L, and the 24-hour nitrate-nitrogen removal rate was calculated. The strain with the best nitrate-nitrogen removal efficiency was screened and identified.

[0032] (3) Identification of bacterial species: Bacterial genomic DNA was extracted using the soil FastDNA SPIN kit (MPbio, America), and 16S rDNA of the strain was amplified by PCR using universal primers (27f and 1492r). The PCR product was sequenced and the sequence data was analyzed using BLAST software. It was found to have high homology with multiple species of the genus Halomonas, and it was preliminarily determined to be a single bacterium. Figure 1 .

[0033] (4) Strain preservation: The highly efficient salt-tolerant aerobic denitrifying strain obtained by screening in the above steps was named HYJ1, which belongs to the alkaliphilic Halomonas species, and its Latin name is: Halomonas alkaliphila It is deposited in the General Microbiology Center of China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The registration number is CGMCC No. 27111 and the collection date is April 13, 2023.

[0034] Example 2: Effect of conventional carbon sources on aerobic denitrification of bacterial strains

[0035] Activation: Prepare 30 mL of liquid salt-tolerant denitrification culture medium, sterilize it at 120 degrees and wait for the liquid to cool to room temperature. Then use a syringe to inject 3 mL of HYJ1 bacterial liquid stored in the refrigerator. Place it in a shaker set at 30°C and 130 r / min for activation culture for 24 hours. Activation is completed when the OD measured at 600 nm is greater than 0.8 (the original transparent liquid becomes milky white and turbid and cannot be seen through).

[0036] Grouping: Clean 12 serum bottles in advance and divide them into 6 groups, with one replicate in each group. Add 30 mL of liquid salt-tolerant denitrification medium to each bottle.

[0037] The carbon sources in each group of bottles were 1.335 g / L methanol, 2.043 g / L sodium citrate, 1.251 g / L glucose, 1.71 g / L sodium acetate, 0.960 g / L ethanol, and 1.6886 g / L sodium succinate, and the other ingredients were the same. After sterilization and cooling to room temperature, 3 mL of activated bacterial solution was inoculated into 12 serum bottles. Then, the culture was maintained at a constant temperature of 30°C and 130 r / min for 30 h, and the nitrate nitrogen content was measured every 2 hours. Note: All operations were performed on a sterile workbench. The results are shown in the figure below. Figure 2 shown.

[0038] The results showed that when sodium acetate was used as the carbon source, the bacteria had the best denitrification performance. After 10 hours, they began to rapidly utilize and degrade nitrates. After 14 hours of culture, all nitrates were removed. When the carbon source components in the culture medium were sodium citrate and ethanol, it took 20 hours to completely remove the nitrate concentration in the culture medium. When sodium succinate was used as the only carbon source, after 26 hours of culture, its nitrate removal rate was only 54%. The results also showed that within 26 hours when methanol and glucose were used as carbon sources, there was no significant change in the nitrate nitrogen content, indicating that the bacteria could not use methanol and glucose for growth and reproduction. So in summary, sodium acetate is the most suitable carbon source.

[0039] Example 3: Effect of different carbon-nitrogen ratios on aerobic denitrification performance of strains

[0040] The activation steps and grouping are the same as in Example 2.

[0041] To investigate the effect of different C / N ratios on the strain's ability to treat saline and high-nitrogen wastewater, the culture media in the 12 serum bottles all used sodium acetate and sodium nitrate as carbon and nitrogen sources, respectively. The C / N ratio was controlled by adjusting the sodium acetate content. The C / N ratios were set to 2, 4, 5, 8, 12, and 20. Activated bacterial solution was then added to each bottle, with a bacterial solution V: medium V ratio of 1:10. The culture was incubated at 130 rpm and 30°C for 26 hours, and the changes in nitrate and nitrogen were monitored every 2 hours. The results are shown in the figure below. Figure 3 shown.

[0042] When the C / N ratio was 4, 5 and 8, the degradation trend of nitrate in nitrogenous wastewater by the strain was basically the same. At 14 h, the degradation of NO3 -The removal rate of NO3-N reached 100%, while when the C / N ratio was 2, the nitrate concentration dropped to 28.42 mg / L after 14 h, and the removal rate was only 70.42%. After that, the nitrate concentration hardly decreased. The possible reason is that the organic matter used for the growth of the strain was exhausted. When the C / N ratio increased to 12, the removal rate of NO3-N reached 100% after 18 h. - The removal rate of -N reached 100%. When the C / N ratio increased from 12 to 20, the nitrate removal rate decreased significantly, and nitrate was completely removed after 26 h. This may be because when the C / N value gradually increased, a large amount of carbon source in the culture medium was utilized, resulting in a rapid decrease in DO and NO3 - -N removal rate decreased. Therefore, for strain HYJ1, the optimal C / N ratio was 4-8.

[0043] Example 4: Effect of different salinities on aerobic denitrification performance of strains

[0044] The activation steps and grouping are the same as in Example 2.

[0045] To investigate the effect of different salinities on the treatment of saline and high-nitrogen wastewater by strain HYJ1, the culture medium components were the same except for the sodium chloride concentration. The sodium chloride concentrations were 0g / L, 10g / L, 20g / L, 30g / L, 40g / L and 50g / L, respectively. At this time, the salinity in the six culture media was guaranteed to be 0%, 1%, 2%, 3%, 4% and 5%, respectively. Then 12 serum bottles were placed in a sterilizer and sterilized at 120℃ for 1h30min. After sterilization, cool to room temperature, and then inoculate 3mL of activated bacterial solution in each serum bottle. Place in a shaker (constant temperature 30℃, 130r / min) and culture for 30h, and measure the nitrate nitrogen content every 2 hours. The results are as follows Figure 4 shown.

[0046] The results showed that when sodium acetate was the only carbon source and the salinity was 1%, 2%, 3%, 4% and 5%, the strain had a strong effect on NO3 - The removal rate of -N can reach 100% within 28 hours, and the denitrification efficiency is the highest when the salinity is 2%. Among them, when the salinity is 2%, the NO3--N concentration in the culture medium has dropped to 0 mg / L within 18 hours, while when the salinity is 1%, the nitrate removal rate reaches 100% after 24 hours of cultivation. Without adding sodium chloride, NO3 - The final removal rate of -N reached 58.8%.

[0047] Example 5: Effect of different pH values ​​on aerobic denitrification of bacterial strains

[0048] The pH value of the environment has a significant impact on the life activities of microorganisms, mainly by causing changes in the charge of the cell membrane, thereby affecting the microorganism's absorption of nutrients. This example examines the ability of bacteria to remove nitrate nitrogen at different times under different pH conditions.

[0049] The activation step was the same as in Example 2, and the grouping was the same as in Example 2. 0.01 mol / L NaOH solution and 3.219% dilute hydrochloric acid were used to adjust the pH. The pH of bottles in groups 1 to 6 were 6, 7, 7.5, 8, 9, and 10, respectively. After sterilization, the bottles were taken out and cooled to room temperature. 3 mL of the activated bacterial solution was inoculated into 12 serum bottles (each bottle contained 30 mL of liquid salt-tolerant denitrification culture medium). The bottles were then placed in a shaker, set to a constant temperature of 30°C and 130 r / min for 30 h, and the nitrate nitrogen content was measured every 2 hours. The results are shown in the figure below. Figure 5 shown.

[0050] The pH range of the culture medium for general bacterial growth is 7.5 to 10. There are significant differences in the degradation rate of nitrate under different pH conditions. In this example, the nitrate removal rate of strain HYJ1 is higher at pH 8.0 to 10.0, and can reach 100% in 28 hours within this range, with the optimal pH being 9.0. When the pH is 6.0 and 7.0, the NO3 - The -N concentration did not change much, further indicating that strain HYJ1 was suitable for survival in an alkaline environment.

[0051] Example 6: Effect of different dissolved oxygen on aerobic denitrification performance of strains

[0052] The activation step was the same as in Example 2, and the grouping was the same as in Example 2. The air in the top of the serum bottle was replaced with pure helium, and then different volume ratios of oxygen were added to the serum bottle to control the dissolved oxygen content in the culture medium, so that the volume of oxygen accounted for 0%, 10%, 20%, 30%, 50%, and 100%, respectively. Similarly, the components of the culture medium were the same, all with sodium acetate as the sole carbon source, a C / N ratio of 5, a pH of 7, and a NaCl concentration of 30 g / L. Only the oxygen ratio was changed. The 12 bottles were placed in a shaker and incubated at 30°C and 130 rpm for 28 hours, and the nitrate concentration was measured.

[0053] Effect of DO on the denitrification performance of strain HYJ1 Figure 6 shown.

[0054] When the oxygen content was 0%, it took 26 hours to completely remove the nitrate concentration. As the oxygen volume ratio increased, the nitrate removal rate also increased significantly. When the oxygen volume ratio was 10%, 20%, and 30%, respectively, the nitrate degradation trend was basically the same, and the nitrate concentration was completely removed after 18 hours of incubation. However, when the oxygen volume ratio increased again, the nitrate removal rate began to decline. Under the conditions of oxygen volume ratios of 50% and 100%, the nitrate removal rate after 18 hours of incubation was only 80.98% and 1.63%. From the above results, it can be seen that different bacteria have different responses to the influence of dissolved oxygen, which reflects the different mechanisms of aerobic denitrification by different bacteria.

[0055] In summary, the optimal oxygen volume ratio range of strain HYJ1 is 10% to 30%.

[0056] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of alkaliphilic Halomonas with salt-tolerant aerobic denitrification characteristics ( Halomonas alkaliphila ), characterized in that, The alkaliphilic halomonas was named HYJ1, and its deposit number was CGMCC No.27111.

2. Use of the alkaliphilic halomonas strain having salt-tolerant aerobic denitrification properties according to claim 1 in wastewater purification.

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