Marinobacter hydrocarbonoides SN-1 as well as culture method and application thereof

Abstract

The invention discloses marinobacter hydrocarbonoclastici SN-1 and a culture method and application thereof, the marinobacter hydrocarbonoclastici SN-1 is marinobacter hydrocarbonoclastici SN-1, is preserved in China Center for Type Culture Collection on August 7, 2024, is located in Wuhan University, Wuhan, China, and has a preservation number of CCTCC NO: M 20241755, and the preservation number of the marinobacter hydrocarbonoclastici SN-1 is CCTCC NO: M 20241755. The invention also discloses application of the marinobacter hydrocarbonoides SN-1 or the microbial inoculum in degradation of ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and / or active phosphate concentration in seawater. The seawater body is a mariculture water body, mariculture tail water or an offshore water body, the salinity of the seawater body is 10-40, and the inoculum size of the mariculture SN-1 is 2%. The marinobacter hydrocarbonoides SN-1 disclosed by the invention can remove ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, total nitrogen, active phosphate and total phosphorus in a seawater water body in an aerobic environment with relatively high salinity, has no toxic effect on aquatic animals, and can be applied to improvement and ecological restoration of a seawater water environment.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a hydrocarbon-removing Marinobacterium SN-1 and a cultivation method and application thereof. Background Art

[0002] At present, the ecological environment of my country's oceans is basically stable, but the pressure of land-based pollution discharge in coastal waters and the frequent occurrence of marine environmental disasters are still prominent. Nitrogen and phosphorus are characteristic pollutants of eutrophication of water bodies, mainly in the form of nitrate (NO3 – ) and phosphate (PO4 3– ) in aquatic ecosystems. The remaining food and excrement in the water are converted into ammonia nitrogen (NH4 + ), nitrate nitrogen (NO3 – ) and nitrite nitrogen (NO2 – ) and other inorganic nitrogen, and excessive nitrogen content will cause serious environmental pollution problems, such as eutrophication of water bodies and red tide disasters, which will not only endanger the growth and reproduction of organisms in the water body, cause environmental pollution and economic losses, but also endanger human life and health and the balance of the ecological environment. It is imperative to study various nearshore seawater pollution treatment and ecological restoration technologies. Eutrophication of water bodies is a key issue in the current water environment protection work. Using microorganisms to remove nitrogen and phosphorus to repair eutrophic water bodies has the characteristics of high efficiency, low consumption and no secondary pollution, and has become an important technical approach for ecological restoration of eutrophic water bodies.

[0003] In recent years, the emergence of heterotrophic nitrification-aerobic denitrification bacteria has broken the limitations of traditional biological processes, especially the discovery of some strains that have the ability to remove phosphorus at the same time, which can achieve the simultaneous and efficient removal of nitrogen and phosphorus pollutants in water bodies under aerobic conditions. However, the current research on heterotrophic nitrification-aerobic denitrification bacteria, especially the ability to simultaneously remove nitrogen and phosphorus, is still in its initial stage, and the understanding of its practical application is not comprehensive enough. Therefore, in order to promote the treatment of excessive discharge of marine aquaculture tail water and the protection and restoration of the ecological environment in coastal waters, it is urgent to develop seawater denitrifying phosphorus-accumulating bacteria that are harmless to aquatic animals. Summary of the invention

[0004] In view of the above technical problems, the present invention provides a hydrocarbon-removing Bacillus SN-1 and a cultivation method and application thereof. The strain can remove ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, total nitrogen, active phosphate and total phosphorus in seawater under high salinity and aerobic environment, and can especially efficiently remove inorganic nitrogen and active phosphate, and is non-toxic to humans and farmed organisms; it has the advantages of good removal effect, high efficiency, and no secondary pollution; at the same time, it has also achieved good application results in the ecological restoration of the water environment and water quality improvement of nearshore bay waters.

[0005] The present invention is achieved in that:

[0006] The first object of the present invention is to provide a hydrocarbon-removing Marinobacter SN-1, wherein the hydrocarbon-removing Marinobacter SN-1 is a hydrocarbon-removing Marinobacter (Marinobacter hydrocarbonoclasticus) SN-1, which was deposited in the China Center for Type Culture Collection on August 7, 2024, located at Wuhan University, Wuhan, China, and its deposit number is CCTCC NO: M 20241755.

[0007] The second object of the present invention is to provide a bacterial agent, which comprises the hydrocarbon-removing Marinobacterium SN-1 or its fermentation liquid as claimed in claim 1.

[0008] The third object of the present invention is to provide the use of the hydrocarbon-removing Marinobacterium SN-1 or the bacterial agent in seawater.

[0009] Furthermore, the hydrocarbon-removing Marinobacterium SN-1 or the bacterial agent is used to degrade the concentration of ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and / or active phosphate in seawater.

[0010] Furthermore, the seawater body is a seawater aquaculture water body, seawater aquaculture tail water or a nearshore water body.

[0011] Furthermore, the salinity of the seawater body is 10-40.

[0012] Furthermore, the salinity of the seawater body is 22±1.

[0013] Furthermore, the inoculation amount of the hydrocarbon-removing Marinobacterium SN-1 is 2%.

[0014] The fourth object of the present invention is to provide the use of the hydrocarbon-removing Marinobacterium SN-1 or the bacterial agent in the preparation of a halophilic microecological preparation for degrading ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and / or active phosphate in seawater.

[0015] The fifth object of the present invention is to provide a method for culturing the hydrocarbon-removing Marinobacterium SN-1, wherein the hydrocarbon-removing Marinobacterium SN-1 is placed in a seawater LB liquid culture medium and cultured continuously for 20 hours at 28°C and 180r / min.

[0016] The main differences and advantages of the hydrocarbon-removing Marinobacterium SD-1 of the present invention and the strains in the prior art are:

[0017] (1) The effect of removing inorganic nitrogen and active phosphate by the hydrocarbon-removing Marinobacterium SN-1 of the present invention is significantly better than that of existing strains, and can achieve the effect of quickly removing inorganic nitrogen and active phosphate in a short time, and can also efficiently remove total nitrogen and total phosphorus in water.

[0018] (2) The hydrocarbon-removing Marinobacterium SN-1 of the present invention has salt-tolerant nitrification-denitrification phosphorus accumulation ability and has a significant effect on nitrogen and phosphorus removal in a high salinity environment.

[0019] (3) The hydrocarbon-removing Bacillus SN-1 of the present invention is isolated from the marine aquatic economic animal breeding environment and is applied to seawater aquaculture water, aquaculture tail water or nearshore waters without any toxic effects on aquatic animals. It can be widely used in more types of marine aquatic economic animal breeding scenarios.

[0020] In summary, the hydrocarbon-removing Sea Bacillus SN-1 of the present invention is an aerobic heterotrophic bacterium, which can have an excellent degradation effect on the concentrations of ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, total nitrogen, active phosphate and total phosphorus in water under seawater conditions, and can especially efficiently remove inorganic nitrogen and active phosphate, has no toxic effect on aquatic animals, has the advantages of wide application conditions, good removal effect, high efficiency, and no secondary pollution. It can be used to improve the environment of marine aquaculture water, aquaculture tail water or nearshore waters under high salinity and alkaline conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a phylogenetic tree of the hydrocarbon-removing Marinobacterium SN-116S rDNA of the present invention.

[0022] Figure 2 This is the growth curve of the hydrocarbon-removing Marinobacterium SN-1 of the present invention. DETAILED DESCRIPTION

[0023] In order to better understand the present invention, the present invention is further described in detail below in conjunction with the embodiments and drawings. However, those skilled in the art understand that the following embodiments are not limitations on the scope of protection of the present invention, and any changes and modifications made on the basis of the present invention are within the scope of protection of the present invention.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0026] Example 1 Isolation and screening of hydrocarbon-removing Marinobacterium SN-1

[0027] S1. Sampling: The strain samples for this experiment were pond activated sludge, collected from a pond in an aquaculture farm (seawater) in Zhangzhou City, Fujian Province. The collected sludge was promptly stored in a 4°C refrigerator for later use.

[0028] S2. Enrichment culture: Weigh 10g of sludge sample, add 100mL of sterile water and stir evenly to mix the sludge and water. Let it stand for 5 minutes, draw 10mL of supernatant, put it into 100mL of YG sterile culture medium, and enrich and culture it at 28℃ and 180r / min for 2 days.

[0029] Enrichment culture medium: MgSO4·7H2O 0.1g, NaH2PO4 0.2 g, K2HPO4 0.5 g, CaCO3 1 g, FeSO4 0.1g, NH4Cl 2.5g, glucose 1.25g, NaCl 30g, H2O 1L, add 2% agar powder to solid culture medium, pH 7.4-7.5, sterilize at 121℃ for 30min.

[0030] S3. Isolation and purification: 100 μL of the enriched culture solution was added to a 1.5 mL centrifuge tube containing 900 μL of sterile saline, mixed, and made into 10 -1 -10 -7 Different dilution gradients of bacterial suspension. Then take 10 -5 , 10 -6 and 10 -7 200 μL of three dilution multiples of bacterial suspension was evenly spread in a solid enrichment medium with a triangular glass rod. The diluted and spread enrichment culture was inverted in a 28°C constant temperature shaker until it grew into obvious colonies visible to the naked eye. Single colonies with different colony morphology, size and color were selected from the culture medium with an inoculation loop. The selected colonies were separated by streaking on a new solid separation medium (DM), and continued to be inverted in a 28°C constant temperature shaker. The streaking separation and purification were repeated many times until a single colony with consistent colony morphology and no infection by other bacteria was obtained. The purified single colony was inoculated into the LB slant solid culture medium and stored in a refrigerator at 4°C.

[0031] Separation medium (DM): NaNO3, 0.84 g, KH2PO4 1.5 g, Na2HPO4 4.0 g, MgSO4·7H2O 0.2 g, glucose 1.5 g, pH 7.4-7.5. For solid culture medium, add 2% agar powder and sterilize at 121°C for 30 min.

[0032] LB medium: NaCl 30 g, yeast extract 5 g, peptone 10 g, H2O 1 L, pH 7.5, sterilization at 121 °C for 30 min.

[0033] S4. Primary screening, rescreening and preservation: Inoculate the single colony stored in the slant culture medium on the bromothymol blue (BTB) culture medium, invert and culture in a constant temperature incubator at 28°C for 2-3 days, observe the color of the colonies, and retain the strains that turn the BTB culture medium blue. Inoculate the retained strains screened by BTB into LB liquid culture medium to make seed liquid, inoculate 2% into 200mL of denitrification polyphosphate culture medium, and culture in a constant temperature shaker at 28°C and 180r / min for 1 day. Determine the OD of the bacterial suspension 600 The bacterial suspension was then centrifuged at 28°C and 8000r / min for 10 min, and the supernatant was taken to detect the removal efficiency of each strain for ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and active phosphate, and the strain with high efficiency was selected as the target strain.

[0034] Bromothymol blue (BTB) medium: glucose 1 g, KNO3 0.75 g, MgSO4·7H2O 0.15 g, NH4Cl 0.2 g, KH2PO4 0.05 g, trace element solution 250 mL, NaCl 30 g, agar 20 g, H2O 950 mL, pH 7.5, sterilization at 121 °C for 30 min.

[0035] Denitrification polyphosphate culture medium: glucose 1g, KNO30.75 g, MgSO4·7H2O 0.15g, NH4Cl 0.2g, KH2PO40.05 g, NaCl 30g, H2O 1L.

[0036] Trace element solution: EDTA 10g, ZnSO4 0.2 g, MnCl2·4H2O 1.2g, FeSO4·7H2O 1.0g, CuSO4·5H2O 0.5g, CoCl2·6H2O 0.3g, Na2MoO4·2H2O 0.2g, CaCl2 0.1 g, H2O 1L.

[0037] Example 2 Identification of the hydrocarbon-removing Marinobacterium SN-1

[0038] The purified bacteria were extracted with TaKaRa MiniBEST Bacteria Genomic DNA Extraction Kit Ver. 3.0 to extract the genomic DNA of the strain. The universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3' (as shown in SEQ ID NO. 1)) and 1492R (5'-GGTTACCTTGTTACGACTT-3' (as shown in SEQ ID NO. 2)) of the 16S rDNA sequence were used as PCR reaction primers, and PCR amplification was performed using the 2×TsingKE Master Mix system. The PCR reaction system was: genomic DNA 1μL, 2×TsingKE Master Mix 25μL, 27F Primer (10μM) 1μL, 1492RPrimer (10μM) 1μL, dH2O 22μL. The PCR reaction conditions were: pre-denaturation at 94°C for 10 min, 30 cycles (94°C for 30 s, 55°C for 30 s, 72°C for 1.5 min), and extension at 72°C for 10 min. The obtained PCR products were purified and sent to Shanghai Biotech Co., Ltd. for sequencing.

[0039] The 16S rDNA sequence of strain SN-1 (1506 bp, as shown in SEQ ID NO.3) was uploaded to the NCBI database and subjected to BLAST homologous sequence comparison. The results showed that the similarity with Marinobacter hydrocarbonoclasticus strain NY-4 (JN 903898.1) and Marinobacter hydrocarbonoclasticus isolate MARC4F (DQ768638.1) was higher than 99.87%. The phylogenetic tree constructed by MEGA11 was shown in Figure 2. Figure 1As shown, the results show that the SN-1 strain is in the same branch of the evolutionary tree as the existing Marinobacter hydrocarbonoclasticus strain NY-4 (JN903898.1) in the NCBI database and is closest. The 16S rDNA sequence of the SN-1 strain was uploaded to the EzBioCloud database (https: / / www.ezbiocloud.net / ), and the results showed that the similarity between the SN-1 strain and the Marinobacterhydrocarbonoclasticus strain NY-4 (JN903898.1) in the 16SrRNA sequence database was as high as 99.87%. Combined with the above analysis results, the strain was identified as Marinobacter hydrocarbonoclasticus SN-1 and named Marinobacter hydrocarbonoclasticus SN-1.

[0040] Example 3 Growth curve of the hydrocarbon-removing Marinobacterium SN-1

[0041] The pure SN-1 strain was selected and inoculated into sterilized LB liquid medium and cultured at 28°C and 180 r / min for 12 h to prepare the seed solution. 600 The value was adjusted to 0.1-0.2, and added to a 96-well plate at a 2% addition amount. Six replicates were set for each sample. The absorbance of the bacterial solution was measured every hour at a wavelength of 600 nm using an ELISA reader. The culture plate was cultured at 28°C for 50 hours. 600 The growth curve is drawn with the value as the ordinate and the culture time as the abscissa. Figure 2 As shown, the growth is slow in 0-3h, the value-added begins to accelerate in 3-5h, and the growth enters the logarithmic proliferation phase from 5-18h, and then the value-added slows down. After reaching the maximum value in 20h, it enters the stable phase and shows signs of death after 30h.

[0042] Example 4 Determination of the removal effect of the hydrocarbon-removing Marinobacterium SN-1 on ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, total nitrogen, active phosphate and total phosphorus in aquaculture water at different time periods

[0043] The pure SN-1 strain was selected and inoculated into the sterilized LB liquid medium, and the seed liquid was prepared at 28°C and 180r / min for 20h. With the tail water of sea eel culture as the background, SN-1 was inoculated into 1L of the tail water at a 2% inoculation amount, and 1g of anhydrous glucose was added per liter of the tail water as the carbon source, pH 7.5. The culture was shaken at 28°C and 180r / min, and samples were collected at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24h, respectively, and centrifuged at 4°C and 8000r / min. The concentrations of ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, total nitrogen, active phosphate and total phosphorus in the supernatant were measured. The results are shown in Tables 1, 2, 3, 4, 5, 6 and 7, among which Table 4 is the result of the determination of inorganic nitrogen in the supernatant.

[0044] As shown in Tables 1 to 7, when the treatment time was 2 hours, the removal rate of ammonia nitrogen by the SN-1 strain reached 92.8%, and the removal rate of ammonia nitrogen reached a maximum of 95.7% after 12 hours; the removal rate of nitrite nitrogen reached 78.8% at 4 hours, and the removal rate of nitrite nitrogen reached a maximum of 97.0% after 16 hours; the removal rate of nitrate nitrogen by the SN-1 strain reached 77.9% at 4 hours, 95.0% at 18 hours, and 98.5% at 22 hours; the removal rate of inorganic nitrogen reached 85.2% at 2 hours, and reached a maximum of 94.1% at 12 hours; the removal rate of total nitrogen reached 61.6% at 10 hours, and reached 92.4% at 20 hours. The removal rate of active phosphate by the SN-1 strain reached 94.5% at 2 hours, and reached a maximum of 97.7% at 12 hours; the removal rate of total phosphorus reached 61.9% at 4 hours, and reached 73.0% at 8 hours.

[0045] Compared with another denitrification and phosphorus reduction strain SD-1 developed in the laboratory, when the treatment time is 2h, the removal rate of ammonia nitrogen is 81.8%; the removal rate of nitrite nitrogen is 75.2% at 4h and 83.6% at 16h; the removal rate of nitrate nitrogen is 68.1% at 4h, 91.0% at 18h and 86.9 at 22h; the removal rate of active phosphate is 90.5% at 6h; the treatment effect of SN-1 strain on ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and active phosphate in water is better than that of strain SD-1, that is, it has a good ability to remove inorganic nitrogen and active phosphate. The oleovorans Pseudomonas (Pseudomonas oleovoranssub sp.oleovorans) YIM B02942 published in patent CN202410830617.X achieved a maximum nitrate nitrogen removal rate of 91.1% in 44 hours. The SN-1 strain has a nitrate nitrogen removal rate of up to 93.2%. Compared with the oleovorans Pseudomonas YIM B02942, it has a better denitrification effect, and can also efficiently remove ammonia nitrogen, nitrite nitrogen and active phosphate. This shows that the SN-1 strain, as a halophilic and alkali-resistant strain, has the function of efficiently and quickly removing ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and active phosphate, and can also efficiently remove total nitrogen and total phosphorus, and has good application prospects.

[0046] Table 1 Determination of the removal effect of ammonia nitrogen in aquaculture water by Marine Bacillus SN-1 at different time periods

[0047]

[0048] Table 2 Determination of the removal effect of nitrite nitrogen in aquaculture water by removing hydrocarbons from Marinobacterium SN-1 at different time periods

[0049]

[0050] Table 3 Determination of the removal effect of nitrate nitrogen in aquaculture water by Marinobacterium SN-1 at different time periods

[0051]

[0052] Table 4 Determination of the removal effect of inorganic nitrogen in aquaculture water by Marinobacterium SN-1 at different time periods

[0053]

[0054] Table 5 Determination of the removal effect of total nitrogen in aquaculture water by Marinobacterium SN-1 at different time periods

[0055]

[0056] Table 6 Determination of the removal effect of active phosphate in aquaculture water by Marinobacterium SN-1 at different time periods

[0057]

[0058] Table 7 Determination of the removal effect of total phosphorus in aquaculture water by removing hydrocarbons by Marinobacterium SN-1 at different time periods

[0059]

[0060] Example 5 Determination of the removal effect of Marinobacterium SN-1 on ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, total nitrogen, active phosphate and total phosphorus in aquaculture water under different pH conditions

[0061] The pure SN-1 strain was selected and inoculated into the sterilized LB liquid medium, and the seed liquid was prepared by culturing at 28°C and 180r / min for 20h. The tail water of sea eel culture was used as the background, and the SN-1 strain was inoculated into 1L of the tail water of culture at a 2% inoculation amount, and 1g of anhydrous glucose was added per liter of the tail water of culture as a carbon source. Seven treatment groups with different pH were set up: 6, 6.5, 7, 7.5, 8, 8.5 and 9, and the samples were collected after culturing at 28°C and 7.0mg / L of dissolved oxygen for 20h, and the samples were centrifuged at 4°C and 8000r / min to measure the concentrations of ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, total nitrogen, active phosphate and total phosphorus in the supernatant. The results are shown in Tables 8, 9, 10, 11, 12, 13 and 14, among which Table 11 is the result of the determination of inorganic nitrogen in the supernatant.

[0062] The denitrification and phosphorus reduction effects of SN-1 strain were also different under the influence of pH. Under the conditions of background ammonia nitrogen concentration of 0.852±0.09mg / L, nitrite nitrogen concentration of 0.095±0.04mg / L, nitrate nitrogen concentration of 2.885±0.11mg / L, inorganic nitrogen concentration of 3.832±0.09mg / L, total nitrogen concentration of 43.090±0.14mg / L, active phosphate concentration of 1.798±0.02mg / L and total phosphorus concentration of 5.101±0.08mg / L, when pH was 6-7.5, SN-1 strain had a significant effect on ammonia nitrogen and nitrite nitrogen. The removal rates of nitrate nitrogen, total nitrogen, active phosphate and total phosphorus all tended to increase with the increase of pH; when the pH was 7.5, the removal rate of ammonia nitrogen by the SN-1 strain reached a maximum of 97.0%, the removal rate of nitrite nitrogen reached a maximum of 93.3%, the removal rate of nitrate nitrogen reached a maximum of 99.9%, the removal rate of inorganic nitrogen reached a maximum of 99.1%, the removal rate of total nitrogen reached 46.2%, the removal rate of active phosphate reached a maximum of 95.4%, and the removal rate of total phosphorus reached a maximum of 76.7%, indicating that pH 7.5 is a suitable culture environment for the SN-1 strain.

[0063] The simultaneous nitrification and denitrification strain LF10 disclosed in Patent No. 202410415484.X can achieve a nitrate nitrogen removal rate of about 90.0% after being cultured for 48 hours under the condition of pH 8.0, and the removal rates of ammonia nitrogen and nitrite nitrogen are 95.4% and 75.5%, respectively. After being cultured for 20 hours under the condition of salinity 7.5, the SN-1 strain of the present invention has a removal rate of 97.0%, 93.3% and 99.9% for ammonia nitrogen, nitrite nitrogen and nitrate nitrogen, and a removal rate of 99.1% for inorganic nitrogen. At the same time, the removal rates of active phosphate, total phosphorus and total nitrogen can reach 95.4%, 50.8% and 76.7%, respectively. This shows that compared with the LF10 strain, the SN-1 strain not only has a significantly higher removal rate of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen, but also achieves efficient removal of active phosphate, total phosphorus and total nitrogen.

[0064] Table 8 Determination of the removal effect of ammonia nitrogen in aquaculture water by Marine Bacillus SN-1 at different pH

[0065]

[0066] Table 9 Determination of the removal effect of nitrite nitrogen in aquaculture water by removing hydrocarbons from Marinobacterium SN-1 at different pH

[0067]

[0068] Table 10 Determination of the removal effect of nitrate nitrogen in aquaculture water by removing hydrocarbons from Marinobacterium SN-1 at different pH values

[0069]

[0070] Table 11 Determination of the removal effect of inorganic nitrogen in aquaculture water by Marine Bacillus SN-1 at different pH

[0071]

[0072]

[0073] Table 12 Determination of the removal effect of total nitrogen in aquaculture water by Marine Bacillus SN-1 at different pH

[0074]

[0075] Table 13 Determination of the removal effect of active phosphate in aquaculture water by removing hydrocarbons from Marinobacterium SN-1 at different pH values

[0076]

[0077] Table 14 Determination of the removal effect of total phosphorus in aquaculture water by removing hydrocarbons by Marinobacterium SN-1 at different pH values

[0078]

[0079] Example 6 Determination of the removal effect of hydrocarbon-removing Marinobacterium SN-1 on ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, total nitrogen, active phosphate and total phosphorus in aquaculture water at different salinities

[0080] The pure SN-1 strain was selected and inoculated into the sterilized LB liquid medium, and the seed liquid was prepared by culturing at 28°C and 180r / min for 20h. The tail water of sea eel culture was used as the background, and the SN-1 strain was inoculated into 1L of the tail water at a 2% inoculation rate. 1g of anhydrous glucose was added per liter of the tail water as the carbon source, pH 7.5, and 7 treatment groups with different salinities were set: 0, 2, 8, 16, 32, 35 and 40. After culturing for 20h under 7.0mg / L dissolved oxygen conditions, the samples were collected and centrifuged at 4°C and 8000r / min to measure the concentrations of ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, total nitrogen, active phosphate and total phosphorus in the supernatant. The results are shown in Tables 15 to 21.

[0081] Under the conditions of background ammonia nitrogen concentration of 0.852±0.09mg / L, nitrite nitrogen concentration of 0.095±0.04mg / L, nitrate nitrogen concentration of 2.885±0.11mg / L, inorganic nitrogen concentration of 3.832±0.09mg / L, total nitrogen concentration of 44.117±0.14mg / L, active phosphate concentration of 1.798±0.02mg / L and total phosphorus concentration of 5.101±0.08mg / L, the SN-1 strain was active in the presence of ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and iodine at salinity below 22. The removal rates of total nitrogen and total phosphorus were both less than 93.0%. With the increase of salinity, the removal rates of ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, total nitrogen, active phosphate and total phosphorus in aquaculture water by SN-1 strain gradually increased. When the salinity was 22, the removal rates of ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, total nitrogen, active phosphate and total phosphorus were 91.5%, 92.4%, 92.8%, 54.4%, 99.1% and 67.0%, respectively. The removal rate of inorganic nitrogen was 94.0%. When the salinity continued to increase, the removal rate tended to be flat.

[0082] Table 15 Determination of the removal effect of ammonia nitrogen in aquaculture water by removing hydrocarbons from Marinobacterium SN-1 at different salinities

[0083]

[0084] Table 16 Determination of the removal effect of nitrite nitrogen in aquaculture water by removing hydrocarbons from Marinobacterium SN-1 at different salinities

[0085]

[0086] Table 17 Determination of the removal effect of nitrate nitrogen in aquaculture water by removing hydrocarbons from Marinobacterium SN-1 at different salinities

[0087]

[0088] Table 18 Determination of the removal effect of hydrocarbon-removing marine bacillus SN-1 on inorganic nitrogen in aquaculture water at different salinities

[0089]

[0090] Table 19 Determination of the removal effect of total nitrogen in aquaculture water by removing hydrocarbons from Marinobacterium SN-1 at different salinities

[0091]

[0092]

[0093] Table 20 Determination of the removal effect of active phosphate in aquaculture water by removing hydrocarbons from Marinobacterium SN-1 at different salinities

[0094]

[0095] Table 21 Determination of the removal effect of total phosphorus in aquaculture water by removing hydrocarbons from Marinobacterium SN-1 at different salinities

[0096]

[0097] Example 7 Safety testing of the hydrocarbon-removing Marinobacterium SN-1 in aquaculture applications

[0098] The pure SN-1 strain was selected and inoculated into the sterilized LB liquid medium, and cultured in a constant temperature oscillator at 28°C and 180 r / min for 20 h to prepare the seed solution. 6 The inoculation rate of 1.5 cfu / L was added to the aquaculture water treatment biofilter of the seawater circulating water Australian eel aquaculture system, and anhydrous glucose was added as a supplementary carbon source. The circulating water Australian eel aquaculture system was fed and managed normally at a feed rate of 1.1% for 15 days. During the Australian eel aquaculture period, the Australian eels grew healthily without illness or death. The results show that Marinobacter hydrocarbonoclasticus SN-1 has a relatively stable safety in the process of nitrogen removal and phosphorus reduction in Australian eel aquaculture. According to relevant studies, the biological hazard level of Marinobacter hydrocarbonoclasticus is: Category 4, no pathogenic objects.

[0099] In summary, the hydrocarbon-removing sea bacillus SN-1 can efficiently remove ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and active phosphate in seawater, and can remove total nitrogen and total phosphorus at the same time, without toxic effects on aquatic animals, and has the advantages of wide application conditions, good removal effect, high efficiency, and no secondary pollution. It can be used to improve the seawater environment under high salinity and alkaline conditions. Compared with the prior art, the denitrification and phosphorus reduction speed and removal rate of the hydrocarbon-removing sea bacillus SN-1 of the present invention are significantly better than similar strains reported in the prior art. The SN-1 strain can achieve rapid and efficient denitrification and phosphorus reduction in a short time, especially the denitrification rate is significantly higher. On the other hand, the hydrocarbon-removing sea bacillus SN-1 has salt tolerance and simultaneous nitrification-denitrification polyphosphate capabilities, and has a significant effect on the removal of nitrogen and phosphorus in the seawater environment. At the same time, it is separated from the aquatic economic animal breeding environment and applied to seawater bodies. It has no toxic effects on aquatic animals and can be widely used in more types of aquatic economic animal breeding scenarios.

[0100] Although the specific implementation modes of the present invention are described above, those skilled in the art should understand that the specific implementation modes described are only illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A hydrocarbon-removing Marinobacterium SN-1, characterized in that: The Marinobacter hydrocarbonoclasticus SN-1 is Marinobacter hydrocarbonoclasticus SN-1, which was deposited in the China Center for Type Culture Collection on August 7, 2024, located at Wuhan University, Wuhan, China, and its deposit number is CCTCC NO: M 20241755.

2. A bacterial agent, characterized in that: The bacterial agent includes the hydrocarbon-removing Marinobacterium SN-1 or its fermentation liquid as described in claim 1.

3. Use of the hydrocarbon-removing Marinobacterium SN-1 according to claim 1 or the bacterial agent according to claim 2 in seawater.

4. The use according to claim 3, characterized in that: The application of the hydrocarbon-removing Marinobacterium SN-1 or the bacterial agent in degrading the concentration of ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and / or active phosphate in seawater.

5. The use according to claim 4, characterized in that: The seawater body is a seawater aquaculture water body, seawater aquaculture tail water or a nearshore sea water body.

6. The use according to claim 4, characterized in that: The salinity of the seawater body is 10-40.

7. The use according to claim 6, characterized in that: The salinity of the seawater body is 22±1.

8. The use according to claim 4, characterized in that: The inoculation amount of the hydrocarbon-removing Marinobacterium SN-1 is 2%.

9. Use of the hydrocarbon-removing Marinobacterium SN-1 according to claim 1 or the bacterial agent according to claim 2 in the preparation of a halophilic microecological preparation for degrading ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and / or active phosphate in seawater.

10. The method for culturing the hydrocarbon-removing Marinobacterium SN-1 according to claim 1, characterized in that: The hydrocarbon-removing Marinobacterium SN-1 was placed in a seawater LB liquid culture medium and cultured continuously for 20 hours at 28° C. and 180 r / min.

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