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

Abstract

The invention discloses marinobacter hydrocarbonoclasticus SD-1 and a culture method and application thereof.The marinobacter hydrocarbonoclasticus SD-1 is preserved in the China Center for Type Culture Collection on August 7, 2024, the address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M 20241756; according to the application of the marinobacter hydrocarbonoides SD-1 or the fungicide thereof in degrading the concentration of nitrogen and phosphorus in a seawater body, the seawater body is a mariculture water body, mariculture tail water or an offshore water body; the salinity of the seawater body is 10 to 40; the inoculum size of the marinobacter hydrocarbonoides SD-1 is 1.5%. The marinobacter hydrocarbonoides SD-1 disclosed by the invention can efficiently remove nitrogen and phosphorus in a seawater water body in a relatively high salinity and aerobic environment, 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 SD-1 and a culture method and application thereof. Background Art

[0002] With the rapid development of marine aquaculture and the widespread application of high-density and intensive models, marine aquaculture tailwater has gradually become an important source of pollution in coastal waters. In the process of marine aquaculture, residual bait and metabolites of seafood remain in the water, producing a large amount of aquaculture tailwater. If it is discharged directly into the marine environment without treatment, it is very easy to cause marine eutrophication and endanger the sustainable development of the marine ecological environment. The main pollutants in marine aquaculture tailwater include total nitrogen, ammonia nitrogen, nitrate, nitrite, total phosphorus, active phosphate and suspended solids, etc. It has the characteristics of large water volume, serious excessive discharge of pollutant concentration and high salinity. The removal of total nitrogen and total phosphorus is the focus and difficulty of marine aquaculture tailwater treatment. In response to the above problems, the new microbial denitrification and phosphorus reduction technology established based on the research and development of efficient denitrification and phosphorus reduction functional bacteria has become one of the important methods for removing nitrogen and phosphorus pollution in aquaculture tailwater due to its economic feasibility, high efficiency and environmental protection, and has achieved good water treatment effects.

[0003] Aerobic denitrifying phosphorus accumulating organisms (ADPAO) have the potential to efficiently remove nitrogen and phosphorus because they can simultaneously carry out heterotrophic nitrification, aerobic denitrification and aerobic phosphorus removal, making the denitrification and phosphorus reduction process simpler, faster and more efficient. They have attracted widespread attention and opened up an opportunity for the denitrification and phosphorus reduction treatment of marine aquaculture tail water. Therefore, it is urgent to develop a seawater denitrifying phosphorus accumulating bacteria that is harmless to aquatic economic animals. Summary of the invention

[0004] In response to the above technical problems, the present invention provides a hydrocarbon-removing Bacillus SD-1 and a cultivation method and application thereof. The strain can remove ammonia nitrogen, nitrite, nitrate, total nitrogen, active phosphate and total phosphorus in seawater under high salinity and aerobic environment, and is especially capable of efficiently removing total nitrogen and total phosphorus, 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 in 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 SD-1, wherein the hydrocarbon-removing Marinobacter SD-1 is Marinobacter hydrocarbonoclasticus SD-1, which was deposited in the China Center for Type Culture Collection on August 7, 2024, located at Wuhan University, Wuhan, China, and its collection number is CCTCC NO:M 20241756.

[0007] The second object of the present invention is to provide a bacterial agent, which includes the hydrocarbon-removing Marinobacterium SD-1 or its fermentation liquid.

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

[0009] Furthermore, the hydrocarbon-removing Marinobacterium SD-1 or the bacterial agent is used to degrade the nitrogen and phosphorus concentrations 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 SD-1 is 1.5%.

[0014] The fourth object of the present invention is to provide the use of the hydrocarbon-removing Marinobacterium SD-1 or the bacterial agent in the preparation of a halophilic microecological preparation for degrading nitrogen and phosphorus in seawater.

[0015] The fifth object of the present invention is to provide a method for culturing the hydrocarbon-removing Marinobacterium SD-1, which comprises placing the hydrocarbon-removing Marinobacterium SD-1 in a seawater LB liquid culture medium and culturing it continuously for 24 hours at 30°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 speed of removing total nitrogen and total phosphorus by the hydrocarbon-removing Marinobacterium SD-1 of the present invention is significantly faster than that of existing strains, and can achieve the effect of rapid denitrification in a short time, while being able to remove total phosphorus, and the nitrogen removal effect is more significant.

[0018] (2) The hydrocarbon-removing marine bacillus SD-1 of the present invention has a salt-tolerant and efficient denitrification and phosphorus accumulation function, and has a significant effect on the removal of nitrogen and phosphorus in a water environment with a relatively high salinity.

[0019] (3) The hydrocarbon-removing Bacillus SD-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 and nearshore waters.

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

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

[0022] Figure 2 This is the growth curve of the hydrocarbon-removing Marinobacterium SD-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 SD-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 enrichment culture medium, and enrich and culture it at 30℃ 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, NaNO3 2.47 g, (NH4)2SO4 2g, glucose 1.25g, NaCl 30g, H2O 1L, add 2% agar powder to solid culture medium, pH 7.4-7.8, 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 solid enrichment medium with a triangular glass rod. The diluted and spread YG culture medium was inverted and cultured in a 30°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, and continued to be inverted in a 30°C constant temperature incubator. The streaking separation was 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 4°C refrigerator.

[0031] Isolation medium: NaNO3 0.84 g, KH2PO4 1.5 g, Na2HPO4 4.0 g, MgSO4 0.2 g, FeSO4·H2O 0.054 g, glucose 1.25 g, agar powder 20 g, H2O 1 L, pH 7.4-7.8, sterilization 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 30°C constant temperature incubator for 2-3 days, observe the color of the culture medium, and retain the strain that turns the BTB culture medium blue. Inoculate the retained strains screened by BTB into LB liquid culture medium to make seed liquid, inoculate 2% inoculum into 200mL of denitrification culture medium, and culture in a constant temperature shaker at 30°C and 180r / min for 1 day. Determine the OD of the bacterial suspension 600The bacterial suspension was then centrifuged at 30°C and 8000r / min for 10 min to detect the total nitrogen and total phosphorus removal efficiency of each strain, and the strain with the highest 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 medium: glucose 1g, KNO30.75 g, MgSO4·7H2O 0.15g, NH4Cl 0.2g, KH2PO40.05 g, trace element solution 2mL, NaCl 30g, H2O 1L, pH 7.5, sterilization at 121℃ for 30min.

[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 SD-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 SD-1 (1504 bp, as shown in SEQ ID NO.3) was uploaded to the NCBI database and BLAST homologous sequence comparison was performed. The results showed that the similarity with strain Marinobacter hydrocarbonoclasticus strain NY-9 (JN903903.1) and strain Marinobacter hydrocarbonoclasticus NBRC 103161 (NR042256.1) was higher than 99.80%. The phylogenetic tree constructed by MEGA11 is shown in Figure 2. Figure 1 As shown, the results show that strain SD-1 and the existing Marinobacter hydrocarbonoclasticus strain NY-9 (JN903903.1) in the NCBI database are in the same branch of the evolutionary tree and are closest. The 16S rDNA sequence of the SD-1 strain was uploaded to the EzBioCloud database (https: / / www.ezbioCloud.net / ), and the results showed that the similarity between strain SD-1 and Marinobacter hydrocarbonoclasticus strain NY-9 (JN903903.1) in the 16S rRNA sequence database was as high as 99.80%. Combined with the above analysis results, the strain was identified as Marinobacter hydrocarbonoclasticus SD-1 and named Marinobacter hydrocarbonoclasticus SD-1.

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

[0041] The pure SD-1 strain was selected and inoculated into the sterilized LB liquid medium, and cultured at 30°C and 180 r / min for 22 h to prepare the seed solution. 600 The value was adjusted to 0.1-0.2, and 1.5% was added to a 96-well plate. 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 30°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 4-8h, and the growth enters the logarithmic proliferation phase from 8-22h, and then the value-added slows down. After reaching the maximum value at 32h, it enters the stable phase and shows signs of death after 36h.

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

[0043] The pure SD-1 strain was selected and inoculated into the sterilized LB liquid medium, and the seed liquid was prepared by culturing at 30°C and 180r / min for 22h. The tail water of sea eel culture was used as the background, and SD-1 was inoculated into 1L of the tail water at a rate of 1.5%, and 1g of anhydrous glucose was added per liter of the tail water as the carbon source, and the pH was 7.5. The culture was shaken at 30°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 high-speed centrifugation was performed at 4°C and 8000r / min. The concentrations of total nitrogen, ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, total phosphorus and active phosphate in the supernatant were measured. The results are shown in Tables 1, 2, 3, 4, 5 and 6.

[0044] As shown in Tables 1, 2, 3, 4, 5 and 6, when the treatment time was 10 h, the removal rate of total nitrogen by the SD-1 strain reached 98.8%, and reached 99.8% at 20 h; the removal rate of ammonia nitrogen reached a maximum of 81.8% at 2 h; the removal rate of nitrate nitrogen reached 68.1% at 4 h, 95.0% at 18 h, and reached a maximum of 98.5% at 22 h; the removal effect of nitrite nitrogen reached 75.2% at a treatment time of 4 h, and reached a maximum of 83.6% at 16 h; the removal rate of total phosphorus reached 75.7% at 2 h, and reached a maximum of 77.0% at 4 h; the removal rate of active phosphate reached 90.5% at 6 h, and reached a maximum of 97.1% at 22 h. Another denitrification and phosphorus reduction strain SN-1 developed by our team has a total nitrogen removal rate of 61.6% at a treatment time of 10 hours and 92.4% at 20 hours; the total phosphorus removal rate is 56.2% at 2 hours and 62.0% at 4 hours. By comparison, the treatment effect of SD-1 strain on total nitrogen and total phosphorus in water bodies is better than that of strain SN-1. Pseudomonas grimontii C18 published in patent CN201010100576.7 reaches the highest denitrification rate of 83.4% after 24 hours of cultivation. Compared with Pseudomonas C18, the denitrification rate of SD-1 is significantly higher than that of Pseudomonas C18, which indicates that SD-1 strain, as a halophilic strain, has the function of efficiently and quickly removing total nitrogen and total phosphorus in water quality, and can also remove ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and active phosphate, and has good application prospects.

[0045] Table 1 Determination of the removal effect of total nitrogen in aquaculture water by Marinobacterium SD-1 at different time periods

[0046]

[0047] Table 2 Determination of the removal effect of ammonia nitrogen in aquaculture water by SD-1 in different time periods

[0048]

[0049] Table 3 Determination of the removal effect of nitrate nitrogen in aquaculture water by SD-1 in different time periods

[0050]

[0051] Table 4 Determination of the removal effect of nitrite nitrogen in aquaculture water by removing hydrocarbons from Marinobacterium SD-1 at different time periods

[0052]

[0053] Table 5 Determination of the removal effect of total phosphorus in aquaculture water by SD-1 in different time periods

[0054]

[0055] Table 6 Determination of the removal effect of active phosphate in aquaculture water by SD-1 in different time periods

[0056]

[0057] Example 5 Determination of the removal effect of the hydrocarbon-removing Marinobacterium SD-1 on total nitrogen, ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, total phosphorus and active phosphate in aquaculture water under different dissolved oxygen conditions

[0058] The pure SD-1 strain was selected and inoculated into the sterilized LB liquid medium, and the seed liquid was prepared by culturing at 30℃ and 180r / min for 22h. The tail water of sea eel culture was used as the background, and SD-1 was inoculated into 1L of the tail water at a rate of 1.5%, and 1g of anhydrous glucose was added per liter of tail water as the carbon source, and the pH was 7.5. Seven treatment groups with different dissolved oxygen were set up: 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5mg / L, and the samples were collected after culturing at 30℃ for 20h, and the samples were centrifuged at high speed at 4℃ and r / min to measure the concentrations of total nitrogen, ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, total phosphorus and active phosphate in the supernatant. The results are shown in Tables 7, 8, 9, 10, 11 and 12.

[0059] The nitrogen removal and phosphorus reduction effects of SD-1 strain were different under the action of different dissolved oxygen. Under the conditions of background total nitrogen concentration of 33.443±0.22mg / L, ammonia nitrogen concentration of 0.856±0.16mg / L, nitrate nitrogen concentration of 2.886±0.18mg / L, nitrite nitrogen concentration of 1.166±0.08mg / L, total phosphorus concentration of 5.101±0.12mg / L and active phosphate concentration of 1.796±0.08mg / L, the removal rates of total nitrogen, ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, total phosphorus and active phosphate by SD-1 strain were all There is a trend of increasing with the increase of dissolved oxygen; when the dissolved oxygen is 6.5mg / L, the removal rate of total nitrogen by SD-1 strain reaches 84.0%, when the dissolved oxygen is 7.0mg / L, the removal rate of ammonia nitrogen reaches 91.0%, the removal rate of nitrate nitrogen reaches 96.7%, and the removal rate of total phosphorus reaches the highest 72.2%; when the dissolved oxygen is 7.5mg / L, the removal rate of nitrite nitrogen reaches 86.4%, and the removal rate of active phosphate reaches 89.2%, indicating that dissolved oxygen 6.5mg / L-7.5mg / L is a suitable culture environment for strain SD-1.

[0060] Table 7 Determination of the removal effect of total nitrogen in aquaculture water by SD-1 under different dissolved oxygen

[0061]

[0062] Table 8 Determination of the removal effect of ammonia nitrogen in aquaculture water by SD-1 under different dissolved oxygen

[0063]

[0064] Table 9 Determination of the removal effect of nitrate nitrogen in aquaculture water by SD-1 of hydrocarbon-removing marine bacillus at different dissolved oxygen levels

[0065]

[0066] Table 10 Determination of the removal effect of nitrite nitrogen in aquaculture water by removing hydrocarbons from marine bacillus SD-1 at different dissolved oxygen levels

[0067]

[0068]

[0069] Table 11 Determination of the removal effect of total phosphorus in aquaculture water by SD-1 of hydrocarbon-removing marine bacillus at different dissolved oxygen levels

[0070]

[0071] Table 12 Determination of the removal effect of active phosphate in aquaculture water by SD-1 of hydrocarbon-removing marine bacillus at different dissolved oxygen levels

[0072]

[0073] Example 6 Determination of the removal effect of the hydrocarbon-removing marine bacillus SD-1 on total nitrogen, ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, total phosphorus and active phosphate in aquaculture water at different salinities

[0074] The pure SD-1 strain was selected and inoculated into the sterilized LB liquid medium, and the seed liquid was prepared by culturing at 30°C and 180r / min for 22h. The tail water of sea eel culture was used as the background, and SD-1 was inoculated into 1L of the tail water at an inoculation rate of 1.5%, and 1g of anhydrous glucose was added per liter of the tail water as the carbon source. The pH was 7.5, and 7 treatment groups with different salinities were set: 10, 16, 22, 28, 32, 36 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. The concentrations of total nitrogen, ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, total phosphorus and active phosphate in the supernatant were measured. The results are shown in Tables 13, 14, 15, 16, 17 and 18.

[0075] Under the conditions of background total nitrogen concentration of 33.238±0.08mg / L, ammonia nitrogen concentration of 2.511±0.08mg / L, nitrate nitrogen concentration of 1.203±0.10mg / L, nitrite nitrogen concentration of 2.630±0.10mg / L, total phosphorus concentration of 1.842±0.11mg / L and active phosphate concentration of 1.999±0.08mg / L, the removal efficiency of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and total phosphorus by SD-1 strain at salinity of 10-16 was Less than 60.0%. With the increase of salinity, the removal rate of total nitrogen, ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, total phosphorus and active phosphate in aquaculture water by SD-1 strain gradually increased. When the salinity was 22, the best effect was achieved, with a total nitrogen removal rate of 99.4%, an ammonia nitrogen removal rate of 73.1%, a nitrate nitrogen removal rate of 83.9%, a nitrite nitrogen removal rate of 92.1%, a total phosphorus removal rate of 73.6%, and an active phosphate removal rate of 94.8%. When the salinity continued to increase, the removal rate gradually decreased. The strain (Bacillus Cereus) disclosed in patent number CN201410084407.7 can achieve a nitrogen removal rate of about 90.0% and a phosphorus removal rate of 70.0% after being cultured for 24 hours under salinity conditions of 7-9. The SD-1 strain of the present invention can achieve a total nitrogen removal rate of 99.5% and a total phosphorus removal rate of 73.7% after being cultured for 22 hours under salinity conditions of 22, indicating that the SD-1 strain has a significantly higher total nitrogen and total phosphorus removal rate than the P1K strain, and also achieves efficient removal of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and active phosphate.

[0076] Table 13 Determination of the removal effect of total nitrogen in aquaculture water by removing hydrocarbons from marine bacillus SD-1 at different salinities

[0077]

[0078] Table 14 Determination of the removal effect of ammonia nitrogen in aquaculture water by SD-1 at different salinities

[0079]

[0080]

[0081] Table 15 Determination of the removal effect of nitrate nitrogen in aquaculture water by removing hydrocarbons from marine bacillus SD-1 at different salinities

[0082]

[0083] Table 16 Determination of the removal effect of nitrite nitrogen in aquaculture water by removing hydrocarbons from marine bacillus SD-1 at different salinities

[0084]

[0085] Table 17 Determination of the removal effect of total phosphorus in aquaculture water by removing hydrocarbons from marine bacillus SD-1 at different salinities

[0086]

[0087] Table 18 Determination of the removal effect of active phosphate in aquaculture water by SD-1 of hydrocarbon-removing marine bacillus at different salinities

[0088]

[0089]

[0090] Example 7 Safety testing of the hydrocarbon-removing Marinobacterium SD-1 in aquaculture applications

[0091] The pure SD-1 strain was selected and inoculated into the sterilized LB liquid medium, and cultured in a constant temperature oscillator at 30°C and 180 r / min for 22 h to prepare the seed solution. 6The 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 the hydrocarbon removal Marinobacter SD-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 hydrocarbon removal Marinobacter (Marinobacter hydrocarbonoclasticus) is: Category 4, no pathogenic objects.

[0092] In summary, the hydrocarbon-removing sea bacillus SD-1 of the present invention can efficiently remove total nitrogen and total phosphorus in seawater aquaculture water, aquaculture tail water or nearshore waters, and can remove ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and active phosphate at the same time, and has no toxic effects on aquatic animals. It 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 conditions. Compared with the prior art, the denitrification and phosphorus reduction speed and removal rate of the hydrocarbon-removing sea bacillus SD-1 of the present invention are significantly better than similar strains reported in the prior art. The SD-1 strain can achieve rapid and efficient denitrification and phosphorus reduction in a short time, especially the nitrogen removal rate is significantly higher. On the other hand, the hydrocarbon-removing sea bacillus SD-1 has salt-tolerant and alkali-loving denitrification and phosphorus 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 aquaculture water, aquaculture tail water or nearshore waters. It has no toxic effects on aquatic animals and can be widely used in more types of aquatic economic animal breeding scenes.

[0093] 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 SD-1, characterized in that: The Marinobacter hydrocarbonoclasticus SD-1 is Marinobacter hydrocarbonoclasticus SD-1, which was deposited in the China Center for Type Culture Collection on August 7, 2024, located at Wuhan University, Wuhan, China, and its collection number is CCTCC NO:M 20241756.

2. A bacterial agent, characterized in that: The bacterial agent includes the hydrocarbon-removing Marinobacterium SD-1 or its fermentation liquid according to claim 1.

3. Use of the hydrocarbon-removing Marinobacterium SD-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 SD-1 or the bacterial agent in degrading the nitrogen and phosphorus concentrations 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 SD-1 is 1.5%.

9. Use of the hydrocarbon-removing Marinobacterium SD-1 according to claim 1 or the bacterial agent according to claim 2 in the preparation of a halophilic microecological preparation for degrading nitrogen and phosphorus in seawater.

10. The method for culturing the hydrocarbon-removing Marinobacterium SD-1 according to claim 1, characterized in that: The hydrocarbon-removing Marinobacterium SD-1 was placed in a seawater LB liquid culture medium and cultured continuously for 24 hours at 30° C. and 180 r / min.

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