Etothiorhodospirillum shakeri HDES1 of penaeus monodon biofloc higher-place pond and application of Etothiorhodospirillum shakeri HDES1 in purification of inorganic nitrogen and phosphorus in water body

Abstract

The invention discloses ectothiorhodospirillum shakeri HDES1 of a penaeus monodon biofloc higher-place pond and application of the ectothiorhodospirillum shakeri HDES1 to purification of inorganic nitrogen and phosphorus in aquaculture water, and belongs to the technical field of microorganisms. The ectothiorhodospirillum shakeri HDES1 is screened from a penaeus monodon biofloc culture higher-place pond, the preservation number is CCTCC NO: M 20191007, and tests show that the strain has a remarkable purification effect on high-concentration inorganic nitrogen and phosphorus in a marine culture water body or tail water, has no adverse effect on cultured penaeus monodon and seawater tilapia mossambica, and can be used for preparing a culture medium for the penaeus monodon and the seawater tilapia mossambica. The fish and shrimp culture yield can be increased, and zero-water-change green ecological culture can be realized.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to a strain of *Ectothiorhodospira shaposhnikovii* HDES1 in a high-level pond of Penaeus monodon bioflocs and its application in purifying inorganic nitrogen and phosphorus in aquaculture water bodies. Background Art

[0002] In an intensive aquaculture system, the efficient management of the water environment is an extremely important technical link, which has important practical significance for enhancing the new quality productivity of the industry and promoting the transformation and upgrading of the industry towards a modern industrial system with high-efficiency output of aquatic products and green environmental protection. Ammonia nitrogen, nitrite nitrogen, phosphate, etc. are likely to accumulate in high-density aquaculture water bodies of fish and shrimp. Among them, high concentrations of ammonia nitrogen and nitrite have serious toxic effects on aquatic animals and endanger the health of cultured animals; phosphate has limited material output channels in an intensive aquaculture environment and is one of the main indicators of water quality eutrophication, bringing great pressure to the purification and up-to-standard discharge of aquaculture tail water.

[0003] Generally common water environment purification methods mainly include physical methods, chemical methods, and biological methods. Among them, physical methods use the adsorption function of water purification materials such as zeolite powder, dolomite powder, and coral sand to adsorb and purify pollutants in water bodies. The adsorption ability is closely related to the physical form of the materials, and there are certain functional limitations. In aquaculture, the method of draining and replacing water is mostly used to reduce water eutrophication. Chemical methods use the strong oxidation reaction of chemical oxidants such as quicklime and chlorine preparations to purify water quality. The purification effect continuously decreases with the consumption of the oxidant; for high-concentration phosphorus substances, phosphorus removal agents such as aluminum salts, iron salts, or polyelectrolytes are mostly used for reaction to form phosphorus chemical sludge. When using chemical methods to purify water quality, improper operation is likely to cause the safety risk of secondary water pollution. Biological methods mostly use specific microorganisms, microalgae, and aquatic animals and plants to transform and recycle eutrophic substances in water bodies, and have the characteristics of environmental friendliness and continuous and stable purification functions compared with physical and chemical methods. Some scholars have applied the multi-biological combination method of marine microalgae + shellfish to remove excess nitrogen and phosphorus nutrients in water bodies; others have used salt-tolerant plants such as Suaeda glauca, Sesuvium portulacastrum, and mangroves to purify the seawater aquaculture water environment in a different location. The water eutrophication level of high-density intensive aquaculture water bodies of fish and shrimp is high, the water consumption is large, and the requirement for the purification efficiency of water quality is relatively high. It is more appropriate to use efficient microbial water purification technology. Microorganisms with nitrification function mainly include autotrophic nitrifying bacteria and heterotrophic nitrifying bacteria. The former commonly includes Nitrococcus, Nitrospira, Nitrobacter, Nitrospina, Nitrosococcus, Nitrosovibrio, Nitrosospira, Nitrosomonas, Nitrosolobus; the latter commonly includes bacteria, fungi, actinomycetes, etc. For example, Arthrobacter globiformis, Pseudomonas aeruginosa, Aspergillus parasiticus, etc. The activated sludge method is a common microbial water purification technology. The commonly used activated sludge cultivation method in water treatment engineering is mostly in-situ enrichment cultivation of microbial communities. The difficulty of separating and purifying nitrifying bacteria is relatively high. Most researchers use activated sludge to enrich and cultivate nitrifying bacteria. There are research reports that when the temperature is 30°C, pH is 6.5 - 8.0, and dissolved oxygen is 2.0 mg / L, the total amount of nitrifying bacteria has increased by nearly 20 times; the ammonia nitrogen removal rate of enriched freshwater nitrifying bacteria is 0.12 mg / g·h, and that of seawater nitrifying bacteria is 0.13 mg / g·h.However, for the application of water environment purification in aquaculture, the activated sludge enrichment culture method has certain shortcomings in ensuring the specificity of dominant bacteria and the safety for aquaculture. The method of expanding culture with purified strains is relatively less applied on a large scale in water treatment projects due to the high requirements for the functions and growth characteristics of strains.

[0004] Excavating microbial strain resources with high-efficiency water quality purification functions from the water ecological environment of healthy cultured fish and shrimp is more conducive to improving the adaptability of strains to the seawater aquaculture environment, the efficiency of ecological function realization, and the safety of cultured organisms. Some scholars have in-situ isolated Rhodococcus and Paracoccus and applied them to aquaculture water with good application effects. Some scholars have reported the physiological and ecological characteristics of Hydrocarboniphaga sp. isolated from Epinephelus merra in Meiji Reef Lagoon and Alkanivorax xenomuta AXMZ1 isolated from Gonostoma elongatum, and confirmed that they have good nitrification or denitrification functions. Sulfurospirillum shawi HDES1 is a purple sulfur bacterium belonging to the phylum Proteobacteria, class Gammaproteobacteria, family Ectothiorhodospiraceae, and genus Ectothiorhodospira. There is a patent report on using Sulfurospirillum shawi as a probiotic to degrade nitrite in the water environment, but it only targets the degradation of low-concentration nitrite. In the water of high-density aquaculture of fish and shrimp, nutrients such as ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, and phosphate are prone to accumulate (their concentrations can reach up to more than a dozen or even dozens of mg / L in aquaculture production), which are harmful to fish and shrimp or cause the water body to be hyper-eutrophic. Screening high-efficiency functional strains under aerobic and anaerobic environments respectively for high-concentration inorganic nitrogen and phosphorus (ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, phosphate) and applying them to high-density aquaculture production of fish and shrimp is more practical and creative. Currently, there is no relevant R & D report on isolating Sulfurospirillum shawi from the high-yield culture system of prawn high-level ponds to purify high-concentration inorganic nitrogen and phosphorus in pond water and applying it to high-density zero-water-exchange aquaculture of fish and shrimp. Summary of the Invention

[0005] The purpose of the present invention is to provide a Sulfurospirillum shawi HDES1 from the biofloc high-level pond of Penaeus monodon and its application in purifying inorganic nitrogen and phosphorus in aquaculture water, so as to solve the problems existing in the above-mentioned prior art. This strain has a strong purification ability for high-concentration inorganic nitrogen and phosphorus in seawater aquaculture water or tail water, has good environmental adaptability, and has no adverse effects on fish and shrimp in high-density zero-water-exchange aquaculture.

[0006] To achieve the above purpose, the present invention provides the following solutions:

[0007] The present invention provides an Ectothiorhodospira shaposhnikovii HDES1, characterized in that the strain is deposited under the accession number CCTCC NO: M 20191007, deposited on December 4, 2019, with the China Center for Type Culture Collection (abbreviated as CCTCC), and the deposit address is Wuhan University, Wuhan, China.

[0008] In the present invention, the water environment of the high-density aquaculture water body for marine fish and shrimps is significantly different from the water environment in the background technology. According to the actual environmental requirements of aquaculture production, indigenous microbial strain resources should be explored, and the ecological adaptability, physiological functions and application safety of the strains should be systematically evaluated to determine that the microbial strains can be effectively applied to the aquaculture production scenario. Simply copying the microbial technology of water treatment projects and ignoring the specific needs of organisms in the aquaculture water environment cannot achieve good strain application effects. The Ectothiorhodospira shaposhnikovii HDES1 in the present invention is screened from the environment of a high-yield shrimp culture pond, has a significant purification effect on high-concentration inorganic nitrogen and phosphorus in the water body, and has no obvious adverse effects on the cultured shrimps and seawater tilapia, which helps to increase the yield of fish and shrimp aquaculture and achieve zero-water-exchange green ecological aquaculture.

[0009] The screening, isolation and identification process of the Ectothiorhodospira shaposhnikovii HDES1 in the present invention is as follows: collect the biofloc sediment in the late-stage culture water body (cultured for 50 - 75 days) of a high-density Penaeus monodon intensive culture high-level pond with an Imhoff cone, filter and remove the excess water with a 0.22 μm filter membrane, place the filter membrane in a photosynthetic bacteria liquid medium and anaerobically culture it for 3 - 7 days at a temperature of 25 - 30 °C and a light intensity of 2000 - 6000 Lx to directionally culture the microbial community in the bioflocs. Streak-culture the cultured bacterial liquid on a photosynthetic bacteria solid plate medium for 3 - 5 days, select the red or orange single colonies with good growth performance, inoculate the strain into the photosynthetic bacteria liquid medium, and anaerobically culture it for 3 - 7 days at 28 - 30 °C and 2000 - 6000 Lx. The cultured bacterial liquid is respectively added to the sterilized aquaculture water body (adjust the ammonia nitrogen concentration in the water body to 10 - 20 mg / L, nitrite nitrogen concentration to 7 - 10 mg / L, and phosphate concentration to 7 - 12 mg / L with NH4Cl, NaNO 2 、KH 2 PO 4 ), and anaerobically culture it for 3 - 7 days at 25 - 30 °C and 1000 - 5000 lx. Select the strains that can effectively reduce the inorganic nitrogen and phosphorus concentration in the water body for strain identification.

[0010] This strain is anaerobic, oxygen-tolerant, can utilize ammonium salts and acetates, and is positive for catalase. It can grow normally at temperatures of 10 - 40°C, salinities of 5 - 40, and pH values of 6 - 10. The optimal conditions are temperatures of 20 - 40°C, salinities of 10 - 40, and pH values of 8 - 10. Under the optimal conditions, it reaches a peak after growing in the nutrient environment of the aquaculture water body for 32 hours and maintains a quantity level of 10 8 CFU / mL for 3 - 9 days. It is suitable for application in most intensive seawater aquaculture ponds.

[0011] The present invention also provides a bacterial agent containing the said Ectothiorhodospira shaposhnikovii HDES1.

[0012] The present invention also provides the application of the said Ectothiorhodospira shaposhnikovii HDES1 in any one of the following:

[0013] (1) Application in purifying high-concentration inorganic nitrogen and phosphorus in seawater aquaculture water bodies or tail waters;

[0014] (2) Application in preparing a bacterial agent for purifying high-concentration inorganic nitrogen and phosphorus in seawater aquaculture water bodies or tail waters.

[0015] The present invention also provides the application of the said bacterial agent in purifying high-concentration inorganic nitrogen and phosphorus in seawater aquaculture water bodies or tail waters.

[0016] Optionally, the high-concentration inorganic nitrogen and phosphorus include ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, and phosphate; the concentration of ammonia nitrogen ≥ 12 mg / L, the concentration of nitrite nitrogen ≥ 7 mg / L, nitrate nitrogen ≥ 20 mg / L, and the concentration of phosphate ≥ 6 mg / L.

[0017] The present invention also provides a method for purifying high-concentration inorganic nitrogen and phosphorus in seawater aquaculture water bodies or tail waters, including the step of putting the said Ectothiorhodospira shaposhnikovii HDES1 into the seawater aquaculture water body or tail water for purification.

[0018] Optionally, the high-concentration inorganic nitrogen and phosphorus include ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, and phosphate; the concentration of ammonia nitrogen ≥ 10 mg / L, the concentration of nitrite nitrogen ≥ 7 mg / L, nitrate nitrogen ≥ 20 mg / L, and the concentration of phosphate ≥ 6 mg / L.

[0019] Optionally, the purification conditions include: water temperature 20 - 40°C, salinity 10 - 40, pH value 8 - 10, and the said Ectothiorhodospira shaposhnikovii HDES1 is reused every 6 - 9 days.

[0020] The Rhodovulum sulfidophilum ESBPM1 provided by the present invention can achieve the effect of nitrogen and phosphorus removal in a seawater environment with a salinity of 10 - 40 within 3 - 6 days. Under aerobic conditions, the phosphate removal rate is 65.5% - 88.1% in 3 days, and the ammonia nitrogen removal rate is 59.5% - 90.4% in 6 days. The nitrite nitrogen removal rate is 99% both under anaerobic or aerobic conditions in 3 days; the nitrate nitrogen removal rate is 99% both under anaerobic or aerobic conditions in 6 days; the total inorganic nitrogen (TIN) removal rate is 6.9% - 66.4% under anaerobic conditions from 3 to 9 days, and the TIN removal rate is 66.3% - 98.7% under aerobic conditions in 3 days. In terms of temperature adaptability, when cultured aerobically at 20 - 30 °C, the phosphate removal rate is 57.4% - 82.4% in 3 days, and the ammonia nitrogen removal rate is 46.4% - 88.4%; the nitrite nitrogen removal rate exceeds 99% both under anaerobic or aerobic conditions from 3 to 6 days; the nitrate nitrogen removal rate exceeds 99% under anaerobic culture for 6 days and exceeds 99% under aerobic culture for 3 days; the TIN removal rate is 76.9% - 94.5% under aerobic culture for 3 days. There are certain differences in the purification effects of the strain on inorganic nitrogen and phosphorus under aerobic and anaerobic conditions. When in use, the specific conditions of the purification target in the aquaculture water body or tail water should be considered, and corresponding purification processes should be adopted to maximize the effect of the strain.

[0021] The present invention discloses the following technical effects:

[0022] (1) The Rhodovulum sulfidophilum HDES1 in the present invention has a significant effect on removing high concentrations of inorganic nitrogen and phosphorus (ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, phosphate) in the high-density culture water body of Penaeus monodon, and has no adverse effects on the cultured Penaeus monodon. However, there are certain differences in the purification effects of the strain on inorganic nitrogen and phosphorus under aerobic and anaerobic conditions. When in use, the specific conditions of the purification target in the aquaculture water body or tail water should be considered, and corresponding purification processes should be adopted to maximize the effect of the strain.

[0023] (2) The Rhodovulum sulfidophilum HDES1 in the present invention is screened from the biofloc microenvironment in the high-level pond culture water body of Penaeus monodon. The strain has good environmental adaptability and is suitable for application in most Penaeus monodon culture pond water bodies.

[0024] (3) The application of the Rhodovulum sulfidophilum HDES1 in the present invention to the purification of intensive aquaculture water quality can achieve good application effects, can significantly reduce the water replacement during the aquaculture production process, and even achieve a high-yield aquaculture effect of zero water replacement under the condition of not being equipped with complex water quality purification equipment. Scientifically applying this strain to further develop the water quality directional regulation technology and tail water purification technology for seawater industrial aquaculture can provide technical support for promoting the transformation and upgrading of the seawater aquaculture industry of fish and shrimp to a modern production mode of ecological environmental protection and high-efficiency output. Description of the Drawings

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0026] Figure 1 It is the growth curve of strain HDES1;

[0027] Figure 2 It is the change of phosphate concentration in water under different salinities;

[0028] Figure 3 It is the change of ammonia nitrogen concentration in water under different salinities;

[0029] Figure 4 It is the change of nitrite nitrogen concentration in water under different salinities;

[0030] Figure 5 It is the change of nitrate nitrogen concentration in water under different salinities;

[0031] Figure 6 It is the change of TIN concentration in water under different salinities;

[0032] Figure 7 It is the change of the amount of strain HDES1 in water under different salinities;

[0033] Figure 8 It is the change of phosphate concentration in water under different temperatures;

[0034] Figure 9 It is the change of ammonia nitrogen concentration in water under different temperatures;

[0035] Figure 10 It is the change of nitrite nitrogen concentration in water under different temperatures;

[0036] Figure 11 It is the change of nitrate nitrogen concentration in water under different temperatures;

[0037] Figure 12 It is the change of TIN concentration in water under different temperatures;

[0038] Figure 13 It is the change of the amount of strain HDES1 in water under different temperatures;

[0039] Figure 14 It is the change of phosphate concentration in water under different pH conditions;

[0040] Figure 15 It is the change of ammonia nitrogen concentration in water under different pH conditions;

[0041] Figure 16 Variation of nitrite nitrogen concentration in water under different pH conditions;

[0042] Figure 17 Variation of nitrate nitrogen concentration in water under different pH conditions;

[0043] Figure 18 Variation of TIN concentration in water under different pH conditions;

[0044] Figure 19 Bacterial quantity variation of strain HDES1 in water under different pH conditions. Detailed implementation manners

[0045] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be considered as a limitation to the present invention, but should be understood as a more detailed description of certain aspects, characteristics and implementation schemes of the present invention.

[0046] It should be understood that the terms described in the present invention are only for describing particular implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0047] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0048] Without departing from the scope or spirit of the present invention, various improvements and variations can be made to the specific implementation manners of the specification of the present invention, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary.

[0049] Regarding "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0050] Example 1 Screening and cultivation of strain HDES1

[0051] 1. Bacterial source and culture medium

[0052] (1) Bacterial source: Collect the biological floc sediment in the high-level pond of cultured Penaeus monodon along the coast of Guangdong, and isolate and culture it on the photosynthetic bacteria culture medium plate.

[0053] (2) Culture medium

[0054] Photosynthetic bacteria liquid culture medium: CH 3 COONa 3g, yeast extract 1g, MgSO 4 ·7H 2 O 0.2g, NH 4 Cl 0.05g, NaCl 28g, NaNO 2 0.05g, KNO 3 0.18g, KH 2 PO 4 0.01g, growth factor solution 1mL. The above drugs are dissolved in distilled water respectively and made up to 1000mL, with pH 7.0.

[0055] Growth factor solution: MnSO 4 ·H 2 O 0.25g, FeSO 4 ·7H 2 O 7g, CaCl 2 5g, glutamic acid 0.02g. The above drugs are dissolved in distilled water respectively and made up to 100mL, with pH 7.0.

[0056] Photosynthetic bacteria solid plate culture medium: Add 20 - 25g / L of agar powder to the photosynthetic bacteria liquid culture medium to prepare a solid plate culture medium.

[0057] 2. Screening and culture of strains

[0058] Collect the biological floc sediment in the later-stage culture water body (cultured for 50 - 75 days) of the high-level pond for intensive culture of high-density Penaeus monodon with an Imhoff cone tube, filter the excess water body with a 0.22μm filter membrane, place the filter membrane in the photosynthetic bacteria liquid culture medium for anaerobic culture for 3 - 7 days, at a temperature of 25 - 30°C and a light intensity of 2000 - 6000Lx, and directionally culture the microbial community in the biological flocs.

[0059] Streak the cultured bacterial liquid on the photosynthetic bacteria solid plate culture medium for 3 - 5 days, select the red or orange single colonies with good growth performance, inoculate the strain into the photosynthetic bacteria liquid culture medium, and culture anaerobically at 28 - 30°C and 2000 - 6000Lx for 3 - 7 days.

[0060] Add the well-cultured bacterial liquid to the sterilized culture water body respectively (using NH 4 Cl, NaNO2 , KH 2 PO 4 Adjust the ammonia nitrogen concentration in the water body to 10 - 20 mg / L, nitrite nitrogen concentration to 7 - 10 mg / L, nitrate nitrogen concentration to 20 - 30 mg / L, and phosphate concentration to 7 - 12 mg / L), and anaerobically culture at 25 - 30 °C, 1000 - 5000 lx for 3 - 7 days. Select strains that can effectively reduce the inorganic nitrogen and phosphorus concentration in the water body for strain identification.

[0061] Example 2 Identification of the strain Rhodovulum sulfidophilum HDES1

[0062] The present invention identifies the 16S rDNA molecule of Rhodovulum sulfidophilum HDES1, and determines the genus and species of the strain from the molecular level in combination with the analysis of bacterial morphological characteristics and physiological and biochemical characteristics. The 16S rDNA sequence analysis is mainly carried out according to the following steps:

[0063] 1. Extraction of bacterial genomic DNA

[0064] Extract according to the instructions of the bacterial genomic DNA extraction kit. The specific operations are as follows:

[0065] (1) Pick a single colony with a sterile toothpick and inoculate it in an enrichment medium for culture;

[0066] (2) Take 1.5 mL of the bacterial culture solution, centrifuge at 10000 rpm (11,500 g) for 1 minute, and aspirate as much supernatant as possible;

[0067] (3) Add 200 μL of buffer GA to the cell pellet, shake until the cells are completely suspended, add 180 μL of lysozyme with a final concentration of 20 mg / mL, and incubate at 37 °C for more than 30 minutes;

[0068] (4) Add 20 μL of proteinase K solution to the tube and mix well;

[0069] (5) Add 220 μL of buffer GB, shake for 15 seconds, place at 70 °C for 10 minutes, the solution becomes clear, and briefly centrifuge to remove the water droplets on the inner wall of the tube cap;

[0070] (6) Add 220 μL of absolute ethanol, shake well for 15 seconds, and briefly centrifuge to remove the water droplets on the inner wall of the tube cap;

[0071] (7) Add the solution and flocculent precipitate obtained in the previous step to an adsorption column CB3 (the adsorption column is placed in a collection tube), centrifuge at 12000 rpm (13,400×g) for 30 seconds, pour out the waste liquid, and place the adsorption column CB3 in the collection tube;

[0072] (8) Add 500 μL of buffer GD to adsorption column CB3, centrifuge at 12,000 rpm (13,400 g) for 30 seconds, discard the waste liquid, and place adsorption column CB3 into the collection tube.

[0073] (9) Add 700 μL of wash buffer PW to adsorption column CB3, centrifuge at 12,000 rpm (13,400 g) for 30 seconds, discard the waste liquid, and place adsorption column CB3 into the collection tube.

[0074] (10) Add 500 μL of wash buffer PW to adsorption column CB3, centrifuge at 12,000 rpm (13,400 g) for 30 seconds, discard the waste liquid, and place adsorption column CB3 into the collection tube.

[0075] (11) Place adsorption column CB3 back into the collection tube, centrifuge at 12,000 rpm (13,400 g) for 2 minutes, discard the waste liquid. Place adsorption column CB3 at room temperature for several minutes to thoroughly dry the residual wash buffer in the adsorption material.

[0076] (12) Transfer adsorption column CB3 into a clean centrifuge tube, suspend and add 50 - 200 μL of elution buffer TE to the middle part of the adsorption membrane, place at room temperature for 2 - 5 minutes, centrifuge at 12,000 rpm (13,400 g) for 2 minutes, and collect the solution into the centrifuge tube.

[0077] (13) Detection of DNA concentration and purity

[0078] The concentration and purity of the recovered DNA fragment were detected by agarose gel electrophoresis and ultraviolet spectrophotometer.

[0079] 2. PCR amplification of 16S rDNA gene

[0080] The universal primers for 16S rDNA bacteria were synthesized by Invitrogen (Shanghai) Trading Co., Ltd. The forward primer (8f) is: 5’-AGAGTTTGATCCTGGCTCAG-3’ (SEQ ID NO: 2); the reverse primer (1492r) is: 5’-GGTTACCTTGTTACGAC TT-3’ (SEQ ID NO: 3). The 50 μL PCR reaction system includes: 37 μL of sterilized double-distilled water, 1 μL of each primer, dNTPs (2.5 mmol / L), 1 μL of Taq enzyme, 5 μL of 10×PCR buffer, and 1 μL of DNA template. PCR conditions: 95°C for 3 minutes, 95°C for 1 minute, 48°C for 1 minute, 72°C for 2 minutes, for a total of 30 cycles; 72°C for 10 minutes.

[0081] 3. 16S rDNA sequence determination

[0082] After the amplification was completed, the PCR products were detected by 1.0% agarose gel electrophoresis and sent to Invitrogen (Shanghai) Trading Co., Ltd. for sequencing. The obtained sequence (SEQ ID NO: 1) is as follows:

[0083]

[0084] 4. Colony Morphology and Physiological Characteristics of Strain HDES1

[0085] The colony morphology and physiological characteristics of strain HDES1 are shown in Table 1 below.

[0086] Table 1 Colony Morphology and Physiological Characteristics of Ectothiorhodospira shaposhnikovii HDES1

[0087]

[0088]

[0089] 5. Identification of Ectothiorhodospira shaposhnikovii HDES1

[0090] The 16S rDNA gene sequence of the strain was compared and analyzed with the registered gene sequences in GenBank. Combining the results of various aspects such as the morphological characteristics of the strain, 16S rDNA gene sequence analysis, and biochemical identification, it was determined that strain HDES1 is Ectothiorhodospira shaposhnikovii. After consulting relevant materials, there is no research report on using Ectothiorhodospira shaposhnikovii HDES1 from the biological flocs in the high-level ponds of Penaeus monodon to purify the inorganic nitrogen and phosphorus in the water body of high-density zero-water-exchange shrimp farming. This strain was deposited in the China Center for Type Culture Collection (abbreviated as CCTCC) on December 4, 2019, with the deposit number: CCTCC NO: M 20191007, and the deposit address: Wuhan University, Wuhan, China.

[0091] Example 3 Small-Scale Application of Ectothiorhodospira shaposhnikovii HDES1

[0092] 1. Growth of the Strain

[0093] The Ectothiorhodospira shaposhnikovii HDES1 strain obtained in Example 1 was inoculated into sterilized aquaculture pond water at a density of 10 5 ~10 6 cells / mL. After 32 h, the cell concentration reached 10 8 cells / mL. The growth curve of strain HDES1 is as shown in Figure 1 .

[0094] 2. Removal Effect of Strain HDES1 on Inorganic Nitrogen and Phosphorus in Water under Different Salinities

[0095] The sterilized water body of the high-level pond of Penaeus monodon (water salinity 25, pH value 7.8 - 8.5) was used as the basic test water body control, and it was incubated at a constant temperature of 30°C without adding strain HDES1. In the bacteria-added group, the strain HDES1 obtained in Example 1 was added at a density of 106 Inoculate at a density of 6 cells / mL into aquaculture waters with different salinities. The water salinities are set at 5, 10, 25, and 40. The light intensity is 2000 - 6000 Lx, and incubate at a constant temperature for 9 days. At the same time, set up two culture groups: an anaerobic culture group (ANO group) and an aerobic incubation group (AEO group). For each salinity test sample in different culture groups, set 3 replicates. Monitor the concentrations of phosphate, ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, and total inorganic nitrogen (TIN) in the water every 3 days.

[0096] As Figure 2 shown, the change in the phosphate concentration of the control group is not significant compared to the initial value, remaining at a relatively high concentration level of 7.328 - 10.572 mg / L. After adding strain HDES1, the phosphate removal rates in each salinity group on the 3rd day of anaerobic culture are 23.3% - 40.6%. Subsequently, the phosphate concentration increased to varying degrees, and on the 9th day, it increased to 7.370 - 9.250 mg / L, showing little difference from the phosphate concentration of the control group. However, on the 3rd day of aerobic culture, the phosphate removal rates in the salinity groups of 5 - 40 reached 65.5% - 88.1%, and the removal effect was best in the salinity group of 25. The results indicate that aerobic culture conditions are more conducive to promoting the removal of phosphate by strain HDES1.

[0097] As Figure 3 shown, during the test period, the ammonia nitrogen concentration of the control group remained at a relatively high concentration level of 12.789 - 19.009 mg / L. On the 9th day of anaerobic culture, the ammonia nitrogen concentrations in each salinity group increased to varying degrees, with the highest increase being 90.8% compared to the initial value. However, on the 6th day of aerobic culture, the ammonia nitrogen removal rates in the salinity groups of 10 - 40 reached 59.5% - 90.4%, and the removal effect increased with the increase in salinity. Generally speaking, in aquaculture seawater with a salinity greater than 10, strain HDES1 can effectively remove ammonia nitrogen in the water under aerobic culture conditions.

[0098] As Figure 4 shown, the change in the nitrite nitrogen concentration of the control group is not significant compared to the initial value, remaining at a relatively high concentration level of 7.219 - 9.765 mg / L. After adding strain HDES1, the removal effect of nitrite nitrogen is best under the condition of salinity 40, and the nitrite nitrogen removal rate can reach 99% on the 3rd day of anaerobic or aerobic culture. Under the condition of salinity 5 - 25, after 3 days of anaerobic culture, the nitrate nitrogen removal rate is 43.6% - 69.1%, and then the nitrite nitrogen concentration increased to varying degrees, with the increase in the salinity group of 5 being particularly obvious. While on the 3rd day of aerobic culture, the nitrite nitrogen removal rates in the salinity groups of 10 - 25 all exceeded 99%. On the 6th day of aerobic culture, the nitrite nitrogen removal rate in the salinity group of 5 reached the best, which was 79.4%.

[0099] As Figure 5 shown, during the test, the nitrate nitrogen concentration in the control group remained at a relatively high level of 30.969 - 39.987 mg / L. After adding strain HDES1, the removal effect of nitrate nitrogen was the best under the condition of salinity 40, and the nitrate nitrogen removal rate could reach 99% after anaerobic or aerobic culture for 6 days; under the condition of salinity 5 - 25, after anaerobic culture for 3 - 6 days, the nitrate nitrogen removal rate was 21.2% - 65.5%, and then the nitrate nitrogen concentration increased to varying degrees. While under aerobic culture, the removal effect of nitrate nitrogen was obvious, and the nitrate nitrogen removal rate on the 6th day was 78.7% - 99.7%.

[0100] As Figure 6 shown, the total inorganic nitrogen (TIN) in the control group was generally at a relatively high level. The concentration was 54.088 - 59.982 mg / L under anaerobic environment for 3 - 9 days, and 41.646 - 66.312 mg / L under aerobic environment for 3 - 6 days. After adding strain HDES1, the TIN removal rate of the salinity 5 - 40 groups under anaerobic culture for 3 - 9 days was 6.9% - 66.4%, which was generally lower than that under aerobic culture conditions; the TIN removal rate under aerobic culture for 3 days could reach 66.3% - 98.7%. Comparatively, the TIN removal effect was better under the condition of salinity 10 - 40, and the removal rate could reach 85.0% - 98.7%.

[0101] As Figure 7 shown, under the condition of salinity 5, during anaerobic culture for 3 - 9 days, strain HDES1 hardly grew, and the bacterial count remained at the order of magnitude of 10 6 cells / mL, but the bacterial count increased to the order of magnitude of 10 8 cells / mL on the 6th day of aerobic culture. Aerobic culture was more conducive to the proliferation of strain HDES1 under low salinity conditions. Under the condition of salinity 10 - 40, the bacterial count in each salinity group showed a continuous increasing trend. Comparatively, the strain grew better under aerobic culture conditions, and the bacterial count reached the order of magnitude of 10 8 cells / mL.

[0102] It can be seen that although strain HDES1 can survive in the low salinity water body with salinity 5 during aerobic culture, its purification effect on water inorganic nitrogen and phosphorus is far less than that in the seawater environment with salinity 10 - 40; generally, the nitrogen and phosphorus removal function can be exerted on the 3rd - 6th day after using the bacteria, and the bacteria can also be reused every 6 days to stably enhance the nitrogen and phosphorus removal effect of the strain.

[0103] 3. Removal effect of strain HDES1 on water inorganic nitrogen and phosphorus at different temperatures

[0104] Using the sterilized water in the high-level pond of Penaeus monodon (water salinity 30, pH value 8.0) as the basic test water control, incubate at a constant temperature of 30°C without adding strain HDES1. In the bacteria-added group, the strain HDES1 obtained in Example 1 was inoculated into the aquaculture water with different pH values at a concentration of 10 5 -10 6 CFU / mL. The culture temperatures were set at 10°C, 20°C, 30°C, and 40°C, the light intensity was 2000 - 6000 Lx, and the incubation was carried out at a constant temperature for 6 days. At the same time, two culture groups were set, namely the anaerobic culture group (ANO group) and the aerobic incubation group (AEO group). Three parallels were set for each pH gradient test sample in different culture groups. The concentrations of phosphate, ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, and total inorganic nitrogen (TIN) in the water were monitored every 3 days.

[0105] As Figure 8 shown, the change in the phosphate concentration in the control group was not significant compared with the initial value, remaining at 8.066 - 9.389 mg / L, at a relatively high concentration level. During anaerobic incubation for 3 - 6 days in the 10°C - 30°C group, the phosphate concentration decreased slightly, with a removal rate of 5.9% - 27.2%; however, during aerobic incubation for 3 days in the 10°C - 30°C group, the phosphate removal rate could reach 57.4% - 82.4%; at 40°C, strain HDES1 could not achieve good removal effect on phosphate under both anaerobic and aerobic conditions.

[0106] As Figure 9 shown, during the test period, the ammonia nitrogen concentration in the control group remained at a relatively high level of 17.724 - 25.549 mg / L. During anaerobic incubation for 3 - 6 days in the 20°C - 40°C group, the ammonia nitrogen concentration showed varying degrees of increase, with the highest increase being 81.2% compared with the initial value; however, during aerobic incubation for 3 days, the ammonia nitrogen removal rate in each temperature group reached 46.4% - 88.4%, and at 6 days of incubation, it was 50.1% - 79.7%. Generally speaking, strain HDES1 can effectively remove ammonia nitrogen in the water under aerobic incubation at various temperature conditions.

[0107] As Figure 10 shown, the change in the nitrite nitrogen concentration in the control group was not significant compared with the initial value, remaining at 9.426 - 11.606 mg / L. After adding strain HDES1, under both anaerobic and aerobic incubation at 10°C - 40°C, the nitrite nitrogen removal rate exceeded 99% within 3 - 6 days, indicating that strain HDES1 has a good removal effect on nitrite nitrogen.

[0108] As Figure 11As shown in the figure, the nitrate nitrogen concentration values of the control group generally showed an upward trend. The concentrations under anaerobic conditions for 3 - 6 days were 20.864 - 33.098 mg / L, and those under aerobic conditions for 3 - 6 days were 29.220 - 45.623 mg / L, both at relatively high concentration levels. After adding strain HDES1, the nitrate nitrogen concentrations decreased significantly at 10℃ - 40℃. The nitrate nitrogen removal rates of each temperature group exceeded 99% after 6 days of anaerobic culture, while the nitrate nitrogen removal rates of each temperature group could exceed 99% after 3 days of aerobic culture, and the overall effect was faster than anaerobic culture. Strain HDES1 could effectively remove nitrate nitrogen in water under anaerobic and aerobic conditions at each temperature.

[0109] As Figure 12 shown in the figure, the TIN concentration values of the control group generally showed an upward trend. The concentrations under anaerobic conditions for 3 - 6 days were 48.014 - 66.457 mg / L, and those under aerobic conditions for 3 - 6 days were 63.137 - 79.328 mg / L, both at relatively high concentration levels. After adding strain HDES1, the TIN concentrations of each temperature group at 10℃ - 40℃ decreased to varying degrees. The TIN removal rates after 6 days of anaerobic culture reached 22.2% - 68.8%, while the TIN removal rates after 3 days of aerobic culture could reach 76.9% - 94.5%. The effect was significantly better than anaerobic culture, and each temperature group could effectively purify TIN in water under aerobic conditions.

[0110] As Figure 13 shown in the figure, during the test period, the bacterial counts of each group continuously increased from the initial 10 5 CFU / mL. The bacterial counts of each temperature group under anaerobic culture for 3 - 6 days stabilized at the order of magnitude of 10 7 CFU / mL, and the bacterial counts of each temperature group under aerobic culture for 3 - 6 days reached 2.6×10 8 CFU / mL - 6.0×10 8 CFU / mL, and there was no obvious difference in bacterial counts among different temperature groups.

[0111] 4. Removal effect of strain HDES1 on inorganic nitrogen and phosphorus in water under different pH conditions

[0112] Using the sterilized water of the high - level pond of Penaeus monodon (water salinity 30, pH value 8.0) as the basic test water control, incubating at a constant temperature of 30℃ without adding strain HDES1. In the bacteria - adding group, strain HDES1 obtained in Example 1 was added at 10 4 -10 5Inoculate at [X] cells / mL into aquaculture water bodies with different pH values. The pH value gradient is set at 6, 8, and 10, the light intensity is 2000 - 6000 Lx, and incubate at a constant temperature of 30 °C for 9 days. At the same time, set up two culture groups: an anaerobic culture group (ANO group) and an aerobic incubation group (AEO group). For each pH gradient test sample in different culture groups, set 3 replicates. Monitor the concentrations of phosphate, ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, and total inorganic nitrogen (TIN) in the water body and the bacterial count every 3 days.

[0113] As Figure 14 shown, the phosphate concentration in the control group remained at 6.604 - 9.528 mg / L; on the 9th day of anaerobic culture, the phosphate removal rate of the strain under the conditions of pH 6 - 10 could reach 59.3% - 95.6%, and the phosphate removal rate in the pH 8 group could reach up to 95.6% at most.

[0114] As Figure 15 shown, the ammonia nitrogen concentration in the control group remained at 14.314 - 18.795 mg / L; during the anaerobic culture process, the concentration in each pH group did not show an obvious downward trend, and even increased to 55.097 mg / L at most, an increase of 141.8%; however, during the aerobic culture process, the ammonia nitrogen concentrations in the pH 8 group and the pH 10 group showed a continuous decreasing trend, and the removal rate reached 71.1% - 95.7% on the 9th day. It shows that aerobic culture is more conducive to the removal of ammonia nitrogen by the strain.

[0115] As Figure 16 shown, the nitrite nitrogen concentration in the control group remained at 7.219 - 9.665 mg / L; on the 3rd day of anaerobic culture, the nitrite nitrogen removal rates in the pH 8 group and the pH 10 group both reached over 99%, and remained until the 9th day; in the pH 6 group during anaerobic culture from 6 - 9 days, the nitrite nitrogen removal rate was 68.0 - 73.3%.

[0116] As Figure 17 shown, the nitrate nitrogen concentration in the control group remained at 27.634 - 31.500 mg / L; on the 3rd day of anaerobic culture, the nitrate nitrogen removal rates in the pH 8 group and the pH 10 group both reached over 99.5%, and remained until the 9th day; in the pH 6 group during anaerobic culture from 6 - 9 days, the nitrate nitrogen removal rate was 59.3% - 64.6%.

[0117] As Figure 18As shown, the TIN concentration values in the control group showed no significant change, remaining at 51.575 - 57.660 mg / L. The TIN removal rates in the pH 8 and pH 10 groups reached 53.5% - 60.1% on the 3rd day of anaerobic cultivation. On the 9th day of anaerobic cultivation, the TIN removal rates in the pH 6 and pH 10 groups reached 40.7% - 68.3%.

[0118] As Figure 19 shown, the bacterial counts during the test for each group continuously increased from the initial 10 5 CFU / mL and reached the order of magnitude of 10 8 CFU / mL at the end of the test. The bacterial counts in the pH 8 and pH 10 groups were slightly higher than those in the pH 6 group.

[0119] It can be seen that although strain HDES1 can survive and grow well in water bodies with pH values ranging from 6 to 10, reaching a level of 10 8 CFU / mL, in terms of its removal effect on inorganic nitrogen and phosphorus, the overall effects in the pH 8 and pH 10 groups were better. Generally, the removal effect on nitrogen and phosphorus in water bodies was better on the 3rd - 6th day of anaerobic cultivation, but aerobic cultivation was required for the removal of ammonia nitrogen. To strengthen or stabilize the nitrogen and phosphorus removal effect, the microbial agent can be reused every 6 - 9 days.

[0120] Example 4 Application effect of strain HDES1 in high - density zero - water - exchange aquaculture production of fish and shrimp

[0121] Application tests of strain HDES1 were carried out in the high - density zero - water - exchange aquaculture production of penaeid shrimp in Jiaxi Town, Shanwei, Guangdong and Dianbai, Maoming, Guangdong. The general microbial agent concentration was 10 4 -10 6 CFU / mL. The microbial agent and an appropriate amount of brown sugar were added to the aquaculture water body within 10 days after fry stocking, and then the microbial agent was regularly added every 6 days. Quicklime water etc. was used to stably adjust the total alkalinity and pH value of the water body, and aeration mechanical equipment was used to increase the dissolved oxygen in the water body. The results showed that the application effect of the microbial agent was good, and strain HDES1 had no adverse effects on the cultured fish and shrimp. Among them, the aquaculture demonstration base in Dianbai, Maoming passed the on - site test by the expert group organized by the Maoming Agricultural Technology Extension Center. The penaeid shrimp was cultured for 90 days and the tilapia was cultured for 120 days. The average weight of the penaeid shrimp was 14.7 g / tail, the body length was 12.1 cm, and the calculated yield per unit area was 10 kg / m 3 ², and the survival rate was 82%; the average weight of the tilapia was 826 g / tail, and the calculated yield per unit area was 23 kg / m 3 ², and the survival rate was 70%. The water body conditions during the aquaculture process are shown in Table 2, with water temperature 23 - 28 °C, salinity 10 - 15, pH value 7.0 - 8.5, dissolved oxygen concentration 5.02 - 6.50 mg / L, ammonia nitrogen 0.145 mg / L, and nitrite nitrogen 0.119 mg / L.

[0122] Table 2 Water quality status of the aquaculture water body using strain HDES1

[0123]

[0124] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Ectothiorhodospira shaposhnikovii HDES1, characterized in that The preservation number of this bacterium is CCTCC NO: M 20191007.

2. A bacterial agent, characterized in that It contains the Exothiorhodospirillum saboriae HDES1 according to claim 1.

3. Use of Exorhodospirillum saboriae HDES1 as claimed in claim 1 in any of the following: (1) Application in purifying high concentrations of inorganic nitrogen and phosphorus in marine aquaculture water or tail water; (2) Application in the preparation of bacterial agents for purifying high concentrations of inorganic nitrogen and phosphorus in marine aquaculture water or tail water.

4. Use of the bacterial agent as claimed in claim 2 in purifying high concentrations of inorganic nitrogen and phosphorus in marine aquaculture water or tail water.

5. The use according to claim 3 or 4, characterized in that: The high concentration inorganic nitrogen and phosphorus include ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and phosphate; the concentration of ammonia nitrogen is ≥10 mg / L, the concentration of nitrite nitrogen is ≥7 mg / L, the concentration of nitrate nitrogen is ≥20 mg / L, and the concentration of phosphate is ≥6 mg / L.

6. A method for purifying high concentrations of inorganic nitrogen and phosphorus in marine aquaculture water or tail water, characterized in that: The method comprises the step of putting the Exorhodospirillum sabii HDES1 described in claim 1 into a marine aquaculture water body or tail water for purification.

7. The method according to claim 6, characterized in that The high concentration inorganic nitrogen and phosphorus include ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and phosphate; the concentration of ammonia nitrogen is ≥12 mg / L, the concentration of nitrite nitrogen is ≥7 mg / L, the concentration of nitrate nitrogen is ≥20 mg / L, and the concentration of phosphate is ≥6 mg / L.

8. The method according to claim 6, characterized in that The purification conditions include: water temperature of 20-40° C., salinity of 10-40, pH value of 8-10, and repeated use of the Exothio-Rhodospirillum saboriae HDES1 every 6-9 days.

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