A strain of high-density bio-floc pond of marbled shrimp, Serratia marcescens HDES1 and its application in purifying inorganic nitrogen and phosphorus in water

Abstract

The application discloses a strain of Sideropenaeus paramamosain bio-floc high-pond Shajia external sulfur red spirillum HDES1 and application of the strain in purifying inorganic nitrogen and phosphorus in aquaculture water, and belongs to the technical field of microorganisms. The Shajia external sulfur red spirillum HDES1 in the application is screened from a Sideropenaeus paramamosain bio-floc aquaculture high-pond, and has a preservation number of CCTCC NO: M 20191007. It is found through experiments that the strain has a remarkable purifying effect on high-concentration inorganic nitrogen and phosphorus in seawater aquaculture water or tail water, has no adverse effect on cultured Sideropenaeus paramamosain and seawater tilapia, and is helpful to improving the yield of fish and shrimp culture and realizing zero-water-change green ecological culture.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a strain of biological floc of Marsdenia tenacissima and its application in purifying inorganic nitrogen and phosphorus in aquaculture water. BACKGROUND

[0002] In the intensive aquaculture system, efficient management of water environment is a very important technical link, which has important practical significance for improving the new quality productivity of the industry and promoting the transformation and upgrading of the industry to a modern industrial system with efficient output and green environmental protection of aquatic products. Ammonia nitrogen, nitrite nitrogen and phosphate are easy to accumulate in high-density fish and shrimp aquaculture water, among which high concentrations of ammonia nitrogen and nitrite have serious toxic effects on aquatic animals, endangering the health of the animals; phosphate has limited material output channels in intensive aquaculture environment, and is one of the main indicators of water eutrophication, which brings great pressure to the purification and discharge of aquaculture tail water.

[0003] The common water environment purification methods mainly include physical method, chemical method and biological method. Among them, the physical method is to use the adsorption function of water purification materials such as zeolite powder, dolomite powder and coral sand to adsorb and purify pollutants in water. The adsorption capacity is closely related to the physical form of the material, and there is a certain functional limitation. Aquaculture industry mostly uses water replacement to reduce water eutrophication. The chemical method uses the strong oxidation reaction of lime, chlorine preparation and other chemical oxidants to purify water quality. The purification effect continues to decrease with the consumption of oxidants. For high concentration of phosphorus, aluminum salt, iron salt or poly electrolyte is used to react with phosphorus chemical sludge. If the operation is not proper, it is easy to cause secondary pollution of water body. The biological method uses specific microorganisms, microalgae and aquatic plants to transform and recycle the eutrophication substances in water. Compared with physical and chemical methods, it has the characteristics of environmental friendliness and stable purification function. Some scholars use marine microalgae + shellfish combination to remove excess nitrogen and phosphorus nutrients in water. Some use alkali, sea grass, mangrove and other salt-tolerant plants to purify the water environment of seawater aquaculture. The water eutrophication level of high-density fish and shrimp intensive aquaculture is high, and the water consumption is large. The water quality purification efficiency is relatively high, and it is more suitable to use efficient microbial water purification technology. The nitrifying microorganisms mainly include autotrophic nitrifying bacteria and heterotrophic nitrifying bacteria. The former includes Nitrococcus, Nitrospira, Nitrobacter, Nitrospina, Nitrosococcus, Nitrosovibrio, Nitrosospira, Nitrosomonas and Nitrosolobus. The latter includes bacteria, fungi and actinomycetes, such as Arthrobacter globiformis, Pseudomonas aeruginosa and Aspergillus parasiticus. Activated sludge method is a common microbial water purification technology. The activated sludge culture method commonly used in water treatment engineering is in-situ microbial community enrichment culture. It is difficult to separate and purify nitrifying bacteria. Most researchers use activated sludge to enrich and culture nitrifying bacteria. Some studies have reported that under the conditions of 30℃, pH 6.5-8.0 and dissolved oxygen 2.0mg / L, the total amount of nitrifying bacteria increased by nearly 20 times. The removal rate of ammonia nitrogen by enriched freshwater nitrifying bacteria was 0.12mg / g·h, and that of seawater nitrifying bacteria was 0.13mg / g·h.However, for water environment purification application of aquaculture, activated sludge enrichment culture method has certain short board in guaranteeing specificity of dominant flora and safety for aquaculture. The purified strain expansion culture method has relatively high requirements for function and growth characteristics of the strain, and is relatively less used in large-scale water treatment engineering.

[0004] It is more beneficial to improve the adaptability of the strain to the seawater aquaculture environment, the ecological function implementation efficiency, the safety of the cultured organisms and the like to excavate microbial strain resources with high-efficiency water quality purification function from the ecological environment of the water body of healthy cultured fish and shrimp. Some scholars have in-situ isolated Rhodococcus and Paracoccus and applied them in the cultured water body to obtain good application effect. Some scholars have reported physiological and ecological characteristics of hydrocarbon-degrading Marinobacter sp. isolated from Epinephelus merra in the lagoon of Meiji Reef and heterotrophic alkane-eating bacteria AXMZ1 of deep-sea Gonostoma elongatum, and confirmed that they have good nitrification or denitrification function. HDES1 is a purple sulfur bacterium belonging to Proteobacteria, Gammaproteobacteria, Ectothiorhodospiraceae and Ectothiorhodospira. Some patents report that HDES1 is used as a microecological preparation to degrade nitrite in the water body environment, but it is only used for low-concentration nitrite degradation. In the high-density aquaculture water body of fish and shrimp, ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and phosphate are easy to accumulate (the concentration thereof can be as high as tens of mg / L in aquaculture production), which is harmful to fish and shrimp or makes the water body super-nutrient. It is more practical and creative to screen high-efficiency functional strains in aerobic and anaerobic environments respectively for high-concentration inorganic nitrogen and phosphorus (ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and phosphate) and apply them to high-density aquaculture production. At present, there is no related research and development report about HDES1 isolated from high-density shrimp pond to purify high-concentration inorganic nitrogen and phosphorus in the pond water body and applied in high-density zero-water-change fish and shrimp culture. SUMMARY

[0005] The purpose of the present application is to provide a HDES1 of HDES1 of Litopenaeus vannamei biofloc high-density pond and its application in purifying inorganic nitrogen and phosphorus in the cultured water body, so as to solve the problems in the prior art. The strain has strong purifying capacity for high-concentration inorganic nitrogen and phosphorus in the seawater aquaculture water body or tail water, and good environmental adaptability, and has no adverse effect on high-density zero-water-change cultured fish and shrimp.

[0006] To achieve the above purpose, the present application provides the following solutions.

[0007] The application provides a Ectothiorhodospira shaposhnikovii HDES1, characterized in that the strain has a preservation number CCTCC NO: M 20191007, a preservation time of December 4, 2019, a preservation unit of China Center for Type Culture Collection (CCTCC) and a preservation address of Wuhan University, Wuhan, China.

[0008] The water environment in the high-density mariculture of sea fish and shrimp in the application is obviously different from the background technology, and the indigenous microbial strain resources should be excavated according to the actual environmental requirements of aquaculture production, the ecological adaptability, physiological function and application safety of the strain should be systematically evaluated, and it is determined that the microbial strain can be effectively applied to the production scene. The microbial technology of the water treatment engineering cannot achieve good application effect of the strain by simply copying and setting, and the specific needs of the organisms in the aquaculture water environment are ignored. The Ectothiorhodospira shaposhnikovii HDES1 in the application is screened from the high-yield shrimp farming pond environment, has a significant purification effect on high-concentration inorganic nitrogen and phosphorus in the water body, has no obvious adverse effect on the farmed shrimp and sea tilapia, and is helpful to improve the yield of fish and shrimp and realize zero water change and green ecological aquaculture.

[0009] The screening, separation and identification process of the Ectothiorhodospira shaposhnikovii HDES1 in the application is as follows: the biological flocculation precipitate in the late aquaculture water body (cultured for 50-75 days) of the high-density intensive culture of high-position ponds of marbled shrimp is collected by an Erlenmeyer flask, the excess water is removed by filtering with a filter membrane with a pore size of 0.22 μm, the filter membrane is placed in a photosynthetic bacterial liquid culture medium for closed anaerobic culture for 3-7 days, the temperature is 25-30 ℃, the light intensity is 2000-6000 Lx, and the microorganism community in the biological flocculation is directionally cultured. The cultured bacterial liquid is streaked on a photosynthetic bacterial solid plate culture medium for 3-5 days, and a red or orange single colony with good growth performance is selected, and the strain is inoculated into a photosynthetic bacterial liquid culture medium, and is subjected to anaerobic culture at 28-30 ℃ for 3-7 days under a light intensity of 2000-6000 Lx. The cultured bacterial liquid is added into sterilized aquaculture water (the aquaculture water is adjusted by NH4Cl, NaNO2 and KH2PO4 to have an ammonia nitrogen concentration of 10-20 mg / L, a nitrite nitrogen concentration of 7-10 mg / L and a phosphate concentration of 7-12 mg / L), and is subjected to anaerobic culture at 25-30 ℃ for 3-7 days under a light intensity of 1000-5000 lx. The strain capable of effectively reducing the inorganic nitrogen and phosphorus concentration of the water body is selected for strain identification.

[0010] The strain is anaerobic and oxygen-tolerant, can utilize ammonium salt and acetate, and is positive for catalase. The strain can normally grow at a temperature of 10-40 ℃, a salinity of 5-40 and a pH of 6-10, and the optimal conditions are a temperature of 20-40 ℃, a salinity of 10-40 and a pH of 8-10. The strain grows in the nutrient environment of the aquaculture water body under the optimal conditions, reaches a peak value after 32 h, and maintains a growth rate of 10 8The quantity level is 10 000 cells / mL, which is suitable for most of the seawater intensive culture pond application.

[0011] The application also provides a bacterial agent containing the said H. salinisoli HDES1.

[0012] The application also provides the application of the said H. salinisoli HDES1 in any of the following aspects:

[0013] (1) the application in purifying high-concentration inorganic nitrogen and phosphorus in seawater culture water or tail water;

[0014] (2) the application in preparing a bacterial agent for purifying high-concentration inorganic nitrogen and phosphorus in seawater culture water or tail water.

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

[0016] Optionally, the high-concentration inorganic nitrogen and phosphorus includes 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.

[0017] The application also provides a method for purifying high-concentration inorganic nitrogen and phosphorus in seawater culture water or tail water, which comprises the step of putting the said H. salinisoli HDES1 into seawater culture water or tail water for purification.

[0018] Optionally, the high-concentration inorganic nitrogen and phosphorus includes 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.

[0019] Optionally, the purification conditions include water temperature of 20-40℃, salinity of 10-40, pH value of 8-10, and the said H. salinisoli HDES1 is used repeatedly once every 6-9 days.

[0020] The present application provides the Shewanella loihica PV-4 strain ESBPM1, which can remove nitrogen and phosphorus in seawater with salinity of 10-40 in 3-6 days, the removal rate of phosphate is 65.5%-88.1% in 3 days under aerobic condition, the removal rate of ammonia nitrogen is 59.5%-90.4% in 6 days, the removal rate of nitrite nitrogen is 99% in 3 days under anaerobic or aerobic condition, the removal rate of nitrate nitrogen is 99% in 6 days under anaerobic or aerobic condition, the removal rate of total inorganic nitrogen (TIN) is 6.9%-66.4% in 3-9 days under anaerobic condition, and the removal rate of TIN is 66.3%-98.7% in 3 days under aerobic condition. In terms of temperature adaptability, the removal rate of phosphate is 57.4%-82.4% in 3 days under aerobic condition at 20-30 DEG C, the removal rate of ammonia nitrogen is 46.4%-88.4%, the removal rate of nitrite nitrogen is more than 99% in 3-6 days under anaerobic or aerobic condition, the removal rate of nitrate nitrogen is more than 99% in 6 days under anaerobic condition and in 3 days under aerobic condition, and the removal rate of TIN is 76.9%-94.5% in 3 days under aerobic condition. There is a certain degree of difference in the purification effect of the strain on inorganic nitrogen and phosphorus under aerobic and anaerobic conditions, and the specific conditions of the purification target in the aquaculture water or tail water should be considered when using, and the corresponding purification process is adopted, so that the strain can achieve maximum effect.

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

[0022] (1) The Shewanella loihica PV-4 strain HDES1 in the present application has remarkable removal effect on high-concentration inorganic nitrogen and phosphorus (ammonia nitrogen, nitrite nitrogen, nitrate nitrogen and phosphate) in high-density marbled shrimp aquaculture water, and has no adverse effect on marbled shrimp, but there is a certain degree of difference in the purification effect of the strain on inorganic nitrogen and phosphorus under aerobic and anaerobic conditions, and the specific conditions of the purification target in the aquaculture water or tail water should be considered when using, and the corresponding purification process is adopted, so that the strain can achieve maximum effect.

[0023] (2) The Shewanella loihica PV-4 strain HDES1 in the present application is screened from the biological floc microenvironment in marbled shrimp high-level pond aquaculture water, and the strain has good environmental adaptability and is suitable for most marbled shrimp pond water application.

[0024] (3) The Shewanella loihica PV-4 strain HDES1 in the present application can achieve good application effect when applied to intensive aquaculture water purification, and can greatly reduce water replacement in the process of aquaculture production, and even realize zero water replacement high-yield aquaculture effect under the condition of not configuring complex water quality purification equipment. Scientific use of the strain can further develop seawater factory aquaculture water directional regulation technology and tail water purification technology, and can provide technical support for promoting the transformation and upgrading of marbled shrimp seawater aquaculture industry to ecological and environmental protection and high-efficiency production mode. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained based on these drawings without creative effort.

[0026] Figure 1 Growth curve of the strain HDES1;

[0027] Figure 2 Change of phosphate concentration in water under different salinity;

[0028] Figure 3 Change of ammonia nitrogen concentration in water under different salinity;

[0029] Figure 4 Change of nitrite nitrogen concentration in water under different salinity;

[0030] Figure 5 Change of nitrate nitrogen concentration in water under different salinity;

[0031] Figure 6 Change of TIN concentration in water under different salinity;

[0032] Figure 7 Change of strain HDES1 amount in water under different salinity;

[0033] Figure 8 Change of phosphate concentration in water under different temperature;

[0034] Figure 9 Change of ammonia nitrogen concentration in water under different temperature;

[0035] Figure 10 Change of nitrite nitrogen concentration in water under different temperature;

[0036] Figure 11 Change of nitrate nitrogen concentration in water under different temperature;

[0037] Figure 12 Change of TIN concentration in water under different temperature;

[0038] Figure 13 Change of strain HDES1 amount in water under different temperature;

[0039] Figure 14 Change of phosphate concentration in water under different pH condition;

[0040] Figure 15 Change of ammonia nitrogen concentration in water under different pH condition;

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

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

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

[0044] Figure 19 The change of strain HDES1 amount in water under different pH conditions. DETAILED DESCRIPTION

[0045] Various exemplary embodiments of the present application will now be described in detail, which should be considered to be illustrative of certain aspects, features and embodiments of the present application, but not limiting of the same.

[0046] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, where particular ranges of values are given, understand that each intervening value, to the upper or lower limit of the ranges is also specifically included. Each smaller range that falls within the broader ranges is also specifically included in the present application. The upper and lower limits of these smaller ranges can independently be included or excluded in the range.

[0047] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in further detail the methods and / or materials associated with the documents. In the event of conflict between the present specification and any incorporated document, the present specification controls.

[0048] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples presented herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0049] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional elements or steps.

[0050] Example 1 Screening and culture of strain HDES1

[0051] 1. Bacterial source and culture medium

[0052] (1) Bacterial source: Collect biological floc precipitate in high-density intensive culture of P. monodon in high-level ponds along the coast of Guangdong, and use photosynthetic bacterial culture medium plate to isolate and culture.

[0053] (2) Culture medium

[0054] Photosynthetic bacterial liquid culture medium: CH3COONa 3g, yeast extract 1g, MgSO4·7H2O 0.2g, NH4Cl 0.05g, NaCl 28g, NaNO2 0.05g, KNO3 0.18g, KH2PO4 0.01g, growth factor solution 1mL, above drugs are dissolved in distilled water, constant volume to 1000mL, pH 7.0.

[0055] Growth factor solution: MnSO4·H2O 0.25g, FeSO4·7H2O 7g, CaCl2 5g, glutamic acid 0.02g, above drugs are dissolved in distilled water, and constant volume to 100mL, pH 7.0.

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

[0057] 2. Screening and culture of strains

[0058] Collect biological floc precipitate in high-density intensive culture of P. monodon in high-level ponds in the later stage of culture (cultured for 50-75 days) with Erlenmeyer tube, filter excess water with 0.22μm filter membrane, place the filter membrane in photosynthetic bacterial liquid culture medium for 3-7 days of anaerobic culture at 25-30℃, light intensity 2000-6000Lx, and direct culture of microbial community in biological floc.

[0059] Culture the bacterial liquid on photosynthetic bacterial solid plate culture medium for 3-5 days of streak culture, select red or orange single colonies with good growth performance, inoculate the strains into photosynthetic bacterial liquid culture medium, 28-30℃, 2000-6000Lx anaerobic culture for 3-7 days.

[0060] Add the cultured bacterial liquid to sterilized culture water (adjust the water body ammonia nitrogen concentration to 10-20mg / L, nitrite nitrogen concentration to 7-10mg / L, nitrate nitrogen concentration to 20-30mg / L, and phosphate concentration to 7-12mg / L with NH4Cl, NaNO2, KH2PO4), 25-30℃, 1000-5000lx, anaerobic culture for 3-7 days. Select strains that can effectively reduce the concentration of inorganic nitrogen and phosphorus in water for strain identification.

[0061] Example 2 Strain identification of HDES1

[0062] The present application identifies HDES1 by 16S rDNA molecule, determines the species of the strain from the molecular level, and combines the analysis of bacterial morphological characteristics and physiological and biochemical characteristics. The 16S rDNA sequence analysis mainly follows the following steps:

[0063] 1. Extraction of bacterial genomic DNA

[0064] According to the bacterial genomic DNA extraction kit instruction, the specific operation is as follows:

[0065] (1) Use a sterile toothpick to inoculate a single colony into an expanded culture medium for culture;

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

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

[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, and place at 70°C for 10 minutes. The solution becomes clear, and a brief centrifugation is performed to remove the water droplets on the inner wall of the tube cap;

[0070] (6) Add 220 μL of anhydrous ethanol, shake thoroughly and mix well for 15 seconds, and perform a brief centrifugation 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 x g) for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in the collection tube;

[0072] (8) Add 500 μL of buffer GD to the adsorption column CB3, centrifuge at 12000 rpm (13,400 g) for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in the collection tube;

[0073] (9) Add 700 μL of rinse solution PW to the adsorption column CB3, centrifuge at 12000 rpm (13,400 g) for 30 seconds, discard the waste liquid, and place the adsorption column CB3 in the collection tube;

[0074] (10) Add 500 μL of rinse solution PW to the adsorption column CB3, centrifuge at 12000 rpm (13,400 g) for 30 seconds, discard the waste solution, and place the adsorption column CB3 in the collection tube;

[0075] (11) Place the adsorption column CB3 back in the collection tube, centrifuge at 12000 rpm (13,400 g) for 2 minutes, discard the waste solution. Place the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material;

[0076] (12) Transfer the adsorption column CB3 into a clean centrifuge tube, and add 50-200 μL of elution buffer TE to the middle of the adsorption membrane, and place it at room temperature for 2-5 minutes, centrifuge at 12000 rpm (13,400 g) for 2 minutes, and collect the solution into the centrifuge tube;

[0077] (13) DNA concentration and purity detection

[0078] The recovered DNA fragments are detected for concentration and purity by agarose gel electrophoresis and ultraviolet spectrophotometry.

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

[0080] The 16S rDNA bacterial universal primers are synthesized by Yingwei Jieji (Shanghai) Trade Co., Ltd., the forward primer (8f) is: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 2), and the reverse primer (1492r) is: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 3). The 50 μL PCR reaction system comprises: 37 μL of sterilized double-distilled water, 1 μL of each primer, 1 μL of Tap enzyme, 5 μL of 10×PCR buffer, and 1 μL of DNA template. The PCR conditions are: 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; and 72°C for 10 minutes.

[0081] 3. 16S rDNA sequence determination

[0082] After amplification, the PCR product is detected by 1.0% agarose gel electrophoresis, and is sent to Yingwei Jieji (Shanghai) Trade Co., Ltd. for sequencing. The sequence (SEQ ID NO: 1) is measured:

[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 gene sequences registered in GenBank, and the results of strain morphology characteristics, 16S rDNA gene sequence analysis, biochemical identification, etc. were comprehensively analyzed. It was determined that strain HDES1 was Ectothiorhodospira shaposhnikovii. According to relevant information, there is no report on the use of Ectothiorhodospira shaposhnikovii HDES1 from the biological floc of high-density ponds of Marsupenaeus japonicus for purifying inorganic nitrogen and phosphorus in the water body of high-density zero-water exchange culture of prawns. The strain was preserved in China Center for Type Culture Collection (CCTCC) on December 4, 2019, with the preservation number CCTCC NO: M 20191007 and the preservation address China. Wuhan. Wuhan University.

[0091] Example 3 Small-scale application of Ectothiorhodospira shaposhnikovii HDES1

[0092] 1. Growth of the strain

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

[0094] 2. Removal effect of strain HDES1 on inorganic nitrogen and phosphorus in water body under different salinities

[0095] The sterilized Marsupenaeus japonicus high-density pond water (water salinity 25, pH value 7.8-8.5) was used as the basic test water control, and the water was incubated at 30°C without adding strain HDES1. The strain HDES1 obtained in Example 1 was inoculated into the water at a concentration of 106 Inoculated at a rate of 1 / mL into aquaculture waters with different salinities (5, 10, 25, and 40), and light intensity of 2000-6000 Lx, the culture was carried out at a constant temperature for 9 days. Two culture groups were established: an anaerobic culture group (ANO group) and an aerobic incubation group (AEO group). For each salinity test sample in each culture group, three replicates were set up. The concentrations of phosphate, ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, and total inorganic nitrogen (TIN) in the water were monitored every 3 days.

[0096] like Figure 2 As shown, the phosphate concentration in the control group remained relatively stable compared to the initial value, ranging from 7.328 to 10.572 mg / L. After adding strain HDES1, the phosphate removal rate in each salinity group was 23.3%–40.6% on day 3 of anaerobic culture, followed by varying degrees of increase in phosphate concentration, reaching 7.370–9.250 mg / L on day 9, not significantly different from the control group. However, after 3 days of aerobic culture, the phosphate removal rate in the salinity groups (5–40) reached 65.5%–88.1%, with the salinity group (25) showing the best removal effect. These results indicate that aerobic culture conditions are more conducive to promoting phosphate removal by strain HDES1.

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

[0098] like Figure 4 As shown, the nitrite nitrogen concentration in the control group did not change significantly compared to the initial value, remaining at a relatively high level of 7.219-9.765 mg / L. After adding strain HDES1, the removal effect of nitrite nitrogen was best at a salinity of 40, with nitrite nitrogen removal rates reaching 99% after 3 days of anaerobic or aerobic culture. Under salinity conditions of 5-25, after 3 days of anaerobic culture, the nitrite nitrogen removal rate was 43.6%-69.1%, after which the nitrite nitrogen concentration increased to varying degrees, with a particularly significant increase in the salinity group of 5. During 3 days of aerobic culture, the nitrite nitrogen removal rate in the salinity groups of 10-25 all exceeded 99%. After 6 days of aerobic culture, the nitrite nitrogen removal rate in the salinity group of 5 reached its optimal level of 79.4%.

[0099] like Figure 5 As shown, the nitrate nitrogen concentration in the control group remained at a relatively high level of 30.969-39.987 mg / L during the test. After adding strain HDES1, the nitrate nitrogen removal effect was best at a salinity of 40, with nitrate nitrogen removal rates reaching 99% after 6 days of anaerobic or aerobic culture. Under salinity conditions of 5-25, the nitrate nitrogen removal rate was 21.2%-65.5% after 3-6 days of anaerobic culture, after which the nitrate nitrogen concentration increased to varying degrees. However, the nitrate nitrogen removal effect was significant during aerobic culture, with a nitrate nitrogen removal rate of 78.7%-99.7% on day 6.

[0100] like Figure 6 As shown, the TIN concentration in the control group was generally high, ranging from 54.088 to 59.982 mg / L under anaerobic conditions for 3-9 days, and from 41.646 to 66.312 mg / L under aerobic conditions for 3-6 days. After adding strain HDES1, the TIN removal rate in the anaerobic culture group with a salinity of 5-40 for 3-9 days was 6.9%-66.4%, which was generally lower than the level under aerobic culture conditions. The TIN removal rate in the aerobic culture group for 3 days reached 66.3%-98.7%. In comparison, the TIN removal effect was better under salinity conditions of 10-40, with a removal rate of 85.0%-98.7%.

[0101] like Figure 7 As shown, under anaerobic culture conditions of salinity 5 for 3-9 days, strain HDES1 showed almost no growth, with the bacterial count maintained at 10. 6 The bacterial count was on the order of magnitude of cells / mL, but it increased to 10 on day 6 of aerobic culture. 8 At the order of magnitude (cFU / mL), aerobic culture under low salinity conditions was more conducive to the proliferation of strain HDES1. Within a salinity range of 10–40, the bacterial count showed a continuous increasing trend across all salinity groups. Comparatively, the strain grew better under aerobic culture conditions, reaching a bacterial count of 10⁻⁶. 8 The number of cells per mL is on the order of magnitude.

[0102] It is evident that although strain HDES1 can survive in low-salinity water with a salinity of 5 during aerobic cultivation, its purification effect on inorganic nitrogen and phosphorus in water is far less than that in seawater environments with a salinity of 10-40. Generally, the strain can exert its nitrogen and phosphorus removal function within 3-6 days of use, and the strain can be reused every 6 days to stably enhance its nitrogen and phosphorus removal effect.

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

[0104] The sterilized Penaeus monodon high pool water (water salinity 30, pH value 8.0) was used as the basic test water control, and incubated at 30°C without adding strain HDES1. The strain HDES1 obtained in Example 1 was inoculated into different pH value aquaculture water at 10 5 -10 6 ML, and the culture temperature was set to 10°C, 20°C, 30°C, and 40°C, the light intensity was 2000-6000Lx, and incubated for 6 days. Meanwhile, an anaerobic culture group (ANO group) and an aerobic incubation group (AEO group) were set up, and each pH gradient test sample of different culture groups was set up in triplicate. 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 shown in Figure 8 , the phosphate concentration of the control group changed little compared with the initial value, and remained at a high concentration level of 8.066-9.389 mg / L. The phosphate concentration in the 10°C-30°C group of the anaerobic culture for 3-6 days decreased slightly, with a removal rate of 5.9%-27.2%; however, the phosphate removal rate in the 10°C-30°C group of the aerobic culture for 3 days reached 57.4%-82.4%; at 40°C, strain HDES1 could not achieve good removal effect of phosphate under anaerobic and aerobic conditions.

[0106] As shown in Figure 9 , the ammonia nitrogen concentration of the control group remained at a high concentration level of 17.724-25.549 mg / L during the test period. The ammonia nitrogen concentration in the 20°C-40°C group of the anaerobic culture for 3-6 days increased to varying degrees, with the highest increase of 81.2% compared with the initial value; however, the ammonia nitrogen removal rate in the 3-day aerobic culture at each temperature reached 46.4%-88.4%, and the 6-day aerobic culture reached 50.1%-79.7%. Overall, strain HDES1 could effectively remove ammonia nitrogen in the water under aerobic culture at each temperature.

[0107] As shown in Figure 10 , the nitrite nitrogen concentration of the control group changed little compared with the initial value, and remained at 9.426-11.606 mg / L. After adding strain HDES1, the nitrite nitrogen removal rate at 10°C-40°C under anaerobic culture or aerobic culture for 3-6 days was more than 99%, indicating that strain HDES1 had good removal effect on nitrite nitrogen.

[0108] As shown in Figure 11As shown, the nitrate nitrogen concentration in the control group generally showed an upward trend, with concentrations ranging from 20.864 to 33.098 mg / L under anaerobic conditions for 3-6 days and from 29.220 to 45.623 mg / L under aerobic conditions for 3-6 days, both at relatively high levels. After adding strain HDES1, the nitrate nitrogen concentration showed a significant decrease at temperatures ranging from 10℃ to 40℃. The nitrate nitrogen removal rate exceeded 99% in all temperature groups after 6 days of anaerobic culture, while the removal rate exceeded 99% in all temperature groups after 3 days of aerobic culture, showing a faster overall effect than anaerobic culture. Strain HDES1 can effectively remove nitrate nitrogen from water under both anaerobic and aerobic conditions at various temperatures.

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

[0110] like Figure 13 As shown, the bacterial count in each group increased from an initial 10 during the test. 5 The bacterial count / mL continued to increase, and after 3-6 days of anaerobic culture, the bacterial count at all temperature groups stabilized at 10. 7 The bacterial count reached the order of magnitude of cells / mL, and after 3-6 days of aerobic culture, the bacterial count at various temperature groups reached 2.6 × 10⁻⁶. 8 6.0 × 10⁻⁶ cells / mL 8 The bacterial count was not significantly different between the different temperature groups.

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

[0112] The sterilized water from the elevated pond of Litopenaeus monodon (salinity 30, pH 8.0) was used as the baseline control, and cultured at a constant temperature of 30°C without the addition of strain HDES1. The group with added strain HDES1 was prepared by adding strain HDES1 obtained in Example 1 at a ratio of 10... 4 -10 5Inoculation was performed at a rate of 1 / mL into aquaculture waters with different pH levels, with pH gradients set at 6, 8, and 10. Light intensity ranged from 2000 to 6000 Lx, and the culture was maintained at a constant temperature of 30℃ for 9 days. Two culture groups were established: an anaerobic culture group (ANO group) and an aerobic incubation group (AEO group). For each pH gradient test in different culture groups, three replicates were prepared. The concentrations of phosphate, ammonia nitrogen, nitrite nitrogen, nitrate nitrogen, and total inorganic nitrogen (TIN), as well as the bacterial count, were monitored every 3 days.

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

[0114] like Figure 15 As shown, the ammonia nitrogen concentration in the control group remained between 14.314 and 18.795 mg / L. During anaerobic culture, the concentration in each pH group did not show a significant decreasing trend, and even increased to a maximum of 55.097 mg / L, an increase of 141.8%. However, during aerobic culture, the ammonia nitrogen concentration in the pH 8 and pH 10 groups showed a continuous decreasing trend, with removal rates reaching 71.1%-95.7% by day 9. This indicates that aerobic culture is more conducive to the removal of ammonia nitrogen by the strain.

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

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

[0117] like Figure 18As shown in Table 1, the TIN concentration value of the control group had no obvious change and remained at 51.575-57.660 mg / L, the TIN removal rate of the pH 8 group and the pH 10 group reached 53.5%-60.1% on the 3rd day of anaerobic culture, and the TIN removal rate of the pH 6 group and the pH 10 group reached 40.7%-68.3% on the 9th day of anaerobic culture.

[0118] As shown in Table 2, the bacterial amount of each group continued to increase from the initial 10 5 8 Figure 19 As shown in Table 1, the TIN concentration value of the control group had no obvious change and remained at 51.575-57.660 mg / L, the TIN removal rate of the pH 8 group and the pH 10 group reached 53.5%-60.1% on the 3rd day of anaerobic culture, and the TIN removal rate of the pH 6 group and the pH 10 group reached 40.7%-68.3% on the 9th day of anaerobic culture.

[0119] As shown in Table 2, the bacterial amount of each group continued to increase from the initial 10 8 8 Figure 19 As shown in Table 1, the TIN concentration value of the control group had no obvious change and remained at 51.575-57.660 mg / L, the TIN removal rate of the pH 8 group and the pH 10 group reached 53.5%-60.1% on the 3rd day of anaerobic culture, and the TIN removal rate of the pH 6 group and the pH 10 group reached 40.7%-68.3% on the 9th day of anaerobic culture.

[0120] Example 4 Application effect of strain HDES1 in high-density zero-water-change aquaculture production

[0121] In the high-density zero-water-change prawn aquaculture production in Jiaxi Town, Shanwei, Guangdong and Dengzhou, Maoming, Guangdong, strain HDES1 was tested for application, and the general bacterial concentration was 10 4 6 4 6 The results showed that the application effect of the bacterial agent was good, and strain HDES1 had no adverse effects on the cultured fish and prawns. Among them, the demonstration base in Maoming Dengzhou passed the on-site test by the expert group organized by Maoming Agricultural Technology Extension Center. The prawns were cultured for 90 days, and the tilapia was cultured for 120 days. The average weight of prawns was 14.7 g per tail, the body length was 12.1 cm, the calculated yield per mu was 10 kg / m 3 , and the survival rate was 82%. The average weight of tilapia was 826 g per tail, the calculated yield per mu was 23 kg / m 3 , and the survival rate was 70%. During the cultivation process, the water conditions were as shown in Table 2, the water temperature was 23-28℃, the salinity was 10-15, the pH value was 7.0-8.5, the dissolved oxygen concentration was 5.02-6.50 mg / L, the ammonia nitrogen was 0.145 mg / L, and the nitrite nitrogen was 0.119 mg / L.

[0122] Table 2 Water quality conditions of the water body in which strain HDES1 was cultivated

[0123]

[0124] The above embodiments are only to describe the preferred modes of the present application, and are not intended to limit the scope of the present application. Any modification and improvement of the technical solutions of the present application made by those skilled in the art without departing from the design spirit of the present application shall fall within the protection scope of the present application.

Claims

1. A strain of Ectothiorhodospira shaposhnikovii HDES1 for purifying high concentration of inorganic nitrogen and phosphorus in seawater culture water or tail water, characterized in that, The bacteria has a preservation number of CCTCC NO: M20191007.

2. An inoculant characterized in that, It contains the Serratia marcescens HDES1 in claim 1.

3. The Serratia marcescens HDES1 in claim 1 is applied in any one of the following: (1) in purifying high-concentration inorganic nitrogen and phosphorus in seawater aquaculture water bodies or tail water; (2) in preparing a bacterial agent for purifying high-concentration inorganic nitrogen and phosphorus in seawater aquaculture water bodies or tail water.

4. The bacterial agent in claim 2 is applied in purifying high-concentration inorganic nitrogen and phosphorus in seawater aquaculture water bodies or tail water.

5. Use according to claim 3 or 4, wherein the compound is ###0002### The high-concentration inorganic nitrogen and phosphorus includes 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 a marine culture water body or tail water, characterized in that, It comprises the step of putting the Serratia marcescens HDES1 in claim 1 into seawater aquaculture water bodies or tail water for purification.

7. The method of claim 6, wherein, The high-concentration inorganic nitrogen and phosphorus includes 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 of claim 6, wherein, The purification conditions include water temperature of 20-40℃, salinity of 10-40, pH value of 8-10, and the Serratia marcescens HDES1 is used repeatedly once every 6-9 days.

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