A strain of aquatic Sphingomonas and its application in degradation of shrimp feces

Abstract

The invention discloses an aquatic sphingomonas and its application in degrading shrimp feces, belonging to the field of microorganisms. The invention separates an aquatic sphingomonas (Sphingomonas aquatilis) from the water of a small shed shrimp breeding pond, and the preservation number is GDMCC No: 64330. The strain ferments and degrades shrimp feces on a fermentation medium with shrimp feces as the only nutrient, and the number of live bacteria in the fermentation liquid is 5.0×10 7 cfu / mL, and the degradation rate of insoluble solids in feces filtration residue reached 50%; the strain can also degrade 30g / L of insoluble solids in shrimp feces to 15g / L within 48-72h, which proves that the strain has excellent shrimp feces degradation ability, can significantly reduce the content of shrimp feces in aquaculture water, and reduce the damage of shrimp feces to shrimp, providing new ideas for the development of safe and ecological purification agents.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, in particular to an aquatic sphingomonas strain and application thereof in degrading shrimp excrement. Background Art

[0002] In aquaculture production, the utilization rate of feed by white shrimp is low. Only a small part of the feed can be effectively utilized by the farmed white shrimp, and most of it is excreted from the body in the form of excrement. In order to make white shrimp grow rapidly, they are usually fed to excess in high-density intensive aquaculture. This feeding method not only easily forms a large amount of residual bait in the aquaculture water, but also increases the food intake of white shrimp accordingly. At the same time, the increase in the feeding level of farmed white shrimp often leads to a decrease in the apparent digestibility of feed and an increase in the content of nutrients in feces. The nutrients in the feed that are not absorbed and utilized by white shrimp will be discharged into the water environment with feces. The greater the amount of feces generated, the more significant the adverse effects on the aquaculture water.

[0003] If the feces discharged by white shrimp maintain their original large particle state in the water, the dissolution rate of nutrients in them will be relatively slow. An appropriate amount of nutrients in the water body can promote the growth of plankton, which is crucial to maintaining the ecological balance of the aquaculture water environment. In aquaculture production, the feces are immediately affected by external forces such as the impact of water flow and the collision of organisms after being discharged, and they are broken into fine particles. The nutrients in them are fully in contact with water and dissolved in the water body, causing the aquaculture water body to accumulate a large amount of nutrients in a short period of time. The dissolved nutrients are difficult to remove, and excessive accumulation will cause harmful algae to grow in large numbers, leading to eutrophication of the water body. Therefore, how to deal with shrimp feces so that it does not affect the water environment is crucial for the aquaculture of white shrimp. Summary of the invention

[0004] The purpose of the present invention is to provide an aquatic sphingomonas strain and its application in degrading shrimp feces to solve the problems existing in the above-mentioned prior art. The strain has excellent shrimp feces degradation ability, can significantly reduce the content of shrimp feces in aquaculture water, reduce the damage of shrimp feces to shrimp, and provide new ideas for the development of safe and ecological purification bacterial agents.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an aquatic Sphingomonas aquatilis, the preservation number of the aquatic Sphingomonas aquatilis is GDMCC No: 64330, the preservation time is May 8, 2024, the preservation unit is Guangdong Microbial Culture Collection Center, and the preservation address is Guangdong Academy of Sciences Institute of Microbiology, 5th Floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou.

[0007] The present invention also provides a method for culturing the aquatic sphingomonas, which comprises culturing the aquatic sphingomonas by shaking at 30-33°C for 70-80h; the liquid fermentation medium is an aqueous solution containing 15-30g / L shrimp feces.

[0008] The present invention also provides the use of the aquatic sphingomonas in degrading shrimp excrement or preparing a bacterial agent for degrading shrimp excrement.

[0009] The present invention also provides the use of the aquatic sphingomonas in purifying shrimp breeding water or preparing a purifying agent for purifying shrimp breeding water.

[0010] The invention also provides a bacterial agent for degrading shrimp excrement, which contains the aquatic sphingomonas.

[0011] The invention also provides a purifier for purifying shrimp breeding water, which contains the aquatic sphingomonas.

[0012] The invention also provides a method for degrading prawn excrement, comprising the steps of inoculating the aquatic sphingomonas into a culture medium containing prawn excrement for mixed culture.

[0013] Preferably, the inoculation amount of the aquatic Sphingomonas is 3%-5% of the culture medium, the temperature of the mixed culture is 30-33° C., and the culture medium is an aqueous solution containing 15-30 g / L.

[0014] The invention also provides a method for purifying a shrimp breeding water body, comprising the steps of inoculating the aquatic sphingomonas into the shrimp breeding water body for mixed culture.

[0015] Preferably, the inoculation amount of the aquatic Sphingomonas is 0.01% of the mass of the shrimp culture water, and the temperature of the mixed culture is 30-33°C.

[0016] The present invention discloses the following technical effects:

[0017] The present invention isolates a strain of aquatic Sphingomonas HD-01 from the water of a small shrimp breeding pond. The HD-01 strain has the ability to significantly reduce shrimp feces. The strain ferments and degrades shrimp feces on a fermentation medium with shrimp feces as the only nutrient. The number of live bacteria in the fermentation liquid is 5.0×10 7cfu / mL, and the degradation rate of insoluble solids in feces filtration residue reached 50%, laying an important foundation for the in-depth research and utilization of shrimp feces degradation. The strain can also degrade 30g / L of insoluble solids in shrimp feces to 15g / L within 48-72h, proving that the strain has excellent shrimp feces degradation ability, which can significantly reduce the content of shrimp feces in aquaculture water, reduce the harm of shrimp feces to shrimp, and provide new ideas for the development of safe and ecological purification bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 This is a graph showing the changes in fecal solids during the fermentation culture of aquatic Sphingomonas strains;

[0020] Figure 2 This is a graph showing the changes in the total number of viable bacteria of aquatic Sphingomonas strains during fermentation culture;

[0021] Figure 3 It is a microscopic picture of aquatic Sphingomonas strain;

[0022] Figure 4 This is the colony morphology of aquatic Sphingomonas strains. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may 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 associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] The feces used in the following examples were collected and processed as follows:

[0029] (1) Feces collection: A nutritional animal protection system (patent number: ZL202221223628.4) is used to collect the feces of whiteleg shrimp by cultivating whiteleg shrimp.

[0030] (2) Feces cleaning: Filter the collected feces with neutral filter paper, add 10 times distilled water to the filtered residue and rinse it (shake on a shaker at 80 rpm for 10 minutes). After rinsing, filter it with neutral filter paper and repeat the rinsing three times.

[0031] (3) Drying of feces: Place the filtered fecal solids into a ventilated drying oven at 55°C and dry to constant weight. Remove and set aside.

[0032] The dried feces are prepared into feces of different concentrations according to the needs.

[0033] Example 1 Isolation, identification and preservation of strains

[0034] 1. Isolation of strains

[0035] The water collected from the small shed shrimp breeding pond in Taishan City, Guangdong Province in February 2024 was screened for microorganisms on the screening medium plate using a 10-fold series gradient dilution separation method. On the screening medium plate, the screened strains were transferred to the liquid fermentation medium at 30°C, 150r / min, and cultured for 72 hours. After the culture was completed, the fermentation broth was filtered with filter paper, the filtrate was tested for the number of viable bacteria (GB 4789.2-2016), the filter residue (insoluble solids) was washed with pure water 3 times, dried in an oven at 105°C to constant weight and weighed. The number of viable bacteria in the filtrate (the detection medium was nutrient agar medium) and the content of the filter residue were finally screened to obtain the strain HD-01 with a high number of viable bacteria and a strong ability to decompose feces under the condition of shrimp feces as the substrate.

[0036] The screening medium is: 20 g / L shrimp feces, 20 g / L agar powder, adjusted to pH 7.5, heated to dissolve, and sterilized at 121°C for 20 min.

[0037] The liquid fermentation medium is: 30 g / L of shrimp feces, make up to 1 L with water, adjust the pH to 7.5, heat to dissolve it (feces is insoluble), and sterilize at 121°C for 30 minutes.

[0038] like Figure 1-Figure 2 The results showed that the isolated strain HD-01 could decompose the macromolecular organic matter in shrimp feces, synthesize the nutrients of the bacteria itself, and finally convert them into bacterial protein. The strain degraded the content of insoluble solids in shrimp feces from 30g / L to 15g / L within 72 hours, and the number of viable bacteria in the fermentation liquid was 5.0×10 7 cfu / mL, which proved that the strain had excellent ability to degrade shrimp feces.

[0039] 2. Biological identification of strain HD-01

[0040] 2.1 Morphological identification and detection of physiological and biochemical characteristics

[0041] The isolated strain HD-01 was inoculated onto a screening culture medium plate and cultured at 30°C for 72 h. The colony growth was observed, and the bacteria were picked to prepare smears for Gram staining. The bacterial morphology and spore formation were observed under a microscope for morphological identification.

[0042] like Figure 3-Figure 4 As shown, the results showed that the colonies were round, with irregular edges and opaque; individual cells were 0.5-0.7×2.0-2.6 μm, Gram-negative, and without spores.

[0043] 2.2 Molecular Biological Identification

[0044] The isolated strain HD-01 was inoculated into a liquid fermentation medium with an inoculation volume of 5% (V / V), a temperature of 33°C, a shaking speed of 150 rpm, and cultured for 24 h. The bacterial genome was extracted using a DNA extraction kit (purchased from Beijing Biotech Biotechnology Co., Ltd.). The 16S rRNA gene was amplified by PCR using the bacterial genome as a template, and the amplified product was submitted to the Guangdong Provincial Microbial Culture Collection Center for sequencing analysis.

[0045] The 16S rRNA gene sequence of the amplified strain is shown in SEQ ID NO: 1:

[0046] CGAGATCTTCGGGTCTAGTGGCGCACGGGTGCGTAACGCGTGGGAATCTGCCCTTTGGTTCGGAATAACAGTTGGAAACGACTGCTAATACCGGATGATGACGAAAGTCCAAAGATTTATCGCCAGAGGATGAGCCCGCGTAGGATTAGCTAGTTGGTGTGGTAAAGGCGCACCAAGGCGACGATCCTTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCAATGCCGCGTGAGTGATGAAGGCCTTAGGGTTGTAAAGCTCTTTTACCCGGGATGATAATGACAGTACCGGGAGAATAAGCTCCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGAGCTAGCGTTGTTCGGAATTACTGGGCGTAAAGCGCACGTAGGCGGCTTTGTAAGTTAGAGGTGAAAGCCTGGAGCTCAACTCCAGAACTGCCTTTAAGACTGCATCGCTTGAATCCAGGAGAGGTGAGTGGAATTCCGAGTGTAGAGGTGAAATTCGTAGATATTCGGAAGAACACCAGTGGCGAAGGCGGCTCACTGGACTGGTATTGACGCTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGATAACTAGCTGTCCGGGGACTTGGTCTTTGGGTGGCGCAGCTAACGCATTAAGTTATCCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCTGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCAGCGTTTGACATGTCCGGACGATTTCCAGAGATGGATCTCTTCCCTTCGGGGACTGGAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCGCCTTTAGTTACCATCATTTAGTTGGGGACTCTAAAGGAACCGCCGGTGATAAGCCGGAGGAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTACGCGCTGGGCTACACACGTGCTACAATGGCGGTGACAGTGGGCAGCAAACTCGCGAGAGTGCGCTAATCTCCAAAAGCCGTCTCAGTTCGGATTGTTCTCTGCAACTCGAGAGCATGAAGGCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCAGGCCTTGTACACACCGCCCGTC(SEQ ID NO:1).

[0047] Alignment of the 16S rRNA gene sequence on NCBI showed that: Alignment of the 16S rRNA sequence of HD-01 sample on NCBI (https: / / blast.ncbi.nlm.nih.gov) showed that: The homology of HD-01 sample with Sphingomonas aquatilis reached 100%, and the homology with Sphingomonas melonis reached 99.92%.

[0048] Comparison on EZBioCloud (https: / / www.ezbiocloud.net) found that the homology of HD-01 sample with Sphingomonas aquatilis reached 100%, the homology with Sphingomonas melonis reached 99.92%, and the homology with Sphingomonas taxi reached 99.30%. Therefore, the isolated strain HD-01 was determined to be Sphingomonas aquatilis, which was deposited in Guangdong Microbial Culture Collection Center on May 8, 2024. The deposit address is: Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is: GDMCC No: 64330.

[0049] Example 2 Detection of the ability of aquatic Sphingomonas HD-01 to degrade shrimp feces

[0050] 1. Preparation of fermentation broth

[0051] Aquatic Sphingomonas HD-01 and commercially available Sphingomonas (Beina Biotech) seed liquid were inoculated into liquid fermentation medium at 5% inoculation rate. Cultured at 30°C, 150r / min for 72 hours and set aside. The liquid fermentation medium was used as a blank control group.

[0052] 2. Filter

[0053] The aquatic Sphingomonas fermentation broth and the commercially available Sphingomonas fermentation broth were filtered with filter paper, and the number of viable bacteria in the filtrate was tested (GB 4789.2-2016). The filter residue was washed three times with pure water, dried in an oven at 105°C to constant weight and weighed.

[0054] 3. Test results:

[0055] Table 1 Degradation results of shrimp feces

[0056] Group Filtrate viable count cfu / mL Filter residue content g / L Control group 0 29.69 Sphingomonas <![CDATA[2.1×10 4 ]]> 29.52 Sphingomonas aquaticus HD-01 <![CDATA[5.0×10 7 ]]> 14.94

[0057] Example 3 Test experiment on degradation of shrimp feces in small shed shrimp farming water by aquatic Sphingomonas HD-01

[0058] Take the water from the small shed shrimp farming (mid-to-late stage of farming, lower layer of the pond), divide the water into 500 mL triangular bottles, 300 mL per bottle, add 15 g / L of white shrimp feces to each bottle, add the aquatic Sphingomonas HD-01 fermentation filtrate obtained in Example 2 (the number of viable bacteria is 5.0×10 7cfu / mL) was added to a conical flask at an addition rate of 0.01% (V / V), and the shaker was operated at 150 rpm and the culture temperature was 30°C for 72 hours. The commercially available water purification Bacillus (100 billion viable bacteria) was used to degrade the feces of shrimp in the water of small shed shrimp farming as a comparison, and the liquid fermentation medium without aquatic sphingomonas inoculation was used as a control. After the culture was completed, the content of solid matter in the feces of shrimp in the water and the total number of viable bacteria in the filtrate were detected (GB4789.2-2016). The results are shown in Table 2.

[0059] Table 2 Results of degradation of shrimp feces in small shed shrimp water by aquatic Sphingomonas HD-01

[0060]

[0061] The experimental results in Table 2 show that under laboratory conditions, aquatic Sphingomonas HD-01 has excellent shrimp feces degradation ability, which significantly and efficiently reduces the content of shrimp feces in aquaculture water, providing a safer and more effective microbial strain for purifying aquaculture water.

[0062] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Sphingomonas aquaticus ( Sphingomonas aquatilis ), characterized in that, The deposit number of the aquatic sphingomonas is GDMCC No: 64330.

2. A method for culturing the aquatic Sphingomonas according to claim 1, characterized in that: The liquid fermentation medium is used for shaking culture at 30-33°C for 70-80 hours; the liquid fermentation medium is an aqueous solution containing 15-30 g / L of shrimp feces.

3. Use of the aquatic Sphingomonas as claimed in claim 1 in degrading shrimp feces or preparing a bacterial agent for degrading shrimp feces.

4. Use of the aquatic sphingomonas according to claim 1 in purifying shrimp aquaculture water or preparing a purifier for purifying shrimp aquaculture water.

5. A microbial agent for degrading shrimp feces, characterized in that: Contains the aquatic Sphingomonas according to claim 1.

6. A purifier for purifying shrimp breeding water, characterized in that: Contains the aquatic Sphingomonas according to claim 1.

7. A method for degrading shrimp feces, characterized in that: The method comprises the steps of inoculating the aquatic sphingomonas described in claim 1 into a culture medium containing shrimp feces for mixed culture.

8. The method according to claim 7, characterized in that The inoculation amount of the aquatic sphingomonas is 3%-5% of the culture medium, the temperature of the mixed culture is 30-33° C., and the culture medium is an aqueous solution containing 15-30 g / L of shrimp feces.

9. A method for purifying shrimp farming water, characterized in that: The method comprises the steps of inoculating the aquatic sphingomonas described in claim 1 into a shrimp breeding water body for mixed culture.

10. The method according to claim 9, characterized in that The inoculation amount of the aquatic sphingomonas is 0.01% of the mass of the shrimp breeding water, and the temperature of the mixed culture is 30-33°C.

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