A strain of Aeromonas bivalvae and its application

Abstract

The invention discloses a strain of Aeromonas bivalvae and application thereof. Aeromonas bivalvium ) was named R‑1, and was deposited in the General Microbiological Center of China Microbiological Culture Collection Committee on March 29, 2024, with the deposit number: CGMCC No. 30186. The bivalve Aeromonas provided by the present invention can effectively remove nitrite nitrogen, nitrate nitrogen and ammonia nitrogen in aquaculture tail water, with good removal effect, and can also be used to prepare shrimp feed, effectively improving the production speed of shrimp.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a strain of Aeromonas bivalveus and application thereof. Background Art

[0002] The rapid development of aquaculture has made an important contribution to meeting the growing demand for aquatic products. Aquaculture tailwater contains a large amount of inorganic nitrogen such as ammonia nitrogen, nitrite nitrogen and nitrate nitrogen. If it is discharged directly without treatment, it will cause eutrophication of the receiving water body and cause ecological disasters such as algal blooms and red tides. In response to this problem, aquaculture tailwater nitrogen removal technology has emerged and is constantly developing. Currently, the commonly used nitrogen removal technologies mainly include: (1) Biological method: using the metabolism of microorganisms to remove nitrogen from water, mainly including nitrification-denitrification method, anaerobic ammonium oxidation method, etc. This is the most widely used method at present, with the advantages of good treatment effect and low operating cost. (2) Physical method: separating or removing nitrogen from water through physical action, such as membrane separation method, adsorption method, ion exchange method, etc. This method is simple to operate, but the cost is relatively high. (3) Chemical method: removing nitrogen from water through chemical reaction, such as chemical precipitation method, advanced oxidation method, etc. This method is fast, but may cause secondary pollution. (4) Ecological method: using aquatic plants, microalgae, etc. to absorb nitrogen in water, such as artificial wetlands, ecological floating beds, etc. This method is environmentally friendly, but is greatly affected by seasons and climate. In order to develop aquaculture tailwater treatment technology in the direction of high efficiency, low consumption, and environmental protection, it is necessary to study a method for aquaculture tailwater denitrification that can improve nitrogen removal efficiency, reduce energy consumption, reduce secondary pollution, and achieve resource utilization. Summary of the invention

[0003] In order to solve the deficiencies of the prior art, the object of the present invention is to provide a strain of Aeromonas bivalveus that can effectively remove nitrite nitrogen, nitrate nitrogen and ammonia nitrogen in aquaculture tail water.

[0004] In order to achieve the above object, the present invention adopts the following technical solution:

[0005] A strain of Aeromonas bivalviae, Aeromonas bivalviae ( Aeromonas bivalvium ) was named R-1 and was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (CGMCC) on March 29, 2024. The deposit number is: CGMCC No.30186, and the deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] A bacterial agent prepared using the aforementioned Aeromonas bivalve bacteria.

[0007] The application of Aeromonas bivalvae or its bacterial agent in the degradation of nitrogen pollutants in aquaculture tail water, wherein the nitrogen pollutants are one or more of nitrite nitrogen, nitrate nitrogen or ammonia nitrogen. The concentration of nitrite nitrogen in aquaculture tail water is 500~1500mg / L, the concentration of nitrate nitrogen is 500~2000mg / L, and the concentration of ammonia nitrogen is 500~1500mg / L; the pH of aquaculture tail water is 7-8, and the temperature is 20~30℃.

[0008] Application of Aeromonas bivalvae in preparing shrimp feed, the content of Aeromonas bivalvae in shrimp feed is 10 6 ~10 8 cfu / g.

[0009] The preparation method of the microbial agent comprises the following specific steps:

[0010] S1, inoculate strain R-1 into LB medium and culture to obtain seed solution;

[0011] S2, inoculating the seed liquid into the fermentation medium for expansion and cultivation to obtain bacterial liquid, and centrifuging and filtering the bacterial liquid to obtain bacterial cells;

[0012] S3. Mix the bacteria with glucose, dry and crush them to make a live bacteria preparation.

[0013] The benefits of the present invention are as follows: the bivalve Aeromonas provided by the present invention can effectively remove nitrite nitrogen, nitrate nitrogen and ammonia nitrogen in aquaculture tail water, with good removal effect, and can also prepare shrimp feed, effectively improve the production speed of shrimp, have a certain ability to promote digestion and absorption, can improve the utilization rate of feed, promote the healthy growth of shrimp, and have an inhibitory effect on Vibrio harveyi and Edwardsiella to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the colony morphology of strain R-1;

[0015] Figure 2 is the phylogenetic tree of strain R-1;

[0016] Figure 3 This is a graph showing the degradation effect of strain R-1 on different concentrations of nitrite nitrogen;

[0017] Figure 4 This is a graph showing the degradation effect of strain R-1 on nitrate nitrogen at different concentrations;

[0018] Figure 5 This is a graph showing the degradation effect of strain R-1 on ammonia nitrogen at different concentrations;

[0019] Figure 6 This is the degradation effect diagram of strain R-1 at different pH;

[0020] Figure 7This is a diagram showing the degradation effect of strain R-1 at different temperatures. DETAILED DESCRIPTION

[0021] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0022] Example 1, Isolation, screening and identification of Aeromonas bivalviae R-1:

[0023] It was isolated from a biological filler treatment system of Nantong Lingyang Agricultural Development Co., Ltd. using the enrichment culture method. The specific steps are as follows:

[0024] (1) Preparation of culture medium: Add 100 mL of inorganic salt culture medium to a 250 mL conical flask, add 1 g / L trace element solution, and sterilize at 121°C for 30 min.

[0025] The inorganic salt culture medium solution is (g / L): 0.3g NaCl, 0.14g MgSO 4 •7H 2 O, 0.03 gFeSO 4 •7H 2 O, 0.136gKH 2 PO 4 , 0.5gNaNO 2 , 1.6 g NaHCO 3 ; Trace element solution (g / L): 1g MnSO 4 •H 2 O, 0.25gNa 2 MoO 4 •2H 2 O, 0.1 g H 3 BO 4 , 0.25 g CuCl 2 •2H 2 O, 0.25 g ZnCl 2 , 0.1 g NH 4 VO 3 , 0.25 g Co(NO 3 ) 2 •6H 2 O, 0.1 g NiSO 4 •6H 2 O, dissolved in 900 mL of distilled water, and 5 mL of concentrated H 2 SO 4 , add distilled water to 1000mL.

[0026] (2) Screening process: Take 5 mL of the collected sludge from the biofiller membrane and inoculate it into 100 mL of inorganic salt culture medium. Oscillate and culture it at 30°C in a shaker with a speed of 180 r / min for 1 day. When the culture medium becomes turbid, take 5 mL of the bacterial solution and inoculate it into 100 mL of new culture medium. Repeat the transfer 3 times. The concentration of nitrate nitrogen in the new culture medium increases successively. Dilute the bacterial solution cultured from the last time in a gradient manner, take 0.1 mL of each concentration of dilution and apply it on a solid culture medium containing nitrate nitrogen. Invert and culture at 30°C for 3 to 5 days, pick a single colony and streak it on a plate again to obtain the purified strain R-1. The colony morphology is shown in the figure below. Figure 1 As shown, the surface is round, yellow-white, smooth, shiny, slightly convex, and has neat edges.

[0027] The 16S rDNA gene sequencing was used to classify and identify the Aeromonas bivalviae R-1 strain, and the steps are as follows 1:

[0028] The DNA of strain R-1 was extracted using a gene extraction kit and used as a template for PCR reaction. The primers for bacterial amplification were bacterial universal primers. 27F: (5-AGAGTTTGATC-CTGGCTCAG-3) and 1492R: (5- GGTTACCTTGTTACGACTT-3). 25 μL 2×Mix Taq was mixed with 2 μL of each forward and reverse primer and 2 μL of bacterial solution, and then water was added to a 50 μL system for PCR amplification. The PCR reaction program was 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s; 55℃ annealing for 1 min; 72℃ extension for 1 min, a total of 30 cycles; 72℃ extension for 10 min. The PCR product was detected by 1% agarose gel electrophoresis and sent to a sequencing company for sequencing. The sequence table is shown in SEQ ID NO: 1. The 16S rDNA sequence of the strain was searched and compared in the NCBI database using the BLAST method. The 16S rDNA sequences of strain R-1 and its related strains were aligned using Mega (Version 7.0) software, and the phylogenetic tree was constructed using the Neighbor-joining method. Figure 2 .

[0029] The above strain was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on March 29, 2024, and its classification name is Aeromonas bivalviae ( Aeromonas bivalvium ), the deposit address is: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the strain deposit number is: CGMCC No.30186.

[0030] Embodiment 2, the preparation method of bacterial agent comprises the following specific steps:

[0031] S1. Inoculate strain R-1 into LB medium. The components of LB medium are: 5 g yeast extract, 10 g peptone, 5 g NaCl and 1 L water. The culture temperature is 25-30 ° C and the pH is 7 for 24 h to obtain seed solution.

[0032] S2, inoculating the seed liquid into a fermentation medium for expansion culture, the components of the fermentation medium are: 15 g / L glucose, 20 g / L soybean meal, 4 g / L yeast extract, 5 g / L mannitol, 1 g / L potassium dihydrogen phosphate and 0.5 g / L magnesium sulfate, the inoculation amount of the seed liquid is 2%-5%, the culture temperature is 25-30°C, and the pH is 7 for 24 hours to obtain a bacterial solution, and the bacterial solution is centrifuged and filtered to collect the bacterial cells;

[0033] S3. Mix the bacteria and glucose in a volume ratio of 1:10, and prepare a live bacteria preparation by drying and crushing.

[0034] Example 3, Identification of the enzyme production ability of Aeromonas bivalviae:

[0035] Prepare amylase selective medium, take Aeromonas bivalvae and inoculate it on solid medium, incubate it at 30°C for 48 hours, and observe whether there is hydrolysis zone around the colony. If there is, it means that the strain has the corresponding enzyme production ability.

[0036] The components of the amylase selection medium are: 10 g soluble starch, 5 g peptone, 1 g yeast extract, 16 g agar, add distilled water to 1000 mL, adjust the pH to 7.4, sterilize at 121° C. for 20 min and set aside.

[0037] The measured ratio of enzyme hydrolysis circle to colony diameter (H / D) is about 5.00, indicating that the bivalve Aeromonas of the present invention can secrete amylase and has a certain ability to promote digestion and absorption. Adding it to feed can improve the utilization rate of feed to a certain extent and promote the healthy growth of shrimp.

[0038] Example 4: Application of Aeromonas bivalvae in feed:

[0039] A control group and an experimental group were set up. The nutritional composition of the control group feed was: 40% fish meal, 26% soybean meal, 8% bran, 14% second flour, 6% rice bran, 2% meat and bone meal, 1% phospholipid oil, 2% additives, and a certain amount of vitamins, minerals and trace elements were added. The experimental group feed was supplemented with Aeromonas bivalve R-1 (concentration of 10 7 cfu / g). The aquatic animals selected were Penaeus vannamei (purchased from Lingyang Agricultural Development Co., Ltd.), which were pre-cultured in the laboratory for three days. 60 Penaeus vannamei were evenly placed in six acrylic fish tanks with a volume of 100L, with three replicates in each group. They were fed at 7:00, 11:00, 18:00, and 23:00 every day for two weeks.

[0040] The results showed that the whiteleg shrimps in both groups grew normally, and the growth of the whiteleg shrimps in the experimental group was slightly stronger than that in the control group. Therefore, it can be seen that the bivalve Aeromonas R-1 of the present invention can increase the growth rate of whiteleg shrimps.

[0041] Example 5, Ability of Aeromonas bivalvae R-1 to resist pathogenic bacteria:

[0042] Take an appropriate amount of bacterial suspension after activation of strain R-1, evenly spread it on LB solid culture medium, and evenly place 4 sterile Oxford cups on the surface of the culture medium to keep the Oxford cups stable; add 200 μl of bacterial suspension of Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, and Edwardsiella into the Oxford cups respectively, and place the culture dish in a 30°C incubator for static culture for 18-24 hours; after the culture is completed, observe whether an antibacterial zone is formed on the culture medium. If a transparent zone appears around the Oxford cup, it means that strain R-1 has antibacterial activity.

[0043] At the end of the experiment, the diameter of the inhibition zone was measured. The results showed that the sizes of the inhibition zones of strain R-1 against Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, and Edwardsiella were 6mm±0.34, 12mm±0.17, 8.7mm±0.28, and 11mm±0.31, respectively. Aeromonas bivalve R-1 had an inhibitory effect on Vibrio harveyi and Edwardsiella to a certain extent.

[0044] Example 6: Degradation effect of Aeromonas bivalviae R-1 on different concentrations of nitrite nitrogen

[0045] The strain obtained in Example 1 was inoculated at a volume ratio of 1% into culture media with initial nitrite nitrogen concentrations of 500 mg / L, 1000 mg / L, 1500 mg / L, 2000 mg / L, 2500 mg / L, 3000 mg / L, and 3500 mg / L, respectively. The culture media used sodium nitrite as the sole nitrogen source, and the culture media components were (g / L): 0.3 g NaCl, 0.14 g MgSO 4 •7H 2 O, 0.03 gFeSO 4 •7H 2 O, 0.136gKH 2 PO 4 , NaNO 2 , 1.6 g NaHCO 3 , 1mL trace element solution; trace element solution is (g / L): 1g MnSO 4 •H 2 O, 0.25 g Na 2 MoO 4 •2H 2 O, 0.1 g H 3 BO4 , 0.25 g CuCl 2 •2H 2 O, 0.25 g ZnCl 2 , 0.1gNH 4 VO 3 , 0.25 g Co(NO 3 ) 2 •6H 2 O, 0.1 g NiSO 4 •6H 2 O, dissolved in 900 mL of distilled water, and 5 mL of concentrated H 2 SO 4 , add distilled water to 1000mL, and after shaking culture at pH 7.0, temperature 30℃, 180rpm for 48h, determine the residual sodium nitrite content and bacterial concentration in the sample. The sodium nitrite content was determined by ultraviolet spectrophotometry. The degradation rate of the strain to different concentrations of nitrite nitrogen is shown in the following table. Figure 3 shown.

[0046] Depend on Figure 3 It can be seen that when the concentration of sodium nitrite is between 500 and 1500 mg / L, the degradation rate of nitrite nitrogen by Aeromonas bivalve R-1 is above 75%. When the concentration is greater than 1500 mg / L, the degradation rate decreases significantly with the increase of concentration.

[0047] Example 7: Degradation effect of Aeromonas bivalviae R-1 on nitrate nitrogen at different concentrations:

[0048] The strain obtained in Example 1 was inoculated with an inoculum volume ratio of 1% into a culture medium with an initial nitrate nitrogen concentration of 500 mg / L, 1000 mg / L, 1500 mg / L, 2000 mg / L, 2500 mg / L, 3000 mg / L, and 3500 mg / L, respectively. The culture medium used potassium nitrate as the sole nitrogen source, and was shaken and cultured at pH 7.0, 30°C, and 180 rpm for 48 hours. The residual potassium nitrate content and bacterial concentration in the sample were determined. The potassium nitrate content was determined by ultraviolet spectrophotometry. The degradation rate of the strain to different concentrations of nitrate nitrogen is shown in the following table. Figure 4 shown.

[0049] Depend on Figure 4 It can be seen that when the concentration of potassium nitrate is between 500 and 2000 mg / L, the degradation rate of potassium nitrate by Aeromonas bivalve R-1 is above 80%. When the concentration is greater than 2000 mg / L, the degradation rate decreases significantly with the increase of concentration.

[0050] Example 8: Degradation effect of Aeromonas bivalviae R-1 on ammonia nitrogen at different concentrations:

[0051] The strain obtained in Example 1 was inoculated with an inoculum volume ratio of 1% into a culture medium with an initial ammonia nitrogen concentration of 500 mg / L, 1000 mg / L, 1500 mg / L, 2000 mg / L, 2500 mg / L, 3000 mg / L, and 3500 mg / L, respectively. The above culture medium used ammonium chloride as the only nitrogen source, and was shaken and cultured at pH 7.0, temperature 30°C, and 180 rpm for 48 hours. The residual ammonium chloride content and bacterial concentration in the sample were determined. The ammonia nitrogen content was determined using a water quality detector, and the results are shown in Table 1. Figure 5 .

[0052] Depend on Figure 5 It can be seen that when the initial concentration of ammonia nitrogen is 500~1500mg / L, the degradation rate of ammonium chloride by Aeromonas bivalve R-1 is above 85%. When the initial concentration of ammonia nitrogen exceeds 1500mg / L, the degradation rate decreases significantly with the increase of concentration.

[0053] Example 9, Degradation Effect of Aeromonas bivalvae R-1 at Different pH:

[0054] The Aeromonas bivalve R-1 strain preserved in Example 1 was activated and cultured to the logarithmic growth phase under suitable conditions. Under the conditions of pH 5, 6, 7, 8, 9, and 10, the strain R-1 was inoculated into an inorganic salt medium containing 2000 mg / L sodium nitrite, 2000 mg / L potassium nitrate, and 2000 mg / L ammonium chloride, respectively, at an inoculation amount of 1% (v / v). The Aeromonas bivalve R-1 in the logarithmic growth phase was inoculated into each culture medium at 1%, and the concentrations of sodium nitrite, potassium nitrate, and ammonium chloride in the sample were determined after shaking culture at a salinity of 1%, a temperature of 30°C, and 180rpm for 48 hours. The degradation rate of nitrogen in each treatment group was calculated based on the measurement results.

[0055] Depend on Figure 6 It can be seen that strain R-1 grows best in the pH range of 7-8, and the degradation rates of sodium nitrite, potassium nitrate and ammonium chloride are all above 70%. After the pH is greater than 8, the degradation effect weakens with the increase of pH value.

[0056] Example 10: Degradation effect of Aeromonas bivalviae R-1 at different temperatures:

[0057] The Aeromonas bivalvae R-1 strain preserved in Example 1 was activated and cultured under suitable conditions to the logarithmic growth phase.

[0058] At 5℃, 8℃, 10℃, 20℃, 30℃, 40℃, and 50℃, strain R-1 was inoculated into inorganic salt medium containing 2000mg / L sodium nitrite, 2000mg / L potassium nitrate, and 2000mg / L ammonium chloride, respectively, at a 1% (v / v) inoculation amount. Aeromonas bivalve R-1 in the logarithmic growth phase was inoculated into each medium at 1%, and the concentrations of sodium nitrite, potassium nitrate, and ammonium chloride in the samples were determined after shaking culture at pH 7.0, salinity 1%, and 180rpm for 48 hours. The degradation rate of nitrogen in each treatment group was calculated based on the measurement results.

[0059] Depend on Figure 7 It can be seen that the optimal growth temperature of strain R-1 is 20~30℃, and it has the best degradation effect on sodium nitrite, potassium nitrate and ammonium chloride, all above 60%, but it cannot survive under low and high temperature conditions and the degradation effect is not good.

[0060] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solution obtained by equivalent replacement or equivalent transformation falls within the protection scope of the present invention.

Claims

1. A strain of Aeromonas bivalvae, characterized in that: The bivalve Aeromonas ( Aeromonas bivalvium ) was named R-1 and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on March 29, 2024, with the deposit number: CGMCC No.30186.

2. A bacterial agent prepared using the bivalve Aeromonas described in claim 1.

3. Use of the bivalve Aeromonas described in claim 1 or the bacterial agent described in claim 2 in degrading nitrogen pollutants in aquaculture tail water, characterized in that: The nitrogen pollutant is one or more of nitrite nitrogen, nitrate nitrogen or ammonia nitrogen.

4. The use according to claim 3, characterized in that: The concentration of nitrite nitrogen in the aquaculture tail water is 500-1500 mg / L, the concentration of nitrate nitrogen is 500-2000 mg / L, and the concentration of ammonia nitrogen is 500-1500 mg / L.

5. The use according to claim 3, characterized in that: The pH of the aquaculture tail water is 7-8 and the temperature is 20-30°C.

6. The use of Aeromonas bivalvae according to claim 1 in preparing shrimp feed, characterized in that: The content of Aeromonas bivalviae in the shrimp feed is 10 6 ~10 8 cfu / g.

7. A method for preparing the bacterial agent according to claim 2, characterized in that: The specific steps include: S1, inoculate strain R-1 into LB medium and culture to obtain seed solution; S2, inoculating the seed liquid into the fermentation medium for expansion and cultivation to obtain bacterial liquid, and centrifuging and filtering the bacterial liquid to obtain bacterial cells; S3. Mix the bacteria with glucose, dry and crush them to make a live bacteria preparation.

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