Bacillus subtilis and application thereof in aquaculture

Abstract

The invention belongs to the technical field of microorganisms, and provides bacillus subtilis and application thereof in aquaculture. The bacillus subtilis provided by the invention is preserved in Guangdong Microbial Culture Collection Center on November 04, 2024, and the preservation number is GDMCC No: 65406. The strain can effectively inhibit excessive propagation of blue-green algae, accelerate decomposition of organic matters in a water body, remarkably degrade harmful metabolites such as ammonia nitrogen and nitrite, and synchronously reduce the chemical oxygen demand (COD) index.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to a Bacillus subtilis and its application in aquaculture. Background Art

[0002] In recent years, the aquaculture industry has developed rapidly, mainly due to the continuous improvement of aquaculture technology, although the aquaculture cost is also increasing continuously. This industry has gradually formed a high-density aquaculture mode mainly based on intensification, scale and industrialization, which greatly meets the demand for aquatic products. Although this aquaculture mode saves costs and resources to a certain extent, the pollution problem in the aquaculture process has become increasingly prominent. The water quality has deteriorated seriously, and the eutrophication phenomenon occurs frequently, resulting in the destruction of the aquaculture ecological environment, frequent diseases of aquatic products such as fish and shrimp, and thus huge economic losses. In severe cases, pollutants will remain in the aquaculture animals, posing a threat to human health.

[0003] Currently, the methods used for water quality treatment in the aquaculture process mainly include physical, chemical and biological remediation, etc., among which the chemical method dominates. Although the chemical method has a significant effect, its duration is short, and long-term and large-scale use will lead to the emergence of drug resistance problems.

[0004] Microbial remediation technology is a method that uses microorganisms to absorb and metabolize and degrade environmental pollutants. The beneficial microorganisms therein can convert complex organic substances into simple inorganic substances for the growth and reproduction of plankton, while purifying the environment and maintaining the ecological balance of the aquaculture environment.

[0005] The principle of microbial remediation, that is, the water purification mechanism of beneficial microorganisms, lies in that the beneficial microorganisms in the water body obtain carbon sources and energy by decomposing organic substances. Under the action of various enzymes of microorganisms, the organic substances undergo anaerobic or aerobic processes and are finally degraded into harmless substances. This process can remove a large amount of residual baits, excretory wastes and animal and plant residues accumulated in the aquaculture environment for a long time without interrupting the aquaculture. These organic substances are first decomposed into small molecules and finally converted into carbon dioxide, nitrates, etc., effectively reducing the chemical oxygen demand (COD), biological oxygen demand (BOD) and the concentrations of ammonia nitrogen and nitrite in the water, thus effectively improving the water quality. Currently, the microorganisms used for water purification include nitrifying bacteria, photosynthetic bacteria, Bacillus, actinomycetes, yeasts and Lactobacillus brevis, etc.

[0006] In traditional aquaculture, although chemical water treatment methods can improve water quality indicators in the short term, their effect is short-lived and they need to be used frequently and in large doses. This mode not only raises the aquaculture cost, destroys the ecological balance of the water body, but also leads to the emergence of drug resistance in pathogens, and long-term accumulation will also cause health damage to cultured organisms. In contrast, Bacillus subtilis, as a typical representative of probiotics, has multiple functions such as water purification, growth promotion, intestinal microecological optimization, and immunomodulation, showing significant advantages in the field of green aquaculture. However, the current industrial application faces double challenges: the complex strain preparation process leads to high production costs, some strains consume too much oxygen during metabolism, which is likely to cause water hypoxia, and there are also technical bottlenecks such as unstable viable bacteria concentration. Therefore, developing probiotic strains with low-cost, low-oxygen consumption characteristics, and capable of maintaining a high viable bacteria concentration and having no toxic or side effects on cultured organisms has important practical significance for realizing the biological prevention and control of organic matter and harmful algae in water bodies. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a strain of Bacillus subtilis and its application in aquaculture. The Bacillus subtilis provided by the present invention has the ability to produce protease, can effectively control the growth of cyanobacteria, can effectively treat organic matter in the water body, and can effectively degrade ammonia nitrogen and COD in the water body.

[0008] The technical solution provided by the present invention is as follows:

[0009] A strain of Bacillus subtilis was deposited at the Guangdong Provincial Microbial Culture Collection Center on November 04, 2024. The deposit address is: 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou. The deposit number is: GDMCC No: 65406, and the taxonomic name is: Bacillus subtilis.

[0010] The application of the above-mentioned Bacillus subtilis in purifying and regulating aquaculture water bodies is also the key content protected by the present invention.

[0011] Preferably, purifying and regulating water includes: Bacillus subtilis inhibiting the growth of cyanobacteria in aquaculture water bodies.

[0012] Preferably, purifying and regulating water includes: Bacillus subtilis decomposing organic matter in aquaculture water bodies.

[0013] Preferably, purifying and regulating water includes: the application of Bacillus subtilis in degrading ammonia nitrogen and nitrite in aquaculture water bodies and reducing the COD in the aquaculture water body.

[0014] Application of Bacillus subtilis in purifying and regulating aquaculture water bodies involves activating Bacillus subtilis and diluting the activated liquid into the aquaculture water body. The volume ratio of the activated liquid to the aquaculture water body is 1:90 - 110. Meanwhile, the aquaculture water body is aerated to keep the dissolved oxygen above 5 mg / L.

[0015] Preferably, the volume ratio of the activated liquid to the aquaculture water body is 1:100.

[0016] Preferably, the activation method is: weigh 5 g of Bacillus subtilis and add 10 g of glucose into 500 mL of natural water body, and use an oxygenation pump to oxygenate and activate for 3 h.

[0017] The content that this invention focuses on protecting also includes:

[0018] The above application of Bacillus subtilis in degrading ammonia nitrogen and chemical oxygen demand in water bodies;

[0019] The above application of Bacillus subtilis in treating organic matter in water bodies;

[0020] The above application of Bacillus subtilis in treating cyanobacteria in water bodies;

[0021] The above application of Bacillus subtilis in producing protease.

[0022] This invention has the following advantages and effects compared with the prior art:

[0023] (1) This strain can effectively inhibit the overgrowth of cyanobacteria, accelerate the decomposition of organic matter in water bodies, significantly degrade harmful metabolites such as ammonia nitrogen and nitrite, and simultaneously reduce the chemical oxygen demand (COD) index; and it does not produce toxic and harmful substances, having no impact on the aquaculture water body and aquaculture animals;

[0024] (2) The viable bacteria content is high, not less than 10 11 CFU / g;

[0025] (3) The Bacillus subtilis provided by this invention has a low use cost and a long-lasting effect after being prepared into a microbial agent. Description of the Drawings

[0026] Figure 1 It is a graph of COD concentration change;

[0027] Figure 2 It is a graph of ammonia nitrogen concentration change;

[0028] Figure 3 It is a graph of nitrite concentration change;

[0029] Figure 4 It is the water body condition of Shed No. 7 in small shed aquaculture before using Baoshuang;

[0030] Figure 5 Water body condition of Shed No. 9 in small shed farming before using Baoshuang

[0031] Figure 6 Water body condition of Shed No. 7 in small shed farming after using Baoshuang

[0032] Figure 7 Water body condition of Shed No. 9 in small shed farming after using Baoshuang

[0033] Figure 8 Water body condition of Shed No. 16 in small shed farming before using Baoshuang

[0034] Figure 9 Water body condition of Shed No. 17 in small shed farming before using Baoshuang

[0035] Figure 10 Water body condition of Shed No. 16 in small shed farming after using Baoshuang

[0036] Figure 11 Water body condition of Shed No. 17 in small shed farming after using Baoshuang

[0037] Figure 12 Blue-green algae condition at the downwind of the pond of Dasyatis akajei before using Baoshuang

[0038] Figure 13 Blue-green algae condition at the downwind of the pond of Dasyatis akajei 2 days after using Baoshuang

[0039] Figure 14 Blue-green algae condition at the downwind of the pond of Dasyatis akajei 5 days after using Baoshuang

[0040] Figure 15 Blue-green algae condition at the downwind of the pond of Dasyatis akajei 8 days after using Baoshuang

[0041] Figure 16 Blue-green algae condition at the downwind of the pond of Litopenaeus vannamei before using Baoshuang

[0042] Figure 17 Blue-green algae condition at the downwind of the pond of Litopenaeus vannamei 2 days after using Baoshuang

[0043] Figure 18 Blue-green algae condition at the downwind of the pond of Litopenaeus vannamei 3 days after using Baoshuang

[0044] Figure 19 Blue-green algae condition at the downwind of the pond of Litopenaeus vannamei 5 days after using Baoshuang

[0045] Figure 20 Blue-green algae condition at the downwind of the pond before using Baoshuang

[0046] Figure 21 Blue-green algae condition at the downwind of the pond 2 days after using Baoshuang

[0047] Figure 22 The cyanobacteria situation at the downwind of the pond 5 days after using Baoshuang

[0048] Figure 23 The experimental results of protease production by Baoshuang Specific implementation manners

[0049] To enable those skilled in the art to better understand the present invention, the present invention will be further described below in conjunction with specific implementation manners.

[0050] Example 1 Analysis of the degradation effect of Bacillus subtilis on ammonia nitrogen, COD, and nitrite in water

[0051] 1.1 Experimental consumables and instruments

[0052] Baoshuang: namely, the Bacillus subtilis provided by the present invention, which was deposited at the Guangdong Provincial Microbial Culture Collection Center on November 04, 2024. Its taxonomic name is: Bacillus subtilis, and the deposit number is: GDMCC No: 65406.

[0053] Experimental instruments: centrifuge (TGL-16MC, Changsha Xiangrui Centrifuge Co., Ltd.), vortex air pump (HG-180, Sensen Group Co., Ltd.), beaker, chemical oxygen demand (COD) rapid detector (5B-3A, Beijing Lianhua Yongxing Technology Development Co., Ltd.), pH meter (SevenDirect SD20, Mettler-Toledo Instruments (Shanghai) Co., Ltd.), spectrophotometer (YP5002, Shanghai Youke Instrument Co., Ltd.)

[0054] 1.2 Experimental content and analysis results

[0055] Experimental group:

[0056] External activation of the system: Weigh 5 g of Baoshuang and add 10 g of glucose to 500 mL of water, activate with oxygen for 3 h. After the activation is completed, measure 10 mL of the activated solution and dilute it to 1 L of water, and measure the COD of the diluted solution at this time as the initial value;

[0057] Internal degradation of the system: Add 1 / 25 of ammonia nitrogen according to the COD value of the diluted solution, activate with oxygen, and measure COD, ammonia nitrogen, and nitrite every 2 h.

[0058] Use a vortex air pump for aeration to keep the dissolved oxygen at 5 mg / L or above (the dissolved oxygen in aquaculture water is generally above 5 mg / L).

[0059] Control group:

[0060] External activation of the system: Weigh 10 g of glucose and add it to 500 mL of water. Activate it with oxygen for 3 h. After the activation is completed, measure 10 mL of the activation solution and dilute it to 1 L of water, and measure the COD of the diluted solution at this time as the initial value.

[0061] Degradation within the system: Add 1 / 25 of ammonia nitrogen according to the COD value of the diluted solution, activate it with oxygen, and measure COD, ammonia nitrogen, and nitrite every 2 h.

[0062] Use a vortex air pump for aeration to keep the dissolved oxygen at 5 mg / L or above (the dissolved oxygen in the aquaculture water is generally above 5 mg / L).

[0063] The analysis results are as follows:

[0064] (1) Experimental results of COD in water

[0065] Table 1-1 Changes in COD concentration

[0066] Group 0h 2h 4h 6h Control group (mg / L) 209.2 216.7 195.6 165.5 Experimental group (mg / L) 246.05 269.35 237.8 186.6

[0067] Combined with the data in Table 1-1, Figure 1 it can be seen that both the COD blank control group and the experimental group in the water show a downward trend within 6 h. The COD degradation rate of the control group is 20.89%, and the degradation rate of the experimental group is 24.17%.

[0068] (2) Experimental results of ammonia nitrogen in water

[0069] Table 1-2 Changes in ammonia nitrogen concentration

[0070] Group 0h 2h 4h Control group (mg / L) 9.28 9.04 7.19 Experimental group (mg / L) 12.42 9.86 4.75

[0071] The changes in ammonia nitrogen values are shown in Table 1-2. The ammonia nitrogen degradation rate of the experimental group within 4 h is 61.76%, and the degradation rate is 1.92 mg / L / h; the degradation rate of the control group within 4 h is 22.52%, and the degradation rate is 0.52 mg / L / h. As Figure 2 shown, the ammonia nitrogen degradation rate of the experimental group is higher.

[0072] (3) Experimental results of nitrite in water

[0073] Table 1-3 Changes in nitrite concentration

[0074] Group 0h 2h 4h Control group (mg / L) 0.04 0.04 0.04 Experimental group (mg / L) 0.03 0.07 0.07

[0075] The changes in nitrite values are shown in Table 1-3, Figure 3 as shown. The nitrite concentrations of both the control group and the experimental group remain at a low level within 4 h. The nitrite concentration of the experimental group is higher than that of the control group, and the nitrite in the experimental group increases by 133%.

[0076] In summary, through the monitoring of the COD value and ammonia nitrogen value in the experimental water body, it is shown that Baoshuang can play a certain role in degrading COD and ammonia nitrogen. COD can be degraded by 24.17% within 6 hours, and ammonia nitrogen can be degraded by 61.76% within 4 hours.

[0077] Combining the changes in COD and ammonia nitrogen values, Baoshuang and nitrifying bacteria agent can be compounded and used in a ratio of 1:1, making the degradation effect of ammonia nitrogen and nitrite more obvious.

[0078] Analysis of the treatment effect of Bacillus subtilis on organic matter in water body in Example 2

[0079] 2.1 Experimental materials and methods

[0080] Baoshuang: taken from the factory warehouse, batch number 20240808

[0081] Experimental method: Fixed-point image collection of organic matter, water color and water transparency on the water surface of the aquaculture pond before and after use.

[0082] 2.2 Experimental process

[0083] Experiment 1

[0084] Table 2-1 Aquaculture overview

[0085]

[0086] Table 2-2 Effect tracking

[0087]

[0088] Experimental information:

[0089] Arrived at the aquaculture pond on August 22. It was found that a large amount of organic matter was floating on the water surface of Shed 7 and Shed 9. The water color was slightly yellowish and the transparency was low. Figure 4 It is the aquaculture water body in Shed 7 before treatment. Figure 5 It is the aquaculture water body in Shed 9 before treatment.

[0090] Started to use Baoshuang for treatment on the same day, 500 g / shed, directly dry spread.

[0091] On the second day (August 23), a return visit was made and it was found that the organic matter on the water surface had basically disappeared, the water color had changed to dark green, and the water transparency had increased, as shown in Figure 6 、 Figure 7 shown.

[0092] Experiment 2

[0093] Table 2-3 Aquaculture overview

[0094]

[0095]

[0096] Table 2-4 Effect Tracking

[0097]

[0098] Experiment Information:

[0099] Arrived at the aquaculture pond on August 25th. It was found that a large amount of organic matter was floating on the water surface of Shed No. 16 and Shed No. 17. The water color was yellowish, the transparency was relatively low, and the air outlets of the air holes were uneven, as Figure 8 、 Figure 9 shown:

[0100] Decided to use Baoshuang for treatment on the same day, 500g (half pack) for each shed, directly broadcast dry.

[0101] On August 26th, a return visit was made. It was found that the organic matter on the water surface of Shed No. 16 and Shed No. 17 was significantly reduced, the yellowing of the water color was alleviated, the water transparency was significantly increased, and the air outlets of the oxygen holes were uniform, as Figure 10 、 Figure 11 shown.

[0102] In summary, Baoshuang has an obvious decomposition effect on the organic matter generated during the aquaculture process. The reason is that the beneficial bacteria contained in Baoshuang have the function of secreting protease, which can decompose proteins, thus achieving the purpose of decomposing organic matter; in addition, the amount of Baoshuang bacteria is rich, which can quickly form an ecological occupancy effect in the water body, inhibit the growth of harmful bacteria, promote the growth and reproduction of beneficial algae, and maintain good water quality and water color.

[0103] Analysis of the Treatment Effect of Bacillus subtilis on Cyanobacteria in Water Body in Example 3

[0104] 3.1 Experimental Materials and Methods

[0105] Baoshuang: Obtained from the warehouse of Tianrunhe Factory, batch number 20240808

[0106] Experimental Consumables: Research Institute Laboratory

[0107] Experimental Method: Collect images of cyanobacteria on the water surface and water color of the aquaculture pond every day, at fixed points.

[0108] 3.2 Experimental Process

[0109] Experiment 1

[0110] Table 3-1 Aquaculture Overview

[0111]

[0112] Table 3-2 Effect Tracking

[0113]

[0114] Experimental information:

[0115] On August 17, the state of the water surface at the downwind: Cyanobacteria gathered severely, emitting a faint stench. On the same day, Baoshuang was activated and sprinkled, as Figure 12 shown.

[0116] On August 19, 2 days after using Baoshuang, the state of the water surface at the downwind: Cyanobacteria gradually dispersed, with reduced activity and a visibly smaller thickness. In the afternoon of the same day, 1 kg of Baoshuang was supplemented for treatment. As Figure 13 shown.

[0117] On August 22, dead algae were observed on the water surface, and the cyanobacteria attached to the shore had disappeared. The color of the water body gradually changed, showing "clear and refreshing". As Figure 14 shown.

[0118] On August 25, the cyanobacteria were basically controlled. A total of 2 kg of Baoshuang was used within a week, and the effect was remarkable. As Figure 15 shown.

[0119] Experiment 2

[0120] Table 3-3 Aquaculture Overview

[0121]

[0122] Table 3-4 Effect Tracking

[0123]

[0124] Experimental information:

[0125] On August 20, the state of the water surface at the downwind: Cyanobacteria showed a gathering phenomenon, and there were sporadic cyanobacteria groups by the pond. On the same day, 2 kg of Baoshuang was activated and sprinkled. As Figure 16 shown.

[0126] On August 22, 2 days after using the product, the state of the water surface at the downwind: There were obvious dead cyanobacteria, but there were still cyanobacteria clusters by the pond. As Figure 17 shown.

[0127] On August 23, 3 days after using the product, the state of the water surface at the downwind: There were more dead cyanobacteria, which gradually turned into yellow dead algae. The cyanobacteria cluster phenomenon by the pond weakened, showing a positive degradation effect. As Figure 18 shown.

[0128] On August 25, 5 days after using the product, the state of the water surface at the downwind: The floating dead algae on the water surface had disappeared, the cyanobacteria on the water surface were almost zero, the water body by the pond became transparent, and there were no cyanobacteria clusters. The effect was remarkable. As Figure 19 shown.

[0129] Experiment 3:

[0130] Table 3-5 Aquaculture Overview

[0131]

[0132] Table 3-6 Effect Tracking

[0133]

[0134] Experiment Information:

[0135] On August 20th, the state of the downwind area under the water surface: The problem of blue-green algae was very serious. Considering that the farmers had not stocked the fry, the product was used with an increased dosage. After activation, 2 kg of Baoshuang was sprinkled. As Figure 20 shown.

[0136] On August 22nd, 2 days after using the product, the state of the downwind area under the water surface: The blue-green algae began to show signs of death, but the scope did not decrease significantly. As Figure 21 shown.

[0137] On August 25th, 5 days after using Baoshuang, the state of the downwind area under the water surface: The death of blue-green algae was obvious, and the dead algae aggregated, and the effect was gradually manifested. As Figure 22 shown.

[0138] In summary, Baoshuang has an obvious effect on clearing the "old green water" outbreak in aquaculture. The reason is that Baoshuang has a rich amount of bacteria, which can form an ecological occupancy effect with blue-green algae (cyanobacteria) in the water body and inhibit its growth.

[0139] Example 4 Analysis of the Protease Production Effect of Bacillus subtilis

[0140] 4.1 Experimental Materials

[0141] Experimental Instruments: Oscillator, Ultra-clean Workbench, Glass Test Tubes, Pipettor, Alcohol Lamp, etc.

[0142] 4.2 Experimental Design

[0143] Activating the Strain: Weigh 1 g of Baoshuang and pour it into a 500 mL glass shaking flask in sequence with 99 mL of normal saline. After activation for 30 minutes, it is ready for use;

[0144] Diluting the Bacterial Solution: Gradiently dilute the activated bacterial solution with a glass test tube to an appropriate concentration;

[0145] Preparing the Milk Plate: Add milk with a final concentration of 1.5% to the ordinary broth peptone solid medium;

[0146] Culturing the Strain: Use a pipettor to aspirate 100 μL of the diluted bacterial solution and spread it on the milk plate, and culture it in a 37°C constant temperature incubator for 1-2 days.

[0147] Brevibacillus brevis was used as a control strain.

[0148] Figure 23 The milk plate showed an obvious clear zone around the colonies, indicating that the bacterium has the ability to produce protease and can decompose the proteins in milk, thus proving that Baoshuang can be used to decompose organic matter, cyanobacteria, dead algae, etc. in water bodies.

[0149] The milk plate of the control group of Brevibacillus brevis showed no clear zone around the colonies.

[0150] In summary, the present invention provides a strain of Bacillus subtilis, which was deposited at the Guangdong Microbial Culture Collection Center on November 04, 2024. Its taxonomic name is: Bacillus subtilis, and the deposit number is: GDMCC No: 65406. This strain can effectively inhibit the overgrowth of cyanobacteria, accelerate the decomposition of organic matter in water bodies, significantly degrade harmful metabolites such as ammonia nitrogen and nitrite, and simultaneously reduce the chemical oxygen demand (COD) index.

[0151] The above are only the preferred embodiments of the present invention, and do not limit the patent scope of the present invention. All equivalent changes and modifications made according to the scope of the present invention should still fall within the scope covered by the present invention.

Claims

1. A Bacillus subtilis strain, characterized in that, The described Bacillus subtilis was deposited at the Guangdong Microbial Culture Collection Center on November 4, 2024, with the deposit number: GDMCC No: 65406.

2. Application of the Bacillus subtilis according to claim 1 in purifying and regulating the water quality of aquaculture water bodies.

3. The application according to claim 2, characterized in that, The purification and regulation of water quality include: the Bacillus subtilis inhibiting the growth of cyanobacteria in aquaculture water bodies.

4. The application according to claim 2, wherein The purification and regulation of water quality include: the Bacillus subtilis decomposing organic matter in aquaculture water bodies.

5. The application according to claim 2, characterized in that The purification and regulation of water quality include: the application of the Bacillus subtilis in degrading ammonia nitrogen and nitrite in aquaculture water bodies and reducing the COD in the aquaculture water body.

6. The application according to claim 2, wherein Activate the Bacillus subtilis, dilute the activation solution into the aquaculture water body, and the volume ratio of the activation solution to the aquaculture water body is 1:90 - 110. At the same time, aerate the aquaculture water body to keep the dissolved oxygen above 5 mg / L.

7. The application according to claim 6, wherein The volume ratio of the activation solution to the aquaculture water body is 1:

100.

8. The application according to claim 6, characterized in that, The activation method is as follows: Weigh 5 g of Bacillus subtilis and add 10 g of glucose to 500 mL of natural water, and activate it with oxygen for 3 h.

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