WO2022088483A1

WO2022088483A1 - Bacillus sp. nb-1, and culture method therefor and use thereof

Info

Publication number: WO2022088483A1
Application number: PCT/CN2020/141242
Authority: WO
Inventors: 江兴龙; 郭少鹏
Original assignee: 集美大学
Priority date: 2020-10-27
Filing date: 2020-12-30
Publication date: 2022-05-05
Also published as: CN112111435B; CN112111435A

Abstract

A Bacillus sp. NB-1, and a culture method therefor and the use thereof. The Bacillus sp. has a deposit number of CCTCC NO: M 2020304, and can significantly reduce ammonia nitrogen and nitrite in aquaculture water and aquaculture tail water.

Description

A kind of Bacillus NB-1 and its cultivation method and application

technical field

The invention relates to the technical field of microorganisms, in particular to a Bacillus NB-1 having both ammonia nitrogen and nitrite degradation functions, a culture method and application thereof.

Background technique

my country is a big country in aquaculture, and its aquaculture production accounts for about 68% of the world's aquaculture production, of which pond aquaculture production accounts for about 45%. The accumulation of nitrogen in the water due to a large amount of feed during the breeding process is also one of the main reasons for the deterioration of aquaculture water. Among them, ammonia nitrogen and nitrite are the most serious physical and chemical factors that endanger the water quality of aquaculture and farmed animals. Animals are highly toxic. Ammonia nitrogen will affect the enzymatic reaction and the stability of cell membranes in farmed animals, and also affect the oxygen transfer of gills, and destroy the excretory system and osmotic balance of aquatic animals. Nitrite is an intermediate product in the cycle of aquatic ecosystems, and is an important environmental factor that induces outbreaks of aquatic animal diseases. In a local hypoxic environment, nitrates will be converted into nitrites. The carcinogenicity of nitrites increases the toxicity of pollutants that were originally low in toxicity, resulting in the deterioration of water quality, accompanied by the breeding of a large number of viruses, bacteria and plankton. and other microorganisms. When the concentration of ammonia nitrogen and nitrite is too high, it is easy to cause a large number of fish and shrimp to become sick or even die, which seriously affects the water quality and the safety of aquatic products. On the other hand, the tail water of pond aquaculture in my country is basically discharged directly without treatment. Because the tail water of aquaculture generally contains high nitrogen and phosphorus, it leads to different degrees of eutrophication pollution to the surrounding water environment. In order to improve the water quality of aquaculture, reduce the occurrence of breeding diseases, reduce the stress of breeding animals, ensure the safety of breeding and protect the ecological environment of the surrounding waters, many aquaculture farmers choose to use microbial preparations to degrade ammonia nitrogen and nitrite in water, and promote the improvement of the water environment. , alleviate the economic loss and environmental pollution caused by the deterioration of water quality. Bacillus is a common probiotic that uses microorganisms to restore the breeding environment. Numerous studies have found that the denitrification performance of indigenous microorganisms screened from aquaculture wastewater or aquaculture sludge is more efficient. However, the current microbial preparation products that degrade ammonia nitrogen and nitrite generally have problems such as high production and application cost, low degradation treatment efficiency and poor treatment effect stability.

Existing Chinese published patents, patent number: CN200610011604.1, CN201810782000.X, CN201811325758.7 and CN201910309817.X all disclose different types of Bacillus, but there are the following problems: 1. High inoculation amount, 2. One type of Bacillus strain can only effectively degrade ammonia nitrogen and nitrite nitrogen A kind of, can not achieve effective degradation of both at the same time, 3, ammonia nitrogen and nitrite degradation effect are low. Therefore, it is urgent to develop a probiotic that can not only efficiently degrade ammonia nitrogen and nitrite in aquaculture water and aquaculture tail water, but also improve the stability of treatment effect and reduce the cost of use.

technical problem

The technical problem to be solved by the present invention is to provide a Bacillus NB-1 and its cultivation method and application, which can not only efficiently degrade ammonia nitrogen and nitrite in aquaculture water and aquaculture tail water at the same time, but also improve the treatment effect. It has the advantages of low operating cost and no secondary pollution.

technical solutions

The present invention is achieved as follows: the first object of the present invention is to provide a Bacillus NB-1, the Bacillus NB-1 is Bacillus sp. NB-1, on July 13, 2020 Deposited in the China Center for Type Culture Collection, the address is China. Wuhan. Wuhan University, the deposit number is CCTCC NO: M 2020304.

The second object of the present invention is to provide a method for culturing Bacillus NB-1, wherein the Bacillus sp.) NB-1 was incubated with LB liquid medium at 30°C for 24h.

The third object of the present invention is to provide an application of Bacillus NB-1, the Bacillus sp.) Application of NB-1 in improving aquaculture water and reducing aquaculture tail water discharge.

Preferably, in the above application, the Bacillus sp. NB-1 is used to degrade ammonia nitrogen and nitrite concentrations in aquaculture water and aquaculture tail water.

Preferably, in the above application, when the Bacillus sp. NB-1 degrades ammonia nitrogen and nitrite in aquaculture water, the dosage of Bacillus sp. NB-1 is 104 cfu/mL; In the case of nitrite, the dosage of Bacillus NB-1 was 105 cfu/mL.

The fourth object of the present invention is to provide the second application of Bacillus NB-1, the Bacillus sp.) NB-1 was used to prepare the degradation preparations of ammonia nitrogen and nitrite in aquaculture water.

The fifth object of the present invention is to provide the third application of Bacillus NB-1, by which the Bacillus sp.) NB-1 was used for the preparation of ammonia nitrogen and nitrite degradation preparations in aquaculture tail water.

beneficial effect

The present invention has the following advantages.

The invention provides a Bacillus sp. NB-1 capable of purifying water quality. The Bacillus sp. NB-1 belongs to autotrophic bacteria and has the ability to degrade ammonia nitrogen and nitrite at the same time, and carry out nitrification. The reaction does not consume the carbon source in the water, and the inoculum is low and the effect of degrading ammonia nitrogen and nitrite is good. It produces secondary pollution, which can be used in large-scale production to improve the aquaculture environment and reduce the discharge of aquaculture tail water.

Embodiments of the present invention

The present invention relates to a Bacillus NB-1, the Bacillus NB-1 is Bacillus sp. NB-1, which was deposited in the China Center for Type Culture Collection on July 13, 2020, and the address is China. Wuhan. Wuhan University, preservation number CCTCC NO: M 2020304.

The present invention also relates to a method for culturing Bacillus sp. NB-1, wherein the Bacillus sp. NB-1 is cultured in LB liquid medium at 30° C. for 24 hours.

The invention also relates to the application of Bacillus sp. NB-1 in improving aquaculture water bodies and reducing the discharge of aquaculture tail water. Specifically, the Bacillus sp.) NB-1 is used to degrade ammonia nitrogen and nitrite concentrations in aquaculture water and aquaculture tail water.

When the Bacillus sp. NB-1 degrades ammonia nitrogen and nitrite in aquaculture water, the dosage of Bacillus sp. NB-1 is 104 cfu/mL; when degrading ammonia nitrogen and nitrite in aquaculture tail water, Bacillus sp. The amount of NB-1 was 105 cfu/mL.

The invention relates to the second application of Bacillus sp. NB-1, which is used for preparing a degrading preparation of ammonia nitrogen and nitrite in aquaculture water.

The invention relates to the third application of Bacillus sp. NB-1. The Bacillus sp. NB-1 is used to prepare a degrading preparation for ammonia nitrogen and nitrite in aquaculture tail water.

The technical solutions of the present invention will be clearly and completely described below with reference to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

Example 1. Isolation and screening of Bacillus NB-1.

Sampling: From the biofilm water purification fence set in the water body of the eel culture pond in the eel breeding workshop of Jimei University Aquatic Products Experiment Field, Jimei District, Xiamen City, Fujian Province, use sterile scissors to cut 10g of nylon wire of the biofilm water purification fence, and put them into a container. In a blue-capped bottle with 100 mL of sterile water, the bacterial suspension was obtained after ultrasonic vibration for 30 min.

Enrichment: The bacterial suspension was added to sterile enrichment medium at a ratio of 10%, incubated at 30°C with constant temperature shaking at 180 r/min, and 2 mL of 5% sodium nitrite solution was added every day for 1 week of enrichment.

Enrichment medium (g/L): glucose 5, sodium chloride 0.1, nitrite 0.2, magnesium sulfate 0.02, potassium dihydrogen phosphate 4, dipotassium hydrogen phosphate 6, prepared with single distilled water, pH 7.4, sterilized at 121 °C bacteria for 30min.

Separation and purification: Take 0.1 mL of the enriched bacterial suspension and dilute it to 10-3, 10-4, 10-5, etc. with sterile water by 10-fold dilution method. Then, take 0.1 mL of the dilution solution and spread it on the common broth solid medium. Each dilution concentration is spread on 3 plates, and cultivated in a constant temperature incubator (30 °C) for 24 hours. After the common broth solid medium grows colonies. Select a medium plate with a suitable number of colonies, scrape a single colony and inoculate it on a fresh common broth solid medium, repeating 3 times. until a pure culture is obtained.

Common broth solid medium: beef extract 0.5%, peptone 1%, sodium chloride 0.5%, agar 1%, prepared with single distilled water, pH 7.4, sterilized at 121 ℃ for 30 min.

Screening and storage: The single colony strain obtained after separation and purification was placed in 50 mL of ordinary broth medium, and heated in a water bath at 80°C for 30 minutes to kill the microbial vegetative cells. Then 30°C, 180r/min constant temperature shaking culture for 24h, the proliferation medium was placed in a 80°C water bath and heated for 30min, and the vegetative cells that did not form spores were killed again. Take 1mL of treatment solution and dilute to 10-3, 10-4, 10-5 and so on by gradient dilution method. Take 0.1 mL of bacterial liquid respectively and spread it on the common broth solid medium, and after culturing it in a 30°C constant temperature incubator for 24 hours, select a single colony, streak it into the common broth solid medium for 24 hours, and separate and purify it three times. . It was inoculated into a slanted common broth solid medium for cultivation and stored at 4°C for later use.

Ordinary broth medium (g/L): beef extract 5, peptone 10, sodium chloride 5, prepared with single distilled water, pH 7.4, sterilized at 121 °C for 30 min.

Example 2. Identification of Bacillus NB-1.

Pick a single colony of the above-preserved strains, inoculate it in LB medium at 30°C for 12 hours with shaking at 170 r/min, collect the bacteria by centrifugation, and use a bacterial DNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.) Extract the genomic DNA of the strain using universal primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') (as shown in SEQ ID No: 1), and 1492R (5'-GGTTACCTTGTTACGACTT-3') (as shown in SEQ ID No: 2), PCR amplification of 16S rRNA gene was carried out, and the PCR reaction conditions were: 95 ℃ pre-denaturation for 5 min, 30 cycles (95 ℃ 1 min , 53°C for 1 min, 72°C for 1.5 min), and extended at 72°C for 10 min. The target fragment of about 1.5 kb was purified with a gel recovery kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the purified PCR product was sent to (Shanghai Meiji Biomedical Technology Co., Ltd.) for sequencing.

LB medium (g/L): peptone 10, yeast extract 5, sodium chloride 10, single distilled water, pH 7.5, sterilized at 121 °C for 30 min.

The obtained gene sequence is shown in SEQ ID No:3.

The above-mentioned sequences obtained were compared and analyzed online by BLAST (http://www.ncbi.nlm.nih.gov/blast) on the NCBI website, and a phylogenetic tree was constructed using the Neighbor-Joining method in MEGA X software to identify strain NB. -1 is Bacillus sp., hence the name Bacillus sp. NB-1.

Example 3. Degradation rate and safety detection of ammonia nitrogen and nitrite in breeding water of Bacillus NB-1.

A single colony of strain NB-1 was picked and inoculated into sterilized LB medium, and cultured with shaking at 170 r/min for 24 h at 30 °C. The inoculum amount of 4 × 104 cfu/mL was added to the plastic bucket of cultured American eel containing 1m3 water body. The control group did not add bacteria. A double parallel control experiment was set up. The weight of each barrel of cultured American eel in the treatment group and the control group was 11.2 kg. , the average size is 140g/tail. During the test, each bucket of culture water samples were collected at 0h, 24h and 168h. The water samples were centrifuged to take the supernatant to measure the concentration of ammonia nitrogen and nitrite nitrogen, and calculate the degradation rate. Both the treatment group and the control group were fed with 1.8% feed rate and managed normally, and the American eel grew healthily without disease and death.

The results (see Table 1) showed that the ammonia nitrogen concentration in the eel culture water in the treatment group added with bacterial solution for 24 hours decreased from 1.363 mg/L to 0.021 mg/L (degradation rate 98.5%), and the nitrite nitrogen concentration decreased from 0.481 mg/L. decreased to 0.003mg/L (degradation rate 99.4%); the ammonia nitrogen concentration of eel culture water in the 168h treatment group was 0.245mg/L, which was significantly lower than that of the control group by 91%, and the nitrite nitrogen concentration was 0.191mg/L, which was significantly lower than that of the control group. 93%. It shows that Bacillus NB-1 can reach more than 91% of the ability to degrade ammonia nitrogen and nitrite nitrogen concentration in aquaculture water, can control ammonia nitrogen and nitrite nitrogen concentration well, stabilize water quality and has good aquaculture safety.

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Example 4. Determination of ammonia nitrogen and nitrite degradation rate and autotrophy in culture tail water of Bacillus NB-1.

A single colony of strain NB-1 was picked and inoculated into sterilized LB medium, and cultured with shaking at 170 r/min for 24 h at 30 °C. The inoculum amount of 4 × 105 cfu/mL was added to 1 L of eel culture tail water, and the water samples were collected and centrifuged for 24 h to obtain the supernatant to determine the degradation rate of ammonia nitrogen and nitrite nitrogen.

The results showed that the ammonia nitrogen concentration in the tail water of eel aquaculture with the addition of bacterial solution for 24 hours decreased from 2.28 mg/L to 0.695 mg/L (degradation rate was 69.5%), and the nitrite nitrogen concentration decreased from 2.28 mg/L to 0.018 mg/L ( The degradation rate was 99.2%); the concentration of CODMn in the tail water changed from 5.30mg/L to 5.16mg/L, and the concentration basically did not change in 24h, indicating that the strain NB-1 did not utilize organic matter and belonged to autotrophic bacteria.

To sum up, the Bacillus NB-1 of the present invention belongs to autotrophic bacteria, has the ability to degrade ammonia nitrogen and nitrite, and does not consume carbon sources in water during nitrification reaction; it is different from the reported screening bacteria that can only degrade singly. Ammonia nitrogen or nitrite, which is different from the reported screening bacteria as heterotrophic bacteria, needs to consume carbon source when degrading; Bacillus NB-1 has the advantages of less inoculum required and lower cost of use, and it is used in degradation culture Ammonia nitrogen and nitrite in the water body only need an inoculum of 4 × 104 cfu/mL, and only 4 × 105 cfu/mL of inoculum is required to degrade ammonia nitrogen and nitrite in aquaculture tail water, which is more than the current domestic The reported dosage of microbial preparations should generally be above the inoculum amount of 106 cfu/mL, which significantly reduces the input dosage; Bacillus NB-1 has efficient ammonia nitrogen and nitrite degradation rates, and the degradation rate of ammonia nitrogen concentration in 24h aquaculture water reaches 100%. 98.5% (from 1.36mg/L to 0.021mg/L), and the degradation rate of nitrite nitrogen concentration reached 99.4% (from 0.48mg/L to 0.003mg/L); the ammonia nitrogen concentration in the 24h aquaculture tail water was reduced from 2.28mg /L decreased to 0.695mg/L (degradation rate 69.5%), and nitrite nitrogen concentration decreased from 2.28mg/L to 0.018mg/L (degradation rate 99.2%).

In addition, the Bacillus NB-1 of the present invention is used for degrading higher ammonia nitrogen and nitrite concentrations in aquaculture tail water, and still has advantages: the required inoculum amount is lower and a higher degradation rate can be achieved.

In conclusion, the Bacillus NB-1 of the present invention has a good market application prospect.

Although the specific embodiments of the present invention are described above, those skilled in the art should understand that the specific embodiments we describe are only illustrative, rather than used to limit the scope of the present invention. Equivalent modifications and changes made by a skilled person in accordance with the spirit of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

A Bacillus NB-1, characterized in that: the Bacillus NB-1 is Bacillus sp. NB -1, which was preserved in the China Center for Type Culture Collection on July 13, 2020, and the address is China . Wuhan. Wuhan University, the deposit number is CCTCC NO: M 2020304.
The method for culturing Bacillus sp. NB-1 according to claim 1, wherein the Bacillus sp. NB -1 is cultured in LB liquid medium at 30° C. for 24 hours.
The application of a Bacillus NB-1 according to claim 1, characterized in that: the application of the Bacillus sp. NB -1 in improving aquaculture water bodies and reducing the discharge of aquaculture tail water.
The application of a Bacillus NB-1 according to claim 3, characterized in that: the Bacillus sp. NB -1 is used to degrade ammonia nitrogen and nitrite concentrations in aquaculture water and aquaculture tail water.
The application of a Bacillus NB-1 according to claim 4, characterized in that: when the Bacillus sp. NB -1 degrades ammonia nitrogen and nitrite in aquaculture water, the Bacillus sp. NB-1 The dosage was 10 4 cfu/mL; the dosage of Bacillus NB-1 was 10 5 cfu/mL when degrading ammonia nitrogen and nitrite in aquaculture tail water.
The application of Bacillus NB-1 according to claim 1, characterized in that: the Bacillus sp. NB -1 is used to prepare a degrading preparation of ammonia nitrogen and nitrite in aquaculture water.
The application of Bacillus NB-1 according to claim 1, characterized in that: the Bacillus sp. NB -1 is used to prepare a degrading preparation of ammonia nitrogen and nitrite in aquaculture tail water.