CN113151122B

CN113151122B - Bacillus subtilis and fermentation method and application thereof

Info

Publication number: CN113151122B
Application number: CN202110665896.5A
Authority: CN
Inventors: 徐虹; 程利芳羽; 雷鹏; 王瑞; 孙良; 李莎
Original assignee: Nanjing Tech University
Current assignee: Nanjing Tech University
Priority date: 2021-06-16
Filing date: 2021-06-16
Publication date: 2022-05-17
Anticipated expiration: 2041-06-16
Also published as: CN113151122A

Abstract

The invention discloses a bacillus subtilis (Bacillus subtilis)Bacillus subtillis) NX-2C, the strain is preserved in China general microbiological culture Collection center (CGMCC) at 1 month and 15 days in 2021, and the preservation number is CGMCC NO. 21625. The invention also discloses a fermentation method and application of the strain. The denitrification strain NX-2C has heterotrophic nitrification capability, is simple in process, can tolerate high-concentration waste nitrogen in aquaculture water, is quick in effect after being put into use, is simple to operate, does not cause secondary pollution, is tolerant to low temperature and low dissolved oxygen, is suitable for a short-term denitrification process, and can achieve high-efficiency denitrification by using the low-concentration denitrification strain.

Description

Bacillus subtilis and fermentation method and application thereof

Technical Field

The invention relates to the technical field of biological environment microorganisms, and relates to bacillus subtilis, a fermentation method thereof and application of bacillus subtilis in denitrification of aquaculture water.

Background

During aquaculture, the nitrogen content in the aquaculture water rises, which can cause various damages to the aquaculture body, such as inflammation, hemorrhagic disease, decreased food intake and even death. Therefore, the nitrogen content in the aquaculture water body is an important detection index of the aquaculture water body. The biological denitrification process is nitrification-denitrification, wherein the nitrification is that ammonia nitrogen is converted into nitrite nitrogen and further into nitrate nitrogen under the action of nitrifying bacteria; the denitrification process is a process of converting nitrate nitrogen into nitrogen and the like under the action of denitrifying bacteria and separating from a water body to achieve thorough purification. Nitrification needs to be carried out under aerobic conditions, but the condition of low dissolved oxygen can exist in the actual aquaculture process, so certain difficulty can be caused to denitrification, and the development and application of certain novel efficient denitrification microbial strains can improve the efficiency of the traditional biological denitrification process.

Gram-positive bacillus subtilis capable of forming spores in growth cycleThe bacteria can produce bacteriocin and have a certain bacteriostatic action. The Bacillus subtilis can also decompose feed bait in aquaculture water by using an enzyme system thereof, and can reduce the feed bait into N by using nitrate and nitrite₂And pollutants such as ammonia nitrogen and the like in the water body are eliminated, and the decomposition of organic matters and the nitrogen circulation process are promoted.

Disclosure of Invention

The invention provides a denitrification bacterial strain applied to an aquaculture water body, which has nitrification capability. The strain of the invention achieves the purification effect of the aquatic product culture water body by using a biological denitrification technology, has high-efficiency denitrification capability in the high-concentration nitrogen-containing aquatic water body, and also has excellent denitrification effect in low-dissolved oxygen and low-temperature environments

Specifically, the invention discloses bacillus subtilis collected by Nanjing industry university in Nanjing City of Jiangsu province 5 months in 2020. The Bacillus subtilis NX-2C is classified and named as Bacillus subtilis, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and has the preservation address: the preservation time of Beijing in China is 1 month and 15 days in 2021, and the preservation number is CGMCC NO. 21625.

The invention further provides a fermentation production method of the Bacillus subtilis NX-2C, which comprises the steps of inoculating the Bacillus subtilis NX-2C into a seed culture medium, carrying out overnight culture to obtain a seed solution, then inoculating the seed solution into a fermentation culture medium, and carrying out shake culture to obtain the Bacillus subtilis NX-2C fermentation liquor.

Wherein, the seed culture medium is peptone 5-10g/L, yeast powder 2-5g/L, and sodium chloride 1-10 g/L; the culture conditions were 30-37 ℃ and 100-.

Specifically, inoculating the seed solution into a fermentation culture medium at 1-10% (v/v), and culturing for 2-3 days at 30-37 ℃ and 200rpm on a shaking table to obtain a denitrification strain NX-2C fermentation broth; the carbon source of the fermentation medium is any one or combination of glucose, sucrose, lactose and sodium acetate, and preferably, the carbon source is one or combination of glucose and sucrose.

The nitrogen source of the fermentation medium is any one or combination of ammonium chloride, ammonium sulfate, sodium nitrite, potassium nitrate, peptone and beef extract. Preferably, the nitrogen source is one or two of ammonium chloride and peptone.

Preferably, the seed culture medium is: 10g/L of peptone, 5g/L of yeast powder and 10g/L of sodium chloride.

Wherein the fermentation medium is: 5g/L of peptone, 5g/L of ammonium sulfate, 5g/L of sodium nitrite, 5g/L of potassium nitrate, 5g/L of beef extract, 5g/L of ammonium chloride, 20g/L of glucose and 20g/L of sucrose.

The invention further provides application of the Bacillus subtilis NX-2C in denitrification of aquaculture water. Preferably, the aquaculture objects to which the denitrification strains are applied are common fish, shrimp and crab.

The denitrogenation rate of the Bacillus subtilis NX-2C is more than 90% when ammonium chloride, sodium nitrite and potassium nitrate are used as unique nitrogen sources. Wherein the denitrification bacterial strain NX-2C can carry out denitrification when the ammonium chloride is 0.1-3000 mg/L; the denitrification bacterial strain NX-2C can carry out denitrification at the concentration of 0.2-2000mg/L of sodium nitrite; the denitrogenation strain NX-2C can denitrify when the potassium nitrate is 0.2-1500 mg/L.

The environmental requirements of the application of the Bacillus subtilis NX-2C are as follows: the temperature is 5-40 ℃, and the dissolved oxygen is 1-8mg/L, pH 5-9. Wherein, the denitrogenation strain NX-2C can still ensure high-efficiency denitrogenation rate at low temperature and low dissolved oxygen. When the temperature is 5-10 ℃, the denitrification rate reaches 90%, the denitrification rate reaches 97% at 10-20 ℃, the denitrification rate reaches 94% at 20-30 ℃ and the denitrification rate reaches 90% at 30-40 ℃. When the dissolved oxygen is 1-3mg/L, the denitrification rate reaches 90%, the denitrification rate reaches 95% and the denitrification rate reaches 97% at 3-5 mg/L.

In the invention, the denitrified strain NX-2C is 10²-10⁷The denitrification capability is good within the range of the inoculation amount of CFU/mL. Adding the bacterial liquid re-suspended by normal saline for 3-5 times into artificially prepared sewage or a simulated ecological experiment, wherein the denitrifying strain NX-2C has good denitrification rate in the experiment.

Has the beneficial effects that: compared with the prior art, the invention has the following advantages:

(1) the denitrification strain NX-2C has heterotrophic nitrification capability and is simple in process;

(2) the high-concentration waste nitrogen in aquaculture water can be tolerated, the effect is quick after the high-concentration waste nitrogen is put into use, the operation is simple, and no secondary pollution is caused;

(3) the low-temperature and low-dissolved oxygen resistance is realized, the method is suitable for a short-term denitrification process, and the high-efficiency denitrification can be realized by using the low-concentration denitrification strain;

(4) the method has the advantages of rapid and stable denitrification, is particularly suitable for short-term denitrification process, can achieve high-efficiency denitrification by using the denitrification strains with lower concentration, can be used as bioremediation to supplement under the condition of unsatisfactory denitrification effect, and has high safety to culture bodies.

Drawings

FIG. 1 is a photograph of a denitrified strain NX-2C under a microscope;

FIG. 2 shows the denitrification effect of the denitrifying strain NX-2C in a simulated ecological experiment.

Detailed Description

The present invention will be described in further detail with reference to specific examples, which will help understanding the present invention, but the scope of the present invention is not limited to the following examples.

Example 1 screening and identification of Bacillus subtilis NX-2C (hereinafter referred to as denitrified strain NX-2C).

The denitrifying strain NX-2C is collected by the inventor in the lake of Jing of Nanjing City of Jiangsu province in 5 months in 2020. The Bacillus subtilis NX-2C is classified and named as Bacillus subtilis, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and has the preservation address: the preservation time of Beijing in China is 1 month and 15 days in 2021, and the preservation number is CGMCC NO. 21625.

The screening process is as follows:

the screening of the solid culture medium is as follows: 5g/L of ammonium chloride, 2g/L of magnesium sulfate heptahydrate, 3g/L of dipotassium phosphate, 1.5g/L of sodium chloride, 20g/L of glucose and 20g/L of agar.

Preferably, if a strain grows on the screening medium, it is judged that the strain can grow with ammonium chloride as the sole nitrogen source.

And (3) judging whether the denitrification bacterial strain NX-2C can grow by taking sodium nitrite or potassium nitrate as the only nitrogen source, and replacing ammonium chloride in the culture medium by sodium nitrite or potassium nitrate.

Preferably, the single colony obtained above is inoculated to a seed medium.

The seed culture medium is as follows: 10g/L of peptone, 5g/L of yeast powder and 10g/L of sodium chloride. Overnight culture, OD₆₀₀About 1.2-1.5, transferred to the fermentation medium at 1-10% (v/v).

The fermentation medium is as follows: 5g/L of peptone, 5g/L of ammonium sulfate, 5g/L of sodium nitrite, 5g/L of potassium nitrate, 5g/L of beef extract, 5g/L of ammonium chloride, 20g/L of glucose and 20g/L of sucrose. Fermenting and culturing for 2-3 days at 30 ℃ and 200rpm/min on a shaking bed to obtain the fermentation liquor of the denitrified strain NX-2C.

Diluting and coating the denitrified strain NX-2C fermentation liquor, and taking 10^-4、10^-5、10^-6The dilutions of the three concentration gradients were spread evenly on solid medium (medium: peptone 5g/L, ammonium chloride 5g/L, glucose 20g/L, agar powder 20 g/L). Culturing in 30 deg.C constant temperature incubator for 24-48h, counting and calculating viable count of the denitrified strain (viable count range is 1 × 10)⁶-2×10⁸CFU/mL)。

The denitrified strain NX-2C has the following properties:

1. morphological characteristics of colonies

After culturing for 24h at 30 ℃ in peptone agar medium, the cells are observed to be single cells and rod-shaped by a microscope, the length of the bacteria is 1.4-2.0 μm, and the width of the bacteria is 0.7-1.0 μm. Belongs to gram-positive bacteria and facultative anaerobe. The thalli can grow in a large amount after being cultured in the culture medium for 24 hours at the temperature of 30 ℃, and bacterial colonies are white and round, have smooth and dry surfaces and are bulged in the middle. The growth temperature range is 5-48 deg.C, the optimum temperature is 25-35 deg.C, the growth pH range is 5.0-9.5, and the optimum pH is 6.5-8.5.

2. 16SrDNA sequence analysis

The length of the 16SrDNA sequence is 1442bp, and the nucleotide sequence is shown in a sequence table. The 16SrDNA sequence was compared to related species in the GeneBank database. The results show that: the denitrification bacterial strain NX-2C and the bacillus subtilis reach 100 percent of homology. Therefore, the strain of the invention is considered to be Bacillus subtilis NX-2C.

Example 2 denitrification effect of the denitrifying strain NX-2C in artificially prepared wastewater.

The sewage is artificially prepared as follows: 0.2g/L of ammonium chloride, 0.1g/L of sodium nitrite, 0.1g/L of potassium nitrate, 20g/L of glucose, 1g/L of sodium chloride, 0.5g/L of dipotassium phosphate, 0.1g/L of magnesium sulfate heptahydrate and 0.2g/L of manganese sulfate; wherein the content of ammonia nitrogen is 68mg/L, the content of nitrite nitrogen is 67mg/L, and the content of nitrate nitrogen is 61 mg/L.

The denitrifying strains NX-2C and the bacillus subtilis CGMCC NO.1.4436 are both 10⁴CFU/mL final concentration was added to the artificially prepared wastewater as experimental group and equal volume of normal saline was added to the control group, each group was repeated 3 times.

Wherein, the bacillus subtilis CGMCC NO.1.4436 is purchased from China general microbiological culture Collection center.

The ammonia nitrogen detection method adopts an indophenol blue colorimetric method to determine the content of residual ammonia nitrogen in a sample, and calculates the degradation rate, as shown in table 1.

The nitrite nitrogen detection method adopts a diazotization coupling spectrophotometry method to determine the content of the residual nitrite nitrogen in the sample and calculate the degradation rate, and is shown in table 1.

The nitrate nitrogen detection method adopts an ultraviolet spectrophotometry method to measure the content of the nitrate nitrogen remained in the sample and calculate the degradation rate, as shown in table 1.

Denitrogenation strain NX-2C and Bacillus subtilis CGMCC NO.1.4436 were tested at different nitrogen source tolerance concentration intervals ([0.1,0.5], [10,50], [100,500], [800,1500], [1500,2000], [2000,3000] mg/L).

In the culture conditions of pH 7, temperature 30 ℃ and dissolved oxygen 5mg/L, the denitrification strain NX-2C and the bacillus subtilis CGMCC NO.1.4436 are inoculated into 100mL of the artificially prepared sewage for shaking culture at 200 rpm/min. The content of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen is detected every 12h, and at least 48h is required.

The denitrification rate of the strain is detected, and the calculation method is as follows:

the specific data are shown in the following table:

TABLE 1 Denitrification Capacity of Denitrification strains NX-2C and NO.1.4436 at different nitrogen source concentration intervals

As shown in Table 1, the denitrifying strain NX-2C has good denitrifying effect in each nitrogen source concentration interval, the denitrifying efficiency is more than 92%, and the denitrifying strain can tolerate 3000mg/L of ammonia nitrogen, 2000mg/L of nitrite nitrogen and 1500mg/L of nitrate nitrogen. Wherein the denitrification rate of the denitrification strain NX-2C is the highest in the concentration interval of [10,50], and the contents of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen in the degradation artificial prepared sewage respectively can reach 98%, 96% and 95%. The bacillus subtilis CGMCC NO.1.443 does not tolerate high-concentration nitrogen and the denitrification effect of the bacillus subtilis is not as good as that of the denitrification strain NX-2C in any interval concentration.

Example 3 denitrification effect of denitrifying strain NX-2C at different temperatures.

The sewage was prepared manually as in example 2.

The denitrifying strains NX-2C and the bacillus subtilis CGMCC NO.1.4436 are both 10⁴CFU/mL was added to the artificially prepared wastewater at final concentration, and an equal volume of saline was added to the control group, 3 replicates per group.

Under the culture conditions of different temperatures (5, 10, 15, 20, 25, 30, 35 and 40 ℃), the denitrogenation strain NX-2C and the bacillus subtilis CGMCC NO.1.4436 are inoculated into 100mL of the artificially prepared sewage and shake culture is carried out at 200 rpm/min. During the period, the contents of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen are detected every 12 hours, and the detection is required for at least 48 hours.

TABLE 2 denitrification rates of denitrifying strains NX-2C and NO.1.4436 at different temperatures

As shown in Table 2, the denitrifying strain NX-2C still maintained high denitrifying ability under a wide range of temperature conditions. When the temperature is lower (5-10 ℃), the denitrification rate of the three nitrogen is over 90 percent, and the denitrification rate of the denitrification bacterial strain NX-2C is the highest at 10-20 ℃, and is over 95 percent. Although the Bacillus subtilis CGMCC NO.1.4436 is not as good as the denitrification strain in denitrification effect, the temperature range with the optimal denitrification effect is 10-20 ℃ which is the same as that of NX-2C. This may indicate that Bacillus subtilis grows at this temperature quickly and therefore has a high denitrification rate.

Example 4 denitrification effect of denitrifying strain NX-2C under different dissolved oxygen.

The sewage was prepared manually as in example 2.

Under the culture conditions of different dissolved oxygen (1-3, 3-5 and 5-8mg/L), the denitrified bacterial strain NX-2C and the bacillus subtilis CGMCC NO.1.4436 are inoculated into 100mL of the artificially prepared sewage for shake culture. During the period, the contents of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen are detected every 12 hours, and the detection is required for at least 48 hours.

In the experimental process, dissolved oxygen is controlled by adjusting the liquid loading amount of the shake flask and the rotating speed of the shaking table, and the dissolved oxygen is detected by a water quality dissolved oxygen detector.

TABLE 3 denitrogenation Capacity of denitrogenation strains NX-2C and NO.1.4436 under different dissolved oxygen

As shown in Table 3, when the dissolved oxygen is low (1-3mg/L), the denitrification rate of the trinitrogen is more than 90%; when the dissolved oxygen is 3-5mg/L, the denitrification rate is improved when the dissolved oxygen is lower, and when the dissolved oxygen is 5-8mg/L, the dissolved oxygen reaches the adaptation range of the dissolved oxygen of the aquaculture water body, so the denitrification rate is optimal. Therefore, the denitrification bacterial strain NX-2C is less limited by factors of temperature and dissolved oxygen in the purification of aquaculture water. The denitrification rate of the bacillus subtilis CGMCC NO.1.4436 is as low as 40% under the condition of low dissolved oxygen, but the denitrification rate is improved under the condition of sufficient dissolved oxygen, but the denitrification rate is far lower than that of the denitrification strain NX-2C.

Example 5 Denitrification Effect of Denitrification Strain NX-2C at different inoculum sizes

The sewage was prepared manually as in example 2.

The denitrified bacterial strain NX-2C and the bacillus subtilis CGMCC NO.1.4436 are respectively inoculated in different amounts (10)²-10⁷CFU/mL) was added to the artificially prepared wastewater, and an equal volume of saline was added to the control group, 3 replicates per group.

In the culture conditions of pH 7, temperature 30 ℃ and dissolved oxygen 5mg/L, the denitrification strain NX-2C and the bacillus subtilis CGMCC NO.1.4436 are inoculated into 100mL of the artificially prepared sewage according to different inoculum sizes in the experiment, and shake culture is carried out at 200 rpm/min. The content of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen is detected every 12 hours, and the detection is required for at least 48 hours.

TABLE 3 Denitrification capacities of denitrifying strains NX-2C and NO.1.4436 at different nitrogen source concentration intervals

As shown in Table 3, the denitrifying strain NX-2C can maintain excellent denitrifying effect even at a low inoculation amount, and maintain a denitrification rate of 90% or more under different inoculation amounts. Wherein the inoculation amount is 10⁷After the CFU/mL experimental group detects for 48 hours, the residual quantity of the trinitrogen is less than 1.5mg/L, and the denitrification rate is 98 percent. Wherein the bacillus subtilis CGMCC NO.1.4436 has the same denitrification performance with the denitrification strain NX-2C under different inoculation amountsThe higher the inoculation amount, the higher the denitrification rate.

In the practical application process, the denitrifying strain NX-2C also has efficient denitrification under the condition of low inoculation amount, so that unnecessary biological pollution to the aquaculture environment is reduced, and the characteristics of environmental friendliness and no secondary pollution of the denitrifying strain NX-2C are reflected.

Example 6 denitrification effect of the denitrifying strain NX-2C in simulated ecological experiments.

Tilapia fry in the simulated ecological experiment is purchased from a lake culture base of Nanjing city, Jiangsu province. Selecting tilapia with strong constitution and basically consistent specification in experiment, randomly dividing initial weight (6.5 + -0.05 g) into 2 groups, setting control group and experimental group, repeating 3 groups in each group, and placing into 6 temperature-controllable circular culture tanks (C)

Height 700mm), and 25 tails are stocked in each storage tank.

Before the experiment, the tilapia fries are pre-cultured in the storage tank for about two weeks. The trinitrogen content is detected every day 3 days before the experiment, and the experiment can be started when the value is maintained in a relatively stable range, wherein the value is the initial nitrogen source content. The initial ammonia nitrogen is detected to be about 26mg/L, the nitrite nitrogen is detected to be about 20mg/L, and the nitrate nitrogen is detected to be about 15mg/L before the experiment.

Repeatedly resuspending the denitrified strain NX-2C for 3-5 times with 10 times after fermentation by normal saline⁴And (3) putting the inoculation amount of CFU/mL into the storage tank, wherein water is not changed during the experiment, feeding is carried out for 2 times every day, the experiment is carried out for one week, and the contents of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen of the bacteria are required to be detected every day.

As can be seen from FIG. 2, the denitrification bacterial strain NX-2C has the advantages of rapid and stable denitrification, the degradation effect on the trinitrogen in the simulated ecological experiment is more than 90%, and no rebound phenomenon occurs after degradation. The method is particularly suitable for a short-term denitrification process, can achieve high-efficiency denitrification by using the denitrification strains with lower concentration, and can be used as bioremediation to supplement under the condition of unsatisfactory denitrification effect. During the experiment, the fry does not die, and the high safety of the denitrified strain NX-2C to the cultured body is inferred.

Sequence listing

<110> Nanjing university of industry

<120> bacillus subtilis and fermentation method and application thereof

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ttgcatgcgg ctgctataca tgcagtcgag cggacagatg ggagcttgct ccctgatgtt 60

agcggcggac gggtgagtaa cacgtgggta acctgcctgt aagactggga taactccggg 120

aaaccggggc taataccgga tggttgtttg aaccgcatgg ttcaaacata aaaggtggct 180

tcggctacca cttacagatg gacccgcggc gcattagcta gttggtgagg taacggctca 240

ccaaggcaac gatgcgtagc cgacctgaga gggtgatcgg ccacactggg actgagacac 300

ggcccagact cctacgggag gcagcagtag ggaatcttcc gcaatggacg aaagtctgac 360

ggagcaacgc cgcgtgagtg atgaaggttt tcggatcgta aagctctgtt gttagggaag 420

aacaagtacc gttcgaatag ggcggtacct tgacggtacc taaccagaaa gccacggcta 480

actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga attattgggc 540

gtaaagggct cgcaggcggt ttcttaagtc tgatgtgaaa gcccccggct caaccgggga 600

gggtcattgg aaactgggga acttgagtgc agaagaggag agtggaattc cacgtgtagc 660

ggtgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcgactctc tggtctgtaa 720

ctgacgctga ggagcgaaag cgtggggagc gaacaggatt agataccctg gtagtccacg 780

ccgtaaacga tgagtgctaa gtgttagggg gtttccgccc cttagtgctg cagctaacgc 840

attaagcact ccgcctgggg agtacggtcg caagactgaa actcaaagga attgacgggg 900

gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccagg 960

tcttgacatc ctctgacaat cctagagata ggacgtcccc ttcgggggca gagtgacagg 1020

tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc gcaacgagcg 1080

caacccttga tcttagttgc cagcattcag ttgggcactc taaggtgact gccggtgaca 1140

aaccggagga aggtggggat gacgtcaaat catcatgccc cttatgacct gggctacaca 1200

cgtgctacaa tggacagaac aaagggcagc gaaaccgcga ggttaagcca atcccacaaa 1260

tctgttctca gttcggatcg cagtctgcaa ctcgactgcg tgaagctgga atcgctagta 1320

atcgcggatc agcatgccgc ggtgaatacg ttcccgggcc ttgtacacac cgcccgtcac 1380

accacgagag tttgtaacac ccgaagtcgg tgaggtaacc ttttaggagc cagccgccga 1440

aa 1442

Claims

1. Bacillus subtilis (B.subtilis)Bacillus subtillis) NX-2C, the strain is preserved in China general microbiological culture Collection center (CGMCC) at 1 month and 15 days in 2021, and the preservation number is CGMCC NO. 21625.

2. The Bacillus subtilis of claim 1 (b), (c), (d) and d)Bacillus subtillis) A process for the fermentative production of NX-2C, which comprises subjecting Bacillus subtilis (Bacillus subtilis) toBacillus subtillis) Inoculating NX-2C to a seed culture medium, culturing overnight to obtain a seed solution, inoculating the seed solution to a fermentation culture medium, and performing shake culture to obtain Bacillus subtilisBacillus subtillis) NX-2C fermentation broth.

3. The method of claim 2, wherein the seed culture medium is peptone 5-10g/L, yeast powder 1-5 g/L, sodium chloride 5-10 g/L; the culture conditions were 30-37 ℃ and 100-.

4. The method as claimed in claim 2, wherein the seed solution is inoculated into the fermentation medium at 1-10% (v/v), and cultured on a shaker at 30-37 ℃ and 200rpm for 2-3 days to obtain Bacillus subtilis (B.), (V/v)Bacillus subtillis) NX-2C fermentation broth; wherein the carbon source of the fermentation medium is any one or a combination of glucose, sucrose, lactose and sodium acetate; the nitrogen source of the fermentation medium is any one or combination of ammonium chloride, ammonium sulfate, sodium nitrite, potassium nitrate, peptone and beef extract.

5. The method according to claim 2, wherein the fermentation medium is peptone 5g/L, ammonium sulfate 5g/L, sodium nitrite 5g/L, potassium nitrate 5g/L, beef extract 5g/L, ammonium chloride 5g/L, glucose 20g/L and sucrose 20 g/L.

6. The Bacillus subtilis of claim 1 (b), (c), (d) and d)Bacillus subtillis) Application of NX-2C in denitrification of aquaculture water.

7. The use according to claim 6, wherein the aquaculture subjects comprise fish, shrimp and crab.

8. The use according to claim 6, wherein Bacillus subtilis (B.) (Bacillus subtillis) The pH condition range of NX-2C denitrification is 5-9, the temperature is 5-40 ℃, and the dissolved oxygen is 1-8 mg/L.

9. The use according to claim 6, wherein Bacillus subtilis (B.) (Bacillus subtillis) The inoculation amount range of NX-2C denitrification is 10²-10⁷ CFU/mL。