CN112410271B

CN112410271B - Bacillus pumilus 3B-1 and application thereof

Info

Publication number: CN112410271B
Application number: CN202011473659.0A
Authority: CN
Inventors: 邹伟; 杜元粉; 叶光斌
Original assignee: Sichuan University of Science and Engineering
Current assignee: Sichuan University of Science and Engineering
Priority date: 2020-12-15
Filing date: 2020-12-15
Publication date: 2022-01-04
Anticipated expiration: 2040-12-15
Also published as: CN112410271A

Abstract

The invention discloses a Bacillus pumilus 3B-1 and application thereof. The strain is preserved in China center for type culture Collection, the preservation time is 2020, 9 and 24 days, and the preservation number is CCTCC NO: m2020538; the nucleotide sequence of the 16S rDNA is shown as SEQID NO. 1. The screened Bacillus pumilus (Rummeliibibacillus subtilis) 3B-1 can perform anaerobic fermentation under various carbon sources to produce high-yield caproic acid, and meanwhile, the Bacillus pumilus can be compounded with clostridium butyricum, and after the bacterium is strengthened, a large amount of caproic acid can be produced through short-term fermentation under an anaerobic environment.

Description

Bacillus pumilus 3B-1 and application thereof

Technical Field

The invention belongs to the technical field of microbial resources and microorganisms, and particularly relates to Bacillus pumilus 3B-1 and application thereof.

Background

The liquor is the traditional distilled liquor in China, wherein the production and sale proportion of the strong aromatic liquor (Lu liquor) accounts for 70% of the whole liquor industry, and the yield and quality of the liquor significantly influence the development of the liquor industry in China. According to analysis, the flavor substances of the Luzhou-flavor liquor are nearly 674, the quantitative detection components are nearly 342, the key flavor substances are ethyl caproate, ethyl butyrate, ethyl lactate, ethyl caproate, caproic acid, butyric acid and caproic acid, wherein the ethyl caproate is a main flavor component, and the typical character and the quality of the Luzhou-flavor liquor style are determined by the content of the ethyl caproate and the proportion of the ethyl caproate in the key flavor substances.

Caproic acid, which is a precursor substance synthesized by ethyl caproate, is mainly synthesized by caproic acid bacteria in pit mud. Caproic acid bacteria are used as key functional bacteria in the strong-flavor pit mud, and the content of caproic acid bacteria in the pit mud determines the quality of the pit mud. The caproic acid-producing bacteria screened from the pit mud at present mainly comprise Clostridium (Clostridium), Bacillus (Bacillus), ruminococcaceae (ruminococcaceae) and the like. The Clostridium kluyveri (C.kluyveri) is a typical Clostridium caproate produced screened from pit mud, and Clostridium kluyveri A-3 is screened from old pit mud by multiple rounds of enrichment culture of the old pit mud by Jie et al, wherein the caproic acid yield reaches 9.77g/L, but the caproic acid produced by the Clostridium has strict requirements on a carbon source and cannot be produced by using saccharides such as glucose. And the single bacterium separated at present generally has the problems of low caproic acid yield, strict anaerobic environment requirement, long acid production period and the like. At present, the mixed fermentation is also widely concerned, researches find that the mixed fermentation of methane bacteria, actinomycetes, saccharomycetes and caproic acid bacteria is beneficial to improving the production of caproic acid and improving the strict anaerobic environment, and better combined strains still need to be screened to overcome the problems of low yield and low stability. The method for improving the caproic acid yield of the strain by metabolic engineering strain still has the problems of instability, low yield and the like at present. Therefore, the breeding of excellent caproic acid-producing strains can not only utilize multiple carbon sources, but also achieve higher and more stable caproic acid yield in a shorter period is still the key point of the whole strong aromatic Chinese spirit industry research.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a Bacillus pumilus aquaponensis 3B-1 and application thereof, the invention obtains a strain capable of highly producing hexanoic acid, namely the Bacillus pumilus aquaponensis 3B-1, the strain can perform anaerobic fermentation on various carbon sources to highly produce hexanoic acid, the strain can be compounded with clostridium butyricum, and after the strain is strengthened, a large amount of hexanoic acid can be produced by short-term fermentation in an anaerobic environment. Can effectively solve the problems of low yield, harsh acid production condition, poor acid production performance, long acid production period and the like of the existing caproic acid producing strains.

In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:

bacillus pumilus 3B-1 (Rummeliibibacillus subtilis 3B-1) is preserved in China center for type culture collection (CCTCC NO): m2020538.

Furthermore, the nucleotide sequence of the 16S rDNA of the strain is shown as SEQ ID NO. 1.

Furthermore, the suitable growth temperature of the strain is 33-37 ℃, the optimum growth temperature is 35 ℃, the pH tolerance range is 1-10, and the optimum pH is 7.

A microbial preparation comprises the above Bacillus pumilus 3B-1.

The Bacillus pumilus 3B-1 or the application of the microbial preparation in preparing a caproic acid preparation.

A microbial preparation for brewing wine comprises the above Bacillus pumilus 3B-1.

Further, Bacillus pumilus 3B-1 can produce hexanoic acid under aerobic or anaerobic conditions.

A complex microbial inoculum capable of improving caproic acid yield comprises the Bacillus rameltus 3B-1 and Clostridium butyricum; the Bacillus pumilus 3B-1 and Clostridium butyricum are mixed in a volume ratio of 1-2: 0.5-1.

Further, the Bacillus pumilus 3B-1 and the clostridium butyricum are compounded and mixed according to the volume ratio of 1: 1.

Further, clostridium butyricum is clostridium butyricum GD 1-1.

Further, the complex microbial inoculum can generate caproic acid under an anaerobic state.

The composite microbial inoculum is applied to wine brewing.

The preparation method of the composite microbial inoculum comprises the following steps:

(1) preparation of seed solution of Bacillus pumilus 3B-1

Selecting a single colony obtained by the growth in the step (2) in the example 1, selecting a proper amount of thalli by using an inoculating loop under the aseptic operation condition, inoculating the selected single colony on a test tube fermentation liquid culture medium, and then culturing the single colony in an anaerobic incubator at 35 ℃ for 3-5 days to obtain a test tube seed solution.

(2) Preparation of seed solution of Clostridium butyricum GD1-1

Activated GD1-1 is inclined plane, activated GD1-1 inclined plane strain is inoculated with 2 rings to a test tube glucose fermentation liquid culture medium, and then the culture is carried out for 3-5 days in an anaerobic culture box at 35 ℃ to obtain seed liquid. Wherein the formula of the glucose fermentation liquid culture medium is as follows: 20g/L glucose, 0.2g/L magnesium sulfate heptahydrate, 7g/L potassium dihydrogen phosphate, 0.5g/L ammonium sulfate, 10g/L yeast extract powder, 0.5g/L cysteine hydrochloride and pH of 7.0.

(3) Bacillus pumilus 3B-1 and clostridium butyricum GD1-1 are compounded and prepared into compound caproic acid bacterial liquid by short-term fermentation of different carbon sources

Respectively inoculating the seed liquid of the Bacillus pumilus 3B-1 in the step (1) and the seed liquid of the Clostridium butyricum GD1-1 in the step (2) into various culture media which take sodium acetate, sodium lactate, glucose and sodium succinate as carbon sources according to the proportion of 1:1 (the inoculation amount is 5 percent), the mixture ratio of the rest components is consistent with that of a fermentation liquid culture medium, carrying out sealed culture at the temperature of 35 ℃ for 5 days to obtain a compound caproic acid bacterial liquid, carrying out aseptic operation, taking a proper amount of fermentation liquid, passing through a 0.2 micron filter membrane, and carrying out gas chromatography to detect the caproic acid yield.

A method for screening the Bacillus pumilus 3B-1 comprises the following steps:

(1) diluting the pit mud enrichment liquid, coating the pit mud enrichment liquid on a sodium caproate enrichment separation solid culture medium to obtain a single bacterial colony of the bacterial strain, purifying for three times, and inoculating the single bacterial colony in a fermentation liquid culture medium to obtain a bacterial strain seed culture solution;

(2) inoculating the seed culture solution into a liquid fermentation culture medium according to the proportion of 5-10%, fermenting and culturing for 10-12 days at the temperature of 30-35 ℃, carrying out primary screening by a copper sulfate color development method, selecting strains with obvious color development reaction, detecting the content of caproic acid in fermentation liquor, and screening to obtain the Rameyer bacillus aquaticum 3B-1.

Further, the preparation process of the solid culture medium for enriching and separating sodium acetate comprises the following steps:

weighing 15g/L of sodium acetate, 0.2g/L of magnesium sulfate heptahydrate, 0.5g/L of ammonium sulfate, 0.04g/L of dipotassium phosphate trihydrate, 10g/L of yeast extract and 0.5g/L of L-cysteine hydrochloride, adding a proper amount of deionized water, and adjusting the pH value to 7.0 by using a sodium hydroxide solution. Adding 20g/L agar, heating to dissolve, metering to appropriate volume, packaging with measuring cylinder, and sterilizing at 121 deg.C for 20 min. After sterilization, the temperature is reduced to 60 ℃, 20mL/L (filtered and sterilized) of absolute ethyl alcohol and 10g/L of calcium carbonate (separately sterilized and added after sterilization) with proper proportion are added and fully shaken for use.

Further, the preparation process of the fermentation liquid culture medium comprises the following steps:

weighing 15g/L of sodium acetate, 0.2g/L of magnesium sulfate heptahydrate, 7g/L of monopotassium phosphate, 0.5g/L of ammonium sulfate, 10g/L of yeast extract powder and 0.5g/L of L-cysteine hydrochloride, adding a proper amount of deionized water, and adjusting the pH value to 7.0 and 7.0 by using a sodium hydroxide solution. Accurately packaging with measuring cylinder, sterilizing at 121 deg.C for 20min, and adding 20mL/L of filtered and sterilized anhydrous ethanol when the temperature is reduced to low temperature.

The invention has the beneficial effects that:

1. the caproic acid production level of the screened Bacillus pumilus (Rummeliibibacillus subtilis) 3B-1 strain is 406.4mg/100mL, the highest butyric acid production level is 455.9mg/100mL, and the higher caproic acid production level is still maintained along with the extension of the fermentation time, which shows that the 3B-1 strain has higher and more stable caproic acid production capacity. Is a strain with higher caproic acid producing capability reported at present and has important application prospect.

2. The 3B-1 strain obtained by screening can utilize different carbon sources to produce hexanoic acid, and can keep higher hexanoic acid yield. Particularly, under the condition that the carbon source is glucose, the yield of the caproic acid is the highest, namely 406.4mg/100mL, and the carbon source is the best carbon source for producing the caproic acid. The method has the capability of utilizing wide substrates to produce hexanoic acid with high yield, and has wide application range.

3. When the 3B-1 strain obtained by screening is subjected to anaerobic fermentation, the caproic acid yield is far greater than that of shake flask aerobic culture, is 406.4mg/100mL and is almost three times as high as that of caproic acid under an aerobic condition, and the strain can produce more caproic acid in an anaerobic environment; in addition, the strain also has the capability of producing hexanoic acid under aerobic conditions, and still has relatively high hexanoic acid production level, which indicates that the 3B-1 strain has relatively high and stable hexanoic acid production capability under anaerobic or aerobic conditions. Compared with the microorganism which strictly anaerobically produces the caproic acid, the 3B-1 strain does not need to be strictly anaerobic all the time, and the operation is more convenient.

4. After the 3B-1 strain obtained by screening is compounded with clostridium butyricum, the caproic acid yield in the composite fermentation broth can be obviously improved in the short-term fermentation process under the anaerobic condition of utilizing different carbon sources, and the composite caproic acid bacterial liquid with high caproic acid content is prepared.

Drawings

FIG. 1 is a plot of the peaks of the gas chromatogram in example 1;

FIG. 2 is a diagram of a colony of hexanoic acid-producing strain 3B-1 cultured anaerobically on a fermentation solid medium (without calcium carbonate addition);

FIG. 3 is a microscopic morphology of a high caproic acid producing strain 3B-1;

FIG. 4 is an electrophoretogram of 16S rDNA PCR products of the high caproic acid producing strain 3B-1; wherein, the strip 1 is DNA Marker, and the strip 2 is 16S rDNA PCR product;

FIG. 5 is a phylogenetic tree of 16SrDNA of strain 3B-1.

Detailed Description

The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.

EXAMPLE 1 obtaining of Bacillus pumilus 3B-1

The high-yield caproic acid jellyfish Bacillus lamill is separated from high-quality aged pit mud in a certain strong-flavor liquor pit pool in Sichuan. The components of the used sodium acetate enrichment and separation culture medium are as follows: 15g/L of sodium acetate, 20mL/L of absolute ethyl alcohol (subjected to filtration sterilization), 0.2g/L of magnesium sulfate heptahydrate, 10g/L of calcium carbonate (subjected to single sterilization and addition after sterilization), 0.5g/L of ammonium sulfate, 0.04g/L of dipotassium hydrogen phosphate trihydrate, 10g/L of yeast extract, 0.5g/L of L-cysteine hydrochloride and pH 7.0. 20g/L of agar is added as a solid medium.

The fermentation liquid culture medium comprises the following components: 15g/L of sodium acetate, 20mL/L of absolute ethyl alcohol (subjected to filtration sterilization), 0.2g/L of magnesium sulfate heptahydrate, 7g/L of monopotassium phosphate, 0.5g/L of ammonium sulfate, 10g/L of yeast extract powder, 0.5g/L of L-cysteine hydrochloride and pH 7.0.

Gas chromatography conditions of DB-WAX (60 m.times.250 μm.times.0.25 μm) column;

the carrier gas is high-purity H₂Initial temperature of 80 deg.C (1min), raising to 200 deg.C at 20 deg.C/min, and raising to 250 deg.C at 50 deg.C (1 min);

SIM (selective ion scan mode), scan ions 60,87,88, 116;

the split ratio is 20:1, and the flow rate of the carrier gas is 1 mL/min;

under the condition, the peak appearance of the standard sample is shown in figure 1, and the peak appearance time of butyric acid and caproic acid is 9.335min and 11.783min respectively.

Sample preparation: preparing the purified single colony into seed liquid with the same concentration by using sterile water, inoculating the seed liquid into 100mL of fermentation liquid culture medium according to the inoculation amount of 5%, fermenting for 10 days at 35 ℃, and taking a proper amount of supernatant to pass through a 0.2-micron microporous filter membrane. And 1mL of the solution is filled in a sample injection bottle for qualitative and quantitative analysis of gas chromatography by adopting an external standard method.

First, isolation of the Strain

1. Enrichment of pit mud

Weighing 10g of pit mud, adding the pit mud into an anaerobic bottle filled with 10mL of sterile water, carrying out water bath treatment at a constant temperature of 80 ℃ for 10min, cooling, inoculating the cooled pit mud into a sterilized sodium acetate enrichment separation culture medium in an inoculation amount of 5%, and carrying out enrichment culture in an anaerobic box at 35 ℃ for 5-7d to obtain an enrichment solution.

2. Separation and purification of bacterial strains

Separating caproic acid bacteria from the enriched liquid by dilution coating method, and diluting the enriched liquid to 10^-1～10^-6 Suction 10^-3～10^-60.1mL of the bacterial liquid is coated on a sodium acetate enrichment and separation solid culture medium (oxygen is removed in an anaerobic incubator for 24 hours), and the solid culture medium is cultured in the anaerobic incubator at 35 ℃ for 3-5 days. Colonies with different morphological characteristics were streaked again at least three times to obtain single colonies.

3. Screening of high-yield caproic acid bacteria

And (3) selecting the single bacterial colony obtained by growth in the step (2), inoculating the single bacterial colony on a test tube fermentation liquid culture medium under an aseptic operation condition by using an inoculating loop, then culturing the single bacterial colony in a 35 ℃ anaerobic incubator for 3-5 days to obtain a first-stage seed solution, then respectively inoculating 5% of the first-stage seed solution of the screened bacteria into 100mL of sterilized fermentation liquid culture medium, standing and culturing for ten days at 35 ℃, primarily screening by a copper sulfate chromogenic method, selecting a bacterial strain with obvious chromogenic reaction, and detecting the caproic acid content in the fermentation liquid by gas chromatography. Finally, a bacterial strain with high caproic acid yield is screened out and named as 3B-1.

Identification of bis, 3B-1

1. Morphological characteristics

The morphology observation of the 3B-1 shows that the strain morphology is as follows: the thalli is thick and long rod-shaped, terminal spores are grown, drumstick-shaped and gram-positive bacteria are cultured in a fermentation solid culture medium (without calcium carbonate) for 24 hours to form irregular round colonies, the colonies are large and opaque, the edges are light yellow, and the middle obvious bulge is milky white. The colony morphology and the bacterial cell microscopic morphology are shown in FIG. 2.

2. Physiological and biochemical identification

The physiological and biochemical identification of 3B-1 was carried out with reference to Bergey's Manual of bacteria identification, ninth edition and Manual of identification of common bacteria systems (scientific Press, division of the east elegance bead), and the results are shown in Table 1.

Table 13B-1 shows the results of physiological and biochemical analyses

Note: + indicates positive reaction; -indicates a negative reaction.

3. 16S rDNA sequence determination and analysis

(1) Extraction of DNA

Inoculating the purified single colonies with different forms into a fermentation liquid culture medium, culturing for 10 days at 35 ℃, sucking 2mL of culture solution into a sterile centrifuge tube, centrifuging for 10min at 13000 r/min, discarding supernatant, and collecting thalli precipitates (2-3 times in total). Bacterial genome DNA rapid extraction kit is used for bacterial precipitation, and genome DNA is extracted according to the operation steps.

(2) Construction of PCR amplification and phylogenetic trees

Taking the DNA solution obtained in the step (1) as an amplification template, and carrying out PCR amplification by adopting a bacterial universal primer 27F-1492R;

the PCR amplification system is as follows: 5 × Buffer (containing Mg)²⁺)10 mu L of 200 mu mol/L dNTPs 1 mu L, 1 mu L of forward and reverse primers respectively, 1 mu L of Taq DNA polymerase and 3 mu L of template DNA, and sterile water is added to make up to 50 mu L;

the amplification conditions were: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 1min, annealing at 55 ℃ for 1min, extension at 72 ℃ for 1.5min for 30 cycles, and final extension at 72 ℃ for 10 min.

The amplified products were detected by 1% agarose gel electrophoresis, and the PCR products were sequenced in Nanjing Pekinsonino Gene technology, Inc. And after the sequencing sequence is sheared by BioEdit, uploading the reliable sequence fragments to an NCBI nucleic acid comparison website for comparison, selecting a sequence with the highest sequence homology, and constructing a phylogenetic tree by using MEGA 5.0 software. A phylogenetic evolutionary tree based on 16SrDNA is shown in figure 5.

The PCR amplification result is shown in figure 4, the size of the PCR product is 1428bp, and the 16SrDNA sequence of the strain 3B-1 is shown as SEQ ID N0.1 through sequencing identification.

3B-1 is identified as the Bacillus pumilus aquaponensis by performing morphological characteristics, physiological and biochemical characteristics and 16S rDNA sequencing on the 3B-1 strain. The suitable growth temperature of the strain is 33-37 ℃, the optimum growth temperature is 35 ℃, the pH tolerance range is 1-10, the optimum pH is 7, and the ethanol tolerance concentration is 5%. The hexanoic acid resistant concentration is as high as 30 g/L.

The strain is cultured in a solid culture medium enriched and separated from sodium acetate at 35 ℃ for 3 days, and is continuously cultured by generations, and the biological characteristics of the strain, such as culture characteristics, morphological characteristics, physiology, biochemistry and the like, have no obvious change, and show good stability of biological properties. The strain is identified as caproic acid-producing bacteria (Rummeliibibacillus suwonensis) by 16S rDNA, is named as Bacillus aquaticus (Rummeliibibacillus suwonensis)3B-1, is preserved in China center for type culture Collection (address: China, Wuhan university) in 9-24 months in 2020, and has the preservation number of CCTCC NO: m2020538.

Example 2 application of Aquifex lanumel 3B-1 to high yield of hexanoic acid

Fermentation culture of 3B-1 and detection of caproic acid production

Inoculating the 3B-1 strain obtained in the step 1 into a test tube filled with a fermentation liquid culture medium by using an inoculating loop sterile operation, and culturing for 3-5 days in an anaerobic incubator at 35 ℃ to obtain a seed solution; inoculating the seed solution with 5% inoculation amount in 100mL sterilized fermentation liquid culture medium, and standing at 35 deg.C for culture. Using a disposable injector to suck a proper amount of fermentation liquor and filter the fermentation liquor through a 0.2 mu m microporous filter membrane when fermenting for 10 days. And 1mL of the solution is filled in a sample injection bottle for qualitative and quantitative analysis of gas chromatography by adopting an external standard method. The yields of butyric acid and caproic acid were determined when 3B-1 utilized sodium acetate as a carbon source. Three replicates were made for each sample. The results are shown in Table 2.

TABLE 23B-1 acid production at different time periods using sodium acetate as carbon source

Carbon source exploration for producing hexanoic acid by secondary and 3B-1 fermentation

The seed solution obtained in the embodiment 1 is inoculated into 100mL of each culture medium which takes sodium acetate (15g/L), sodium lactate (20g/L), glucose (20g/L) and sodium succinate (20g/L) as carbon sources according to the inoculation amount of 5 percent, the mixture ratio of the rest components is consistent with that of the fermentation liquid culture medium, the seed solution is hermetically cultured for 10 days at the temperature of 35 ℃, and then an appropriate amount of fermentation liquid is taken in an aseptic operation, passes through a 0.2 mu m filter membrane and is subjected to gas chromatography to detect the yield of butyric acid and caproic acid. The detection results are shown in table 3, and it can be seen from the table that the yield of hexanoic acid is the highest under the condition that the carbon source is glucose, and the carbon source is the best carbon source for producing hexanoic acid; secondly, the strain also shows higher caproic acid production capacity by utilizing sodium lactate, and the caproic acid production yield is stable.

TABLE 33 acid production by fermentation for ten days with different carbon sources for B-1

Oxygen condition exploration for producing hexanoic acid by using three, 3B-1 fermentation

Inoculating the seed solution obtained in the embodiment 1 into 100mL of fermentation liquid culture medium with glucose as a substrate according to the inoculation amount of 5%, carrying out sealed culture and triangular flask shake flask culture (200r/min) at 35 ℃, taking a proper amount of fermentation liquor in an aseptic operation at 10 days, passing through a 0.2 mu m filter membrane, and carrying out gas chromatography to detect the yield of butyric acid and caproic acid. Three replicates were made for each sample.

The detection results are shown in table 4, and it can be seen from the table that during anaerobic fermentation, the caproic acid yield is far greater than that of shake flask aerobic culture, almost three times as much as that of caproic acid under aerobic condition, and the strain can produce more caproic acid in anaerobic environment; in addition, the strain also has the capability of producing hexanoic acid under aerobic conditions, and still has relatively high hexanoic acid production level, which indicates that the 3B-1 strain has relatively high and stable hexanoic acid production capability under anaerobic or aerobic conditions.

TABLE 43B-1 fermentation of acid production in aerobic and anaerobic environments for ten days with glucose as carbon source

Example 3 preparation of Compound caproic acid bacterial liquid by compounding Bacillus pumilus 3B-1 and Clostridium butyricum GD1-1

(1) Preparing a seed solution of the Bacillus pumilus 3B-1:

(2) Preparation of seed solution of clostridium butyricum GD 1-1: activated GD1-1 is inclined plane, activated GD1-1 inclined plane strain is inoculated with 2 rings to a test tube glucose fermentation liquid culture medium, and then the culture is carried out for 3-5 days in an anaerobic culture box at 35 ℃ to obtain seed liquid. Wherein the formula of the glucose fermentation liquid culture medium is as follows: 20g/L glucose, 0.2g/L magnesium sulfate heptahydrate, 7g/L potassium dihydrogen phosphate, 0.5g/L ammonium sulfate, 10g/L yeast extract powder, 0.5g/L cysteine hydrochloride and pH of 7.0.

The results of the detection of caproic acid production by the single bacteria and the composite caproic acid bacteria liquid are shown in table 5, the caproic acid yield of the composite caproic acid bacteria liquid prepared by the two bacteria is obviously higher than that of the single bacteria, and the caproic acid bacteria liquid with high caproic acid yield can be obtained within a short fermentation time.

TABLE 5 acid production in five days by anaerobic fermentation of single and mixed bacteria using different carbon sources

Sequence listing

<110> Sichuan university of light chemical industry

<120> Bacillus pumilus 3B-1 and application thereof

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ggtttcttaa gtctgatgtg aaagcccacg gcttaaccgt ggagggtcat tggaaactgg 600

gagacttgag tgcagaagag gaaagtggaa ttccaagtgt agcggtgaaa tgcgtagaga 660

tttggaggaa caccagtggc gaaggcgact ttctggtctg caactgacgc tgaggcgcga 720

aagcatgggg agcaaacagg attagatacc ctggtagtcc atgccgtaaa cgatgagtgc 780

taagtgttag ggggtttccg ccccttagtg ctgcagctaa cgcattaagc actccgcctg 840

gggagtacga ccgcaaggtt gaaactcaaa ggaattgacg ggggcccgca caagcggtgg 900

agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc aggtcttgac atcccgatga 960

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gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc cttaatctta 1080

gttgccatca tttagttggg cactctaagg tgactgccgg tgataaaccg gaggaaggtg 1140

gggatgacgt caaatcatca tgccccttat gacctgggct acacacgtgc tacaatggac 1200

ggtacaaaga gtcgctaact cgcgagagta tgctaatctc ataaaaccgt tctcagttcg 1260

gattgtaggc tgcaactcgc ctacatgaag ccggaatcgc tagtaatcgc ggatcagcat 1320

gccgcggtga atacgttccc gggccttgta cacaccgccc gtcacaccac gagagtttgt 1380

aacacccgaa gtcggtgagg taaccttggg agccagccgc cgaaggtg 1428

Claims

1. Bacillus pumilus (A) for treating waterRummeliibacillus suwonensis)3B-1, characterized in that the strain is preserved in China center for type culture Collection with a preservation time of 2020, 9 and 24 days, and the preservation number is CCTCC NO: m2020538.

2. A microbial preparation comprising Bacillus pumilus 3B-1 of claim 1.

3. Use of the Bacillus pumilus 3B-1 of claim 1 or the microbial preparation of claim 2 for the preparation of a caproic acid-producing preparation.

4. Use of the Bacillus pumilus 3B-1 of claim 1 or the microbial preparation of claim 2 for brewing wine.