CN113502242A - Bacillus firmus and application thereof - Google Patents

Bacillus firmus and application thereof Download PDF

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Publication number
CN113502242A
CN113502242A CN202110714782.5A CN202110714782A CN113502242A CN 113502242 A CN113502242 A CN 113502242A CN 202110714782 A CN202110714782 A CN 202110714782A CN 113502242 A CN113502242 A CN 113502242A
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firmus
bacillus firmus
pha
cytobacillus
bacillus
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CN113502242B (en
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郑维爽
张梦君
黄艺
于盛洋
李康
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Mibei Shenzhen Biotechnology Co ltd
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Peking University Shenzhen Graduate School
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids

Abstract

The invention relates to the technical field of microorganisms, and particularly relates to bacillus firmus and application thereof. The bacillus firmus is obtained by separating 10cm of soil on the surface layer of the mangrove protection region in the Shenzhen Futian region and named as bacillus firmus MN 36-4. The bacillus firmus MN36-4 has better capacity of producing PHA, the PHA yield reaches 1.71g/L, wherein the PHB and the PHV respectively account for 97.1 percent and 2.9 percent, and the bacillus firmus has good industrial application prospect.

Description

Bacillus firmus and application thereof
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to bacillus firmus and application thereof.
Background
Petroleum-based plastic products are used on a large scale in the prior art, but due to the non-biodegradable nature of petroleum-based plastics, extensive use of petroleum-based plastics can pose serious threats to the natural ecosystem and to human health. Polyhydroxyalkanoates (PHA) are a class of natural polymer-based materials synthesized by microorganisms, have physicochemical properties comparable to synthetic plastics, and are biodegradable and biocompatible, and thus are becoming important substitutes for synthetic plastics.
Mangrove is a population of woody plants growing in tropical and subtropical intertidal zones, with soil in the intertidal zone periodically flooded with seawater. The microorganism of mangrove ecosystem develops unique physiological and biochemical characteristics to adapt to the special environment. Researches show that mangrove as a high-salt, high-carbon and low-nutrient habitat contains rich PHA-producing microbial resources. Therefore, PHA microbial strains with different functions can be produced by utilizing cheap carbon sources in the mangrove forest, and the method has important industrial application value.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides bacillus firmus and application thereof.
In a first aspect, the present invention provides a Bacillus firmus MN 36-4. The invention separates a bacillus firmus named MN36-4 from the 10cm soil on the surface layer of the mangrove protection area in the Shenzhen Futian area, and the preservation information is as follows:
the name of the depository: china general microbiological culture Collection center, preservation number: CGMCC No.22298, preservation date: no. 5/10 in 2021, category name: cytobacillus firmus, preservation address: west road No.1, north west of the township, beijing, ministry of sciences, china, institute of microbiology, zip code: 100101.
the invention carries out morphological observation, physiological and biochemical identification and 16S rDNA sequence (the 16S rDNA sequence is shown as SEQ ID NO. 1) which are consistent with the bacillus firmus.
In a second aspect, the invention provides a microbial inoculum comprising said bacillus firmus MN36-4 or a fermentation broth thereof.
Further, the PHA concentration in the microbial inoculum reaches more than 1.71 g/L.
Further, PHB and PHV accounted for 97.1% and 2.9%, respectively, in the PHA.
In a third aspect, the present invention provides a method of producing PHA, comprising: the Bacillus firmus (Cytobacillus firmus) MN36-4 or the microbial inoculum is used for fermentation culture.
Further, the fermentation culture is as follows:
culturing for 3-5 days at the temperature of 25-35 ℃ and at the speed of 150-200 r/min.
Further, the culture medium used in the fermentation culture is one or more of a nutrient broth culture medium, an inorganic salt culture medium with glucose as a single carbon source or an inorganic salt culture medium with sodium pyruvate as a single carbon source.
Further, the components of the nutrient broth are as follows:
10g/L of peptone, 5g/L of beef extract and 5g/L of NaCl;
the inorganic salt culture medium with glucose as a single carbon source comprises the following components:
10g/L glucose, 9g/L Na2HPO4KH of 1.5g/L2PO41g/L NH4Cl, 0.2g/L MgSO40.02g/L of CaCl20.0012g/L ferric ammonium citrate, 100 μ L microelement liquid (1g/L ZnSO)4,0.3g/L MnCl2,3g/L H3BO3,2 g/L CoCl2,0.1g/L CuCl2,0.2g/L NiCl2,0.3g/L NaMoO4)。
The inorganic salt culture medium with sodium pyruvate as a single carbon source comprises the following components:
10g/L of sodium pyruvate, 9g/L of Na2HPO4KH of 1.5g/L2PO41g/L NH4Cl, 0.2g/L MgSO40.02g/L of CaCl20.0012g/L ferric ammonium citrate, 100 μ L microelement liquid (1g/L ZnSO)4,0.3g/L MnCl2,3g/L H3BO3,2 g/L CoCl2,0.1g/L CuCl2,0.2g/L NiCl2,0.3g/L NaMoO4)。
Further, all media had a pH of 7.0 and were autoclaved at 121 ℃ for 25 min.
The invention further provides the Bacillus firmus (Cytobacillus firmus) MN36-4 and application of the microbial inoculum in PHA production.
The invention further provides the Bacillus firmus (Cytobacillus firmus) MN36-4 and application of the microbial inoculum in improving PHA yield.
Further, the PHA comprises poly-3-hydroxybutyrate and/or poly-hydroxyvalerate.
The invention further provides application of the bacillus firmus (Cytobacillus firmus) MN36-4 and the microbial inoculum in production of packaging materials, bonding materials or spraying materials.
The invention has the following beneficial effects:
the invention separates and obtains a bacillus firmus (Cytobacillus firmus) MN36-4 in the mangrove soil of coastal intertidal zone, which has stronger PHA production capacity when utilizing an inorganic salt culture medium (M1) with glucose as a single carbon source for fermentation culture, the total yield of the produced PHB and PHV reaches 1.71g/L, provides important strain resources and technical means for producing natural polymer biological materials for replacing synthetic plastics, and has good industrial application prospect.
Drawings
FIG. 1 is a flow chart of the preparation of Bacillus firmus MN36-4 provided in example 1 of the present invention.
FIG. 2 is a gel diagram of a DNA product of Bacillus firmus MN36-4 according to example 1 of the present invention, which has been separated by agarose gel electrophoresis, after amplification of the phaC synthetase gene by Polymerase Chain Reaction (PCR).
FIG. 3 is a colony diagram of Bacillus firmus MN36-4 provided in example 2 of the present invention cultured at 30 ℃ for 1 day.
FIG. 4 is a gram stain plot of Bacillus firmus MN36-4 provided in example 2 of the present invention.
FIG. 5 is a phylogenetic tree diagram constructed from the 16S sequence of Bacillus firmus MN36-4 provided in example 2 of the present invention.
FIG. 6 is a graph showing the results of PHA production assay of Bacillus firmus MN36-4 provided in example 3 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Media used in the examples: an oil-containing and salt-containing enrichment culture medium for separating bacterial strains, which is prepared by adding 1-10% (v/v) of mixed vegetable oil (goldfish: peanut oil: rapeseed oil: 1:1) and 35-125% of NaCl into aged seawater. The peanut oil comprises more than 5 percent of components according to the GB/T1534-2017 specification: palmitic acid (C16:0, 8.0-14.0), oleic acid (C18:1, 35.0-69.0) and linoleic acid (C18:2, 13.0-43.0). The rapeseed oil has the composition of more than 5 percent according to the GB/T1536-2004 specification: palmitic acid (C16:0, 1.5-6.0), oleic acid (C18:1, 8.0-60.0), linoleic acid (C18:2, 11.0-23.0), linolenic acid (C18:2, 5.0-13.0), arachidonic acid (C20:1, 3.0-15.0) and erucic acid (C22:1, 3.0-60.0).
Ordinary seawater culture medium (2216E) for purifying and culturing bacterial strain, which comprises peptone 5.0g/L, yeast extract 1.0g/L, ferric citrate 0.1g/L, NaCl 19.45 g/L, and MgCl 5.98g/L23.24g/L of Na2SO4, 1.8g/L CaCl2KCl 0.55g/L, Na 0.16g/L2CO3KBr of 0.08g/L and SrCl of 0.034g/L20.022g/L of H3BO30.004g/L of Na2O·nSiO20.0024g/L NaF, 0.0016 g/L NaNO30.008g/L of Na2HPO4Sterilizing with high pressure steam at 121 deg.C for 25min, with pH of 7.6 + -0.2.
The PHA-producing liquid culture medium mainly comprises a nutrient broth culture medium (NB), an inorganic salt culture medium (M1) taking glucose as a single carbon source and an inorganic salt culture medium (M2) taking sodium pyruvate as a single carbon source, and comprises the following components:
NB: 10g/L peptone, 5g/L beef extract, 5g/L NaCl, pH 7.0, and sterilization at 121 ℃ for 25 min.
M1: 10g/L glucose, 9g/L Na2HPO4, KH 1.5g/L2PO41g/L NH4Cl, 0.2g/L MgSO40.02g/L of CaCl20.0012g/L ferric ammonium citrate, 100 μ L microelement liquid (1g/L ZnSO)4,0.3g/L MnCl2,3g/L H3BO3,2g/L CoCl2,0.1g/L CuCl2,0.2g/L NiCl2,0.3g/L NaMoO4) Sterilizing at 121 deg.C for 25min and pH 7.0.
M2: 10g/L of sodium pyruvate, 9g/L of Na2HPO4KH of 1.5g/L2PO41g/L NH4Cl, 0.2g/L MgSO40.02g/L of CaCl20.0012g/L ferric ammonium citrate, 100 μ L microelement liquid (1g/L ZnSO)4,0.3g/L MnCl2,3g/L H3BO3,2g/L CoCl2,0.1g/L CuCl2,0.2g/L NiCl2,0.3g/L NaMoO4) Sterilizing at 121 deg.C for 25min and pH 7.0.
Example 1 isolation and screening of Bacillus firmus MN36-4
The present embodiment provides a preparation process of bacillus firmus MN36-4, and with reference to fig. 1, the specific steps are as follows:
1. isolation of culturable strains
Collecting 10cm of soil on the surface layer of the mudflat of the mangrove protection area in the Shenzhen province and the Futian; placing the collected soil into a conical flask filled with 100mL of sterilized oil-containing salt-containing enrichment medium, carrying out constant-temperature shaking culture at 30 ℃ and 160r/min for 77 days, transferring the soil into the enrichment medium containing fresh medium by 1% of inoculum size every 7 days, simultaneously increasing the oil content by 1% and the salinity by 10 per mill, and taking 1mL of soil suspension liquid after 7, 42 and 77 days of culture; carrying out gradient dilution on the bottom mud suspension to prepare a soil suspension with the concentration of 10 < -3 > to 10 < -5 >; adding the diluted bottom mud suspension into a 2216E culture medium plate for coating treatment, and culturing for 48h in a constant-temperature incubator at 30 ℃ to obtain bacterial colonies; selecting single colonies with different forms, streaking, purifying and culturing, and preserving strains at low temperature.
haC Gene identification, the strain with the band is PHA-producing positive bacteria and is named as MN 36-4.
2. Screening of PHA-producing strains
And identifying the phaC gene of the strain by adopting a colony PCR method. And (3) selecting a single colony, adding the single colony into a sterilized PCR tube containing 50 mu L of sterile water, and obtaining a colony PCR template at 95 ℃ for 10 min. The phaC gene forward primer is BmphaC015 (5'-CGTGCAAGAGTGGGA AAAAT-3') (SEQ ID NO.2), and the reverse primer is BmphaC931R (5'-TCGC AATATGATCACGGCTA-3') (SEQ ID NO. 3).
The PCR reaction system is as follows: 2 XPCR Master Mix 25 uL; 1 μ L each of the forward primer and the reverse primer; 1 mu L of template; ddH2O make up to 50. mu.L.
The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 6 min; denaturation at 94 ℃ for 45s, and annealing at 54 ℃ for 30 s; extension at 72 ℃ for 90s, and 31 cycles; extending for 10min at 72 ℃, and storing at 4 ℃. The obtained PCR product is subjected to 120V, 30min and 1% agarose gel electrophoresis, a blue light color-permeable instrument observes colloid, a sample with strips is the phaC gene positive strain, the phaC gene positive strain is named as MN15-19, and an electrophoresis chart refers to FIG. 2.
Example 2 identification of Bacillus firmus MN36-4
In this example, the bacillus firmus MN36-4 obtained in example 1 was identified, including morphological observation, physiological and biochemical identification, and 16S rDNA sequence analysis, as follows:
1. morphological observation
The strain MN36-4 provided in example 1 was streaked onto a 2216E medium plate, which was then inverted, cultured in an incubator at 30 ℃ for 24 hours, and the growth of colonies on the plate was observed and recorded. The colony morphology of strain MN36-4 is shown in FIG. 3. As can be seen from FIG. 3, the bacterial colony of the strain is light yellow, oval, flat, irregular in edge, slightly turbid, and moist and smooth in surface.
The bacterial strain MN36-4 provided in the invention example was gram-stained with the kit, and the bacterial strain was observed under an oil lens, and the gram stain pattern of the bacterial strain is shown in FIG. 4. From FIG. 4, it can be seen that the strain is purple, and is a gram-positive bacterium.
2. Physiological and biochemical identification
The bacterial strain MN36-4 provided by the embodiment of the invention is subjected to physiological and biochemical identification by referring to physiological and biochemical identification indexes in a common bacterial system identification manual.
The indexes of physiological and biochemical identification of the strain provided by the embodiment comprise catalase ability, methyl red MR experiment, VP experiment, oxidase ability, starch hydrolysis ability, hydrogen sulfide production ability, nitrate reduction ability, malonate utilization ability, citrate utilization ability and gelatin liquefaction ability. The results of physiological and biochemical assays are shown in Table 1.
TABLE 1 physiological and biochemical identification results of the present strains
Characterization of the properties of a sheet Reaction characteristics Characterization of the properties of a sheet Reaction characteristics
Catalase enzyme + Production of hydrogen sulfide -
MR experiment - Nitrate reduction +
VP experiment - Utilization of malonic acid salt -
Oxidase enzyme - Citric acid salt -
Starch hydrolysis - Liquefaction of gelatin +
In the table, + indicates that the present strain reacted or could be used, and-indicates that the present strain did not react or could not be used.
3.16S rDNA sequence analysis
In the embodiment, DNA in a strain MN36-4 is extracted by an Ezup bacterial genome DNA extraction kit. The forward primer for the PCR amplification was 27F (5'-AGAGTTTGATCCTGGC TCAG-3') and the reverse primer was 1492R (5'-GGTTACCTTGTTACGACTT-3').
The PCR reaction system is as follows: 2 XPCR Master Mix 25 uL; 1 μ L each of the forward primer and the reverse primer; 1 mu L of template; ddH2O make up to 50. mu.L.
The PCR reaction system is as follows: pre-denaturation at 94 ℃ for 4min for 1 cycle; denaturation at 94 ℃ for 45s, and annealing at 55 ℃ for 45 s; extension at 72 ℃ for 90s for 30 cycles; storing at 4 ℃.
The PCR product was sequenced by Shanghai Bioengineering Co., Ltd, and the sequencing result was shown as SEQ ID NO. 1. And performing Blast similarity comparison on the obtained sequence in GenBank to obtain a sequence with higher similarity. The MEGA7.0 software is used for constructing a phylogenetic tree of the strain, the homology of a 16S rDNA sequence of the strain and bacillus firmus (Bacillus firmus) reaches 99.9 percent, and the phylogenetic tree of the strain is shown in figure 5.
By combining the results of the morphological observation, the physiological and biochemical identification and the 16S rDNA sequence analysis, the strain MN36-4 can be determined to be Bacillus firmus (Cytobacillus firmus), the strain is named as Bacillus firmus MN36-4 and is preserved, and the preservation information is as follows:
the preservation number is: CGMCC No.22298, preservation date: no. 5/10 in 2021, category name: cytobacillus firmus, preservation address: west road No.1, north west of the township, beijing, ministry of sciences, china, institute of microbiology, zip code: 100101.
EXAMPLE 3 PHA-Productivity assay of Bacillus firmus MN36-4
The embodiment of the invention carries out the measurement of the PHA production capacity of the bacillus firmus MN36-4, and the measurement comprises the following contents:
extraction of PHA
The Bacillus firmus MN36-4 provided in example 1 was inoculated into a liquid medium and shake-cultured at a constant temperature of 150r/min at 30 ℃ for 4 days. After the culture is finished, centrifuging the fermentation liquor for 20min at 5000r/min to obtain cell precipitates, and then carrying out freeze drying treatment; weighing 10mg of bacteria freeze-dried sample, putting the bacteria freeze-dried sample into a lipidization tube, adding 1mL of chloroform (containing 0.5mg/mL of methyl benzoate) and 1mL of methanol solution containing 15% (v/v) of concentrated sulfuric acid, sealing for 2.5h under 100 ℃ oil bath, and carrying out methyl esterification reaction; after the reaction is finished, the sample is cooled for 5min in ice bath, then 0.5mL of deionized water is added, the mixture is fully and uniformly mixed for 30s, the mixture is centrifuged and layered for 10min at 3500 r/min, and the lower chloroform phase is taken for chromatographic analysis.
The liquid culture medium comprises a nutrient broth culture medium (NB), an inorganic salt culture medium (M1) with glucose as a single carbon source and an inorganic salt culture medium (M2) with sodium pyruvate as a single carbon source, and comprises the following components:
NB: 10g/L peptone, 5g/L beef extract, 5g/L NaCl, pH 7.0, and sterilization at 121 ℃ for 25 min.
M1: 10g/L glucose, 9g/L Na2HPO4KH of 1.5g/L2PO41g/L NH4Cl, 0.2g/L MgSO40.02g/L of CaCl20.0012g/L ferric ammonium citrate, 100 μ L microelement liquid (1g/L ZnSO)4,0.3g/L MnCl2,3g/L H3BO3,2g/L CoCl2,0.1g/L CuCl2,0.2g/L NiCl2,0.3g/L NaMoO4) pH 7.0, sterilization at 121 ℃ for 25min。
M2: 10g/L of sodium pyruvate, 9g/L of Na2HPO4KH of 1.5g/L2PO41g/L NH4Cl, 0.2g/L MgSO40.02g/L of CaCl20.0012g/L ferric ammonium citrate, 100 μ L microelement liquid (1g/L ZnSO)4,0.3g/L MnCl2,3g/L H3BO3,2g/L CoCl2,0.1g/L CuCl2,0.2g/L NiCl2,0.3g/L NaMoO4) Sterilizing at 121 deg.C for 25min and pH 7.0.
Determination of PHA content
The monomeric composition of PHAs determined in this example included poly-3-hydroxybutyrate (PHB) and Polyhydroxyvalerate (PHV).
In the embodiment, a gas chromatograph is adopted to analyze a methyl esterification product sample to determine the content of PHA, a DB-WAX model chromatographic column is selected as a stationary phase, an inert gas helium is used as a mobile phase, the sample injection amount is 1 mu L, the sample injection temperature is 250 ℃, and the flow rate is 0.7 mL/min. PHA synthesized by strain MN36-4 was qualitatively analyzed using analytically pure grades of poly-3-hydroxybutyrate (PHB) and Polyhydroxyvalerate (PHV) as standards, with methyl benzoate as internal standard and quantitatively analyzed by the internal standard method. Weighing PHA products with certain gradient mass, performing methyl esterification pretreatment, after gas phase analysis, reading the ratio of the PHA monomer peak area/internal standard peak area and the data of the monomer mass/internal standard substance mass to make a standard curve, wherein the standard curve is used for quantitatively analyzing the PHA content in stem cells.
4. Measurement results
The results of this example on the measurement of PHA-producing ability of Bacillus firmus MN36-4 are shown in FIG. 6, and it can be seen from FIG. 6 that when they were fermentatively cultured using a Nutrient Broth (NB), an inorganic salt medium (M1) containing glucose as a single carbon source, and an inorganic salt medium (M2) containing sodium pyruvate as a single carbon source, the strains synthesized two different kinds of PHA: poly 3-hydroxybutyrate (PHB) and Polyhydroxyvalerate (PHV).
After 4 days of culture in Nutrient Broth (NB), the PHA yield reached 0.84g/L, with a relative PHB content of 92.4% and a relative PHV content of 7.6%.
After 4 days of culture in an inorganic salt culture medium (M2) with sodium pyruvate as a single carbon source, the PHA yield reaches 0.30g/L, wherein the relative proportion of PHB is 51.2 percent, and the relative proportion of PHV is 48.8 percent.
After the strain MN36-4 is cultured for 4 days in an inorganic salt culture medium (M1) taking glucose as a single carbon source, the PHA production capacity is the strongest, the yield reaches 1.71g/L, wherein the relative ratio of PHB is 97.1%, and the relative ratio of PHV is 2.9%, so that the strain has a good industrial application prospect.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
<110> Shenzhen institute of Beijing university
<120> Bacillus firmus and application thereof
<130> KHP211115302.6
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cagcggcgga cgggtgagta acacgtgggc aacctgcctg taagactggg ataactccgg 120
gaaaccgggg ctaataccgg ataactcttt tcctcacatg aggaaaagct gaaagatggt 180
ttcggctatc acttacagat gggcccgcgg cgcattagct agttggtgag gtaacggctc 240
accaaggcca cgatgcgtag ccgacctgag agggtgatcg gccacactgg gactgagaca 300
cggcccagac tcctacggga ggcagcagta gggaatcttc cgcaatggac gaaagtctga 360
cggagcaacg ccgcgtgagt gatgaaggtt ttcggatcgt aaaactctgt tgtcagggaa 420
gaacaagtac cggagtaact gccggtacct tgacggtacc tgaccagaaa gccacggcta 480
actacgtgcc agcagccgcg gtaatacgta ggtggcaagc gttgtccgga attattgggc 540
gtaaagcgcg cgcaggcggt tccttaagtc tgatgtgaaa gcccccggct caaccgggga 600
gggtcattgg aaactgggga acttgagtgc agaagagaag agtggaattc cacgtgtagc 660
ggtgaaatgc gtagagatgt ggaggaacac cagtggcgaa ggcgactctt tggtctgtaa 720
ctgacgctga ggcgcgaaag cgtggggagc aaacaggatt agataccctg gtagtccacg 780
ccgtaaacga tgagtgctaa gtgttagagg gtttccgccc tttagtgctg cagcaaacgc 840
attaagcact ccgcctgggg agtacggccg caaggctgaa actcaaagga attgacgggg 900
gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa ccttaccagg 960
tcttgacatc tcctgacaac cctagagata gggcgttccc cttcggggga caggatgaca 1020
ggtggtgcat ggttgtcgtc agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag 1080
cgcaaccctt gatcttagtt gccagcattc agttgggcac tctaaggtga ctgccggtga 1140
caaaccggag gaaggtgggg atgacgtcaa atcatcatgc cccttatgac ctgggctaca 1200
cacgtgctac aatggatggt acaaagggct gcaagaccgc gaggttaagc gaatcccata 1260
aaaccattct cagttcggat tgcaggctgc aactcgcctg catgaagccg gaatcgctag 1320
taatcgcgga tcagcatgcc gcggtgaata cgttcccggg ccttgtacac accgcccgtc 1380
acaccacgag agtttgtaac acccgaagtc ggtggggtaa ccttttggag ccagccgcta 1440
agggacagga gt 1452
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tcgcaatatg atcacggcta 20

Claims (10)

1. The bacillus firmus (Cytobacillus firmus) MN36-4 is characterized in that the preservation number of the bacillus firmus (Cytobacillus firmus) MN36-4 is CGMCC No. 22298.
2. The Bacillus firmus (Cytobacillus firmus) MN36-4 of claim 1, wherein the sequence of 16s rDNA of the Bacillus firmus (Cytobacillus firmus) MN36-4 is shown in SEQ ID No. 1.
3. A microbial preparation comprising the Bacillus firmus (Cytobacillus firmus) MN36-4 or a fermentation liquid thereof according to claim 1 or 2.
4. The microbial inoculum of claim 3, wherein the concentration of PHA in the microbial inoculum is more than 1.71 g/L.
5. A method of producing PHA, comprising:
the use of the Bacillus firmus (Cytobacillus firmus) MN36-4 of claim 1 or 2 or the microbial agent of claim 3 or 4 for fermentation culture.
6. The method of claim 5, wherein the fermentation culture is:
culturing for 3-5 days at the temperature of 25-35 ℃ and at the speed of 150-200 r/min.
7. The method according to claim 5 or 6, wherein the medium used in the fermentation culture is one or more of a nutrient broth medium, an inorganic salt medium with glucose as a single carbon source, or an inorganic salt medium with sodium pyruvate as a single carbon source.
8. Use of the Bacillus firmus (Cytobacillus firmus) MN36-4 of claim 1 or 2 or the microbial agent of claim 3 or 4 for the production of PHA.
9. The use as claimed in claim 8, wherein the PHA comprises poly-3-hydroxybutyrate and/or polyhydroxyvalerate.
10. Use of the Bacillus firmus (Cytobacillus firmus) MN36-4 of claim 1 or 2 or the microbial agent of claim 3 or 4 for producing packaging materials, adhesive materials or spray materials.
CN202110714782.5A 2021-06-26 2021-06-26 Bacillus firmus and application thereof Active CN113502242B (en)

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