CN116574645A - Bacillus cereus MG1 and application thereof - Google Patents
Bacillus cereus MG1 and application thereof Download PDFInfo
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- CN116574645A CN116574645A CN202310499735.2A CN202310499735A CN116574645A CN 116574645 A CN116574645 A CN 116574645A CN 202310499735 A CN202310499735 A CN 202310499735A CN 116574645 A CN116574645 A CN 116574645A
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- 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
- C12N1/205—Bacterial isolates
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- 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
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- 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
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/085—Bacillus cereus
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Abstract
The invention provides Bacillus cereus MG1 and application thereof, belongs to the technical field of microorganism application, and particularly provides Bacillus cereus MG1 which is preserved in China center for type culture collection (China center for type culture collection) at 5-13 days of 2022, wherein the preservation address is as follows: eight paths of Lopa nationality university of mountain and Wuhan in Wuchang district of Wuhan, hubei province have a preservation number of CCTCC NO: m2022620; the bacillus cereus MG1 provided by the invention can only use sugar residues as nutrients to produce PHA, has a certain salt tolerance, can treat industrial wastes with high salt content, such as sugar residues and the like, is used for producing high value-added compound PHA, and avoids resource waste and environmental pollution caused by improper treatment of industrial wastes.
Description
Technical Field
The invention belongs to the technical field of microorganism application, and particularly relates to bacillus cereus MG1 and application thereof.
Background
With the development of society and the improvement of living standard, conventional plastics such as Polyethylene (PE) produced from non-renewable fossil resources have been widely used in various aspects of national production and living, but the large-scale use of plastics causes serious "white pollution" problems. PHA is a linear polyester composed of 3-hydroxy fatty acid in microorganisms, can be compared with traditional thermoplastics in certain properties, has unique biodegradability, biocompatibility and optical isomerism, improves the stress resistance of microorganisms and the like, and is considered as a green plastic with the most development potential. In nature, many microorganisms have been demonstrated to synthesize Polyhydroxyalkanoates (PHA) and store them in the body. Although PHAs have advantages over conventional plastics in terms of biodegradation and biocompatibility, PHAs are produced at a higher cost than conventional plastics, limiting the commercial production and use of PHAs. At present, commercial production prices of PHA are as high as 2.2-5.0 Euro per kilogram, while the price of polypropylene plastics is only 1.0 Euro per kilogram. In order to reduce the cost of PHA production, researchers have sought to explore new fermentation modes (mixed bacteria fermentation, extreme microbial fermentation) to reduce the cost of fermentation feedstock and fermentation process. Meanwhile, genetic engineering and metabolic engineering techniques are also used to improve the PHAs synthesis ability and accumulation efficiency of bacteria to reduce production costs.
Most of PHA synthetic strains reported at present are gram negative bacteria, glucose, fructose and the like are used as substrates, and PHA accumulation amount can reach 50% -80% of cell dry weight. However, there are certain drawbacks to using gram-negative bacteria to produce PHA. Gram negative bacteria contain Lipopolysaccharide (LPS), thus interfering with the purification of PHA, increasing the complexity and cost of the extraction process; secondly, LPS produced by gram-negative bacteria can induce a strong immune response in humans, so PHA containing LPS cannot be applied to the field of biological medicine. In contrast, the use of gram-positive bacteria represented by bacillus for producing PHA has the advantages of wide sources of raw materials, strong stress resistance, simple extraction process and the like, but no report on the use of sugar residues as nutrients for producing PHA is known, especially the use of waste residues (i.e. sugar residues) generated by saccharification reaction of corn starch as nutrients in the production of citric acid is known, and no report on the use of sugar residues for producing PHA is known.
Chinese patent document CN102119220a (application No. 200880130671.2) discloses the preparation of PHA (polyhydroxyalkanoate) using lemon residue, which discloses the use of citrus processing residue (named lemon residue) to obtain a squeeze liquid and/or lemon molasses by grinding, treating with calcium oxide and squeezing the lemon residue; and adding trace element nutrient solution, and then culturing and producing PHA by using microorganisms.
In the prior art, when citric acid is produced, waste residues (namely sugar residues) generated by saccharification reaction of corn starch are used for selling feed, and some of the waste residues are stacked randomly without being treated, so that certain pollution to the environment and resource waste are caused due to improper treatment. Accordingly, there is also an urgent need for research on converting waste residues into high value-added compounds.
Because the total content of saccharides exceeds 200g/kg, but the protein content is low and is lower than 0.05g/kg, the carbon-nitrogen ratio of the sugar slag is far higher than that of microbial cells, and only the sugar slag is used as a nutrient component for culturing microbial strains, so that the growth and metabolism of the microbial strains are greatly influenced, and some strains possibly cannot grow normally and the generation of metabolic substances is also influenced.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides bacillus cereus MG1 and application thereof.
The sugar slag refers to waste slag generated after saccharification reaction of corn starch by a double-enzyme method.
The technical scheme of the invention is as follows:
bacillus cereus MG1 deposited at the China center for type culture Collection, accession number: eight paths of Lopa nationality university of mountain and Wuhan in Wuchang district of Wuhan, hubei province have a preservation number of CCTCC NO: m2022620.
The culture method of Bacillus cereus MG1 comprises the following steps:
(1) Inoculating bacillus cereus MG1 on a solid culture medium, and performing activation culture at 30-37 ℃ for 12-14h to obtain activated thalli;
(2) Inoculating the activated thallus into liquid culture medium, culturing at 30-37deg.C and 180-200rpm for 12-14 hr to obtain seed solution;
(3) Inoculating 1-3% of the prepared seed liquid into a liquid culture medium according to the volume percentage, and culturing for 12-24 hours at the temperature of 30-37 ℃ and the speed of 180-200rpm to obtain bacillus cereus MG1 bacterial liquid.
According to a preferred embodiment of the present invention, in step (1), the culture is activated at 37℃for 12 hours.
According to the present invention, in the step (2), the culture is preferably carried out at 37℃and 200rpm for 12 hours.
According to the present invention, in the step (3), the culture is preferably carried out at 37℃and 200rpm for 24 hours.
According to the present invention, preferably, the solid medium in the step (1) is LB solid medium; the liquid culture medium in the step (2) and the step (3) is LB liquid culture medium.
The use of the above Bacillus cereus MG1 for the production of PHA.
According to a preferred aspect of the invention, the use of the above Bacillus cereus MG1 for the production of PHA uses sugar residues.
Further preferably, the sugar slag is waste slag generated after saccharification reaction of corn starch by a double-enzyme method.
The beneficial effects of the invention are as follows:
1. bacillus cereus MG1 has the characteristics of easy culture, rapid growth and simple requirement on environmental nutrition.
2. Bacillus cereus MG1 can produce PHA from sugar residue, has cheap raw materials, can obtain 17.5MG/L PHA at a sugar residue concentration of 40g/L, can obtain 25.5MG/L PHA by additionally using glucose as a carbon source, and can obtain 27.5MG/L PHA by additionally using glycerol as a carbon source.
Drawings
FIG. 1 is a gram stain of Bacillus cereus MG1.
FIG. 2 is a diagram of the evolved tree of Bacillus cereus MG1 system.
FIG. 3 is a graph showing the effect of different concentrations of carbon sources on PHA production;
in the figure: a is the effect of different concentrations of glucose on PHA production; b is the effect of glycerol at various concentrations on PHA production.
FIG. 4 is a chart showing the hydrogen nuclear magnetic resonance spectrum of Bacillus cereus MG1 fermented with different carbon sources to yield PHA;
in the figure: a is nuclear magnetic resonance hydrogen spectrum of PHA obtained by fermenting bacillus cereus MG1 with glucose; b is nuclear magnetic resonance hydrogen spectrum of PHA obtained by fermenting bacillus cereus MG1 with glycerol.
FIG. 5 is a graph showing PHA production by Bacillus cereus MG1 at various salt concentrations.
Detailed Description
The technical scheme of the present invention is further described in conjunction with the following examples, but the scope of the present invention is not limited thereto.
The experimental methods in the following examples are all conventional methods unless otherwise specified; the test materials used in the examples described below, unless otherwise specified, were all conventional commercial biochemical reagents.
Source of main materials
The sugar residue used in the invention is derived from the solar Jin Hebo source biochemical company; when the sugar slag is used for producing citric acid, the waste slag is produced after the saccharification reaction of corn starch by a double-enzyme method.
Example 1
The bacillus cereus MG1 provided by the invention is screened from activated sludge, and specific strains are identified as follows:
(1) Gram staining
As a result of gram staining of Bacillus cereus MG1, the size of Bacillus cereus is about 1.0-1.4X13.0-5.0 μm, and gram positive bacteria is facultative aerobic bacteria, which can form spores in vivo, and the cells of the bacteria are rod-shaped with flat two ends and arranged in short or long chain.
(2) Identification of species
Taking genome extracted from bacillus cereus MG1 as a template, and 27F, a nucleotide sequence shown as SEQ ID NO.2 and 1492R, and a nucleotide sequence shown as SEQ ID NO. 3; as primers, 16S rDNA sequences were obtained by PCR. The PCR reaction was as follows (50. Mu.L) and shown in Table 1.
AGAGTTTGATCCTGGCTCAG SEQ ID NO.2；
TACGGCTACCTTGTTACG ACTT SEQ ID NO.3。
TABLE 1 PCR reaction System
The PCR program parameters were set as follows:
the PCR product is recovered by using an Omega gel recovery kit, is connected with a T vector, is sent to Qingdao biological technology Co., ltd. For sequencing, and the 16S rDNA sequence contains 1540bp, and the nucleotide sequence is shown as SEQ ID NO. 1.
BLAST (website: http:// BLAST. Ncbi. Nlm. Nih. Gov/BLAST. Cgi) alignment of the sequence was performed in a GeenBank database, and a phylogenetic tree thereof was constructed, as shown in FIG. 2, and the result showed that the bacterium belongs to Bacillus cereus and is named Bacillus cereus MG1.
Bacillus cereus MG1 deposited at the China center for type culture Collection, accession number: eight paths of Lopa nationality university of mountain and Wuhan in Wuchang district of Wuhan, hubei province have a preservation number of CCTCC NO: m2022620.
Example 2
The capacity of bacillus cereus MG1 for producing PHA by utilizing sugar residues with different concentrations is as follows:
streaking bacillus cereus MG1 on an LB solid medium, and culturing for 12 hours at 37 ℃ in an inverted way to obtain an activated strain; inoculating the activated strain into LB liquid medium, shake culturing at 37deg.C and 200rpm for 12 hr to obtain seed solution; inoculating the prepared seed liquid into a sugar residue culture medium according to the inoculum size with the volume fraction of 1%, and fermenting at 37 ℃ and 200rpm for 24 hours, wherein the sugar residue concentration is 20g/L, 40g/L, 50g/L, 100g/L and 150g/L respectively. After fermentation, standing for 5min, collecting supernatant, centrifuging at 10000rpm for 10min, collecting centrifuged thallus, and freezing thallus at-80deg.C for more than 12 hr.
LB liquid medium: 10g of peptone, 5g of yeast powder and 10g of sodium chloride, and the pH is natural after the volume is fixed to 1L.
LB solid medium: 10g of peptone, 5g of yeast powder, 10g of sodium chloride, 20g of agar powder, and naturally pH, and keeping the volume to 1L.
Sugar residue culture medium: sugar residues with different concentrations and distilled water, wherein the specific main components of the sugar residues are shown in table 2, and the pH value is 6.5.
TABLE 2 sugar slag specific main ingredients
Freeze-drying the bacterial cells, weighing and grinding, putting 0.05g of each bacterial cell into an esterification pipe, adding 2mL of chloroform, 850 mu L of methanol and 150 mu L of sulfuric acid, carrying out oil bath for 1h, cooling, adding 1mL of distilled water, standing for layering, and sucking the lower layer by a syringe for filtration and collection. The esterified samples were analyzed for PHA production by gas chromatography, and the results are shown in Table 3.
TABLE 3 influence of sugar residues of different concentrations on PHA production by Bacillus cereus MG1
According to the experimental results, the bacillus cereus MG1 can utilize sugar residues to produce PHA, and the influence of sugar residues with different concentrations on the PHA yield is measured, so that the PHA production capacity of the bacillus cereus MG1 is found to be strongest when the sugar residue concentration is 40g/L, and a foundation is laid for subsequently improving the PHA yield.
Example 3
Ability of bacillus cereus MG1 to produce PHA with different carbon sources added
Inoculating single bacterial colony of bacillus cereus MG1 into LB liquid culture medium, culturing at 37deg.C and 200rpm for 12 hr to obtain activated bacterial liquid; inoculating the activated bacterial liquid into 40g/L sugar residue culture medium according to 1% of inoculation amount by volume fraction, and respectively adding glucose (10 g/L,20g/L, 30g/L, 40g/L, 50g/L, 60 g/L) with different concentrations, and performing shake fermentation culture at 37 ℃ for 24h with different concentrations of glycerol (25.2 g/L, 50.4g/L, 75.6g/L, 100.8g/L, 126g/L, 151.2 g/L) at 200 rpm. The effect of these two carbon sources on PHA production by Bacillus cereus MG1 was compared. After fermentation, standing for 5min, centrifuging the supernatant at 10000rpm for 10min, collecting the centrifuged thallus, and freezing the thallus at-80deg.C for more than 12 hr.
Sugar residue culture medium: mixing sugar residues with different concentrations with distilled water, and packaging into 100mL bottles.
Glucose medium: after sterilizing the sugar residue culture medium, glucose with different concentrations is added.
Glycerol medium: after sterilizing the sugar residue culture medium, glucose with different concentrations is added.
Freeze-drying thallus, weighing and grinding, respectively taking 0.05g in an esterification pipe, adding 2mL of chloroform, 850 mu L of methanol and 150 mu L of sulfuric acid, carrying out oil bath for 1h, cooling, adding 1mL of distilled water, standing for layering, and sucking the lower layer by a syringe for filtering and collecting. The esterified samples were analyzed for PHA production by gas chromatography, and the results are shown in FIG. 3.
According to the above experimental results, it was shown that the PHA yield can be significantly improved when glucose or glycerol is added as a carbon source to the medium. Glucose and glycerol, each having a different concentration gradient, were set, and it was found that the PHA yield obtained by fermentation of Bacillus cereus MG1 was highest (27.2 MG/L) when the glucose concentration was 30 g/L; when 25.2g/L glycerol was added to the medium, PHA in the broth reached the highest value (25.5 mg/L) and the variation in PHA production was not particularly pronounced as the glycerol concentration was continued to be increased.
Example 4
Structure for measuring PHA by nuclear magnetic resonance hydrogen spectrum
The experiment is mainly carried out by nuclear magnetic resonance hydrogen spectrum 1 H-NMR) to measure the structure of the polymer, and using electromagnetic waves of a specific frequency to cause energy level transition of hydrogen atoms, namely nuclear resonance, and amplifying the generated magnetic frequency signals by conversion to be reflected in the form of peaks on a spectrogram. Firstly extracting PHA by an organic solvent, weighing 5mg of PHA, adding 1mL of deuterated chloroform for dissolution, and then adding the PHA into a nuclear magnetic resonance tube. 1 Reference solvent Signal CDCl at 7.26ppm in H-NMR spectra 3 As shown in FIG. 4, wherein the peaks at 0.89ppm,1.26 to 1.28ppm represent methyl groups of 3HV and 3HB, respectively (numerals 9 and 4), a methylene peak of 3HV appears in the vicinity of 1.56ppm (numeral 8), and the diastereoisomeric protons at 2.46 to 2.49ppm and 2.59 to 2.62ppm show two bimodal signals corresponding to the methylene groups of 3HV and 3HB, respectively (numerals 2 and 6), and the methines (-CH, numerals 3 and 7) of 3HV and 3HB show the highest degree of de-screening at 5.13 to 5.18ppm and 5.23 to 5.28ppm, respectively, which demonstrates that the copolymers produced by fermentation of Bacillus cereus MG1 with glucose or glycerol in example 3 are the same structures, both being PHBV.
Example 5
PHA production by Bacillus cereus MG1 in mineral salt Medium
Single colony of bacillus cereus MG1 is selected and inoculated into LB culture medium, and is cultured for 12 hours at 37 ℃ and 200rpm overnight to prepare seed liquid; inoculating the seed solution into an inorganic salt culture medium according to the volume ratio of 3%, and fermenting and culturing at 37 ℃ for 3 days at 200 rpm. Finally, 19.56mg/L PHA can be obtained.
Inorganic salt medium composition：Na 2 HPO 4 9g/L，KH 2 PO 4 1.5g/L，NH 4 Cl 1g/L，MgSO 4 0.2g/L，CaCl 2 0.02g/L, 0.0012g/L ferric ammonium citrate, 20g/L glucose, and 100 mu L of trace element solution are added.
Trace element solution components: znSO (ZnSO) 4 1g/L，MnCl 2 0.3g/L，H 3 BO 3 3g/L，CoCl 2 2g/L，CuCl 2 0.1g/L，NiCl 2 0.2g/L，NaMoO 4 0.3g/L。
Example 6
PHA production by Bacillus cereus MG1 at different salt concentrations
Inoculating 0.1mL bacillus cereus MG1 into LB culture medium, and culturing at 37 ℃ and 200rpm for 12h to obtain activated bacterial liquid; and (3) inoculating the prepared activated bacterial liquid into different LB liquid culture media according to the volume percentage of 1%. The salinity is 1%, 3%, 5%, 7% by mass, respectively, and the salinity is cultivated at 37 ℃ for 24 hours by shaking at 200 rpm. The cells were collected and the PHA content was measured as shown in FIG. 5. The yield of PHA of the bacillus cereus MG1 is increased along with the increase of the salt concentration when the salt concentration is between 1% and 5%, and the bacillus cereus MG1 does not grow any more when the salt concentration is 7%, which indicates that the MG1 has certain salt tolerance property.
Example 7
PHA production by Bacillus cereus MG1 in LB Medium
Inoculating 0.1mL bacillus cereus MG1 into LB culture medium, and culturing at 37 ℃ and 200rpm for 12h to obtain activated bacterial liquid; the prepared activated bacterial liquid is inoculated into different LB liquid culture media according to the volume percentage of 1 percent, namely, the LB liquid culture media is added with glucose with the final concentration of 40g/L, and the culture is carried out for 24 hours by a shaking table at 37 ℃ and 200 rpm. The cells were collected to measure the PHA content, and 0.7g/L of PHA was obtained.
Comparative example 1
Meanwhile, the comparison of Bacillus subtilis 168 produced PHA using sugar residues of different concentrations under the same conditions as in example 2, and the results are shown in Table 4. As a result, the PHA yield obtained by using sugar residues of different concentrations by Bacillus subtilis 168 was significantly lower than that obtained by Bacillus cereus MG1.
TABLE 4 influence of sugar residues of different concentrations on PHA production by Bacillus subtilis 168
As can be seen from the experimental data, the bacillus cereus MG1 provided by the invention can only use sugar residues as nutrients to produce PHA, and when the concentration of the sugar residues is 40g/L, the PHA yield is highest, and meanwhile, the bacillus cereus MG1 provided by the invention has a certain salt tolerance, and when the salt concentration reaches 5%, the PHA yield is highest. The copolymer produced by bacillus cereus MG1 provided by the invention is PHBV; according to the strain characteristics of bacillus cereus MG1, industrial wastes with high salt content, such as sugar residues and the like, can be treated, are used for producing the compound PHA with high added value, and avoid resource waste and environmental pollution caused by improper treatment of the industrial wastes.
Claims (9)
1. Bacillus cereus MG1 deposited at the China center for type culture Collection, accession number: eight paths of Lopa nationality university of mountain and Wuhan in Wuchang district of Wuhan, hubei province have a preservation number of CCTCC NO: m2022620.
2. The method for culturing Bacillus cereus MG1 of claim 1, comprising the steps of:
(1) Inoculating bacillus cereus MG1 on a solid culture medium, and performing activation culture at 30-37 ℃ for 12-14h to obtain activated thalli;
(2) Inoculating the activated thallus into liquid culture medium, culturing at 30-37deg.C and 180-200rpm for 12-14 hr to obtain seed solution;
(3) Inoculating 1-3% of the prepared seed liquid into a liquid culture medium according to the volume percentage, and culturing for 12-24 hours at the temperature of 30-37 ℃ and the speed of 180-200rpm to obtain bacillus cereus MG1 bacterial liquid.
3. The method according to claim 2, wherein in the step (1), the culture is activated at 37℃for 12 hours.
4. The method according to claim 2, wherein in the step (2), the culture is performed at 37℃and 200rpm for 12 hours.
5. The method according to claim 2, wherein in the step (3), the culture is performed at 37℃and 200rpm for 24 hours.
6. The culture method according to claim 2, wherein the solid medium in the step (1) is LB solid medium; the liquid culture medium in the step (2) and the step (3) is LB liquid culture medium.
7. Use of Bacillus cereus MG1 of claim 1 in the production of PHA.
8. The use as claimed in claim 7, characterized in that Bacillus cereus MG1 uses sugar residues in the production of PHA.
9. The use of claim 8, wherein the sugar residue is waste residue produced after a double enzyme corn starch saccharification reaction.
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| CN117286061A (en) * | 2023-09-20 | 2023-12-26 | 河北牧群生物科技有限公司 | Geobacillus and application thereof |
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| CN116904376B (en) * | 2023-09-13 | 2023-11-14 | 广州大学 | High-salt-tolerance bacillus cereus strain, microbial agent and application thereof |
| CN117286061A (en) * | 2023-09-20 | 2023-12-26 | 河北牧群生物科技有限公司 | Geobacillus and application thereof |
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