CN108865950B - Bacillus megaterium and application thereof - Google Patents
Bacillus megaterium and application thereof Download PDFInfo
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- CN108865950B CN108865950B CN201810806567.6A CN201810806567A CN108865950B CN 108865950 B CN108865950 B CN 108865950B CN 201810806567 A CN201810806567 A CN 201810806567A CN 108865950 B CN108865950 B CN 108865950B
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- bacillus megaterium
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- polyhydroxyalkanoate
- xylose
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- C12R2001/07—Bacillus
- C12R2001/11—Bacillus megaterium
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Abstract
本发明公开了一株利用木糖生产聚羟基脂肪酸酯的巨大芽孢杆菌(Bacillus megaterium)B‑10及其应用，该菌株保藏编号CGMCC No.15753。本发明的菌株为利用木糖产聚羟基脂肪酸酯菌株，当培养基中添加3～20g/L木糖时，聚羟基脂肪酸酯的产量可以达到1.906g/L，其转化率高，是一株优良的合成聚羟基脂肪酸酯的菌株。The invention discloses a strain of Bacillus megaterium B-10 that utilizes xylose to produce polyhydroxyalkanoates and its application, and the strain has a deposit number of CGMCC No. 15753. The strain of the present invention utilizes xylose to produce polyhydroxyalkanoate. When 3-20g/L xylose is added to the medium, the yield of polyhydroxyalkanoate can reach 1.906g/L, and the conversion rate is high, which is An excellent strain for synthesizing polyhydroxyalkanoates.
Description
Technical Field
The invention relates to the technical field of microorganisms and production thereof, in particular to bacillus megaterium for producing polyhydroxyalkanoate and application thereof, and particularly provides a strain capable of producing polyhydroxyalkanoate by using xylose and application thereof in production of polyhydroxyalkanoate.
Background
"Plastic" is a non-naturally occurring polymeric material that is currently produced in an annual volume of about 3 hundred million tons and is increasing. Petrochemical plastics are widely used in applications such as packaging materials, parts in the automotive industry, biomedical devices, electronic parts, and the like. While petrochemical plastics provide convenience, serious drawbacks exist, such as continuous depletion of fossil resources, white waste composed of nondegradable all-carbon backbone plastics, and increased levels of carbon dioxide and toxins in the atmosphere caused by burning petrochemical plastics.
In order to overcome the above disadvantages, we have searched for a sustainable development material that can replace petrochemical plastics. Such materials are wholly or partially derived from organisms and are therefore referred to as biobased materials. Moreover, most of the bio-based materials cannot be directly synthesized by animals, but are prepared by providing monomers by organisms and polymerizing the monomers into high polymer materials through chemical polymerization at a later stage. For example, in the production and extraction of lactic acid monomers from corn, polylactic acid is obtained through polymerization. And a bio-based material, Polyhydroxyalkanoates (PHA), which needs to be obtained through microbial synthesis attracts attention, and the renewable resource of the biosynthesis is a synthetic monomer of bio-based plastics, and accordingly, the conversion of plastics based on gasoline into a bio-substitute with plastic properties based on renewable resources is proposed, so that the difficulty in degrading petrochemical plastics is overcome, and the bio-based material is considered to be one of promising strategies.
The polyhydroxyalkanoate is a natural biological polymer material produced by bacteria in response to environmental and nutritional stress conditions, the physiological function of the polyhydroxyalkanoate is firstly used as a carbon source and an energy storage substance in bacteria, and the polyhydroxyalkanoate also has basic characteristics of common polymers, such as thermoplastic or hot workability, and meanwhile, the polyhydroxyalkanoate also has some special material characteristics, such as: nonlinear optical activity, piezoelectricity, gas barrier property, and the like. The polyhydroxyalkanoate is a biomaterial, has good biodegradability and good biocompatibility, and is widely considered as a renewable material for replacing traditional plastics. Because the polyhydroxy fatty acid ester exists as a biological metabolism intermediate, the premise of synthesizing the polyhydroxy fatty acid ester material is to screen and obtain a bacterial strain capable of efficiently synthesizing the polyhydroxy fatty acid ester.
Lignocellulose is the most abundant polysaccharide substance on earth and mainly comprises cellulose, hemicellulose and lignin. After the hemicellulose was degraded, 90% of its hydrolysate was xylose. Xylose is a five-carbon sugar in the hydrolysate of lignocellulosic feedstocks, the most abundant monosaccharide from renewable biomass resources in addition to glucose. If xylose can be efficiently utilized to synthesize polyhydroxyalkanoate through the metabolism of bacteria to realize the resource utilization of lignocellulose, the method is also the technical key for converting lignocellulose into polyhydroxyalkanoate material. Therefore, screening strains that efficiently utilize xylose for conversion to produce polyhydroxyalkanoates is a necessary condition for advancing bioplastics to replace petroleum-based plastics.
Disclosure of Invention
The invention aims to provide a novel bacterium for efficiently producing polyhydroxyalkanoate. The strain is Bacillus megaterium B-10 with the preservation number of CGMCC No. 15753.
The Bacillus megaterium (Bacillus megaterium) is separated from fresh soil collected from the Yuenu mountain of the Yuenu area in Changsha city, Hunan, is named as B-10, and is preserved in the China general microbiological culture Collection center (CGMCC) on 5-11 months in 2018 with the preservation number of CGMCC No. 15753.
The biological characteristics of the strain are as follows: on LB solid plate culture medium, the colony surface is smooth, light yellow in the initial period, and then gradually becomes wax white, the edge is neat, and spores are formed when the colony is arranged singly.
The bacillus megaterium strain is obtained by separating and screening through the following method:
(1) taking a fresh soil sample collected from the Yuenu mountain of the Yuenu area of Changsha city in Hunan province, adding 10mL of sterile water into 5g of fresh soil in the sample to prepare a soil solution; 1mL of soil solution is taken to be put into an ion culture medium prepared by the straw acid treatment waste liquid rich in sugar, the temperature is 30 ℃, and the vibration frequency is 150rpm, and the soil solution is cultured for 36 hours in a shaking incubator.
(2) And (2) dipping the bacterial liquid obtained in the step (1) by using an inoculating loop sterilized by an alcohol lamp, streaking and inoculating on an LB solid culture medium, and culturing for 2d in a constant-temperature incubator at the temperature of 30 ℃. As a result, a wax white colony was found in LB solid medium, which was Bacillus megaterium B-10 proposed in the present invention.
(3) And (3) picking individual wax white colonies from the LB solid medium in the step (2) by using the sterilized inoculating loop, respectively streaking the wax white colonies in the LB solid medium, and culturing the wax white colonies for 2d at the temperature of 35 ℃ to find that a single colony is grown. Culturing the purified single bacterium in an LB liquid culture medium to obtain a bacterium liquid, partially preserving the bacterium liquid in a refrigerator at minus 80 ℃, and partially preserving the bacterium liquid in a refrigerator at 4 ℃ for subsequent experiments.
(4) The 16S rDNA sequence obtained by PCR of the general primer of the above-mentioned Bacillus strain has 99% similarity with the 16SrDNA sequence (MF614908.1) of Bacillus megaterium in the database, but is not completely identical.
The second purpose of the invention is to provide the application of the strain in the production of polyhydroxyalkanoate.
Further, xylose is used to produce polyhydroxyalkanoate.
Furthermore, the culture medium contains 3-20 g/L of xylose when the polyhydroxyalkanoate is produced.
Further, the strain B-10 of the present invention was inoculated into a xylosyl basal medium at an inoculum size of 10%, and polyhydroxyalkanoate was accumulated. The culture medium comprises the following components: 3-20 g/L xylose (NH)4)2SO4 2g/L，K2HPO4 1g/L，MgSO4 0.2g/L，CaCl2 0.01g/L，FeSO4 0.015g/L，MnSO4 0.01g/L，KH2PO41g/L, and the pH value is 7.0-7.4.
The culture conditions for producing the polyhydroxyalkanoate are 30-35 ℃, the rotation speed is 120-.
The bacillus megaterium B-10 provided by the invention is a new strain capable of utilizing xylose to biosynthesize polyhydroxyalkanoate, provides possibility for directly utilizing lignocellulose to degrade products to produce polyhydroxyalkanoate, and provides technical support for the existing biosynthesis technology taking lignocellulose as a raw material and the co-utilization of cellulose and hemicellulose.
Because the main component of the product obtained after the lignocellulose is treated by dilute acid is xylose, the strain can also be used for treating waste liquid after the lignocellulose is treated by acid. The waste of available carbon sources in the waste liquid and the pollution to the environment are avoided, and the method has the advantages of simple operation, mild reaction conditions, high carbon source utilization rate and the like.
Specifically, Bacillus megaterium (Bacillus megaterium) B-10 with the preservation number of CGMCC No.15753 is inoculated into waste liquid obtained by lignocellulose acid method treatment, and the cultured thalli contains polyhydroxyalkanoate.
Because acid hydrolysis of the hemicellulose and cellulose portions of lignocellulose, hemicellulose and cellulose are sugar polymers. Therefore, the main component of the waste liquid obtained by the acid treatment of lignocellulose is a reducing sugar mixture with xylose as the main component.
The acid treatment of the waste liquid requires sterilization.
Preferably: the initial inoculation amount is 5-20% of the volume ratio of the seed liquid to the waste liquid treated by the acid method.
Preferably: the temperature range is 30-35 ℃.
Preferably: the time range is 30-60 h.
Preferably: the rotation speed range is 120-150 rpm.
Preferably: inorganic salt for ensuring the growth of bacteria is added into the waste liquid generated by the acid treatment of lignocellulose.
Further preferably, the inorganic salt is added in a concentration of (NH)4)2SO4 2g/L，K2HPO4 1g/L，MgSO4 0.2g/L，CaCl20.01g/L，FeSO4 0.015g/L，MnSO4 0.01g/L，KH2PO41 g/L. The pH value is 7.0-7.4.
The lignocellulose is derived from one or more of rice straw, corn straw, bamboo and bagasse.
Preferably: the lignocellulose is crushed, cleaned and dried for acid treatment.
Further preferably: pulverizing lignocellulose, sieving with 60 mesh sieve, cleaning with ultrapure water twice, and oven drying at 60 deg.C to constant weight.
The acid used in the acid method treatment comprises the following steps: sulfuric acid, phosphoric acid or hydrochloric acid.
The mass concentration of the sulfuric acid used for the acid method treatment is 0.5-2%. Preferably 0.5-1.5%. Further preferably 0.5 to 1%.
The solid-to-liquid ratio of the lignocellulose to the acid during the acid treatment is 3-8g/100 mL.
Heating is required for the acid treatment. Preferably at 100 ℃ and 121 ℃.
The acid treatment time is at least 15 min. Preferably 15-45 min. Further preferably 15-20 min.
No matter what kind of acid, concentration of the acid, solid-liquid ratio of lignocellulose to the acid, treatment temperature and time are selected in the acid method treatment, the hemicellulose is dissolved out to the maximum extent. However, if the acid concentration is too high and the treatment time is too long, the dissolved sugar may be further converted into a toxic substance such as furfural furan, which may inhibit the metabolism of cells and may cause death, so that the acid type, concentration, solid-to-liquid ratio, treatment temperature and time may be appropriately selected according to actual conditions.
Inoculating Bacillus megaterium (Bacillus megaterium) B-10 with the preservation number of CGMCC No.15753 to the waste liquid obtained by the acid method, culturing, collecting the thallus, and preferably but not limited to extracting polyhydroxyalkanoate in the thallus by a methanol-chloroform method.
Alkaline pretreatment of lignocellulose, which is one of the most widely used pretreatment techniques, works by dissolving and destroying the lignin components. Therefore, the alkaline pretreatment waste liquid mainly contains a large amount of lignin-related products (the main component obtained by treating the waste liquid with an alkaline method is a lignin fragment compound). It has been reported earlier that bacteria such as Pseudomonas putida KT2440 can utilize lignin components in the alkaline process pretreatment waste liquid and accumulate in the bacteria to form a bioplastic precursor, namely polyhydroxyalkanoate, thereby realizing the recycling of the alkaline process pretreatment waste liquid.
However, unlike alkaline pretreatment, dilute acid pretreatment acts mainly on hemicellulose and a small amount of lignin components in lignocellulose, and thus a hemicellulose-rich lysate (mainly xylose), and a small amount of lignin-related products, are present in the dilute acid pretreatment waste stream. If the same bacteria as those used in the alkaline method are used, the amount of the carbon source of lignin that can be utilized is small, and the amount of the accumulated product cannot meet the demand.
If bacteria capable of utilizing dilute acid to treat the obtained hemicellulose product solution are adopted, the related products of lignin can inhibit the bacteria, and the ideal effect is still difficult to achieve. Therefore, at present, no effective method is available for well recycling the dilute acid pretreatment waste liquid.
The occurrence of the strain can well solve the problem of resource utilization of the dilute acid pretreatment waste liquid.
The preservation information of the strain of the invention is as follows:
the preservation unit: china general microbiological culture Collection center
And (4) storage address: microbial research institute of western road 1 institute No. 3 of China academy of sciences, Beijing, Chaoyang
Preservation time: 5 and 11 months in 2018
The preservation number is: CGMCC No. 15753.
And (3) classification and naming: bacillus megaterium (Bacillus megaterium).
Drawings
FIG. 1 is a characteristic diagram of a colony of Bacillus megaterium B-10 on an LB solid medium;
FIG. 2 is a fluorescent microscopic view of polyhydroxyalkanoate;
FIG. 3 is a diagram of a polyhydroxyalkanoate product;
FIG. 4 is a monomer composition characterization chart of polyhydroxyalkanoate;
FIG. 5 is an appearance diagram of a culture medium prepared from a rice straw acid pretreatment waste liquid;
FIG. 6 is a fluorescent microscope photograph of polyhydroxyalkanoates formed after treatment of a lignocellulose acid treatment waste liquid by Bacillus megaterium B-10;
FIG. 7 is a GC-MS spectrum of polyhydroxyalkanoate formed after treatment of a lignocellulose acid treatment waste liquid by Bacillus megaterium B-10;
FIG. 8 is a digital photograph of a white polyhydroxyalkanoate formed after treatment of a lignocellulose acid treatment waste liquid by Bacillus megaterium B-10;
FIG. 9 is a graph B-10 showing growth curves in ionic media containing the lignin model compounds ferulic acid, sinapic acid and p-coumaric acid alone, and in xylose-ionic media with the addition of ferulic acid, sinapic acid and p-coumaric acid.
Detailed Description
The following examples are intended to further illustrate the invention, but are not intended to limit the invention.
Example 1: strain screening
(1) Taking a fresh soil sample collected from feet of Yuenu mountain in Yuenu area of Changsha city in Hunan province, adding 10mL of sterile water into 5g of fresh soil in the sample to prepare a soil solution; 1mL of soil solution is taken to be put into an ion culture medium prepared by 100mL of straw acid treatment solution (containing rich glycogen), the temperature is 30 ℃, and the vibration frequency is 150rpm, and the culture is carried out for 36h in a shaking incubator.
(2) The bacterial liquid is dipped by an inoculating loop sterilized by an alcohol lamp, streaked and inoculated on an LB solid culture medium, and cultured in a constant temperature incubator at the temperature of 30 ℃ for 2 d. As a result, a white and a waxy white colony were found in LB solid medium, and the yellow colony was Bacillus megaterium B-10 proposed in the present invention.
(3) Wax white independent colonies were picked from the LB solid medium in step (2) with a sterilized inoculating loop, streaked on 2 LB solid media, and cultured at 35 ℃ for 2d, and a single colony was found to grow. Culturing the purified single bacterium in an LB liquid culture medium. The obtained inoculum was partially stored in a refrigerator at-80 deg.C, and partially stored in a refrigerator at 4 deg.C for subsequent experiments.
(4) Liquid culture medium for producing polyhydroxyalkanoate: 3-20 g/L xylose (NH)4)2SO4 2g/L，K2HPO4 1g/L，MgSO4 0.2g/L，CaCl2 0.01g/L，FeSO4 0.015g/L，MnSO4 0.01g/L，KH2PO41g/L, and the pH value is 7.0-7.4.
Example 2: strain identification
(1) Culturing bacillus megaterium in an LB culture medium, wherein the LB culture medium comprises the following components: 10g/L of tryptone and 5g/L, Nacl 5g/L of yeast extract.
(2) And (3) taking the cultured bacillus megaterium seed solution for 16s rDNA identification.
(3) The 16s rDNA identification method in the step (2) comprises the following steps: extraction of genomic DNA, and growth of the cells in LB liquid medium (about 12 to 24 hours). 1 mul of bacterial liquid is taken to carry out PCR gene amplification, 0.5 mul of PCR template DNA is added to adjust to proper concentration, the concentration of a primer is 0.4 mul, the concentration of dNTP is 0.2mM, DNA polymerase is about 2.5U, the total volume of the reaction is 50 mul, the denaturation temperature is 94 ℃, the annealing temperature is 55 ℃, the extension temperature is 72 ℃, and 30 cycles are total. The PCR product was about 1.5kb, and then DNA was electrophoresed by 1% agarose gel electrophoresis. And (3) carrying out agarose gel electrophoresis, ethidium bromide staining and ultraviolet analyzer detection, and sending the qualified DNA to a gene platform for sequencing. The 16S rDNA sequence of the strain enterobacter is subjected to homologous comparison with the 16S rDNA sequence registered in GenBank (gb: MF614908.1), and the 16S rDNA sequence is compared with the 16S rDNA of several strains of the bacillus megaterium, so that the similarity is up to 99 percent but is not completely the same. In combination with morphological observation of the bacterium: the characteristics of the strain in LB plate culture and colony are that the surface is smooth, the strain is white and yellow, spores are produced, and the strain belongs to the genus of Bacillus megaterium (figure 1).
(4) Wherein the TAE adopted in the step (3): 50-fold tris-acetate-EDTA (2M tris-acetate, 0.05M EDTA, pH 8.3.) agarose electrophoresis gel: 0.6g agarose, 1.2ml TAE buffer, 59ml H2O。
Example 3: production of polyhydroxy fatty acid ester by bacillus megaterium using xylose
(1) Inoculating the bacillus megaterium stored on the LB inclined plane into an LB liquid culture medium, and culturing at the temperature of 30 ℃ for 18h to obtain a seed solution of the bacillus megaterium; wherein the LB liquid culture medium comprises the following components in percentage by weight: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 1L of distilled water; the LB inclined plane is formed by adding 15g/L agar on the basis of the formula;
(2) centrifuging the bacillus megaterium seed solution obtained in the last step for 5 minutes at 12000rpm, removing supernatant, and collecting thalli;
(3) inoculating the collected bacillus megaterium thallus into a xylose sterile culture medium according to the inoculation amount of the bacterium liquid accounting for 20 percent (volume ratio) of the culture medium before centrifugation, culturing at 30 ℃ and 150rpm for 48 hours, and collecting the thallus. Wherein the xylose sterile culture medium comprises the following components: xylose 5g/L, (NH)4)2SO4 2g/L，K2HPO4 1g/L，MgSO4 0.2g/L，CaCl2 0.01g/L，FeSO4 0.015g/L，MnSO40.01g/L，KH2PO 41 g/L, and pH value of 7.0-7.4.
By the practice of this example, 2mL of sample was collected, centrifuged at 12000rpm for 5min, the supernatant was discarded, and the cells were resuspended in 150. mu.L of deionized water and 50. mu.L of dimethyl sulfoxide. mu.L of nile red dye (0.15mg/mL) was added to the suspension and stained for 30 min. Then, the formation of inclusion bodies of polyhydroxyalkanoate in the bacterial cells was confirmed by observation with a fluorescence microscope, as shown in FIG. 2. And 0.883g/L of polyhydroxyalkanoate (FIG. 3) was obtained, and the conversion rate of polyhydroxyalkanoate obtained by this example has a significant advantage.
Example 4: production of polyhydroxy fatty acid ester by bacillus megaterium using xylose
(1) Inoculating the bacillus megaterium stored on the LB inclined plane into an LB liquid culture medium, and culturing at the temperature of 30 ℃ for 18h to obtain a seed solution of the bacillus megaterium; wherein the LB liquid culture medium comprises the following components in percentage by weight: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 1L of distilled water; the LB inclined plane is formed by adding 15g/L agar on the basis of the formula;
(2) centrifuging the bacillus megaterium seed solution obtained in the last step for 5 minutes at 12000rpm, removing supernatant, and collecting thalli;
(3) inoculating the collected bacillus megaterium thallus into a xylose sterile culture medium according to the inoculation amount of the bacterium liquid accounting for 20 percent (volume ratio) of the culture medium before centrifugation, culturing at 35 ℃ and 120rpm for 60 hours, and collecting the thallus. Wherein the xylose sterile culture medium comprises the following components: xylose 10g/L, (NH)4)2SO4 2g/L，K2HPO4 1g/L，MgSO4 0.2g/L，CaCl2 0.01g/L，FeSO4 0.015g/L，MnSO40.01g/L，KH2PO 41 g/L, and pH value of 7.0-7.4.
The polyhydroxy fatty acid ester conversion rate obtained by the example of 1.333g/L of the polyhydroxy fatty acid ester obtained by the example has obvious advantage.
Example 5: production of polyhydroxy fatty acid ester by bacillus megaterium using xylose
(1) Inoculating the bacillus megaterium stored on the LB inclined plane into an LB liquid culture medium, and culturing at the temperature of 30 ℃ for 18h to obtain a seed solution of the bacillus megaterium; wherein the LB liquid culture medium comprises the following components in percentage by weight: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 1L of distilled water; the LB inclined plane is formed by adding 15g/L agar on the basis of the formula;
(2) centrifuging the bacillus megaterium seed solution obtained in the last step for 5 minutes at 12000rpm, removing supernatant, and collecting thalli;
(3) inoculating the collected bacillus megaterium thallus into a xylose sterile culture medium according to the inoculation amount of the bacterium liquid accounting for 20 percent (volume ratio) of the culture medium before centrifugation, culturing at 30 ℃ and 150rpm for 60 hours, and collecting the thallus. Wherein the xylose sterile culture medium comprises the following components: xylose 15g/L, (NH)4)2SO4 2g/L，K2HPO4 1g/L，MgSO4 0.2g/L，CaCl2 0.01g/L，FeSO4 0.015g/L，MnSO40.01g/L，KH2PO 41 g/L, and pH value of 7.0-7.4.
By the implementation of the embodiment, the obtained polyhydroxyalkanoate is 1.906mg/L, and the conversion rate of the polyhydroxyalkanoate obtained by the embodiment has obvious advantages.
Example 6: polyhydroxyalkanoate identification
(1) Selecting single colony, inoculating into the above 50ml culture medium of example 3, shaking 250ml flask, culturing at 30-35 deg.C for about 36 hr, collecting 100ml bacterial solution, centrifuging at 12000rpm at 4 deg.C for 10min, discarding supernatant, precipitating bacterial solution in N2Rapidly cooling and storing in-80 deg.C refrigerator for one night, and freeze drying.
(2) The lyophilized cells were collected in a screw-top bottle and 1.7mL of methanol, 0.3mL of 98% concentrated H2SO4After slight shaking, 2mL of chloroform was added, the screw bottle was tightened, vortexed for 30 seconds, shaken for 3-10 minutes, and pyrolyzed at 100 ℃ for 140 minutes.
(3) After cooling the sample to room temperature, 1mL of deionized water was added, vortexed and shaken for 5-10 minutes, centrifuged at 3000rpm for 5 minutes, and the aqueous phase and the organic phase were separated. The lower organic phase was extracted through a 0.45 μm organic filter and subjected to GC-MS detection.
(4) The GC-MS detection method in the step (3) comprises the following steps: the column box temperature 50 ℃, the injection port temperature 275 ℃, the hold time 20 minutes, the total run time 30 minutes, the detector temperature 250 ℃, and the vaporization chamber temperature 240 ℃ were used with GC QP2010MS (Shimadzu, Kyoto, Japan) and HP-5MS UI columns (30m, 0.32mm ID, 0.25 μm df). The identified products are 3-hydroxybutyric acid and 2-hydroxybutyric acid (which are monomers constituting polyhydroxyalkanoate), and the gas phase map and the mass spectrum map are shown in FIG. 4.
Example 7 production of polyhydroxyalkanoates by Bacillus megaterium Using diluted Lignocellulosic acid treatment solution
(1) Crushing rice straws, sieving by a 60-mesh sieve, cleaning twice by ultrapure water, and drying at 60 ℃ to constant weight.
(2) Adding rice straw into 0.5% dilute sulfuric acid solution according to solid-to-liquid ratio of 5g/100mL, standing at 121 ℃ for 40min, filtering and separating to obtain acid treatment waste liquid.
(3) Inoculating the bacillus megaterium B-10 stored on the LB inclined plane into an LB liquid culture medium, and culturing at the temperature of 30 ℃ for 18h to obtain a seed solution of the bacillus megaterium; wherein the LB liquid culture medium comprises the following components in percentage by weight: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 1L of distilled water; the LB inclined plane is formed by adding 15g/L agar on the basis of the formula;
(4) centrifuging the bacillus megaterium seed solution obtained in the last step for 5 minutes at 12000rpm, removing supernatant, and collecting thalli;
(5) inoculating the collected bacillus megaterium thallus into a sterile culture medium containing acid-treated waste liquid according to the inoculation amount of the bacteria liquid accounting for 10 percent (volume ratio) of the culture medium before centrifugation, culturing for 48h at 30 ℃, and collecting the thallus. Wherein the sterile culture medium containing the acid-treated waste liquid is prepared by adding inorganic salt into the acid-treated waste liquid, wherein the type and concentration of the inorganic salt are (NH)4)2SO4 2g/L，K2HPO4 1g/L，MgSO4 0.2g/L，CaCl2 0.01g/L，FeSO4 0.015g/L，MnSO4 0.01g/L，KH2PO41g/L, and the pH value is 7.0-7.4.
(6) And extracting the polyhydroxy fatty acid ester accumulated in the thallus by a methanol-chloroform method from the collected thallus.
By the implementation of the embodiment, the removal rate of the total sugar in the acid treatment waste liquid reaches 59.59%. 2mL of the sample was collected, centrifuged at 12000rpm for 5min, the supernatant was discarded, and the cells were resuspended in 150. mu.L of deionized water and 50. mu.L of dimethyl sulfoxide. mu.L of nile red dye (0.15mg/mL) was added to the suspension and stained for 30 min. Then, the observation was carried out by a fluorescence microscope, and as a result, as shown in FIG. 6, it was confirmed that inclusion bodies of polyhydroxyalkanoate were formed in the bacterial cells. The polyhydroxy fatty acid ester accumulated in the bacteria is extracted by a methanol-chloroform method to obtain 1.493g/L of polyhydroxy fatty acid ester (shown in figure 7), and the polyhydroxy fatty acid ester is determined by a gas chromatography-mass spectrometer, and is a copolymer of 3-hydroxybutyric acid (3HB) and 2-hydroxybutyric acid (2 HB).
The methanol-chloroform method is specifically operated as follows: and (3) taking the bacterial liquid into a centrifugal tube, centrifuging at the low temperature of 4 ℃ for 10 minutes at 1200rpm, taking out supernatant, keeping the thalli, putting the thalli into a refrigerator at the temperature of-80 ℃, freeze-drying for 24 hours after cooling, taking out dried powder, adding 1mL of trichloromethane, adding trichloromethane to 7mL after vigorous stirring, putting the mixture into a shaking table at the temperature of 60 ℃ and at the speed of 150rpm overnight. After overnight, 2mL of water was added and mixed for 5min, and the mixture was centrifuged at 2500rpm to separate layers. Taking out the lower organic phase after layering and filtering the lower organic phase with a 0.45 mu m filter membrane to obtain N2Adding precooled methanol with the volume of 10 times that of the mixture into 1mL, mixing for 30min to dissolve out the polyhydroxyalkanoate, centrifuging and drying to obtain the polyhydroxyalkanoate pure substance.
Example 8 production of polyhydroxyalkanoates by Bacillus megaterium Using diluted Lignocellulosic acid treatment solution
(1) Crushing rice straws, sieving by a 60-mesh sieve, cleaning twice by ultrapure water, and drying at 60 ℃ to constant weight.
(2) Adding rice straw into 0.5% dilute sulfuric acid solution according to solid-to-liquid ratio of 5g/100mL, standing at 121 deg.C for 20min, filtering, and separating to obtain acid treatment waste liquid.
(3) Inoculating the bacillus megaterium B-10 stored on the LB inclined plane into an LB liquid culture medium, and culturing at the temperature of 30 ℃ for 18h to obtain a seed solution of the bacillus megaterium; wherein the LB liquid culture medium comprises the following components in percentage by weight: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 1L of distilled water; the LB inclined plane is formed by adding 15g/L agar on the basis of the formula;
(4) centrifuging the bacillus megaterium seed solution obtained in the last step for 5 minutes at 12000rpm, removing supernatant, and collecting thalli;
(5) inoculating the collected bacillus megaterium thallus into a sterile culture medium containing acid-treated waste liquid according to the inoculation amount of the bacterium liquid accounting for 20 percent (volume ratio) of the culture medium before centrifugation, culturing for 36h at 30 ℃, and collecting the thallus. Wherein the sterile culture medium containing the acid-treated waste liquid is prepared by adding inorganic salt into the acid-treated waste liquid, wherein the inorganic salt is added in a concentration of (NH)4)2SO4 2g/L，K2HPO4 1g/L，MgSO4 0.2g/L，CaCl2 0.01g/L，FeSO4 0.015g/L，MnSO4 0.01g/L，KH2PO41g/L, and the pH value is 7.0-7.4.
(6) And extracting the polyhydroxy fatty acid ester accumulated in the thallus by a methanol-chloroform method from the collected thallus.
By the implementation of the embodiment, the removal rate of the total sugar in the acid treatment waste liquid reaches 61.62%. The obtained polyhydroxyalkanoate 1.395g/L has obvious advantage in the conversion rate of the polyhydroxyalkanoate obtained by the example.
Example 9 production of polyhydroxyalkanoates by Bacillus megaterium Using diluted Lignocellulosic acid treatment solution
(1) Crushing rice straws, sieving by a 60-mesh sieve, cleaning twice by ultrapure water, and drying at 60 ℃ to constant weight.
(2) Adding rice straws into a 1% dilute sulfuric acid solution according to the solid-to-liquid ratio of 3g/100mL, standing in a constant temperature environment of 121 ℃ for 20min, and filtering and separating to obtain acid treatment waste liquid.
(3) Inoculating the bacillus megaterium B-10 stored on the LB inclined plane into an LB liquid culture medium, and culturing at the temperature of 30 ℃ for 18h to obtain a seed solution of the bacillus megaterium; wherein the LB liquid culture medium comprises the following components in percentage by weight: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 1L of distilled water; the LB inclined plane is formed by adding 15g/L agar on the basis of the formula;
(4) centrifuging the bacillus megaterium seed solution obtained in the last step for 5 minutes at 12000rpm, removing supernatant, and collecting thalli;
(5) inoculating the collected bacillus megaterium to an acid-containing place according to the inoculation amount of the bacterial liquid accounting for 20 percent (volume ratio) of the culture medium before centrifugationCulturing in sterile culture medium at 30 deg.C for 48 hr, and collecting thallus. Wherein the sterile culture medium containing the acid-treated waste liquid is prepared by adding inorganic salt into the acid-treated waste liquid, wherein the inorganic salt is added in a concentration of (NH)4)2SO4 2g/L，K2HPO4 1g/L，MgSO4 0.2g/L，CaCl2 0.01g/L，FeSO4 0.015g/L，MnSO4 0.01g/L，KH2PO41g/L, and the pH value is 7.0-7.4.
(6) And extracting the polyhydroxy fatty acid ester accumulated in the thallus by a methanol-chloroform method from the collected thallus.
By the practice of this example, the removal rate of total sugars in the acid-treated waste liquid reached 57.32%, and 0.933g/L of polyhydroxyalkanoate was obtained. The solid-liquid ratio, the time and the acid concentration of the acid treatment all influence the dissolution of the hemicellulose, the solid-liquid ratio of the embodiment is small, and similarly, the dissolved sugar is less because the 100ml acid liquor is only 3g of biomass, and the yield is correspondingly reduced compared with other embodiments.
Example 10: model Compound culture experiment
(1) And (3) picking a single colony from a solid culture medium by using an inoculating loop to a 20mL LB culture medium for activation, inoculating the activated B-10 into a 100mLLB culture medium, putting the culture medium into a shaking table at the temperature of 30 ℃ and the rotating speed of 120rpm for culturing, and waiting for a subsequent experiment for use.
(2) Preparing a xylose-ion culture medium: xylose 0.2g/L, (NH)4)2SO4 2g/L，K2HPO4 1g/L，MgSO40.2g/L，CaCl2 0.01g/L，FeSO4 0.015g/L，MnSO4 0.01g/L，KH2PO 41 g/L, and the pH value is 7.0-7.4; preparing an ion culture medium: (NH)4)2SO4 2g/L，K2HPO4 1g/L，MgSO4 0.2g/L，CaCl2 0.01g/L，FeSO40.015g/L，MnSO4 0.01g/L，KH2PO 41 g/L, and the pH value is 7.0-7.4;
(3) the lignin model compounds ferulic acid, sinapic acid and p-coumaric acid are respectively added into the xylose-ion culture medium prepared in the last step, and the concentration is 100 mg/L. And (3) centrifugally collecting the cultured B-10 bacterial liquid, adding the bacterial liquid into a xylose-ion culture medium containing a model compound, culturing for 48 hours at 30 ℃, recording OD600 values at 0 hour, 6 hour, 12 hour, 24 hour, 36 hour and 48 hour respectively, and drawing a growth curve.
(4) The lignin model compounds ferulic acid, sinapic acid and p-coumaric acid are respectively added into the ion culture medium, and the concentration is 100 mg/L. And (3) centrifugally collecting the cultured B-10 bacterial liquid, adding the bacterial liquid into an ion culture medium containing a model compound, culturing for 48 hours, recording OD600 values at 0 hour, 6 hours, 12 hours, 24 hours, 36 hours and 48 hours, and drawing a growth curve.
(5) The growth curves are shown in FIG. 9, B-10 shows no growth behavior in the ion medium containing the model lignin compounds ferulic acid, sinapic acid and p-coumaric acid alone, whereas addition of ferulic acid, sinapic acid and p-coumaric acid B-10 to xylose-ion medium maintained growth trends and metabolic capacity.
Example 11: production of polyhydroxyalkanoates by using glucose by bacillus megaterium
(1) Inoculating the bacillus megaterium stored on the LB inclined plane into an LB liquid culture medium, and culturing at the temperature of 30 ℃ for 18h to obtain a seed solution of the bacillus megaterium; wherein the LB liquid culture medium comprises the following components in percentage by weight: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 1L of distilled water; the LB inclined plane is formed by adding 15g/L agar on the basis of the formula;
(2) centrifuging the bacillus megaterium seed solution obtained in the last step for 5 minutes at 12000rpm, removing supernatant, and collecting thalli;
(3) inoculating the collected bacillus megaterium thallus into a glucose sterile culture medium according to the inoculation amount of the bacterium liquid accounting for 20 percent (volume ratio) of the culture medium before centrifugation, culturing at 35 ℃ and 120rpm for 36h, and collecting the thallus. Wherein the glucose sterile culture medium comprises the following components: glucose 10g/L, (NH)4)2SO4 2g/L，K2HPO4 1g/L，MgSO4 0.2g/L，CaCl2 0.01g/L，FeSO4 0.015g/L，MnSO4 0.01g/L，KH2PO 41 g/L, and pH value of 7.0-7.4.
By the practice of this example, 2mL of sample was collected, centrifuged at 12000rpm for 5min, the supernatant was discarded, and the cells were resuspended in 150. mu.L of deionized water and 50. mu.L of dimethyl sulfoxide. mu.L of nile red dye (0.15mg/mL) was added to the suspension and stained for 30 min. And then observed with a fluorescence microscope. Accumulation of polyhydroxyalkanoate was observed in the bacterial body, demonstrating that it also has the ability to convert glucose.
Comparative example 1
(1) Crushing rice straws, sieving by a 60-mesh sieve, cleaning twice by ultrapure water, and drying at 60 ℃ to constant weight.
(2) Adding rice straw into 2% dilute sulfuric acid solution according to solid-to-liquid ratio of 5g/100mL, standing at 121 deg.C for 60min, filtering, and separating to obtain acid treatment waste liquid.
(3) Inoculating the bacillus megaterium stored on the LB inclined plane into an LB liquid culture medium, and culturing at the temperature of 30 ℃ for 18h to obtain a seed solution of the bacillus megaterium; wherein the LB liquid culture medium comprises the following components in percentage by weight: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 1L of distilled water; the LB inclined plane is formed by adding 15g/L agar on the basis of the formula;
(4) centrifuging the bacillus megaterium seed solution obtained in the last step for 5 minutes at 12000rpm, removing supernatant, and collecting thalli;
(5) inoculating the collected bacillus megaterium thallus into a sterile culture medium containing acid-treated waste liquid according to the inoculation amount of the bacterium liquid accounting for 20 percent (volume ratio) of the culture medium before centrifugation, culturing for 12h at 30 ℃, and collecting the thallus. Wherein the sterile culture medium containing the acid-treated waste liquid is prepared by adding inorganic salt into the acid-treated waste liquid, wherein the inorganic salt is added in a concentration of (NH)4)2SO4 2g/L，K2HPO41g/L，MgSO4 0.2g/L，CaCl2 0.01g/L，FeSO4 0.015g/L，MnSO4 0.01g/L，KH2PO41g/L, and the pH value is 7.0-7.4.
By the implementation of the present comparative example, the removal rate of total sugars in the acid-treated waste liquid was only 2.5%, and polyhydroxyalkanoate could not be accumulated in the bacillus megaterium body. The comparative example has high acid treatment strength and long treatment time, so that the dissolved sugar is further converted into toxic substances such as furfural furan and the like, thereby inhibiting the metabolism of cells and possibly killing the cells.
Comparative example 2
(1) Crushing rice straws, sieving by a 60-mesh sieve, cleaning twice by ultrapure water, and drying at 60 ℃ to constant weight.
(2) Adding rice straw into 2% dilute sulfuric acid solution according to solid-to-liquid ratio of 1g/100mL, standing at 121 deg.C for 40min, filtering, and separating to obtain acid treatment waste liquid.
(3) Inoculating the bacillus megaterium stored on the LB inclined plane into an LB liquid culture medium, and culturing at the temperature of 30 ℃ for 18h to obtain a seed solution of the bacillus megaterium; wherein the LB liquid culture medium comprises the following components in percentage by weight: 10g of peptone, 5g of yeast powder, 10g of sodium chloride and 1L of distilled water; the LB inclined plane is formed by adding 15g/L agar on the basis of the formula;
(4) centrifuging the bacillus megaterium seed solution obtained in the last step for 5 minutes at 12000rpm, removing supernatant, and collecting thalli;
(5) inoculating the collected bacillus megaterium thallus into a sterile culture medium containing acid-treated waste liquid according to the inoculation amount of the bacterium liquid accounting for 20 percent (volume ratio) of the culture medium before centrifugation, culturing for 60 hours at 30 ℃, and collecting the thallus. Wherein the sterile culture medium containing the acid-treated waste liquid is prepared by adding inorganic salt into the acid-treated waste liquid, wherein the inorganic salt is added in a concentration of (NH)4)2SO4 2g/L，K2HPO41g/L，MgSO4 0.2g/L，CaCl2 0.01g/L，FeSO4 0.015g/L，MnSO4 0.01g/L，KH2PO41g/L, and the pH value is 7.0-7.4.
By the implementation of the present comparative example, the removal rate of total sugars in the acid-treated waste liquid was only 0.94%, and polyhydroxyalkanoate could not be accumulated in the bacillus megaterium body. Since the strength of the acid treatment is high in this comparative example, the amount of lignocellulose treated is small, so that the further conversion of the dissolved sugars into toxic substances such as furfural furan inhibits the metabolism of cells and may cause death.
Comparative example 3
This comparative example, i.e., the recycling method according to the present invention, is compared with the patent (application No. 200510133634.5).
The patent application No. 200510133634.5 discloses a recombinant polyhydroxyfatty acid ester-producing bacterium GBR008 and an expression vector thereof, wherein the yield of the polyhydroxyfatty acid ester produced by the recombinant dalf bacterium in glucose utilization is 0.991g/L in terms of oxygen deficiency. Compared with the method of the patent, the resource method has the following advantages: (1) the bacillus megaterium adopted in the invention does not need to be genetically modified, so that the investment cost of the process is greatly reduced. (2) The yield of the polyhydroxyalkanoate obtained by the resource method adopted in the invention is high, the conversion rate has obvious advantages (3) the carbon source adopted in the invention is waste lignocellulose acid treatment solution. Compared with glucose, the waste lignocellulose acid treatment solution has complex components, high treatment difficulty and high conversion difficulty. Meanwhile, the waste lignocellulose acid treatment solution is used as a substrate, so that the investment cost of the process can be greatly reduced.
Claims (7)
1. Bacillus megaterium with preservation number of CGMCC number 15753Bacillus megaterium)B-10。
2. Use of the strain of claim 1 for the production of polyhydroxyalkanoates.
3. The use according to claim 2, wherein the strain is inoculated into a waste liquid obtained by treating a lignocellulose with an acid method, and the cultured cells contain polyhydroxyalkanoate.
4. Use of the strain of claim 1 for the production of polyhydroxyalkanoates using xylose.
5. The use of claim 4, wherein the medium contains 3-20 g/L xylose for the production of polyhydroxyalkanoate.
6. According to claim5, characterized in that the culture medium for the production of polyhydroxyalkanoates comprises: 3-20 g/L xylose (NH)4)2SO4 2g/L，K2HPO4 1g/L，MgSO4 0.2g/L，CaCl2 0.01g/L，FeSO4 0.015g/L，MnSO4 0.01g/L，KH2PO41g/L, and the pH value is 7.0-7.4.
7. The use as claimed in claim 4, wherein the polyhydroxyalkanoate is produced under the culture conditions of 30-35 ℃, the rotation speed of 120-150rpm, and the culture period of 30-60 h.
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| EP2301958A1 (en) * | 2004-01-30 | 2011-03-30 | Novozymes Inc. | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
| CN102056962A (en) * | 2008-06-05 | 2011-05-11 | 国立大学法人东京工业大学 | Polyhydroxyalkanoate copolymer and manufacturing method therefor |
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| CN1302823A (en) * | 2000-11-17 | 2001-07-11 | 西北大学 | Process for preparing polyhydroxyl alkanoic acid |
| EP2301958A1 (en) * | 2004-01-30 | 2011-03-30 | Novozymes Inc. | Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same |
| CN102056962A (en) * | 2008-06-05 | 2011-05-11 | 国立大学法人东京工业大学 | Polyhydroxyalkanoate copolymer and manufacturing method therefor |
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