Method for extracting polyhydroxyalkanoate through enzymolysis of bamboo powder

Abstract

The invention discloses a method for extracting polyhydroxyalkanoate through enzymolysis of bamboo powder, and belongs to the technical field of extraction. Comprising the following steps that whole bamboos are taken and processed, and bamboo powder is obtained; bamboo powder is subjected to ozone pretreatment and enzymolysis, and enzymolysis residues are obtained; carrying out lignin and hemicellulose separation treatment on the enzymolysis residues to obtain lignin; grafting epoxy chloropropane with lignin to obtain modified lignin; grafting cyclodextrin to the modified lignin to obtain cyclodextrin grafted lignin; grafting a lignin enveloped dioxolane polymer with cyclodextrin to obtain a composite carbon source; culturing a strain by using a composite carbon source, extracting and purifying to obtain polyhydroxyalkanoate; the cyclodextrin grafted high-purity lignin enveloped dioxolane polymer is used as a composite carbon source, so that the yield of polyhydroxyalkanoate produced by the strain is increased.

Description

Technical Field

[0001] The present invention relates to the technical field of extraction, and particularly relates to a method for extracting polyhydroxyalkanoates by enzymatic hydrolysis of bamboo powder. Background Art

[0002] Polyhydroxyalkanoates (PHA for short) are a kind of biopolymer materials synthesized by microorganisms through the fermentation process under specific nutritional conditions, such as when the carbon source is rich while other nutrient elements such as nitrogen and phosphorus are relatively scarce. This material has attracted much attention because it has physical and chemical properties similar to those of traditional plastics, such as good mechanical properties and processing properties. At the same time, polyhydroxyalkanoates also have excellent biocompatibility and biodegradability, and are environmentally friendly materials that can be decomposed by microorganisms in the natural environment and will not cause long-term pollution to the environment. Therefore, the research on polyhydroxyalkanoates not only helps to solve the increasingly serious plastic pollution problem, but also has far-reaching significance for alleviating the global energy crisis and promoting the sustainable utilization of resources.

[0003] With the rapid development of synthetic biology technology and the strong support of national policies, significant progress has been made in the production process of polyhydroxyalkanoates. Despite many achievements in the production process, relatively few studies have been conducted on the extraction technology of polyhydroxyalkanoates, which has limited its large-scale production and industrial application process to a certain extent. Therefore, future research needs to pay more attention to how to optimize the extraction and purification processes in order to achieve the commercialization and wide application of polyhydroxyalkanoates, so as to better serve the goals of environmental protection and resource sustainable development.

[0004] Bamboo is rich in lignocellulosic biomass, mainly composed of cellulose, hemicellulose, lignin, etc. Lignin is a three-dimensional network macromolecular polymer with a three-dimensional structure. It is widely present in plant cells, combines with hemicellulose, and the two together fill the microfibrils in the cell wall, playing a role in compression resistance and support. As a by-product of the pulp and paper making, fuel ethanol and biomass refining industries, lignin is usually used for burning to supply heat energy or directly landfilled, which not only causes waste of resources but also pollutes the environment. Therefore, the high-value bioconversion of lignin is of great significance for environmental protection and energy security. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for extracting polyhydroxyalkanoates by enzymatic hydrolysis of bamboo powder, which is used to improve the bioconversion rate of lignin in bamboo and promote the bioconversion of lignin into high-yield polyhydroxyalkanoates.

[0006] The purpose of the present invention can be achieved by the following technical solutions: A method for extracting polyhydroxyalkanoates from enzymatically hydrolyzed bamboo powder, comprising the following steps: S1. Take the whole bamboo, process it, crush it, and grind it to obtain bamboo powder; S2. Take the bamboo powder and pretreat it in an ozone atmosphere to obtain pretreated bamboo powder; S3. Take the pretreated bamboo powder and enzymatically hydrolyze it to obtain enzymolysis residue; S4. Take the enzymolysis residue and perform lignin and hemicellulose separation treatment to obtain lignin; S5. Take the lignin and graft it with epichlorohydrin to obtain modified lignin; S6. Take the modified lignin and graft it with cyclodextrin to obtain cyclodextrin-grafted lignin; S7. In a nitrogen atmosphere, add 1,3-dioxolane, initiator, and catalyst to a polymerization reaction kettle, mix evenly, heat and stir, add ammonia water to terminate the reaction, precipitate the product at low temperature, filter, wash, and vacuum dry to obtain a polymer material; S8. Take the cyclodextrin-grafted lignin and deionized water, mix them, add the polymer material, stir ultrasonically, let stand, filter, take the solid phase, wash, and dry to obtain a composite carbon source; S9. Pick the strain and streak-inoculate it on a solid medium for activation, pick the activated strain and inoculate it in a liquid medium, culture to obtain a liquid seed, inoculate the liquid seed into a fermentation medium to culture to obtain a fermentation broth, and take the fermentation broth after fermentation to perform extraction and purification to obtain the polyhydroxyalkanoates; The carbon source in the fermentation medium includes glucose and a composite carbon source.

[0007] As a further scheme of the present invention, step S1 specifically includes: taking the whole bamboo, cleaning it to remove surface impurities, flattening it after splitting the whole bamboo, peeling it, cutting the peeled bamboo along the length direction into bamboo strips, and then cutting it into bamboo blocks. The bamboo blocks are dried after being centrifuged to remove water, crushed into particles with a size of 2-4 mm, and ground to obtain bamboo powder with a particle size of 300-500 meshes.

[0008] As a further scheme of the present invention, the whole bamboo is any one of Bambusoideae, such as but not limited to black bamboo, yellow-striped bamboo, Sasa fortunei, cinnamon bamboo, and phoenix-tail bamboo.

[0009] As a further scheme of the present invention, in step S2, the preparation method of the pretreated bamboo powder specifically includes the following steps: Take the bamboo powder and deionized water, mix them, disperse ultrasonically, adjust the pH to 1.8-3.0, heat and stir, transfer to a reaction kettle, and perform treatment in an ozone atmosphere, vacuum filter and wash to obtain pretreated bamboo powder; The adsorption of cellulase onto the cellulose surface is the first step of enzymatic hydrolysis. However, lignin can undergo irreversible non-productive adsorption with cellulase through hydrophobic, hydrogen bonding, and electrostatic interactions, etc., and coat the cellulose surface as a physical barrier, forming steric hindrance to cellulase. Cellulase is more likely to adsorb on the lignin surface in the enzymatic hydrolysis system, which has an adverse effect on the enzymatic hydrolysis of cellulose. Through ozone pretreatment, the physical barrier of the substrate cellulose is reduced, the enzymatic hydrolysis resistance is lowered, the accessibility of cellulase hydrolysis is increased, and the lignin removal rate is improved; Preferably, the mass ratio of the bamboo powder to deionized water is 1.8:100. Under this ratio condition, cellulose is more likely to undergo water absorption and swelling, resulting in a decrease in the internal cohesion within the fiber, making it easier for ozone molecules to penetrate into the fiber interior, and the lignin removal effect is better, reducing the dissolution of hemicellulose and cellulose, and improving the selectivity of lignin removal; Preferably, anhydrous acetic acid is used to adjust the pH value to 2.2, which can reduce the generation of reactive oxygen free radicals, protect carbohydrates from being damaged, and thus is beneficial to lignin removal.

[0010] As a further scheme of the present invention, in step S3, the preparation method of the enzymatic hydrolysis residue specifically includes the following steps: Mix the pretreated bamboo powder, deionized water, cellulase, and additive, adjust the pH to 5 - 6, perform enzymatic hydrolysis, and filter and separate to obtain an enzymatic hydrolysis residue rich in lignin; The additive includes Tween 80, polyethylene glycol 4000, bovine serum albumin, and dodecylbenzenesulfonic acid; The mass ratio of Tween 80, polyethylene glycol 4000, bovine serum albumin, and dodecylbenzenesulfonic acid is 0.08 - 0.25:0.08 - 0.18:0.06 - 0.12:0.02 - 0.05; Preferably, the dosage ratio of the pretreated bamboo powder, deionized water, cellulase, and additive is 25 g:500 mL:1100 FPU:0.45, and citrate buffer is used to adjust the pH to 5.5; Preferably, the cellulase selected is Cellulase RS cellulase, CAS number 9012 - 54 - 8 (purchased from Beijing BioLabo Technology Co., Ltd.); By adding non-ionic surfactants Tween 80 and polyethylene glycol 4000, they can bind to lignin through hydrophobic interactions and hydrogen bonding, occupy the adsorption sites on the surface of lignin, and reduce the adsorption between cellulase and lignin; adding bovine serum albumin, the hydrophobic structures on its surface can have hydrophobic adsorption with lignin and occupy the hydrophobic adsorption sites of lignin, reducing the adsorption of lignin on cellulase; adding anionic surfactant dodecylbenzenesulfonic acid to adsorb on the surface of lignin to form a hydration film and steric hindrance, reducing the adsorption on cellulase, and improving the dissolution of lignin through the synergistic effect among Tween 80, polyethylene glycol 4000, bovine serum albumin and dodecylbenzenesulfonic acid.

[0011] As a further scheme of the present invention, in step S4, the preparation method of the lignin specifically includes the following steps: Take the enzymatic hydrolysis residue, zinc chloride and methanol and mix them, add hydrochloric acid aqueous solution and formic acid aqueous solution, heat and stir in an oil bath under sealed conditions, then cool in an ice-water bath, and separate to obtain lignin; Through the above extraction steps, by the synergistic effect of the zinc chloride-methanol-hydrochloric acid-formic acid mixed system, the connection between lignin and hemicellulose is cut off, so as to dissolve lignin relatively uniformly.

[0012] As a further scheme of the present invention, in step S7, the initiator includes at least one of perchloric acid, perchlorate and perchlorate hydrate.

[0013] As a further scheme of the present invention, in step S7, the catalyst includes at least one of boron trifluoride, boron trifluoride ethyl ether complex, boron trifluoride n-butyl ether complex, tin tetrachloride, titanium tetrachloride, aluminum trichloride, zinc chloride, vanadium trichloride, antimony trichloride, phosphorus pentafluoride and antimony pentafluoride.

[0014] As a further scheme of the present invention, in step S9, the preparation method of the solid medium is as follows: peptone 5 g, yeast extract 1 g, sodium chloride 27.5 g, potassium chloride 0.7 g, magnesium chloride hexahydrate 5.4 g, magnesium sulfate heptahydrate 6.8 g, calcium chloride dihydrate 1.4 g, sodium bicarbonate 0.2 g, potassium bromide 0.07 g, boric acid 20 mg, sodium silicate 5 mg, sodium fluoride 3 mg, ammonium nitrate 2 mg, potassium dihydrogen phosphate 1.5 g, agar 15 g, add distilled water to make up to 1 L, pH is 7.5, sterilize at 121 °C for 20 min.

[0015] As a further solution of the present invention, in step S9, the method for preparing the liquid medium is as follows: 5 g of peptone, 1 g of yeast extract, 90 g of sodium chloride, 0.7 g of potassium chloride, 5.4 g of magnesium chloride hexahydrate, 6.8 g of magnesium sulfate heptahydrate, 1.4 g of calcium chloride dihydrate, 0.2 g of sodium bicarbonate, 0.07 g of potassium bromide, 20 mg of boric acid, 5 mg of sodium silicate, 3 mg of sodium fluoride, 2 mg of ammonium nitrate, 1.5 g of potassium dihydrogen phosphate, add distilled water to make up to 1 L, pH is 7.5, sterilize at 121 °C for 20 min.

[0016] As a further solution of the present invention, in step S9, the method for preparing the fermentation medium is as follows: 5 g of peptone, 1 g of yeast extract, 27.5 g of sodium chloride, 0.7 g of potassium chloride, 5.4 g of magnesium chloride hexahydrate, 6.8 g of magnesium sulfate heptahydrate, 1.4 g of calcium chloride dihydrate, 0.2 g of sodium bicarbonate, 0.07 g of potassium bromide, 20 mg of boric acid, 5 mg of sodium silicate, 3 mg of sodium fluoride, 2 mg of ammonium nitrate, 1.5 g of potassium dihydrogen phosphate, 10 g of glucose, 10 - 15 g of composite carbon source, add distilled water to make up to 1 L, pH is 7.5, sterilize at 121 °C for 20 min.

[0017] As a further solution of the present invention, the strain is Pseudomonas putida KT2440, purchased from Shanghai Jiachu Bioengineering Co., Ltd., and the strain number is SHMCC(SHBCC)D25212.

[0018] As a further solution of the present invention, the extracted polyhydroxyalkanoate is specifically poly-3-hydroxybutyrate.

[0019] Advantages of the present invention: A method for extracting polyhydroxyalkanoate by enzymatic hydrolysis of bamboo powder disclosed by the present invention uses the enzymatic hydrolysis of bamboo powder to extract high-purity lignin as a carbon source for the strain to synthesize polyhydroxyalkanoate, thereby increasing the yield of polyhydroxyalkanoate by the biological conversion of lignin; Furthermore, in order to obtain high-purity lignin, ozone pretreatment of bamboo powder reduces the physical barrier of the substrate cellulose, reduces the enzymatic hydrolysis resistance, increases the accessibility of cellulase hydrolysis, and improves the lignin removal rate; the combined synergistic effect among Tween 80, polyethylene glycol 4000, bovine serum albumin and dodecylbenzenesulfonic acid synergistically improves the dissolution of lignin; Furthermore, cyclodextrin-grafted lignin encapsulates dioxolane polymer, and the affinity of the ether bond of the dioxolane polymer for Pseudomonas putida strains enables the strains to adhere to the surface of cyclodextrin-grafted lignin, thereby avoiding the phenomenon of serious adherent growth, extrusion death, or sedimentation or stratification caused by the reproduction of the strains, resulting in uneven growth, and improving the yield of polyhydroxyalkanoate. Description of the drawings

[0020] The present invention will be further described below in conjunction with the accompanying drawings.

[0021] Figure 1 It is the infrared spectrum analysis diagram of the polyhydroxyalkanoate extracted in Example 2 of the present invention; Figure 2 It is the nuclear magnetic resonance carbon spectrum analysis diagram of the polyhydroxyalkanoate extracted in Example 2 of the present invention; Figure 3 It is the colony morphology of Pseudomonas putida KT2440 cultured in Example 2 of the present invention. Detailed implementation manners

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0023] Example 1 A method for extracting polyhydroxyalkanoate by enzymatic hydrolysis of bamboo powder, comprising the following steps: S1. Take the whole purple bamboo, clean it to remove surface impurities, flatten it after splitting the whole bamboo, remove the skin, cut the skinned bamboo into bamboo strips along the length direction, and then cut it into bamboo blocks. After the bamboo blocks are centrifuged to remove water, they are dried, crushed into 2 mm particles, and ground to obtain bamboo powder with a particle size of 300 mesh; S2. Take the bamboo powder and deionized water, mix them, disperse them ultrasonically for 20 min, add anhydrous acetic acid to adjust the pH to 1.8, stir at 60 °C for 1 h, transfer to a reaction kettle, and treat it in an ozone atmosphere for 1.5 h. Then perform vacuum filtration and washing to obtain pretreated bamboo powder; the mass ratio of the bamboo powder to deionized water is 1.2:100; the ozone concentration in the ozone atmosphere is 90 mg / L, and the ozone flow rate is 3 L / min; S3. Take the pretreated bamboo powder, deionized water, cellulase, and auxiliary agent, mix them, add citrate buffer solution to adjust the pH to 5, and perform enzymatic hydrolysis at 50 °C and 200 rpm in a water bath oscillator for 60 h, and then perform filtration and separation to obtain an enzyme hydrolysis residue rich in lignin; the dosage ratio of the pretreated bamboo powder, deionized water, cellulase, and auxiliary agent is 20 g:500 mL:800 FPU:0.16 g; The cellulase selected is Cellulase RS cellulase; The auxiliary agent includes Tween 80, polyethylene glycol 4000, bovine serum albumin, and dodecylbenzenesulfonic acid, and the mass ratio of Tween 80, polyethylene glycol 4000, bovine serum albumin, and dodecylbenzenesulfonic acid is 0.08:0.08:0.06:0.02; S4. Mix the enzymatic hydrolysis residue, zinc chloride, and methanol, add hydrochloric acid aqueous solution and formic acid aqueous solution, seal and heat with stirring in an oil bath at 45 °C for 12 min, then cool in an ice-water bath, and transfer the mixed material to a separatory funnel with deionized water. Add ethyl acetate to extract lignin, collect the ethyl acetate phase, filter by suction, wash the filter residue, and dry at 80 °C for 10 h to obtain lignin. The dosage ratio of the enzymatic hydrolysis residue, zinc chloride, hydrochloric acid aqueous solution, formic acid aqueous solution, and methanol is 2 g: 22 g: 2 mL: 2 mL: 30 mL. The concentration of the hydrochloric acid aqueous solution is 2.5 mol / L. The concentration of the formic acid aqueous solution is 3 mol / L. S5. Mix the lignin and sodium hydroxide aqueous solution, stir for 5 min, add epichlorohydrin, and stir at 80 °C for 2 h. After the product is vacuum filtered to dryness, wash it and dry it under vacuum to obtain modified lignin. The dosage ratio of the lignin, sodium hydroxide aqueous solution, and epichlorohydrin is 1 g: 6 mL: 12 g. The concentration of the sodium hydroxide aqueous solution is 1 mol / L. S6. Mix the modified lignin, β-cyclodextrin, and sodium hydroxide aqueous solution and stir evenly, stir at 35 °C for 2 h, filter, wash the solid phase, and dry it under vacuum to obtain β-cyclodextrin grafted lignin. The dosage ratio of the modified lignin, β-cyclodextrin, and sodium hydroxide aqueous solution is 2 g: 4 g: 30 mL. The concentration of the sodium hydroxide aqueous solution is 1 mol / L. S7. In a nitrogen atmosphere, add 1,3-dioxolane, perchloric acid, and boron trifluoride diethyl ether complex (purchased from Shandong Yinglang Chemical Co., Ltd.) to a polymerization reaction kettle and mix evenly. Stir at 40 °C for 15 min, add ammonia water to terminate the reaction, precipitate the product in methanol at 0 °C, filter, wash, and dry it under vacuum to obtain a polymer material. The dosage ratio of 1,3-dioxolane, perchloric acid, boron trifluoride diethyl ether complex, and ammonia water is 3 g: 0.0014 g: 0.0004 g: 10 mL. The concentration of the ammonia water is 1 mol / L. S8. Mix the β-cyclodextrin grafted lignin and deionized water, add the polymer material, stir ultrasonically for 10 min, let it stand for 24 h, filter, wash the solid phase, and dry it to obtain the composite carbon source. The mass ratio of the β-cyclodextrin grafted lignin, deionized water, and polymer material is 1: 50: 4. S9. Specifically, it includes the following process: S91. Strain activation: Take the Pseudomonas putida KT2440 strain stored at -80 °C, scrape the ice chips with a sterile pipette tip, pick the strain and streak inoculate it on a solid medium, and culture it in a constant temperature incubator at 30 °C for 20 h. The preparation method of the solid medium is as follows: 5 g of peptone, 1 g of yeast extract, 27.5 g of sodium chloride, 0.7 g of potassium chloride, 5.4 g of magnesium chloride hexahydrate, 6.8 g of magnesium sulfate heptahydrate, 1.4 g of calcium chloride dihydrate, 0.2 g of sodium bicarbonate, 0.07 g of potassium bromide, 20 mg of boric acid, 5 mg of sodium silicate, 3 mg of sodium fluoride, 2 mg of ammonium nitrate, 1.5 g of potassium dihydrogen phosphate, 15 g of agar. Add distilled water to make up to 1 L, with a pH of 7.5, and sterilize at 121 °C for 20 min.

[0024] S92. Prepare the seed liquid: Pick an activated strain and inoculate it into the liquid medium, and shake and culture it at 30 °C and 200 rpm for 12 h to obtain the liquid seed. The preparation method of the liquid medium is as follows: 5 g of peptone, 1 g of yeast extract, 90 g of sodium chloride, 0.7 g of potassium chloride, 5.4 g of magnesium chloride hexahydrate, 6.8 g of magnesium sulfate heptahydrate, 1.4 g of calcium chloride dihydrate, 0.2 g of sodium bicarbonate, 0.07 g of potassium bromide, 20 mg of boric acid, 5 mg of sodium silicate, 3 mg of sodium fluoride, 2 mg of ammonium nitrate, 1.5 g of potassium dihydrogen phosphate. Add distilled water to make up to 1 L, with a pH of 7.5, and sterilize at 121 °C for 20 min.

[0025] S93. Liquid fermentation: According to the inoculation amount of the liquid seed and the fermentation medium at a volume ratio of 5:100, shake and culture it at 30 °C and 150 rpm for 72 h to obtain the fermentation broth. The preparation method of the fermentation medium is as follows: 5 g of peptone, 1 g of yeast extract, 27.5 g of sodium chloride, 0.7 g of potassium chloride, 5.4 g of magnesium chloride hexahydrate, 6.8 g of magnesium sulfate heptahydrate, 1.4 g of calcium chloride dihydrate, 0.2 g of sodium bicarbonate, 0.07 g of potassium bromide, 20 mg of boric acid, 5 mg of sodium silicate, 3 mg of sodium fluoride, 2 mg of ammonium nitrate, 1.5 g of potassium dihydrogen phosphate, 10 g of glucose, 10 g of composite carbon source. Add distilled water to make up to 1 L, with a pH of 7.5, and sterilize at 121 °C for 20 min.

[0026] S94. Extraction and purification: Take the fermentation broth after fermentation and centrifuge it at 12000 rpm for 15 min to collect the thallus. Resuspend the thallus with deionized water and wash it 3 times, then freeze-dry it. Grind the freeze-dried thallus thoroughly with a mortar. Add 10 mL of chloroform to each gram of the bacterial powder, extract it at 60 °C for 10 h, filter by suction, take the chloroform phase, concentrate it in a rotary evaporator, add 5 times the volume of absolute ethanol, let it stand at 4 °C for 20 h, then centrifuge to collect the solid precipitate, and dry it under vacuum to obtain polyhydroxyalkanoate. [[ID=I5]]

[0027] Example 2 A method for extracting polyhydroxyalkanoate by enzymatic hydrolysis of bamboo powder, comprising the following steps: S1. Take the whole black bamboo, clean it to remove surface impurities, split the whole bamboo and press it flat, remove the skin, cut the bamboo material along the length direction into bamboo strips, and then cut it into bamboo blocks. After the bamboo blocks are dried by centrifugation to remove moisture, they are dried, crushed into 3 mm particles, and ground to obtain bamboo powder with a particle size of 400 mesh. S2. Take the above-mentioned bamboo powder and deionized water, mix them, disperse ultrasonically for 25 min, add anhydrous acetic acid to adjust the pH to 2.2, stir at 60 °C for 1.5 h, transfer to a reaction kettle, and treat in an ozone atmosphere for 2 h, then perform vacuum filtration and washing to obtain pretreated bamboo powder; the mass ratio of the bamboo powder to deionized water is 1.8:100; the ozone concentration in the ozone atmosphere is 90 mg / L, and the ozone flow rate is 3 L / min. S3. Take the pretreated bamboo powder, deionized water, Cellulase RS cellulase, and auxiliary agent, mix them, add citrate buffer solution to adjust the pH to 5.5, and perform enzymatic hydrolysis at 50 °C and 200 rpm in a water bath oscillator for 72 h, then filter and separate to obtain enzymatic hydrolysis residue rich in lignin; the dosage ratio of the pretreated bamboo powder, deionized water, Cellulase RS cellulase, and auxiliary agent is 25 g:500 mL:1100 FPU:0.45 g. The cellulase used is Cellulase RS cellulase. The mass ratio of Tween 80, polyethylene glycol 4000, bovine serum albumin, and dodecylbenzenesulfonic acid in the auxiliary agent is 0.16:0.13:0.09:0.??? S4. Take the enzymatic hydrolysis residue, zinc chloride, and methanol, mix them, add hydrochloric acid aqueous solution and formic acid aqueous solution, heat and stir in an oil bath at 52 °C for 15 min while sealing, then cool in an ice-water bath, and then transfer the mixed material to a separatory funnel with deionized water, add ethyl acetate to extract lignin, collect the ethyl acetate phase, perform suction filtration, take the filter residue and wash it, and dry it at 80 °C for 12 h to obtain lignin; the dosage ratio of the enzymatic hydrolysis residue, zinc chloride, hydrochloric acid aqueous solution, formic acid aqueous solution, and methanol is 2.5 g:26 g:3 mL:3 mL:30 mL; the concentration of the hydrochloric acid aqueous solution is 3 mol / L; the concentration of the formic acid aqueous solution is 4 mol / L. S5. Take the lignin and sodium hydroxide aqueous solution, stir for 6 min, add epichlorohydrin, and stir at 80 °C for 3 h. After the product is vacuum filtered to dryness, wash it, and perform vacuum drying to obtain modified lignin; the dosage ratio of the lignin, sodium hydroxide aqueous solution, and epichlorohydrin is 1.1 g:7 mL:14 g; the concentration of the sodium hydroxide aqueous solution is 1 mol / L. It should be noted that there is an unclear value "0.035" in the mass ratio of the auxiliary agent in step S8 in the original text. I have translated it as "0.035" as it is, but you may need to check and correct it according to the actual situation. Also, there seems to be a missing value in the English translation of step S8 where it says "0.16:0.13:0.09:0.???".S6. Mix the modified lignin, β-cyclodextrin, and sodium hydroxide aqueous solution evenly by stirring, stir at 40 °C for 3 h, filter, wash the solid phase, and dry it under vacuum to obtain cyclodextrin-grafted lignin; the dosage ratio of the modified lignin, β-cyclodextrin, and sodium hydroxide aqueous solution is 2.5 g: 4.5 g: 30 mL; the concentration of the sodium hydroxide aqueous solution is 1 mol / L; S7. In a nitrogen atmosphere, add 1,3-dioxolane, perchloric acid, and boron trifluoride diethyl ether complex to a polymerization reaction kettle and mix evenly, stir at 50 °C for 20 min, add ammonia water to terminate the reaction, precipitate the product in methanol at 0 °C, filter, wash, and dry it under vacuum to obtain a polymer material; the dosage ratio of 1,3-dioxolane, perchloric acid, boron trifluoride diethyl ether complex, and ammonia water is 4 g: 0.0016 g: 0.0005 g: 11 mL; the concentration of the ammonia water is 1 mol / L; S8. Mix the cyclodextrin-grafted lignin and deionized water, add the polymer material, stir ultrasonically for 15 min, let stand for 24 h, filter, wash the solid phase, and dry to obtain the composite carbon source; the mass ratio of the cyclodextrin-grafted lignin, deionized water, and polymer material is 2: 50: 6; S9. Specifically, it includes the following processes: S91. Strain activation: Take the Pseudomonas putida KT2440 strain stored at -80 °C, scrape the ice chips with a sterile pipette tip, pick the strain and streak it on a solid medium, and culture it in a constant temperature incubator at 30 °C for 30 h; The preparation method of the solid medium is as follows: 5 g of peptone, 1 g of yeast extract, 27.5 g of sodium chloride, 0.7 g of potassium chloride, 5.4 g of magnesium chloride hexahydrate, 6.8 g of magnesium sulfate heptahydrate, 1.4 g of calcium chloride dihydrate, 0.2 g of sodium bicarbonate, 0.07 g of potassium bromide, 20 mg of boric acid, 5 mg of sodium silicate, 3 mg of sodium fluoride, 2 mg of ammonium nitrate, 1.5 g of potassium dihydrogen phosphate, 15 g of agar, add distilled water to make up to 1 L, pH is 7.5, sterilize at 121 °C for 20 min.

[0028] S92. Prepare the seed liquid: Pick the activated strain and inoculate it into a liquid medium, and shake it at 30 °C and 200 rpm for 15 h to obtain a liquid seed; The preparation method of the liquid medium is as follows: 5 g of peptone, 1 g of yeast extract, 90 g of sodium chloride, 0.7 g of potassium chloride, 5.4 g of magnesium chloride hexahydrate, 6.8 g of magnesium sulfate heptahydrate, 1.4 g of calcium chloride dihydrate, 0.2 g of sodium bicarbonate, 0.07 g of potassium bromide, 20 mg of boric acid, 5 mg of sodium silicate, 3 mg of sodium fluoride, 2 mg of ammonium nitrate, 1.5 g of potassium dihydrogen phosphate, add distilled water to make up to 1 L, pH is 7.5, sterilize at 121 °C for 20 min.

[0029] S93. Liquid fermentation: According to the inoculation amount of liquid seeds and fermentation medium at a volume ratio of 8:100, shake-culture at 30 °C and 150 rpm for 72 h to obtain the fermentation broth; The preparation method of the fermentation medium is as follows: peptone 5 g, yeast extract 1 g, sodium chloride 27.5 g, potassium chloride 0.7 g, magnesium chloride hexahydrate 5.4 g, magnesium sulfate heptahydrate 6.8 g, calcium chloride dihydrate 1.4 g, sodium bicarbonate 0.2 g, potassium bromide 0.07 g, boric acid 20 mg, sodium silicate 5 mg, sodium fluoride 3 mg, ammonium nitrate 2 mg, potassium dihydrogen phosphate 1.5 g, glucose 10 g, composite carbon source 13 g, add distilled water to make up to 1 L, adjust the pH to 7.5, and sterilize at 121 °C for 20 min.

[0030] S94. Extraction and purification: Take the fermentation broth after fermentation, centrifuge at 12000 rpm for 15 min to collect the bacterial cells, resuspend the bacterial cells with deionized water, wash 3 times, and then freeze-dry. Grind the freeze-dried bacterial cells thoroughly with a mortar. Add 10 mL of chloroform to each gram of the bacterial powder, extract at 60 °C for 10 h, filter by suction, take the chloroform phase, concentrate it in a rotary evaporator, add 5 times the volume of absolute ethanol, let it stand at 4 °C for 20 h, then centrifuge to collect the solid precipitate, and dry it under vacuum to obtain polyhydroxyalkanoate.

[0031] As Figure 1 shown, in the infrared spectrum of the polyhydroxyalkanoate extracted in this example, the absorption peak at 1720 cm -1 is the stretching vibration absorption peak of C=O, which is the characteristic absorption peak of polyhydroxyalkanoate.

[0032] As Figure 2 shown, in the nuclear magnetic resonance carbon spectrum of the polyhydroxyalkanoate extracted in this example, the peaks at 19.746 ppm, 40.757 ppm, 77.000 ppm and 169.135 ppm correspond to methyl (-CH3), methylene (-CH2-), methine (-CH-) and carbonyl (C=O) respectively, indicating that its intracellular polymer is specifically poly-3-hydroxybutyrate.

[0033] As Figure 3 shown, the colony morphology of Pseudomonas putida KT2440 growing on the fermentation medium.

[0034] Example 3 A method for extracting polyhydroxyalkanoate by enzymatic hydrolysis of bamboo powder, comprising the following steps: S1. Take the whole purple bamboo, clean it to remove surface impurities, flatten it after splitting the whole bamboo, remove the skin, cut the skinned bamboo along the length direction into bamboo strips, and then cut them into bamboo blocks. After the bamboo blocks are centrifuged to remove water, they are dried, broken into 4 mm particles, and ground to obtain bamboo powder with a particle size of 500 mesh; S2. Mix the bamboo powder and deionized water, ultrasonically disperse for 30 min, add anhydrous acetic acid to adjust the pH to 3.0, stir at 60 °C for 2 h, transfer to a reaction kettle, and treat in an ozone atmosphere for 2.5 h. Then perform vacuum filtration and washing to obtain pretreated bamboo powder. The mass ratio of the bamboo powder to deionized water is 2.5:100. The ozone concentration in the ozone atmosphere is 90 mg / L, and the ozone flow rate is 3 L / min. S3. Mix the pretreated bamboo powder, deionized water, Cellulase RS cellulase, and additive, add citrate buffer solution to adjust the pH to 6, and perform enzymatic hydrolysis at 50 °C and 200 rpm in a water bath oscillator for 80 h. Then filter and separate to obtain enzymolysis residue rich in lignin. The dosage ratio of the pretreated bamboo powder, deionized water, Cellulase RS cellulase, and additive is 30 g:500 mL:1500 FPU:0.75 g. The mass ratio of Tween 80, polyethylene glycol 4000, bovine serum albumin, and dodecylbenzenesulfonic acid in the additive is 0.25:0.18:0.12:0.05. S4. Mix the enzymolysis residue, zinc chloride, and methanol, add hydrochloric acid aqueous solution and formic acid aqueous solution, seal and heat with stirring in an oil bath at 58 °C for 18 min, then cool in an ice-water bath. Next, transfer the mixed material to a separatory funnel with deionized water, add ethyl acetate to extract lignin, collect the ethyl acetate phase, perform suction filtration, wash the filter residue, and dry at 80 °C for 15 h to obtain lignin. The dosage ratio of the enzymolysis residue, zinc chloride, hydrochloric acid aqueous solution, formic acid aqueous solution, and methanol is 3 g:30 g:4 mL:4 mL:30 mL. The concentration of the hydrochloric acid aqueous solution is 3.5 mol / L. The concentration of the formic acid aqueous solution is 5 mol / L. S5. Mix the lignin and sodium hydroxide aqueous solution, stir for 7 min, add epichlorohydrin, and stir at 80 °C for 4 h. After the product is vacuum filtered to dryness, wash it, and perform vacuum drying to obtain modified lignin. The dosage ratio of the lignin, sodium hydroxide aqueous solution, and epichlorohydrin is 1.2 g:8 mL:16 g. The concentration of the sodium hydroxide aqueous solution is 1 mol / L. S6. Mix the modified lignin, β-cyclodextrin, and sodium hydroxide aqueous solution and stir evenly, stir at 45 °C for 4 h, filter, wash the solid phase, and perform vacuum drying to obtain β-cyclodextrin grafted lignin. The dosage ratio of the modified lignin, β-cyclodextrin, and sodium hydroxide aqueous solution is 3 g:5 g:30 mL. The concentration of the sodium hydroxide aqueous solution is 1 mol / L. S7. In a nitrogen atmosphere, 1,3-dioxolane, perchloric acid, and boron trifluoride ether complex were added together to a polymerization reactor and mixed evenly. The mixture was stirred at 60 °C for 25 min, and then ammonia water was added to terminate the reaction. The product was precipitated in methanol at 0 °C, filtered, washed, and dried under vacuum to obtain a polymer material. The dosage ratio of 1,3-dioxolane, perchloric acid, boron trifluoride ether complex, and ammonia water was 5 g: 0.0018 g: 0.0006 g: 12 mL. The concentration of the ammonia water was 1 mol / L. S8. The cyclodextrin-grafted lignin and deionized water were mixed, and the polymer material was added thereto. The mixture was ultrasonically stirred for 20 min, allowed to stand for 24 h, filtered, and the solid phase was taken, washed, and dried to obtain the composite carbon source. The mass ratio of cyclodextrin-grafted lignin, deionized water, and polymer material was 3: 50: 8. S9. Specifically, it includes the following processes: S91. Strain activation: The Pseudomonas putida KT2440 strain stored at -80 °C was taken, and ice chips were scraped with a sterile pipette tip. The strain was picked and streaked on a solid medium, and cultured in a constant temperature incubator at 30 °C for 40 h. The preparation method of the solid medium was as follows: 5 g of peptone, 1 g of yeast extract, 27.5 g of sodium chloride, 0.7 g of potassium chloride, 5.4 g of magnesium chloride hexahydrate, 6.8 g of magnesium sulfate heptahydrate, 1.4 g of calcium chloride dihydrate, 0.2 g of sodium bicarbonate, 0.07 g of potassium bromide, 20 mg of boric acid, 5 mg of sodium silicate, 3 mg of sodium fluoride, 2 mg of ammonium nitrate, 1.5 g of potassium dihydrogen phosphate, 15 g of agar, made up to 1 L with distilled water, pH 7.5, sterilized at 121 °C for 20 min.

[0035] S92. Preparation of seed liquid: The activated strain was picked and inoculated into a liquid medium, and cultured by shaking at 30 °C and 200 rpm for 16 h to obtain a liquid seed. The preparation method of the liquid medium was as follows: 5 g of peptone, 1 g of yeast extract, 90 g of sodium chloride, 0.7 g of potassium chloride, 5.4 g of magnesium chloride hexahydrate, 6.8 g of magnesium sulfate heptahydrate, 1.4 g of calcium chloride dihydrate, 0.2 g of sodium bicarbonate, 0.07 g of potassium bromide, 20 mg of boric acid, 5 mg of sodium silicate, 3 mg of sodium fluoride, 2 mg of ammonium nitrate, 1.5 g of potassium dihydrogen phosphate, made up to 1 L with distilled water, pH 7.5, sterilized at 121 °C for 20 min.

[0036] S93. Liquid fermentation: According to the inoculation amount of liquid seed and fermentation medium at a volume ratio of 5 - 10: 100, the mixture was cultured by shaking at 30 °C and 150 rpm for 72 h to obtain a fermentation broth. The preparation method of the fermentation medium is as follows: 5 g of peptone, 1 g of yeast extract, 27.5 g of sodium chloride, 0.7 g of potassium chloride, 5.4 g of magnesium chloride hexahydrate, 6.8 g of magnesium sulfate heptahydrate, 1.4 g of calcium chloride dihydrate, 0.2 g of sodium bicarbonate, 0.07 g of potassium bromide, 20 mg of boric acid, 5 mg of sodium silicate, 3 mg of sodium fluoride, 2 mg of ammonium nitrate, 1.5 g of potassium dihydrogen phosphate, 10 g of glucose, 15 g of complex carbon source, add distilled water to make up to 1 L, adjust the pH to 7.5, and sterilize at 121 °C for 20 min.

[0037] S94. Extraction and purification: Take the fermentation broth after fermentation and centrifuge at 12000 rpm for 15 min to collect the thalli. Resuspend the thalli with deionized water, wash 3 times, and then freeze-dry. Grind the freeze-dried thalli thoroughly with a mortar. Add 10 mL of chloroform to each gram of the bacterial powder, extract at 60 °C for 10 h, filter by suction, take the chloroform phase, concentrate it in a rotary evaporator, add 5 volumes of absolute ethanol, let it stand at 4 °C for 20 h, then centrifuge to collect the solid precipitate, and dry it under vacuum to obtain polyhydroxyalkanoate.

[0038] Comparative Example 1 The difference from Example 2 is that the bamboo powder obtained by grinding in Step S1 is directly subjected to enzymatic hydrolysis without ozone pretreatment.

[0039] Comparative Example 2 The difference from Example 2 is that in the auxiliary agent described in Step S3, the mass ratio of Tween 80, polyethylene glycol 4000, bovine serum albumin, and dodecylbenzenesulfonic acid is 0:0.29:0.09:0.035.

[0040] Comparative Example 3 The difference from Example 2 is that in the auxiliary agent described in Step S3, the mass ratio of Tween 80, polyethylene glycol 4000, bovine serum albumin, and dodecylbenzenesulfonic acid is 0.29:0:0.09:0.035.

[0041] Comparative Example 4 The difference from Example 2 is that in the auxiliary agent described in Step S3, the mass ratio of Tween 80, polyethylene glycol 4000, bovine serum albumin, and dodecylbenzenesulfonic acid is 0.16:0.22:0:0.035.

[0042] Comparative Example 5 The difference from Example 2 is that in the auxiliary agent described in Step S3, the mass ratio of Tween 80, polyethylene glycol 4000, bovine serum albumin, and dodecylbenzenesulfonic acid is 0.16:0.13:0.125:0.

[0043] Comparative Example 6 The difference from Example 2 lies in that the preparation method of the fermentation medium is as follows: 5 g of peptone, 1 g of yeast extract, 27.5 g of sodium chloride, 0.7 g of potassium chloride, 5.4 g of magnesium chloride hexahydrate, 6.8 g of magnesium sulfate heptahydrate, 1.4 g of calcium chloride dihydrate, 0.2 g of sodium bicarbonate, 0.07 g of potassium bromide, 20 mg of boric acid, 5 mg of sodium silicate, 3 mg of sodium fluoride, 2 mg of ammonium nitrate, 1.5 g of potassium dihydrogen phosphate, 23 g of glucose. Add distilled water to make up the volume to 1 L, adjust the pH to 7.5, and sterilize at 121 °C for 20 min.

[0044] Comparative Example 7 The difference from Example 2 lies in that the cyclodextrin-grafted lignin prepared in step S6 is the composite carbon source.

[0045] Test Example 1 The extraction rate and purity of the lignin obtained from Examples 1-3 and Comparative Examples 1-5 were determined according to the standard method of the National Renewable Energy Laboratory of the United States (NREL). The data results are shown in Table 1 below.

[0046] Table 1 As can be seen from Table 1, the method for extracting lignin from enzymatically hydrolyzed bamboo powder used in Examples 1-3 of the present application has a high extraction rate and purity; in Comparative Example 1, no ozone pretreatment was carried out, and the enzymatic hydrolysis effect of cellulase on cellulose in bamboo powder was reduced, resulting in a significant reduction in both the purity and extraction rate of the prepared lignin; in Comparative Example 2, Tween 80 was not added as an auxiliary agent, and cellulase adsorbed on the surface of lignin, resulting in a reduction in the enzymatic hydrolysis effect of cellulase on cellulose in bamboo powder, and thus a reduction in the purity and extraction rate of the prepared lignin; in Comparative Example 3, polyethylene glycol 4000 was not added as an auxiliary agent, and cellulase adsorbed on the surface of lignin, resulting in a reduction in the enzymatic hydrolysis effect of cellulase on cellulose in bamboo powder, and thus a reduction in the purity and extraction rate of the prepared lignin; in Comparative Example 4, bovine serum albumin was not added as an auxiliary agent, and cellulase adsorbed on the surface of lignin, resulting in a reduction in the enzymatic hydrolysis effect of cellulase on cellulose in bamboo powder, and thus a reduction in the purity and extraction rate of the prepared lignin; in Comparative Example 5, dodecylbenzenesulfonic acid was not added as an auxiliary agent, and cellulase adsorbed on the surface of lignin, resulting in a reduction in the enzymatic hydrolysis effect of cellulase on cellulose in bamboo powder, and thus a reduction in the purity and extraction rate of the prepared lignin.

[0047] Test Example 2 The yields of polyhydroxyalkanoates obtained after culturing for 72 h and extracting and purifying in Examples 1-3 and Comparative Examples 1-7 are shown in Table 2 below.

[0048] Table 2 As shown in Table 2, a relatively large amount of polyhydroxyalkanoates was extracted in Examples 1-3 of this application. In Comparative Examples 1-5, due to the low purity of lignin, the yield of polyhydroxyalkanoates decreased; in Comparative Example 6, no lignin-containing composite carbon source was added, resulting in a significant decrease in the yield of polyhydroxyalkanoates; in Comparative Example 7, no p-dioxolane polymer was encapsulated, resulting in a decrease in the yield of polyhydroxyalkanoates.

[0049] The above has described in detail some embodiments of the present invention, but the content described is only the preferred embodiments of the present invention and cannot be considered as limiting the scope of implementation of the present invention. All equivalent changes and improvements made within the scope of the application of the present invention should still fall within the scope covered by the patent of the present invention.

Claims

1. A method for extracting polyhydroxyalkanoates by enzymatic hydrolysis of bamboo powder, characterized in that, It includes the following steps: S1. Take whole bamboo for processing to obtain bamboo powder; S2. Take the bamboo powder and pretreat it in an ozone atmosphere to obtain pretreated bamboo powder; S3. Take the pretreated bamboo powder and carry out enzymatic hydrolysis to obtain enzymatic hydrolysis residue; S4. Take the enzymatic hydrolysis residue and carry out lignin and hemicellulose separation treatment to obtain lignin; S5. Take the lignin and graft epichlorohydrin to obtain modified lignin; S6. Take the modified lignin and graft cyclodextrin to obtain cyclodextrin-grafted lignin; S7. Use 1,3-dioxolane as the raw material, add an initiator and a catalyst to prepare a dioxolane polymer; S8. Use cyclodextrin-grafted lignin to encapsulate the dioxolane polymer to obtain a composite carbon source; S9. Pick a strain and streak-inoculate it on a solid medium for activation. Pick the activated strain and inoculate it into a liquid medium, culture to obtain a liquid seed. Inoculate the liquid seed into a fermentation medium and culture to obtain a fermentation broth. Take the fermentation broth after fermentation and carry out extraction and purification to obtain the polyhydroxyalkanoate; The carbon source in the fermentation medium includes glucose and the composite carbon source.

2. The method for extracting polyhydroxyalkanoates by enzymatic hydrolysis of bamboo powder according to claim 1, wherein In step S2, the preparation method of the pretreated bamboo powder specifically includes the following steps: Take the bamboo powder and deionized water, mix and disperse ultrasonically, adjust the pH to 1.8 - 3.0, heat and stir, transfer to a reaction kettle, carry out treatment in an ozone atmosphere, and carry out vacuum filtration and washing to obtain pretreated bamboo powder.

3. A method for extracting polyhydroxyalkanoates by enzymatically hydrolyzing bamboo powder according to claim 1, characterized in that, In step S3, the preparation method of the enzymatic hydrolysis residue specifically includes the following steps: Take the pretreated bamboo powder, deionized water, cellulase, and an auxiliary agent, mix, adjust the pH to 5 - 6, carry out enzymatic hydrolysis, and filter and separate to obtain an enzymatic hydrolysis residue rich in lignin; The auxiliary agent includes Tween 80, polyethylene glycol 4000, bovine serum albumin, and dodecylbenzenesulfonic acid; The mass ratio of Tween 80, polyethylene glycol 4000, bovine serum albumin, and dodecylbenzenesulfonic acid is 0.08 - 0.25:0.08 - 0.18:0.06 - 0.12:0.02 - 0.

05.

4. A method for extracting polyhydroxyalkanoates by enzymatic hydrolysis of bamboo powder according to claim 1, characterized in that, In step S4, the preparation method of the lignin specifically includes the following steps: Take the enzymatic hydrolysis residue, zinc chloride, and methanol, add hydrochloric acid aqueous solution and formic acid aqueous solution, heat and stir in an oil bath under sealed conditions, then cool in an ice-water bath, and separate to obtain lignin.

5. A method for extracting polyhydroxyalkanoates by enzymatic hydrolysis of bamboo powder according to claim 1, characterized in that, In step S7, the initiator includes at least one of perchloric acid, perchlorate, and perchlorate hydrate.

6. The method for extracting polyhydroxyalkanoates by enzymatic hydrolysis of bamboo powder according to claim 1, characterized in that, In step S7, the catalyst includes at least one of boron trifluoride, boron trifluoride ethyl ether complex, boron trifluoride n-butyl ether complex, tin tetrachloride, titanium tetrachloride, aluminum trichloride, zinc chloride, vanadium trichloride, antimony trichloride, phosphorus pentafluoride, and antimony pentafluoride.

7. A method for extracting polyhydroxyalkanoates by enzymatically hydrolyzing bamboo powder according to claim 1, characterized in that, In step S9, the preparation method of the solid medium is as follows: peptone 5g, yeast extract 1g, sodium chloride 27.5g, potassium chloride 0.7g, magnesium chloride hexahydrate 5.4g, magnesium sulfate heptahydrate 6.8g, calcium chloride dihydrate 1.4g, sodium bicarbonate 0.2g, potassium bromide 0.07g, boric acid 20mg, sodium silicate 5mg, sodium fluoride 3mg, ammonium nitrate 2mg, potassium dihydrogen phosphate 1.5g, agar 15g, add distilled water to make up to 1L, pH is 7.5, sterilize at 121°C for 20 min.

8. A method for extracting polyhydroxyalkanoates by enzymatic hydrolysis of bamboo powder according to claim 1, characterized in that, In step S9, the preparation method of the liquid culture medium is as follows: 5 g of peptone, 1 g of yeast extract, 90 g of sodium chloride, 0.7 g of potassium chloride, 5.4 g of magnesium chloride hexahydrate, 6.8 g of magnesium sulfate heptahydrate, 1.4 g of calcium chloride dihydrate, 0.2 g of sodium bicarbonate, 0.07 g of potassium bromide, 20 mg of boric acid, 5 mg of sodium silicate, 3 mg of sodium fluoride, 2 mg of ammonium nitrate, 1.5 g of potassium dihydrogen phosphate. Add distilled water to make the volume up to 1 L, adjust the pH to 7.5, and sterilize at 121 °C for 20 min.

9. A method for extracting polyhydroxyalkanoates by enzymatically hydrolyzing bamboo powder according to claim 1, characterized in that, In step S9, the preparation method of the fermentation medium is as follows: 5 g of peptone, 1 g of yeast extract, 27.5 g of sodium chloride, 0.7 g of potassium chloride, 5.4 g of magnesium chloride hexahydrate, 6.8 g of magnesium sulfate heptahydrate, 1.4 g of calcium chloride dihydrate, 0.2 g of sodium bicarbonate, 0.07 g of potassium bromide, 20 mg of boric acid, 5 mg of sodium silicate, 3 mg of sodium fluoride, 2 mg of ammonium nitrate, 1.5 g of potassium dihydrogen phosphate, 10 g of glucose, 10 - 15 g of composite carbon source. Add distilled water to make the volume up to 1 L, adjust the pH to 7.5, and sterilize at 121 °C for 20 min.

10. A method for extracting polyhydroxyalkanoates from enzymatically hydrolyzed bamboo powder according to claim 1, characterized in that, The extracted polyhydroxyalkanoate is specifically poly-3-hydroxybutyrate.

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