| With the growth of population and the intensification of industrialization,the consumption of traditional plastics produced from fossil fuels is increasing.These plastics can not be degraded in a short time after being discarded.It will also bring serious "white pollution" and "microplastic pollution" problems.Incineration will cause air pollution,water pollution and other environmental problems.Moreover,with the continuous depletion of fossil energy,there is an urgent need to find a "green plastic" to replace petroleum-based plastics.Poly-β-hydroxybutyrate(PHB)is a biodegradable polymer,which can be used as a substitute for fossil fuel-derived plastics because of its good biocompatibility.However,because of its high price and the disadvantage of non-renewable matrix,the output of industrial PHB has been lower than that of traditional plastics.Therefore,the synthesis of PHB using sustainable and cheap substrates is of great significance to reduce the production cost of PHB.Agricultural and forestry by-products and industrial by-products can be converted into carbon sources for microbial growth through treatment.Molasses is an industrial by-product that contains high concentrations of fermentable carbon sources.Lignocellulose from agroforestry by-products is a kind of renewable resource,and the fermentable sugar produced by pretreatment,detoxification and enzymatic hydrolysis can be used as a substrate for microbial growth.However,the low enzymatic hydrolysis efficiency of cellulase is the key factor to prevent its biotransformation to achieve large-scale,and the hydrophobic structure on the surface of protein auxiliaries can combine with the hydrophobic part of lignin to reduce the adsorption of lignin to cellulase.In this paper,protein auxiliaries were used to improve the efficiency of enzymatic hydrolysis of poplar fiber by cellulase,and the synthesis of PHB by Aeromonas hydrophila using poplar fiber and molasses as substrate was systematically studied.the main results are as follows:(1)Explore the effect of protein additive on the enzymatic hydrolysis of alkali-treated poplar fiber.The contents of three major components of NaOH treated poplar fiber were determined,and then casein,bovine serum protein,collagen and peptone were added during the enzymatic hydrolysis of alkali treated poplar fiber,and their effects on enzymolysis efficiency and cellulase thermal stability were discussed.It was found that all the four proteins could improve the enzymolysis efficiency and enhance the thermal stability of cellulase,among which casein had the best effect,and the yield of reducing sugar was 11.94%higher than that of the control group.The reaction conditions for the degradation of poplar fiber by casein combined with commercial cellulase were optimized by single factor experiment and response surface optimization method.The optimum reaction conditions were as follows:enzymatic hydrolysis pH5.0,enzymatic hydrolysis time 66 h,enzyme addition 37mg/g,temperature 50℃.Under the optimum conditions,the conversion amount of reducing sugar was 417.65mg/g.(2)The fermentation process for the synthesis of PHB by Aeromonas hydrophila based on poplar fiber and molasses was optimized.Fermentable sugar was prepared by hydrolysis of poplar fiber with sulfuric acid pretreatment molasses and cellulase,and Aeromonas hydrophila used it to synthesize biodegradable PHB.It was found that the mixed carbon source formed by poplar fiber and molasses was more suitable for PHB accumulation.The fermentation conditions(carbon source ratio,carbon source concentration,ammonium sulfate concentration,pH,inoculum size)were optimized,and the growth curve and PHB accumulation curve of Aeromonas hydrophila under the optimum fermentation conditions were determined.The optimum fermentation conditions are as follows:carbon source ratio 2:3,carbon source concentration 15g/L,ammonium sulfate concentration 1.5g/L,pH7.0,inoculation amount 7%,and the highest yield is 0.32g/L.(3)In order to reduce the extraction cost of PHB,the extraction method of PHB was discussed.The results showed that freezing and thawing could make cells break up quickly,and SDS could degrade protein and nucleic acid in cell fragments,and the extraction conditions were discussed,and it was found that freeze-thaw times,acetone treatment time and SDS concentration had little effect on the extraction amount of PHB,but the extraction temperature had a great effect on it,and the optimum extraction temperature was 60℃. |
