Breeding Of Strains For Efficiently Producing PHAs From Xylose And Study Its Application

The large-scale use of plastic products brings convenience to people's lives on the one hand,and causes serious environmental pollution on the other.The use of degradable plastics to replace non-degradable petrochemical plastics is a very important measure to solve the problem of plastic pollution,which has attracted more and more attention.As a new type of biological material,Polyhydroxyalkanoates(PHAs)can be degraded in a short period of time in the natural environment in addition to the characteristics of traditional plastics,and hardly cause environmental pollution,so it is a new type of excellent environmental protection material with great potential It is expected to replace petrochemical plastics and reduce the pollution and damage of plastic waste to the environment.However,traditional PHAs fermentation has high raw material cost,which greatly limits the large-scale production of PHAs.Using cheap and renewable lignocellulosic raw materials such as crop straws and forestry wastes as fermentation carbon sources can effectively reduce the production cost of PHAs and realize the reuse of waste resources.Whilein the process of producing PHAs by fermenting lignocellulose as raw material,the xylose produced by its hydrolysis is difficult to be used by microorganisms,and xylose is a very important component in the lignocellulose hydrolysate.Therefore,it is necessary to improve the effect of microorganisms on xylose.The conversion efficiency of lignocellulose is the key to realize the efficient utilization of lignocellulose fermentation to produce PHAs.This dissertation will focus on this problem.First,isolate strains that can efficiently use xylose to synthesize PHAs from soil samples,and then use screening strains as starting strains to use UV and methyl sulfonate compound mutagenesis methods to further improve The strain uses the ability of xylose to produce PHAs,and then optimizes the fermentation process parameters of the strain to obtain the best fermentation conditions.Finally,lignocellulose is used as a raw material for fermentation.The specific research content and results are as follows:(1)Screening,isolation,identification and characterization of strains that efficiently use xylose to produce PHAs: Using xylose as the sole carbon source,using Nile red fluorescent staining and Sudan black staining methods,a strain of wood that can be fermented efficiently is selected from the natural environment The sugar-producing PHAs strain3Y-17,using 16 S r DNA sequence homology comparison combined with morphological observation and related physiological and biochemical tests,preliminarily identified the bacteria as Paraburkholderia.Further fermentation studies on the bacteria showed that the cell growth rate and PHAs yield(YP/S)of the bacteria were 0.03 g/L/h and 0.15 g/g xylose,respectively.Finally,Fourier infrared spectroscopy(FIRT)and nuclear magnetic(NMR)were used to analyze the products obtained from the fermentation of the bacteria,and the main components of the PHAs products obtained were preliminarily identified as PHB.(2)Mutagenesis breeding of Paraburkholderia sp.3Y-17:Paraburkholderia sp.3Y-17 was mutagenized by ultraviolet,methyl sulfonate(EMS)and the compound mutagenesis method of the two respectively.Based on the wild strain Paraburkholderia sp.3Y-17,a xylose strain was obtained.Utilizing the better mutant strain Paraburkholderia sp.UVEM-20,compared with the original starting strain,the mutant strain's biomass and PHAs yield per unit time are increased by 76.67% and 42.49%,respectively,and the genetic characteristics are stable.(3)Optimization of fermentation parameters of the mutant strain Paraburkholderia sp.UVEM-20 to produce PHAs from xylose:Taking the mutant strain Paraburkholderia sp.UVEM-20 as the research object,the best fermentation nitrogen source was determined by single factor experiment as 1g/L urea,and on this basis,4 important factors affecting PHV production were determined by single factor experiment.For temperature,p H,inoculum volume,and rotational speed,the Box-behnken four-factor three-level response surface test was designed to optimize the optimal culture conditions as rotational speed 210.07rpm/min,initial p H 6.97,inoculation volume 14.97%,and temperature31.05?,and the fermentation verification experiment proved that the optimized conditions are beneficial to increase the fermentation capacity of the strains,and finally the output of PHAs was increased by 18.23%compared with that before optimization.(4)Study on the production of PHAs by the mutant strain Paraburkholderia sp.UVEM-20 by fermentation of corn stover hydrolysate:First,the pretreatment conditions of corn stalks were optimized,and the best pretreatment conditions were determined as follows: liquid-to-material ratio 1:10,hydrolysis time 120 min,hydrolysis temperature 120?,and sulfuric acid concentration 1.4%.Under these conditions,the hydrolysate was prepared to investigate the use of the hydrolysate to produce PHAs by fermentation of Paraburkholderia sp.UVEM-20.The results showed that when the bacteria directly used the hydrolyzed liquid without detoxification for fermentation,its PHAs yield was much lower than that of pure sugars.After the hydrolysate was detoxified,the dry cell weight and PHAs yield increased from 1.21 g/L and 0.39 g/L to 5 g/L and 3.1 g/L,respectively.The result proves that detoxification treatment is a necessary step when the corn stover hydrolysate is reused for fermentation.Finally,the performance of the fermentation product was tested,and it was found that the physicochemical and thermal properties of PHB produced by Paraburkholderia sp.UVEM-20 can satisfy the processing and modification of a variety of commercial products.The conclusions obtained in this study will provide technical and theoretical reference for the efficient use of lignocellulosic raw materials to produce PHAs.