The Preliminary Study Of Poly-β-Hydroxybutyrate Producing Strain And Optimization Of Fermentation Conditions

Due to the increasing depletion of oil resources and the growing problem of "white pollution" caused by oil plastic products, Therefore, to accelerate the research and development of environment-friendly biodegradable plastic to achieve a breakthrough in the process, degradable plastics industry and practical, and has important practical significance for the prevention and control of environmental pollution and sustainable development.PHA, one of the three biodegradable plastics (starch-based plastic, PLA, PHA), is a kind of plastic produced in the condition that carbon and nitrogen are out of imbalance. As part of the PHA family, PHB has been the focus and hot spot of the study. In this study, soil samples were collected from the Nanning and other regions screened high-yielding strains of high-yield PHB, screened strain CK-5by Nile blue staining, PHB yield is3.4g/L of CK-5.In this experiment, by UV mutagenesis method, lithium chloride mutagenesis, UV joint lithium chloride mutagenesis strain CK-5obtained strain CK-U76, PHB yield is4.6g/L of CK-U76, than the strain CK-5PHB production increased by35.3%., identified by morphological observation and molecular biology, CK-U76was identified as Bacillus sp.Box-Behnken Design and response surface analysis is applied to optimize the fermentation conditions of CK-U76.Optimal fermentation conditions of CK-U76is:glycerol as the carbon source, carbon source concentration of10.98g/L, yeast extract and peptone as a combined nitrogen source, nitrogen source concentration of25.25g/L, pH8.05, fermentation temperature of20℃fermentation time of72h, the speed of180r/min, the liquid volume of150mL, inoculum size of10%. Under these conditions, the PHB yield of CK-U76reached15.9g/L, compared to un optimized4.6g/L increased246%.This experiment also makes an initial inquiry about the extraction and analysis methods of mutagenesis strain CK-U76. From the point of view of economic security, the freeze-thaw method is more appropriate, Extraction of PHB PHB purity PHB molecular weight on SDS method is most suitable for extraction method, and therefore this method is selected as the extraction method of the PHB. This experiment will make qualitative and quantitative analysis of CK-U76by UV spectrophotometry, high performance liquid chromatography,gas chromatography. UV spectrophotometry is a sensitive method for qualitative and quantitative analysis of PHB, at a wavelength of235nm.Quantitative analysis can be given to PHB when it exists at a lower concentration. Owing to the complex sample processing, the analysis result is susceptible to organic impurities, thus affecting its accuracy. Efficient liquid chromatography method is suitable for the orientation quantitative analysis of PHB with easy operation and high accuracy. The advantage of gas chromatography method lies in that the cells do not need to be processed and the peak figure is better, but the reaction time is relatively long and the middle of the process is difficult to control.This experiment breeding a strain of PHB CK-U76, identified by morphological observation and molecular biology, CK-U76was identified as Bacillus sp. Optimize the use of response surface methodology on CK-U76fermentation conditions, PHB production increased from4.6g/L to15.9g/L A preliminary study of its extraction conditions and analysis methods can provide certain reference value for the industrial production of PHB.