2,3-Butanediol Production By Klebsiella Oxytoca ZU-03 Fermentation

2,3-butanediol is a widely-applied chemical and fuel.Lignocellulosic biomass is a cheap,renewable,abundantly available resource,and its bioconversion to 2,3-butanediol is very meaningful to the sustaining development of our society and economy.In this dissertation,key technologies in 2,3-butandiol fermentation from glucose,xylose and lignocellulosic biomass including pretreatment,enzymatic hydrolysis of corncob and domestication of Klebsiella oxytoca ZU-03 were investigated.The parameters of Klebsiella oxytoca ZU-03 fermentation on glucose were studied in shaking flask.Under optimized conditions:glucose 160g/L,corn liquor 6g/L,inoculum 10%(V/V),initial pH 6.5 and 30℃,the concentration of 2,3-butanediol reached 74.2g/L and the yield of 2,3-butanediol from glucose was 0.46g/g.2,3-butanediol could be also produced by Klebsiella oxytoca ZU-03 fermentation on xylose,the main reducing sugar in hemicellulosic hydrolysate.Under optimized conditions:xylose 90g/L,corn liquor 6g/L,inoculum 10%(V/V),initial pH 5.5 and 30℃,the concentration of 2,3-butanediol reached 36.2g/L and the yield of 2,3-butanediol from xylose was 0.40g/g.The research results are meaningful in bioconversion of renewable hemicellulosic resources.However,Klebsiella oxytoca ZU-03 was sensitive to inhibitors such as 0.25 g/L acetic acid or higher.It was found that the recombinant Klebsiella oxytoca ZU-03 could effectively ferment 80 g/L glucose and 50 g/L xylose to 2,3-butandiol,and the consuming rate of xylose was increased from 87.0%to 96.6%.Corncob was pretreated by diluted acid and sodium hydroxide,respectively,to enhance its enzymatic susceptibility before cellulase hydrolysis.It was found that the reducing sugar concentration in hydrolysate reached 61.9g/L,yielding 84.5%under acid pretreatment,while 73.5g/L,yielding 81.2%under sodium hydroxide pretreatment.Pretreatment by sodium hydroxide was chosed.Fed-batch hydrolysis was performed to increase the reducing sugar concentration,which could help to obtain higher 2,3-butanediol concentration in subsequent fermentation,while maintaining a relatively acceptable hydrolysis yield.High concentration of salt in hydrolysate had inhibiting effects on producing 2,3-butanediol.The domestication of Klebsiella oxytoca ZU-03 could enhance its tolerance to inhibitors.By domestic Klebsiella oxytoca ZU-03 fermentation on hydrolysate with 121.8g/L reducing sugar(76.8 g/L glucose and 45 g/L xylose)at initial pH 6.0 for 108h,the concentration of 2,3-butanediol could reach 55.4g/L, which yields 0.45g/g.Producing 2,3-Butanediol by Klebsiella oxytoca ZU-03 fermentation on cellulase hydrolysate had high economical and social value,which had never been studied in our country until now.By domestic Klebsiella oxytoca ZU-03 fermentation on hydrolysate,yields of 2,3-butanediol reach 0.45g/g.Still,researches in this dissertation were very meaningful to bioconversion of renewable cellulosic resources.