Strain Development And Process Optimization For Butanol Production From Corn Stover

Butanol is regarded as one of the most promising biofuels due to its superior properties.Butanol is typically produced by acetone-butanol-ethanol?ABE?fermentation from renewable resources.Although ABE fermentation is one of the oldest large-scale fermentative processes,biorefining of butanol is limited to high cost of raw materials,low sugar utilization as well as low butanol yield,making ABE fermentation not economically competitive.The research optimized the hybrid pretreatment strategy of corn stover to improve sugar yield and transformed Clostridium beijerinckii BA101 by genetic modification to explore ABE fermentation performance from corn stover hydrolysate.Due to the high recovery rate of hemicellulose by dilute H₂SO₄ and high removal rate of lignin by aqueous ammonia,corn stover was first pretreated by dilute H₂SO₄ and then by aqueous ammonia.Xylose and arabinose were retained in the acid hydrolysate after first step of pretreatment,and the lignin was removed during the second step of pretreatment,leading to a sharp increase on cellulose content of corn stover residuals.In order to improve the sugar yield,the research optimized the conditions of aqueous ammonia pretreatment.It was found that when the dilute acid pretreated corn stover was pretreated by 10wt.%aqueous ammonia at80°C for 24 h,a relatively high reducing sugar concentration of 51.27 g/L was obtained.At the same time,the maximum concentration of butanol and ABE were obtained by ABE fermentation,which were 9.36 g/L and 13.96 g/L,respectively.Under optimal hybrid pretreatment conditions,the recovery rate of hemicellulose and lignin removal rate reached to82.89%and 86.77%,respectively,and the cellulose content of copretreated corn stover improved to 76.14%.Most of recent researches neutralize the pretreated lignocellulose by washing,resulting in the generation of plenty of wastewater and high cost.Therefore,it's crucial to optimize the washing times of pretreated corn stover.The research found that the optimization of washing times can not only enhance enzymatic hydrolysis and ABE fermentation,but reduce the amount of water usage.Compared to unwashing,the sugar yield increased from 346.04 mg/g-corn stover to 401.76 mg/g-corn stover and the butanol yield improved from 9.33 g/L to 10.89 g/L,when the corn stover was washed once after each step of pretreatment.On the other hand,compared to the conventional dilute acid or dilute alkali pretreatment method,the amount of wastewater generated during the optimized hybrid pretreatment was reduced from 2.11 g/L-butanol and 3.46 g/L-butanol to 0.83 g/L-butanol,respectively.To enhance ABE fermentation of Clostridium beijerinckii BA101,gene cbei₀109,adhE,ald-ctfAB were overexpressed,and gene cbei₃304 was inactivated in BA101 respectively.The research investigated the engineered strains in fermenting hydrolysate,obtained from hybrid pretreated corn stover,and found that the knockout stain BA101-?3304 had a similar fermentation performance to BA101.The overexpressed strains BA101-0109,BA101-adhE and BA101-ald-ctfAB produced more by-products of ethanol and acetone,and obtained enhanced capacities of acetic acid synthesis and butyric acid reabsorption.