Microbial Production And Aqueous Two-phase Extraction Of 2,3-Butanediol From Dioscorea Zingiberensis

Bioconversion of Dioscorea zingiberensis to produce 2,3-butanediol and its separation were explored in this thesis.Firstly,the conditions for liquefaction and saccharification of Dioscorea zingiberensis were studied.The optimal liquefaction conditions were Dioscorea zingiberensis particle(≤60 mesh),ratio of 1:4(w/w) for materials to water,α-anylase 10 U/g,80～90℃(30min),and the optimal saccharification conditions were pH 4.0～4.5,glucoamylase 120 U/g,58～60℃(120 min).High concentration of reducing sugar was obtained from the liquedied material. and the diosgenin was obtained by the microbial transformation from saccharified residue of Dioscorea zingiberensis.The dioscin loss was only 1.3%in the saccharification process. which was important for the decrease of pollution in industrial diosgenin manufacturing and the value-increment of Dioscorea zingiberensis.Secondly,the production of 2,3-butanediol by Klebsiella pneumoniae CICC 10011 with the hydrolyzate of Dioscorea zingiberensis was investigated.The results showed that the hydrolyzate of Dioscorea zingiberensis was good substrate for 2,3-butanediol production by K.pneumoniae.The organic acids in Dioscorea zingiberensis could promote the metabolic flux in TCA and acetic acid production pathway and decrease that in butane diacid production pathway,thus,the 2,3-butanediol yield was increased.80.2 g/L of 2,3-butanediol,86.2 g/L of 2,3-butanediol and acetoin,and 1.54 g/(L·h) of productivity were achieved using the hydrolyzate of Dioscorea zingiberensis as substrate and feeding glucose during fed-batch fermentation,with an enhancement of 8.5%,7.4%and 7.7%,respectively,compared with that using glucose as sole substrate.Finally,the separation of 2,3-butanediol from fermentation broth was studied.Aqueous two-phase systems of ethanol and potassium carbonate,and salting-out system of potassium carbonate were applied.The optimal conditions for aqueous two-phase extraction of 2,3-butanediol were ethanol 22%(w/w),potassium carbonate 26%(w/w) for Dioscorea zingiberensis fermentation broths,ethanol 25%(w/w),potassium carbonate 28%(w/w) for glucose broth.The recovery ratios of 2,3-butanediol,acetoin,reducing sugar were 97.2%. 100%,87.1%respectively,the removal ratios of cells and proteins were 98.9%and 94.23%in the former broth,while 98.2%,100%,92.2%,99.5%,95.4%with the latter broth in the optimal conditions respectively.The optimal condition for salting-out of 2,3-butanediol was potassium carbonate 49.5%(w/w).Under this condition,the recovery ratio of 2,3-butanediol was found to be 93%and a concentration of 724.8 g/L 2,3-butanediol in top phase was obtained with the former broth by membrane.With potassium carbonate 48.5%(w/w),90.0% 2,3-butanediol(713.6 g/L) was recovered using the latter broth by membrane.These results demonstrated that aqueous two-phase extration with ethanol and potassium carbonate,and salting-out with potassium carbonate were effective for the separation of 2,3-butanediol.Furthermore,the recovery of potassium carbonate was tried.Over 88.6%potassium carbonate was precipitated by pumping CO₂ into salt-rich phase,followed by 40%(w/w) methanol.If recovering potassium carbonate with vented gas during the 2,3-butanediol fermentation process,it is promising both for treatment of vented gas and preparation of pure CO₂.