Development And Optimization Of Integreted Process For Production Of Xylose And Ethanol Using Xylose Crystallization Mother Liquor

The xylose crystallization mother liquor is the main byproduct in the production of xylose.Its main components are xylose,arabinose,glucose and galactose.Further comprehensive utilization of xylose crystallization mother liquor has important industrial demand and application value for the healthy development of the industrial system of producing xylose and xylitol with corn cob as the main raw material,improving its economic benefits and improving its environmental friendliness.For this reason,this paper explored the use of newly constructed strains to bio-transform the main monosaccharide from xylose crystallization mother liquor(except xylose),into ethanol and recover the xylose at the same time,which can form a new technical solution for the comprehensive process of xylose crystallization mother liquor.This article obtained following beneficial results:(1)Analysis of monosaccharide metabolism characteristics of ethanol-producing E.coli without consuming xylose.A series of evaluations were performed on the transport and metabolism of xylose in the recombinant E.coli B0013-2012PA.It was confirmed that the strain did not grow on the M9 plate with xylose as the sole carbon source.In the liquid M9 medium containing xylose and glucose as a carbon source,Xylose was not utilized and cell break analysis showed that the xylose in the medium was not transported into the cell.A series of evaluations were performed on the metabolism of recombinant E.coli B0013-2012PA using glucose,arabinose,and galactose.It can grow on M9 plates with glucose,arabinose and galactose as sole carbon sources.Under aerobic shake flask culture conditions,the growth rate of the bacterial cells in M9 liquid medium with glucose,arabinose,and galactose as sole carbon sources was 0.28 g/(L·h),0.26 g/(L·h),0.31 g/(L·h),respectively.And specific growth rates were 0.243 h-1,0.296 h-1 and 0.237 h-1,respecti-vely.A series of evaluations were performed on the characteristics of the metabolism of recombinant E.coli B0013-2012PA using glucose,arabinose and galactose to produce ethanol by anaerobic fermentation.After aerobic incubation for 12 h to obtain highly active bacteria,the ethanol productivity of glucose,arabinose,and galactose were 0.48 g/(L·h),0.39g/(L·h),and 0.31 g/(L·h),respectively.The inhibition of the growth of xylose,glucose,arabinose,galactose,xylose crystallization mother liquor and ethanol was investigated.The minimum inhibitory concentrations were 13.7%,16.3%,11.9%,15.6%,14.1%and 4.3%respectively.Combined with the process characteristics,the tolerability of xylose,xylose crystallization mother liquor and ethanol under the anaerobic fermentation conditions of recombinant E.coli B0013-2012PA was investigated.Concentrations were 16.7%,14.6%,and 6.3%,respectively.(2)Development and optimization of ethanol fermentation process.A series of evaluations were performed on the process parameters of the xylose crystallization mother liquor as raw materials produce xylose and ethanol under shake flask condition.The optimum culture medium was determined as follows:M9 basic medium was added with final concentration of 2.0 g/L ammonium chloride and 5.0 g/L yeast extract,the initial amount of xylose crystallization mother liquor was 1.5%of the fermentation volume;the optimum process was determined under aerobic culture conditions,37?,200 r/min,cultured for 12 hours;After cultivation,the culture was continued at 40? until the end of fermentation.Under optimal conditions,the amount of xylose mother liquor supplemented was 10%of fermentation volume.The strain consumed 14.1 g/L glucose,19.4 g/L arabinose,and 5.6 g/L glucose to produce ethanol 18.0 g/mL,the conversion rate of mixed sugar to ethanol was 45.9%,and ethanol productivity was 1.19 g/(L·h),which was 3.5 times higher than control group(initial ethanol productivity was 0.34 g/(L·h));The xylose content retained in the fermentation broth was 33.0 g/L;the proportion of xylose in the total sugar in the fermentation broth increased from 57%to 97%,which contributed to the separation and extraction of xylose as a product.Combining the medium composition and fermentation process parameters determined under shake flask conditions,under the 5 L fermenter system,a dual-phase fed-batch feeding process was used.The initial amount of xylose crystallization mother liquor was 10%of the fermentation volume and aerobic culture at 37?.The cells were cultured for approximately 8 h,and then transferred to the anaerobic ethanol fermentation stage.The xylose crystallization mother liquor totaling 900 mL was added three times,and the ethanol accumulation amount was 32 h at 40?.The yield and ethanol productivity were 51.0 g/L and 1.59 g/(L·h),respectively.The conversion rate of total sugar to ethanol was 47.2%except for xylose,and the accumulation of xylose was 113 g/L.Xylose occupied ratio of total sugar in fermentation broth is 98.2%.The experimental results show that the xylose crystallization mother liquor as raw material can be produced by the recombinant E.coli B0013-2012PA fermentation.