| Lignocellulose is the most abundant and inexpensive renewable resources.Bio-ethanol produced from lignocellulosic feedstock has been greatly concerned.Xylose is the second largest sugar in lignocellulosic hydrolysates,therefore,its effective utilization is one of the prerequisites to achieve the industrialization of lignocellulosic ethanol.Spathaspora passalidarum,a recently isolated native xylose-fermenting yeast,can ferment xylose faster than glucose,and can co-ferment glucose,xylose,and cellobiose.In this study,the productivity of Spathaspora passalidarum for ethanol production with xylose was examined,besides,the genetic genetic transformation system for Spathaspora passalidarum was also established.Furthermore,the gene expression in Spathaspora passalidarum by this system was conducted,which lays a foundation for bio-transformation of xylose to ethanol by Spathaspora passalidarum.The main research contents were listed as follows:(1)The influence of the fermentation medium and fermentation conditions on xylose fermentation to ethanol was explored,the optimized fermentation medium were:xylose of 100 g/L,corn steep of 15 g/L;the fermentation conditions were:temperature of 30?,inoculation size of 7%(v/v),initial pH of 5.5,rotation rate of 120 r/min,and liquid volume 100 mL/250 mL.Under the optimized conditions,39.04 g/L of ethanol production was obtained,the ethanol yield was 0.40 g/g,xylose utilization rate was 100%,the ethanol production rate was 0.325 g/(L·h).(2)On the base of fermentation medium optimization in the shake flask,the effects of pH,ventilation ratio and rotational speed on xylose fermentation to ethanol by U-30 in the 1 L fermentor were further discussed.There was few difference of the biomass and ethanol production when controlled the pH of 5.5 and controlled the initial pH of 5.5,while the ventilation ratio and rotational speed have great influence on the xylose fermentation.When the conditions were:rotational speed of 150 r/min,ventilation ratio of 0.125 vvm,40.2 g/L of ethanol production was obtained,the ethanol yield was 0.406 g/g,the ethanol production rate was 0.744 g/(L·h).Compared with the fermentation in the shake flask,the fermentation period was obviously shortened in the fermentor.(3)A genetic transformation system using KanMX resistance gene as a selectable marker and the Saccharomyces cerevisiae 2? replicon as a replication origin via the lithium acetate transformation method for Spathaspora passalidarum was established.This paper proved that the 2? replicon can be used as replication origin for Spathaspora passalidarum.Several important parameters affecting transformation efficiency were optimized;these parameters included the strain growth period,heat shock time,lithium acetate concentration,and the amount of plasmid DNA.With the cells in the mid-logarithmic growth phase(OD600 about 15 at this time),plasmid added in an amount of 2.5 ?g,the heat shock time of 20 min,lithium acetate at a concentration of 0.5 mol/L,the transformation efficiency reached 170±30 cfu/?g.(4)The research of XUT1 gene expression in Spathaspora passalidarum was conducted by the established system.The recombinant strains obtained were compared with the parent strain for the xylose fermentation.Fermentation test showed that basic fermentation performances such as xylose utilization rate and ethanol production were not affected.In addtion,the mRNA levels of XUT1 in U-TXK3 measured by qRT-PCR were 4.6 times larger than that of parental strain U1,which preliminary confirmed the effectiveness of this transformation system. |
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