| The use of bioethanol is an ideal solution for energy shortage and environment pollution. The problems in production are the cost and the shortage of raw material. In this study, two key technical improvements in bioethanol fermentation were addressed, very high gravity ethanol fermentation to reduce energy consumption and using lignocellulose as raw material.1. The influence of aeration conditions on very high gravity ethanol fermentation by flocculating yeastIn this work, flocculating yeast was used to convert300g/L glucose medium under five aeration conditions, including non-aeration, controlled-aeration regulated by redox potential (ORP) at-150mV and-100mV, constant aeration by pumping air at the rate of0.05vvm and0.2vvm. ORP was monitored under all conditions and taken as a criterion to distinct anaerobic, microaerobic and aeraobic conditions. The results showed that the least ethanol was produced under anaerobic condition and left the highest glucose unconverted in72h. Microaerobic fermentation achieved the accurate air supply depending on the real-time cell oxygen demand, which lead to higher ethanol productivity and yield. Aerobic fermentation brought about a quick biomass formation, and corresponding fast substrate utilization. However, too large aeration rate like0.2vvm caused the decrease of final ethanol and low yield due to the huge formation of biomass and by-product such as glycerol, reduced the carbon flux to ethanol. On the other hand, the lost of ethanol by air flow was highest under this condition. Moreover, it was observed that the flocculation quantified as particle size of floes was promoted by increasing the air supply. Oxygen may involve in the synthesis of cell plasma membrane which help cells to form big floes. In order to undertake a comprehensive evaluation for ethanol production and flocculation characteristic under different aeration schemes, a series of radar plots were illustrated based on data normalization. Constant aeration at0.05vvm was the preferable aeration condition thanks to its performance balance at all investigated aspects.2. Selected the strain to ferment the mixed sugar (glucose and xylose) and studied the effects of lignoeellulose degradation products on its mixed sugar fermentationK. marxianus1727which can ferment glucose and xylose was selected through reading a lot of literature and simple fermenting property evaluation. The inhibitory effects of four lignoeellulose degradation products and ethanol on glucosc and xylose fermentation by K. marxianus1727were studied in batch cultures. Acetic acid and formic acid were shown to be strong inhibitors of both glucose and xylose fermentation when they were added to the culture medium at the concentration of5.0and0.5g/L. Furfural and5-hydroxylmethyl furfural at the concentration of1g/L didn’t affect glucose fermentation. Ethanol was also shown to be strong inhibitor of xylose fermentation, when ethanol concentration was more than20g/L, biomass and xylose transformation efficiency were44%and73.9%of the control.3. Improved the xylose utilization rate by heterologous expression of SUT2in K. marxianus1727Sugar transporter gene (SUT2) was obtained by PCR amplification using Pichia stipitis genome as template. And the recombinant expression plasmid pH1O/pgk-SUT2was constructed using phosphoglycerate kinase (pgk) promoter to direct the expression of Sut2. After linearization by Not I, the expression plasmids were transformed into K. marxianus1727through electric shock and two clones HPS2/1and HPS2/2were selected. The glucose and xylose utilization rate by HPS2/2were improved by10%respectively than wild type. And IlPS2/2can ferment xylose under41℃. In batch cultures under anaerobic condition ethanol was4.81g/L, about1.4times to xylitol, and xylose utilization was speeded up under aeration condition. |
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