| The output of lignocelluloses materials, which is renewable, is huge. To use it produce uesful compound describes a beautiful picture of green economy. It is necessory to develop the relevant technologies and patterns. Oil palm empty fruit bunches (EFB) and xylose mother liquor are readily available industrial waste that are cheap and easier to obtain. Its comprehensive utilization can improve the value of waste and increase the economic benefit. Saccharomyces cerevisiae is a widely used microorganism in food grade industrial fermentation, which has several obvious advantages such as widely substrate range, strong fermenting power, robustness, food stuff security, and mature genetic operation tools. The S. cerevisiae as cell factories were constructed by introducing appropriate metabolic pathways to realize high value conversion of industrial waste materials after optimizing the fermentation process. In this thesis, the technologies of producing the the second generation fuel ethanol using EFB and producing the xylitol using xylose mother liquor were studied in three levels: the selection of suitable strain, the introduction of metabolic pathway, and the optimization of the fermentation process. The result will promote the industrialization.1. Study of fuel ethanol production process using Oil palm empty fruit bunches (EFB) as raw materialThe abundant EFB is produced with the operation of large-scale factories that produce palm oil for each year. The comprehensive utilization of palm fruit can not only improve the economic benefits of palm oil production factory but also reduce the pressure of environmental protection. Cooperating with the Malaysian Palm Oil Board (MPOB), the first part of this thesis foucos on evaluating the toxicity of different methods pretreated EFB and the feasible fermentation process. The component analysis of dilute-acid plus sodium sulflte, alkali soaking, and formic acid pretreated EFB results show that the formic acid pretreatment could remove most of hemi-celluloses and lignin in the EFB, only left the cellulose. The final ethanol concentration is high in the fermented broth. However, the fermentation raw material obtained by this method has relative high toxicity, therefore it needs a lot of water to wash for detoxification. Alkaline pretreated EFB change the pH during the saccharification by cellulase, which affects the fermentation efficiency. Sodium sulfite pretreatment removed part of lignin, increase proportion of cellulose, but lost a part of hemi-cellulose. An industrial S. cerevisiae strain RC212 was chosen because of it high tolerance to pretreated EFB. The initial xylose metabolic pathway was introduced into RC212 result in RC212-127. The condition of 10% concentration of substrate 30 FPU/(g substrate)-1 cellulase was used in the further evaluation experiments. The effects to the final ethanol concentration of fermentation process, including SHF, SSF and semi-SSF were compared each other using RC212-127. The result suggested that semi-SSF has highest ethanol yield. In the semi-SSF with 10%(w/v) substrate concentration of sodium sulfite pretreated EFB, the final ethanol concentration achieved 16 g L-1, and the ethanol yield is 0.16 g g-1 after 60 hours fermentation. When the additional material was feed at 6 and 12 hours after the fermention start in the feed-semi-SSF of formic acid pretreated EFB, the final ethanol production reached to 56 g L-1, which could meet the needs of industry to ethanol distillation. For the other two kinds of methods pretreated meterials, the more efficient xylose utilization strain is necessory to increase the final ethanol concentration.2. High-value utilization of of xylose mother liquorXylose mother liquor was the by-products of xylose production from corn cob material. Cooperating with Shandong Longlive Bio-Technology Co.,Ltd, the second part of this thesis is focus on the high value utilization of xylose mother liquor. The component analysis result shows that the xylose mother liquor contains as high as 400 g L-1 xylose and additional high concentrate glucose, arabinose, and galactose. In our design, the fermention will keep the arabinose and galactose components, consume the glucose component, and convert the xylose component to xyltiol, so that recover the high value sugars in the xylose mother liquor. The S.cerevisiae GZ-5 has high tolerance to dilute xylose mother liquor was selected for this work. The gene GAL1, which encoding the first enzyme in galactose metabolic pathway, was knockout to block the galactose metabolism of GZ-5.Then several copies of the Scheffersomyces(Pichia) stipitis XYL1 gene encoding the xylose reductase was integrated into the chromosome of GAL1 delected GZ-5. After seven times of integration, the XR activity of the result strain DX7 reached to 0.5 U/mg. The fermentation process for xylitol production was optimized. With constant feeding of 2.25 g L-1h-1 glucose after 36 hours fermentation of simulative 20% of xylose mother liquor (containingy100 g L-1 xylose, ～15 g L-1 glucose),85% xylose was consumed and the xylitol yield was 0.88 g g-1, when the fermentation finished at 60 hours. With constant feeding of 0.75 g L-1h-1 glucose after 8 hours fermentation of simulative 20% of xylose mother liquor, similar xylose was consumed and the xylitol yield was 0.76 g g-1, when the fermentation finished at 60 hours. |
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