【作者】 邹韫智；

【导师】 钟建江；

【作者基本信息】 上海交通大学 ， 生物化工， 2009， 硕士

【摘要】 木质纤维素是自然界含量丰富的可再生资源,可在水解后被微生物发酵生产木糖醇、乙醇等有用产品。然而其水解产生的抑制物阻碍微生物的生长与发酵,限制了该资源的有效利用。我们利用从土壤中筛选得到的耐受木质纤维素水解液抑制物的季氏毕赤酵母来发酵木糖产木糖醇,在添加了较高浓度的木质纤维素水解液主要抑制物的培养基中考察了接种量、碳源以及抑制物浓度对其木糖醇发酵的影响。同时研究了不同初始KLa对于季氏毕赤酵母生长、木糖消耗和木糖醇产量的影响,并考察了木糖代谢的关键酶——木糖还原酶、木糖醇脱氢酶和葡萄糖-6-磷酸脱氢酶的酶活性变化。研究结果显示,在添加木质纤维素水解液抑制物(糠醛、羟甲基糠醛和乙酸)的培养基中,接种量约1.8g l-1与葡萄糖:木糖比例(g:g)小于1:10的条件有利于季氏毕赤酵母发酵木糖生产木糖醇。即使在较高浓度抑制物(2g l-1糠醛、2g l-1羟甲基糠醛和3g l-1乙酸)存在条件下,该菌也能迅速降解糠醛和羟甲基糠醛,与无抑制物添加的对照相比,其木糖醇生产能力仍能维持90%以上。同时发现该酵母对氧供应敏感,在一个极低的供氧条件下(初始KLa=0.075h-1),木糖醇的产量(12.0g l-1)和对木糖的得率(0.61g g-1)达到最大。在低初始KLa(0.033-0.37h-1)范围内,葡萄糖-6-磷酸脱氢酶的酶活随KLa增大而提高;低初始KLa条件使木糖醇脱氢酶活性降低,从而抑制了木糖醇降解。另外,在该季氏毕赤酵母中观察到木糖还原酶的酶活低于木糖醇脱氢酶的酶活。与此同时,我们还筛选获得了一株遗传背景较为清晰能耐受木质纤维素水解液抑制物的细菌,成功构建了带有Zymomonas mobilis的pdc基因和adhB基因的质粒,为今后对其进行面向产乙醇的代谢工程改造奠定了一定基础。更多还原

【Abstract】 Lignocellulosic materials are cost-effective resources for bio-production of value-added products such as xylitol and ethanol. However, the microbial utilization of lignocellulose is limited because of the toxic compounds present in the hydrolysate. In this work, a lignocellulosic inhibitory compounds-resistant strain, Pichia guilliermondii, was isolated and was used for xylitol production from xylose. Different inoculation sizes, mixed carbon sources, and concentrations of inhibitors were investigated. After optimization of these fermentation conditions, oxygen supply conditions, i.e., various initial volumetric oxygen transfer coefficient (KLa) values, and the behavior of key enzymes of xylose metabolism, i.e., xylose reductase (XR), xylitol dehydrogenase (XDH) and glucose-6-phosphate dehydrogenase (G6PD), were studied in detail. The results indicated that an inoculation size of 1.8g l-1 and a glucose:xylose ratio (g:g) of less than 1:10 was favorable to the P. guilliermondii fermentation in the presence of lignocellulosic inhibitory compounds. In culture medium with high concentration of inhibitors (2g l-1 furfural, 2g l-1 5-hydroxymethyl furfural and 3g l-1 acetic acid), this yeast degraded furfural and HMF rapidly and maintained the fermentation ability of more than 90% compared to the control without inhibitors addition. This strain was found to be very sensitive to oxygen supply during xylose fermentation. A very low initial KLa value, i.e., within the range of 0.033-0.37h-1, showed a significant effect on the cell growth, substrate uptake, xylitol production as well as the activities of key enzymes (XR, XDH and G6PD). The highest xylitol yield on xylose (0.61gg-1) was attained at an initial KLa value of 0.075h-1. The G6PD specific activity was enhanced with the increase of intail KLa; the very low initial KLa inhibited the activity of XDH, thus inhibited the degradation of xylitol. Furthermore, a XR/XDH ratio <1 was found in this yeast during xylitol production.At the same time, a lignocellulosic inhibitory compounds-resistant bacterium was screened out with its known genomic background. A plasmid with pdc and adhB from Zymomonas mobilis was constructed in the aim of its genetic engineering towards ethanol production.更多还原