【作者】 刘佳；

【导师】 袁兴中； 曾光明；

【作者基本信息】 湖南大学 ， 环境科学与工程， 2008， 博士

【摘要】 针对木质纤维素资源生物转化制取酒精技术实用化的一个主要的障碍——纤维素酶活性不高、生产成本过高而导致其应用仍受到限制的问题,本研究首次将由微生物产生的生物表面活性剂鼠李糖脂用于木质纤维素原料制取酒精的几个主要环节,即液态发酵和固态发酵生产纤维素酶、纤维素酶法水解及同步糖化发酵制酒精的过程,目的是为了促进微生物产酶,并进一步提高酶活,从而提高木质纤维素的水解效率和酒精的转化率,并降低成本。同时与化学合成的非离子表面活性剂Tween 80作了比较。在液态发酵过程中,加入少量的鼠李糖脂和Tween 80就能对绿色木霉的产酶起到促进作用,尤其在产酶高峰期。添加鼠李糖脂分别使滤纸酶活(FPA)、羧甲基纤维素酶活(CMC酶活)、微晶纤维素酶活最大提高了1.08倍,1.6倍和1.03倍。添加Tween 80也能使酶活增大,效果略逊于鼠李糖脂,并且测得的酶活值整体上均比添加鼠李糖脂的样低。与Tween 80相比,加入鼠李糖脂使溶液的表面张力降得更低,并且在整个发酵过程中都将发酵液的表面张力保持在较低的水平,说明鼠李糖脂不会作为优先降解对象被过早降解,可以在整个发酵过程中发挥作用。固态发酵过程中添加不同浓度的鼠李糖脂对绿色木霉所产的纤维素酶活和木聚糖酶活有不同程度的促进作用。对纤维素酶系中的三种主要成分,绿色木霉所产的CMC酶活最高,但是表面活性剂对其的影响最小。鼠李糖脂和Tween 80对微晶纤维素酶和纤维二糖酶的促进作用较为明显。另外,与对纤维素酶的影响不同,两种表面活性剂在产酶高峰期对木聚糖酶的影响就很明显。在纤维素酶促水解的过程中,探讨了添加不同浓度的鼠李糖脂和Tween 80对稻草酶解过程的糖产率、酶稳定性、以及对动力学特征和酶在纤维素上吸附的影响。另外,还采用荧光探针法,以芘为荧光探针,研究了表面活性剂和纤维素酶之间的相互作用。结果表明,表面活性剂有利于增强酶的稳定性,能不同程度地提高反应过程的CMC酶活和FPA,而且对FPA的作用效果更好。加入表面活性剂能提高酶促反应的最大速度和米氏常数,降低了底物与酶的亲和力,提高了纤维素酶的利用效率。表面活性剂能显著减少酶在纤维素上的吸附,从而降低无效吸附酶的数量,增加游离酶与纤维素反应,同时也使纤维素酶更易于回收。另外,通过采用芘作为荧光探针,研究了表面活性剂和纤维素酶之间的作用关系。纤维素酶分子与表面活性剂分子之间有较强的作用力,从而有利于表面活性剂胶团的形成。和鼠李糖脂相比,Tween 80和纤维素酶之间的作用力相对较小,但随着Tween 80的浓度的增大,它和纤维素酶之间的作用力越来越大,尤其在浓度超过临界胶束浓度时。研究在同步糖化发酵反应条件最优的条件下添加表面活性剂的影响,因此,采用响应面法中的中心组合设计来对实验条件进行优化,得到影响同步糖化发酵进行的主要的四个影响因子的最优组合:酶用量为25FPU/gds,底物浓度为2.5%,pH为5.3,反应温度为39℃,其影响显著性为:酶用量>底物浓度>反应温度>pH值。在此条件下,酒精转化率的最大值为78.83%。对得到的回归方程进行方差分析,结果显示方程拟合度高,说明这个实验方法是可靠的。最后将两种表面活性剂应用在同步糖化发酵纤维素制酒精的过程中,验证了表面活性剂同样能在同步糖化发酵这个复杂的反应体系中起到促进作用。结果显示在反应的最优条件下,分别添加不同浓度的鼠李糖脂和Tween 80,酒精的转化率均有不同程度的提高。提高最大的是反应进行到第72h时,添加1cmc(临界胶束浓度,critical micelle concentration简写为cmc)鼠李糖脂和Tween 80,酒精转化率分别达到84.8%和91.7%,比对照样提高了12.2%和21.3%。总之,鼠李糖脂和Tween 80都能提高绿色木霉所产的纤维素酶和木聚糖酶的活性,有利于增强酶的稳定性,降低酶和底物的亲和力,减少酶在底物上的无效吸附,提高纤维素酶的利用率。应用在同步糖化发酵过程中,添加表面活性剂在最优的实验条件下仍然能进一步提高酒精的转化率。生物表面活性剂与化学表面活性剂相比具有一些特殊的优点,因此将生物表面活性剂应用于酶的生产及木质纤维素生物转化制酒精有很大的潜力。更多还原

【Abstract】 The main obstacle of bioconversion of lignocellulose into ethanol is the low activity of cellulase, which result in the high production cost. For the first time, in the research the biosurfactant rhamnolipid was applied in the main process of bioconversion of lignocellulose into ethanol, that is, the production of cellulase by Trichoderma viride in liquid substrate fermentation and solid state fermentation, the enzymatic hydrolysis of cellulose, and Simultaneous saccharification and fermentation (SSF). The purpose of the research is to enhance the activity of cellulase, and then the degradation rate of lignocellulose and the conversion rate of ethanol can be increased. In this research, the effects of rhamnolipid were also compared with those of chemical nonionic surfactant Tween 80.The effects of rhamnolipid and Tween 80 on cellulase production by T. viride in liquid substrate fermentation process were investigated. The results showed that the surfactants could enhance the cellulase activity by T. viride. The Filter paper activity (FPA), Sodium carboxymethylcellulose activity (CMCase activity) and Avicelase activity were promoted 1.08, 1.6 and 1.03 times higher than the controls by rhamnolipid. The enhancement of the enzyme activity by rhamnolipid was much higher than that of Tween 80. At the same time, rhamnolipid could decrease the surface tension to a lower level than Tween 80 did, and the surface tension was kept at the lower level during the fermentation process, indicating that rhamnolipid was not degraded prior to other substrate.The addition of rhamnolipid and Tween 80 resulted in better cellulase and xylanase production and more efficient lignocellulose degradation during SSF, and rhamnolipid was more effective than Tween 80. The effects of the same surfactant on enzyme production varied for different enzymes of the same organism. Among three major components of cellulase system, CMCase activity secreted by T. viride was the highest, but the stimulatory effects of surfactants on CMCase were the least. Rhamnolipid and Tween 80 were more effective to enhance the activities of Avicelase and cellobiase. Furthermore, the two surfactants had a pronounced stimulatory effect on xylanase activity during the peak phase of enzyme production.Effects of rhamnolipid and Tween 80 on enzymatic hydrolysis of straw were studied. The effects of different concentrations of surfactants on reducing sugar yield, enzymatic stability and cellulose contents during the course of enzymatic hydrolysis as well as the impacts of maximal reaction rate and adsorption of cellulase on cellulose were evaluated. The results indicated that rhamnolipid and Tween 80 improved enzymatic stability effectively and promoted the enzyme activity of CMCase and FPA to different extents and the effects on FPA was more efficiency. Surfactants also enhanced conversion rate of cellulose, and that in the present of rhamnolipid, the conversion rate of cellulose was higher than that with Tween 80 distinctly. Moreover, surfactants promoted maximal velocity of enzymatic reaction and helped to reduce adsorption of cellulase on cellulose remarkably. Therefore the efficiency of cellulase was increased.In addition, the reciprocity between surfactants and cellulase was studied by fluorescence probe method with pyrene as the probe. The results showed that there was action force between the sufactant molecule and the enzyme molecule. Compared with rhamnolipid, the action force between Tween 80 and enzyme was on the small side, but with the increasing of concentration, the action force increased, especially the concentration going over the critical micelle concentration.The response surface methodology was used to optimize the four main factors affecting SSF. The optimal combination of the conditions was obtained, that is the enzyme dosage of 25FPU/gds, the substrate concentration of 2.5%, pH value of 5.3, and reaction temperature of 39℃. The impact prominence of the four factors is enzyme dosage > substrate concentration > reaction temperature > pH value. The model predicts that the maximum conversion rate of ethanol that can be obtained under the above optimum conditions of the variables is 78.83%. The variance of regression equation was analyzed. The results show that the models provide a better fit to the real data and the method is effective and reliable.According to the effects of surfactants on the main processes of bioconversion of lignocellulose into ethanol, the rhamnolipid and Tween 80 was applied into the production of ethanol from lignocellulose by SSF. The results showed that the conversion rate of ethanol was increased to different extents by the two surfactants at the optimal condition. The maximum enhancement was obtained at the reaction time of 72 hours, at which time the conversion rates of ethanol were 84.8% and 91.7% with presence of 1cmc (critical micelle concentration) of rhamnolipid and Tween 80, and the conversion rates were12.2% and 21.3% higher than the control respectively. The experiment proved that rhamnolipid and Tween 80 could also improve the complex system of SSF.In conclusion, surfactants could enhance the enzyme activities by Trichoderma viride and improve enzymatic stability and reduce ineffective adsorption of cellulase on lignocellulose. Therefore the efficiency of cellulase was increased. Two surfactants were applied in the main process of SSF and the conversion rate of ethanol also was increased under the optimum conditions of the variables. Biosurfactants are of some special advantages over chemical surfactants, therefore it is of a great potential to be applied in enzyme production and lignocellulose bioconversion into ethanol.更多还原