| 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.
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