Fermentation Of Lignocellulose At High Temperature For Ethanol And Lactate Production

One of the major technical barriers in the simultaneous saccharification and fermentation (SSF) of lignocellulose is the mismatch of the optimal temperature between the fermenting microorganims and the cellulolytic enzymes. In the present tudy, a smiplied method of cellulase activity determination based HPLC assay was proposed. And, effects of different fermentation parameters on the SSF of the dilute acid pretreated corn stover using Saccharomyces cerevisiae DQ1at high solids loadings at elevated temperatures were detailed carried out. Futhermore, a lignocellulose-dependent themophilic lactate producing strain Pediococcus acidilactici DQ2was discovered and employed in the SSF of corn stover for lactate production. High titre ethanol and lactate were successfully achieved by the SSF of lignocellulose at high temperatures.At the part one of the research work, a simplied cellulase assay method was presented based on HPLC assay. The method was according to the good linear relationships between logarithms of filter paper units measured by DNS method and sum concentrations of glucose and cellobiose measured by HPLC. Furthermore, the ratio of glucose to cellobiose could be obtained to characterize a cellulase for the capacity of enzymatic hydrolysis of cellulose into monomeric sugars. The innovative point of the method was the application of the specifically analytical tool HPLC for measuring the sugar concentrations which could give a meaningful index-the ratio of glucose to cellobiose.The performance of commercial cellulases had significant difference when enzymatic hydrolysis of the corn stover. The ratio of glucose to cellobiose in the hydrolysate could be increased with supplementation of cellobiase to the cellulase.At the part two of the research work was on the SSF of corn stover for ethanol production at elevated temperatures. The corn stover before use was pretreated with a method of dilute acid pretreatment and then washed with water once to reduce the inhibitors. The SSF experiments were conducted with the strain Saccharomyces cerevisiae DQ1in a5L helical stirring reactor. Temperature characteristics of S. cerevisiae DQ1was firstly evaluated in YPD medium. The results indicated the strain could grow at44℃while the ethanol yield and grow ability of the strain were reduced greatly when cultured at the temperature over40℃. SSF experiments at different temperature without prehydrolysis were conducted when solid loadings and enzyme loading were20%(w/w),5FPU/g DM. The results indicated that S. cerevisiae could be employed in SSF at40℃which gave better performance than SSF at30-37℃. However, the fermentation was slowed or stuck when SSF experiments at40℃without prehydrolysis as increased solid loadings (～30%) and/or increased enzyme loadings (～15FPU/g DM). This may due to the weak growth ability of S. cerevisiae DQl at40℃, and the inhibition of the inhibitors and cellulase were augmented as increased solid and/or enzyme loadings. Even supplementation with external nutrients into SSF experiments could not enhance the fermentation performance of S. cerevisiae DQ1at40℃. The modes of SSF could significantly affect the fermentation performance. And SSF with temperature swing could overcome the problems related with SSF without prehydrolysis at40℃. SSF with temperature swing conducted at30%solids loading with an enzyme loading of15FPU/g DM gave ethanol concentration48g/L and ethanol yield61%. The research demonstrated that S. cerevisiae DQ1could be used for ethanol fermentation from lignocellulose at elevated temperatures. Furthermore, the stain for cellulosic ethanol fermentation at elevated temperatures should be capable of robust growth and stress tolerance at high temperature and tested in the practical lignocellulose and conditions. The main innovative point of the research was using a thermotolerant Saccharomyces strain for SSF of high loadings of the practical lignocellulosic substrate at elevated temperatures.At the part three of the research was on the discovery and isolation of a thermophilic lactate producing bacterium and its application in the SSF of corn stover for lactate production. The phenomomena of lactate formation was observed when studying the ethanol fermentation from the bioabated dilute acid pretreated corn stover at high temperature. A thermophilic lactate producing was isolated from cellulosic ethanol broth and identified belong to Pediococcus acidilactici, named as DQ2. The SSF of bioabated corn stover using P. acidilactici DQ2for lactate production could be conducted at the temperature up to48℃, and lactate concentration73g/L and yield63%were obtained. With the characteristics of high temperature tolerance, anaerobic fermentation, low nutrients requirements and lignocellulose-dependence, P. acidilactici DQ2brought a great opportunity for the SSF of lignocellulose for lactate production. The main innovative point of the research was the first report of a thermophilic lactate producing Pediococcus strain souring from the lignocellulose and high titre lactate procuction by SSF of the dilute acid corn stover using P. acidilactici DQ2at high temperature.In this thesis, the SSFof the dilute acid pretreated corn stover at high temperatures using S. cerevisiae DQ1and P. acidilactici DQ2was carried out. The ethanol titer of48g/L and the lactate titre of75g/L were obtained, respectively, which provided the fundamental technology for the biorefinery of the lignocellulose and had great significance for application to the high temperature fermentation of lignocellulose.