Bioethanol from lignocellulosic biomass is among the green alternatives to fossil fuels, but as the processing techniques are today, gasolin is still heaper than bioethanol. Lignocellulose, which remains the primary resource for ioethanol production in Denmark, is complex when it comes to release of
fermentable sugars (glucose) as cellulose is tightly linked to hemicellulose and lignin. Lignocellulose is disrupted during pretreatment, but to degrade cellulose to single sugars, lignocellulolytic enzymes such as cellulases and hemicellulases are needed. Lignocellulolytic enzymes are costly for the ioethanol production, but the expenses can be reduced by using thermostable enzymes, which are known for their increased stability and inhibitor olerance. However, the advantage of using thermostable enzymes has not been studied thoroughly and more knowledge is needed for development of bioethanol processes. Enzymes are added to the bioethanol process after pretreatment. For an efficient sugar and ethanol yield, the solids content of biomass is normally increased, which results in highly viscous slurries that are difficult to mix. Therefore, the first enzymatic challenge is to ensure rapid reduction of viscosity. During the course of this study, the thermostable enzymes have had their benefits compared to mesophilic enzymes when studying the acroscopic changes in biomass during liquefaction (chapter 4 and 5). The reduction in viscosity was more pronounced when increasing temperature, which could be due to a more active thermostable enzyme or a more viscous biomass. Thermostable endoglucanase and endoxylanase were also shown to speed up the liquefaction process, which was observed by measuring viscosity and fibre length. In addition, the severity of pretreatment of biomass affected the initial viscosity. When the solids are increased, the overall enzymatic degradation of biomass decreases due to product inhibition and less free water for enzyme transport. During liquefaction and simultaneous saccharification and fermentation (SSF) using mesophilic enzymes it was seen that a long liquefaction step resulted in less glucan conversion after SSF (chapter 6). It was speculated that the high temperature during liquefaction caused inactivation and degradation of the mesophilic enzymes before SSF, which probably could be avoided by using thermostable enzymes. After unning thermostable enzymes at high solids content trough liquefaction and SSF, the cellulase and xylanase activity remained at 80% of the initial value. During distillation, the thermostable enzymes were more temperature and ethanol tolerant compared to mesophilic enzymes (chapter 7). For both mixtures it was seen that an increasing ethanol concentration reduced the stability of the enzymes, while addition of polyethylene glycol (PEG) had the opposite effect. In addition, thermostable enzymes retained more activity when tested in a pilot distillation setup, where the enzymes were exposed to increasing temperature and air-liquid interfaces (chapter 8). It was possible to recover more thermostable activity after distillation meaning that the nzymes could be recycled more efficiently resulting in a net savings of enzymes used.
By testing different enzyme cocktails through the bioethanol process, specially during liquefaction and distillation, thermostable enzymes were shown to be more efficient than mesophilic enzymes when it came to temperature, inhibitor tolerance and stability against air-liquid forces.