The enzymatic hydrolysis of the pretreated raw material and the fermenta­tion of the hydrolysed sugars can be performed separately or simultaneously, commonly referred to as SHF (separate hydrolysis and fermentation) or as SSF (simultaneous saccharification and fermentation). The SSF process con­figuration has been generally considered more favourable for reducing the ethanol production costs [72,81]. The hydrolysis rate in the separate hydro­lysis is strongly inhibited by the accumulation of the end products, cellobiose and glucose [60]. In the simultaneous hydrolysis and fermentation, the end product inhibition is alleviated by the continuous removal of glucose by the fermenting organism. In the separate hydrolysis and fermentation the most severe end product inhibition caused by cellobiose has been overcome by adding an adequately high amount of в-glucosidase. For the same reason, the enzyme dosage needed is obviously lower in the SSF. Other claimed ad­vantages of the SSF are the lower risk of contamination and reduction of investment costs by combined reactors. The low concentration of free glucose and the presence of ethanol make it more difficult for contaminating micro­organisms to take over the fermentation and decrease the ethanol yield. The drawback of the SSF is that the conditions, i. e. the pH and temperature of the hydrolysis and fermentation, are suboptimal in a combined process. The optimal temperature for the enzymatic hydrolysis is clearly higher than that of the presently used fermenting organisms. Another drawback of the SSF is the difficulty in optimising the fermentation of techniques, i. e. by running continuous fermentation or recirculating and reusing the yeast due to the presence of the solid residues from the hydrolysis.

To improve the overall process economics and to achieve a faster hydrolysis rate by using thermostable enzymes, various modifications of the present process configurations can be considered (Fig. 1). After the pretreatment, the temperature of the substrate is high, and is reduced to achieve the operat­ing temperature in the following process stages. In the traditional SSF, the temperature is about 35 °C. In a separate hydrolysis and fermentation pro­cess, the first total hydrolysis stage is carried out at about 45-50 °C with the present commercial enzymes, or above 60 °C with novel thermostable

enzymes. Other options include a partial prehydrolysis at higher tempera­tures, denoted as liquefaction, where the viscosity of the substrate is de­creased using a chosen composition of thermostable cellulases based on one or several enzymes. The liquefaction stage, i. e. an enzymatic treatment improving the rheological properties (improved flowability, reduced viscos­ity) of the slurry, can significantly improve the mixing properties of the substrate slurry [83]. This partial hydrolysis can be carried out even with the limited number of thermostable cellulolytic and hemicellulolytic en­zymes available. Using a set of thermoactive enzymes in the prehydrolysis, it was possible to reduce the viscosity and increase the sugar formation [83]. The high viscosity is a consequence of a high initial substrate consistency, needed to achieve a high final sugar and ethanol concentration and to de­crease the distillation costs [69]. With a theoretical ethanol yield of 25-30% of the raw material, the raw material consistency should be at least 15% (d. w.) to reach an ethanol concentration of 4-5%. Some of the technical obstacles related to high consistency can thus be overcome by a rapid de­crease of viscosity. After a liquefying partial hydrolysis, the saccharification stage using a complete or complementary set of hydrolytic enzymes, ei­ther simultaneously or separately from the fermentation (SSF or SHF), can be carried out. A separate hydrolysis stage (SHF) can be carried out at el­evated temperatures with the complete set of hydrolytic thermostable en­zymes needed for a chosen substrate. Finally, thermostable enzymes could be supplemented to bacterial fermentations using anaerobic, ethanol pro­ducing strains, such as Clostridia, to improve their conversion rate of cellu — losic substrates into sugars (SSF or consolidated bioprocessing). Thus, new thermostable enzymes would allow the design of more flexible process con-

figurations, based on the availability of novel thermostable lignocellulolytic enzymes.

The performance of chosen thermostable cellulolytic enzymes with present commercial fungal enzymes was compared in this paper. The refer­ence enzyme preparations contain the whole set of cellulolytic enzymes, i. e. cellobiohydrolases and endoglucanases, as well as several hemicellulolytic ac­tivities and в-glucosidases. These enzymes work at temperatures up to about 45 °C in long-term hydrolysis conditions and up to 50 °C in short-term con­ditions. New enzyme compositions were designed and tested in the hydrolysis of various steam pretreated raw materials.

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