Enzyme Production from Trichoderma reesei and Aspergillus Strain


Ethanol is an attractive alternative to the fossil fuels. Conventional production of ethanol from sugar- and starch-based feedstock such as corn and sugarcane competes with the food supply. To avoid this food competition, lignocellulosic biomass has been perceived as an excellent biorefinery resource to produce ethanol and other value chemicals.

Lignocellulosic biomass is mainly composed of cellulose, hemicellulose, and lignin (Lee et al. 2009). Conversion of lignocellulosic biomass to fermentable sugars is the concerted action of pretreatment and catalytic degradation. There are two methods of catalytic degradation: acidic and enzymatic. Problem associated with the hazardous acid use and the recycling prevents it from industrial use. Another method of biomass conversion is enzymatic hydrolysis. Despite that enzymatic hydrolysis result in higher sugar extraction and production of lower amount of inhibitors, cellulolytic enzymes can account for $0.10-0.40/gal of total ethanol production cost (Aden and Foust 2009; Dutta et al. 2010; Kazi et al. 2010). One of the ways to reduce the cost of the enzymes required for hydrolysis is to produce them on-site using inexpensive carbon source found in the biorefinery such as sidestreams of pretreated materials.

The cellulosic enzyme from Trichoderma reesei is one of the most efficient and extensively used cellulolytic enzyme systems used for lignocellulosic biomass hydrolysis to fermentable sugars. The overall extracellular matrix of these filamentous fungi is composed of 60-80 % cellobiohydrolases (1,4-p-D-glucan cellobiohy- drolase, EC, 20-36 % p-1,4-endoglucanases (1,4-p-D-glucan

4-glucanohydrolase, EC, cellulase), and 1 % p-glucosidases ф-D-glucoside glucohydrolase, EC, cellulase 1,4-p-glucosidase) (Zaldfvar et al. 2001).

These enzymes act synergistically to convert cellulose to glucose where product of one enzymes’ action becomes substrate for another enzyme (Ryu and Mandels 1980). Trichoderma reesei RUT-C30 is known as the hyperproducer of cellulolytic enzymes.

© The Author(s) 2017

V. Rana, D. Rana, Renewable Biofuels, SpringerBriefs in Applied Sciences and Technology, DOI 10.1007/978-3-319-47379-6_3

In addition to cellulases, T. reesei also produces xylanases (Foreman et al. 2003; Martinez et al. 2008). Several factors influence the enzyme production such as the type and concentration of carbon source, pH, aeration, agitation, and temperature (Bailey and Tahtiharju 2003; Haki and Rakshit 2003). It has been previously reported that cellulase production is induced by different carbon sources such as cellulose, or dimers such as sophorose, lactose, and cellobiose (Hari Krishna et al. 2000). It has also been shown that fungus cultivated on a specific lignocellulosic biomass with different ratio of cellulose to hemicellulose would produce enzyme mixture suitable for degrading that particular lignocellulosic material (Olsson et al. 2003; Juhasz et al. 2005; Sipos et al. 2010). As reported in the past, an enzyme concentration of 0.5-18 FPU/mL from T. reesei RUT-C30 has been achieved by using a variety of carbon sources, substrate concentrations, and growth conditions (Kovacs et al. 2009; Wu et al. 2011; Alvira et al. 2013). Previously, a significant number of studies were conducted on cellulase and xylanase production with T. reesei RUT-C30. However, a systematic study was not found to compare the efficiency of enzymatic hydrolysis of woody biomass and agricultural residues on on-site produced enzymes compared to commercial enzymes. Therefore in the current study, the goal was set to (1) evaluate the potential of cellulase production from T. reesei RUT-C30 using carbon sources from the mixture of pretreated corn stover and corn mash and p-glucosidase production from A. saccharolyticus using xylan-rich liquid stream of wet-exploded corn stover and (2) to compare the efficiency of enzyme cocktails produced by T reesei RUT-C30 and A. saccharolyticus on the hydrolysis of softwood and corn stover compared with that of commercial enzyme.

As previously reported, the supplementation of p-glucosidase to T. reesei RUT-C30 resulted in higher cellulose conversion (Tabka et al. 2006; Bey et al. 2011). T. reesei RUT-C30 generally show low p-glucosidase activity resulting in accumulation of cellobiose and subsequent product inhibition of endo- and exoglucanases (Stockton et al. 1991). In order to overcome this limitation, we supplemented T. reesei RUT-C30 enzyme complex with p-glucosidase produced from A. saccharolyticus, a novel Aspergillus strain containing p-glucosidase with a hydrolytic efficiency comparable to P-glucosidase derived from A. niger (S0rensen et al. 2011). Enzyme mixtures produced on-site from T. reesei RUT-C30 and A. saccharolyticus were characterized based on their activities and evaluated for hydrolytic efficiency on wet-exploded corn stover and loblolly pine. A comparative study of hydrolysis was performed in parallel with commercial enzymes Celluclast 1.5 L and Novozym 188.

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