Three-stage hydrolysis to enhance enzymatic saccharification of steam-exploded corn stover

Abstract

The objective of the present research was to explore new approach to reduce the hydrolysis time and to enhance the productivity of enzymatic saccharification. One-stage hydrolysis of steam-exploded corn stover required 72 h to reach a yield of 62.8%, while multi-stage hydrolysis could reduce the time to 24 h. A concept of three-stage hydrolysis was therefore proposed in which cellulosic substrate was hydrolyzed for 6, 6, and 12 h, respectively. High hydrolysis yields, 70.2% with enzyme recycling and 76.1% with the supplement of fresh enzyme to eliminate enzyme recovery procedure, were obtained in 24 h. Analysis indicated that short-time hydrolysis and the removal of end products at each stage improved cellulase activities and benefited the adsorption of cellulase enzyme to the solid substrate. When steam-exploded corn stover was used as the substrate for cellulase synthesis, a hydrolysis yield of 88.6% was achieved in 24 h.

Introduction

Lignocellulosic materials are widely considered as an important source for the production of sugar streams that can be fermented to ethanol and other organic chemicals (Cardona and Sánchez, 2007). Corn stover is one of the most abundant agricultural wastes; it is estimated that about 150 million tons are produced annually in China. Currently due to lack of effective utilization, corn stover is predominantly disposed of by direct burning in open field, which also causes serious environmental pollution. Conversion of this residue into fuel ethanol and other organic chemicals provides an attractive opportunity for more sustainable development of agricultural resources.

To get more soluble sugars, enzymatic hydrolysis of cellulosic materials has been extensively studied in the last decades (Mooney et al., 1998, Sun and Cheng, 2002, Wilkins et al., 2007). However, high enzyme loading and long incubation time are generally required for effective cellulose saccharification to be achieved (Gregg and Saddler, 1996, Kuo and Lee, 2009). During enzymatic hydrolysis, there is an initial logarithmic phase followed by an asymptotic phase. A rapid increase of hydrolysis rate is normally observed in the initial phase, and the conversion of the remaining cellulose requires more than three days. Several possible mechanisms to the declining hydrolysis rate have been proposed, including enzyme adsorptive loss to lignin, loss of enzymatic synergism, deactivation of the enzyme (Berlin et al., 2005, Gunjikar et al., 2001), the intrinsic structural features of the substrate (Zhu et al., 2008), and the end-product inhibition.

The end-product inhibition has been shown to play a major role in hindering a continuously fast hydrolysis rate (Xiao et al., 2004). Many methods have been put forward to overcome the end-product inhibition during hydrolysis. These include the use of high concentrations of cellulases (Chen et al., 2008), the supplementation of extra β-glucosidase (Tengborg et al., 2001), the elimination of sugars during hydrolysis by ultrafiltration (Knutsen and Davis, 2005, Mores et al., 2001, Xiao et al., 2004), and simultaneous saccharification and fermentation (SSF) (Öhgren et al., 2006). The high dosage of cellulases and supplementation of extra β-glucosidases would add to the already substantial cost for enzymes in the bioconversion process. The removal of sugars by ultrafiltration might be a high-cost process; this restricts its large-scale applications. During the SSF process, there are some disadvantages should be faced with, such as incompatible temperature of hydrolysis and fermentation, ethanol tolerance of microbes, and inhibition of enzymes by ethanol (Hari Krishna et al., 2001).

The objective of the present research was to explore new approach to shorten the hydrolysis time and to enhance the productivity of enzymatic saccharification. The main concern was the hydrolysis time, because long-time hydrolysis would bring on deactivation of the enzyme through thermal, mechanical and chemical actions. A concept of three-stage hydrolysis was therefore proposed in which cellulosic substrate was hydrolyzed for 6, 6, and 12 h, respectively, with a total hydrolysis time of 24 h. The hydrolytic performance of various enzyme preparations was also evaluated.