Effect of pretreatment and enzymatic hydrolysis of wheat straw on cell wall composition, hydrophobicity and cellulase adsorption

Abstract

The present study aimed to determine the impact of cell wall composition and lignin content on enzyme adsorption and degradability. Thioacidolysis analysis of residual lignins in wheat straw after steam-explosion or organosolv pretreatment revealed an increase in lignin condensation degree of 27% and 33%, respectively. Surface hydrophobicity assessed through wettability tests decreased after the pretreatments (contact angle decrease of 20–50%), but increased with enzymatic conversion (30% maximum contact angle increase) and correlatively to lignin content. Adsorption of the three major cellulases Cel7A, Cel6A and Cel7B from Trichoderma reesei decreased with increasing hydrolysis time, down to 7%, 31% and 70% on the sample with the highest lignin content, respectively. The fraction of unspecifically bound enzymes was dependent both on the enzyme and the lignin content. Adsorption and specific activity were shown to be inversely proportional to lignin content and hydrophobicity, suggesting that lignin is one of the factors restricting enzymatic hydrolysis.

Introduction

The biological conversion of lignocellulosic feedstocks to ethanol involves pretreatment, enzymatic hydrolysis of its cellulose fraction (saccharification step), glucose fermentation and ethanol recovery.

The pretreatment step is essential to improve cellulose accessibility to enzymes and has been intensively studied as reviewed by Hendriks and Zeeman (2009). The pretreatment by steam explosion has proven to be efficient to process a wide range of lignocellulosic substrates (Kumar et al., 2010). This process consists of heating the biomass with high pressurized steam (20–50 bar) at high temperature and for a few minutes, then rapidly releasing the pressure (Mason, 1928 cited by Sun et al., 2005). The addition of an acid catalyst such as sulfuric acid improves the hydrolysis efficiency of hemicelluloses (Kumar et al., 2010). In addition to the substantial hydrolysis of hemicelluloses, steam explosion induces the melting of lignins and their partial depolymerization by hydrolysis of the predominant β-O-4 bonds, with the concomitant formation of new free phenolic groups (Excoffier and Vignon, 1991). An alternative pretreatment process consists of treating the lignocellulosic biomass with an aqueous organic solvent at high temperature (organosolv delignification process, Zhao et al., 2009). By contrast to the steam explosion process, most of the degraded lignin molecules are readily solubilized in the organosolv cooking liquor. The organosolv process thereby allows the recovery of isolated lignins, of hemicelluloses-derived sugars and of a cellulose-rich final residue used either as conventional pulp or as a saccharification substrate.

The saccharification step is catalysed by multienzyme systems generally produced by fungi such as Trichoderma reesei and is a slow and inefficient process, due to the insoluble and recalcitrant nature of the lignocellulosic substrate. Understanding the molecular bases of the substrate–enzyme interactions is therefore a prerequisite to develop appropriate pretreatments for efficient degradation and enzymes with improved hydrolytic activity on the pretreated substrates. Enzyme adsorption to cellulose might be a determining factor for efficient hydrolysis as suggested by the correlations between adsorption of cellulolytic enzymes and hydrolysis rates or yields (Medve et al., 1994, Hong et al., 2007). Adsorption and enzymatic cellulose hydrolysis are greatly influenced by substrate characteristics such as cellulose crystallinity (Hall et al., 2010), pore volume and accessible surface area (Grethlein, 1985, Hong et al., 2007). In lignocellulosic substrates, additional factors such as lignin content and distribution or hemicellulose content also determine enzyme adsorption and, in turn, degradability (Jeoh et al., 2007, Kumar and Wyman, 2009). The cellulose accessibility to cellulase was shown to be a major factor for digestibility of pretreated lignocellulosic substrates (Zhu et al., 2009). Lignins can impact hydrolysis rates by acting as a physical barrier and restricting enzyme access (Mooney et al., 1998), by undergoing unproductive binding with the enzymes (Nakagame et al., 2010a, Smith et al., 2010) and by inhibiting the enzymatic reaction (Pan, 2008). These interactions with lignins lead to reduced hydrolysis rates and yields. They depend on the type of substrate and pretreatment, which largely determine lignin structure (Nakagame et al., 2010a, Piccolo et al., 2010). Consequently, it seems important to study cellulase adsorption and hydrolysis rates in substrates with limited variation of physico-chemical parameters.

The interaction of the two cellobiohydrolases Cel7A (CBH1) and Cel6A (CBH2) with cellulose and lignocellulosic substrates has been studied extensively (Medve et al., 1994, Jeoh et al., 2007, Kumar and Wyman, 2009), but not the interaction of other individual enzymes from T. reesei with lignocellulosic substrates. Binding of CBH1 to steam pretreated willow was found to correlate well with hydrolysis, indicating specific binding (Kotiranta et al., 1999). More recently, the binding of different individual T. reesei enzymes were for the first time followed during hydrolysis of lignocellulosic substrates and suggested substantial binding of the enzymes to hydrolyzed, lignin-rich substrates (Varnai et al., 2011). Although hydrophobic interactions are likely to be involved in the unproductive adsorption phenomenon between cellulases and lignocellulosic substrates (Eriksson et al., 2002, Tu et al., 2009), the relationship of cellulase adsorption to wheat straw wettability has never been examined so far.

In the present study, the adsorption and binding specificity of the three major T. reesei cellulases, CBH1, CBH2 and EG1, were evaluated on lignocellulosic substrates at different stages of hydrolysis. Adsorption was determined as a function of the enzyme type, cell wall composition and lignin content. The surface hydrophobicity of wheat straw was investigated using wettability tests. A delignified organosolv wheat straw sample and a series of steam exploded wheat straw samples, progressively hydrolyzed and enriched in lignins, were prepared here, providing six different substrates covering a wide range of lignin contents. These substrates were submitted to structural characterization, to wettability tests, and to cellulases adsorption.