Liquefaction of hydrothermally pretreated wheat straw at high-solids content by purified Trichoderma enzymes

Abstract

Enzymatic liquefaction was studied by measuring continuously the flowability change of high-solids lignocellulose substrates using a real time viscometric method. Hydrolysis experiments of hydrothermally pretreated wheat straw were carried out with purified enzymes from Trichoderma reesei; Cel7A, Cel6A, Cel7B, Cel5A, Cel12A and Xyn11A. Results obtained at 15% (w/w) solids revealed that endoglucanases, in particular Cel5A, are the key enzymes to rapidly reduce the viscosity of lignocellulose substrate. Cellobiohydrolases had only minor and the xylanase practically no effect on the viscosity. Efficient, fast liquefaction was obtained already at a dosage of 1.5 mg of Cel5A/g dry solids. Partial replacement or supplementation of Cel5A by the other major hydrolytic enzymes did not improve the liquefaction. The reduction of viscosity did not correlate with the saccharification obtained in the same reaction, suggesting that efficient liquefaction is rather dependent on the site than the frequency of enzymatic cleavages.

Introduction

The increasing demand for environmentally benign, secure and competitive transportation fuels has reinforced interest in the production of fuel ethanol from renewable resources. The primary interest is to use lignocellulose biomass as feedstock, preferably wastes and by-products of existing forest or agricultural crop production, not competing with food supplies or with land where food crops can be grown. However, to become economically competitive, the production of lignocellulosic ethanol still needs technological improvements. As the enzyme cost still accounts for a considerable proportion of the total production costs (Hahn-Hägerdal et al., 2006), one obvious target of process optimization is to achieve efficient solubilization of fermentable sugars from the polysaccharide substrates at reduced enzyme use. Another objective is to increase the final ethanol concentration by using high-solids raw material treatments, most importantly because the costs of down-stream processing are sharply reduced as the product concentration is increased. Distillation can be considered economical if the fermentation broth contains more than 4% (w/w) ethanol (Zacchi and Axelsson, 1989). To reach an ethanol concentration of higher than 4% (w/w), for most types of lignin containing biomasses the initial solids content needs to be above 15% (Larsen et al., 2008).

Recently, research to develop conversion processes performing enzymatic hydrolysis of lignocelluloses at high substrate concentrations has gained growing interest (Varga et al., 2004, Jørgensen et al., 2007, Georgieva et al., 2008). Although the flowability of these substrates improves as a result of cellulolytic activity, the initially high viscosity of concentrated lignocelluloses currently prevents efficient mixing. Therefore, one of the most important breakthroughs in this area has been the replacement of conventional stirring systems with gravimetric mixing, enabling liquefaction, saccharification, and fermentation of pretreated biomass at up to 40% initial solids content (Jørgensen et al., 2007, Larsen et al., 2008). Enzymatic hydrolysis at high-solids creates new challenges for the enzymes and understanding the rheological behaviour of high-solids raw materials during the enzymatic hydrolysis becomes more important.

To investigate the material properties of high-solids lignocelluloses most authors have used pretreated corn stover as substrate. Using this material at 5–30% (w/w) solids and the helical impeller technique as the rheological measurement Pimenova and Hanley (2004) first reported that the suspensions exhibited plastic-type rheological behaviour with an apparent yield stress. Corn stover suspensions (20–35%) were also studied by using torque rheometry and the shear-thinning behaviour of the substrate was confirmed (Ehrhardt et al., 2010). It was found that the rheological properties of these slurries were well described by the Bingham model, with the yield stress increasing with solids concentration. Also other models, the Casson and the Herschel–Buckley models, have been used for describing the rheology of 10–40% biomass suspensions (Houchin and Hanley, 2004, Pimenova and Hanley, 2004). The most comprehensive study available to date on the rheology of high-solids biomass suspensions is the recent inter-laboratory report by Stickel et al. (2009). A variety of instruments and tools (vanes, roughened plates and a torque rheometer) at different substrate concentrations up to 30% insoluble solids were used to measure the rheology of dilute acid treated corn stover. Previously reported characteristics, such as the strongly shear-thinning and viscoelastic behaviour, and concentration-dependent yield stress, were confirmed. In another study the advantages and disadvantages of several rheological techniques in various flow regimes (shear-flow using a vane, torsional flow between parallel plates, and biaxial extensional flow between plates) using the same substrate were evaluated, concluding that the vane geometry provided the simplest methodology and the most reproducible results (Knutsen and Liberatore, 2009).

To follow the reduction of viscosity during enzymatic hydrolysis, all studies so far have used traditional rheological approaches, i.e., samples collected at specific time points in the hydrolysis reactions have been subjected to various measurements in a separate instrument. Rosgaard et al. (2007) studied the effect of commercial cellulases on the viscosity of 5–15% (w/w) steam-pretreated barley straw using the vane spindle method and confirmed that the apparent viscosity increased with solids concentration and decreased with time during the enzymatic hydrolysis. Roche et al. (2009) used parallel plate and vane-in-cup geometries to study the rheology of dilute acid pretreated corn stover at solids loadings of 20%, and developed a semi-empirical relationship to connect the progress of enzymatic hydrolysis with particle concentration and yield stress. They concluded that the dominant effect on the rheology of saccharifying biomass was material dilution as the hydrolyzed biomass was transferred from solid to liquid phase. The importance of particle size and shape affecting the rheology has also been recognized using parallel plate (Viamajala et al., 2009) or vane-in-cup systems (Dasari and Berson, 2007).

The current starch-based processes are conducted at substrate loadings as high as 33–37% dry matter (w/w) (Thomas and Ingledew, 1992) and are initialized with a high-temperature (∼90 °C) liquefaction of starch followed by saccharification and fermentation carried out at moderate temperatures depending on the temperature optima of the biocatalysts and yeast used. There are several approaches to apply this process concept also for the treatment of lignocellulose biomass (Viikari et al., 2007). The well studied filamentous fungus Trichoderma reesei is one of the most efficient producers of biomass degrading enzymes. Many of the commercially available cellulase preparations suitable for moderate temperatures are produced with this organism. The fungus produces a compliment of cellulases, including two cellobiohydrolases (CBHs) and five endoglucanases (EGs). The goal of the present study was to indentify the key enzyme(s) needed for an efficient liquefaction of high-solids pretreated biomasses. To obtain detailed information on the time course of liquefaction, a new rheological measurement method using a rotational viscometer was developed, which allows real time monitoring of substrate viscosity during the enzymatic action under fully controlled temperatures and agitation rates. In particular, the technique was focused on the early stage of hydrolysis where the changes in the rheological behaviour of high-solids substrates are most pronounced. The developed system was used to evaluate the liquefaction properties of enzymes derived from various glycosyl hydrolase families and possessing different hydrolytic properties.