Ali R. Esteghlalian

Enhancing the Enzymatic Hydrolysis of Cellulosic Materials Using Simultaneous Ball Milling

One of the limiting factors restricting the effective and efficient bioconversion of softwood-derived lignocellulosic residues is the recalcitrance of the substrate following pretreatment. Consequently, the ensuing enzymatic process requires relatively high enzyme loadings to produce monomeric carbohydrates that are readily fermentable by ethanologenic microorganisms. In an attempt to circumvent the need for larger enzyme loadings, a simultaneous physical and enzymatic hydrolysis treatment was evaluated. A ball-mill reactor was used as the digestion vessel, and the extent and rate of hydrolysis were monitored. Concurrently, enzyme adsorption profiles and the rate of conversion during the course of hydrolysis were monitored. alpha-Cellulose, employed as a model substrate, and SO2-impregnated steam-exploded Douglas-fir wood chips were assessed as the cellulosic substrates. The softwood-derived substrate was further posttreated with water and hot alkaline hydrogen peroxide to remove >90% of the original lignin. Experiments at different reaction conditions were evaluated, including substrate concentration, enzyme loading, reaction volumes, and number of ball beads employed during mechanical milling. It was apparent that the best conditions for the enzymatic hydrolysis of alpha-cellulose were attained using a higher number of beads, while the presence of air-liquid interface did not seem to affect the rate of saccharification. Similarly, when employing the lignocellulosic substrate, up to 100% hydrolysis could be achieved with a minimum enzyme loading (10 filter paper units/g of cellulose), at lower substrate concentrations and with a greater number of reaction beads during milling. It was apparent that the combined strategy of simultaneous ball milling and enzymatic hydrolysis could improve the rate of saccharification and/or reduce the enzyme loading required to attain total hydrolysis of the carbohydrate moieties.

The effect of shaking regime on the rate and extent of enzymatic hydrolysis of cellulose

In an attempt to elucidate the effect of mixing on the rate and extent of enzymatic hydrolysis of cellulosic substrates, alpha-cellulose was hydrolysed using a commercial cellulase preparation at varying levels of substrate concentration (2.5,5 and 7.5% (w/v)) and by using three shaking regimes: continuous at low-speed (25 rpm), continuous at high-speed (150 rpm) and an intermittent regime comprised of high and low-speed shaking intervals. The continuous, high-speed shaking produced the highest conversion yields, whereas the intermittent and low-speed shaking regimes resulted in lower conversions. After 72 h, at all shaking regimes (150 rpm,25 rpm and intermittent), using a low substrate concentration (2.5%) produced conversion yields (82,79 and 80%) higher than those obtained at high (7.5%) substrate concentration (68,63 and 68%). As the substrate concentration increased, the conversion yields at intermittent shaking gradually approached those resulting from high-speed shaking. Thus, it appears that intermittent shaking could be a beneficial process option as it can reduce the mixing energy requirements while producing reasonably high conversion yields.

Steam pretreatment of Douglas-fir wood chips. Can conditions for optimum hemicellulose recovery still provide adequate access for efficient enzymatic hydrolysis?

Douglas-fir sapwood and heartwood were impregnated with SO2 and steam exploded at three severity levels, and the cellulose-rich, water-insoluble component was enzymatically hydrolyzed. The high-severity conditions resulted in near complete solubilization and some degradation of hemicelluloses and a significant improvement in the efficiency of enzymatic digestibility of the cell ulose component. At lower severity, some of the hemicellulose remained un hydrolyzed, and the cellulose present in the pretreated solids was not readily hydrolyzed. The medium-severity pretreatment conditions proved to be a good compromise because they improved the enzymatic hydrolyzability of the solids and resulted in the recovery of the majority of hemicellulose in a monomeric form within the water-soluble stream. Sapwood-derived wood chips exhibited a higher susceptibility to both pretreatment and hydrolysis and, on steam explosion, formed smaller particles as compared to heartwood-derived wood chips.

Steam Pretreatment of Douglas-Fir Wood Chips

Douglas-fir sapwood and heartwood were impregnated with SO2 and steam exploded at three severity levels, and the cellulose-rich, water-insoluble component was enzymatically hydrolyzed. The high-severity conditions resulted in near complete solubilization and some degradation of hemicelluloses and a significant improvement in the efficiency of enzymatic digestibility of the cellulose component. At lower severity, some of the hemicellulose remained unhydrolyzed, and the cellulose present in the pretreated solids was not readily hydrolyzed. The medium-severity pretreatment conditions proved to be a good compromise because they improved the enzymatic hydrolyzability of the solids and resulted in the recovery of the majority of hemicellulose in a monomeric form within the water-soluble stream. Sapwood-derived wood chips exhibited a higher susceptibility to both pretreatment and hydrolysis and, on steam explosion, formed smaller particles as compared to heartwood-derived wood chips.

Influence of Mixing Regime on Enzymatic Saccharification of Steam-Exploded Softwood Chips

In an attempt to elucidate the effect of reduced mixing on the enzymatic hydrolysis of lignocellulosic feedstocks, a pretreated softwood substrate was hydrolyzed under various mixing regimes using a commercial cellulase mixture. The substrate was generated by SO2-catalyzed steam explosion of Douglas fir wood chips followed by alkali-peroxide treatment to remove lignin. Three mixing regimes were tested; continuous mixing at low (25 rpm) and high (150 rpm) speeds, and mixing at low-speed interspersed with 5-min intervals of high-speed agitation at 150 rpm. At both substrate concentrations (7.5 and 10% [w/w]), the mixed-speed mixing was able to produce sufficiently high conversion rates and yields (93% after 96 h), close or slightly better than those obtained under vigorous mixing (150 rpm). The low-speed shaking produced appreciably lower conversion yields at both levels of substrate concentration. Therefore, the mixed-speed regime may be a viable process option, because it does not seem to have an adverse impact on the cellulose conversion yield and can be an effective means of reducing the mixing energy requirements of an enzymatic hydrolysis process.