Alex Berlin

Substrate Pretreatment: The Key to Effective Enzymatic Hydrolysis of Lignocellulosics?

Although the structure and function of cellulase systems continue to be the subject of intense research, it is widely acknowledged that the rate and extent of the cellulolytic hydrolysis of lignocellulosic substrates is influenced not only by the effectiveness of the enzymes but also by the chemical, physical and morphological characteristics of the heterogeneous lignocellulosic substrates. Although strategies such as site-directed mutagenesis or directed evolution have been successfully employed to improve cellulase properties such as binding affinity, catalytic activity and thermostability, complementary goals that we and other groups have studied have been the determination of which substrate characteristics are responsible for limiting hydrolysis and the development of pretreatment methods that maximize substrate accessibility to the cellulase complex. Over the last few years we have looked at the various lignocellulosic substrate characteristics at the fiber, fibril and microfibril level that have been modified during pretreatment and subsequent hydrolysis. The initial characteristics of the woody biomass and the effect of subsequent pretreatment play a significant role on the development of substrate properties, which in turn govern the efficacy of enzymatic hydrolysis. Focusing particularly on steam pretreatment, this review examines the influence that pretreatment conditions have on substrate characteristics such as lignin and hemicellulose content, crystallinity, degree of polymerization and specific surface, and the resulting implications for effective hydrolysis by cellulases.

Weak lignin-binding enzymes: a novel approach to improve activity of cellulases for hydrolysis of lignocellulosics.

Economic barriers preventing commercialization of lignocellulose-to-ethanol bioconversion processes include the high cost of hydrolytic enzymes. One strategy for cost reduction is to improve the specific activities of cellulases by genetic engineering. However, screening for improved activity typically uses “ideal” cellulosic substrates, and results are not necessarily applicable to more realistic substrates such as pretreated hardwoods and softwoods. For lignocellulosic substrates, nonproductive binding and inactivation of enzymes by the lignin component appear to be important factors limiting catalytic efficiency. A better understanding of these factors could allow engineering of cellulases with improved activity based on reduced enzyme-lignin interaction (“weak lignin-binding cellulases”). To prove this concept, we have shown that naturally occurring cellulases with similar catalytic activity on a model cellulosic substrate can differ significantly in their affinities for lignin. Moreover, although cellulose-binding domains (CBDs) are hydrophobic and probably participate in lignin binding, we show that cellulases lacking CBDs also have a high affinity for lignin, indicating the presence of lignin-binding sites on the catalytic domain.

Weak Lignin-Binding Enzymes

Economic barriers preventing commercialization of lignocellulose-to-ethanol bioconversion processes include the high cost of hydrolytic enzymes. One strategy for cost reduction is to improve the specific activities of cellulases by genetic engineering. However, screening for improved activity typically uses “ideal” cellulosic substrates, and results are not necessarily applicable to more realistic substrates such as pretreated hardwoods and softwoods. For lignocellulosic substrates, nonproductive binding and inactivation of enzymes by the lignin component appear to be important factors limiting catalytic efficiency. A better understanding of these factors could allow engineering of cellulases with improved activity based on reduced enzyme-lignin interaction (“weak lignin-binding cellulases”). To prove this concept, we have shown that naturally occurring cellulases with similar catalytic activity on a model cellulosic substrate can differ significantly in their affinities for lignin. Moreover, although cellulose-binding domains (CBDs) are hydrophobic and probably participate in lignin binding, we show that cellulases lacking CBDs also have a high affinity for lignin, indicating the presence of lignin-binding sites on the catalytic domain.

Updates on softwood-to-ethanol process development.

Softwoods are generally considered to be one of the most difficult lignocellulosic feedstocks to hydrolyze to sugars for fermentation, primarily owing to the nature and amount of lignin. If the inhibitory effect of lignin can be significantly reduced, softwoods may become a more useful feedstock for the bioconversion processes. Moreover, strategies developed to reduce problems with softwood lignin may also provide a means to enhance the processing of other lignocellulosic substrates. The Forest Products Biotechnology Group at the University of British Columbia has been developing softwood-to-ethanol processes with SO2-catalyzed steam explosion and ethanol organosolv pretreatments. Lignin from the steam explosion process has relatively low reactivity and, consequently, low product value, compared with the high-value coproduct that can be obtained through organosolv. The technical and economic challenges of both processes are presented, together with suggestions for future process development.

Evaluation of Cellulase Preparations for Hydrolysis of Hardwood Substrates

Seven cellulase preparations from Penicillium and Trichoderma spp. were evaluated for their ability to hydrolyze the cellulose fraction of hardwoods (yellow poplar and red maple) pretreated by organosolv extraction, as well as model cellulosic substrates such as filter paper. There was no significant correlation among hydrolytic performance on pretreated hardwood, based on glucose release, and filter paper activity. However, performance on pretreated hardwood showed significant correlations to the levels of endogenous β-glucosidase and xylanase activities in the cellulase preparation. Accordingly, differences in performance were reduced or eliminated following supplementation with a crude β-glucosidase preparation containing both activities. These results complement a previous investigation using softwoods pretreated by either organosolv extraction or steam explosion. Cellulase preparations that performed best on hardwood also showed superior performance on the softwood substrates.

An Evaluation of British Columbian Beetle-Killed Hybrid Spruce for Bioethanol Production

The development of bioconversion technologies for production of fuels, chemicals, and power from renewable resources is currently a high priority for developed nations such as the United States, Canada, and the European Union as a way to improve national energy security and reduce greenhouse gas emissions. The widespread implementation of such technologies will require a sustainable supply of biomass from forestry and agriculture. Forests are a major source of feedstocks for biofuels production in Canada. Woody biomass includes residues from logging and forest thinning, and from wood processing and pulp production.More recently, damaged wood caused by beetle infestations has become available on a large scale in Western Canada. This study evaluates beetle-killed British Columbian hybrid spruce (HS) (Picea glauca × P. engelmannii) as a feedstock for the production of bioethanol. In the past 30 yr, attack by the beetle Dendroctonus rufipennis and associated fungi has resulted in estimated losses of more than three billion board feet in British Columbia alone. Here we describe the chemical and some physical characteristics of both healthy (HHS) and beetle-killed (BKHS) British Columbian HS and evaluate the technical feasibility of using these feedstocks as a source of biomass for bioethanol production. Untreated HHS and BKHS did not differ significantly in chemical composition except for the moisture content, which was significantly lower in BKHS (approx 10%) compared with HHS (approx 18%). However, the yields of carbohydrates in hydrolyzable and fermentable forms were higher at mild pretreatment conditions (H-Factor <1000) for BKHS compared with HHS. At medium (H-Factor 1000–2000) and severe (H-Factor >2000) pretreatment conditions HHS and BKHS behaved similarly. Organosolv pretreated HHS and BKHS demonstrated good ethanol theoretical yields, approx 70 and 80%, respectively.