The ability of a commercial Trichoderma reesei cellulase preparation (Celluclast 1.5L), to hydrolyze the cellulose and xylan components of pretreated corn stover (PCS) was significantly improved by supplementation with three types of crude commercial enzyme preparations nominally enriched in xylanase, pectinase, and beta-glucosidase activity. Although the well-documented relief of product inhibition by beta-glucosidase contributed to the observed improvement in cellulase performance, significant benefits could also be attributed to enzymes components that hydrolyze non-cellulosic polysaccharides. It is suggested that so-called "accessory" enzymes such as xylanase and pectinase stimulate cellulose hydrolysis by removing non-cellulosic polysaccharides that coat cellulose fibers. A high-throughput microassay, in combination with response surface methodology, enabled production of an optimally supplemented enzyme mixture. This mixture allowed for a approximately twofold reduction in the total protein required to reach glucan to glucose and xylan to xylose hydrolysis targets (99% and 88% conversion, respectively), thereby validating this approach towards enzyme improvement and process cost reduction for lignocellulose hydrolysis.

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.

The conversion of lignocellulosic biomass to fuel ethanol typically involves a disruptive pretreatment process followed by enzyme-catalyzed hydrolysis of the cellulose and hemicellulose components to fermentable sugars. Attempts to improve process economics include protein engineering of cellulases, xylanases and related hydrolases to improve their specific activity or stability. However, it is recognized that enzyme performance is reduced during lignocellulose hydrolysis by interaction with lignin or lignin-carbohydrate complex (LCC), so the selection or engineering of enzymes with reduced lignin interaction offers an alternative means of enzyme improvement. This study examines the inhibition of seven cellulase preparations, three xylanase preparations and a beta-glucosidase preparation by two purified, particulate lignin preparations derived from softwood using an organosolv pretreatment process followed by enzymatic hydrolysis. The two lignin preparations had similar particle sizes and surface areas but differed significantly in other physical properties and in their chemical compositions determined by a 2D correlation HSQC NMR technique and quantitative 13C NMR spectroscopy. The various cellulases differed by up to 3.5-fold in their inhibition by lignin, while the xylanases showed less variability (< or = 1.7-fold). Of all the enzymes tested, beta-glucosidase was least affected by lignin.

Current attempts to produce ethanol from lignocellulosic biomass are focused on the optimization of pretreatment to reduce substrate recalcitrance and the improvement of enzymes for hydrolysis of the cellulose and hemicellulose components to produce fermentable sugars. Research aimed at optimizing both aspects of the bioconversion process involves assessment of the effects of multiple variables on enzyme efficiency, resulting in large factorial experiments with intensive assay requirements. A rapid assay for lignocellulose hydrolysis has been developed to address this need. Pretreated lignocellulose is formed into handsheets, which are then used to prepare small disks that are easily dispensed into microtiter plates. The hydrolysis of cellulose to glucose is estimated using an enzyme-coupled spectrophotometric assay. Using disks prepared from ethanol organosolv pretreated yellow poplar, it is shown that the assay generates data comparable with those produced by hydrolysis of pretreated yellow poplar pulp in Erlenmeyer flasks, followed by HPLC analysis of glucose. The assay shows considerable time and cost benefits over the standard assay protocol and is applicable to a broad range of lignocellulosic 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.

Seven fungal cellulase preparations from Trichoderma and Penicillium spp. were evaluated for their ability to hydrolyze the cellulose component of lignocellulose samples prepared from softwoods by steam explosion or organosolv pretreatment. Hydrolysis was quantified using two indices: mean specific rate and specific conversion. The activities of the cellulase preparations on model cellulosic substrates (filter paper, carboxymethylcellulose and Avicel) were also determined, together with their β-glucosidase, xylanase, pectinase, galactomannanase and β-glucanase activities. The ability of a cellulase preparation to hydrolyze pretreated softwood showed little correlation with its activity on filter paper, carboxymethylcellulose and Avicel; however, there was a significant correlation with its level of endogenous β-glucosidase and xylanase activity. Differences in the performance of the various cellulase preparations were substantially reduced following supplementation with a commercial β-glucosidase preparation from Aspergillus niger. In addition to β-glucosidase, this preparation was shown to contain significant levels of endogenous xylanase activity. It appears that the levels of endogenous β-glucosidase and xylanase are important factors in determining the ability of a cellulase preparation to hydrolyze pretreated softwood and that deficiencies in the levels of both enzymes can be compensated by supplementation with corresponding activities present in the β-glucosidase preparation. Presumably, β-glucosidase improves cellulose hydrolysis by reducing end-product inhibition by cellobiose, while xylanase activity increases the accessibility of cellulose to cellulases.