David J. Gregg

Effects of Sugar Inhibition on Cellulases and β-Glucosidase During Enzymatic Hydrolysis of Softwood Substrates

A quantitative approach was taken to determine the inhibition effects of glucose and other sugar monomers during cellulase and beta-Glucosidase hydrolysis of two types of cellulosic material: Avicel and acetic acid-pretreated softwood. The increased glucose content in the hydrolysate resulted in a dramatic increase in the degrees of inhibition on both beta-Glucosidase and cellulase activities. Supplementation of mannose, xylose, and galactose during cellobiose hydrolysis did not show any inhibitory effects on beta-Glucosidase activity. However, these sugars were shown to have significant inhibitory effects on cellulase activity during cellulose hydrolysis. Our study suggests that high-substrate consistency hydrolysis with supplementation of hemicellulose is likely to be a practical solution to minimizing end-product inhibition effects while producing hydrolysate with high glucose concentration.

Factors affecting cellulose hydrolysis and the potential of enzyme recycle to enhance the efficiency of an integrated wood to ethanol process.

Past technoeconomic modeling work has identified the relatively large contribution that enzymatic hydrolysis adds to the total cost of producing ethanol from lignocellulosic substrates. This cost was primarily due to the high concentration of enzyme and long incubation time that was required to obtain complete hydrolysis. Although enzyme and substrate concentration and end‐product inhibition influenced the rate of hydrolysis, the effect was less pronounced during the initial stages of hydrolysis. During this time most of the cellulases were adsorbed onto the unhydrolyzed residue. By recycling the cellulases adsorbed to the residual substrate remaining after an initial 24 h, a high rate of hydrolysis, with low overall residence time and minimal cellulase input, could be achieved for several rounds of enzyme recycle. A comparison of the front end (pretreatment, fractionation, and hydrolysis) of a softwood/hardwood to ethanol process indicated that the lignin associated with the softwood‐derived cellulose stream limited the number of times the cellulose containing residue could be recycled.

Strategies to Enhance the Enzymatic Hydrolysis of Pretreated Softwood with High Residual Lignin Content

Pretreatment of Douglas-fir by steam explosion produces a substrate containing approx 43% lignin. Two strategies were investigated for reducing the effect of this residual lignin on enzymatic hydrolysis of cellulose: mild alkali extraction and protein addition. Extraction with cold 1% NaOH reduced the lignin content by only approx 7%, but cellulose to glucose conversion was enhanced by about 30%. Before alkali extraction, addition of exogenous protein resulted in a significant improvement in cellulose hydrolysis, but this protein effect was substantially diminished after alkali treatment. Lignin appears to reduce cellulose hydrolysis by two distinct mechanisms: by forming a physical barrier that prevents enzyme access and by non-productively binding cellulolytic enzymes. Cold alkali appears to selectively remove a fraction of lignin from steam-exploded Douglas-fir with high affinity for protein. Corresponding data for mixed softwood pretreated by organosolv extraction indicates that the relative importance of the two mechanisms by which residual lignin affects hydrolysis is different according to the pre- and post-treatment method used.

Cellulase Adsorption and an Evaluation of Enzyme Recycle During Hydrolysis of Steam-Exploded Softwood Residues

The sugar yield and enzyme adsorption profile obtained during the hydrolysis of SO2-catalyzed steam-exploded Douglas-fir and posttreated steam-exploded Douglas-fir substrates were determined. After hot alkali peroxide posttreatment, the rates and yield of hydrolysis attained from the posttreated Douglas-fir were significantly higher, even at lower enzyme loadings, than those obtained with the corresponding steam-exploded Douglas-fir. The enzymatic adsorption profiles observed during hydrolysis of the two substrates were significantly different. Ultrafiltration was employed to recover enzyme in solution (supernatant) and reused in subsequent hydrolysis reactions with added, fresh substrate. These recycle findings suggested that the enzyme remained relatively active for three rounds of recycle. It is likely that enzyme recovery and reuse during the hydrolysis of posttreated softwood substrates could lead to reductions in the need for the addition of fresh enzyme during softwood-based bioconversion processes.

Techno-economic evaluations of a generic wood-to-ethanol process: effect of increased cellulose yields and enzyme recycle.

A previously described techno-economic model (Gregg and Saddler, 1995a,b; Gregg and Saddler, 1996) was used to evaluate the effects of enzyme recycling and hydrolysis time on the production cost of ethanol from either hardwood or softwood substrates. Despite a significant reduction in the enzyme requirement the enzyme cost still represented about 18.5% and 22.7% of the total ethanol production cost when using either a hardwood or softwood feedstock. Enzyme recycling using the hydrolysis reactors (both hydrolysis and enzyme recycle combined in the same vessels) for two cycles reduced the ethanol cost by about 12% for both wood substrates. However, the enzyme recycling gain was less than 5% when implemented with dedicated enzyme recycle equipment (reactors and pumps). Doubling the hydrolysis time from 24 to 48 h decreased the ethanol cost by 18% for hardwoods and 27% for softwoods. The combination of enzyme recycling and doubling the hydrolysis time further decreased the ethanol cost by 11%. Consequently the lignin extraction and recovery component became the main contributor to ethanol production cost. It is probable that future benefits will likely come from incremental advances in each process step and/or a combination of a number of steps i.e., process integration, to reduce the production cost to marketable levels.

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.

A Techno-Economic Assessment of the Pretreatment and Fractionation Steps of a Biomass-to-Ethanol Process

It is generally recognized that the front-end (pretreatment, fractionation, enzymatic hydrolysis) steps of a lignocellulose-to-ethanol process are both technologically immature and represent a large component (∼60%) of the total product cost. In the past, we have tried to itemize the process steps and equipment for a complete plant. It was evident that, owing to the complexity and interrelated nature of this process, it was difficult to determine the influence of even minor changes to the process on the overall production cost of the product. We had originally developed a techno-economic model, based on spreadsheets, as a computational and assessment tool. However, our more recent work, which has looked at various process options such as hardwood vs softwoods, SO2 pretreatment of softwoods, and enzyme recycling, indicated that the model required greater flexibility if it was to assess a “generic” biomass-to-ethanol process. The model is currently being modified to address both the flexibility issues, through the incorporation of flowsheeting concepts, as well as including the most recent work on the various process options. In this article, we have described some of the pretreatment and fractionation issues that are being addressed in the updated model.

Fermentability of the hemicellulose‐derived sugars from steam‐exploded softwood (douglas fir)

Steam explosion ofDouglas fir wood chips under low-severity conditions (log Ro = 3.08 corresponding to 175 degrees C, 7.5 min, and 4.5% SO2) resulted in the recovery of around 87% of the original hemicellulose component in the water-soluble stream. More than 80% of the recovered hemicellulose was in a monomeric form. As the pretreatment severity increased from 3.08 to 3.76, hemicellulose recovery dropped to 43% of the original hemicellulose found in Douglas fir chips while the concentration of glucose originating from cellulose hydrolysis increased along with the concentration of sugar degradation products such as furfural and hydroxymethylfurfural. Despite containing a higher concentration of hexose monomers (mainly glucose originating from cellulose degradation), the water-soluble fraction prepared under high-severity conditions (log Ro = 3.73 corresponding to 215 degrees C, 2.38 min, and 2.38% SO2) was not readily fermented. Only the two hydrolyzates obtained at low and medium (195 degrees C, 4.5 min, and 4.5% SO2) severities were fermented to ethanol using a spent sulfur liquor adapted strain of Saccharomyces cerevisiae. High ethanol yields were obtained for these two hydrolyzates with 0.44 g of ethanol produced per gram of hexose utilized (86% of theoretical). However, the best results of hemicellulose recovery and fermentability were obtained for the low-severity water-soluble fraction which was fermented significantly faster than the fraction obtained after medium-severity treatment probably because it contained higher amounts of fermentation inhibitors. Copyright 1999 John Wiley & Sons, Inc.

Optimization of Steam Explosion to Enhance Hemicellulose Recovery and Enzymatic Hydrolysis of Cellulose in Softwoods

A combination of Douglas fir heartwood and sapwood chips were steam pretreated under three conditions as measured by the Severity Factor (log Ro), which incorporated the time, temperature/pressure of pretreatment. By adjusting the steam pretreatment conditions, it was hoped to recover the majority of the hemicellulose component as monomers in the water-soluble stream, while providing a cellulosic-rich, water-insoluble fraction that could be readily hydrolyzed by cellulases. These three conditions were chosen to represent either high hemicellulose sugar recovery (low severity [L], log Ro=3.08), high-enzyme hydrolyzability of the cellulosic component (high severity [H], log Ro=4.21), and a compromise between the two conditions (medium severity [M], log Ro=3.45). The medium-severity pretreatment conditions (195°C, 4.5 min, 4.5% SO2 logRo=3.45) gave the best compromise in terms of relatively high hemicellulose recovery after stream pretreatment and the subsequent efficiency of enzymatic hydrolysis of the water-insoluble cellulosic fraction. The percent recovery of the original hemicellulose in the water-soluble fraction dropped significantly when the severity was increased (L-76.8%, M-64.7%, and H-37.5%). However, the ease of enzymatic hydrolysis of the cellulose-rich, water-insoluble fraction increased with increasing severity (L-24%, M-86.6%, and H-97.9%). Although more severe pretreatment conditions provided optimum hydrolysis of the cellulosic component, less severe conditions resulted in better recovery of the combined hemicellulose and cellulosic components.

Assessing the emerging biorefinery sector in Canada

The biorefinery is a key concept used in the strategies and visions of many industrial countries. The potential for Canadian biorefineries based on lignocellulosic forest and agricultural residues is examined. The sector is described in terms of research interests, emerging companies, and established corporate interests. It is found that the Canadian biorefining sector currently has an emphasis on specific bioproduct generation, and the process elements required for a true sugar-based process are in the research phase. A Canadian national strategy should focus on increasing forest industry participation, and increasing collaboration with the provinces, particularly in western Canada.