The influence of lignin migration and relocation during steam pretreatment on the enzymatic hydrolysis of softwood and corn stover biomass substrates.

To be effective, steam pretreatment is typically carried out at temperatures/pressures above the glass transition point (Tg) of biomass lignin so that it can partly fluidize and relocate. The relocation of Douglas-fir and corn stover derived lignin was compared with the expectation that, with the corn stover lignin's lower hydrophobicity and molecular weight, it would be more readily fluidized. It was apparent that the Tg of lignin decreased as the moisture increased, with the easier access of steam to the corn stover lignin promoting its plasticization. Although the softwood lignin was more recalcitrant, when it was incorporated onto filter paper, it too could be plasticized, with its relocation enhancing enzymatic hydrolysis. When lignin recondensation was minimized, the increased hydrophobicity suppressed lignin relocation. It was apparent that differences in the accessibility of the lignin present in Douglas-fir and corn stover to steam significantly impacted lignin fluidization, relocation, and subsequent cellulose hydrolysis.

Horizontal gene transfer and gene dosage drives adaptation to wood colonization in a tree pathogen.

Some of the most damaging tree pathogens can attack woody stems, causing lesions (cankers) that may be lethal. To identify the genomic determinants of wood colonization leading to canker formation, we sequenced the genomes of the poplar canker pathogen, Mycosphaerella populorum, and the closely related poplar leaf pathogen, M. populicola. A secondary metabolite cluster unique to M. populorum is fully activated following induction by poplar wood and leaves. In addition, genes encoding hemicellulose-degrading enzymes, peptidases, and metabolite transporters were more abundant and were up-regulated in M. populorum growing on poplar wood-chip medium compared with M. populicola. The secondary gene cluster and several of the carbohydrate degradation genes have the signature of horizontal transfer from ascomycete fungi associated with wood decay and from prokaryotes. Acquisition and maintenance of the gene battery necessary for growth in woody tissues and gene dosage resulting in gene expression reconfiguration appear to be responsible for the adaptation of M. populorum to infect, colonize, and cause mortality on poplar woody stems.

The effects of increasing swelling and anionic charges on the enzymatic hydrolysis of organosolv-pretreated softwoods at low enzyme loadings.

Organosolv-pretreated Lodgepole pine substrates were physically and chemically treated to increase their hydrophilicity and swelling as these are two substrate attributes which have been shown to improve cellulolytic hydrolysis. Surprisingly, mechanical treatment of the organosolv-treated substrates by PFI-mill refining did not significantly increase hydrolysis yields despite decreases in particle size and crystallinity and increases in swelling. However, sulfonation of the substrate did, significantly, increase enzymatic hydrolysis at loadings of both 5 and 2.5 FPU g(-1) cellulose (from 80% to 95% and from 35% to 80%, respectively). In addition, sulfonation resulted in an increase in the amount of free enzymes detected during the course of hydrolysis to a maximum of 80% after 72 h. This suggested that the beneficial effects of sulfonation were primarily due to a decrease in the non-specific binding of the cellulases to the lignin.

The influence of lignin on the effectiveness of using a chemithermomechanical pulping based process to pretreat softwood chips and pellets prior to enzymatic hydrolysis.

Over the last century the pulp and paper sector has assessed various technologies to fractionate woody biomass to produce strong, bright fibers. Several of these processes have also been assessed for their potential to pretreat and fractionate biomass to enhance the subsequent enzymatic hydrolysis of the cellulosic component. Although many of these pretreatments are effective on agricultural residues, softwoods have proven more recalcitrant, primarily due to their high lignin content and structure. As delignification is too expensive to be used routinely a more economically attractive approach might be to alter the lignin. Recent work has shown that, using a modified chemithermomechanical pulping (CTMP) "front end", lignin can be modified and relocated. This significantly enhanced hemicellulose recovery and enzyme-mediated cellulose hydrolysis of woody biomass. As well as being effective on wood chips, the modified CTMP pretreatment process also enhanced the bioconversion of densified feedstocks such as pellets.

The Potential of Using Immobilized Xylanases to Enhance the Hydrolysis of Soluble, Biomass Derived Xylooligomers.

Earlier work had indicated that enzyme-mediated hydrolysis of xylooligomer-rich water-soluble streams (derived from steam pre-treated wheat straw) resulted in the effective production of xylose which was subsequently used to produce bio-glycol. In the work reported here, both the thermostability and recyclability of xylanases were significantly improved by covalent immobilizing the enzymes onto alginate beads. The immobilized xylanases showed a lower hydrolytic potential (~55% xylooligomer conversion) compared to the commercial xylanase cocktail HTec3 (~90% xylooligomer conversion) when used at the same protein loading concentration. This was likely due to the less efficient immobilization of key higher molecular weight enzymes (>75 kDa), such as ß-xylosidases. However, enzyme immobilization could be improved by lowering the glutaraldehyde loading used to activate the alginate beads, resulting in improved hydrolysis efficacy (~65% xylooligomer conversion). Enzyme immobilization improved enzyme thermostability (endoxylanase and ß-xylosidase activities were improved by 80% and 40%, respectively, after 24 h hydrolysis) and this allowed the immobilized enzymes to be reused/recycled for multiple rounds of hydrolysis (up to five times) without any significant reduction in their hydrolytic potential.

Enhanced delignification of steam-pretreated poplar by a bacterial laccase.

The recalcitrance of woody biomass, particularly its lignin component, hinders its sustainable transformation to fuels and biomaterials. Although the recent discovery of several bacterial ligninases promises the development of novel biocatalysts, these enzymes have largely been characterized using model substrates: direct evidence for their action on biomass is lacking. Herein, we report the delignification of woody biomass by a small laccase (sLac) from Amycolatopsis sp. 75iv3. Incubation of steam-pretreated poplar (SPP) with sLac enhanced the release of acid-precipitable polymeric lignin (APPL) by ~6-fold, and reduced the amount of acid-soluble lignin by ~15%. NMR spectrometry revealed that the APPL was significantly syringyl-enriched relative to the original material (~16:1 vs. ~3:1), and that sLac preferentially oxidized syringyl units and altered interunit linkage distributions. sLac's substrate preference among monoaryls was also consistent with this observation. In addition, sLac treatment reduced the molar mass of the APPL by over 50%, as determined by gel-permeation chromatography coupled with multi-angle light scattering. Finally, sLac acted synergistically with a commercial cellulase cocktail to increase glucose production from SPP ~8%. Overall, this study establishes the lignolytic activity of sLac on woody biomass and highlights the biocatalytic potential of bacterial enzymes.

Canadian biomass reserves for biorefining.

A lignocellulosic-based biorefining strategy may be supported by biomass reserves, created initially with residues from wood product processing or agriculture. Biomass reserves might be expanded using innovative management techniques that reduce vulnerability of feedstock in the forest products or agricultural supply chain. Forest-harvest residue removal, disturbance isolation, and precommercial thinnings might produce 20-33 x 10(6) mt/yr of feedstock for Canadian biorefineries. Energy plantations on marginal Canadian farmland might produce another 9-20 mt. Biomass reserves should be used to support first-generation biorefining installations for bioethanol production, development of which will lead to the creation of future high-value coproducts. Suggestions for Canadian policy reform to support biomass reserves are provided.

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 beta-glucosidase and xylanase activities in the cellulase preparation. Accordingly, differences in performance were reduced or eliminated following supplementation with a crude beta-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.

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.

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.

Enhanced enzymatic hydrolysis of steam-exploded Douglas fir wood by alkali-oxygen post-treatment.

Good enzymatic hydrolysis of steam-exploded Douglas fir wood (SEDW) cannot be achieved owing to the very high lignin content ( >40%) that remains associated with this substrate. Thus, in this study, we investigated the use of alkali-oxygen treatment as a posttreatment to delignify SEDW and also considered the enzymatic hydrolyzability of the delignified SEDW. The results showed that under optimized conditions of 15% NaOH, 5% consistency, 110 degrees C, and 3 h, approx 84% of the lignin in SEDW could be removed. The resulting delignified SEDW had good hydrolyzability, and cellulose-to-glucose conversion yields of over 90 and 100% could be achieved within 48 h with 20 and 40 filter paper units/g of cellulose enzyme loadings, respectively. It was also indicated that severe conditions, such as high NaOH concentration and high temperature, should not be utilized in oxygen delignification of SEDW in order to avoid extensive condensation of lignin and significant degradation of cellulose.

Effects of sugar inhibition on cellulases and beta-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.

SO2-catalyzed steam explosion of corn fiber for ethanol production.

Corn fiber, a by-product of the corn wet-milling industry, represents a renewable resource that is readily available in significant quantities and could potentially serve as a low-cost feedstock for the production of fuel-grade alcohol. In this study, we used a batch reactor to steam explode corn fiber at various degrees of severity to evaluate the potential of using this feedstock in the bioconversion process. The results indicated that maximum sugar yields (soluble and following enzymatic hydrolysis) were recovered from corn fiber that was pretreated at 190 degrees C for 5 min with 6% SO2. Sequential SO2-catalyzed steam explosion and enzymatic hydrolysis resulted in very high conversion (81%) of all polysaccharides in the corn fiber to monomeric sugars. Subsequently, Saccharomyces cerevisiae was able to convert the resultant corn fiber hydrolysates to ethanol very efficiently, yielding 90-96% of theoretical conversion during the fermentation process.

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.

Optimization of SO2-catalyzed steam pretreatment of corn fiber for ethanol production.

A batch reactor was employed to steam explode corn fiber at various degrees of severity to evaluate the potential of using this feedstock as part of an enzymatically mediated cellulose-to-ethanol process. Severity was controlled by altering temperature (150-230 degrees C), residence time (1-9 min), and SO2 concentration (0-6% [w/w] dry matter). The effects of varying the different parameters were assessed by response surface modeling. The results indicated that maximum sugar yields (hemicellulose-derived water soluble, and cellulose-derived following enzymatic hydrolysis) were recovered from corn fiber pretreated at 190 degrees C for 5 minutes after exposure to 3% SO2. Sequential SO2-catalyzed steam explosion and enzymatic hydrolysis resulted in a conversion efficiency of 81% of the combined original hemicellulose and cellulose in the corn fiber to monomeric sugars. An additional posthydrolysis step performed on water soluble hemicellulose stream increased the concentration of sugars available for fermentation by 10%, resulting in the high conversion efficiency of 91%. Saccharomyces cerevisiae was able to ferment the resultant corn fiber hydrolysates, perhydrolysate, and liquid fraction from the posthydrolysis steps to 89, 94, and 85% of theoretical ethanol conversion, respectively. It was apparent that all of the parameters investigated during the steam explosion pretreatment had a significant effect on sugar recovery, inhibitory formation, enzymatic conversion efficiency, and fermentation capacity of the yeast.

Wood-ethanol for climate change mitigation in Canada.

The impetus for this paper is Canada's commitment under the United Nations Framework Convention on Climate Change to reduce national greenhouse gas emissions as well as reducing our dependency on fossil fuels. Wood-based ethanol offers an excellent opportunity for greenhouse gas mitigation due to market potential, an ability to offset significant emissions from the transportation sector, a reduction of emissions from CO2-intensive waste-management systems, and carbon sequestration in afforested plantations. While there are technological and economic barriers to overcome, using wood-biomass as a source of ethanol can be an economically viable tool for reducing greenhouse gas levels in the atmosphere. This paper examines the costs and mitigation potential of the production of ethanol from biomass supplied from industrial wood waste as well as from trees harvested from afforested land.

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.

Industrial sustainability of competing wood energy options in Canada.

The amount of sawmill residue available in Canada to support the emerging cellulosic ethanol industry was examined. A material flow analysis technique was employed to determine the amount of sawmill residue that could possibly be available to the ethanol industry per annum. A combination of two key trends--improved efficiency of lumber recovery and increased uptake of sawmill residues for self-generation and for wood pellet production--have contributed to a declining trend of sawmill residue availability. Approximately 2.3 x 106 bone-dry tons per year of sawmill residue was estimated to be potentially available to the cellulosic ethanol industry in Canada, yielding 350 million liters per year of cellulosic ethanol using best practices. An additional 2.7 billion liters of cellulosic ethanol might be generated from sawmill residue that is currently used for competing wood energy purposes, including wood pellet generation. Continued competition between bioenergy options will reduce the industrial sustainability of the forest industry. Recommendations for policy reforms towards improved industrial sustainability practices are provided.