Formulation of an optimized synergistic enzyme cocktail, HoloMix, for effective degradation of various pre-treated hardwoods.

In this study, two selected hardwoods were subjected to sodium chlorite delignification and steam explosion, and the impact of pre-treatments on synergistic enzymatic saccharification evaluated. A cellulolytic core-set, CelMix, and a xylanolytic core-set, XynMix, optimised for glucose and xylose release, respectively, were used to formulate HoloMix cocktail for optimal saccharification of various pre-treated hardwoods. For delignified biomass, the optimized HoloMix consisted of 75%:25% protein dosage, CelMix: XynMix, while for untreated and steam exploded biomass the HoloMix consisted of 93.75%:6.25% protein dosage. Saccharification by HoloMix (27.5mgprotein/gbiomass) for 24h achieved 70-100% sugar yields. Pre-treatment of the hardwoods (especially those with a higher proportion of lignin) with a laccase, improved saccharification by HoloMix. This study provided insights into enzymatic hydrolysis of various pre-treated hardwood substrates and showed the same lignocellulolytic cocktail comparable to/if not better than commercial enzyme preparations can be used to efficiently hydrolyse different hardwood species.

A two-stage pretreatment approach to maximise sugar yield and enhance reactive lignin recovery from poplar wood chips.

A two-stage pretreatment approach, employing steam followed by organosolv treatment, was assessed for its ability to fractionate and recover most of the hemicellulose, lignin and cellulose components of poplar wood chips. A mild steaming stage was initially used to maximise hemicellulose sugar recovery, with 63% of the original xylan solubilised and recovered after this stage and close to 90% recovered in total. Rather than hindering subsequent organosolv delignification, the prior steam treatment enhanced lignin solubilisation with more than 66% of the original lignin removed after the two-stage pretreatment. The extracted lignin contained at least equal or greater amounts of functional groups as compared to the lignin solubilised after a single-stage organosolv pretreatment. More than 98% of the original cellulose was recovered after the two-stage pretreatment and 88% of the cellulose could be hydrolysed to glucose at enzyme loading of 5FPU/g cellulose after 72h.

Bioethanol from lignocellulosics: Status and perspectives in Canada.

Canada has invested significantly in the development of a domestic bioethanol industry, and it is expected that bioethanol from lignocellulosics will become more desirable to the industry as it expands. Development of the Canadian industry to date is described in this paper, as are examples of domestic research programs focused on both bioconversion and thermochemical conversion to generate biofuels from lignocellulosic biomass. The availability of lignocellulosic residues from agricultural and forestry operations, and the potential biofuel production associated with these residues, is described. The policy tools used to develop the domestic bioethanol industry are explored. A residue-based process could greatly extend the potential of the bioethanol industry in Canada. It is estimated that bioethanol production from residual lignocellulosic feedstocks could provide up to 50% of Canada's 2006 transportation fuel demand, given ideal conversion and full access to these feedstocks. Utilizing lignocellulosic biomass will extend the geographic range of the bioethanol industry, and increase the stability and security of this sector by reducing the impact of localized disruptions in supply. Use of disturbance crops could add 9% to this figure, but not in a sustainable fashion. If pursued aggressively, energy crops ultimately could contribute bioethanol at a volume double that of Canada's gasoline consumption in 2006. This would move Canada towards greater transportation fuel independence and a larger role in the export of bioethanol to the global market.

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.

An ethanologenic yeast exhibiting unusual metabolism in the fermentation of lignocellulosic hexose sugars.

Three lignocellulosic substrate mixtures [liquid fraction of acid-catalyzed steam-exploded softwood, softwood spent sulfite liquor (SSL) and hardwood SSL] were separately fermented by the industrially employed SSL-adapted strain Tembec T1 and a natural galactose-assimilating isolate (Y-1528) of Saccharomyces cerevisiae to compare fermentative efficacy. Both strains were confirmed as S. cerevisiae via molecular genotyping. The performance of strain Y-1528 exceeded that of Tembec T1 on all three substrate mixtures, with complete hexose sugar consumption ranging from 10 to 18 h for Y-1528, vs 24 to 28 h for T1. Furthermore, Y-1528 consumed galactose prior to glucose and mannose, in contrast to Tembec T1, which exhibited catabolite repression of galactose metabolism. Ethanol yields were comparable regardless of the substrate utilized. Strains T1 and Y-1528 were also combined in mixed culture to determine the effects of integrating their distinct metabolic capabilities during defined hexose sugar and SSL fermentations. Sugar consumption in the defined mixture was accelerated, with complete exhaustion of hexose sugars occurring in just over 6 h. Galactose was consumed first, followed by glucose and mannose. Ethanol yields were slightly reduced relative to pure cultures of Y-1528, but normal growth kinetics was not impeded. Sugar consumption in the SSLs was also accelerated, with complete utilization of softwood- and hardwood-derived hexose sugars occurring in 6 and 8 h, respectively. Catabolite repression was absent in both SSL fermentations.

The influence of bark on the fermentation of Douglas-fir whitewood pre-hydrolysates.

Douglas-fir ( Pseudotsuga menziesii) whitewood was supplemented with increasing concentrations of bark (0-30%) and was pretreated using SO(2)-catalysed steam explosion. The presence of bark in the feedstock resulted in the decreased recovery of total sugars, furfural and 5-hydroxymethylfurfural in the resultant pre-hydrolysate. No detrimental impact on monomer sugar recovery was observed. The concentration of lipophilic extractives present in the pre-hydrolysate increased with increasing bark loading, to a maximum of 0.43 g x l(-1). The water-soluble pre-hydrolysates were fermented by Saccharomyces cerevisiae to determine the impact of bark on sugar consumption and ethanol production. Despite the inclusion of bark, fermentation of all pre-hydrolysates resulted in the complete consumption of hexose sugars within 48 h. Ethanol yields were greater than 0.43 g x g(-1) for all pre-hydrolysates regardless of bark content, indicating that, up to a content of 30%, bark had a negligible impact on the fermentation of the pre-hydrolysates to ethanol.

Degradation of trilinolein by laccase enzymes.

Laccase enzymes were investigated for their potential to catalyze the oxidation of trilinolein and methyl linoleate. This study demonstrates that laccase enzymes can oxidize unsaturated fatty acid esters and their associated lipids. The reaction products resulting from laccase-catalyzed reactions with trilinolein were analyzed using combined reversed-phase high-performance liquid chromatography and mass spectrometry via an atmospheric pressure chemical ionization source. The dominant oxidation products detected were monohydroperoxides, bishydroperoxides, and epoxides. This paper presents the first detailed investigation into the interaction between laccase enzymes and lipids containing unsaturated fatty acids.

Do enzymatic hydrolyzability and Simons' stain reflect the changes in the accessibility of lignocellulosic substrates to cellulase enzymes?

In an attempt to elucidate the impact of substrate accessibility to cellulases on the susceptibility of lignocellulosic substrates to enzymatic hydrolysis, a hydrogen peroxide treated, Douglas fir kraft pulp was dried using several methods with varying levels of intensity. Oven-drying at 50 and 100 degrees C, air-drying, and freeze-drying methods were employed to remove the interfibrillar water from the pulp samples. Subsequently, the never-dried and variably dried pulps were hydrolyzed using a commercial cellulase preparation supplemented with additional beta-glucosidase. Drying reduced the susceptibility of the substrates to enzymatic hydrolysis, which can be attributed to the hornifying effect that drying has on fibers. This effect was more pronounced for the fibers that were oven-dried at 100 degrees C (23% reduction) and 50 degrees C (15% reduction), and there was a good correlation between the Simons's stain results and the enzymatic digestibility of the dried pulps. These observations indicated that drying significantly reduced the population of larger pores and that the partial closure of larger pores created a large number of smaller pores that were not accessible to the displacement dye molecules (orange dye). The inaccessibility of the cellulose to the enzymes, due to the collapse or closure of the large pores, appears to be the primary reason for the lower susceptibility of the dried pulps to enzymatic hydrolysis.

Sugar recovery and fermentability of hemicellulose hydrolysates from steam-exploded softwoods containing bark.

The hemicellulose sugar recovery and ethanol production obtained from SO2-catalyzed steam explosion of a mixed white fir (70%) and ponderosa pine (30%) feedstock containing bark (9% dry weight/dry weight) was assessed. More than 90% of the available hemicellulose sugars could be recovered in the hydrolysate obtained after steam explosion at 195 degrees C, 2.38 min, and 3.91% SO2, with 59% of the original hemicellulose sugars detected in a monomeric form. Despite this high sugar recovery, this hydrolysate showed low ethanol yield (64% of theoretical yield) when fermented with a spent sulfite liquor-adapted strain of Saccharomyces cerevisiae. In contrast, most hydrolysates prepared at higher steam explosion severity showed comparable or higher ethanol yields. Furthermore, the hydrolysates prepared from bark-free feedstock showed better fermentability (87% of theoretical yield) despite containing higher concentration of known inhibitors. The ethanol yield from the hydrolysate prepared from a bark-containing wood sample could be improved to 81% by an extra stage acid hydrolysis (121 degrees C for 1 h in 3% sulfuric acid). This extra stage acid hydrolysis and steam explosion at higher severity conditions seem to improve the fermentability of the hydrolysates by transforming certain inhibitory compounds present in the hydrolysates prepared from the bark-containing feedstock and thus lowering their inhibitory effect on the yeast used for the ethanol fermentation.

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 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.

The nature of lignin from steam explosion/ enzymatic hydrolysis of softwood: structural features and possible uses: scientific note.

Effective utilization of the lignin by-product is a prerequisite to the commercial viability of ethanol production from softwood wastes using a steam explosion (SE)/enzymatic hydrolysis (EH)/fermentation process. Changes in the chemical composition of Douglas fir wood on SO2-catalyzed SE followed by EH were assessed using conventional analytical methods and new halogen-probe techniques. A significant solubilization of hemicelluloses was observed in the SE stage, the severity of which affected subsequent fermentation of cellulose and sorption of enzymes. SE of softwood resulted in dramatic changes in the chemical structure of lignin in the residual material involving chemical reactions via the benzyl cation. This leads to a more condensed lignin with partly blocked alpha-reaction centres. Possible uses for this lignin are discussed.

Analysis of molecular size distributions of cellulose molecules during hydrolysis of cellulose by recombinant Cellulomonas fimi beta-1,4-glucanases.

Four beta-1,4-glucanases (cellulases) of the cellulolytic bacterium Cellulomonas fimi were purified from Escherichia coli cells transformed with recombinant plasmids. Previous analyses using soluble substrates had suggested that CenA and CenC were endoglucanases while CbhA and CbhB resembled the exo-acting cellobiohydrolases produced by cellulolytic fungi. Analysis of molecular size distributions during cellulose hydrolysis by the individual enzymes confirmed these preliminary findings and provided further evidence that endoglucanase CenC has a more processive hydrolytic activity than CenA. The significant differences between the size distributions obtained during hydrolysis of bacterial microcrystalline cellulose and acid-swollen cellulose can be explained in terms of the accessibility of beta-1,4-glucan chains to enzyme attack. Endoglucanases and cellobiohydrolases were much more easily distinguished when the acid-swollen substrate was used.

Microbiology and biodegradation of resin acids in pulp mill effluents: a minireview.

Resin acids, a group of diterpenoid carboxylic acids present mainly in softwood species, are present in many pulp mill effluents and toxic to fish in recipient waters. They are considered to be readily biodegradable. However, their removal across biological treatment systems has been shown to vary. Recent studies indicate that natural resin acids and transformation products may accumulate in sediments and pose acute and chronic toxicity to fish. Several resin acid biotransformation compounds have also been shown to bioaccumulate and to be more resistant to biodegradation than the original material. Until recently, the microbiology of resin-acid degradation has received only scant attention. Although wood-inhabiting fungi have been shown to decrease the level of resin present in wood, there is no conclusive evidence that fungi can completely degrade these compounds. In contrast, a number of bacterial isolates have recently been described which are able to utilize dehydroabietic or isopimaric acids as their sole carbon source. There appears to be an unusually high degree of substrate specificity with respect of the utilization of abietane congeners and the presence of substituents. Pimaranes do not appear to be attacked to the same extent as the abietanes. This paper reviews the occurrence, chemistry, toxicity, and biodegradation of resin acids in relation to the biological treatment of pulp and paper mill effluents.

Bleach boosting and direct brightening by multiple xylanase treatments during peroxide bleaching of kraft pulps.

The effects of multiple xylanase treatments were assessed during the peroxide bleaching of three pulps: Douglas-fir (kraft); Western hemlock (oxygen delignified kraft); and trembling Aspen (kraft). The addition of a xylanase treatment stage, either before or after the peroxide bleaching stage(s), resulted in the enhanced brightening of all pulps. A higher brightness was achieved using two enzyme treatments, one before and one after the peroxide stage(s). Both bleach boosting and direct brightening seemed to contribute to the enhancement of the peroxide bleaching. Compared to xylanase prebleaching, xylanase posttreatment of peroxide bleached pulps solubilized less lignin and chromophores and made smaller amounts of these materials alkaline soluble. Nevertheless, the final brightness achieved by xylanase posttreatment was similar or superior to that achieved with xylanase prebleaching of the corresponding unbleached pulps. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 312-318, 1997.

A review of techno-economic modeling methodology for a wood-to-ethanol process.

Techno-economic modeling has been a valuable tool in directing and assessing the research and development efforts for biomass-to-ethanol processes. In developing a techno-economic model of a "generic" wood-to-ethanol process, we decided to follow a three-pronged design approach. This initially consisted of a detailed review of the current definition and technical maturity of the process, which concluded that the process remains complex and immature. More recently, we have critically assessed/compared two inherited models, and examined the historical and current trends in modeling design. We confirmed that process complexity and immaturity, in association with the capabilities of the available modeling tools and the ease with which they can be used, influenced the design and implementation of past models. We have discussed these influences with reference to our own model development decisions. For example, on review of two inherited techno-economic models, we decided that our new model would require a greater degree of flexibility in its structure and user interface.

Cellobiose dehydrogenase, an active agent in cellulose depolymerization.

The ability of cellobiose dehydrogenase purified from Phanerochaete chrysosporium to modify a Douglas fir kraft pulp was assessed. Although the addition of cellobiose dehydrogenase alone had little effect, supplementation with cellobiose and iron resulted in a substantial reduction in the degree of polymerization of the pulp cellulose. When the reaction was monitored over time, a progressive depolymerization of the cellulose was apparent with the concomitant production of cellobiono-1,5-lactone. Analysis of the reaction filtrates indicated that glucose and arabinose were the only neutral sugars generated. These sugars are derived from the degradation of the cellobiose rather than resulting from modifications of the pulp. These results suggest that the action of cellobiose dehydrogenase results in the generation of hydroxyl radicals via Fenton's chemistry which subsequently results in the depolymerization of cellulose. This appears to be the mechanism whereby a substantial reduction in the degree of polymerization of the cellulose can be achieved without a significant release of sugar.

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. (c) 1996 John Wiley & Sons, Inc.

Growth, induction, and substrate specificity of dehydroabietic acid-degrading bacteria isolated from a kraft mill effluent enrichment.

We investigated resin acid degradation in five bacteria isolated from a bleach kraft mill effluent enrichment. All of the bacteria grew on dehydroabietic acid (DHA), a resin acid routinely detected in pulping effluents, or glycerol as the sole carbon source. None of the strains grew on acetate or methanol. Glycerol-grown, high-density, resting-cell suspensions were found to undergo a lag for 2 to 4 h before DHA degradation commenced, suggesting that this activity was inducible. This was further investigated by spiking similar cultures with tetracycline, a protein synthesis inhibitor, at various times during the DHA disappearance curve. Cultures to which the antibiotic was added prior to the lag did not degrade DHA. Those that were spiked with the antibiotic after the lag phase (4 h) degraded DHA at the same rate as did controls with no added tetracycline. Therefore, de novo protein synthesis was required for DHA biodegradation, confirming that this activity is inducible. The five strains were also evaluated for their ability to degrade other resin acids. All strains behaved in a similar fashion. Unchlorinated abietane-type resin acids (abietic acid, DHA, and 7-oxo-DHA) were completely degraded within 7 days, whereas pimarane resin acids (sandaracopimaric acid, isopimaric acid, and pimaric acid) were poorly degraded (25% or less). Chlorination of DHA affected biodegradation, with both 12,14-dichloro-DHA and 14-chloro-DHA showing resistance to degradation. However, 50 to 60% of the 12-chloro-DHA was consumed within the same period.

Evaluation of cellulase recycling strategies for the hydrolysis of lignocellulosic substrates.

Recycling of cellulases should lower the overall cost of lignocellulosiic bioconversion processes. In this study, three recycling strategies were evaluated to determine their efficiencies over five successive rounds of hydrolysis. The effect of lignin on recycling was examined by comparing water-washed, steam-exploded birch (WB; 32% lignin) and WB which had been further extracted with alkali and peroxide (PB; 4% lignin). When the cellulases were recovered from the residual substrates after partial hydrolysis of both substrates, the recovered cellulase activity toward the mixture of fresh and residual substrates decreased after each recycling step. When the cellulases in the supernatants were also recycled, up to 20% more activity could be recovered. In both of these cases, the recovered activities did not correspond to the activities expected from the amount of cellulase protein recovered during recycling. The best recovery was obtained when the cellulases were recovered from both the residue and the supernatant after complete hydrolysis of the PB substrate. In this case, all of the originally added cellulase activity could be recovered for four consecutive hydrolysis rounds. However, when the same recycling strategy was carried out using the WB substrate, the recovered cellulase activity declined quickly with each recycling round. In all three of the recycling strategies, lower cellulase activities were recovered from the substrates with higher lignin contents. (c) 1995 John Wiley & Sons, Inc.