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 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.

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.

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.

Anaesthetic management of hip arthroplasty in an individual with trisomy 21 and Eisenmenger's syndrome.

A 49-year-old man with trisomy 21 and Eisenmenger's syndrome presented for hip arthroplasty. Eisenmenger's syndrome is defined by the presence of obstructive pulmonary vascular disease secondary to long-standing left-to-right shunt causing pulmonary hypertension, eventually leading to shunt reversal in to right-to-left direction. Patients with Eisenmenger's syndrome pose a significant perioperative risk because of the physiological alterations induced by anaesthetic agents. The choice of anaesthetic technique in these patients is therefore not straightforward. A successful outcome was achieved with general anaesthesia supplemented with nerve blocks.

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.

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.

High consistency enzymatic hydrolysis of hardwood substrates.

The feasibility of using a laboratory peg mixer to carry out high consistency enzymatic hydrolysis of lignocellulosic substrates was investigated. Two hardwood substrates, unbleached hardwood pulp (UBHW) and organosolv pretreated poplar (OPP), were used in this study. Hydrolysis of UBHW and OPP at 20% substrate consistency led to a high glucose concentration in the final hydrolysate. For example, a 48 h enzymatic hydrolysis of OPP resulted in a hydrolysate with 158 g/L of glucose. This is the highest glucose concentration ever obtained from enzymatic hydrolysis of lignocellulosic substrates. Fermentation of UBHW and OPP hydrolysates with high glucose content led to high ethanol concentrations, 50.4 and 63.1 g/L, respectively after fermentation. Our results demonstrate that using common pulping equipment to carry out high consistency hydrolysis can overcome the rheological problems and greatly increase the sugar and ethanol concentrations after the hydrolysis and fermentation.

Comparative sugar recovery and fermentation data following pretreatment of poplar wood by leading technologies.

Through a Biomass Refining Consortium for Applied Fundamentals and Innovation among Auburn University, Dartmouth College, Michigan State University, the National Renewable Energy Laboratory, Purdue University, Texas A&M University, the University of British Columbia, and the University of California at Riverside, leading pretreatment technologies based on ammonia fiber expansion, aqueous ammonia recycle, dilute sulfuric acid, lime, neutral pH, and sulfur dioxide were applied to a single source of poplar wood, and the remaining solids from each technology were hydrolyzed to sugars using the same enzymes. Identical analytical methods and a consistent material balance methodology were employed to develop comparative performance data for each combination of pretreatment and enzymes. Overall, compared to data with corn stover employed previously, the results showed that poplar was more recalcitrant to conversion to sugars and that sugar yields from the combined operations of pretreatment and enzymatic hydrolysis varied more among pretreatments. However, application of more severe pretreatment conditions gave good yields from sulfur dioxide and lime, and a recombinant yeast strain fermented the mixed stream of glucose and xylose sugars released by enzymatic hydrolysis of water washed solids from all pretreatments to ethanol with similarly high yields. An Agricultural and Industrial Advisory Board followed progress and helped steer the research to meet scientific and commercial needs.