John Saddler
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Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
Several oil refineries have repurposed their operations to “stand-alone” biorefineries that upgrade oleochemical feedstocks to produce lower carbon-intensive fuels. The refining of biogenic feedstocks produces a range of lower-carbon intensive fuels with the nature of the feedstock, the refinery configuration, the source of hydrogen, all contributing to the carbon intensity of the process and the final fuels. Policies have also encouraged some refineries to co-process biogenic feedstock rather than to repurpose. This has proven to be a less capital-intensive way of reducing the carbon intensity of the refinery and its products. Although, at this time, oleochemical feedstocks predominate as co-processing feedstocks, in the longer term, biocrudes are expected to predominate. The work primarily focused on co-processing of oleochemical feedstocks with the recognition that any results will be needed for future biocrude co-processing. Initial work assessed if a stable baseline could be established and whether a 6% co-processing ratio would allow any changes in the yields of the various process streams to be readily detected. However, the minimum changes detected indicated that the “signal” resulting from processing biogenic feedstocks was not strong enough to overcome the background “noise” of normal petroleum refining. Thus, to amplify detection, multiple linear regression models which made use of year-long commercial operational data, while normalizing critical changing variables, were developed. These models were successfully used to quantify the impact of co-processing lipid feedstocks on the yields of existing petroleum operations.However, it was apparent that the results from modelling were not as consistent as desired for all fractions. The difficulties in obtaining direct measurements highlighted the practical problems in sampling the various fuel streams. A combination of direct measurements and long-term process data provided the most practical solution, particularly for hard-to-measure fractions such as the “green coke” that was generated during co-processing of biogenic feedstocks. Although tracking the “green molecules” proved challenging, these methods will be needed to improve the quality of needed data (e.g., mass balance and hydrogen consumption) if we are to determine the carbon intensity of the various fuels produced as a result of co-processing and for the refinery to obtain “policy credits”.
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To try to take advantage of existing infrastructure and experience a chemi-thermomechanical pulping (CTMP) process was assessed for its potential as a “front-end” for a biochemical-based bioconversion process. It had been shown that biomass, after mechanical pulping treatment, remained highly recalcitrant to enzymatic hydrolysis. This was largely due to the presence of lignin restricting enzyme accessibility to cellulose. Considering the high costs related to complete delignification, mild chemical treatment such as sulfonation and oxidation under neutral/alkaline conditions were assessed to minimize lignin’s inhibitory influence while maximizing the recovery of hemicellulose in the water-insoluble component. Sulfonation and oxidation were able to incorporate acid groups onto the lignin macromolecule, consequently enhancing substrate swelling. This increased enzyme accessibility to the cellulose while reducing non-productive lignin binding via increased lignin hydrophilicity.CTMP-based pretreatment was shown to be effective on agricultural and hardwood substrates. Mild alkali treatment of agricultural residues induced deacetylation of the hemicellulose and partial delignification. This resulted in enhanced enzyme accessibility to the hemicellulose and cellulose and increased enzymatic hydrolysis. Although hardwood lignin was more resistant to delignification, the incorporation of oxygen treatment into the CTMP treatment of the hardwood substrate substantially reduced the negative effects of lignin on enzymatic hydrolysis. As the lignin present in the CTMP treated substrate was enriched in acid groups, this resulted in increased substrate swelling and a decrease in the non-productive binding of enzymes to the lignin (via hydrophobic interactions).Both softwood chips and pellets where pretreated using the adapted CTMP process to provide both a comparison with hardwood and agriculture feedstocks and to assess any differences between pellets and chips. Alkali addition prior to CTMP pulping enhanced lignin sulfonation. This predominantly occurred within the secondary-cell-wall, consequently increasing cellulose accessibility. However, the pretreated softwood chips and pellets remained relatively recalcitrant to enzymatic hydrolysis. Although the reduced particle size of softwood pellets was anticipated to facilitate chemicals and enzyme access, the high temperatures used during pelletisation resulted in lignin condensation. This was indicated by higher molecular weight and lower β-O-4 linkages of pellet-derived lignin, probably contributing to this substrate’s higher recalcitrance.
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Cellulose is a structural material that, through its association with lignin and hemicellulose, is recalcitrant to degradation. Although the effectiveness of cellulose hydrolysis is usually assessed via glucose release, typically, cellulase accessibility to the cellulosic substrate is the key limitation that restricts effective enzymatic hydrolysis and has proven much harder to quantify. A novel method, which has the potential to better elucidate the mechanisms involved, involves the use of carbohydrate-binding modules (CBMs). In the work described here, CBM production was optimized, yielding g.L-1 quantities of the specific proteins, which were subsequently used to both characterize the surface morphology of lignocellulosic substrates and functionalize cellulose surfaces. A combination of type A and type B CBMs (CBM2a and CBM17) were primarily employed, as they showed binding preferences towards different morphologies within the cellulosic structure. Compared to more established methods the CBM method more accurately predicted enzyme accessibility, indicating that refining did not significantly improve enzyme accessibility at the microfibril level of the cellulosic substrate. In subsequent work, fluorescence-tagged carbohydrate binding modules (CBMs), which specifically bind to crystalline (CBM2a-RRedX) and paracrystalline (CBM17-FITC) cellulose, were used to differentiate the supramolecular cellulose structures in bleached softwood Kraft fibers during enzyme-mediated hydrolysis. Quantitative image analysis, supported by 13C NMR, SEM imaging, and fiber length distribution analysis, indicated that enzymatic degradation predominated in the more disorganized zones during the initial phase of the hydrolysis reaction. This resulted in rapid fiber fragmentation and an increase in cellulose surface crystallinity. Drying decreased the accessibility of enzymes to these disorganized zones, resulting in a delayed onset of degradation and fragmentation. The use of fluorescence-tagged CBMs with specific recognition sites provided a quantitative way to elucidate cellulose morphology and its impact on enzyme accessibility. This in turn provided novel insights into the mechanisms involved in enzyme-mediated cellulose deconstruction. As well as using CBMs as an analytical tool, the affinity of CBMs for cellulosic surfaces was also used to introduce functionality. When CBM2a-alkyne bioconjugation was used to link polyethylene glycol (PEG) to CNC surfaces via Click reactions, the CBM-PEG modification of cellulosic surfaces increased CNC redispersion after drying and improved suspension stability.
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To try to compete with conventional bioethanol production from sugar/starch feedstocks, “whole-slurry”, high solids loading hydrolysis and fermentation of steam pretreated softwood biomass was employed to obtain high sugar, high ethanol concentrations. However, two major challenges were encountered; substrate recalcitrance limiting effective enzymatic hydrolysis and high concentrations of inhibitors inhibiting effective sugar fermentation. The major focus of the work described in this thesis was to assess the benefits of integrating sulphite treatments prior, during and after steam pretreatment to enhance the effectiveness of the enzymatic hydrolysis of the whole cellulose and hemicellulose derived slurry and the fermentation of the biomass derived sugars.Initial work focused on improving fermentation at high sugar concentrations (up to 25% w/v) by improved strain selection, nutrient supplementation and the use of high cell density growth Subsequently, sulphite post treatment was assessed to see if it could improve both the hydrolysis of the whole slurry, containing both cellulose and hemicellulose, and the fermentation of the softwood derived sugars. To try to maximize the beneficial influence of sulphite treatment the softwood chips were treated prior to steam pretreatment, to result in sulphonation ahead of lignin condensation, hopefully improving the extent of sulphonation with less sulphite loading. This approach resulted in some improvements. However, it proved difficult to balance some of the factors that influence the effectiveness of enzyme mediated hydrolysis, such as the extent of suphonation, substrates size reduction and lignin condensation, although an improvement in whole slurry fermentation was observed. To try to further improve this strategy a two stage, alkali- followed by acid-sulphite approach was assessed, using sulphite in the first stage to sulphonate the lignin and SO₂ in the second stage to further sulphonated the lignin while decreasing particle size. This resulted in high degree of sulphonation, enhanced delignification and substantial substrate size reduction. Minimum lignin condensation and fermentation inhibitors were detected. More than 160 g/L fermentable sugars and 80 g/L ethanol could be achieved when using the two stage (alkali and acid) sulphite pretreatment of lodgepole pine approach to generate a hemicellulose and cellulose whole slurry that could be readily hydrolysed and fermented.
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Enzyme-mediated hydrolysis of lignocellulosic materials for their conversion into bioproducts would benefit significantly if high solids concentrations (low water-to-biomass ratios) could be processed effectively. However, the fibrous nature of lignocellulosic biomass makes effective reaction mixing difficult, resulting in mass transfer limitations that reduce process yields. Overcoming these rheological challenges will require a better understanding of the substrate properties that influence slurry rheology, and in particular, how the action of carbohydrate-active enzymes can better facilitate slurry viscosity reduction, or ‘enzymatic liquefaction’. To this end, the work described in this thesis assessed: the underlying causes of the rheological challenge of high-solids bioconversion; the possible mechanisms of enzymatic liquefaction; how liquefaction is influenced by the nature of the substrate; and the roles the various enzymes play in liquefaction. It was apparent that the relationship between substrate properties, slurry rheology and high-solids hydrolysis kinetics is complex and multifaceted. Substrate–water interactions were shown to be a key determinant that influenced the mass transfer boundary and the scaling of yield stress with solids loading. The yield stress profiles of the various pretreated substrates varied extensively, indicating that ‘high solids’ is a relative, substrate-dependent quality. Substrate rheological characteristics, especially slurry yield stress, were shown to be directly linked to liquefaction efficiency and to reductions in hydrolysis yield with increasing solids loading. It appeared that enzyme-mediated liquefaction of biomass was achieved through a combination of material dilution, particle fragmentation and alteration of interparticle interactions. Cellobiohydrolases and endoglucanases were shown to be the key enzymes involved in these mechanisms. However, the effectiveness of the various enzymes was strongly influenced by the substrate’s physicochemical properties and concentration. Notably, an enzyme’s low-solids slurry viscosity-reducing capacity did not necessarily reflect its capacity to catalyze liquefaction at high solids loadings. Furthermore, reaction efficiencies tested at low solids loadings did not reliably predict efficiency at high solids due to substrate-specific rheological differences. In summary, this work provided rheological and enzymological insights into the highly disparate reaction kinetics and rheological challenge prevailing at commercially relevant substrate concentrations.
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Any pretreatment process used to enhance the enzymatic deconstruction of lignocellulosic substrates, although opening up and enhancing access to the cellulose, will typically generate inhibitory compounds (i.e. soluble mono/oligomeric sugars, phenolics, furans, extractives, etc.) that will limit or restrict the efficiency of cellulose hydrolysis. To develop more effective inhibition mitigation strategies, it would be beneficial if we had a better understanding of the inhibitory mechanisms of these soluble compounds on the enzyme components of cellulase cocktails. Most of the previous studies which have tried to assess the effects of inhibitors on cellulase enzymes, have used “synthetic mixtures of inhibitors” and “traditional” cellulase preparations such as Celluclast. The work presented in this thesis assessed the major inhibitory compounds derived from a range of “real-life” lignocellulosic biomass substrates that were steam pretreated at various severities. The major inhibitory mechanisms, such as reversible/irreversible inhibition of the major enzyme activities (e.g. exo/endo-glucanase, β-glucosidase, xylanase activities, etc.), were investigated and potential inhibitor mitigation strategies were evaluated. Initial work showed that, although the more recent cellulase mixture CTec3 was more inhibitor tolerant than the older Celluclast enzyme preparation, they were still strongly inhibited by pretreatment derived inhibitors. Of the various inhibitors, sugars and phenolics were shown to be the major groups that significantly contributed to the observed decrease in cellulose hydrolysis. This was mostly because of the strong inhibition and deactivation of β-glucosidase and cellobiohydrolase activities present within the cellulase mixture. Surprisingly, although hemicellulose derived sugars did not appear to inhibit individual enzyme activities, they did inhibit overall cellulose hydrolysis. Subsequent work suggested that hemicellulose-derived sugars inhibited the processive movement of cellobiohydrolase Cel7A and thus restricted cellulose hydrolysis. In addition to sugars, pretreatment derived phenolics were shown to be more influential, with the molecular size and carbonyl content of the phenols playing major roles in influencing the extent of phenolic inhibition. However, the phenolics could be modified to minimize enzyme inhibition and allow the use of lower enzyme loadings.
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Dissolving pulp is characterized by its high cellulose/low hemicellulose content, minor amounts of residual lignin/extractives, high brightness and a uniform molecular weight distribution. Dissolving pulp can be produced through acid sulfite cooking or alkaline Kraft cooking. However, due to issues with chemical recovery and pollution, the predominant pulping process has globally shifted to the Kraft process. Kraft pulps retain hemicellulose and high molecular weight cellulose, which are undesirable for dissolving pulps. Therefore, steps such as prehydrolysis (PHK) and cold caustic extraction (CCE) aimed at removing hemicellulose and decreasing cellulose molecular weight are typically employed. However, these processes are chemically intensive, non-specific and pose operational challenges for mills. The use of enzymes (hemicellulases and cellulases) is one potential alternative to chemical methods of facilitating mill conversion due to the high specificity of enzymes and their ability to function under more benign conditions. Initially, xylanase and oxalic acid treatments were assessed for their potential to convert Kraft-to-dissolving pulp. It was apparent that the accessibility of hemicellulose and cellulose to chemical or enzymatic reagents was critical. Compared to oxalic acid, enzymes were more specific in removing hemicellulose while boosting cellulose reactivity. Model substrates, varying in their hemicellulose accessibility and cellulose properties, were used to assess the influence of various pulp characteristics on enzymatic pulp modification. The influence of pulp characteristics imparted by PHK and CCE on the ease of enzymatic modification was also assessed. It appeared that CCE negatively impacted the accessibility of hemicellulose due to the solubilisation of low molecular weight carbohydrates fragments which acted as “spacers” between cellulose microfibrils, preventing fibril aggregation. Lowering the acidity of the prehydrolysis or the alkalinity of Kraft pulping conditions increased the ease of enzymatic removal of the hemicellulose, presumably by increasing hemicellulose accessibility. Separating the fibres into various size fractions indicated that the shorter fibres within the Kraft pulp were more susceptible to enzymatic modification, likely due to their increased porosity. It was apparent that Kraft pulping conditions played a significant role in governing enzyme accessibility to the various pulp carbohydrates and thus the potential of using enzymes to enhance dissolving pulp production and properties.
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Relatively high enzyme loadings are required for the bioconversion of lignocellulosic biomass, impeding the economical production of cellulosic sugars. The relative stability and robustness of these enzymes make enzyme recycling an attractive cost-reduction strategy. However, the efficiency of enzyme recycling has been limited by the complexity of enzyme-substrate interactions, which are influenced by enzyme, substrate, and physical factors. A lack of techniques to probe specific enzyme adsorption further limits our understanding of these interactions. Therefore, overcoming these challenges to better understand enzyme-substrate interactions is crucial if we are to improve the effectiveness of enzyme recycling strategies.Initial work compared various ways to assess enzyme adsorption during hydrolysis of steam pretreated corn stover (SPCS) using a complete commercial cellulase mixture. While the distribution of six individual enzymes could be followed, the initial approach used was laborious, highlighting the limitations of techniques used to quantify individual enzyme adsorption profiles. A quicker, more sensitive double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was subsequently developed, to follow Cel7A, Cel6A, and Cel7B adsorption during hydrolysis, and shown to agree with earlier results.As enzyme, substrate, and physical factors were known to affect enzyme recycling performance, their influence on individual enzyme adsorption was evaluated. Although the lignin present in the SPCS did not appear to influence enzyme adsorption (although Cel6A adsorbed more readily to the lignin-containing SPCS), cellulose allomorphs and crystallinity did appear to influence enzyme adsorption. The addition of Auxiliary Activity (AA) family 9, an oxidative enzyme, increased desorption of Cel7A, likely by increasing the substrate’s negative charge. The AA9 itself remained primarily in the supernatant, which highlighted the importance of recovering enzymes from both the liquid and solid phases of the reaction. The influence of glucose and ethanol on enzyme adsorption was evaluated, and a reduction in enzyme adsorption was observed at high glucose but not ethanol concentrations.When the addition of fresh substrate was assessed as one way to recover enzymes, by combining enzyme recycling at low glucose concentrations with enzyme supplementation, good overall cellulose hydrolysis (~70%) over 5 rounds of enzyme recycle could be achieved with a 50% reduction in enzyme loading.
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To achieve effective cellulose hydrolysis requires the synergistic cooperation of various cellulases and accessory enzymes/proteins. Most of previous synergism studies have used “model” cellulosic substrates, such as cotton or Avicel, and focused on the initial stages of hydrolysis. Previous studies have also demonstrated that the extent of synergism was influenced significantly by the composition and concentration of “cellulase” mixture and the nature of cellulosic substrate. To gain a better understanding of “cellulase synergism”, the actions of individual and combinations of cellulases, β-glucosidase and “accessory” enzymes (such as xylanases, xyloglucanases and AA9) were assessed on various pretreated lignocellulosic substrates at different enzyme loadings. The synergistic cooperation between cellulases and xylanases was found to enhance the extent of hydrolysis of steam pretreated corn stover and dramatically reduced the required cellulase dosage (about 7 times) needed to achieve reasonable cellulose hydrolysis (>70%). Xylanases appeared to act cooperatively with cellulases by solubilising the xylan and consequently increasing fibre swelling and cellulose accessibility. However, the observed synergism between the cellulase monocomponents and hemicellulases was highly substrate dependent. Those hemicellulases with broader substrate specificities, such as family 10 xylanase and family 5 xyloglucanase, promoted the greatest improvement in the hydrolytic performance of cellulases on a broader range of substrates. The “boosting effect” of AA9 on cellulase hydrolytic performance was highest on substrates showing a higher degree of accessible crystalline, rather than amorphous cellulose. The synergistic cooperation was probably, at least in part, due to AA9s oxidative cleavage, resulting in negatively charged sites on the cellulose. This likely increased the more rapid turn-over of processive enzyme CBHI. A greater degree of synergism among cellulase components was demonstrated at lower enzyme concentrations and on pretreated substrates containing relatively accessible/disordered cellulose. Higher xylanase loadings were required to derive an “optimized mixture” when high solid loadings and substrates with a higher xylan content were used, while the addition of low amounts of AA9 (2mg/g cellulose) was beneficial in all cases. Determining the optimum enzyme composition for a particular substrate was shown to be a key strategy for reducing the protein loading required to achieve effective hydrolysis of pretreated biomass substrates.
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Agricultural and forestry-derived fibres can be converted into fuels and chemicals via a biorefinery. However, the densely-packed fibrillar architecture of lignocellulosic biomass makes the cellulose inherently inaccessible to the enzymes involved in this bioconversion process. This limits the efficiency of enzymatic deconstruction and necessitates relatively high enzyme/protein loadings, which decreases the economic viability of the overall process. It has previously been suggested that the rate-limiting step of cellulose hydrolysis is not the depolymerisation of the carbohydrate chains, but rather the rate at which the enzymes can gain access to the cellulose buried within the biomass. Recently, several proteins such as the Expansins, Swollenin and Loosenin have been shown to disrupt the cellulosic structure without directly depolymerizing the carbohydrates. This protein-induced “amorphogenesis” is thought to occur as a delamination, splitting, peeling, swelling, or decrystallizing of the biomass, thereby enhancing accessibility of the entrenched carbohydrates to the depolymerizing enzymes. However, a key challenge when studying these amorphogenesis-inducing proteins involves quantifying their disruptive effects. While depolymerizing enzymes can be readily quantified by measuring the amount of liberated soluble sugars, amorphogenesis-inducing proteins are thought to promote a variety of disruptive effects without releasing soluble products. As the undefined nature of the amorphogenesis end product makes quantification challenging, one of the initial goals of the work was to refine/develop techniques to better quantify amorphogenesis. Two distinct carbohydrate binding modules (CBMs), one of which preferentially binds to crystalline cellulose and the other to amorphous cellulose were used to track changes in cellulose accessibility and surface morphology. When various substrates were treated with the amorphogenesis-inducing protein, Swollenin, CBM adsorption revealed that Swollenin promoted the dispersal and disruption of the more amorphous regions of biomass, increasing the access of the depolymerizing enzymes to the cellulose component. Subsequent work involving the fluorescent tagging of these CBMs and confocal microscopy further suggested that Swollenin was targeting the less-ordered regions of the cellulosic substrate. When Swollenin was assessed for its ability to disrupt an industrially-relevant substrate, steam pretreated corn stover, it primarily targeted amorphous regions where it synergised strongly with xylanases (~300%), promoting the release of hemicellulosic oligomers.
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For any bioconversion/biorefinery process, the nature of the pretreatment process used has a significant influence on all of the subsequent process steps. Although steam pretreatment has proven effective on agricultural residues and hardwoods, softwoods are considerably more recalcitrant, usually requiring an acid catalyst to ensure effective pretreatment. One of the initial objectives of the work was to assess how effective acid catalysed steam pretreatment would be on a range of softwood substrates as past work had utilized wood chips that were obtained from one tree. It was apparent that similar pretreatment conditions could be used for a range of softwood substrates, resulting in comparable hemicellulose recovery while providing a cellulosic component which could be readily hydrolysed, but at the expense of using high enzyme loadings. To try to enhance cellulose hydrolysis we assessed the role of the various substrate components that are thought to limit hydrolysis. Lignin was shown to restrict hydrolysis at low enzyme loadings (5 – 10 FPU/g glucan), primarily by limiting the accessible cellulose surface area, but also by unproductive binding of the enzymes. To achieve effective hydrolysis at low enzyme loadings, a post-treatment step that removed/modified lignin to enhance the cellulose accessibility was assessed. Steam pretreatment and post-treatment were further optimised to result in a >85% cellulose hydrolysis at an enzyme loading of 10 FPU/g glucan. To try to increase the concentration of final sugars obtained we next evaluated the use of high substrate concentrations. Increased biomass loading during steam pretreatment not only minimised steam and SO₂ consumption, it also resulted in good recovery of the sugars at high concentration. However this was done at the expense of high enzyme loadings. Past work has primarily utilised pulp chips as the feedstock. However, they are unlikely to be used as a commercial bioconversion feedstock. A more likely feedstock, wood pellets were presoaked and steam pretreated. Surprisingly, little hemicellulose loss occurred while the cellulosic rich, water insoluble fraction was readily enzymatically hydrolysed. It was also possible to apply a single steam pretreatment to facilitate both pelletisation and subsequent enzymatic hydrolysis without the need for subsequent steam pretreatment.
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Lignocellulosic biomass is a potential source of sugars for the production of fuels and chemicals. However, its resistance to chemical and biological degradation poses a significant challenge. Consequently, a pretreatment is required to increase the accessibility of cellulose to cellulases. The organosolv process is one of the few pretreatments that can process softwoods to generate substrates that are readily hydrolyzed by cellulases. However, because the residual lignin and hemicelluloses can restrict cellulose accessibility, obtaining significant cellulose conversion at low enzyme loadings (
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The competitiveness and operational viability of a wood-based lignocellulosic ethanol biorefinery located in Canada were assessed using techno-economic and logistical models, combined with fuel and feedstock market data analyses. Scenario analyses included the ability to compete for forest feedstocks with alternative bioenergy options; for market share with ethanol producers using alternative feedstocks (corn, sugarcane, Brazilian eucalyptus); and for financing, given the market risks and alternative investor options. The model variables used to identify the most competitive lignocellulosic ethanol production conditions included feedstock type and properties, feedstock logistics system design, facility site, facility scale, capital cost, pretreatment technology, operating tactics, co-products, ethanol and co-product revenues, enzymes and other process inputs, ethanol and co-product yields, and taxes and renewable energy support policies. It was determined that a wood-based lignocellulosic ethanol facility in Canada is technically viable, but will find sustainable profitability difficult at current energy prices and without a change in the historical volatility of those prices. Using scenario analyses, it was determined that the minimum ethanol selling price (MESP) of lignocellulosic ethanol produced from Canadian forest biomass is $0.80-1.10 L⁻¹, with most scenarios in the $0.90-0.95 L⁻¹ range. This compares with recent corn and sugarcane ethanol MESP of $0.30-0.40 L⁻¹, highlighting the difficulty of competing with these conventional biofuels. In addition, other bioenergy products, such as wood pellets and combined heat and power, will compete with lignocellulosic ethanol facilities for feedstock but offer more stable markets. Largely due to the lack of correlation between transportation fuel markets and forest feedstock costs, the gross processing margin of lignocellulosic ethanol production was shown to be decidedly volatile. This volatility results in an anticipated cost of capital (>11%) that exceeds other fuel production facilities. Although supplying large (e.g., 800 ML yr⁻¹) lignocellulosic ethanol facilities that maximize economies-of-scale (and minimize cost per unit) with feedstock is logistically possible, high feedstock costs, exceeding $100 bdt⁻¹, are projected to put Canadian producers at a disadvantage relative to tropical country producers. Significant production cost reductions must occur before lignocellulosic ethanol can compete for market share with gasoline and conventional ethanol.
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During the “enzymatic hydrolysis of the cellulose” component of the overall lignocelluloses-to-bioethanol process, lignin has been shown to be a very influential factor, acting as both a physical barrier and limiting hydrolysis through the adsorption of cellulases. Although hydrophobic, electrostatic and hydrogen bonding interactions between lignin and cellulases have been suggested to influence the hydrolysis efficiency, comparative studies using isolated lignins from different types of biomass which have been pretreatment in different ways have not been done. To gain a better understanding of the effects of lignin on enzymatic hydrolysis, six different substrates: steam and organosolv pretreated softwood (lodgepole pine), hardwood (poplar) and an agricultural residue (corn stover), were prepared. Lignin was isolated from the pretreated substrates by two methods. The lower lignin yields obtained with corn stover when compared to poplar and lodgepole pine suggested that the hydrophobicity of the corn stover derived lignin was lower than the lignin from poplar and lodgepole pine. The characterization of the physical and chemical properties of the isolated lignins showed that the carboxylic acid present in the isolated lignin had a significant influence on the enzymatic hydrolysis yields when lignin was added to pure cellulose. Dehydrogenative polymers (DHP) from ferulic acid adsorbed lower amounts of cellulases and did not decrease hydrolysis yields when compared to the DHP from coniferyl alcohol, showing that the increased carboxylic acid content of the lignin alleviated the non-productive binding of cellulases and increased the enzymatic hydrolysis of the cellulose. Douglas-fir was next steam pretreated at different severities and the lignin was isolated from the water insoluble fraction. The lower hydrolysis yields obtained with the substrates pretreated at 190⁰C when compared with those treated at 200 and 210⁰C was attributed to the lower accessible surface area of the substrate pretreated at 190⁰C rather than lignin-enzyme interactions. Isoelectric focusing analysis after incubation of cellulases with the lignin showed that the positively charged cellulases were preferentially adsorbed, indicating that electrostatic interaction was involved in cellulase adsorption onto the lignin. It was also apparent that the hydrophobicity of the lignin also played a role in the adsorption of cellulases.
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Master's Student Supervision
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
The conversion of abundant under-utilized forest residues into biofuels is a promising strategy for transitioning the energy structure and for decarbonizing the transportation sector. As one of the emerging thermo-chemical conversion technologies, microwave-assisted catalytic pyrolysis (MACP) is able to efficiently convert solid biomass into valuable products, including bio-oil (~36 wt%), biochar (~28 wt%) and non-condensable gas (NCG) (~36 wt%). As well as resolving technical obstacles, MACP was also assessed for its economic and environmental impact, at a systematic level, for the purpose of commercialization. The work described in this study involved a techno-economic assessment (TEA) and a life cycle assessment (LCA) based on process integration. This was used to evaluate the economic feasibility and environmental impact of a hypothetical MACP system for the co-production of biofuel and biochar from forest residues in British Columbia (BC). The minimum selling price (MSP) of MACP biofuel was shown to be $1.01/L, indicating that MACP biofuel was still not priced competitively to petroleum fuels. The on-site utilization of NCG and integration of an upgrading process helped achieve self-sufficiency in heat and hydrogen supply, but raised concerns about high capital costs. Sensitivity analysis suggested that future research efforts should focus on improving the process performance and reducing the capital investment to bring down the MSP. However, LCA results suggested that an MACP system could potentially make a considerable contribution to reducing greenhouse gas (GHG) emissions of transportation fuels. The cradle-to-gate (CTG) carbon intensity (CI) of MACP biofuel was shown to be -57.6 g CO2-eq/MJ, indicating that a carbon-negative system could be achieved with a GHG emission reduction of 162% compared to petroleum fuels. The key reasons were the green electricity mix and carbon sequestration of co-product biochar. The dominant influence was shown to be biomass-to-biofuel conversion step which accounted for 47.3% of the GHG emissions produced. Besides, the conversion efficiencies and location specific parameters also had significant impacts on the CI of MACP derived biofuels.
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The demand for novel cellulosic material, such as micro/nanofibrillated cellulose (MNFC) is expected to face increasing growth due to its unique properties and evolving, high value applications in packaging, biomedical and nanocomposite materials. However, the hydrophilicity of MNFC has limited some of its applications, particularly in the packaging sector. Common modifications to improve hydrophobicity of MNFC often involves costly and environmentally challenging chemicals. Lignin is naturally hydrophobic, environmentally benign and as described herein, could be an effective agent to improve the hydrophobicity of MNFC. Softwood Kraft lignin (SWKL) was first considered due to its commercial availability. When SWKL was dissolved in alkaline solution and acid precipitated onto MNFC, substantial amounts of lignin were deposited on the surface, resulting in a two-fold increase in initial water contact angle as compared to the control. However, no significant improvement on MNFC hydrophobicity was observed as the contact angle was unstable over time. It was apparent that the contact angle measurements were strongly influenced by the roughness of the paper, the porous nature of cellulose and the extent of lignin homogeneity on cellulose surface. To enhance the efficacy of the approach, hot pressing lignin-containing papers near lignin’s glass transition temperature was assessed. It was hoped that this would help redistribute the lignin, resulting in better lignin homogeneity on the fiber’s surface. Initial results seemed promising as the contact angle was stable over a period of two minutes after hot pressing. Other attempt to incorporate lignin, such as spray coating with hot pressing, was evaluated to enhance homogenous lignin coverage on the paper. Contact angles as high as 85° and 95° were achieved, for SWKL and organosolv lignin respectively. Lignin coverage as low as 1% was able to impart hydrophobicity using the spray coating method. Although the water vapor transmission rate (WVTR) could be substantially reduced by hot pressing, the incorporation of lignin onto the paper did not reduce the WVTR significantly. This work showed that hot pressing lignin-containing papers resulted in improved hydrophobicity, likely due to the redistribution of lignin on fiber surfaces and the formation of a denser fiber network.
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Predicting the hydrolytic potential of a “cellulase enzyme cocktail” on lignocellulosic substrates is an ongoing challenge, particularly as enzyme companies try to reduce the required enzyme loading to achieve rapid and complete cellulose hydrolysis. The filter paper assay (FPA) is still widely used to assess the hydrolytic potential of cellulase enzyme preparations, as the method is clearly documented and Whatman No.1 paper is a universally available and consistent substrate. However, characteristics of filter paper such as its high cellulose content and dried nature, as well as the short (1 hour) duration of the assay, etc., all compromise the ability of the FPA to predict how enzyme cocktails will hydrolyze lignocellulosic substrates. To assess the influence of factors such as drying and the presence of lignin and hemicellulose on the FPA, “model/paper sheet” substrates were prepared and substituted for filter paper. When a paper sheet prepared from never-dried pulp was used in the assay, it was apparent that drying was a major, negative influence. Using sheets prepared from pretreated substrates containing lignin and hemicellulose resulted in a 53% decrease in the measured filter paper activity, illustrating the detrimental effects of lignin and hemicellulose on cellulase activity. Therefore, several realistic substrates were prepared that were rich in either xylan, mannan and/or lignin. These substrates were used to assess the beneficial effects of substituting cellulases with accessory enzymes (e.g. xylanases, mannanases). In many cases up to 50% of the cellulases in the enzyme cocktail could be substituted with accessory enzymes to achieve hydrolysis yields >70%. However, when the substituted enzyme cocktail was assessed via the filter paper assay, the resulting activity decreased by up to 58%. It was apparent that the predictability of the FPA was highly dependent on the composition/characteristics of both the substrate and the enzyme cocktail. The results indicated a potentially more useful method to predict the effectiveness of a cellulase mixture on a given substrate may be to perform a longer-time hydrolysis of the actual biomass substrate while using the filter paper assay to provide an estimate of the initial protein/activity loading.
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The production of monomeric sugars from lignocellulosic feedstocks is challenging, partly due to the high enzyme loadings that are typically required to effectively break down biomass substrates. One pretreatment approach is to try to keep most of the sugars associated with the solid fraction where cellulose and hemicellulose degrading enzymes will be needed to both open-up the lignocellulosic matrix and hydrolyse the polymeric matrix. Past work has shown that the addition of “accessory enzymes” to the “cellulase” cocktail can enhance the hydrolytic performance of enzyme mixture while reducing the protein/enzyme loading required to hydrolyse pretreated biomass substrates.The potential of novel “hemicellulose-specific” enzymes to work synergistically with traditional (Celluclast) and more recent (CTec series) cellulase mixtures was assessed on a range of pretreated and “model” cellulosic substrates. Xyloglucanases showed a higher degree of cooperation than did mixed-linkage glucanases. However, although they generally enhanced cellulose hydrolysis, this synergistic cooperation was strongly influenced by the type of enzyme activity, substrate composition and enzyme and substrate concentration. The backbone-acting xyloglucanase from Bacteroides ovatus, BoGH5, demonstrated a broader specificity compared to other accessory enzymes, resulting in a higher degree of cooperation with cellulase enzymes and enhanced cellulose hydrolysis. Supplementing Celluclast with both backbone acting and debranching enzyme α-xylosidase, resulted in an over 10% improvement in cellulose hydrolysis for substrates with a higher hemicellulose content. This also correlated with an increase in the release of xyloglucan-derived oligomers. As a result of this enhanced synergism, it was possible to reduce the overall protein/enzyme loading by 35% when a goal of 80% cellulose hydrolysis of alkali pretreated corn stover within 72 hrs was targeted.The thesis work suggested that xyloglucan plays a role in limiting enzyme access to the cellulose and therefore the xyloglucan must be disrupted in order to facilitate effective cellulose hydrolysis. It is likely that xyloglucan acts as a physical barrier, possibly coating cellulose microfibrills and/or restraining cellulose fiber swelling.
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Groups such as the International Energy Agency have predicted that increased bioenergy and biofuels production will be needed to reduce greenhouse gas (GHG) emissions and fossil fuel use globally. British Columbia (BC) has a world-renowned forest sector and the highest percentage of third party certified sustainable forests in the world. Therefore, BC is well positioned to supply sustainable forest biomass for bioenergy/biofuels.Currently, underutilized forest residues could provide a major source of biomass for bioenergy/biofuels. However, the use of forest residues under current BC forest management standards does not fulfill some sustainability requirements defined by trade policies. Therefore, an improved sustainability verification system would support the growth of bioenergy/biofuels globally. Most forest certification systems were initially developed for traditional forest products such as lumber and pulp. In contrast, the evolving bioenergy sector uses biomass-sourcing certification standards that have limited connection to in-forest certification procedures. As a result, gaps between these certification standards challenge the potential of forest residues being used as sustainable feedstocks for the current and future bioeconomy.A partnership between forest and biomass-sourcing sustainability standards is likely, to connect GHG emissions data and other key metrics along the supply chain. The Programme of Endorsed Forest Certifications has begun developing a GHG tracking system for forest managers and is considering partnership opportunities with the Sustainable Biomass Program, a prominent wood pellet certification organization.However, there are significant economic challenges limiting the increased use of BC forest residues. To reduce the economic challenges of using forest residues, BC’s forest and climate policies need to be modified, while making sure any unintended negative stakeholder impacts are considered. The thesis work indicates that a combination of policy and economic incentives will be required for commercial-scale use of forest residues. One way to enhance forest residue use is through the use of regulatory incentives. As described in the thesis, it is likely that any increased use of BC forest residues will require significant government support via policies that will ensure the sustainable management of BC’s forest while further developing markets for the various biomaterials derived from forest residues.
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Aviation is the fastest growing industry in the transport sector and its GHG emissions are expected to increase 7-fold over the next 35 years. To achieve the industry’s goals of a 50% emission reduction by 2050, groups such as the International Civil Aviation Organisation (ICAO) have stated that biofuels will play an essential role. Although various methods of producing biojet fuel have been proposed, the specific GHG emission reductions that might be achieved have yet to be fully elucidated. This thesis explores the Lifecycle Assessment (LCA) of biojet fuel production via thermochemical and oleo-chemical means through the review of biojet LCA literature. By comparing the assumptions used within, it became apparent that the nature of the LCA model had a significant impact on the carbon intensity results. Results using the GHGenius model were found to be significantly different than results from GREET or SimaPro, likely due to the inclusion of land use change and the use of the displacement allocation method in the GHGenius model. Although these two variables influenced the results more than any other variable, the location of production also had a significant impact on the oleo-chemical and pyrolysis methods, as did the source of hydrogen. Even with these differences, all models agreed that biojet fuel produced by gasification provided the lowest greenhouse gas emissions.In the second part of this thesis, the LCA of B.C. forest biomass-to-biojet pyrolysis scenarios were modeled, assessing three possible biomass supply chains: (Vancouver Mainland (forest residue), Vancouver Island (forest residue) and Prince George (wood pellets)). The GHG emission reductions of each supply chain scenario compared to petroleum jet fuel were 71.1%, 70.6%, and 68.2%, respectively. A sensitivity analysis of the Prince George scenario indicated that the results were most sensitive to the type of feedstock used for pellet production, the allocation method used, the moisture content of the feedstock and the source of hydrogen. It was shown that, independently, these variables can change the GHG emission results by 10% - 60% or, combined, could reduce the overall GHG emissions to - 22.13 gCO₂eq/MJ biojet fuel (125% reduction).
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In order to improve the economic viability of a bioconversion process it would be extremely beneficial to maximize the recovery of all of the lignocellulosic components while enhancing enzyme accessibility to the cellulosic component. Wood residue derived pellets are already a prominent Canadian commodity and the existing supply-chain produces a high density, low moisture feedstock suitable for mass collection and transport. However, pellets are almost exclusively used for combustion and not as a possible biorefinery feedstock. As a result, there is limited information on the influence of the pelletization process (e.g. grinding, drying, compressing) on the susceptibility of pellets, as opposed to chips, to the various pretreatment, fractionation and cellulose hydrolysis steps that are components of a typical bioconversion process. The work described in the thesis assessed the potential of a two-stage steam/organosolv pretreatment process to fractionate and isolate the hemicellulose and lignin components from softwood pellets, yielding a more accessible, cellulose-rich substrate. Various steam pretreatment conditions were compared for their ability to enhance hemicellulose solubilisation while minimizing lignin condensation (first-stage), to improve subsequent organosolv delignification (second-stage). Carbocation scavengers were compared for their ability to minimize lignin condensation during either stage. When softwood chips and pellets were compared, the effectiveness of the pretreatment was determined by hemicellulose solubilisation, delignification capability and the ease of enzymatic hydrolysis of the cellulosic component. It was apparent that pellets were more responsive than chips to pretreatment due to their smaller particle size, which facilitated both hemicellulose solubilisation and delignification. At conditions that solubilized and recovered hemicellulose, acid-catalyzed steam pretreatment induced lignin condensation. This impeded subsequent organosolv delignification and enzymatic hydrolysis. The addition of lignosulfonates as a potential carbocation scavenger during acid-catalyzed steam pretreatment resulted in increased hemicellulose solubilisation and carbohydrate recovery while improving delignification during subsequent organosolv treatment. Adding lignosulfonates during acid-catalyzed steam pretreatment also enhanced enzymatic hydrolysis. It was likely that the added lignosulfonates increased lignin hydrophilicity which facilitated lignin dissolution and decreased non-productive enzyme inhibition. It was apparent that the addition of lignosulfonates prior to pretreatment reduced the detrimental effects of lignin condensation which benefited subsequent fractionation of the pretreated biomass.
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Biomass is the world’s largest source of renewable energy and it is likely to remain so until at least 2035 (IEA, 2013a). Globally, there should be enough biomass available to meet growing demand. However, biomass is predisposed to being used locally possibly resulting in limited domestic supply in countries where biomass is already used extensively (IEA, 2009). This could potentially result in competition between bioenergy or biofuels applications. The work described here explored the current and potential bioenergy/biofuel uses of biomass both globally as well as regionally, with a focus on Brazil, Denmark, Sweden and the United States. In each of these countries, biofuels or bioenergy are already important parts of their energy mix. For all of the countries studied the major drivers to use biomass for energy/fuels were: energy security; the desire to mitigate climate change; prevailing regional economic interests, and; the potential that bioenergy/biofuels are cheaper than fossil derived alternatives. Government support policies for bioenergy and biofuels are examined within the context of each of the four drivers. It was apparent that there is limited competition for biomass between bioenergy and transportation biofuel applications. This situation is likely to continue until advanced biofuels technologies become much more commercially established. In each of the four countries biomass is predominantly used to produce bioenergy (heat and power), even in those regions where biofuels are significant component of their transportation sector (United States, Brazil and Sweden). The vast majority of biofuel production continues to be based on conventional sugar, starch and oil rich feedstocks, while bioenergy (heat, power, residential, industrial) is produced almost exclusively from forest biomass with agricultural biomass playing a small, but increasing, secondary role. As current and proposed commercial scale biomass-to-ethanol facilities almost exclusively use agriculture derived residues (corn stover, wheat straw, sugar cane bagasse), it is likely that, if there is ever to be competition for biomass feedstock’s for bioenergy/biofuel applications, it will be for agricultural based biomass with co-product lignin and other residues used to concomitantly produce heat-and-electricity on site at biofuel production facilities.
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The cost-effective production of sugars from biomass continues to remain challenging, partly due to the relatively high enzyme/protein loading required to effectively hydrolyze pretreated lignocellulosic substrates. Previous works have shown conflicting observations regarding the correlation between enzyme adsorption and the hydrolytic performance of an enzyme mixture. Unfortunately, it has proven difficult to accurately determine the roles of adsorbed enzymes during the hydrolysis of lignocellulosic substrates, in part because of the interference that protein determination methods encounter from the release of sugars and other biomass derived materials, the lack of a hydrolysis strategy for hydrolysis with only adsorbed enzymes and the use of “model” substrates in many studies. To better understand the role that adsorbed enzymes play in cellulose deconstruction, it is important that we are able to accurately quantify protein distribution and enzyme performance. Various protein quantification assays were initially assessed for their ability to accurately and reproducibly quantify protein/enzymes during typical biomass hydrolysis conditions. However, the ninhydrin assay, which was the most promising assay due to its specificity for protein and compatibility with most compounds derived from lignocellulosic samples, still suffered from the incompatibility with sugar degradation products, long hydrolysis times and potentially wide-ranging standard deviations. To overcome these limitations, an accurate and rapid modified ninhydrin assay was developed which employed a sodium borohydride treatment to eliminate sugar interference followed by acid hydrolysis at 130ºC, reducing the overall reaction time to 4 hours. Utilizing the modified ninhydrin assay, the role of adsorbed enzymes in determining the rate and extent of hydrolysis of several different pretreated biomass substrates was then assessed. Once the distribution of enzymes reached equilibrium, after 60 minutes, those enzymes that were adsorbed or free in solution were separated by centrifugation and subsequently assessed for their ability to hydrolyze various cellulosic substrates at different enzyme loadings. It was apparent that the adsorbed enzymes were critically important as the removal of those enzymes in solution resulted in no significant decrease in the rate and extent of hydrolysis. By using the adsorbed enzymes, enzyme loadings could be reduced by up to 53% while resulting in similar hydrolysis yields.
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For a biochemically based biomass-to-ethanol process, one of the advantages of using softwoods as the substrate is the predominance of hexose sugars, which means that most of the sugars should be readily fermented by Saccharomyces cerevisiae. However, one of the biggest challenges with fermenting softwood derived sugars is the presence of both process derived and naturally occurring inhibitory compounds that are detrimental to both the growth and metabolism of yeasts. The presence of inhibitory compounds together with “low” initial sugar concentrations typically result in poor ethanol yields and titres which limit the economic viability of the process. In the work reported here, we tried to improve the fermentation of Douglas-fir derived sugar streams by enhancing the sugar concentration of the upstream processes (steam pretreatment and enzymatic hydrolysis) while using a combination of strategies to efficiently ferment the resulting liquor. These included the use of industrially relevant Saccharomyces cerevisiae strains, high yeast cell density, nutrient supplementation and liquor detoxification. To obtain as high a sugar concentration as possible, a high consistency steam pretreatment and subsequent enzymatic hydrolysis of the combined cellulose and hemicellulose fractions was carried out. Although this “softwood derived liquor” had a final sugar concentration of 18% wt/wt, it also had a very high concentration of inhibitory compounds including phenolics, furan derivatives and organic acids. When the fermentation profile obtained after growth on this liquor was compared to those obtained after growth on glucose and an enzymatically hydrolysed dissolving pulp, it was apparent that these inhibitory compounds severely restricted the growth and fermentation of all of the S. cerevisiae strains. Although the Tembec T2 strain that had previously been adapted to growth on spent sulfite liquor demonstrated the best fermentation performance, a detoxification stage was still required before reasonable (77.2%) ethanol yields could be obtained. Even with a prior detoxification stage, a high initial cell density of OD=13 was required before effective fermentation could be achieved. A combination of sulfite detoxification and high cell density fermentation resulted in a final ethanol concentration of about 5.0% (wt/vol) and volumetric productivity 4.9g/l/h.
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From 1980 to 2010 China’s energy use increased six fold to 2430 Mtoe/yr and it is projected to further increase by about 50% to 3359 Mtoe/yr by 2020. Currently renewable energy, such as bioenergy, hydro, solar and wind, contributes less than 9% of this total. During China’s “industrial revolution” phase of economic growth (in the 1970/80’s), coal was the major source of power and electricity with oil and natural gas playing a much lesser role. More recently, due to the rapid increase in the number of motorized vehicles, the country has gone from oil self-sufficiency in the early 1960’s to importing more than 271 Mt/yr of oil in 2012. China’s biofuels industry is in its infancy with its current bioethanol production (primarily from corn) at 2.5 GL/yr and biodiesel production (primarily from waste cooking oil) at 0.4 GL/yr. Although the national goal is to produce 12.7 GL bioethanol and 2.3 GL of biodiesel by 2020, the potential for growth of so-called first generation or conventional biofuels is very limited due to food-vs-fuels concerns and China’s desire to be as self-sufficient as possible in food production. Thus, research, development and demonstration (RD&D) is being encouraged to grow and process so-called one-and-a-half generation crops such as sweet sorghum with a goal of producing 9 GL of ethanol by utilizing 40% of the available marginal land. However, to date, few plantation or conversion facilities have been built. Regarding so-called second-generation facilities, China has the potential to annually produce 22 GL of cellulosic ethanol by utilizing 15% of its 874 Mt agricultural residues. This could increase to 29 GL bioethanol by 2020, using 15% of the 1150 Mt residue that is anticipated to be available at that time. Biodiesel growth is expected to be achieved by growing oil-bearing trees with the potential of producing 2.5-6.7 GL/yr grown on 10% of the available marginal land. However, it is unlikely that biofuels will contribute substantially to China’s transport sector and that, even with aggressive importation, biofuels will play a relatively minor role for quite some time.
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Ethanol, an alternative liquid fuel, can be produced from sugars derived from lignocellulosic biomass in a bioconversion process that involves pretreatment, enzymatic hydrolysis, and fermentation. Among the different types of biomass investigated for bioconversion, softwoods are readily available in Canada, the US, and Scandinavia. Acid catalyzed steam pretreatment is a preferred method for softwoods due to its ability to effectively recover hemicellulose-derived sugars at moderate operating conditions. More severe conditions are generally required to produce a substrate readily hydrolyzed by enzymes, but because sugar degradation also occurs at these conditions, steam pretreatment is essentially a compromise.Prediction of sugar recoveries from steam pretreated and enzymatically hydrolyzed softwood is desirable for the purposes of process control and steam pretreatment reactor design. In this thesis, efforts were made to determine whether response surface methodology or the thermal severity factors Ro and CS were better suited to the development of empirical models of steam pretreatment. The construction of the thermal severity factor models highlighted the predominance of temperature and time in determining the direct outcomes of the acid catalyzed steam pretreatment of radiata pine. Within a comparison of several response surface methodology models, a hybrid experimental design produced the most robust model because it was developed in conjunction with a narrow process space. Moreover, it was apparent that the response surface methodology models possessed the greater capacity for predicting the direct outcomes of steam pretreatment.In an attempt to overcome limitations identified in the first portion of this thesis, the predictive capability of response surface methodology was further tested using lodgepole pine ranging in chip size and moisture content. The additional model created demonstrated that response surface methodology could successfully account for feedstock characteristics as well as steam pretreatment operating conditions. Moisture content, but not chip size, was shown to have a significant influence on the combined sugar recovery obtained after SO2 catalyzed steam pretreatment and subsequent enzymatic hydrolysis. In addition, model development was conducted in this portion of the thesis such that the model could form the basis of a more dynamic simulation of the entire softwood to bioethanol process.
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Forest residues represent an abundant and potentially sustainable source of biomass which could be used as a feedstock for a biomass-to-chemicals-and-fuels process (biorefinery). However, due to the heterogeneity of forest residues, one of the expected challenges will be to obtain an accurate material balance of both the starting and pretreated material. As current compositional analysis methods have been developed to quantify more homogenous feedstocks such as whitewood and agricultural crops, it is likely that they will have difficulty in providing a complete material balance for these more diverse substrates. The research work initially assessed the robustness of established methods to quantify a variety of forest residues (bark, hog fuel, forest thinnings, logging residue, disturbance wood) before and after steam pretreatment. It was anticipated that the diverse chemistry and heterogeneity of forest residues would make it difficult to obtain an accurate material balance. Although the NREL recommended methods provided a reasonable estimate of carbohydrate components of the various feedstocks, method revision was necessary to accurately quantify the non-carbohydrate components and thus obtain an acceptable summative mass closure. This was particularly evident for high-extractive containing residues such as bark. After steam pretreatment, the incomplete removal of extractives from the pretreated material proved to be more problematic. The refined material balance methods were subsequently used to evaluate the potential of using pretreated forest residues as a biorefinery feedstock. Acid catalysed steam pretreatment was not as effective on forest residues and poor sugar yields were obtained despite using high enzyme loadings. It was likely that, in the acidic medium resulting from SO₂ catalysed steam pretreatment, the extractives reacted with the lignin and consequently restricted enzyme accessibility to the cellulose. In contrast, an alkaline pretreatment effectively removed most of the extractives and lignin from cellulosic components of the bark. The resulting cellulose-rich, water insoluble component could be almost completely hydrolyzed. It was apparent that established analytical methods will have to be modified to obtain a representative material balance of both the starting and pretreated material and that, even with “tailoring” pretreatment/fractionation strategies, forest residues will prove to be challenging feedstocks for any potential bioconversion process.
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Wheat straw is available in western Canada and it is a potential feedstock for bio-ethanol production as it can be effectively fractionated into simple sugars using acid catalyzed, steam pretreatment followed by enzymatic hydrolysis. Steam pretreatment is usually a compromise whereby conditions that facilitate effective enzymatic hydrolysis at low enzyme loadings usually sacrifice the recovery of the hemicellulose component. Previous work that tried to optimize the pretreatment to maximize hemicellulose recovery was usually done at the expense of using unacceptably high enzyme loadings to hydrolyze the cellulosic fraction. The goal in this thesis was to determine the highest possible amount of sugar that could be solubilized after both pretreatment and enzymatic hydrolysis while using low enzyme loadings and high solids concentration. It was anticipated that the optimum conditions for maximizing the total soluble sugar yield would still result in the degradation of a portion of the hemicellulose. The biomass handling conditions were first investigated to identify the best possible conditions to maximize sugar recovery. An optimized moisture content combined with the explosive decompression resulted in the highest xylose recovery. It was also found that H₂SO₄ could be used at a loading of 1.5% w/w to produce a substrate with similar chemical composition, sugar recovery and ease of enzymatic hydrolysis to what was obtained when using 3% SO₂ as the catalyst.The pretreatment conditions were then varied to determine the effect of pretreatment severity on the recovery of total soluble sugars. The highest soluble sugar yield of 75% was obtained after pretreatment at 190°C, 8 min and 1.5% H₂SO₄. This is among the highest sugar yields that have been reported and comparable to those reported when using a three-fold higher enzyme loading. However, at these conditions only 52% of the original xylan was recovered. A less severely treated substrate with 70% xylan recovery achieved a total soluble sugar yield of 72% when the “cellulase mixture” was supplemented with xylanases. Thus, pretreatments at lower severities followed by enzymatic hydrolysis using a “cellulase mixture” with xylanase supplementation may be an effective approach to improve the total soluble sugar yield when processing wheat straw.
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One of the main challenges in the bioconversion of lignocellulosics into bioethanol is tomaximize the recovery of hemicellulosic sugars while increasing ethanol productionthrough fermentation of these sugars. Steam pretreatment of Douglas-fir (DF) andLodgepole pine (LPP) at a severity factor of logR₀ = 3.64 resulted in water solublefractions (WSFs) containing monomeric hexose sugars up to 86 g/L. The crude WSFswere not fermentable by four yeast strains: T₁ and T₂ (spent sulfite liquor adapted strains),Y1528 (haploid strain that preferentially ferment galactose first) and BY4742 (haploidlaboratory strain). Dilution of fermentation inhibitors in crude WSFs led to appreciableimprovements in their fermentability, especially by the SSL-adapted yeast strain T₂.The four yeast strains were tested against several model furan and phenolic compounds toexamine their tolerance to these fermentation inhibitors. All four yeast strains producedcomparable ethanol productivity when 3 g/L of HMF or 0.8 g/L of furfural were added tomedium containing 2% glucose. However, T₁ and T₂ exhibited higher ethanolproductivity compared to Y1528 and BY4742 when 5 g/L of 4-hydroxybenzoic acid and5 g/L of vanillic acid were added to media as supplements. This provides evidence thatthe robustness of SSL-adapted T₁ and T₂ yeast strains probably originates from theirtolerance to certain phenolic compounds.Overliming improved ethanol production from Douglas-fir 1 (DF1) WSF by T₂ from 1.7g/L to 13 g/L. When DF1 WSF was spiked with glucose up to 100 g/L, it producedethanol yields similar to that of the glucose reference fermentation media. Since it is notpractical to spike WSF with glucose in an industrial process, we investigated theapplicability of separate hydrolysis and fermentation (SHF) and hybrid hydrolysis andfermentation (HHF) with the whole slurry to achieve higher initial fermentable sugarconcentration. SHF of combined WSF and hydrolysates recovered after enzymatichydrolysis of water insoluble fraction (WIF) by T₂ produced up to 90% ethanol yield.HHF produced ethanol concentrations comparable to those of SHF with or withoutoverliming. This result indicated that SHF and HHF of the whole slurry can helpimprove the fermentability of WSFs.
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The work described in this thesis focused on the development of a practical, high consistency hydrolysis and fermentation processes utilizing existing pulp mill equipment. Carrying out enzymatic hydrolysis at high substrate loading provided a practical means of reducing the overall cost of a lignocellulose to ethanol bioconversion process. A laboratory peg mixer was used to carry out high consistency hydrolysis of several lignocellulosic substrate including an unbleached hardwood pulp (UBHW), an unbleached softwood pulp (UBSW), and an organosolv pretreated poplar (OPP) pulp. Enzymatic hydrolysis of OPP for 48 hours resulted in a hydrolysate with a glucose content of 158 g/L. This is among the highest glucose concentration reported for the enzymatic hydrolysis of lignocellulosic substrates. The fermentation of UBHW and OPP hydrolysates with high glucose content led to high ethanol concentrations in the final fermentation broth (50.4 and 63.1 g/L, respectively). These values were again as high as any values reported previously in the literature. To overcome end-product inhibition caused by the high glucose concentration resulting from hydrolysis at high substrate concentration, a new hydrolysis and fermentation configuration, (liquefaction followed by simultaneous saccharification and fermentation (LSSF)), was developed and evaluated using the OPP substrate. Applying LSSF led to a production of 63 g/L ethanol from OPP. The influence of enzyme loading and β-glucosidase addition on ethanol yield from the LSSF process was also investigated. It was found that, at higher enzyme loading (10FPU or higher), the ethanol production from LSSF was superior to that of the SHF process. It was apparent that the LSSF process could significantly reduce end-product inhibition when compared to a Separate Hydrolysis and Fermentation (SHF) process. It was also apparent that β-glucosidase addition was necessary to achieve efficient ethanol production when using the LSSF process. A 10CBU β-glucosidase supplement was enough for the effective conversion of the 20% consistency OPP by LSSF. The rheological property change of the different substrates at the liquefaction stage was also examined using the rheometer technique.The use of a fed-batch hydrolysis process to further improve the high consistency hydrolysis efficiency was also assessed.
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