The production of biofuels from non-edible plant biomass has been necessitated by the concern for the environmental consequences of fossil fuel use and the tightening of supply and demand for liquid fuels. In contrast to first generation biofuels which rely on crops used for food supplies, second generation biofuels, derived from lignin-containing feedstocks, completely eliminate the competition for food. The major challenges associated with second generation biofuels are both technical and economic. Due to the recalcitrant nature of the raw biomass materials to further biological conversion, their structural degradation often requires severe and costly pretreatment processes such as heat, physical and chemical treatments to disturb and fractionate the biomass. Significant research effort has been devoted to understanding the recalcitrant nature and to accelerate the commercialization process of second generation biofuels. In this thesis, three pretreatment methods that belong to different categories have been investigated to understand their impacts on cellulose and/or lignocellulose and the subsequent hydrolysis steps. Physicochemical pretreatments, such as steam explosion, have been identified as one of the most effective and cost-efficient pretreatment methods for lignocelluosic materials. In Chapter 2, SO2-catalyzed steam explosion will be discussed and the effect of pretreatment severity on the substrate characteristics and degradation efficiency is also elucidated. Although the crystallinity index (CrI) of cellulose decreases as the severity increases, significant non-specific degradation and low yield of cellulose was observed at high severity. A new method for cellulose CrI determination has been developed with least squares curve fitting and validated with mechanically mixed cellulose samples. Biological pretreatment is another pathway through which the biomass structure can be modified to obtain a more amenable state for enzymatic degradation. Cellulose-binding domain (CBD) originated from Trichoderma reesei Cel7A (i.e. Tr cellobiohydrolase I) has been discovered as a potential biological pretreatment agent which is capable of modifying cellulose crystal structure. An extensive study on the protein engineering, expression, purification and functionalities of Cel7A CBDs was carried out (Chapter 3). The target mutations were identified with a computational protein engineering method involving principal component analysis (PCA). Due to the lack of catalytic activity and high throughput screening method, the library size was limited to nine. The wild-type and mutated CBDs were compared for their adsorption behavior and decrystallization effect on cellulose. Resulting saccharification efficiency after CBD pretreatment were studied and a possible explanation for the rate enhancement was proposed. In addition to physicochemical and biological pretreatment methods, chemical pretreatment is also a commonly employed method to overcome the recalcitrance of lignocellulosic materials. The most widely studied include dilute acid, alkaline, and organosolv processes. Inspired by the rapidly growing green solvent ionic liquid (IL) researches in biomass pretreatment, substituted imidazoles have been investigated in this thesis to assess their potential as pretreatment agents for lignocelluloses (Chapter 4). 1-Methylimidazole (MI), a precursor to some ILs, has been determined to be the most promising agent for lignocellulose pretreatment due to its exceptional delignification and cellulose expansion efficiency. The chemical recovery and MI process development will also be discussed in Chapter 4. In order to understand pretreatment effect, a semi-quantitative assay utilizing low molecular weight direct dyes and cellulases to estimate the accessibility and pore size distribution has been developed for application on pure cellulose substrates in Chapter 5. Finally, main conclusions as well as future perspectives for this work will be discussed in Chapter 6.

The global biofuels industry is growing fast, involving many different actors, such as producers, forestry companies, biofuel producers and others. This development forces industry actors and policy makers to take biofuels into consideration as a source of energy and to also consider arising and shifting stakeholder interests. Experience has shown that the role of stakeholders can be critical to commercial success especially where environmentally sensitive activities are involved or when strong lobby groups exist. The purpose of the first part of this study is to identify the interests of different stakeholder groups involved in the biofuel industry in Canada. This study is based on primary data collected from representatives of each stakeholder group. It follows a framework developed by Turcksin et al. (2011), who use a similar stakeholder analysis as input to a Multi-Actor Multi-Criteria Analysis (MAMCA) to assess different biofuel alternatives and opportunities. This study draws on the definition of stakeholders and their interests, and uses pairwise comparisons of the interests for each stakeholder group. The responses are analyzed using a methodology commonly used in the Analytic Hierarchy Process (AHP, Saaty, 1990) to derive a ranking of stakeholder interests for each group. The key results of this study are the weighted rankings of interests for each stakeholder group. These results also allow for a comparison between Canada and Belgium, based on the earlier work of Turcksin et al. (2011), which shows noticeable differences between the priorities of stakeholders in Canada and Belgium. The second part of this study explores first the potential impact of public research on stakeholders and then the opinions of all stakeholders on public policies and programs of relevance to the development of the biofuels industry. The results suggest that researchers generally expect positive impacts of their work on all stakeholder groups. They anticipate that the greatest impact of their work will be on end-users, in terms or allowing them to project a green image. The second highest impact is anticipated on increasing the production capacity for biofuels producers. In terms of the importance of public policies and programs on biofuels commercialization, respondents generally anticipate tax measures and research and development support to facilitate the commercialization of biofuels. Agricultural and trade policies are considered less important. However, there are differences between the stakeholder groups. For example, government respondents are least optimistic about the effectiveness of research and development measures, yet most optimistic about biofuel mandates. Biofuel producers show the greatest appreciation for agricultural and trade measures, and consider tax measures as less important than all other stakeholders. Comparing the results from all three parts of the study, the results document considerable differences between the stakeholder groups, and they suggest that the main contributions of researchers to the different stakeholder groups are not necessarily aligned with the priorities stakeholders have for their interests in biofuels.

The ability of micro–algae to respond to diverse and often rapidly changing habitats has been attributed to the versatility of their cellular lipids. Amongst these the energy rich triacylglycerols (TAG) have attracted considerable attention due to their potential use as feedstock for renewable biofuel. Although micro-algae have considerable advantages over other biofuel sources there are constraints to their utilisation. Improvements are required in certain areas including efficiencies in production and enhanced lipid yields if micro-algal biofuels are to become commercially feasible. To achieve this, genetic and metabolic manipulation will be essential and therefore a greater understanding of the lipid biosynthetic pathways is required. In this study the expression of genes putatively involved in TAG biosynthesis in the diatom Phaeodactylum tricornutum was investigated, for the first time, with CO2 supplementation and low pH stress over the entire growth cycle. This molecular analysis was combined with physiochemical examination of the lipid accumulation in the micro-algal model. The results indicated that TAG accumulation was enhanced by CO2 supplementation and occurred predominantly during the stationary growth phase. The molecular analysis revealed increased expression for three genes of interest, encoding enzymes involved in the acyl dependent pathway: Glycerol-3-phosphate acyl transferase (GPAT) -7198728 (Phatrdraft_50031), lysophosphatidic acyl transferase (LPAAT) -7196550 (Phatrdraft_42446) and phosphotidic acid phosphatase (PAP) -7195747 (Phatrdraft_40261) in cultures supplemented with CO2. Under the same conditions up-regulation of a gene involved in the first committed step of fatty acid biosynthesis, Acetyl CoA carboxylase (ACC) -7194806 (Phatrdraft_54926) was also observed. Overall, this study provides further insight on the specific genes linked with increased TAG production in P. tricornutum and the identification of references genes suitable for normalisation of qPCR data across the growth cycle and under CO2 supplementation, thus providing the tools needed for future molecular studies of P. tricornutum lipid production.

No description available.

This research explores the ion chemistry and pyrolysis of a selection of formates. Alternative fuels have become increasingly popular over the past decade, one of particular interest is biofuels. Biofuels contain a variety of oxygenated compounds that are not present in traditional crude-oil fuels such as formates. Therefore, knowing the pyrolysis and ion chemistry of a selection of formates is of particular interest in being able to determine the environmental implications of switching to biofuels. The pyrolysis in particular for these formates have largely been studied using shock-tube experiments where both unimolecular and bimolecular reactions occur. This study employs the use of a micro-reactor coupled to imaging photoelectron photoion coincidence spectroscopy (iPEPICO) using synchrotron vacuum ultra-violet (VUV) radiation at the Swiss Light Source (SLS). The sample is introduced in the dilute-gas phase to optimise for unimolecular reactions. This thesis therefore presents the unimolecular pyrolysis chemistry of methyl formate, ethyl formate and methyl chloroformate and the ion chemistry of methyl chloroformate, phenyl formate and phenyl chloroformate. In chapter 3, the thermal dissociation of the atmospheric constituent methyl formate was probed by coupling pyrolysis with iPEPICO. The pyrolysis products of dilute methyl formate, CH₃OC(O)H, were elucidated to be CH₃OH⁺, CO, 2 CH₂O and CH₄ + CO₂ as in part distinct from the dissociation of the radical cation (CH₃OH⁺˙ + CO and CH₂OH⁺ + HCO). Density functional theory, CCSD(T), and CBS-QB3 calculations were used to describe the experimentally observed reaction mechanisms, and the thermal decomposition kinetics and the competition between the reaction channels are addressed in a statistical model. One result of the theoretical model is that CH₂O formation was predicted to come directly from methyl formate at temperatures below 1200 K, while above 1800 K, it is formed primarily from the thermal decomposition of methanol. Chapter 4 utilises the same techniques but expands on it by taking advantage of threshold photoionization and ion imaging, parent ions of neutral pyrolysis products and dissociative photoionization products could be distinguished, and multiple spectral carriers could be identified in several ms-TPES. The TPES and mass-selected TPES for ethyl formate are reported for the first time and appear to correspond to ionization of the lowest energy conformation having a cis configuration of the O=C(H)-O-C(H₂)-CH₃ and trans configuration of the O=C(H)-O-C(H₂)-CH₃ dihedral angles. We observed the following ethyl formate pyrolysis products: CH₃CH₂OH, CH₃CHO, C₂H₆, C₂H₄, HC(O)OH, CH₂O, CO₂, and CO, with HC(O)OH and C₂H₄ pyrolyzing further, forming CO + H₂O and C₂H₂ + H₂. The reaction paths and energetics leading to these products, together with the products of two homolytic bond cleavage reactions, CH₃CH₂O˙ + ˙CHO and CH₃CH₂˙ + HC(O)O˙, were studied computationally at the M06-2X-GD3/aug-cc-pVTZ and SVECV-f12 levels of theory, complemented by further theoretical methods for comparison. The calculated reaction pathways were used to derive Arrhenius rate parameters for each reaction. The reaction rate constants and branching ratios are discussed in terms of the residence time and suggest carbon monoxide as a competitive primary fragmentation product at high temperatures. Chapter 5 explores the pyrolysis chemistry of methyl chloroformate (MCF) in a similar manner but also introduces the study of the ion chemistry. The TPES for MCF was acquired for the first time; the geometry change upon ionization of MCF results in a broad, poorly defined TPES. Franck-Condon simulations are consistent with an ionisation energy (IE) of 10.90 ± 0.05 eV. Ionized MCF dissociated by the expected loss of Cl with a measured appearance energy (AE) of 11.30 ± 0.01 eV. Together with the above IE, this AE suggests a reaction barrier of 0.40 eV, consistent with that found from SVECV-f12 calculations (0.41 eV). At higher internal energies, the loss of CH₃O˙ becomes competitive due to its more favourable entropy of activation. Pyrolysis of neutral MCF formed the anticipated major products of CH₃Cl + CO₂ (R1) and the minor products HCl + CO + CH₂O (R2), all species being confirmed by their mass-selected TPES. Several possible reactions were computationally explored but these two were confirmed to be the dominant reaction channels. R1 proceeds by a concerted Cl atom migration via a 4-membered transition state in agreement with that proposed in the literature. R2 is a two-step reaction proceeding first by loss of HCl to make 2-oxiranone which then decomposes to CH₂O and CO. Kinetic modelling of the neutral decomposition could be made to simulate the observed reactions only if the vibrational temperature of the MCF was assumed not to cool during the expansion. Chapter 6 further expands on the ion chemistry study by exploring the ion dissociation of phenyl formate (PF) and phenyl chloroformate (PCF). Imaging photoelectron photoion coincidence (iPEPICO) spectroscopy and tandem mass spectrometry were employed to explore the ionisation and dissociative ionisation of phenyl formate (PF) and phenyl chloroformate (PCF). The threshold photoelectron spectra of both compounds are featureless and lack a definitive origin transition, owing to the internal rotation of the formate functional group relative to the benzene ring, active upon ionisation. CBS-QB3 calculations yield ionisation energies of 8.88 and 9.03 eV for PF and PCF, respectively. Ionised PF dissociates by the loss of CO via a transition state composed of a phenoxy cation and a HCO moieties. The dissociation of PCF ions involves the competing losses of CO (m/z 128/130), Cl (m/z 121), and CO₂ (m/z 112/114), with Cl loss also shown to occur from the second excited state in a non-statistical process. The primary CO- and Cl-loss fragment ions undergo sequential reactions leading to fragment ions at m/z 98 and 77. The mass-analysed ion kinetic energy (MIKE) spectrum of PCF⁺ showed that the loss of CO₂ occurs with a large reverse energy barrier, which is consistent with the computationally derived minimum energy reaction pathway. Chapter 7 highlights the conclusions drawn from across the chapters 3-6 and brings chapter 1 back into focus with how these findings can help to inform future studies on pyrolysis. Furthermore, chapter 7 discusses how future technologies for biofuels can be shaped with the understanding of the pyrolysis products of these biofuel related compounds.

Biodiesel is a renewable, biodegradable, clean burning fuel, produced from vegetable oils and animal fat. It is a mixture of fatty acid alkyl esters, products that result from the catalytic transesterification of lipids. The first part of this research describes the development of a new and direct method used to rapidly and quantitatively determine the amount of free methanol in biodiesel samples. The analytical method developed is different from the current standards for methanol determination, and it is the first headspace-SPME method used to extract methanol from biodiesel as matrix. The second part of this research describes the direct analysis of acetic acid and 2uraldehyde in an aqueous mixture using headspace SPME. The direct and accurate determination and quantitation of these two analytes is very important, as they can be inhibitors or food sources for microorganisms capable of producing lipids or ethanol.

The carboxylate biofuels platform (CBP) involves the conversion of cellulosic biomass into carboxylate salts by a mixed microbial community. Chemical engineering approaches to convert these salts to a variety of fuels (diesel, gasoline, jet fuel) are well established. However, prior to initiation of this project, little was known about the influence of inoculum source on platform performance. The studies in this dissertation test the hypothesis that microbial communities from particular environments in nature (e.g. saline and/or thermal sediments) are pre-adapted to similar industrial process conditions and, therefore, exhibit superior performances. We screened an extensive collection of sediment samples from extreme environments across a wide geographic range to identify and characterize microbial communities with superior performances in the CBP. I sought to identify aspects of soil chemistry associated with superior CBP fermentation performance. We showed that CBP productivity was influenced by both fermentation conditions and inocula, thus is clearly reasonable to expect both can be optimized to target desired outcomes. Also, we learned that fermentation performance is not as simple as finding one soil parameter that leads to increases in all performance parameters. Rather, there are complex multivariate relationships that are likely indicative of trade-offs associated within the microbial communities. An analysis of targeted locus pyrosequence data for communities with superior performances in the fermentations provides clear associations between particular bacterial taxa and particular performance parameters. Further, I compared microbial community compositions across three different process screen technologies employed in research to understand and optimize CBP fermentations. Finally, we assembled and characterized an isolate library generated from a systematic culture approach. Based on partial 16S rRNA gene sequencing, I estimated operational taxonomic units (OTUs), and inferred a phylogeny of the OTUs. This isolate library will serve as a tool for future studies of assembled communities and bacterial adaptations useful within the CBP fermentations. Taken together the tools and results developed in this dissertation provide for refined hypotheses for optimizing inoculum identification, community composition, and process conditions for this important second generation biofuel platform.

The present work, where additional value-creating processes in existing combined heat and power (CHP) structures have been examined, is motivated by a political- and consumer-driven strive towards a bioeconomy and a stagnation for the existing business models in large parts of the CHP sector. The research is based on cases where the integration of flash pyrolysis for co-production of bio-oil, co-gasification for production of fuel gas and synthetic biofuels as well as leaching of extractable fuel components in existing CHP plants have been simulated. In particular, this work has focused on the CHP plants that utilize boilers of fluidized bed (FB) type, where the concept of coupling a separate FB reactor to the FB of the boiler forms an important basis for the analyses. In such dual fluidized bed (DFB) technology, heat is transferred from the boiler to the new rector that is operating with other fluidization media than air, thereby enabling other thermochemical processes than combustion to take place. The result of this work shows that broader operations at existing CHP plants have the potential to enable production of significant volumes of chemicals and/or fuels with high efficiency, while maintaining heat supply to external customers. Based on the insight that the technical preconditions for a broader operation are favourable, the motivation and ability among the incumbents in the Swedish CHP sector to participate in a transition of their operation towards a biorefinery was examined. The result of this assessment showed that the incumbents believe that a broader operation can create significant values for their own operations, the society and the environment, but that they lack both a strong motivation as well as important abilities to move into the new technological fields. If the concepts of broader production are widely implemented in the Swedish FB based CHP sector, this can substantially contribute in the transition towards a bioeconomy. / Bioeconomy has been identified to hold a great potential for reducing fossil fuel dependence and for maintaining and creating economic growth. Large parts of the combined heat and power (CHP) sector, which successfully have contributed in the transition towards a fossil free society, are at present facing stagnation. District heating actors are facing challenges due to warmer climate, better insulated buildings and competition from heat pumps. The forest industry where CHP plants supplies processes with heat is facing structural changes foremost in the graphic segments. The emerging bioeconomy and the stagnation for the existing business models in large parts of the CHP sector form the background for the examination of additional value-creating processes in the existing CHP structure presented in this thesis. The technical viability for integration of fast pyrolysis, gasification and leaching with existing CHP plants has been analysed as well as the motivation and ability of the CHP incumbents to participate in a transition towards the bioeconomy by developing their plants to biorefineries.

Oleaginous yeasts are able to produce a high percentage of their weight as lipids, which can be used as the starting material for biodiesel production, producing a fuel with many of the same properties as petroleum-based diesel. The objective of this research was to compare three oleaginous yeast species, Rhodosporidium babjevae, Rhodosporidium diobovatum, and Yarrowia lipolytica to determine which species would be the best candidate for larger-scale production. Following the comparison work, it was determined that R. diobovatum was the best candidate for scale-up. Subsequent experiments used batch cultures in bioreactors at a volume of 3.5 L, followed by a 25x fold increase to 90 L production. The results of this scale-up showed that the high levels of production and growth continued in a reactor system. As such, R. diobovatum could be a possible organism to use in the production of lipids from waste glycerol for biodiesel production. / October 2015

OleTJE (CYP152L1) is a P450 peroxygenase that was first isolated from Jeotgalicoccus sp. 8456 in 2011. OleTJE is primarily a fatty acid decarboxylase, converting mid-chain fatty acids (C10:0 to C22:0) to terminal alkenes, which are industrially useful petrochemicals. Terminal alkenes are hydrophobic with high energy density, and are compatible with existing transportation infrastructure. Thus OleTJE has attracted considerable interest due to potential applications for generating "drop-in" biofuels. As a P450 peroxygenase, OleTJE is able to utilise H2O2 as a sole oxygen and hydrogen donor. This is atypical of P450s, which usually require electron transfer from redox partners to perform substrate oxidation. Other P450 peroxygenases have previously been characterised, including fatty acid hydroxylases P450 Spalpha (CYP152B1) from Sphingomonas paucimobilis and P450 BSβ (CYP152A1) from Bacillus subtilis. In addition to decarboxylation, OleTJE also hydroxylates fatty acids, generating 2-OH and 3-OH fatty acids as minor products. P450 BSβ has also been reported to perform low levels of decarboxylation. However, OleTJE has superior decarboxylase activity, posing questions about the mechanism of OleTJE. This thesis describes initial structural and biochemical characterisation of OleTJE. These data highlighted three amino acid residues thought to be key for effective catalysis: His85, Phe79 and Arg245. We hypothesised that the active site His85 could act as a proton donor to thereactive ferryl-oxo species compound I, allowing homolytic scission of the substrate C-Calpha bond to form the alkene product. Phe79 sandwiches His85 between the heme, and Arg245 co-ordinates the fatty acid carboxylate moiety. I performed mutagenesis studies to probe the roles of these residues, creating H85Q, F79A, F79W, F79Y, R245L and R245E OleTJE mutants, and characterised them by a combination of spectroscopic, analytical and structural methods. I also developed a novel system, where OleTJE was fused to alditol oxidase (AldO) from Streptomyces coelicolor, creating a fusion protein where addition of glycerol drives hydrogen peroxide production and the decarboxylation of fatty acids. Finally, studies showed that OleTJE is capable of performing secondary oxidation of hydroxylated products, which has expanded our knowledge of OleTJE's catalytic repertoire. This thesis also describes the initial characterisation of the OleTJE orthologue P450 KR from Kocuria rhizophila, which is also a terminal alkene-forming fatty acid decarboxylase. The crystal structure of P450 KR revealed an unusual dimeric state, with structural interactions unprecedented for a P450 enzyme. These data thus provide characterisation of two P450 peroxygenases involved in the production of terminal alkenes and which are of great interest as tools for the development of alternative sources of advanced biofuels.