The role of liquid biofuels in transportation to minimize the effects of climate change is  evident and has led to a number of studies on finding effective solutions to replace fossil fuels. Liquid biofuels are especially important for heavy duty transports as the effective ‘green’ alternatives are not as many compared to light duty vehicles; for which for e.g. electrification is an option. This thesis presents a comparison study of 8 liquid biofuels with a total of 13 different fuel pathways for use in road freight transports; both current and potential future fuels are assessed in terms of their environmental effects, fuel properties and compatibility with the heavy duty vehicle engines (see Table 10, page 36). Furthermore, a case study is performed to assess the practicality of the results of the study.  Hydro-treated vegetable oil, Bio Dimethyl ether, Liquefied Bio Methane/ ED95 are identified as fuels with considerable potential in the shorter term. Algal biofuel and Biomass to liquid (BTL) fuels from synthesis gas, if realized commercially would be a breakthrough for biofuels in overall transportation sector. However, life cycle analysis has to be performed for the different fuel pathways to completely understand the various impacting factors.

Instrumental accounts of policy start at the policy document—the framework for action— and move on from there, identifying gaps, criticising shortcomings or praising proposals. Critical and interpretive reviews of policymaking regard it as a process to be examined rather than an outcome to be managed. At the core of this thesis, the Biofuels Industrial Strategy of South Africa presents a new terrain in which to examine the policy process before such instrumental approaches become pertinent. In doing so, pervasive underlying 'win-win' and 'pro-poor' narratives and associated discourses articulated and legitimised by constituent vested interests, global and local networks (the biofuels assemblage) and the power relations between them are scrutinised as part of the 'messy politics' of policymaking. Through such an investigation, the thesis adds to the understanding of policymaking in South Africa and seeks to instil the importance of interpretive approaches to analysing policymaking. Ultimately, decisions around biofuels highlight the importance of meaning and cognitive frameworks that policymakers bring to the table and the symbolic nature of policy. It must, for example be made clear what purpose policy actually fulfils rather than simply subscribing to social constructions of instrumental success or failure. There is a lingering if not hegemonic supposition that although South Africa has 'good' policy, implementation 'fails' due to capacity. While this may be the case, it is inadequate as an explanation of 'policy failure', where remedial action then becomes more about improving capacity, which may only serve to reify the abstract disjuncture between policy and practice. Rather an attempt should be made to 'problematise' what makes policy either 'good' or 'bad' but more so unpack the taken for granted in policymaking and how policy itself is part of wider sense making processes whilst also fulfilling symbolic roles beyond the merely instrumental. Given an inescapable reality in which politics and knowledge share a dialectic relationship in policymaking, we should rethink the veracity and technocratic assumptions of evidence-based policymaking and the value of 'knowledge' in policymaking processes over and above the way policymakers frame and interpret issues themselves. Considering 'evidence' to be a deus ex machina or panacea, as it is in New Public Management proposals, may very well be short sighted. Neglecting the interpretive and political aspects of policymaking, especially within the technical realms of renewable energy in general and biofuels in particular is equally myopic. Deconstructing the nature of the policymaking process around biofuels has wider implications or findings for the South African context. One can see, for example, the perseverance but slight reconfiguration of the Minerals-Energy Complex (MEC) and a largescale technological fetish that continues to control the vision and direction of renewable energy transitions (and policies thereof) in the country. Corporate networks are, however, only part of the picture and decisions and decision makers involved in the process extend beyond an 'MEC elite', but increasingly include ANC political gatekeepers who inscribe their own ideologies and meanings into policy. These are especially acute in the form of narratives surrounding decisions made, such as the broad-brush exclusion of maize in the face of an emotive and racially politicised food-versus-fuel storyline.

Current research efforts are focused on 'second generation biofuels', which includes biofuels produced from lignocellulosic material. Lignocellulosic material is primarily composed of cellulose, a glucose polymer, xylose rich hemicellulose and non-fermentable lignin. Saccharomyces cerevisiae is widely used on an industrial scale for the production of ethanol from glucose; however, native S. cerevisiae does not contain the genes required for fermentation of xylose into ethanol. Others have sequentially expressed trans-genes from xylose fermenting organisms to engineer strains of S. cerevisiae capable of fermenting this pentose. The goal of this thesis was to generate a single cassette of 9 genes which have been shown to ferment xylose and arabinose. The 17 kb DNA fragment harboring all the genes necessary was introduced into the yeast genome using one-step homologous recombination based transformation. Expression of this cassette was verified by demonstrating that the first and last genes on this cassette were transcribed. The modified strain exhibited xylose utilization under microaerobic fermentation conditions. Further genetic and process engineering methods may be employed to improve the yield. The experiments described here demonstrate that generating a functional cassette of pentose fermenting genes is still achievable.

To fulfil global energy demand and to mitigate economical, geopolitical and ecological challenges associated with fossil fuel utilisation, the energy sector is moving towards greater use of sustainable and environmentally friendly energy sources, including biofuels. The ideal transport biofuel would be hydrocarbons that are identical to fossil petroleum. However, to date characterised hydrocarbon biosynthetic pathways include a decarbonylation or decarboxylation reaction, which involves the loss of one carbon resulting in odd-numbered carbon chain hydrocarbons. This carbon loss decreases carbon efficiency for alkane production, which reduces microbial fuel economic competitiveness. Therefore, it is key that new pathways for alkane production are identified. The sulphate-reducing bacteria genus Desulfovibrio was previously reported to synthesise even-numbered carbon chain alkanes, which suggests an alternative route for alkane production without carbon loss. This investigation aimed to verify Desulfovibrio alkane biosynthesis and characterise the possible synthetic pathway. Ten Desulfovibrio strains, representing seven species, were screened for alkane synthesis using isotopically labelled growth media. The ability to produce alkanes within the Desulfovibrio genus was confirmed and was shown to be strain-specific under a set of culture conditions. The biogenic alkanes detected were octadecane (C18), nonadecane (C19) and eicosane (C20), with a predominance of even-numbered carbon chain alkanes. Fatty acid analysis of Desulfovibrio strains showed an alkane biosynthetic pathway was unlikely to involve a decarbonylation or decarboxylation step. A novel hypothesis was therefore proposed that alkane biosynthesis by Desulfovibrio follows a metabolic route, which has not previously been characterised, involving a series of reduction reactions from the fatty acid pool. The characterisation of the putative Desulfovibrio hydrogenation pathway for alkane biosynthesis was undertaken via a target-directed genome mining approach. The genomic DNA of nine Desulfovibrio spp. was purified, sequenced, de novo assembled and annotated. Seven of these nine genomes are unpublished to date. No homologs of previously characterised alkane biosynthetic enzymes from bacteria were in silico identified in the genomes and proteomes of alkane producing Desulfovibrio spp., suggesting that Desulfovibrio alkane biosynthetic pathway is likely to be catalysed by currently uncharacterised enzymes. The 16S rRNA-based phylogeny of Desulfovibrio spp. supported the hypothesis that the Desulfovibrio alkane biosynthetic pathway was acquired by a common ancestral strain via horizontal gene transfer. The ability of Desulfovibrio to produce alkanes was therefore hypothesised to be due to the presence of recruited genes encoding enzymes involved in alkane synthesis. A comparative genomic analysis intersecting six-alkane producing and four non-alkane producing Desulfovibrio genomes resulted in the in silico identification of 33 hypothetical proteins considered with high confidence to be exclusive to alkane producing Desulfovibrio strains. A novel hypothetical Desulfovibrio alkane biosynthetic pathway was proposed involving a V-type ATPase, an uncharacterised protein, named as a putative reductase in this investigation, and a putative methyltransferase, which were predicted to be exclusive to alkane producing Desulfovibrio spp. The inorganic phosphates resulting from the ATP hydrolysis catalysed by the V-type ATPase would be involved in a reaction with fatty alcohols to form alkyl phosphates, which are putative activated intermediates required for the hydrogenation route from fatty alcohols to alkanes. The putative reductase and the methyltransferase, predicted to share similar structural features with known alkane-binding proteins, would subsequently reduce alkyl phosphates to alkanes and to iso-alkanes respectively. Empirical investigation of the candidate molecular basis function in Desulfovibrio alkane biosynthesis was undertaken. The Desulfovibrio alkane biosynthetic pathway remains to be fully characterised.

Resumo: A necessidade de se reduzir o consumo de energia se fundamenta nas questões ambientais uma vez que a poluição atmosférica, chuva ácida e alteração climática global estão intimamente relacionadas com a produção e utilização de energia. É com esse desafio que essa investigação buscou, por meio de um estudo sobre biocombustíveis na educação básica, promover a formação do indivíduo com competências básicas, contribuindo para que esse indivíduo saiba lidar com a tecnologia e adquira senso crítico para atuar por uma sociedade melhor e intervir nos problemas ambientais relativos a questões energéticas. Para tanto, a contribuição foi feita por meio do processo educativo, com o olhar dirigido ao educador. Por meio de formação continuada de professores, a pesquisa promoveu a intervenção nas propostas de projetos de ensino dos educadores com diferentes estratégias de ensino para abordar o tema biocombustíveis entre outros relativos à energia. As estratégias citadas foram: o trabalho de campo, muito utilizado em projetos de educação ambiental; registros do professor e do aluno, denominados diários de aprendizagem; planejamento do projeto de ensino ou do plano de trabalho docente para a condução de trabalhos de alunos por meio da Aprendizagem Baseada em Projetos (ABP); e a avaliação de desempenho com critérios de excelência definidos entre professores e alunos na forma de rubricas. O desenvolvimento de projetos no contexto escolar, conduzidos por meio da ABP, demonstrou ser uma estratégia de ensino indicada à formação integral do aluno, em especial, nos conteúdos atitudinais, o que foi possível verificar na reflexão dos atores (alunos e professores) envolvidos nesse processo / Abstract: The need to reduce energy consumption is based on environmental issues since air pollution, acid rain and global climate change are closely related to the production and use of energy. It is with this challenge that this research sought, through a study of biofuels in high education, to promote the formation of the individual with basic skills, contributing so that that individual can handle the technology and acquire critical thinking to work for a better society and intervene in environmental problems related to energy issues. With a view to this purpose, the contribution was made through the educational process, with a focus on the educator. Through the continued formation of the teachers, the research has promoted the intervention in the educator‟s teaching projects, emphasizing different teaching strategies to address the issue of biofuels and other energy-related issues. The strategies mentioned were: the field work, often used in environmental education projects; records of the teacher and the student, known as learning logs; planning of the teaching project or of the teaching plan for driving the students‟ work through Project Based Learning (PBL); and the performance evaluation based on excellence criteria established between teachers and students in the form of rubrics. The development of projects in schools, conducted by means of a PBL approach, proved to be a teaching strategy indicated to the formation of the student as a whole, especially in the attitudinal contents, which could be seen from the reflection of the actors (students and teachers) involved in this process / Orientador: Galeno José de Sena / Coorientador: Jânio itiro Akamatsu / Banca: Leonardo Mesquita / Banca: Rubens Alves Dias / Banca: Silvio Henrique Fiscarelli / Banca: Edson da Costa Bortoni / Doutor

Toxicity of organic solvents to microbial hosts is a major consideration in the economical production of biofuels such as ethanol and especially butanol, with low product concentrations leading to high recovery costs. The key to rational engineering of solvent tolerant microorganisms for such processes lies in obtaining appropriate tolerance genes (modules) suited for different compounds. In this project, a synthetic biology approach was adopted to generate a library of standardised BioBrick parts involved in different stress responses. Using a multiple-assay approach, including a bioluminescence assay, these stress response genes were tested individually and in combination to determine their effects on survival in ethanol, nbutanol, acetone and fermentation inhibitors produced by biomass pre-treatment. A set of tolerance modules was obtained for ethanol and n-butanol. Proof-of-concept tests suggested that ethanol and n-butanol toxicity was mainly due to damage to membrane, cellular proteins and DNA possibly by oxidative stress. No synergistic interactions were observed from a combination of different tolerance genes. Further tests carried out using enzyme and fluorescence-based assays to elucidate the effect of n-butanol on the cell envelope showed that the solvent released lipopolysaccharides from the outer membrane of E. coli and also caused both outer and inner membranes to be leaky. Very high n-butanol concentrations resulted in an altered cell shape and bleb formation suggesting an impairment in cell division and peptidoglycan biosynthesis. The cell membrane was modified by cis-trans isomerisation of unsaturated fatty acids in the phospholipids resulting in a reduction of membrane leakage which in effect, increased n-butanol tolerance in E. coli. In conclusion, results from this research suggest that strategies to protect the membrane and cellular proteins should be included in rational engineering of n-butanol tolerant bacteria.

In the world today there is a massive dependency on fossil fuels as they are currently used to provide around 80% of the world’s energy. This is hugely detrimental to the environment and is a major contributory factor in climate change. Biofuel is a renewable energy source that is already being used to lessen some of the fossil fuel dependency. 2nd generation biofuels, by using non-food parts of plants, circumvent the food vs fuel argument, and by using farming waste or surplus can also avoid changing land use problems. Additionally liquid biofuels can use existing infrastructure for storage and delivery, and also fit into current lifestyles. Cost-effective 2nd generation biofuel production is directly affected by the presence of the polymer lignin in plant biomass, as it has been shown to impede enzymatic sugar release (saccharification) that is used for biofuel production. The work undertaken in this project developed a high-throughput methodology for the assessment of straw lignin content and composition across a large population of elite varieties in the economically important cereal crop, barley. Saccharification yield was also measured across the same population along with a number of other agronomically important traits, such as thousand-grain weight, biomass, mechanical stem properties and height. The data provided by these measurements allowed correlations between traits to be identified and their strength gauged. Genome wide association studies (GWAS) were also carried out and identified influential regions of the genome for each trait. The results revealed varying levels of association between measured traits and lignin content and monomeric constituents. Importantly a negative connection was shown between lignin content and saccharification yield, with lignin content being responsible for approximately 1/5th of the variation seen. Interestingly there was no correlation between lignin content and mechanical stem properties, an important factor in the agronomically important trait, lodging. GWAS results revealed a number of genomic regions that were influential across several traits indicating regions that would be difficult to separate through breeding due to their close proximities. However, unique QTL were identified for saccharification yield and lignin content providing candidates for breeding or genetic manipulation to improve the crop for biofuel production.

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mso-tstyle-colband-size:0 / mso-style-noshow:yes / mso-style-priority:99 / mso-style-parent:"" / mso-padding-alt:0cm 5.4pt 0cm 5.4pt / mso-para-margin-top:0cm / mso-para-margin-right:0cm / mso-para-margin-bottom:10.0pt / mso-para-margin-left:0cm / line-height:115% / mso-pagination:widow-orphan / font-size:11.0pt / font-family:"Calibri","sans-serif" / mso-ascii-font-family:Calibri / mso-ascii-theme-font:minor-latin / mso-hansi-font-family:Calibri / mso-hansi-theme-font:minor-latin / mso-bidi-font-family:"Times New Roman" / mso-bidi-theme-font:minor-bidi / mso-fareast-language:EN-US / } </style> <![endif]--> <p style="margin-bottom:0cm / margin-bottom:.0001pt / text-align: justify / line-height:normal / mso-layout-grid-align:none / text-autospace:none" class="MsoNormal"><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">There has been a decline in the global fossil fuel reserves, due to an increasing demand for petroleum. Biofuels can be used as an alternative source of energy whereby biomass is converted to liquid fuels such as bioethanol. There is considerable interest in lipolytic enzymes because of their broad substrate range and for this purpose these enzymes have potential in a variety of biotechnological application. Lipolytic enzymes include esterases (E.C 3.1.1.1) and lipases (E.C3.1.1.3). Esterases preferentially hydrolyse short chain (&lt / C10) ester-containing molecules that are partly soluble in water, while lipases hydrolyse a broad range of substrates preferably water-insoluble fatty acyl molecules (&gt / C10). The aim of this study was to express, purify and characterise the lipolytic enzyme present on afosmid, Try 11, previously isolated from a metagenomic library of a Malawian hotspring, which conferred activity on tributyrin and ethyl ferulate. Bioinformatic analysisof the fosmid insert sequence predicted an open reading frame consisting of 951 bp,designated RHgene34, encoding a 317 amino acid protein with 41 % similarity to the</span><span style="font-size: 10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / mso-fareast-font-family:ArialOOEnc">&alpha / /&beta / hydrolase fold-3 domain protein of </span><i><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">Burkholderia </span></i><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">sp. The RHgene34 protein </span><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">contains conserved motifs of esterases/lipases, such as HGGG (residues 95-98),GxSxG (residues 167 - 171) and the putative catalytic triad composed of Ser157,Asp255 and His285. The gene was cloned and expressed in pET21a(+), and transformed into <i>Escherichia coli </i>Rosetta. <i>p</i>-Nitrophenol (<i>p</i>-Np) fatty acyl esters of different carbon chain lengths were used for kinetic characterisation of RHgene34. </span><span style="font-size:10.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / ">Kinetic analysis revealed that RHgene34 had a broad range activity on the <i>p</i>-Npesters, from C2 - C14. RHgene34 operates optimally at 45 &ordm / C, pH 9.0 and has a half life of 30 mins at 45 &ordm / C. This study demonstrates that functional screening combined with the sequence analysis is a useful approach for isolating novel enzymes from ametagenome.</span></p> <p style="margin-bottom:0cm / margin-bottom:.0001pt / text-align: justify / line-height:normal / mso-layout-grid-align:none / text-autospace:none" class="MsoNormal"><span style="font-size:12.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / "><br type="_moz" /> </span></p> <br /> <p style="margin-bottom:0cm / margin-bottom:.0001pt / text-align: justify / line-height:normal / mso-layout-grid-align:none / text-autospace:none" class="MsoNormal"><span style="font-size:12.0pt / font-family:&quot / Arial&quot / ,&quot / sans-serif&quot / "><br type="_moz" /> </span></p> <br />

The use of bioenergy has been increasing due to efforts in fossil fuels replacement. Modern bioenergy technologies aim for high efficiency and low pollution levels, which increases the need for methods for the on-line characterization of biofuels. Dielectric methods have been identified as useful for the sensing of solid biofuels because they allow for rapid, nonhazardous, nondestructive, and bulk determination of material properties. The dielectric properties describe the interaction between the material and the electromagnetic waves. Dielectric properties are intrinsic of the materials and can therefore be used for the development of prediction models that can be applied regardless of the measurement technique. The study of the dielectric properties is also important as it improves the understanding of the dielectric behavior of the materials. This thesis focuses on the dielectric properties of solid biofuels and their use in the characterization of these materials. The work presented includes the development of new methods permitting the determination of the dielectric properties of solid biofuels with large particle size (waveguide method), broadband measurement of the dielectric properties (coaxial-line probe), and the use of a previously developed method for the accurate determination of the dielectric properties (free-space method). The results includes the dielectric properties of solid biofuels and their dependence on parameters such as frequency, moisture, density, and temperature. This thesis also presents semi-theoretical models for the determination of moisture content, which obtained a RMSEP of 4% for moisture contents between 34 and 67%, and an empirical model that resulted in a RMSEC of 0.3% for moisture contents between 4 and 13%. Finally, this thesis includes measurements of the influence of salt content on the dielectric properties and a discussion of its use for estimation of the ash content of solid biofuels. 

This thesis contains three essays on topics in agricultural economics. The research is focused on the economic effects of different trade policies applied within the US, Canada and the European Union. <p>Essay one evaluates the accession of Austria, Finland and Sweden to the single EU common market. The degree of integration of these three countries in agricultural trade in the EU has not previously been evaluated. Trade theory suggests that one of the outcomes resulting from a regional trade agreement is increased market integration among markets in member states. The cointegration of the commodity prices across countries is tested using time-series techniques. This method is important as it can be applied to questions relating to globalization. <p>Essay two examines the biofuel industry in Canada and US from a trade perspective. The development of a large market for biofuels is judged to have two main benefits for North America: environmental benefits in Canada and energy security in the US. A theoretical model is developed using the option value theory to determine whether the two distinct motivating factors can lead to different levels of optimal subsidies in each country. While the development of a biofuel industry is viewed as extremely important in a number of countries, the trade laws on subsidies with respect these products lacks clarity. This research represents an important step in understanding the economics of biofuels and the situations where trade disputes can be expected to appear in the future.<p>Dumping is the subject of the third essay where the strategies of firms in the face of an anti-dumping action are examined using game theory. The possibility of free riding in case of an anti-dumping petition is investigated in two situations: the benefits of the anti-dumping case are considered either a public good or a joint product. The second situation can be applied only for US, because of so-called Byrd Amendment. The theoretical model developed represents an important contribution to trade policy and it can be easily applied when examining the effects of other trade or domestic policies.