Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Lignocellulose, which is composed of cellulose, hemicellulose and lignin, is the main structural component of plant cell walls. Xylan is the main structural component of hemicellulose. Xylan is a complex heteropolysaccharide and, therefore, requires numerous synergistically acting enzymes for its complete hydrolysis. The focus of this study was on xylanases, which is a main chain cleaving enzyme required for xylan hydrolysis. Xylanases have numerous industrial applications and are commonly used in the biofuels, pulp and paper, food, animal feed and textile industries. Of particular interest is the use of xylanases in the biofuels industry due to the depletion of fossil fuels. A major bottleneck is, however, the low yield and high cost of the enzymatic hydrolysis process. In this study, three different xylanase genes from Aspergillus fumigatus, isolated from a triticale compost heap, were cloned and expressed in Saccharomyces cerevisiae. This yeast is an attractive host for the expression of these heterologous proteins, since A. fumigatus is considered a human pathogen and would not be suited for large-scale enzyme production. The recombinant xylanases obtained in this study were functional after expression in the yeast host and yielded high levels of enzyme activity, ranging from 100 to 300 nkat/mg dry cell weight (DCW). Higher enzyme yields will reduce the overall cost of the enzymatic hydrolysis process, making these enzymes attractive to the biofuels industry. The recombinant xylanases obtained in this study were also free of other cellulases. This characteristic makes these enzymes attractive to the pulp and paper industry as cellulose fibres are required to remain intact. Two of the recombinant xylanases, F10 and F11, were relatively stable at a temperature of 50°C with pH optima at pH 6, while the recombinant xylanase G1 only maintained half of its activity at this temperature and displayed pH optimum at pH 5. No synergistic effect was observed between the recombinant xylanases in this study. Future studies could investigate the synergistic interaction between these recombinant xylanases and other accessory enzymes used for the degradation of xylan, such as the esterases. Xylan hydrolysis levels could increase significantly due to a synergistic effect, which would further reduce the overall cost of the lignocellulose enzyme hydrolysis process. / AFRIKAANSE OPSOMMING: Lignosellulose, saamgestel uit sellulose, hemisellulose en lignien, vorm die hoof strukturele bestanddeel van plantselwande. Xilaan is die hoof strukturele komponent van hemisellulose. Xilaan is ʼn komplekse hetero-polisakkaried en verskeie saamwerkende ensieme vir volledige hidroliese hiervan word benodig. Die fokus van hierdie studie is op xilinases, die hoof kettingbrekende-ensiem vir xilaan hidroliese. Xilinases het verskeie industriële toepassings onder meer in die biobrandstof-, papier en pulp-, voedsel-, dierevoeding- en tekstielindustrieë. Weens die uitputting van fossielbrandstofreserwes word xilinases in die biobrandstof industrie van groot waarde geag. Lae opbrengste en hoë kostes van die ensiemhidroliese proses bly egter ʼn knelpunt. In hierdie studie is drie verskillende xilinase gene vanuit ʼn tritikale komposhoop Aspergillus fumigatus isolaat gekloneer en in Saccharomyces cerevisiae uitgedruk. Gis is ʼn aanloklike gasheer vir die uitdrukking van hierdie heteroloë proteïne aangesien A. fumigatus as menspatogeen nie vir grootskaalse ensiemproduksie geskik is nie. Die rekombinante xilinases verkry in hierdie studie is funksioneel in die gis gasheer uitgedruk en hoë vlakke ensiemaktiwiteit is verkry, van 100 tot 300 nkat/mg droë sel massa (DSM). In die lig van hoër ensiemopbrengste wat die totale koste van die ensiem hidroliese proses verlaag, word die ensieme in hierdie studie aanloklik vir die biobrandstof industrie. Die rekombinante ensieme in hierdie studie verkry is ook vry van ander sellulases, ʼn eienskap wat van waarde is vir die papier en pulp industrie waar die sellulose vesels intak moet bly. Twee van die rekombinante xilinases, F10 en F11, was relatief stabiel by ʼn temperatuur van 50°C met ‘n pH optimum van pH 6, terwyl die rekombinante xilinase G1 slegs die helfte van sy aktiwitieit by hierdie temperatuur kon behou met ʼn pH optimum van pH 5. Geen samewerkende effek kon tussen die drie rekombinante xilinases waargeneem word nie. Toekomstige studies kan die samewerkende effek tussen hierdie rekombinante xilinases en bykomstige ensieme betrokke by xilaanafbraak, soos byvoorbeeld die esterases, ondersoek. Xilaanhidroliese vlakke kan aansienlik as gevolg van hierdie samewerkende effek verhoog, wat die koste van ensiem hidroliese van lignosellulose verder kan verlaag. / Stellenbosch University and the Technology Innovation Agency for financial support

Texas – the energy center of the world – is emerging as a pioneer in algae biodiesel research and production. There are a number of reasons for this. Texas is the largest emitter of CO₂ in the country, and efforts are being made to reduce the state's dependence on fossil fuels. Also, algae – robust and promising organisms – need non-arable land, lots of sunlight and brackish/waste water, along with CO₂. Texas has all of these in abundance, plus universities and algae start-ups that are doing crucial R / text

Many developed nations depend on oil for the production of gasoline, diesel, and natural gas. Meanwhile, oil shortages progress and bottlenecks in oil productions continue to materialize. These and other factors result in an energy crisis, which cause detrimental social and economic effects. Because of the impending energy crisis, various potential energy sources have developed including solar, wind, hydroelectric, nuclear, and biomass. Within the biomass sector for renewable energy sources, algae-based biofuels have become one of the most exciting, new feedstocks. Of the potential plant biofuel feedstocks, microalgae is attractive in comparison to other crops because it is versatile and doesn’t pose a threat to food sources. Despite its many advantages, the process to convert the microalgae into a biofuel is very complex and inefficient. All steps within the algae to biofuel production line must be optimized for microalgal biofuel to be sustainable. The production of biofuels from algae begins with selecting and cultivating an algae strain and giving it all the necessities to grow. The algae is then harvested and processed for specific uses. It is the harvesting or lysing step, which includes the extraction of the algal lipids, which is the biggest hindrance of algae being used as a cost effective energy source. The lysing step within the microalgal biofuel processing is of particular interest and will be the focus of this work. This work discusses the optimization of the biofuel production from microalgae biomass through computational and experimental approaches. With atomic force microscopy (AFM), a key mechanical property that would aid in the computational modeling of mechanical lysis in the in-house computational fluid dynamics (CFD) code, Particle-Surface Analysis Code (P-STAC), was determined. In P-STAC, various flow patterns were modeled that would most effectively lyse microalgal cells based on the shear stresses placed on the cells, which will be compared against microfluidic experiments using lipid specific dyes. These results would be influential in developing an energy-efficient method of processing microalgae for biofuel.

Biofuels have caught the attention of the world as a source of renewable energy which can provide energy security, advance rural development, mitigate climate change, and foster international trade. India developed the National Mission on Biodiesel (NMB) as a rural development policy option to produce biodiesel from Jatropha curcas and promoted it as a pro-poor and pro-growth initiative. This thesis examines the emergence, trajectory, and the consequences of the NMB to assess how the NMB worked as a test development policy programme in India. The thesis focuses on the policy-making process in India, particularly the role of narratives in development policy-making and how it leads to blueprint development. It argues that the narratives supporting the NMB were based on shaky scientific facts and did not represent the needs of the rural people. The thesis takes into account that policy processes involve various actors, networks, their interactions and their knowledge, communication of knowledge and politics. It traces the role of various actors such as policy-makers, bureaucrats, researchers, professionals from private companies and NGOs, farmers, and landless labourers involved in the biodiesel mission. This thesis is anchored in the discipline of Science and Technology Studies (STS) and it draws from Actor Network Theory (ANT) and Social Construction of Technology (SCOT) to analyse how the NMB progressed as a test policy model and whether it really was a ‘pro-poor’, ‘pro-growth’ development initiative. Hence this thesis studies how development narratives were used to promote the biodiesel initiative, how networks were created to establish the biodiesel mission as a policy option and advocate its adoption, and in turn how the NMB progressed as a development initiative. As the thesis draws from SCOT the discussion will emphasise on the practices of a society adopting a technology/development initiative, the importance of users (scientists, policy-makers, farmers, labourers, representatives from the industry and NGOs), how users are represented, and in turn how the NMB had an impact on the people adopting it. This thesis contributes to the understanding of the policy-making process of development renewable policies in India and it also examines the apparent inevitability of technological solutions to development challenges. It also contributes to the literature of narratives serving as ‘blueprints’ for development policy-making. Additionally it adds to the literature on biofuels and reveals the complex nature of regional and national networks that comprise a part of the rising Global Biofuel Network.

Lignocellulosic materials provide a raw material source for biofuel conversion and offer several advantages over fossil fuels- usage of a renewable resource, reduced greenhouse emissions, a decreased dependence on foreign oil, and stimulation of the agricultural sector. However, a primary technological challenge in converting lignocellulosic biomass into fuel is overcoming the recalcitrance of its matrix to enzymatic hydrolysis. To overcome these problems for chemical processing, naturally occurring cellulose biomass must be pretreated before it can be further processed using enzymatic hydrolysis or bioconversion. The goal of this work was to develop a model that predicts the glucose yield (pretreatment and enzymatic digestibility) of dilute acid pretreated switchgrass as a function of pretreatment process conditions (acid loading, 0-1.5 vol%, temperature, 165-195oC, and residence time, 1-10 min). This project was the first study that used a multivariable design experimental series to directly compare the pretreatment effectiveness (product yield, biomass composition and appearance, pH, etc) of using conventional and microwave heated reactors. Microwave-pretreated switchgrass afforded up to a 100% higher total glucose yield (combined pretreatment and enzymatic-hydrolysis liquor yields) at equivalent pretreatment severity and at one tenth of the reaction time, relative to conventional pretreatment. Under best pretreatment conditions of 0.75 vol% acid, 195oC, 1 min residence time, 99% glucose yield and 99% hemicellulose removal were achieved. Kinetic parameters were estimated for the cellulose and xylan hydrolysis reactions in the pretreatment liquor and the solid residue. The kinetic model gave an average correlation coefficient of 0.93 for all reactions. In addition, the combined severity factors (CSF) were also determined for each experiment. Highest observed enzymatic glucose yield corresponded to a CSF of 1.7. A mass and energy balance, and economic analysis based on production scale was developed for both reactor systems. The microwave pretreatment process theoretically yielded 48% more ethanol relative to the conventional process. For microwave pretreatment to be commercially viable, two criteria must be met. One, the cost for largescale continuous microwave reactors would need to be significantly lower than current estimates. And second, higher solids content must be used (>20 wt% in the slurry) to maximize output.

Microalgae are widely viewed as a potential source of renewable biofuels. Microalgae are highly productive and can be cultured in recycled water on margial or non-agricultural land. Despite their advantages, the industrial scale deployment of microalgae faces numerous challenges including relatively little knowledge of the algae themselves and the comparatively expensive infrastructures required for culture. The green microalga, Botryococcus braunii is particularly interesting because it synthesizes long-chain (C30- C40) hydrocarbons that can be converted to liquid fuel by hydrogenation and catalytic cracking. Moreover, B. braunii is the major fossil present in the Ordovician oil shales and kerogen deposits. Although studied since the 1970s, very little is known regarding critical aspects of B. braunii, notably its molecular biology. In higher plants molecular clocks have been well defined and transcript profiling has revealed a sophisticated network of circadian scheduling of metabolic processes. Characterization of temporal controls over hydrocarbon synthesis is therefore of importance to optimization of biofuel production from B. braunii. In this project B. braunii (Race B, strain Guadeloupe) were cultured in a 12-hour photoperiod and either maintained in that regime or transferred to constant light. Algae were sampled every 4 hours, during a 28-hour time-course and mRNA extracted. mRNA was reverse-transcribed to cDNA and sequenced using a paired-end protocol on an Illumina HiSeq 2000 platform. Over 2 billion sequence reads of 100 bp were generated and assembled de novo, into a complete transcriptome for B. braunii. The transcriptome was comprehensively annotated using global and targeted protocols and differential expression and co-expression analyses were performed. Metabolic pathway analysis confirmed the presence, and photoperiodic regulation of the MEP/DOXP Terpenoid Backbone synthesis pathway. Targeted annotation and expression analysis revealed two predicted B. braunii circadian clock components, which were incorporated into a B. braunii circadian clock model. In non-hierarchical cluster analysis, contigs of the B. braunii transcriptome clustered under four distinct patterns of diel expression. Networks of co- and anti-expressed contigs were elucidated by hierarchical clustering. These results demonstrate the exquisite control over metabolism in B. braunii. Such knowledge is essential for the industrial applications of B. braunii, either directly or through the engineering of selected B. braunii genes or molecular pathways into alternative chassis.

Doctor of Philosophy / Department of Agronomy / Scott Alan Staggenborg / Ethanol from cellulose and biodiesel are both advanced biofuels according to the renewable fuel standard version two (RFS2) as part of the Energy Independence and Security Act of 2007. Agricultural production of feedstocks for these fuels can occur as co-products from the primary use of the crops. Use of cellulosic material produced from annual grain and sugar crops does not displace land use from grain and sugar production. Production of corn (Zea mays L.), grain sorghum, dual purpose forage sorghum, sweet sorghum, and photoperiod sensitive sorghum (Sorghum bicolor (L.) Moench) are all primarily driven for products other than cellulosic ethanol. Corn production if driven by grain and silage markets with fodder occasionally used for forage. Grain sorghum production is driven by grain markets and grown primarily in semi arid regions. Dual purpose forage sorghum is used for forage both as baled hay and as silage. Sweet sorghum is produced for sugar and molasses production. Photoperiod sensitive sorghum is produced for baled hay. The current study tests the effect of seeding rate on cellulosic ethanol on each crop. Yellow grease is the most common source of oil for biodiesel production. Intensification of oil crop production may increase the feedstock availability for biodiesel. The current study uses double cropping of spring camelina (Camelina sativa (L.) Crantz), spring canola (Brassica napus L.), sesame (Sesamum indicum L.), safflower (Carthamus tinctorius Mohler, Roth, Schmidt and Bourdeux), soybean (Glycine max L.), and sunflower (Helianthus annuus L.) to search for cropping system options that will produce more oil on an annual basis than full season crops. The full season crop options used were maturity group IV soybean, maturity group V soybean, and full season sunflower. Fertility inputs are inherently less for the non legume crops due to the N fixation ability of symbiotic rhizobium. Canola and camelina are also more sensitive to sulfur deficiency than many crops. Long chain and polyunsaturated fatty acids have higher market values than biodiesel. Separation of these fatty acids from the lipid profile of oil seed crops provides additional demand for oil seed crops. Demand for the crops will drive commodity prices and move land use into oil crop production. The second year of oilseed production provided an opportunity to look at lipid profiles of successfully produced crops during a drought year. Three new discoveries were concluded. Grams cellulosic ethanol g[superscript]-1 stover is not affected by density within the densities considered. Among the double crop options tested only sesame after spring crops was viable in normal years and none were viable in an extreme drought year. Lipid profiles are provided for crops produced in concurrent field growing conditions.

Today, most transportation fuels are derived from crude oil. However, fossil fuels are limited resources and contribute to climate change, and are therefore not considered as sustainable. Biofuels are highly relevant candidates for replacing fossil fuels and research has gone into butanol as a biofuel. It has a high energy density, is less hygroscopic and can be blended up to 85% with gasoline. The yeast Saccharomyces cerevisiae is considered a good host for bio- butanol production; it produces small amounts of isobutanol naturally through the Ehrlich pathway, is easy to manipulate genetically and can therefore be engineered to produce higher titres of butanol. End-product toxicity, however, is a problem that needs to be solved to make butanol production in S. cerevisiae more effective, since the organism cannot tolerate higher concentrations of butanol than 2%. Four different S. cerevisiae strains were cultivated in 1.5%, 2%, 3% and 4% isobutanol by spot tests and in liquid media to evaluate their tolerance. Gene expression was measured for genes RPN4, RTG1 and ILV2 to examine their up-regulation and relevance in butanol tolerance. S. cerevisiae strain Saflager 34/70 was determined as the most tolerant strain and was able to grow in 2% liquid isobutanol and 3% isobutanol on agar plates. A three-fold up-regulation of RPN4, a transcription factor involved in the regulation of proteasome gene expression, was observed. These results contribute to the progress of genetic engineering of butanol host organisms, which is needed to create a more effective production of butanol as a biofuel.

Aerobic activated sludge membrane bioreactors (AS-MBR) in municipal wastewater treatment are compact systems that can efficiently perform biological organic oxidation. However, aerobic processes require mechanical aeration accounting for over 40% of total expenditure of a wastewater facility. Additionally, a global urgency for nutrient (Nitrogen/Phosphorus) removal strategies due to surges of eutrophication events requires complex MBR configurations. An innovative and cost-effective process was developed with a dual income-stream: high-quality treated effluent and value-added microalgal biomass for several applications. The proposed process involved several integrated components; an ultrafiltration AS-MBR for organic oxidation followed by a microalgal membrane photobioreactor (MPBR) to remove nutrients (N/P) through assimilation while simultaneously photosynthetically generating dissolved oxygen effluent that was recirculated back into the AS-MBR, thereby reducing the need for mechanical aeration for oxidation. A lab-scale system was fed with a synthetic medium-strength municipal wastewater. The microalgal species C. vulgaris was initially tested in batch trials as a proof-of-concept study on its potential as a photosynthetic oxygenator for the AS-MBR and identify its nutrient utilization efficiencies. The MPBR and MBR were later constructed for continuous operation, with the aim to identify an optimal process configuration. The unit processes were subsequently isolated, where the AS-MBR was subjected to a modelled algal effluent to assesses the impact of varying influent characteristics and effluent recycle rates. A microbial community analysis was performed by high-throughput sequencing and a statistical data-driven modeling approach to assess treatment performances. The MPBR stage was then subjected to the effluent achieved by the AS-MBR stage under varying operating conditions to assess its treatment performance and the resulting algal biomass biochemical composition to identify its suitability for bioethanol, biodiesel, or animal feed production. The findings of this study ultimately confirmed the ability of C. vulgaris to support the AS-MBR for organic removal and fractional nutrient removal by supplying the oxygen demand, and further achieve an effluent polish stage for nutrient removal. The process configuration also demonstrated the ability to achieve a high microalgal biomass production with the potential of extracting valuable products as an added benefit of the wastewater treatment.

A Colômbia, a partir de 2003, prevendo um déficit na sua produção de combustíveis e tentando reativar a economia rural, iniciou seu programa nacional de biocombustíveis apoiado em políticas, regulamentos e incentivos econômicos para o novo setor energético. É assim que esta dissertação tem como objetivo descrever e analisar o processo de implementação do programa de biodiesel na Colômbia a partir de óleo de palma, determinando a capacidade produtiva do país e discutindo as barreiras existentes para o seu desenvolvimento. Para isto são descritos aspectos relacionados à produção de biodiesel na Colômbia como: a fase agrícola e industrial, a produção de biodiesel, o setor de combustíveis na Colômbia, a legislação criada para o biodiesel, assim como os mecanismos econômicos para sua continuidade. A partir dessas informações foram construídos três cenários possíveis até 2020, nos quais são analisados os objetivos do programa e o potencial de produção. Conclui-se que o biodiesel de óleo de palma oferecerá benefícios econômicos, sociais e ambientais caso o governo oriente esforços e recursos às populações pobres e à agricultura familiar. Adicionalmente, a Colômbia tem potencial para substituir a totalidade do diesel consumido no país e ter excedentes de dois milhões de litros de biodiesel para exportação. / Since 2003, Colombia, predicting a deficit in fuel production and trying to revive the rural economy, begins its national biofuels program based on policies, regulations and economic incentives for the new energy sector. This dissertation aims to describe and to analyze the process of implementing the biodiesel program from palm oil in Colombia, determining the country\'s productive capacity and discussing the barriers to its development. Therefore describes the different areas related to the biodiesel production in Colombia as agricultural and industrial stage; biodiesel production; the Colombian fuel sector; legislation developed for biodiesel; as well as economic mechanisms for continuity. From this information were built three possible scenarios until 2020, which analyze the program\'s objectives and production potential. It is concluded that biodiesel from palm oil offers economic, social and environmental benefits if the focus would be in poor population and small farms. In addition, Colombia has the potential to replace all diesel consumed in the country and have over two million liters of biodiesel for export.