The application of microalgae for biofuel production is a subject of increasing interest as fuel prices continue to fluctuate and the United States aims to secure a reliable, domestic fuel source. Though microalgae have proven to be very efficient at producing oil-rich lipids, the optimum conditions for algae cultivation and methods for harvesting and oil extraction have not been determined. In particular, the harvesting component is especially important to the effectiveness of the overall process because of the large volumes of algae-rich water that must be processed, the strict requirements for downstream lysing, oil extraction and fuel production and the necessity to generate algae biomass with significant post-extraction byproduct value. A number of solid-liquid separation technologies have shown some potential for achieving microalgae/water separation; however, application of these processes to biofuel production requires an evaluation of treatment effectiveness as a function of water quality, algae particle characteristics, and process chemistry. The goal of this research was to identify and evaluate several potentially viable harvesting methods that could be incorporated into end-to-end algae to biofuel production. To achieve this goal, a literature review was conducted to identify the most promising harvesting methods for biofuel applications, and bench scale tests were performed for several harvesting processes. A number of significant findings were identified. Batch algae coagulation experiments with ferric chloride, chitosan, and pH-induced autoflocculation suggest that coagulants can provide effective treatment, but the effectiveness is dependent on water composition and pH. Electrocoagulation experiments indicated that dissolution of the sacrificial electrode led to high metal concentrations in the algae. Pre-oxidation with ozone increased the removal of freshwater Neochloris oleobundans by 20-80% after subsequent flocculation and sedimentation compared with non-ozonated samples. Most notably, this research identified the importance of optimizing water quality and algae particle characteristics for a particular algae harvesting process. Implementing a harvesting process that takes advantage of the natural constituents of a water and the surface characteristics of an algae culture, minimize treatment requirements and enables smoother integration with subsequent processing steps. / text

The fields of biodiesel and bioethanol research and development have largely developed independently of one another. Opportunities exist for greater integration of these processes that may result in decreased costs of production for both fuels.To that end, this work addresses the use of the starches and glycerol from processed algal biomass as substrates for fermentation by the yeasts <ital>Saccharomyces cerevisiae</ital> and <ital>Pachysolen tannophilus,</ital> respectively. Ethanol producers commonly employ the former yeast for ethanol production and include the latter yeast among candidate species for cellulosic ethanol production.A simple 95% ethanol extraction at 70°C followed by sulfuric acid hydrolysis at 121°C and 2 atm proved a sufficient pretreatment for <ital>S. cerevisiae</ital> fermentation of starch from <ital>Chlamydomonas reinhardtii</ital> mutant <ital>cw15.</ital> The maximum rate of ethanol production was observed as 14 mL/g-h and a maximum concentration of 0.9±0.01% (m/v) was observed by 28 hours. Some starch appeared invulnerable to hydrolysis.<ital>P. tannophilus</ital> fermentation of glycerol, both independently and among mixed substrates, was likewise demonstrated. It was found that glucose consumption preceded that of glycerol and xylose, but that the latter two substrates were consumed concurrently. Under aerobic, batch conditions, the maximum specific growth rate of the species on a 2% glycerol substrate was observed as 0.04/hr and the yield coefficient for conversion of glycerol to ethanol was 0.07 g/g. While the maximum observed concentration of ethanol in the glycerol-only fermentation was 0.1% m/v, that in mixed media containing 2% each glucose, xylose, and glycerol was 1.5%.Also investigated here was the flocculation of a mutant species of the algae <ital>C. reinhardtii</ital> by a combination of methanol and calcium. Algae harvest is typically an energy-intensive process, but the technique demonstrated here is not. Complete flocculation of cells was observed with only 5 minutes of mixing and less than 10 minutes of settling using 12 mM CaCl₂ and 4.6% methanol. Ethanol was observed to operate in the same capacity, intimating another area in which yeast bioethanol and algal biodiesel processes might enable one another. During growth, either an inhibitor of flocculation was produced or a facilitator was consumed.

Current biofuel production methods use engineered bacteria to break down cellulose and convert it to biofuel. However, this production is limited by the toxicity of the biofuel to the organism that is producing it. Therefore, to increase yields, microbial biofuel tolerance must be increased. Tolerant strains of bacteria use a wide range of mechanisms to counteract the detrimental effects of toxic solvents. Previous research demonstrates that efflux pumps are effective at increasing tolerance to various biofuels. However, when overexpressed, efflux pumps burden cells, which hinders growth and slows biofuel production. Therefore, the toxicity of the biofuel must be balanced with the toxicity of pump overexpression. We have developed a mathematical model and experimentally characterized parts for a synthetic feedback loop to control efflux pump expression so that it is proportional to the concentration of biofuel present. In this way, the biofuel production rate will be maximal when the concentration of biofuel is low because the cell does not expend energy expressing efflux pumps when they are not needed. Additionally, the microbe is able to adapt to toxic conditions by triggering the expression of efflux pumps, which allows it to continue biofuel production. The mathematical model shows that this feedback loop increases biofuel production relative to a model that expresses efflux pumps at a constant level by delaying pump expression until it is needed. This result is more pronounced when there is variability in biofuel production rates because the system can use feedback to adjust to the actual production rate. To complement the mathematical model, we also constructed a whole cell biosensor that responds to biofuel by expressing a fluorescent reporter protein from a promoter under the control of the sensor.

Recent advances in synthetic biology have enabled the construction of non-native metabolic pathways for production of next-generation biofuels in microbes. One such biofuel is the jet-fuel precursor α-pinene, which can be processed into high-energy pinene dimers. However, accumulation of toxic biofuels in the growth medium limits the possible fuel yield. Overexpression of transporter proteins such as efflux pumps can increase tolerance to biofuels by pumping them out of the cell, thus improving fuel yields. However, too many efflux pumps can compromise the cell as well, creating a trade-off between biofuel toxicity and pump toxicity. In this work we improve the conditions of this trade-off in order to increase pinene tolerance in E. coli. We do so by constructing strains incorporating multiple efflux pumps from a variety of organisms and then testing them for tolerance in growth assay experiments. Previous research has suggested that certain combinations of efflux pumps can confer additional tolerance compared to the individual pumps themselves. However, the functional form of the combination of the tolerance provided by each pump and the toxicity due to their simultaneous activity is unknown. Using differential equations, we developed a growth model incorporating the trade-offs between toxicity of α-pinene and efflux pump activity to describe the dynamics of bacterial growth under these conditions. By analyzing biofuel toxicity and the effects of each efflux pump independently through a series of experiments and mathematical models, we propose a functional form for their combined effect on growth rate. We model the mean exponential growth rate as a function of pump induction and biofuel concentration and compare these results to experimental data. We also apply this technique to modeling toxicity of ionic liquids, a class of corrosive salts that has emerged as and effective chemical for pretreatment of biofuel production feedstock. We compare a model for a variety of ionic liquid responsive efflux pump controllers to that of an IPTG inducible controller and show agreement with experimental data, supporting the model's utility to test control schemes before conducting experiments. The overall goal of this project is to use modeling to guide design of tolerance mechanisms to improve overall biofuel yield.

Currently, one of the hurdles hindering efficient production of cellulosic biofuel is the recalcitrant nature of cellulose to hydrolysis. A wide variety of cellulase enzymes are found natively in microorganisms that can potentially be used to effectively hydrolyze cellulose to fermentable sugars. Thermobifida fusca is a high G-C content, thermophilic, gram-positive soil actinobacterium with high cellulolytic activity. The phenomenological and mechanistic parameters affecting cellulase activity were studied in T. fusca and two mechanisms have been found: 1) transcriptions of cellulase-related genes were not closely associated with measured differences in cellulase activity and 2) cellular energetics (intracellular ATP) correlated more closely to changes in specific cellulase activity. In T. fusca, CelR is thought to act as the primary regulator of cellulase gene expression by binding to a 14-bp inverted repeat: 5’-(T)GGGAGCGCTCCC(A) that is upstream of many known cellulase genes. An efficient procedure for creating precise chromosomal gene replacements has been developed and this procedure was demonstrated by generating a celR deletion strain. Measurements of mRNA transcript levels in both the celR deletion strain and the wild-type strain indicated that the CelR potentially acts as a repressor for some cellulase genes and as an activator for other cellulase genes. Based on the protocol of disrupting celR gene, the direct conversion of untreated cellulosic biomass to 1-propanol in aerobic growth conditions using an engineered strain of T. fusca was demonstrated. Based upon computational predictions, a bifunctional butyraldehyde/alcohol dehydrogenase (encoded by adhE2) was added to T. fusca leading to production of 1-propanol during growth on glucose, cellobiose, cellulose (Avicel), switchgrass, and corn stover. The highest 1-propanol titer (0.48 g/L) was achieved for growth on switchgrass. The adaptive evolution of T. fusca was conducted to find a high cellulase-yield strain. The evolved strains of T. fusca were generated for two different scenarios: continuous exposure to cellobiose (strain muC with specialist phenotype) or alternating exposure to cellobiose and glucose (strain muS with generalist phenotype). Characterization of cellular phenotypes and whole genome re-sequencing were conducted for both the muC and muS strains and 18 and 14 point mutations in the muC and muS strains, respectively were verified. Among these mutations, the site mutation of Tfu_1867 was found to contribute the specialist phenotype and the site mutation of Tfu_0423 was found to contribute the generalist phenotype. The experiment results were used to test genome-scale metabolic model of T. fusca built in this study.

>Magister Scientiae - MSc / The yeast Saccharomyces cerevisiae is considered a good host used for heterologous protein production due to the organism’s microbial safety, rapid growth and eukaryotic post- translational processing. As a fermentative organism, S. cerevisiae is thus not only a useful platform for the production of biopharmaceuticals and industrial enzymes, but also a promising organism for second-generation biofuel production. Substantial effort has been focused on alleviating the many bottlenecks in recombinant gene expression, as well as in the secretory pathway to enhance heterologous protein titres. It was recently shown that highly active endocytosis could decrease the overall secreted protein titre in the supernatant. In this study, we aimed to block endocytotic and vacuolar complexes to ultimately disrupt, or impair, the endocytotic and vacuolar mechanisms of proteolysis and test the effect that this would have on secreted heterologous protein titres. This was accomplished by knocking out various genes involved in endocytosis and transforming the strains with genes encoding various hydrolases including β-glucosidase (Bgl), xylanase (Xyn2), endoglucanase (Eg2) and cellobiohydrylase (Cbh1). Our study demonstrated that genetic blocking of endocytotic mechanisms as well as vacuolar complexes could theoretically improve heterologous protein secretion in S. cerevisiae. Endoglucanase (Eg2) titres displayed improvement of 26% and 30% in strains which had the RVS161 and VRP1 genes deleted and xylanase titres displayed an improvement of 71% and 143% in strains with the END3 and SSA4 gene deletions. Several of the gene knockouts tested improved Xyn2 and Eg2 titres but the effect of the different gene targets varied widely. A double knock-out strain with deletions in CLC1 and RVS161 secreted 104% more Eg2 than its parental control strain on a per dry cell weight basis, a significant synergistic improvement. Other double knock-out strains displayed additive or similar activities when compared to their controls. Cbh1 secretion could not be improved through the gene deletions tested in our study and Bgl activity could not be measured in our transformants. These results demonstrate the different relationships of various heterologous proteins with various components of the secretion machinery and may also imply how endocytosis as well as vacuolar complexes affect the level of secreted protein.

Dois exemplos de combustíveis renováveis em uso atualmente são bioetanol e biodiesel. Novas alternativas de combustíveis incluem etanol celulósico e biobutanol. Estes apresentam vantagens pois contribuem para uma melhor produtividade e otimização do uso de biomassa. Possuem ainda boas propriedades que garantem o bom desempenho como combustíveis. A pesquisa e interesse industrial têm crescido sobre o biobutanol, com melhorias no processo tradicional de fermentação ABE (Acetona-Butanol-Etanol), desenvolvimento de novos microorganismos para aumentar o rendimento e técnicas de separação para isolar o solvente do meio fermentativo. Algumas companhias anunciaram planos para a introdução de biobutanol em misturas com gasolina no mercado norte-americano. O interesse por biobutanol no Brasil como combustível ainda é limitado, pois a infraestrutura de comercialização já é adaptada ao uso de bioetanol, e a maior parte da frota de carros circulante utiliza motores adaptados ao uso do bioetanol. A cana-de-açúcar pode ser utilizada como matéria prima no processo produtivo do biobutanol, capacitando o Brasil a tornar-se um importante exportador para suprir o biobutanol para outros países. Em curto prazo, o biobutanol poderá ser produzido no Brasil para substituir o petro-butanol como solvente em aplicações industriais ou para o mercado de exportação como combustível. O presente trabalho propõe o uso da técnica technology roadmapping para o planejamento estratégico do desenvolvimento do biobutanol, alinhando os objetivos de longo prazo com os recursos, linhas de financiamento e prioridades para atender as necessidades do processo de desenvolvimento. / Two examples of renewable fuels in use today are bioethanol and biodiesel. New alternatives on biofuels include cellulosic ethanol and biobutanol. They present several advantages over the conventional biofuels, either in terms of better productivity and optimization of the use of biomass, as well as higher performance attributes. The research and industrial interest has grown on biobutanol, with improvements on the traditional ABE fermentation process, on the development of new microorganism strains to improve yield, and separation techniques to isolate the solvent. Companies have announced plans for the introduction of biobutanol in blends with gasoline in the north-american market. The interest on biobutanol as a fuel in Brazil is still limited, since the infrastructure is tailored to bioethanol already, and most of the car fleet uses engines adapted to this fuel. Sugar cane can be used as a potential feedstock in the butanol production process, enabling Brazil to become a key exporter to supply biobutanol to other countries. For the short future biobutanol will be produced in Brazil to replace petro-butanol as a solvent in industrial applications only, or for the export market as a fuel. This work proposes the use of technology roadmapping as a technique for long term strategic planning of the biobutanol development, aligning long term goals with the resources, funding, and priorities to fulfill the needs in the development process.

More deaths are caused every year by indoor air pollution than malaria, HIV/AIDS and tuberculosis combined. Cooking with traditional fuels such as charcoal and fuelwood with poor ventilation causes the single most important environmental health risk factor worldwide. It also contributes to environmental issues such as deforestation as traditional biomass fuels and cooking stoves are inefficient and requires large quantities of wood. This is especially critical in Africa where the largest regional population growth in the world is expected to occur. A solution to these issues was realized through fuel pellets and modern cooking stoves by Emerging Cooking Solutions, a company started by two Swedes and based in Zambia. The production of fuel pellets in Zambia is dependent on pine sawdust from small sawmills and is a declining source of raw material. However, other sources of biomass are available in Zambia such as pigeon pea stalk, an agricultural waste product, and sicklebush, an invasive tree species. If these species are viable for pelletization, the production of pellets can increase while reducing issues with sicklebush and promoting cultivation of pigeon pea. The aim of this work is to evaluate if pigeon pea stalk and sicklebush are viable to pelletize in Zambia and how the press is affected by the different raw materials. A pilot study is done at Karlstad University with a single unit press, hardness tester and soxhlet extractor to evaluate how the material constituents correlate to friction in the press channel and hardness of the pellets. The results of the pilot study provide support for full scale tests done in a pellet plant in Zambia. The normal production of pellets from pine sawdust is used as quality and production reference for the tests with pigeon pea stalk, sicklebush, and different mixes of the raw materials. The properties used to evaluate the quality of the pellets are hardness, durability, moisture content, bulk density, and fines. The press configuration is evaluated by logging the electricity consumption by the press motor, calculating the power and specific energy consumption from the logs, and observations during the tests. The results show that sicklebush, and mixes of sicklebush with pigeon pea stalk can produce pellets with better quality than the reference pine pellets. An interesting composition is a mix of 80% pigeon pea and 20% sicklebush that produces pellets with the best quality of all the tests. However, pellets produced from sicklebush and pigeon pea show a larger variation in hardness as compared to the reference pellets from pine sawdust. Mixing pigeon pea with pine reduces these variations but reduces the hardness of the pellets below the reference. The press struggles to process sicklebush and pigeon pea stalk with fluctuating power consumption that causes the motor to trip. The inhomogeneity of the materials in sicklebush and pigeon pea are identified to cause the issues in the press. Production improvements are discussed to facilitate the production of pigeon pea stalk and sicklebush pellets.

Dois exemplos de combustíveis renováveis em uso atualmente são bioetanol e biodiesel. Novas alternativas de combustíveis incluem etanol celulósico e biobutanol. Estes apresentam vantagens pois contribuem para uma melhor produtividade e otimização do uso de biomassa. Possuem ainda boas propriedades que garantem o bom desempenho como combustíveis. A pesquisa e interesse industrial têm crescido sobre o biobutanol, com melhorias no processo tradicional de fermentação ABE (Acetona-Butanol-Etanol), desenvolvimento de novos microorganismos para aumentar o rendimento e técnicas de separação para isolar o solvente do meio fermentativo. Algumas companhias anunciaram planos para a introdução de biobutanol em misturas com gasolina no mercado norte-americano. O interesse por biobutanol no Brasil como combustível ainda é limitado, pois a infraestrutura de comercialização já é adaptada ao uso de bioetanol, e a maior parte da frota de carros circulante utiliza motores adaptados ao uso do bioetanol. A cana-de-açúcar pode ser utilizada como matéria prima no processo produtivo do biobutanol, capacitando o Brasil a tornar-se um importante exportador para suprir o biobutanol para outros países. Em curto prazo, o biobutanol poderá ser produzido no Brasil para substituir o petro-butanol como solvente em aplicações industriais ou para o mercado de exportação como combustível. O presente trabalho propõe o uso da técnica technology roadmapping para o planejamento estratégico do desenvolvimento do biobutanol, alinhando os objetivos de longo prazo com os recursos, linhas de financiamento e prioridades para atender as necessidades do processo de desenvolvimento. / Two examples of renewable fuels in use today are bioethanol and biodiesel. New alternatives on biofuels include cellulosic ethanol and biobutanol. They present several advantages over the conventional biofuels, either in terms of better productivity and optimization of the use of biomass, as well as higher performance attributes. The research and industrial interest has grown on biobutanol, with improvements on the traditional ABE fermentation process, on the development of new microorganism strains to improve yield, and separation techniques to isolate the solvent. Companies have announced plans for the introduction of biobutanol in blends with gasoline in the north-american market. The interest on biobutanol as a fuel in Brazil is still limited, since the infrastructure is tailored to bioethanol already, and most of the car fleet uses engines adapted to this fuel. Sugar cane can be used as a potential feedstock in the butanol production process, enabling Brazil to become a key exporter to supply biobutanol to other countries. For the short future biobutanol will be produced in Brazil to replace petro-butanol as a solvent in industrial applications only, or for the export market as a fuel. This work proposes the use of technology roadmapping as a technique for long term strategic planning of the biobutanol development, aligning long term goals with the resources, funding, and priorities to fulfill the needs in the development process.

This thesis looked at a biorefinery pilot plant that converted lignin in black liquor into biofuel. A heat/mass balance was made which was used to create a heat/mass balance for a theoretical large-scale plant. This then created the CAPEX for building the plant. OPEX for the largescale plant and income from sold biofuels was calculated and payback time found. This was done for three different cases with different flows and yield to optimize the plant. A sensitivity analysis was then made to find the most important parameters regarding CAPEX, OPEX and payback time.