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

Doctor of Philosophy / Department of Grain Science and Industry / Praveen V. Vadlani / Microbial lipids derived from oleaginous yeasts are a promising alternative source of edible oils due to the following advantages: no requirement of broad lands; availability of year-round production; and no food versus fuels controversy. Oleaginous yeast has an inherent ability to accumulate lipids inside cells and their lipids are preferable as starting materials in oleo-chemical industries because of their distinct fatty acid composition. Lignocellulosic biomass is a promising substrate to supply carbon sources for oleaginous yeast to produce lipids due to the high content of polysaccharides and their abundancy. Lignocellulosic-based sugar streams, which can be generated via pretreatment and enzymatic hydrolysis, contained diverse monosaccharides and inhibitors. The major objectives of this study were: 1) to develop a novel purification method to generate clean sugar stream using sorghum stalks after acid pretreatment; 2) to optimize fermentation conditions for Trichosporon oleaginosus to achieve high yields and productivity of microbial lipids using lignocellulosic hydrolysates; 3) to investigate the potentials of sorghum stalks and switchgrass as feedstocks for microbial lipid production using oleaginous yeast strains, such as T. oleaginosus, Lipomyces starkeyi, and Cryptococcus albidus; 4) to develop an integrated process of corn bran based-microbial lipids production using T. oleaginosus; and 5) to develop bioconversion process for high yields of lipids from switchgrass using engineered Escherichia coli. In our investigation, major inhibitory compounds of lignocellulosic hydrolysates induced by pretreatment were acetic acid, formic acid, hydroxymethyl furfural (HMF) and furfural. The activated charcoal was effective in removing hydrophobic compounds from sorghum stalk hydrolysates. Resin mixtures containing cationic exchangers and anionic exchangers in 7:3 ratio at pH 2.7 completely removed HMF, acetic acid, and formic acid from sorghum stalk hydrolysates. T. oleaginosus was a robust yeast strain for lipid production. In the nitrogen-limited synthetic media, total 22 g/L of lipid titers were achieved by T. oleaginosus with a lipid content of 76% (w/w). In addition, T. oleaginosus efficiently produced microbial lipids from lignocellulosic biomass hydrolysates. The highest lipid titers of 13 g/L lipids were achieved by T. oleaginosus using sorghum stalk hydrolysates with a lipid content of 60% (w/w). L. starkeyi and C. albidus also successfully produced microbial lipids using lignocellulosic hydrolysate with a lipid content of 40% (w/w). Furthermore, corn bran was a promising feedstock for microbial lipid production. The highest sugar yields of 0.53 g/g were achieved from corn bran at the pretreatment condition of 1% acid and 5% solid loading. Microbial lipids were successfully produced from corn bran hydrolysates by T. oleaginosus with lipid yields of 216 mg/g. Engineered E. coli also effectively produced lipids using switchgrass as feedstocks. E. coli ML103 pXZ18Z produced a total of 3.3 g/L free fatty acids with a yield of 0.23 g/g. The overall yield of free fatty acids was 0.12 g/g of raw switchgrass and it was 51 % of the maximum theoretical yield. This study provided useful strategies for the development of sustainable bioconversion processes for microbial lipids from renewable biomass and demonstrated the economic viability of a lignocellulosic based-biorefinery.

Atualmente há uma busca crescente por fontes energéticas renováveis motivada principalmente por razões ambientais e estratégicas, tais como o acúmulo de poluentes na atmosfera decorrente do uso de combustíveis fósseis e a diminuição de suas reservas. O biodiesel tem atraído atenção cada vez maior por sua capacidade em substituir o diesel do petróleo. A maior parte do biodiesel produzido utiliza como matéria-prima os óleos vegetais, os quais apresentam como principais desvantagens a sazonalidade na produção devido aos períodos de safra e a competição no uso de terras agrícolas com a produção de alimentos. Uma alternativa capaz de possibilitar a redução do uso de óleos vegetais são os óleos microbianos, como os produzidos por microalgas, fungos e leveduras. Os lipídeos produzidos por leveduras constituem uma fonte interessante de matéria-prima renovável para a produção de biocombustíveis, pois é independente das mudanças climáticas e sazonalidade, como as culturas vegetais. Desta forma, o presente trabalho tem por objetivo contribuir para o desenvolvimento de tecnologias voltadas para produção de óleos microbianos em cultivos heterotróficos. Neste sentido foram desenvolvidos estudos buscando selecionar leveduras capazes de produzir lipídeos tendo glicerol como substrato, bem como estabelecer as condições de cultivo mais favoráveis para a produção e acúmulo de lipídeos pela levedura selecionada. Para a seleção de microrganismos foram realizados ensaios visando comparar cinco diferentes leveduras (Cryptococcus curvatus Y -1511, Lipomyces starkeyi Y-27493, Rhodotorula glutinis Y- 12905, Rhodosporidium toruloides Y-1091 e Rhodotorula mucilaginosa Y-27053), neste estudo verificou-se que as cinco leveduras avaliadas foram capazes de consumir substrato, crescer e produzir lipídeos durante os cultivos. Dentre as leveduras avaliadas a Rhodotorula glutinis Y- 12905 e Cryptococcus curvatus Y -1511 foram as que apresentaram as maiores produtividades volumétricas em lipídeos, alcançando valores entre 0,20 e 0,23 g/L.dia, respectivamente. Quanto à capacidade de consumo de substrato a R. glutinis se destacou em relação as demais leveduras tendo sido capaz de consumir cerca de 59% do glicerol após 120 horas de cultivo, consumo este que representa quase duas vezes o valor alcançado pela C. curvatus, que apresentou o segundo maior consumo de substrato. Devido a sua elevada capacidade de assimilação de glicerol e apresentando uma das maiores produtividades volumétricas de lipídeos entre as leveduras avaliadas a levedura Rhodotorula glutinis foi considerada como uma das mais promissoras para a obtenção de óleos microbianos empregando glicerol como substrato. Nos ensaios realizados com diferentes agitações e razões de volume de frasco por volume de meio verificou-se que quanto maior a disponibilidade de oxigênio, maior foi o acúmulo de lipídeos, o crescimento celular e o consumo de glicerol. Quanto à avaliação da composição nutricional a presença de fontes de nitrogênio orgânico, tais como extrato de levedura e peptona mostraram-se fundamentais para o processo de produção de óleos microbianos juntamente com MgSO4.7H2O. Quando estes nutrientes estavam presentes de forma combinada foram obtidas concentrações de até 4,4 g/L de lipídeos e produtividades de 0,43 g/L.dia. Na avaliação da influência do pH inicial e da razão carbono/nitrogênio (C:N), verificou-se que o acúmulo de lipídeos pela levedura é favorecido para cultivos realizados em pH inicial entre 6 e 7, e com razão C:N entre 30:1 e 50:1. Em tais condições a levedura foi capaz de acumular até 8 g/L de lipídeos com produtividade de até 0,82 g/L.dia. A composição do óleo microbiano obtido pela levedura revelou elevados teores dos ácidos graxos palmítico (C16:0), esteárico (C18:0), oleico (C18:1) e linoleico (C18:2), os quais correspondem a 99% de sua composição. O óleo apresentou um comportamento de fluido newtoniano com viscosidade de 39,3 cP à 50ºC, tendo um índice de acidez de 5,8±0,2 mgKOH/góleo e teor de ácido graxos de 1,93±0,08 %. / Currently, there is a search for renewable energy sources motivated mainly by environmental and strategic reasons, such as the concept of solutions for the development of fossil fuel use and a decrease of its reserves. Biodiesel has attracted increasing attention for its ability to replace diesel oil. Most of the biodiesel produced uses vegetable oils as raw material, which has as main disadvantages the seasonality and a competition in the use of agricultural land with a food production. An alternative capable of reducing the use of vegetable oils is the use of microbial oils, such as those produced by microalgae, fungi and yeasts. The lipids produced by yeasts are an interesting source of renewable raw material for biofuel production, because it is independent of climatic changes and seasonality, such as vegetable crops. In this way, the present study aims to contribute to the development of technologies of microbial oils production in heterotrophic cultures. In this sense, studies were developed to select yeasts capable of producing lipids having glycerol as substrate, as well as to establish the culture conditions more favorable for the production and accumulation of lipids by the selected yeast. For a selection of microorganisms assays were carried out aiming to compare five different yeasts (Cryptococcus curvatus Y-1511, Lipomyces starkeyi Y-27493, Rhodotorula glutinis Y-12905, Rhodosporidium toruloides Y-1091 and Rhodotorula mucilaginosa Y-27053). In this step it was verified that the five yeasts evaluated were able to consume substrate, grow and produce lipids during the cultures. Among the evaluated yeasts, Rhodotorula glutinis Y-12905 and Cryptococcus curvatus Y-1511 has presented the highest volumetric lipid yields, reaching values between 0.20 and 0.23 g/L.day, respectively. Regarding the substrate consumption capacity, R. glutinis stood out in relation to other yeasts, being able to consume about 59% of glycerol after 120 hours of cultivation. Due to its high capacity for assimilation of glycerol and presenting one of the highest volumetric productivities of lipids among evaluated yeasts, Rhodotorula glutinis was considered as one of the most promising for the obtaining of microbial oils from glycerol as substrate. In the assays performed with different oxygenation levels, it was verified that higher oxygen availability higher the volume of lipids, cell growth and glycerol consumption. In the nutritional composition evaluation it was observed that the organic nitrogen source, such as yeast extract and peptone are essential for the production process of microbial oils together with MgSO4.7H2O. When these nutrients were presents in combination, concentrations of 4.4 g/L lipids and productivity of 0.43 g/L.day were obtained. In the evaluation of the influence of the initial pH and the carbon/nitrogen ratio (C:N), it was verified that the accumulation of lipids by yeast is favored for the cultures at initial pH between 6 and 7, and with C:N ratio between 30:1 and 50:1. In such conditions, it was obtained by yeast 8 g/L of lipids and volumetric productivity of 0.82 g/day. The composition of the microbial oil obtained by the yeast revealed high levels of palmitic (C16:0), stearic (C18:0), oleic (C18:1) and linoleic (C18:2) fatty acids, which correspond to 99% of composition. The oil exhibited a Newtonian fluid behavior and a viscosity of 39.3 cP at 50 °C, having an acid number of 5.8 ± 0.2 mgKOH/goil and a fatty acid content of 1.93 ± 0.08%.

Este trabalho teve como objetivos avaliar as condições de cultivo para a produção de lipase e acúmulo de lipídio pela levedura Candida viswanathii utilizando diferentes fontes de carbono e nitrogênio em condições submersas, imobilizar a lipase e avaliar o seu potencial em reação de hidrólise de gordura de frango em biorreator tipo cesto. Os cultivos submersos em condições limitantes de nitrogênio com diferentes fontes de lipídios puros ou complexos mostraram que C. viswanathii acumulou 44% de lipídio intracelular em trioleína e 39% em azeite de oliva utilizando extrato de levedura como fonte de nitrogênio. Nestas condições, a produção de lipase foi 26,78 U/ml em azeite de oliva e 23,6 U/ml em trioleína. O nitrato de amônio inibiu a produção de lipase em 48% em trioleína e 69% em azeite de oliva. Além disso, a análise dos ácidos graxos revelou predominância de ácido oleico (C18:1), em proporções de 69,31, 60,69 e 68,84%, para azeite de oliva, glicose e azeite/glicose, respectivamente. Quanto à imobilização, estudou-se a influência do diâmetro da esfera e da concentração de alginato, obtendo-se o melhor diâmetro de 2 mm e concentração de 2% de alginato, com atividade enzimática de 13,42 U/g e 28 U/g, respectivamente. Utilizou-se alginato de sódio em sua forma convencional e modificado com glutaraldeído, álcool polivinílico e carboximetilcelulose, sendo que o alginato na forma convencional foi o que apresentou melhores resultados, obtendo-se 28,6 U/g e mais de 50% de atividade após 15 ciclos de reuso. A caracterização da lipase livre e encapsulada de C. viswanathii revelou temperatura ótima de atividade de 35 ºC para a enzima encapsulada e na faixa de 40 – 45 ºC para a lipase livre sobre a hidrólise do p-NPP. Sobre a hidrólise de gordura de frango, tanto enzima livre quanto encapsulada apresentaram temperatura ótima de atividade à 40 ºC. Quanto à estabilidade térmica, a enzima livre apresentou boa estabilidade até 12h de incubação nas temperaturas de 30 e 40 ºC. Já a enzima encapsulada mostrou-se estável em até 72 horas de incubação nas mesmas temperaturas. A enzima livre apresentou aumento de atividade na presença de NH4Cl e CaCl2 (117,50% e 111,25%, respectivamente). Já a lipase encapsulada apresentou aumento na atividade com todos os íons analisados, sendo CaCl2 (152,94%), NH4Cl (145,88%), BaCl2 (144,12%) e NaCl (138,24%). Em relação à hidrólise, observou-se que tanto a enzima livre como encapsulada apresentaram maior atividade hidrolítica após 96 horas de incubação, em que a enzima encapsulada obteve 34,66% de hidrólise e a enzima livre obteve 17,91% de hidrólise. Assim, a levedura Candida viswanathii mostrou-se eficiente tanto para o acúmulo de lipídio, quanto para a produção de lipase nas condições estabelecidas. Além disso, a enzima encapsulada apresentou boa estabilidade e potencial para aplicação na hidrólise de gordura de frango. / The aim of this work was to evaluate the culture conditions for lipase production and lipid accumulation by yeast Candida viswanathii using different carbon sources and nitrogen under submerged conditions; and to immobilize the lipase produced in alginate and to evaluate its potential for poultry fat hydrolysis in basket type bioreactor. Submerged cultures under nitrogen-limiting conditions with different sources of pure or complex lipids showed that C. viswanathii accumulated 44% intracellular lipid in triolein and 39% in olive oil using yeast extract as a source of nitrogen. Under these conditions, lipase production was 26.78 U/ml using olive oil and 23,6 U/ml with triolein. Ammonium nitrate inhibited lipase production in 48% with triolein and 69% wiht olive oil. In addition, analysis of fatty acids showed a predominance of oleic acid (C18:1), in proportions of 69.31, 60.69 and 68.84%, for olive oil, glucose and olive oil/glucose, respectively. The influence of drop diameter and alginate concentration was studied, obtaining the best 2 mm diameter and 2% alginate concentration, with enzymatic activity of 13.42 U/g and 28.0 U/g , respectively. Sodium alginate was used in its conventional form and modified with glutaraldehyde, polyvinyl alcohol and carboxymethylcellulose, and the alginate in the conventional form gave the best results, obtaining 28.6 U/g and more than 50% activity after 15 cycles of reuse. The free and encapsulated lipase characterization of C. viswanathii revealed an optimal activity temperature of 35 °C for the encapsulated enzyme and in the 40-45 °C range for free lipase on the hydrolysis of p-NPP. On the hydrolysis of poultry fat, both free and encapsulated enzyme presented optimum temperature of activity at 40 ºC. As for thermal stability, the free enzyme presented good stability up to 12 h of incubation at temperatures of 30 and 40 ºC. The encapsulated enzyme was stable in up to 72 hours of incubation at the same temperatures. The free enzyme showed increased activity in the presence of NH4Cl and CaCl2 (117.50% and 111.25%, respectively). In addition, the encapsulated lipase presented increased activity with all the ions analyzed, being CaCl2 (152.94%), NH4Cl (145.88%), BaCl2 (144.12%) and NaCl (138.24%). In relation to the hydrolysis, it was observed that both the free and encapsulated enzyme presented higher hydrolytic activity after 96 hours of incubation, in which the encapsulated enzyme obtained 34.66% hydrolysis and the free enzyme obtained 17.91% hydrolysis. Thus, yeast Candida viswanathii proved to be efficient both for lipid accumulation and for lipase production under the established conditions. In addition, the encapsulated enzyme presented good stability and potential for application in poultry fat hydrolysis.

Atualmente há uma busca crescente por fontes energéticas renováveis motivada principalmente por razões ambientais e estratégicas, tais como o acúmulo de poluentes na atmosfera decorrente do uso de combustíveis fósseis e a diminuição de suas reservas. O biodiesel tem atraído atenção cada vez maior por sua capacidade em substituir o diesel do petróleo. A maior parte do biodiesel produzido utiliza como matéria-prima os óleos vegetais, os quais apresentam como principais desvantagens a sazonalidade na produção devido aos períodos de safra e a competição no uso de terras agrícolas com a produção de alimentos. Uma alternativa capaz de possibilitar a redução do uso de óleos vegetais são os óleos microbianos, como os produzidos por microalgas, fungos e leveduras. Os lipídeos produzidos por leveduras constituem uma fonte interessante de matéria-prima renovável para a produção de biocombustíveis, pois é independente das mudanças climáticas e sazonalidade, como as culturas vegetais. Desta forma, o presente trabalho tem por objetivo contribuir para o desenvolvimento de tecnologias voltadas para produção de óleos microbianos em cultivos heterotróficos. Neste sentido foram desenvolvidos estudos buscando selecionar leveduras capazes de produzir lipídeos tendo glicerol como substrato, bem como estabelecer as condições de cultivo mais favoráveis para a produção e acúmulo de lipídeos pela levedura selecionada. Para a seleção de microrganismos foram realizados ensaios visando comparar cinco diferentes leveduras (Cryptococcus curvatus Y -1511, Lipomyces starkeyi Y-27493, Rhodotorula glutinis Y- 12905, Rhodosporidium toruloides Y-1091 e Rhodotorula mucilaginosa Y-27053), neste estudo verificou-se que as cinco leveduras avaliadas foram capazes de consumir substrato, crescer e produzir lipídeos durante os cultivos. Dentre as leveduras avaliadas a Rhodotorula glutinis Y- 12905 e Cryptococcus curvatus Y -1511 foram as que apresentaram as maiores produtividades volumétricas em lipídeos, alcançando valores entre 0,20 e 0,23 g/L.dia, respectivamente. Quanto à capacidade de consumo de substrato a R. glutinis se destacou em relação as demais leveduras tendo sido capaz de consumir cerca de 59% do glicerol após 120 horas de cultivo, consumo este que representa quase duas vezes o valor alcançado pela C. curvatus, que apresentou o segundo maior consumo de substrato. Devido a sua elevada capacidade de assimilação de glicerol e apresentando uma das maiores produtividades volumétricas de lipídeos entre as leveduras avaliadas a levedura Rhodotorula glutinis foi considerada como uma das mais promissoras para a obtenção de óleos microbianos empregando glicerol como substrato. Nos ensaios realizados com diferentes agitações e razões de volume de frasco por volume de meio verificou-se que quanto maior a disponibilidade de oxigênio, maior foi o acúmulo de lipídeos, o crescimento celular e o consumo de glicerol. Quanto à avaliação da composição nutricional a presença de fontes de nitrogênio orgânico, tais como extrato de levedura e peptona mostraram-se fundamentais para o processo de produção de óleos microbianos juntamente com MgSO4.7H2O. Quando estes nutrientes estavam presentes de forma combinada foram obtidas concentrações de até 4,4 g/L de lipídeos e produtividades de 0,43 g/L.dia. Na avaliação da influência do pH inicial e da razão carbono/nitrogênio (C:N), verificou-se que o acúmulo de lipídeos pela levedura é favorecido para cultivos realizados em pH inicial entre 6 e 7, e com razão C:N entre 30:1 e 50:1. Em tais condições a levedura foi capaz de acumular até 8 g/L de lipídeos com produtividade de até 0,82 g/L.dia. A composição do óleo microbiano obtido pela levedura revelou elevados teores dos ácidos graxos palmítico (C16:0), esteárico (C18:0), oleico (C18:1) e linoleico (C18:2), os quais correspondem a 99% de sua composição. O óleo apresentou um comportamento de fluido newtoniano com viscosidade de 39,3 cP à 50ºC, tendo um índice de acidez de 5,8±0,2 mgKOH/góleo e teor de ácido graxos de 1,93±0,08 %. / Currently, there is a search for renewable energy sources motivated mainly by environmental and strategic reasons, such as the concept of solutions for the development of fossil fuel use and a decrease of its reserves. Biodiesel has attracted increasing attention for its ability to replace diesel oil. Most of the biodiesel produced uses vegetable oils as raw material, which has as main disadvantages the seasonality and a competition in the use of agricultural land with a food production. An alternative capable of reducing the use of vegetable oils is the use of microbial oils, such as those produced by microalgae, fungi and yeasts. The lipids produced by yeasts are an interesting source of renewable raw material for biofuel production, because it is independent of climatic changes and seasonality, such as vegetable crops. In this way, the present study aims to contribute to the development of technologies of microbial oils production in heterotrophic cultures. In this sense, studies were developed to select yeasts capable of producing lipids having glycerol as substrate, as well as to establish the culture conditions more favorable for the production and accumulation of lipids by the selected yeast. For a selection of microorganisms assays were carried out aiming to compare five different yeasts (Cryptococcus curvatus Y-1511, Lipomyces starkeyi Y-27493, Rhodotorula glutinis Y-12905, Rhodosporidium toruloides Y-1091 and Rhodotorula mucilaginosa Y-27053). In this step it was verified that the five yeasts evaluated were able to consume substrate, grow and produce lipids during the cultures. Among the evaluated yeasts, Rhodotorula glutinis Y-12905 and Cryptococcus curvatus Y-1511 has presented the highest volumetric lipid yields, reaching values between 0.20 and 0.23 g/L.day, respectively. Regarding the substrate consumption capacity, R. glutinis stood out in relation to other yeasts, being able to consume about 59% of glycerol after 120 hours of cultivation. Due to its high capacity for assimilation of glycerol and presenting one of the highest volumetric productivities of lipids among evaluated yeasts, Rhodotorula glutinis was considered as one of the most promising for the obtaining of microbial oils from glycerol as substrate. In the assays performed with different oxygenation levels, it was verified that higher oxygen availability higher the volume of lipids, cell growth and glycerol consumption. In the nutritional composition evaluation it was observed that the organic nitrogen source, such as yeast extract and peptone are essential for the production process of microbial oils together with MgSO4.7H2O. When these nutrients were presents in combination, concentrations of 4.4 g/L lipids and productivity of 0.43 g/L.day were obtained. In the evaluation of the influence of the initial pH and the carbon/nitrogen ratio (C:N), it was verified that the accumulation of lipids by yeast is favored for the cultures at initial pH between 6 and 7, and with C:N ratio between 30:1 and 50:1. In such conditions, it was obtained by yeast 8 g/L of lipids and volumetric productivity of 0.82 g/day. The composition of the microbial oil obtained by the yeast revealed high levels of palmitic (C16:0), stearic (C18:0), oleic (C18:1) and linoleic (C18:2) fatty acids, which correspond to 99% of composition. The oil exhibited a Newtonian fluid behavior and a viscosity of 39.3 cP at 50 °C, having an acid number of 5.8 ± 0.2 mgKOH/goil and a fatty acid content of 1.93 ± 0.08%.

The production of biofuels and oleochemicals from renewable sources offers an opportunity to reduce our dependence on fossil fuels. The work contained in this dissertation has focused on developing and improving methods for the production of biodiesel from non-traditional feedstocks and understanding biosynthetic pathways that result in the production of oleochemicals and fuels. Pure vegetable oil can account for 70-80% of the total cost of biodiesel production. Many low-cost oils contain high amounts of free fatty acids, which are unsuitable for base-catalyzed transesterification. Herein an approach is described that efficiently accomplishes the simultaneous esterification and transesterification of both free fatty acids and triglycerides found in low-cost oils. The approach utilizes an acid catalyst and longer-chain alcohols to improve biodiesel yields from oils high in free fatty acids. Microalgae are a promising biodiesel feedstock, due to its high lipid productivity and its ability to be cultivated using resources, land and water, unsuitable for agriculture. As part of this work, reaction conditions were optimized for the direct (or in situ) transesterification of algal biomass to biodiesel. This approach accomplishes the simultaneous extraction and conversion of the total lipids from microalgae and results in increased yields compared to extraction followed by conversion. The use of this process to effectively produce biodiesel from wet algal biomass is also discussed. Wax esters are a class of oleochemicals that can be used for a wide range of applications in diverse industries. The chemical composition of native wax esters from the bacterium Marinobacter aquaeolei was determined. It was found that including small alcohols in the growth medium resulted in the in vivo formation of esters similar to biodiesel. All of the proteins involved in the wax ester biosynthetic pathway are not known. The cloning, purification, and characterization of a putative fatty aldehyde reductase from M. aquaeolei, believed to be involved in the production of wax esters, is reported. Finally, the expression of a ws/dgat (wax ester synthase) gene from M. aquaeolei in the cyanobacterium Synechocystis sp. PCC 6803 is discussed as an approach to producing biodiesel in vivo from sunlight and CO2.

Doctor of Philosophy / Department of Grain Science and Industry / Praveen V. Vadlani / As the world population continues to grow and the uncertainty of petroleum and food availability transpires, alternative resources will be needed to meet our demands. Single cell oil (SCO) from oleaginous yeast is a renewable noncrop-based resource that can be used for the production of petroleum counterparts. Currently, commercial production is limited, mainly due to high production costs and competition from cheaper alternatives. As a result, improved fermentation techniques, utilization of low-valued feedstocks and efficient downstream processing would be highly valuable. The major objectives of this study were to: 1) optimize fermentation conditions for the development of a novel fed-batch fermentation to enhance oil production using Lipomyces starkeyi, 2) determine the major lipids produced by L. starkeyi, 3) utilize low-valued hemicellulose-rich feedstocks for oil production, and 4) demonstrate the use of 2-methyltetrahydrofuran (2-MeTHF) and cyclopentyl methyl ether (CPME) as greener solvents for oil extraction. Under optimized fermentation conditions, the oil yield increased from 78 to 157 mg oil/g sugar when supplying xylose rather than glucose as the major carbon source. A novel repeated fed-batch fermentation supplying glucose for growth and xylose for lipid accumulation generated the highest oil yield of 171 mg oil/g sugar, oil content of 60% (dry mass basis) and oil productivity of 143 mg oil/L/hr. Oleic acid accounted for 70% of the total fatty acid profile indicating that oil from L. starkeyi is a naturally high source of oleic acid; an added benefit for the biofuel, cosmetic, food, and oleochemical industries. Hemicellulose-rich corn bran and wheat bran were successfully used to produce oil; oil yields of 125 and 71 mg oil/g sugar were reported for whole and de-starched bran hydrolysates, respectively. Compared to traditional methods, biphasic oil extraction systems of 2-MeTHF and CPME had an 80 and 53% extraction efficiency and 64 and 49% selectivity, respectively. The information from this study will be useful for the development of an integrated approach to improve the viability of SCO biochemical platforms for the production of advanced biofuels and renewable chemicals.

Over recent years microbial oil has attracted much attention due to its potential to replace traditional oil sources in the production of biofuels and nutraceuticals. Its advantages arise from its independence of the food supply chain and its ease of production compared to conventional plant oils. Also, as concerns for the environment grow, microbially-synthesized oil emerges as potential competitor for the sustainable production of biodiesel. However, the high cost of its production currently hinders its large scale application. The bottlenecks to industrial microbial oil production are the cost of substrate and cultivation. Current research is focusing on process improvements to make microbial oil more competitive and worthwhile to produce. Several types of microorganisms have been explored so far and waste substrates have been utilised as cheap feedstocks. The overall cost is affected by the fermentation stage, therefore it is imperative to design cultivations with little operating requirements and high yields. Consequently, the present thesis aims to contribute to the field by developing and investigating a simple process for oleaginous yeast cultivation, focusing mainly on enhancing the yields during the bioreactor stage. Oleaginous yeasts were screened for their ability to grow on glycerol and the most promising strain was selected for further research. Then, the necessary conditions for its growth and oil accumulation were defined. Shake-flask cultivations showed that the specific growth rate and glycerol consumption of Rh. glutinis were higher at lower glycerol concentrations (smaller or equal to40 g/L), while higher C/N elemental ratios enhanced oil content. Experimental data were used to construct an unstructured kinetic model to describe and predict the system's behaviour. The Monod-based model took into account double substrate growth dependence and substrate inhibition. Following that, bioreactor cultivations extended the range of parameters studied, to include the influence of aeration rate and oxygen supply on cellular growth and microbial oil production. Cultivations at different air flow rates were performed in a 2 L bioreactor and showed that a low aeration rate of 0.5 L/min gave the best glycerol and nitrogen uptake rates, resulting in a concentration of biomass of 5.3 g/L with oil content of 33% under simple batch operation. This was improved by 68% to 16.8 g/L (cellular biomass) with similar oil content (34%) by applying a fed-batch strategy. Finally, different glycerol feeding schemes were evaluated in terms of their effect on oil accumulation. The concept of targeting first a cell proliferation stage, limited by the availability of nitrogen, followed by a lipid accumulation stage, fuelled by glycerol was tested. Continual feeding and pulsed feedings, delivering the same total amount of nitrogen (and glycerol), resulted in similar elevated values of both cellular biomass (~25 g/L) and oil content (~40%). Addition of glycerol at higher rates but giving the same total amount of nitrogen led to a further increase in oil content to 53%, resulting in an overall oil yield of more than 16 g/L (the highest achieved throughout the project). With comparable yields to those reported in the literature but achieved with a much poorer medium, there is every reason to be optimistic that microbial oil production from glycerol could be commercially viable in the future.