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

The potential of oleaginous yeast Rhodotorula glutinis for the sustainable production of biodiesel feedstock via fermentation of lignocellulosic biomass in pulp and paper wastewater as a fermentation media was investigated. The overall objective was to increase the levels of lipid feedstock in oleaginous yeast in order to reduce biodiesel production cost. To meet sustainable production of biodiesel, industrial wastewater and waste lignocellulose biomass were used as cultivation media and carbon source, respectively. Pulp and paper wastewater effluent was selected as a source of water and nutrients for the production of microbial lipids due to its environmental pollution as it creates large volume of wastewater discharge with high chemical oxygen demand (COD). Since medium composition and process fermentation condition can significantly affect the fermentative performance of oleaginous microorganisms, to find the optimum cultivation condition, design of experiment combined with RSM optimization technique was performed, which has been shown to be successful to predict the optimum condition for the biomass and lipid production in batch fermentation. In addition, lignocellulosic biomass hydrolysate was used as a substrate to improve the cost associated with feedstock fermentation. Lignocellulosic hydrolysate, a product of degradation of lignocelllosic biomass, contains degradation by-products such as 5- hydroxymethylfurfural (HMF), furfural and acetic acid that are known as major inhibitors that influences microorganism growth process. Therefore, their impacts on the fermentative performance and lipid productivity of oleaginous yeast were explored. A detailed operating condition and equipment design for the process of biocrude production from pulp and paper wastewater on a commercial scale was developed. A technological assessment of the process was performed to evaluate their technical benefits and limitations. Results show that pulp and paper wastewater can be used as a cultivation media for the production of microbial lipids using R. glutinis. However, its carbon content has to be improved. Analysis of the design and cost of the process showed that acid hydrolysis process using paper mill sludge as lignocellulosic biomass required the smallest process equipment units but at a higher raw material cost compared to fermentation process.

Wastewater has been shown to contain the necessary nutritive requirements for the growth of microorganisms. The term, oleaginous, has been given to a classification of microorganisms know to produce up to twenty percent of the weight as oil. This study is designed to examine the potential accumulation of lipids within an oleaginous consortium grown on synthetic wastewater. Potential of the fluorescent stain, Nile red, as a lipid detector is also emphasized. Percentages of extractables greater than thirtyive percent were achieved within the oleaginous consortium using a nitrogen-limited medium. Low pH was found to increase the percentages of extractables. Xylose was shown to be a more optimal carbon source for accumulation than glucose. Nile red was shown to bind to intracellular inclusions and may be useful in monitoring lipid accumulation in industrial settings.

Driven by the rising costs, decreasing convenience, and increased demand of fossil fuels, the need for alternative, sustainable energy sources has caused a spark in interest in biomass-based fuels. Oleaginous organisms such as yeast, algae, and bacteria have been considered as microscopic biofactories for oils that can be converted into biodiesel. The process of growing such organisms using current technology requires an alarming amount of freshwater, which is another resource of growing concern. The research detailed within explains how several sources of underutilized wastewater can serve as growth medium in the biodiesel production process. Using only nitrogen and in one case phosphorus as external supplements, algae were shown to grow on produced water from oil and gas industry waste, local municipal wastewater, environmental brackish water from the Great Salt Lake, and wastewater from the potato processing industry. In each case, growth and biodiesel production in wastewaters was as good as or better than laboratory media. The bacterial organism Rhodococcus opacus PD630 and the yeast organism Cryptococcus curvatus were also used to grow on the dairy manufacturing wastewater whey permeate, a large source of underutilized fixed carbon, with successful lipid production. C. curvatus was also used to successfully grow and form large amounts of biodiesel from ice cream factory wastewater and from wheat straw hydrolysate. In each case, the need for freshwater and outside nutrients was nearly entirely replaced, with the exception of some nitrogen supplementation, with a wastewater nutrient source, thus adding to the sustainability of biomass-based fuels.

Oleaginous microbes can accumulate over 20% of their cell dry weight as lipids that are stored as intracellular energy reserves. The characterization of other oleaginous bacteria creates opportunities for the development of alternative feedstocks and technologies. Rhodococcus rhodochrous is a gram-positive bacterium known for its biodegradation capabilities, but little is known about its ability to accumulate lipids. As R. rhodochrous is capable of degrading hydrocarbon gasses and other aromatics, this study aims to investigate any associated lipid production during the conversion of waste and nontraditional carbon sources, such as model lignocellulosic inhibitors. Lignocellulosic biomass is the most abundant and renewable organic material in the world and is composed of cellulose, hemicellulose, and lignin, which can be pretreated to release sugars from the complex, and often recalcitrant, lignin polymer for microbial fermentation. R. rhodochrous was cultivated with various carbon sources, including glucose, xylose, acetic acid, furfural, phenol, vanillic acid, hydroxybenzoic acid, and propane. The results suggest that R. rhodochrous can survive in the presence of these compounds, achieving almost 7g/L cell dry weight after 168 hours and still accumulate up to 40-50% of cell dry weight as lipid in glucose supplemented media. Furthermore, the aromatic compounds are undetected after 48 hours indicating that R. rhodochrous was able to tolerate these compounds and accumulate lipids. Fatty acid methyl ester profiles show a prevalence of palmitic and oleic methyl esters. Overall, these studies are contributing to a better understanding and characterization of another oleaginous Rhodococcus species, Rhodococcus rhodochrous.

Alternative fuels are necessary to meet the increasing demands for fuels. Alternative fuels such as biodiesel are produced using vegetable oils, which are prominentt in the food industry. An alternate feedstock could be oil-producing microorganisms. These oleaginous microorganisms are defined as accumulating more than 20% of their weight in oil as lipids. Cultivating these microorganisms for oil production is not economical due to the high production costs from the sugars in the culture medium. Municipal wastewater could be a potential growth medium that has not previously been considered for cultivating oleaginous microorganisms. However, municipal wastewater contains a low concentration of carbon, which does not promote oil accumulation in the oleaginous microorganisms. To increase the carbon concentration in the wastewater, lignocellulosic sugars could be added to the municipal wastewater. These sugars are a potential alternative to sugars that are in the food industry. The goal of this research is to determine the efficacy of using municipal wastewater to cultivate a consortium of oleaginous microorganisms, thus, producing oil for biodiesel production. First, a consortium of oleaginous microorganisms was cultivated on autoclaved wastewater to determine if the wastewater contains any inhibiting substances for the microorganisms. In addition to the substances in the wastewater, indigenous microorganisms are possible inhibitors to the consortium. Therefore, to determine the effect these indigenous microorganisms have on the oleaginous microorganisms, the consortium was cultivated on raw municipal wastewater amended with varying amounts of sugar. Since the municipal wastewater can be used as a cultivation medium, the effect of the addition of lignocellulosic sugars was determined. During the production of lignocellulosic sugars, furfural and acetic acid, known microbial inhibitors, are formed. The effect of these inhibitors on the consortium’s growth and oil accumulation ability was ascertained, and inhibition models were developed to describe their impact. With these results, SuperPro Designer v6.0 was used to perform simulations and economic analyses to determine the efficacy of incorporating an oleaginous microorganism consortium in a wastewater treatment facility.

2-phenylethanol (2PE) is a valuable fragrance compound which gives a rose-like aroma. As such 2PE is one the highest used fragrances globally. While 2PE is predominantly produced from petrochemical resources, there is a growing market for a naturally derived alternative for food products. 2PE from natural sources is priced so highly due to limited supply from rose petals. Recently, a few reports have demonstrated the production in yeasts through both the de novo production from glucose and ex novo biosynthesis with L-phenylalanine as a precursor. While these are promising most of the yeasts used can only produce low titres under optimal conditions, and the fermentation still appears to be too expensive. In this investigation the wine yeast M. pulcherrima was selected to be assessed for 2PE production. M. pulcherrima is known to produce 2PE in small titres in wine production though has yet to be explored as a platform for this product. M. pulcherrima has several advantages as a yeast platform, in that it produces a range of antimicrobials which can ward off invasive species, allowing for less sterile control in any large scale fermentation. M. pulcherrima was demonstrated to be able to produce 2PE in high titres in the batch mode through de novo synthesis of glucose, producing up to 1 g/L in shake flasks on the lab scale. Arabitol was also observed in the fermentation broth and was produced up to 20 g/L. The fermentation was then scaled up to 2L in batch mode. From these experiments, up to 700 mg/L of 2PE was produced. This is substantially more than any other yeast in the literature to date. Though when xylose or glycerol was present then 2PE production was severely limited. M. pulcherrima was also demonstrated to be able to produce 2PE by bioconversion from phenylalanine up to 1.5 g/L. This 2PE concentration is suggested to be threshold of toxicity to M. pulcherrima by the toxicity study. The production of 2PE could be increased substantially by introducing an absorbent into the process. Liquid solvents and solid adsorbents were assessed to increase 2PE production, used as in-situ 2PE adsorbents. Oleyl alcohol was found to be a good solvent for in-situ extractive solvent in M. pulcherrima culture and increase the production to 3.3 g/L which is higher than 2PE tolerance threshold of the yeast. Activated carbon was also found to be an excellent 2PE adsorbents, with maximum Langmuir adsorption capacity up to 0.807 g/g. 2PE synthesis with activated carbon as an in-situ adsorbent can increase 2PE production to 14 g 2PE/L. Finally, the process was scaled to 2L and run in batch, continuous and semi-continuous modes. This study demonstrates that not only is M. pulcherrima a viable organism to produce 2PE but it has the potential to be scaled up and run in a more cost effective semi-continuous mode when coupled to a continuous extraction technique.

Currently, the production of palm oil leads to the destruction of rainforest. A more sustainable source of lipids could be obtained using abundant lignocellulosic waste (e.g. wheat straw) as a source of carbon in the form of polysaccharides. Some species of oleaginous yeast, grown on sugars, can be made to accumulate between 20-80% of their biomass as oil and so offer a promising alternative to terrestrial crops. In this thesis, the yeast Metschnikowia pulcherrima was selected for its resilience to contamination. Although not previously classified as oleaginous, a combination of low temperature and restricted nutrient availability prevented sporulation and consequently triggered levels of oil production in M. pulcherrima cultures of up to 47%. The potential of this yeast to produce lipids inexpensively on waste resources was investigated. This yeast was grown under non-sterile conditions at pilot scale with minimal temperature control. The possibility of growing M. pulcherrima on lignocellulose was studied on models and showed that it was tolerant to a range of sugars and inhibitors commonly found in hydrolysed lignocellulose. The yeast produced 6.04 g L-1 lipid when cultivated on a mixture of hexoses and pentoses. This was corroborated by demonstrating that the yeast could be cultured on oligomers and sugars produced by hydrolysing wheat straw. Evidence of cellulase production was observed, and this was utilised in a process involving mildly pretreated wheat straw, using a range of pretreatment processes and culture conditions to yield a maximum of 1.12 g L-1 lipid. The usefulness of the products of this cultivation in forming oil in water emulsions was evaluated, and some evidence of surface-active effects was found. Overall, M. pulcherrima was found to have phenotypes that would be highly beneficial in reducing the capital and running costs of a putative lipid production process.

Palm Oil is the highest produced edible oil globally, with over 66 million tonnes produced annually. It has been estimated that up to 50% of all products sold in the supermarket contain palm oil in some form. Palm oil has attractive properties such as a high melting point and texture due to a balanced ratio of unsaturated and saturated fatty acids. It contains approximately 40% oleic acid (monounsaturated fatty acid), 10% linoleic acid (polyunsaturated fatty acid), 45% palmitic acid and 5% stearic acid (saturated fatty acid), that results in an edible oil that is suitable for use in a variety of food, detergent and cosmetics products. In addition, palm oil is the least expensive oil produced due to its high productivity and extensive production. Due to the high demand for the product, vast amounts of rainforest have been cleared to make way for more plantations, reducing biodiversity and releasing huge levels of carbon dioxide into the atmosphere. There is a clear need for an alternative lipid that can match palm oils properties but can be produced sustainably. Recent work suggests that some yeasts are capable of producing a similar oil to palm oil and can be grown on waste resources. In this thesis a novel bioprospecting protocol was developed to isolate yeasts that can survive the harsh conditions necessary for industrial biotechnology. In this way a vineyard and the local area was sampled for yeasts which were then cultured under extremes of pH, multiple sugars and inhibitors caused from the breakdown of lignocellulose. The wild yeast were cultured in four stages: minimal medium with Lysine; minimal medium with inhibitors; minimal medium with xylose as sole carbon-source; and lastly minimal medium with only arabinose and cellobiose as carbon-sources. Only strains that survived each stage were taken forward to the next, to isolate species that were truly suited to these conditions. Out of the estimated 1000s of strains screened this resulted in 12 strains of yeast, mostly in the Metschnikowia pulcherrima, group being able to cope with the conditions. The 12 strains were further analyzed by culturing them in an array of 4 different model lignocellulosic feedstocks namely wheat straw, corn Stover, sugarcane bagasse, and palm kernel cake hydrolysates. Other conditions incorporated in these analysis were a range of pH from pH 1.5 to pH 7.0; four levels of a mixture of 5 inhibitors; and two different temperatures. All of the 12 strains showed similar behaviour where inhibitor tolerance was only marked at higher pH, and at low pH the strains could not grow at all. Though all strains were able to grow on the hydrolysate models, even those with little glucose and/or xylose content. The lipid profile of the strains was also assessed and proved to be similar to most terrestrial crops, with suitable lipid profiles for a rapeseed oil, and in some cases palm oil substitute. Lastly, to further evaluate the accurate identification of the strains as there are some ambiguity in the Metschnikowia pulcherrima group, we applied an approach only widely used for Pathogenic Bacteria/Yeast identification, Multilocus Sequence Typing (MLST). Using 25 strains (7 of this collection), 6 type species and some isolates from the original culture collection in Bath. Sequences of 6 genes was analysed using the Bayesian statistical method. The result showed grouping of M. pulcherrima into 3-4 groups 9 different for each gene. M. Corniflorae being the outgroup. In all 3 genes successfully sequenced: M. Fruticola; R6; Mp DAH 3; and ICS48 were consistently shown to be clonal. The work presented here demonstrates a new method for bioprospecting strains capable of isolating strains for industrial biotechnology, and for characterisation of the yeast in the Metschnikowia genus. Some of the yeasts identified were oleaginous, and could potentially be used as a novel source of palm oil substitute.

Made available in DSpace on 2014-06-11T19:31:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-12-01Bitstream added on 2014-06-13T19:41:53Z : No. of bitstreams: 1 fernandes_jc_dr_botfca.pdf: 1115073 bytes, checksum: 2801ee5f0b4afe076eb6fd7a690f282d (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O girassol é uma oleaginosa de reconhecida importância na alimentação animal, humana, sendo, atualmente, uma das principais matérias-prima para produção de biocombustível. Existem poucos estudos relacionados aos sistemas de manejo do solo adotados para esta cultura. Objetivou-se no presente trabalho quantificar e analisar os efeitos dos sistemas de manejo nos atributos físicos e químicos de um Nitossolo Vermelho Distroférrico (NVdf), após a colheita do girassol, em uma área cultivada durante 11 anos sob o sistema de semeadura direta, e identificar a velocidade na operação de semeadura que permite o melhor desenvolvimento do girassol. O experimento foi instalado na Fazenda Experimental do Lageado, pertencente à Faculdade de Ciências Agronômicas da UNESP, localizada no município de Botucatu-SP. O delineamento experimental foi em blocos ao acaso, seguindo o esquema de parcela subdividida, com quatro repetições. Os tratamentos efetuados nas parcelas foram: os sistemas de manejo do solo (semeadura direta com mecanismo sulcador tipo haste- SDh; semeadura direta com mecanismo sulcador tipo disco duplo-SDd; cultivo reduzido-CR e cultivo convencional-CC). Nas subparcelas, as velocidades utilizadas para a semeadura foram de 3; 5; 6 e 8 km h-1. Amostras de solo foram coletadas na camada superficial, após a colheita do girassol e analisou-se a textura do solo, densidades, porosidade, diâmetro médio ponderado, percentagem de agregados, resistência do solo à penetração e análise química do solo. Para o desempenho operacional dos equipamentos de preparos do solo e semeadura, foram analisadas as seguintes variáveis: velocidade de semeadura, patinagem das rodas motrizes, força média de tração na barra, resistência específica operacional, potência média de tração na barra, consumo horário e operacional de combustível, capacidade de campo teórica e área de solo... / The sunflower is an oleaginous plant of recognized importance in the animal and human feeding, being, at present, one of the main raw materials for bio-fuels production. There are few researches to the soil management systems for this crop. It was objectified in the present work to quantify and to analyze the effect of the management systems in the physical and chemical attributes of a Paleudult, after the harvest of the sunflower, in an area cultivated during 11 years under the system of direct sowing, identify the speed in the sowing operation that allows the best development in the productivity of the sunflower. The experiment was installed in “UNESP – São Paulo State University”, in the Experimental Farm of the Lageado of the Agronomic Sciences College- Botucatu-SP. The experimental was in randomized block design in split plot, with four repetitions. The effected treatments in the plots were: the soil management systems (no-till using furrow opener with mechanism type rod-SDh; no-till using furrow opener with mechanism type double disk-SDd record; minimum tillage-CR and conventional tillage-CC). In the sub plots, the used speeds for the sowing were 3; 5; 6 and 8 km h-1. Soil samples were collected in the surface layer, after the harvest of sunflower and analyzed to soil texture, density, porosity, mean weight diameter, percentage of aggregates, soil resistance to penetration and soil chemistry analysis. For the operational performance of equipment for soil preparation and seeding, were analyzed the following variables: speed of sowing, skating wheels driving, the average tensile strength in the bar, resistance specific operational, average power of the traction bar, time consumption and operational fuel, field capacity and theoretical area of soil mobilized. The variables of the culture of sunflower were evaluated: plant population, longitudinal distribution of plants... (Complete abstract click electronic access below)