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Thesis--Auburn University, 2009. / Abstract. Includes bibliographical references.

The growth of Rhodotorula glutinis in sweet sorghum juice in three levels of three factors of temperature, carbon to nitrogen ratio and pH was evaluated. Accompanying of this growth was the generation of lipids converted to fatty acid methyl ester (FAMEs) and carotenoids. The optimized condition for maximum biomass and carotenoid accumulation was determined to be at 25C, pH of 5.5 and carbon to nitrogen ratio of 10. This condition yielded 22.7 g/L biomass with specific growth rate of 0.213 hr-1. At this condition the carotenoids generation was also maximum with 2.6 mg/gram biomass, comprising of torularhodin, beta-carotene and torulene. The accumulation of lipids following generation of biodiesel was highest at same temperature and pH but carbon to nitrogen ratio of 70, generating 96.3 mg of FAMEs/gram of biomass containing methyl ester of oleic acid, linoleic acid, palmitic acid, stearic acid and linolenic acid.

Developing microbial systems capable of converting low cost lipids into value added products depends on the ability to acquire substrates from the growth media. Saccharomyces cerevisiae can acquire free fatty acids from the growth media and a portion of these lipids can be converted into new lipid products. However, they cannot acquire complex lipids from the growth media unless a nonspecific lipase is included. To circumvent lipase addition, we are genetically engineering S. cerevisiae to secrete a lipase into the growth media. We selected the LIP2 gene from Yarrowia lipolytica, which encodes a nonspecific lipase. Several modifications were made to the LIP2 gene to improve processing. Results identified strains secreting the most lipase. From these results, high producing strains were inserted into an oil inducible vector. Halo assays confirmed lipase secretion, while measuring the fatty acid composition confirmed triacylglycerol breakdown, and yeast uptake of the free fatty acids released.

The focus of this dissertation is centered on the mass spectral analysis of lipids and changes occurring in keeping with the concept of homeoviscous adaptation [1]. Homeoviscous adaptation is the process of modification of membrane lipids in response to environmental stimuli [1]. Dissertation investigations applied this concept to prokaryotic and eukaryotic organisms, and expanded the perception of environmental factors from exogenous organic solvents to intracellular environment. The field of lipidomics deals with the analysis of phospholipid and fatty acid components of membranes the changes that occur due to environmental stimuli and their biological significance [2-6]. The high sensitivity of mass spectrometry (MS) is an ideal tool for lipidomics allowing detection, quantification and structural elucidation [6]. Coupling of a mass spectrometer to a chromatographic system, such as gas chromatograph (GC), allows the separation of fatty acid methyl esters analytes prior to analysis [7]. The research investigations that comprise this dissertation are divided into three interrelated projects. The first project involved the analysis of composition and structure of Clostridium thermocellum membranes from wild-type and ethanol-adapted strains in response to adaptation of cultures to growth in ethanol. The hypothesis being that adaptation of cultures to growth in ethanol would result in compensatory change to the membrane composition. Rat mitochondrial fatty acid profiles isolated from brain, liver, kidney and heart tissues were compared. The hypothesis being that differences in cellular environments found among various tissues would be reflected in the mitochondrial membrane composition. These data support the concept that variations to the lipid content of neurological mitochondria may increase susceptibility to the products of oxidative stress. Lastly, changes in neurological mitochondria as a function of Alzheimer’s disease progression were studied. The hypothesis being that changes to the mitochondrial lipidome would be significantly reflected during advanced stages of AD, in addition to being more prevalent in regions displaying greater pathology. The three interrelated projects increased our understanding of the boundaries established by the concept of homeoviscous adaptation. Project specific hypotheses were supported by data obtained from these investigations.

No description available.

Biodiesel use and production has significantly increased in the United States and in other parts of the world in the past decade. This change is driven by energy security and global climate legislation mandating reductions in the use of petroleum-based diesel. Recent air quality research has shown that emission of some pollutants such as CO, particulate matter (PM), SO2, hydrocarbons, and carcinogenic polycyclic aromatic hydrocarbons (PAHs) is greatly reduced with biodiesel. However, studies have also shown that some unregulated emissions, such as gas-phase carbonyls, are increased with biodiesel combustion. Very limited research has been done to investigate the particle-phase carbonyl and quinone emissions from biodiesel combustion. Also, very limited studies have investigated the ozone oxidation of biodiesel exhaust PM. Fatty acid methyl esters (FAMEs) are found in high abundance in biodiesel exhaust PM. The presence of these FAMEs in biodiesel exhaust PM can potentially alter the kinetics of the reactions between ozone and particle-phase PAHs. In this study, an Armfield CM-12 automotive light-duty diesel engine operated on a transient drive cycle was used to generate PM from various waste vegetable oil (WVO) and soybean biodiesel blends (containing 0%, (B00), 10% (B10), 20% (B20), 50% (B50), and 100% (B100) biodiesel by volume). The primary PM emissions were sampled using Teflon-coated fiberfilm filters. Laboratory PAHs, FAMEs, and B20 exhaust PM were exposed to ~0.4 ppm ozone for time periods ranging from 0-24 hours in order to study the effect of FAMEs and biodiesel exhaust PM on the ozonolysis of PAHs. Organic chemical analysis of samples was performed using gas chromatography/mass spectrometry (GC/MS). PAHs, carbonyls, FAMEs, and n-alkanes were quantified in the exhaust PM of petrodiesel, WVO and soybean fuel blends. The emission rates of the total PAHs in B10, B20, B50, and B100 exhaust PM decreased by 0.006-0.071 ng/µg (5-51%) compared to B00, while the emission rates for the FAMEs increased with increasing biodiesel content in the fuel. The emission rates of the total n-alkanes in B10, B20, B50, and B100 exhaust PM decreased by 0.5-21.3 ng/µg (4-86%) compared to B00 exhaust PM. The total emission rates of the aliphatic aldehydes in biodiesel exhaust PM (B10, B20, B50, and B100) increased by 0.019-2.485 ng/µg (36-4800%) compared to petrodiesel. The emission rates of the total aromatic aldehydes, total aromatic ketones, and total quinones all generally decreased with increasing biodiesel content in the fuel. With the exception of benzo[a]pyrene, the pseudo-first order ozone reaction rate constants of all the PAHs decreased by 1.2-8 times in the presence of the FAMEs. Phenanthrene, fluoranthene, and pyrene were the only PAHs detected in the B20 exhaust PM, and their ozone reaction rate constants were about 4 times lower than those obtained when the PAHs alone were exposed to ozone. The findings of this study indicate that there are both positive and negative effects to emissions associated with biodiesel use in light-duty diesel engines operating on transient drive cycle.

Made available in DSpace on 2015-05-14T13:21:49Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 3150509 bytes, checksum: cf7874482452595653f1051a5680bdf0 (MD5) Previous issue date: 2010-09-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / In this work, computational chemistry calculations and thermal analysis experiments were performed in order to determine the oxidative stability of four fatty acid methyl esters (stearate, oleate, ricinoleate and linoleate), whose fatty chains may be inserted in oils and biodiesel. In the computational chemistry investigation the sequence of stabilities, based on the dissociation energy of the C-H bond was: C18:2 < C18:1 < C18:1;12-OH < C18:0, for the B3LYP 6-31G(d) and MP2 6-311++G(2d,p); and C18:2 < C18:1;12-OH < C18:1 < C18:0, for the B3LYP 6-311++G(2d,p). The spin density analysis allowed stating that the ricinoleate hydroxyl does not act as a pro-oxidizing, as the radicals formed in C-12 or OH are not stabilized by the unsaturation in C9, showing, thus, the behavior of a secondary alkyl alcohol in relation to these sites, whereas their allylic hydrogen display an energy similar to the oleate hydrogens. In the experimental investigation carried out TG, it was possible to observe the formation of hydroperoxides by means of the mass gain in an oxygen atmosphere for oleate, linoleate and ricinoleate, but only volatilization for the stearate. In this investigation, a small heating rate (2 ºC/min) was utilized. The kinetic calculations based on PDSC, in the dynamic and isothermal modes showed that the oxidation susceptibility is quite dependent of temperature, atmosphere and the method employed, being more critical in relation to the methyl ricinoleate. In the dynamic mode, in an air atmosphere at 110ºC, the relative susceptibility was 1 : 17 : 17 : 226 (C18:0 : C18:1 : C18:1;12-OH : C18:2). In an O2 atmosphere this proportion was 1 : 11 : 1 : 102. In the isothermal mode PDSC, at the same temperature, the proportion was 1 : 1230 : 1585 : 23001 in an air atmosphere, and 1 : 33 : 40 : 445 in an O2 atmosphere. Performing a structure/property relationship, the oxidation temperature determined at a heating rate of 10 ºC/min was shown to be strongly correlated with the BDE (C-H) obtained by DFT and MP2, confirming the relationship between the first exothermic event of PDSC in the dynamic mode and the C-H bond strength. Therefore, PDSC is shown as a accelerated testing technique able to determine the true oxidative stability of lipids, as it supplies information on the rate controlling step of auto-oxidation (L-H + R1● → L● + R1-H), whereas the Rancimat method does not supply such information. Ternary ester blends were made and their oxidative stabilities were assessed by means of PDSC in a synthetic air atmosphere. Four equations were obtained with high linear correlation coefficients (R2 > 0.98). A biodiesel representation model was also developed, expressing its main oxidation sites and molecular descriptors for several physico-chemical properties. This representation is expressed by the molecular formula Ca Hb H*c Hd** He***(O2)f (0H)g and shows as one of its advantages the easy display of biodiesel data, what makes more evident the study of structure/property relationship. Its application for the four FAME s and twenty-three blends allowed determining the oxidation temperature (OT) in an air atmosphere, based on the descriptors for allylic hydrogen (H*) and bis-allylic hydrogens (H**). From such model a program in language C was elaborated, whose input is the FAME mole fraction and whose output is the OT in a synthetic air atmosphere. Keywords: Auto-oxidation, FAME (fatty acid methyl esters), Biodiesel, PDSC, DFT. / Nesse trabalho, cálculos de química computacional e experimentos de análise térmica foram realizados para determinar a estabilidade oxidativa de quatro ésteres metílicos de ácidos graxos (estearato, oleato, ricinoleato e linoleato), cuja cadeia graxa pode estar inserida em óleos e biodiesel. Na investigação por química computacional a seqüência de estabilidade com base na energia de dissociação da ligação C-H foi: C18:2 < C18:1 < C18:1;12-OH < C18:0, para o B3LYP 6-31G(d) e MP2 6-311++G(2d,p); e C18:2 < C18:1;12-OH < C18:1 < C18:0, para o B3LYP 6-311++G(2d,p). A análise da densidade de spin permitiu afirmar que a hidroxila do ricinoleato não age como pró-oxidante, pois os radicais formados no C-12 ou OH não são estabilizados pela insaturação no C9, comportando-se, portanto, como um álcool alquílico secundário em relação a esses sítios, enquanto que seus hidrogênios alílicos possuem energia próxima aos do oleato. Na investigação experimental por TG foi possível observar a formação dos hidroperóxidos através do ganho de massa em atmosfera de oxigênio para o oleato, linoleato e ricinoleato, mas apenas volatilização para o estearato. Para tanto, uma pequena taxa de aquecimento (2 ºC/min) foi utilizada. Os cálculos cinéticos obtidos por PDSC nos modo dinâmico e isotérmico mostraram que a susceptibilidade relativa à oxidação é bastante dependente da temperatura, da atmosfera e do método empregados, sendo mais crítica em relação ao ricinoleato de metila. No modo dinâmico, em atmosfera de ar à 110ºC, a susceptibilidade relativa foi de 1 : 17 : 17 : 226 (C18:0 : C18:1 : C18:1;12-OH : C18:2). Em atmosfera de O2 essa proporção foi de 1 : 11 : 1 : 102. Na PDSC modo isotérmico nessa mesma temperatura a proporção foi de 1 : 1230 : 1585 : 23001 em atmosfera de ar, e 1 : 33 : 40 : 445 em atmosfera de O2. Fazendo uma relação estrutura-propriedade, a temperatura de oxidação na taxa de aquecimento de 10 ºC/min mostrou-se bastante correlacionada com a BDE (C-H) obtidas por DFT e MP2, confirmando a relação entre o primeiro evento exotérmico da PDSC no modo dinâmico e a força da ligação C-H. Nesse sentido, a PDSC apresenta-se como a técnica de ensaio acelerado capaz de determinar a verdadeira estabilidade oxidativa de lipídeos, pois fornece informações sobre a etapa contraladora da velocidade de autoxidação (L-H + R1● → L● + R1-H), enquanto que o método Rancimat não fornece essa informação. Foram realizadas misturas ternárias dos ésteres e verificadas suas estabilidades oxidativas por PDSC em atmosfera de ar sintético. Quatro equações foram obtidas com elevada correlação linear (R2 > 0.98). Foi desenvolvido também um modelo de representação do biodiesel expressando seus principais sítios de oxidação e descritores moleculares para diversas propriedades físico-químicas. Essa representação é dada pela fórmula molecular Ca Hb H*c Hd** He***(O2)f (0H)g e tem como uma das vantagens a simplificação de apresentação dos dados para biodieseis, o que torna mais palpável o estudo de relação estrutura-propriedade. Sua aplicação para os quatro FAMEs e vinte e três misturas permitiu determinar a temperatura de oxidação (OT) em atmosfera de ar com base nos descritores para hidrogênios alílicos (H*) e bis-alílicos (H**). A partir desse modelo foi elaborado um programa em linguagem C, tendo como dados de entrada a fração molar dos FAMEs e como saída a OT em atmosfera de ar sintético.

Due to a number of factors, the biodiesel industry in the United States is surging in growth. Traditionally, oil seed crops such as soybean are used as the feedstock to create biodiesel. However, the crop production can no longer safely keep up with the demand for the growing biodiesel industry. Using algae as a feedstock has been considered for a number of years, but it has always had limitations. These limitations were mainly due to the production methods used to grow and harvest the algae, rather than the reaction methods of creating the biodiesel, which are the same as when using traditional crops. Algae is a promising alternative to other crops for a number of reasons: it can be grown on non arable land, is not a food crop, and produces much more oil than other crops. In this project, we propose a novel attached growth method to produce the algae while recycling dairy farm wastewater using the microalga Chlorella sp. The first part of the study provided a feasibility study as the attachment of the alga onto the supporting substrate as well as determining the pretreatment options necessary for the alga to grow on wastewater. The results showed that wastewater filtered through cheesecloth to remove large particles was feasible for production of Chlorella sp, with pure wastewater producing the highest biomass yield. Most importantly, the attached culture system largely exceeded suspended culture systems as a potentially feasible and practical method to produce microalgae. The algae grew quickly and were able to produce more than 3.2 g/m2-day with lipid contents of about 9% dry weight, while treating dairy farm wastewater and removing upwards of 90% of the total phosphorus and 79% of the nitrogen contained within the wastewater. Once the "proof-of-concept" work was completed, we investigated the effects of repeat harvests and intervals on the biomass and lipid production of the microalgae. The alga, once established, was harvested every 6, 10, or 15 days, with the remaining algae on the substrate material functioning as inoculums for repeated growth. Using this method, a single alga colony produced biomass and lipids for well over six months time in a laboratory setting. The second part of this study investigated another aspect of biodiesel production from algae. Rather than focus solely on biomass production, we looked into biodiesel creation methods as well. Biodiesel is created through a chemical reaction known as transesterification, alcoholysis, or commonly, methylation, when methanol is the alcohol used. There are several different transesterification methods. By simplifying the reaction conditions and examining the effects in terms of maximum fatty acid methyl esters (FAME) produced, we were able to determine that a direct transesterification with chloroform solvent was more effective than the traditional extraction-transesterification method first popularized by Bligh & Dyer in 1959 and widely used. This synergistic research helps to create a more complete picture of where algal biodiesel research and development is going in the future. / Master of Science