The growth and physiology of <i>Saccharomyces cerevisiae</i> GB4918 (baker’s yeast) was studied under glucose-limitation in chemostat culture. Levels of lg/1 (0.1% w /v) glucose allowed cell growth while preventing fermentation in a defined medium (QEMM3). Metabolism of glucose by respiration or fermentation was shown to affect the mean cell volume, with fermentative use of glucose causing an increase in cell size. This was also a major physiological difference between <i>S. cerevisiae </i>GB4918 (a Crabtree positive yeast) and <i>Kliiyveromyces marxianus</i> DBVPG 6165 (a Crabtree negative yeast). The ability of the Crabtree positive yeast to substantially increase its mean cell volume was also reflected in a 5-fold greater consumption of glucose, reduced biomass yield and increased ethanol yield, compared with the Crabtree negative <i>K . marxianus</i>. Growth of both these yeasts was seen in 50g/l glucose in the presence of the respiratory inhibitor, antimycin A. This was evident by the switching to fermentation in <i>K . marxianus</i>, and the complete fermentation of glucose by <i>S. cerevisiae</i>. The growth and physiology of <i>S. cerevisiae</i> GB4918 was also established in glucose-limited chemostat cultures, with special regard to the intracellular macromolecular compounds that are relevant to industrial yeast biomass production. This showed that in respiring cultures of <i>S. cerevisiae</i>, increasing growth rate resulted in decrease in both trehalose and glycogen content, while increasing protein and RNA. This is true until μ_max (in this context the growth rate at which respiro-fermentativemetabolism occurs) when accumulation of trehalose and glycogen is apparent. Once μ_erit (growth rate at which washout of the culture begins) was reached then biomass significantly reduced. In describing the steady-state condition of baker’s yeast it was then possible to describe changes occurring in yeast when subjected to a variety of nutrient perturbations. With a lactic acid (2% v/v) perturbation there were dramatic effects on both growth and metabolism at a growth rate of 0.12h_1, but significant decreases in biomass and protein, and significant increases in trehalose and glycogen. At a higher growth rate (0.22h_1) the effect was much severer on protein content, and on reduced levels of trehalose and glycogen. The effect of perturbing the cultures with elevated levels of calcium was also most significant on reducing yeast trehalose and glycogen levels, probably due to inhibition of the biosynthesis of these compounds. Zinc additions to chemostat cultures acted to increase the levels of protein in the cells,while having little effect on any of the other cellular macromolecules. This suggests that increasing calcium levels during the latter stages of yeast propagations may produce a yeast with reduced stress responses. Increased zinc may also encourage a greater protein content, which would, in turn, provide a better nutritive content for both protein and amino acids in yeasts destined for use as a food additive.

Dissertação(mestrado) - Universidade Federal do Rio Grande, Programa de Pós-Graduação em Engenharia e Ciência de Alimentos, Escola de Química e Alimentos, 2010. / Submitted by Caroline Silva (krol_bilhar@hotmail.com) on 2012-08-13T19:45:26Z No. of bitstreams: 1 dissertao - francisco-1.pdf: 1598375 bytes, checksum: 011e4e3014589fe2de68fb5af51b8aaf (MD5) / Approved for entry into archive by Bruna Vieira(bruninha_vieira@ibest.com.br) on 2012-12-02T12:20:25Z (GMT) No. of bitstreams: 1 dissertao - francisco-1.pdf: 1598375 bytes, checksum: 011e4e3014589fe2de68fb5af51b8aaf (MD5) / Made available in DSpace on 2012-12-02T12:20:25Z (GMT). No. of bitstreams: 1 dissertao - francisco-1.pdf: 1598375 bytes, checksum: 011e4e3014589fe2de68fb5af51b8aaf (MD5) Previous issue date: 2010 / A crescente demanda de energia em tempos de diminuição no fornecimento de combustível fóssil tem atraído a atenção para a busca por fontes alternativas, que venham a substituir o uso do petróleo, do carvão e do gás natural. Neste contexto, o biodiesel produzido a partir de óleos vegetais aparece como uma alternativa para substituição ao óleo diesel. Esta possibilidade de utilização de combustíveis de origem agrícola vem apresentando um potencial promissor no mundo inteiro, sendo um mercado que cresce aceleradamente devido à sua enorme contribuição ao meio ambiente, com a redução qualitativa e quantitativa dos níveis de poluição ambiental, principalmente nos grandes centros urbanos, e também servindo como fonte estratégica de energia renovável em substituição ao óleo diesel e outros derivados do petróleo. A produção de biodiesel a partir dos óleos vegetais fornece um sistema bifásico, sendo uma fase não polar de ésteres de ácidos graxos e outra mais densa constituída por glicerina e outros componentes residuais do processo. Considerando que na produção de biodiesel há geração de aproximadamente 10% de glicerina, com a mistura constituída de 5% de biodiesel ao diesel (B5) estimativas apontam, em 2013, uma geração de cerca de 150 mil toneladas por ano de glicerina. Uma vez que os mercados tradicionais de glicerina não conseguirão absorver esta oferta de produto neste cenário, este trabalho vem contribuir em inovações tecnológicas relacionadas ao aproveitamento da glicerina, mais especificamente na conversão por via biotecnológica da glicerina gerada na síntese de biodiesel em biomassa de interesse comercial, como fonte de nutrientes. Os efeitos da composição do meio de preparo de inóculo e da temperatura de incubação sobre o crescimento da levedura Yarrowia lipolytica NRRL YB – 423, cultivada em meio à base de glicerina residual, foram estudados em incubadora rotatória, verificando-se que a temperatura de 25°C e utilizando um meio de inóculo com mesma composição do meio de produção foram mais adequados ao crescimento microbiano, atingindo uma concentração de biomassa de 17,7 g.L-1. Em biorreator de bancada, um planejamento fatorial para avaliar a aeração e agitação (22 ensaios mais 3 pontos centrais) foi realizado. Os ensaios indicaram agitação de 200 rpm e aeração de 1 vvm como a melhor condição de cultivo, atingindo 19,14 g.L-1 de concentração de biomassa máxima média, conteúdo protéico médio de 13,55% e teor lipídico de 7,87%. Nestas condições, o cultivo em biorreator de bancada, em relação ao cultivo em frascos agitados, levou a incrementos significativos na biomassa máxima, velocidade específica máxima de crescimento celular e produtividade, respectivamente de 1,22, 1,53 e 2,36 vezes. Com base na determinação do perfil de ácidos graxos e aminoácidos, a biomassa mostrou-se fonte promissora de ácidos graxos essenciais, em particular o ácido linoléico (49,16%) e aminoácidos essenciais como isoleucina, valina, treonina e lisina, que apresentaram escores químicos superiores ao padrão FAO/WHO, respectivamente 1,42, 1,42, 1,30 e 1,17. Portanto a biomassa mostrou-se promissora para utilização como fonte de nutrientes, em particular para alimentação animal. / The crescent demand of energy in times of decreasing in the supply of fossil fuel has been attracting the attention for the search for alternative sources, which will come to substitute the use of the petroleum, coal and natural gas. In this context, the vegetable oils appear as an alternative for substitution to the diesel oil. This possibility of using fuels from agricultural sources has shown a promising potential in the whole world, with a market that is growing rapidly because of its enormous contribution to the environment, with the qualitative and quantitative reduction of the levels of environmental pollution, mainly in the great urban centers, and also serving as strategic source of renewable energy in substitution to the diesel oil and others derived of the petroleum. The production of biodiesel from vegetable oils provides a biphasic system, being a non-polar phase of esters of fatty acids and other more dense consisting of glycerin and other waste components of the process. Considering that in the biodiesel production there is a generation of approximately 10% of glycerin, with the mixture consisting of 5% of biodiesel to diesel (B5) estimates indicate, in 2013, the generation of 150,000 tons per year. Since the traditional markets of glycerin can not absorb this additional supply of product on this scene, this work will contribute in technological innovations related to the use of glycerin, more specifically in the biotechnology conversion of glycerin generated in the synthesis of biodiesel in biomass of commercial interest, as source of nutrients. The effects of temperature and inoculum medium composition on the performance of yeast Yarrowia lipolytica NRRL YB – 423, growing on a glycerin-based medium, were studied in shaken flasks, verifying that the temperature of 25°C and the inoculum medium with the same composition of production medium were the most appropriate conditions for microorganism growth, reaching a biomass concentration of 17.7 g.L-1. In the bioreactor bench scale, a factorial design involving two variables (22 assays plus three central points) was performed, where the studied variables were agitation and aeration. The assays showed agitation of 200 rpm and aeration of 1 vvm as the best culture conditions, reaching a concentration of 19.14 g.L-1 of biomass with protein content of 13.55% and lipid content of 7.88 %. In these conditions, the cultivation in the bioreactor, in relation to the shaken flasks cultivation, leaded to significant increases in the maximum biomass concentration, maximum specific growth rate and productivity, respectively 1.22, 1.53 and 2.36 times. Based on the fatty acid and aminoacid profiles, the biomass showed to be a promising source of essential fatty acids, in particular linoleic acid (49.16%), and essential aminoacids such as isoleucine, valine, threonine and lysine, which presented chemical scores superior than FAO/WHO standard, respectively 1.42, 1.42, 1.30 and 1.17.Therefore biomass showed to be promise for use as a source of nutrients, particularly for animal feed.