Dissertations / Theses on the topic 'High sugar fermentation; Saccharomyces cerevisiae'

O aumento da participação dos biocombustíveis na matriz energética mundial pode ajudar a prolongar a existência das reservas de petróleo, mitigar as ameaças representadas pela mudança climática e permitir melhor segurança do fornecimento de energia em uma escala global. Neste cenário, o processo brasileiro da produção de etanol a partir da cana-de-açúcar tem ganhado papel de destaque, pelo alto rendimento e baixo custo da produção. Linhagens de S. cerevisiae são amplamente empregadas nas fermentações industriais e, embora sejam consideradas mais tolerantes em relação a outras, o processo brasileiro impõe uma variedade de fatores estressantes sob a mesma, afetando o seu metabolismo e crescimento. A fermentação com alto teor alcoólico, realizada a partir da utilização de mostos contendo altas concentrações de açúcares, é uma das maneiras mais eficientes de se obter elevados níveis de etanol. No entanto, tal tecnologia procede ocasionando efeitos deletérios adicionais à levedura. Neste contexto, aumentar a tolerância da levedura é de fundamental importância para alcançar um desempenho fermentativo satisfatório. Neste estudo foram avaliadas linhagens de S. cerevisiae, isogênicas a linhagem industrial CAT-1, com a sobre-expressão dos genes TRP1 e MSN2, envolvidos na biossíntese de triptofano e na resposta geral ao estresse, respectivamente. Tais linhagens foram avaliadas quanto ao seu potencial para realizar fermentações com alto teor alcoólico, simulando as condições industriais brasileiras. Os resultados revelaram que o gene MSN2, na versão truncada, favoreceu a linhagem principalmente com relação ao estresse osmótico, aumentando a velocidade de fermentação e o consumo de açúcares. O gene TRP1 promoveu maior crescimento da linhagem em meio YEPD com 8% de etanol, contudo, tornou a linhagem menos viável em concentrações acima deste nível. No presente trabalho também foi avaliado o efeito da suplementação de aminoácidos na fisiologia da linhagem CAT-1 em meio YNB e em mostos de melaço e xarope de cana-de-açúcar. A suplementação com histidida promoveu maior crescimento e viabilidade celular nos diferentes meios testados. Além de histidina, os aminoácidos lisina e alanina aumentaram o crescimento da CAT-1 em mosto de melaço. A suplementação de triptofano e asparagina também promoveu aumento da viabilidade celular em mosto de xarope. Por outro lado, nos testes em microplacas a suplementação com cisteína depreciou o crescimento da linhagem em meio YNB com 10 e 12% de etanol e em mosto de melaço com 20% de ART. Os resultados obtidos indicam que tanto a engenharia genética, quanto a suplementação de aminoácidos podem ser alternativas viáveis para aumentar a tolerância de S. cerevisiae, para suportar condições de múltiplo estresse, encontradas em destilarias brasileiras.
The expansion biofuels participation in the world energy matrix would help to extend the existence of fossil fuel reservoirs, mitigate the threats of climate change, and enable a better security of energy supply. The Brazilian process of ethanol production from sugarcane has gained an important role in the global energy scenario, for the high yield and low production cost. S. cerevisiae species is widely used in industrial fermentations for being resistant, but the Brazilian process imposes a variety of stressing factors to the yeast, affecting its metabolism and growth. The Very High Gravity Fermentation is performed by the utilization of musts with high sugar concentration and is one of the most efficient ways for obtaining high ethanol levels. However, this technology causes additional deleterious effects to the yeast. In this context, increasing yeast tolerance is of fundamental importance for a satisfactory fermentative performance. In this study we assessed S. cerevisiae strains - isogenic to the industrial strain CAT-1 - with over expression of TRP1 and MSN2 genes involved to tryptophan biosynthesis and in general stress response, respectively. These strains were evaluated for their potential to perform fermentations with high ethanol content, simulating the conditions of Brazilian distilleries. The results showed that the MSN2 gene in the truncated version improved strain mainly to respond to the osmotic stress, increasing in fermentation velocity and the consumption of sugars. The TRP1 gene overexpression promoted higher growth in YEPD medium with 8% ethanol, however, decreased viability at concentrations above this level. The present work also evaluated the effect of amino acid supplementation on the physiology of the CAT-1 strain in YNB medium and in molasses and syrup of sugarcane. Histidide supplementation increased the growth and cell viability in the different media tested. In addition to histidine, the amino acids lysine and alanine increased the growth of CAT-1 in molasses. Supplementation of tryptophan and asparagine also promoted increased cell viability in sugarcane syrup. On the other hand, in microplate assays, cysteine supplementation decreased growth in YNB medium with 10 and 12% ethanol, and in molasses with 20% ART. The results indicate that both genetic engineering and amino acid supplementation may be viable alternatives to increase tolerance of S. cerevisiae to supporting multiple stress conditions typical in Brazilian distilleries.

A linhagem Pedra 2 (PE-2) de Saccharomyces cerevisiae destaca-se por ser o organismo mais comumente utilizado no processo industrial de produção de biocombustíveis. Com a descoberta das linhagens selvagens, alcançou-se uma maior tolerância ao etanol permitindo o desenvolvimento de uma nova tecnologia: a fermentação com alto teor alcoólico. Desta forma, foi possível aumentar tanto a concentração de açúcares totais do mosto quanto o volume final de etanol purificado. No entanto, esse novo processo de fermentação tem causado um agravamento nos diversos estresses aplicados à levedura. No presente trabalho, é apresentado o perfil transcricional da linhagem PE-2 sob condições de alto etanol em fermentadores industriais através da tecnologia de microarranjo de DNA. Com a utilização desta, analisou-se o perfil global da expressão da levedura, identificando grupos de genes de interesse e vias metabólicas correguladas no processo de adaptação e sobrevivência às diferentes condições de estresses impostas a levedura pela fermentação industrial. Mais especificamente, 5860 genes foram estudados nesse trabalho e tiveram as suas variações de expressão quantificadas ao longo dos tempos 0, 6, 12 e 18 horas do ciclo fermentativo industrial. Em particular, algumas vias metabólicas associadas a compostos-chave no processo fermentativo tiveram seus genes diferenciamente expressos mapeados. Além disso, identificou-se vários grupos de genes altamente correlacionados a diferentes processos biológicos em S. cerevisiae, como por exemplo, a atividade de biossíntese de etanol. Por fim, espera-se que estes resultados forneçam bases para a realização de estudos mais direcionados no intuito de obter uma maior eficiência fermentativa e adaptação a estresses gerados durante durante o processo industrial.
The Pedra-2 (PE-2) strain of Saccharomyces cerevisiae is commonly used in the industrial process for biofuel production. In studies of wild type strains of S. cerevisiae, a wider tolerance to ethanol was achieved, which allowed for the development of a new technology of high alcohol percentage fermentation. This process made possible the increase of total sugar concentration in the mixture, and the volume of purified ethanol, although the new process has caused increase in the stresses applied to the yeast. In this study, the transcriptional profile of the PE-2 strain in high ethanol conditions is presented using DNA microarray. The global expression profile was used to identify groups of genes of interest and to analyze metabolic pathways that became co-regulated in adaptation to stress conditions imposed to the yeast by the industrial fermentation. In particular, 5860 genes were studied in this work and were detailed according to their expression profiles belong the fermentation cicle (0, 6, 12 and 18 hours). Moreover, metabolic pathways associated to key compounds in the fermentative process were described in terms of the composition of the differentially expressed genes. In addition, groups of genes highly correlated to different biological process in S. cerevisiae were identified. Finally, it is expected that this work could provide new directions in the study of fermentative efficiency and induced stress adaptation during the industrial fermentative process.

O etanol contribui significativamente para que a matriz energética do país se apresente extremamente favorável quanto à participação da energia renovável. A demanda por este biocombustível é crescente e tecnologias que permitam a sua produção de forma sustentável é de suma importância, como a fermentação com alto teor de etanol, já empregada em alguns países. Linhagens de leveduras com tolerância a múltiplos estresses muito contribuíram para a implantação de tal tecnologia no Brasil. Neste contexto se insere o presente trabalho, o qual busca linhagens de leveduras capazes de suportar os estresses impostos por uma fermentação com alto teor alcoólico. Para tal, 3 entre as melhores linhagens industriais de Saccharomyces cerevisiae atualmente disponíveis (CAT-1, PE-2 e SA- 1), foram utilizadas num programa para a seleção de híbridos para tolerância múltipla aos estresses etanólico, osmótico, ácido, além de outros, inerentes à fermentação com alto teor de etanol. Estas linhagens foram esporuladas e dissecadas para obtenção de células haplóides, as quais foram submetidas a cruzamentos entre- e intra-linhagens. Foram conduzidos cruzamentos massais (aleatórios) cujos produtos foram submetidos à evolução adaptativa em meios com os fatores estressantes em intensidades crescentes no transcorrer de 80 gerações. Ao final da evolução buscou-se variantes prevalentes na população de híbridos, os quais foram submetidos a novos procedimentos seletivos com imposições de várias condições estressantes. Igualmente foram conduzidos cruzamentos direcionados entre haplóides mediante micromanipulação, sendo tais híbridos submetidos ao mesmo procedimento seletivo nos meios estressantes. Assim, a partir de 230 haplóides das 3 linhagens, 174 isolados (120 oriundos dos cruzamentos massais e 54 dos cruzamentos direcionados) foram pré-selecionados pela maior tolerância aos meios seletivos, tendo os seus cariótipos estabelecidos mediante a cariotipagem eletroforética. Os isolados com maior tolerância (27) foram novamente avaliados em fermentações com reciclo de células e sob condições de elevado teor alcoólico (até 14,5% v/v) em mosto de melaço e água. Em todas as etapas da seleção os isolados foram comparados com as linhagens parentais (CAT-1, PE-2 e SA-1), sendo que ao final do processo seletivo destacou-se a linhagem 35B (híbrido entre CAT-1 e PE-2) com atributos fermentativos superiores aos exibidos pelos parentais. Tais atributos fermentativos contemplaram parâmetros bioquímicos, fisiológicos e tecnológicos (rendimento em etanol, viabilidade celular, crescimento em biomassa, formação de glicerol e teores celulares de carboidratos de reserva glicogênio e trealose). Os resultados permitem sugerir que devido às características fermentativas desejáveis do híbrido 35B, o mesmo possa ser empregado no processo industrial para ser avaliado como uma promissora linhagem a conduzir a fermentação com alto teor alcoólico.
Ethanol contributes significantly to the energy country matrix, which presents itself as extremely favorable to the share of renewable energy. The demand for this biofuel is increasing and technologies for its production in a sustainable way is of paramount importance such as fermentation with very high gravity, already used in some countries. Yeast strains tolerant to multiple stresses greatly contributed to the deployment of such technology in Brazil. In this context the present work is inserted, which seeks yeast strains capable of withstanding the stresses imposed by high ethanol content fermentation. In order that, three of the best industrial strains of Saccharomyces cerevisiae currently available (CAT-1, PE-2 and SA-1), were used in a program to select hybrids with tolerance towards multiples stresses: ethanolic, osmotic and acid, besides other factors involved in a high ethanol content fermentation. These strains were sporulated and dissected to obtain haploid cells, which were submitted to inter- and intra-strains crossings. Hybrids from polycrossings (random crossings) were subjected to an adaptative evolution in media with increasing stressing action over the course of 80 generations. At the end of evolution, prevalent variants were sought in the hybrids population, and submitted to new selective procedures with several stressing conditions. In the same way, directed crossings (between identified haplois) were performed by micromanipulation, and the resulting hybrids were subjected to the same selective procedure. Therefore, from 230 haploid from the 3 parent strains, 174 were isolated (120 from polycrossings and 54 from directed crossings) and pre-selected for higher tolerance in selective media; moreover their karyotypes were established by electrophoretic karyotyping. Strains showing greater tolerance (27) were again evaluated during cell recycling fermentations with high ethanol content (up to 14.5% v/v) using must formulated with water and molasses. At all stages, the isolates were compared with the parental strains (CAT-1, PE-2 and SA-1), and at the end of the selection process, the strain 35B (hybrid between CAT-1 and PE-2) standed out with fermentative attributes higher than the parentals. The fermentative performance was assessed by biochemical, physiological and technological parameters (ethanol efficiency, cell viability, biomass gain, glycerol formation and cellular levels of reserve carbohydrates - glycogen and trehalose). The results suggest that due to desirable fermentation traits, the hybrid 35B, could be used as starter in industrial fermentation process with high ethanol content.

This study aimed to evaluate the influence of concentrations of sugars in the must, on the performance of ethanol fermentation conducted in fed batch with variable flow of power to define the best concentrations of ART in the must (juice, molasses and mixed) that lead to improved efficiencies and productivity in ethanol fermentation.In the preparation of mash mix were used the following proportions (20% molasses + 80% broth, 40% molasses + 60% broth, 50% molasses + 50% broth, 60% molasses + 40% broth, 80% molasses + 20% broth ). The profile power was declining, varying the flow rate from 0.75 to 0.25 Lh-1, with time filling the fermenter 3 hours for all tests, ranging from 30 to 30 minutes to feed flow wort in fermenter 4L workload (3 liters of wine and 1 liter of inoculum), evaluating diferente concentrations of ART in three types of wine studied. We evaluated performance parameters such as fermentation and process efficiencies and productivity in ethanol. Musts were quantified in pH, sulfuric acid, Brix and ART.In the middle fermented (wine), pH, acidity, residual sugar and ethanol content and quantity of cells. The kinetic profile was defined by quantifying the concentrations of cells, substrate and ethanol (in 1 hour). The figures in this study as a starting point for industrial use are 16 to 18 Brix (ART 114.25 to 125.86 g / L), 16 to 18 ° Brix (ART 127.70 to 141.24 g / L) and around 16 ° Brix (ART 113.68 g / L to 123.30), respectively, for juice of molasses, juice and mix (juice + molasses).The fermentation efficiencies were 77.17 to 90.30% for grape juice, from 74.4 to 86.51% for wine and mixed wine from 61.84 to 84.06 for molasses. Yields were obtained from 6.85 to 8.21 g / Lh for wine broth, 5.90 to 7.77 g / Lh for wine mixed and 4.04 to 6.72 g / Lh for grape molasses. These tracks serve to subsidize recommended as a starting point, the conduct of industrial ethanol fermentation conducted in fed batch with variable flow supply, since the conditions for conducting the tests, as well as the raw materials used in the preparation of musts were similar to those used industrially.
Fundação de Amparo a Pesquisa do Estado de Alagoas
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Este estudo objetivou avaliar a influência das concentrações de açúcares nos mostos, sobre o desempenho da fermentação etanólica conduzida em batelada alimentada com vazão variável de alimentação, para a definição das melhores concentrações de ART nos mostos (de caldo, de melaço e misto) que conduzam a melhores eficiências de fermentação e produtividade em etanol. Na preparação do mosto misto foram utilizadas as seguintes proporções (20% melaço + 80% caldo, 40% melaço + 60% caldo, 50% melaço + 50% caldo, 60% melaço + 40% caldo, 80% melaço + 20% caldo). O perfil de alimentação foi decrescente, variando-se a vazão de 0,75 a 0,25 L.h-1, com tempo de enchimento do fermentador de 3 horas para todos os ensaios, variando-se de 30 em 30 minutos a vazão de alimentação de mosto, em fermentador de 4L de volume de trabalho (3 litros de mosto e 1 litro de inoculo), avaliando-se diferentes concentrações de ART nos 3 tipos de mosto estudados. Foram avaliados parâmetros de desempenho, como eficiências fermentativa e de processo e produtividade em etanol. Nos mostos foram quantificados pH, acidez sulfúrica, Brix e ART. No meio fermentado (vinho), pH, acidez, Açúcares Residuais e teor de etanol e quantidade de células. O perfil cinético foi definido, quantificando-se as concentrações de células, substrato e etanol (em intervalos de 1 hora). Os valores indicados neste estudo, como ponto de partida para utilização industrial, são Brix de 16 a 18 (ART 114,25 a 125,86 g/L), de 14 a 18 °Brix (ART de 112,90 a 141,24 g/L) e próximo de 16 °Brix (ART de 113,68 g/L a 123,30), respectivamente para mostos de melaço, caldo e misto (caldo + melaço). As eficiências de fermentação foram: 77,17 a 90,30%, para mosto de caldo, 74,4 a 86,51% para mosto misto e 61,84 a 84,06 para mosto de melaço. As produtividades obtidas foram 6,85 a 8,21g/L.h, para mosto de caldo, 5,90 a 7,77g/L.h para mosto misto e 4,04 a 6,72g/L.h para mosto d melaço. Estas faixas recomendadas servem para subsidiar, como ponto de partida, a condução da fermentação etanólica industrial conduzida em batelada alimentada com vazão variável de alimentação, visto que as condições de condução dos ensaios, assim como as matérias-primas utilizadas na preparação dos mostos, foram semelhantes às utilizadas industrialmente.

Yeast (Saccharomyces cerevisiae) plays a key role in the completion of several fermentations including those used for beverage and bioethanol production. In the wine industry, slow or incomplete alcoholic fermentation is still a challenging problem and often results in increased costs of production and decreased wine quality. One of the reasons for the persistence of this problem could be the trend towards rising sugar concentrations in grape musts. What is already a high sugar concentration fermentation (~200 g L⁻¹ or more) has increased by some 20 – 40 g L⁻¹ due to climate warming and winemaker pursuit of ripeness. In this project we aim to gain a better understanding of how wine yeast cope in high sugar fermentations (HSF) to help develop strategies for managing these types of grape musts. With the availability of collections of laboratory yeast including gene deletion and overexpression libraries and the development of techniques used for whole genome analysis, it is now possible to investigate yeast biology under oenological conditions with a systems biology approach. A number of genome-wide studies of yeast have previously been conducted to identify yeast genes involved in sensitivity to individual stresses present during fermentation. However, in reality many of these stresses are often present at the same time, or sequentially throughout the phases of fermentation. This highlights an important gap in current research, that being identification of those genes important for maintenance of fermentation efficiency throughout a complete cycle of fermentation, and in particular an environment which has high initial sugar content such as that found in grapes used to make quality wines. We expected these genes to be related to wine yeast adaption, survival and maintenance of fermentative metabolism. In this study 93 genes were identified as important for the successful completion of high sugar fermentation as deletants of these resulted in either protracted or incomplete fermentation. We have named this gene set the Fermentation Essential Genes (FEGs). A gene ontology (GO) analysis of these revealed that vacuolar acidification (VA) is an important biological process required for efficient completion of a high sugar fermentation: 20 of the 93 FEGs annotate to this GO term (vacuolar acidification). Also, this gene set is highly represented in the FEGs since these 20 FEGs represent 77% of all genes annotated to this same GO term. In this study we also report 18 genes (also all FEGs), not previously associated with VA, of which deletants have VA defects. This was achieved through examination of the VA of 93 FEGs using the vacuolar specific probe 6-carboxyfluorescein diacetate (6-CFDA), microscopic and Fluorescence Activated Cell Sorting (FACS) analysis. It was shown that, nine FEGs were seen to be particular critically to fermentation progression and completion. Their deletion result in the extreme phenotype of arrested or ‘stuck’ fermentation. Amongst these, featured two genes involved in trehalose biosynthesis. The disaccharide trehalose is an enigmatic compound accumulated in Saccharomyces and known to be associated with survival under environmental stress conditions. Deletion of either TPS1 or TPS2, encoding enzymes involved in trehalose biosynthesis, resulted in incomplete fermentation. This phenotype could be reversed by the over-expression of HXK2 (a paralog of HXK1 encoding hexokinase isomer 2) in Δtps1 and introduction of the phosphotrehalase gene (TreA), from Bacillus subtilis, in Δtps2. HXK2 over-expression increased the fermentation rate of Δtps1 and the parent BY4743 which actually demonstrated a shorter fermentation duration than the parent having blank plasmid. To further investigate fermentation of yeast in HSF we sought to examine the fermentation performance of a gene overexpression library, which was constructed in this study by transformation of a Yeast Genomic Tiling Collection into a haploid wine yeast strain; ISOC9dΔleu2. The construction of this library was performed in collaboration with two other PhD students (Mrs Jade Haggerty and Ms Jin Zhang). The clonal identity, degree of plasmid retention and development of methodologies to allow fermentation in high sugar chemically defined grape juice medium (CDGJM) were achieved. However, due to time constraints further evaluation of this library was not possible within the current project. The collective findings from this project have provided greater insight into the mechanism by which yeast cope with HSF as well as providing direction if not specific gene targets for exploitation in strain improvement programs.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2014

Based on ethanol fermentation kinetics and bioreactor engineering theory, a system composed of a continuously stirred tank reactor (CSTR) and three tubular bioreactors in series was established for continuous very high gravity (VHG) ethanol fermentation with Saccharomyces cerevisiae. Sustainable oscillations of residual glucose, ethanol, and biomass characterized by long oscillation periods and large oscillation amplitudes were observed when a VHG medium containing 280 g/L glucose was fed into the CSTR at a dilution rate of 0.027 h1. Mechanistic analysis indicated that the oscillations are due to ethanol inhibition and the lag response of yeast cells to ethanol inhibition. A high gravity (HG) medium containing 200 g/L glucose and a low gravity (LG) medium containing 120 g/L glucose were fed into the CSTR at the same dilution rate as that for the VHG medium, so that the impact of residual glucose and ethanol concentrations on the oscillations could be studied. The oscillations were not significantly affected when the HG medium was used, and residual glucose decreased significantly, but ethanol maintained at the same level, indicating that residual glucose was not the main factor triggering the oscillations. However, the oscillations disappeared after the LG medium was fed and ethanol concentration decreased to 58.2 g/L. Furthermore, when the LG medium was supplemented with 30 g/L ethanol to achieve the same level of ethanol in the fermentation system as that achieved under the HG condition, the steady state observed for the original LG medium was interrupted, and the oscillations observed under the HG condition occurred. The steady state was gradually restored after the original LG medium replaced the modified one. These experimental results confirmed that ethanol, whether produced by yeast cells during fermentation or externally added into a fermentation system, can trigger oscillations once its concentration approaches to a criterion. The impact of dilution rate on oscillations was also studied. It was found that oscillations occurred at certain dilution rate ranges for the two yeast strains. Since ethanol production is tightly coupled with yeast cell growth, it was speculated that the impact of the dilution rate on the oscillations is due to the synchronization of the mother and daughter cell growth rhythms. The difference in the oscillation profiles exhibited by the two yeast strains is due to their difference in ethanol tolerance. For more practical conditions, the behavior of continuous ethanol fermentation was studied using a self-flocculating industrial yeast strain and corn flour hydrolysate medium in a simulated tanks-in-series fermentation system. Amplified oscillations observed at the dilution rate of 0.12 h1 were postulated to be due to the synchronization of the two yeast cell populations generated by the continuous inoculation from the seed tank upstream of the fermentation system, which was partly validated by oscillation attenuation after the seed tank was removed from the fermentation system. The two populations consisted of the newly inoculated yeast cells and the yeast cells already adapted to the fermentation environment. Oscillations increased residual sugar at the end of the fermentation, and correspondingly, decreased the ethanol yield, indicating the need for attenuation strategies. When the tubular bioreactors were packed with ½” Intalox ceramic saddles, not only was their ethanol fermentation performance improved, but effective oscillation attenuation was also achieved. The oscillation attenuation was postulated to be due to the alleviation of backmixing in the packed tubular bioreactors as well as the yeast cell immobilization role of the packing. The residence time distribution analysis indicated that the mixing performance of the packed tubular bioreactors was close to a CSTR model for both residual glucose and ethanol, and the assumed backmixing alleviation could not be achieved. The impact of yeast cell immobilization was further studied using several different packing materials. Improvement in ethanol fermentation performance as well as oscillation attenuation was achieved for the wood chips, as well as the Intalox ceramic saddles, but not for the porous polyurethane particles, nor the steel Raschig rings. Analysis for the immobilized yeast cells indicated that high viability was the mechanistic reason for the improvement of the ethanol fermentation performance as well as the attenuation of the oscillations. A dynamic model was developed by incorporating the lag response of yeast cells to ethanol inhibition into the pseudo-steady state kinetic model, and dynamic simulation was performed, with good results. This not only provides a basis for developing process intervention strategies to minimize oscillations, but also theoretically support the mechanistic hypothesis for the oscillations.

The stoichiometric relationship between carbon dioxide (CO2) generated and glucose consumed during fermentation can be utilized to predict glucose consumption as well as yeast growth by measuring the CO2 concentration. Dissolved CO2 was chosen as opposed to off-gas CO2 due to the high solubility of CO2 in the fermentation broth as well as its ability to reflect on yeast growth more accurately than off-gas CO2. Typical very-high-gravity (VHG) ethanol fermentation is plagued by incomplete glucose utilization and longer durations. Aiming to improve substrate utilization and enhance VHG fermentation performance, characteristics of dissolved CO2 concentration in fermentation broths using Saccharomyces cerevisiae were studied under batch conditions. Based on this study a novel control methodology based on dissolved CO2 was developed and its effectiveness on enhancing VHG fermentation was evaluated by measuring the fermentation duration, glucose conversion efficiency and ethanol productivity. Four different initial concentrations 150, 200.05±0.21, 250.32±0.12, and 300.24±0.28 g glucose/L were used for batch ethanol fermentation without control. Zero substrate was indicated for 150 and 200.05±0.21 g glucose/L by a characteristic abrupt drop in dissolved CO2 concentration. On the other hand sluggish fermentation and incomplete substrate utilization were witnessed for 250.32±0.12, and 300.24±0.28 g glucose/L. A material balance equation was developed to compensate for the inability of the dissolved CO2 profiles to accurately predict the different growth phases of yeast. Dissolved CO2 was controlled at three distinct levels of 500, 750 and 1000 mg/L using aeration rates of 820 and 1300 mL/min for initial concentrations of 259.72±7.96 and 303.92±10.66 g glucose/L. Enhancement of VHG fermentation was achieved in the form of complete glucose utilization and higher ethanol productivities and shorter fermentation duration in comparison to batches without control. Complete glucose utilization was facilitated under ~250 and ~300 g glucose/L in 27.02±0.91 and 36.8±3.56 h respectively. Irrespective of the control set points and aeration rates, ethanol productivities of 3.98±0.28 g/L-h and 3.44±0.32 g/L-h were obtained for 259.72±7.96 and 303.92±10.66 g glucose/L respectively. The glucose conversion efficiencies for both 259.85±9.02 and 299.36±6.66 g glucose/L when dissolved CO2 was controlled were on par with that of batches without control.

碩士
大同大學
生物工程學系(所)
97
Controlling the level of dissolved oxygen in fermenting medium is essential in ethanol production. Since the traditional DO sensor failed to monitor anaerobic fermentation by detecting the significant value at low amount of dissolved oxygen, ORP sensor was utilized as a new delicate method to monitor the slight change of oxygen level, furthermore, to control the level of dissolved oxygen during yeast fermentation. Comparing to DO sensor, ORP sensor offered more flexibility and the management to control different ORP levels which played a significant role in ethanol industry by increasing activity of yeast, enhancing ethanol yield coefficient and average ethanol productivity, and reducing residual glucose concentration. Nine groups of ethanol fermentation studies were conducted using glucose baths of the concentrations of 300, 250, and 200 g/L, parameters of ORP -150, -100 mV by injecting slight different amount of air and uncontrolled ORP as negative control group. The results showed the best ethanol yield coefficient was achieved at -150 mV of ORP, and average ethanol productivity was increased at -100 mV of ORP. Taken both factors into consideration, the group using 250 g/L glucose with -100 mV of ORP had better performance, in which the ethanol yield coefficient was increased by 2% to 0.477, comparing to that of control group 0.467. Average ethanol productivity was increased by 25% to 4.073 g/L/hr, comparing to that of the control group 3.262 g/L/hr. During very-high-gravity ethanol fermentation using 300 g/L glucose, considering the influence of aeration in different phases of yeast growth and the different injection levels of air on the productivity of ethanol, it was found in log phase of cell growth (after 8-18 hr fermentation), with 820 ml/min air injection, ethanol yield coefficient maintained the same as that of control group (0.439) and average ethanol productivity was increased by 142 % to 3.737 g/L/hr, comparing to that of the control group 2.624 g/L/hr.

碩士
國立臺灣科技大學
化學工程系
92
High cell density fermentation of recombinant yeasts Saccharomyces cerevisiae and Pichia pastoris were studied. Both recombinant yeasts strains carry glucose oxidase(GOD) gene of Aspergius niger and constitutively express this extracellular enzyme. Among the carbon sources screened, fructose is the one very suitable for cultivating both GOD expressing recombinant yeasts. Fed-batch cultivation was employed to achieve high cell density fermentation. When 20﹪(w/v) fructose was used in the feeding nutrient and exponential feeding strategy was employed for S. cerevisiae high cell density cultivation, 200 OD600 was obtained after 66 hours cultivation and the final GOD activity was 107 U/ml. Under the same cultivation condition, cell concentration of P. pastoris could reach 220 OD600 and the GOD activity could only reach 40 U/ml. In order to prevent the extracellular GOD from being degraded by protease, 1﹪ casmino acid was employed in the medium. The cell density of P. pastoris could be increased further to 365 OD600 , when 40﹪ fructose was used in the feeding medium. The GOD activity of 85 U/ml was obtained. GOD from S. cerevisiae and P. pastoris had very different glycosylation extent. Glycosylation degree of GOD(S. cerevisiae) and GOD(P. pastoris) were 104﹪ and 25﹪, respectively. The high glycosylation extent of GOD(S. cerevisiae) make it has broader pH and temperature stability range than that of GOD(P. pastoris). The molecular weight of GOD(S. cerevisiae) and GOD(P. pastoris) were determined to be about 140 KDa and 90 KDa , respectively.

碩士
國立中正大學
化學工程研究所
105
In industry, simultaneous saccharification and fermentation (SSF) is considered to be an important process for the production of alcohol. Because it can efficiently reduce the cost of the production of cellulosic bioethanol. Unfortunately, the temperature for cellulose saccharification is higher than that for yeast fermentation. Therefore, it is desirable to develop a thermotolerance yeast strain to be adopted in the SSF process. When exposed to a variety of environmental stresses such as high temperature, alcoholic or oxidative stress, yeasts rapidly produces trehalose and heat shock proteins (HSPs). In the case of high temperature stress, the hydrogen bonding and Van der Waals force inside proteins can be destroyed then leading their denaturation. The increased misfolding proteins then induced expression of HSPs to help protein refolding and the survival of the cells. Among HSPs, HSP104 is essential for cell survival under high temperature. However, the induction of HSP104 is only transient by which the cells would not be able to survive after a long period of high-temperature fermentation. To prolong the expression of HSP104 at high temperature, plasmid pTEF1-2μ ori HSP26P ISA1 40homo was constructed and the NDA fragment 40homo-FRT-HSP26p-40homo amplified using polymerase chain reaction transformed to Saccharomyces cerevisiae for substituting the HSP104 promoter for the HSP26 promoter using homologous recombination.

Tese de Doutoramento em Engenharia Química e Biológica
This thesis is focused on physiological aspects of the yeasts used in two alcoholic fermentation processes: primary brewing fermentation and fermentation of lactose (particularly lactose derived from cheese whey) to ethanol by recombinant Saccharomyces cerevisiae flocculent strains. The brewing fermentation is probably the most extensively studied alcoholic fermentation process. Nevertheless, developments in brewing technology demand deeper understanding of yeast physiology under process conditions. The studies with brewer’s yeast reported in this thesis addressed two specific questions that had not yet been effectively investigated. First, it is here directly demonstrated for the first time that the brewer’s yeast lipid composition, particularly the amounts of sterols and unsaturated fatty acids, affects the activity of maltose transporters. The maltose uptake rates were correlated with the amount of ergosterol in yeast, showing that proper function of the maltose permeases requires adequate amounts of ergosterol in the plasma membrane. This effect may partly explain the low maltose (and maltotriose) transport rates at the beginning and during the second half of brewery fermentations, when the sterol content of the yeast is low. Second, the energetic state of the yeast was studied under the specific environments of high- and very high-gravity brewing fermentations. The adenylate energy charge (EC) of the yeast was high (>0.8) throughout fermentation until residual sugar concentrations became low and specific rates of ethanol production became less than 5% of the maximum values observed in early fermentation. At that point, the EC fell to around 0.5 – 0.6. The results suggest that the ethanol tolerance of brewer’s yeast is high so long as fermentation continues. However, when residual α-glucoside concentrations no longer support adequate rates of fermentation both the EC and the yeast viability collapse. The development of microorganisms that efficiently ferment lactose has a high biotechnological value for the design of processes for the bioremediation of cheese whey with simultaneous production of bio-ethanol. A new lactose-fermenting flocculent S. cerevisiae recombinant strain is described here. This strain (T1-E) was obtained by evolutionary engineering of an original recombinant (T1, constructed in previous work) that had shown rather poor lactose fermentation and flocculation performances. The new strain T1-E consumed lactose 2-fold faster producing 30% more ethanol than T1. Its flocculation performance was also significantly better than that of T1. A series of physiological and genetic studies were done to compare T1 and T1-E. The contribution of the identified molecular differences to the improved lactose fermentation phenotype of strain T1-E is discussed. In batch fermentations with mineral medium, the new strain T1-E consumed rapidly and completely lactose at initial concentrations up to 150 g•L-1. The maximum ethanol titre reached was 8% (v/v) and the highest ethanol productivity was 1.5 – 2 g•L-1•h-1. T1-E was also able to ferment 3-fold concentrated cheese whey (about 150 g•L-1 of lactose), producing 7% (v/v) of ethanol. These results demonstrate that T1-E is the most efficient lactosefermenting S. cerevisiae recombinant strain reported in the literature. Being highly flocculent, this strain is suitable for developing high cell density fermentation systems that, when operated in continuous with flocculated biomass retention, can reach very high productivities. Sugar transport is a key factor determining fermentation efficiency in both processes studied in this thesis. Studies of lactose transport by the recombinant S. cerevisiae strains (T1 and T1-E) and by Kluyveromyces lactis revealed that zero-trans uptake rates of lactose measured by standard methodology (i.e. using suspensions of yeast harvested from fermentation, washed and stored in nutrient-free buffer at low temperature before they are assayed using radiolabeled sugar) were too small (by factors of 3 to 8) to account for the lactose consumption rates observed during fermentations. A short incubation (1 – 7 min) with glucose (10 – 30 mM) increased the low intracellular ATP and EC of cells in the starved yeast suspensions to the levels found in actively fermenting yeast cells, and simultaneously increased the activity (Vmax) of the lactose transporters by factors of 1.5 to 5. Similar observations were made for maltose transport in brewer’s yeasts. These results suggest that the electrochemical proton potential that drives transport through sugar/H+ symports is significantly smaller in the starved yeast suspensions used for the zero-trans assays than in actively metabolising cells. Short exposure of the starved cells to glucose is suggested as a quick method to approach more closely the sugar/H+ symport capacity of the actively fermenting cells.
Esta tese foca-se em aspectos relacionados com a fisiologia de leveduras utilizadas em dois processos de fermentação alcoólica: a fermentação primária da cerveja e a fermentação de lactose (em particular a lactose derivada do soro do queijo) para produção de etanol por estirpes floculantes de Saccharomyces cerevisiae geneticamente modificadas. A fermentação da cerveja é provavelmente o processo de fermentação alcoólica mais extensivamente estudado. Todavia, o desenvolvimento da tecnologia para o fabrico de cerveja requer uma compreensão mais aprofundada da fisiologia da levedura nas condições específicas deste processo. Os estudos com levedura de cerveja descritos nesta tese abordaram duas questões específicas que não haviam ainda sido efectivamente investigadas. Em primeiro lugar, é aqui demonstrado pela primeira vez que a composição lipídica da levedura de cerveja, em particular o seu conteúdo em esteróis e ácidos gordos insaturados, afecta a actividade dos transportadores de maltose. Verificou-se uma relação entre as taxas de transporte de maltose e a quantidade de ergosterol na levedura, demonstrando que o funcionamento adequado das permeases da maltose requer quantidades adequadas de ergosterol na membrana plasmática. Este efeito poderá explicar parcialmente as baixas taxas de transporte de maltose (e maltotriose) no início e durante a segunda metade das fermentações de cerveja, fases em que o conteúdo de esteróis da levedura é baixo. Em segundo lugar, o estado energético da levedura foi estudado nas condições específicas de fermentações de cerveja com mostos de alta e muito alta gravidade. A carga energética (EC) da levedura foi elevada (>0.8) durante a fermentação até a concentração residual de açúcar se tornar baixa e as taxas específicas de produção de etanol baixarem para menos de 5% dos valores máximos observados na fase inicial da fermentação. Nesse ponto, a EC decaiu para cerca de 0.5 – 0.6. Os resultados sugerem que a tolerência da levedura de cerveja ao etanol é elevada enquanto a fermentação prosseguir. No entanto, quando as concentrações residuais de α-glucosídeos não mais sustentam taxas de fermentação adequadas tanto a EC como a viabilidade da levedura colapsam. O desenvolvimento de microrganismos que fermentem de forma eficiente a lactose tem um elevado valor em termos biotecnológicos para o desenho de processos para a biorremediação do soro do queijo com produção de bio-etanol em simultâneo. Uma nova estirpe recombinante de S. cerevisiae capaz de fermentar a lactose e floculante é descrita nesta tese. Esta estirpe (T1-E) foi obtida por evolução dirigida a partir de uma estirpe recombinante original (T1, construída em trabalho prévio) que havia demonstrado um fraco desempenho em termos de fermentação de lactose e de floculação. A nova estirpe, T1-E, foi capaz de consumir a lactose duas vezes mais rapidamente e de produzir 30% mais etanol do que a estirpe T1. Foram efectuados vários estudos fisiológicos e genéticos para comparar as estirpes T1 e T1-E. A contribuição das diferenças identificadas ao nível molecular para o fenótipo melhorado da estirpe T1-E em termos de fermentação de lactose é aqui discutida. Em fermentações descontínuas com meio mineral, a nova estirpe T1-E consumiu rapidamente e por completo lactose em concentrações iniciais até 150 g•L-1. A concentração máxima de etanol atingida foi 8% (v/v) e a produtividade máxima foi 1.5 – 2 g•L-1•h-1. A estirpe T1-E foi também capaz de fermentar soro do queijo três vezes concentrado (cerca de 150 g•L-1 de lactose), produzindo 7% (v/v) de etanol. Estes resultados demonstram que a estirpe T1-E é a estirpe recombinante de S. cerevisiae mais eficiente para fermentação de lactose descrita na literatura. Sendo floculante, esta estirpe é adequada para desenvolver sistemas de fermentação de alta densidade celular que, quando operados em continuo com retenção da biomassa floculada, permitem atingir produtividades muito elevadas. O transporte de açúcares é um factor chave na determinação da eficiência de fermentação em ambos os processos estudados nesta tese. Estudos do transporte de lactose nas estirpes recombinantes de S. cerevisiae (T1 e T1-E) e em Kluyveromyces lactis revelaram que as taxas iniciais de transporte de lactose medidas pela metodologia padrão (i.e. usando suspensões de leveduras colhidas da fermentação, lavadas e guardadas em tampão sem nutrientes a baixa temperatura antes de serem utilizadas para ensaios com açúcar marcado radioactivamente) eram demasiado baixas (por factores de 3 a 8) para explicar as taxas de consumo de lactose observadas durante as fermentações. Uma breve incubação (1 – 7 min) com glucose (10 – 30 mM) aumentou a baixa EC e os baixos níveis de ATP intracelulares encontrados nas suspensões de leveduras guardadas sem nutrientes para os níveis encontrados em células de leveduras a fermentar activamente. Simultaneamente, essa incubação aumentou a actividade (Vmax) dos transportadores de lactose por factores de 1.5 a 5. Foram feitas observações semelhantes para o transporte de maltose em leveduras de cerveja. Estes resultados sugerem que o potencial electroquímico de protões responsável pelo transporte por mecanismos de simporte açúcar-protão é significativamente mais reduzido nas células guardadas em tampão sem nutrientes, utilizadas para os ensaios de transporte, do que nas células a fermentar activamente. Um breve tratamento das células guardadas sem nutrientes com glucose é sugerido como um método rápido para abordar de forma mais aproximada a capacidade dos sistemas de simporte açúcar-protão das leveduras a fermentar activamente.
Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/13463/2003.