Добірка наукової літератури з теми "High sugar fermentation; Saccharomyces cerevisiae"

In the tequila industry, fermentation is traditionally achieved at sugar concentrations ranging from 50 to 100 g·L–1. In this work, the behaviour of the Saccharomyces cerevisiae yeast (isolated from the juices of the Agave tequilana Weber blue variety) during the agave juice fermentation is compared at different sugar concentrations to determine if it is feasible for the industry to run fermentation at higher sugar concentrations. Fermentation efficiency is shown to be higher (above 90%) at a high concentration of initial sugar (170 g·L–1) when an additional source of nitrogen (a mixture of amino acids and ammonium sulphate, different than a grape must nitrogen composition) is added during the exponential growth phase.Key words: Saccharomyces cerevisiae, fermentation efficiency, nitrogen source, tequila.

ABSTRACTDuring alcoholic fermentation,Saccharomyces cerevisiaeis exposed to a host of environmental and physiological stresses. Extremes of fermentation temperature have previously been demonstrated to induce fermentation arrest under growth conditions that would otherwise result in complete sugar utilization at “normal” temperatures and nutrient levels. Fermentations were carried out at 15°C, 25°C, and 35°C in a defined high-sugar medium using threeSaccharomyces cerevisiaestrains with diverse fermentation characteristics. The lipid composition of these strains was analyzed at two fermentation stages, when ethanol levels were low early in stationary phase and in late stationary phase at high ethanol concentrations. Several lipids exhibited dramatic differences in membrane concentration in a temperature-dependent manner. Principal component analysis (PCA) was used as a tool to elucidate correlations between specific lipid species and fermentation temperature for each yeast strain. Fermentations carried out at 35°C exhibited very high concentrations of several phosphatidylinositol species, whereas at 15°C these yeast strains exhibited higher levels of phosphatidylethanolamine and phosphatidylcholine species with medium-chain fatty acids. Furthermore, membrane concentrations of ergosterol were highest in the yeast strain that experienced stuck fermentations at all three temperatures. Fluorescence anisotropy measurements of yeast cell membrane fluidity during fermentation were carried out using the lipophilic fluorophore diphenylhexatriene. These measurements demonstrate that the changes in the lipid composition of these yeast strains across the range of fermentation temperatures used in this study did not significantly affect cell membrane fluidity. However, the results from this study indicate that fermentingS. cerevisiaemodulates its membrane lipid composition in a temperature-dependent manner.

<p style="text-align: justify;">An approach consisting of controlling yeast inoculum to minimize volatile acidity production by Saccharomyces cerevisiae during the alcoholic fermentation of botrytized must was investigated. Direct inoculation of rehydrated active dry yeasts produced the most volatile acidity, while a yeast preparation pre-cultured for 24 hours reduced the final production by up to 23 %. Using yeasts collected from a fermenting wine as a starter must also reduced volatile acidity production. The conditions for preparing the inoculum affected the fermentation capacity of the first generation yeasts: fermentation duration, sugar to ethanol ratio, and wine composition. A pre-culture medium with a low sugar concentration (&lt; 220 g/L) is essential to limit volatile acidity production in high sugar fermentations.</p>

In order to screen out Saccharomyces cerevisiae suitable for table grape fermentation, and compare it with commercial Saccharomyces cerevisiae in terms of fermentation performance and aroma producing substances, differences of fermentation flavor caused by different strains were discussed. In this experiment, yeast was isolated and purified from vineyard soil, 26s rDNA identification and fermentation substrate tolerance analysis were carried out, and the causes of flavor differences of wine were analyzed from three aspects: GC-MS, PCA and sensory evaluation. The results showed that strain S1 had the highest floral aroma fraction, corresponding to its high production of ethyl octanoate and other substances, and it had the characteristics of high sugar tolerance. The fruit sensory score of S3 wine was the highest among the six wines. Through exploration and analysis, it was found that compared with commercial Saccharomyces cerevisiae, the screened strains had more advantages in fermenting table grapes. The flavor of each wine was directly related to the growth characteristics and tolerance of its strains.

Saccharomy cescerevisiae D8 isolated from Queen pineapple extract and selected for high ethanol fermentation activity (12.37% v/v ethanol concentration in fermentation media with a total sugar content of 200 g/L) has been reported previously (Hoang Thi Le Thuong et al., 2017). In this paper, the strain was subjected to random mutagenesis by N-methyl-N-nitro-N-nitrosoguanidine (NTG) and ultraviolet (UV) in order to enhance its ethanol fermentation. The results showed that 1% NTG was more lethal than UV (260 nm, 50 V) to S. cerevisiae D8 at the same time of treatment. The combination of NTG and UV was found to increase the mortality of S. cerevisiae D8. Surviving cells after treatment with NTG and UV combination were identified for ethanol fermentation. Thirteen clones were cabable of fermenting higher ethanol concentration than S. cerevisiae D8 was. Especially, mutant clone NU 120.4 was able to ferment glucose up to the highest ethanol concentration (22% higher than that of S. cerevisiae D8). This mutant clone also showed more tolerant to high ethanol and sugar concentrations in the fermentation medium than that of S. cerevisiae D8. The ethanol fermentation of this mutant was relatively stable in Queen pineapple extract (ethanol concentration of about 15.07 ± 0.12%) and the yield of ethanol fermentation was 92.62 ± 0.2%, while S. cerevisiae D8 gained maximum alcohol concentration of 12.37 ± 0.2%. In the context of the availability of pineapple used as primary source of food processing in Vietnam nowadays, these results showed a potential application of this mutant clone NU 120.4 in brandy production from pineapple extract.

In the current scenario of climatic warming, the over-ripening of grapes increases the sugar content, producing flat and alcoholic wines with low acidity, high pH and low freshness. Additionally, a high pH makes wines more chemically and microbiologically unstable, requiring a higher sulphite content for preservation. Some strains of Lachancea thermotolerans can naturally lower the pH of wine by producing lactic acid from sugars; this pH reduction can reach 0.5 units. The industrial performance of four selected strains has been compared with that of two commercial strains and with that of Saccharomyces cerevisiae. The yeasts were assessed under variable oenological conditions, measuring lactic acid production and fermentative performance at two fermentation temperatures (17 and 27 °C), and in the presence or absence of sulphites (25 and 75 mg/L). Lactic acid production depends on yeast populations, with higher concentrations being reached when the microbial population is close to or above 7-log CFU/mL. A temperature effect on acidification can also be observed, being more intense at higher fermentation temperatures for most strains. Ethanol yield ranged from 7–11% vol., depending on the fermentation conditions (temperature and SO2) at day 12 of fermentation, compared with 12% for the S. cerevisiae control in micro-fermentations. The production of fermentative esters was higher at 27 °C compared with 17 °C, which favoured the production of higher alcohols. Volatile acidity was moderate under all fermentation conditions with values below 0.4 g/L.

Aroma profile is one of the main features for the acceptance of wine. Yeasts and bacteria are the responsible organisms to carry out both, alcoholic and malolactic fermentation. Alcoholic fermentation is in turn, responsible for transforming grape juice into wine and providing secondary aromas. Secondary aroma can be influenced by different factors; however, the influence of the microorganisms is one of the main agents affecting final wine aroma profile. Saccharomyces cerevisiae has historically been the most used yeast for winemaking process for its specific characteristics: high fermentative metabolism and kinetics, low acetic acid production, resistance to high levels of sugar, ethanol, sulfur dioxide and also, the production of pleasant aromatic compounds. Nevertheless, in the last years, the use of non-saccharomyces yeasts has been progressively growing according to their capacity to enhance aroma complexity and interact with S. cerevisiae, especially in mixed cultures. Hence, this review article is aimed at associating the main secondary aroma compounds present in wine with the microorganisms involved in the spontaneous and guided fermentations, as well as an approach to the strain variability of species, the genetic modifications that can occur and their relevance to wine aroma construction.

Natural adhesion of Saccharomyces cerevisiae onto sugar beet pulp (SBP) is a very simple and cheap immobilization method for retaining high cells density in the ethanol fermentation system. In the present study, yeast cells were immobilized by adhesion onto SBP suspended in the synthetic culture media under different conditions such as: glucose concentration (100, 120 and 150 g/l), inoculum concentration (5, 10 and 15 g/l dry mass) and temperature (25, 30, 35 and 40?C). In order to estimate the optimal immobilization conditions the yeast cells retention (R), after each immobilization experiment was analyzed. The highest R value of 0.486 g dry mass yeast /g dry mass SBP was obtained at 30?C, glucose concentration of 150 g/l, and inoculum concentration of 15 g/l. The yeast immobilized under these conditions was used for ethanol fermentation of sugar beet molasses containing 150.2 g/l of reducing sugar. Efficient ethanol fermentation (ethanol concentration of 70.57 g/l, fermentation efficiency 93.98%) of sugar beet molasses was achieved using S. cerevisiae immobilized by natural adhesion on SBP.

Starmerella bacillaris (synonym Candida zemplinina) is an important non-Saccharomyces yeast in winemaking with valuable oenological properties, accompanying Saccharomyces species in sweet wine fermentation, and has also been suggested for application as combined starter culture in dry or sweet wines. In this study, the major metabolites and nitrogen utilization of these yeasts are evaluated in the musts with high or extremely high sugar concentration. The change in the metabolic footprint of Saccharomyces cerevisiae, Saccharomyces uvarum and Starmerella bacillaris strains was compared when they were present as pure cultures in chemically defined grape juice medium with 220 and 320 g/L of sugar, to represent a fully matured and an overripe grape. Surprisingly, the extreme sugar concentration did not result in a considerable change in the rate of sugar consumption; only a shift of the sugar consumption curves could be noticed for all species, especially for Starmerella bacillaris. At the extreme sugar concentration, Starmerella bacillaris showed excellent glycerol production, moderate nitrogen demand together with a noticeable proline utilisation. The change in the overall metabolite pattern of Starmerella bacillaris allowed clear discrimination from the change of the Saccharomyces species. In this experiment, the adequacy of this non-Saccharomyces yeast for co-fermentation in juices with high sugar concentration is highlighted. Moreover, the results suggest that Starmerella bacillaris has a more active adaptation mechanism to extremely high sugar concentration.

ABSTRACTIn this study we investigated the possibility of usingCandida zemplinina, as a partner ofSaccharomyces cerevisiae, in mixed fermentations of must with a high sugar content, in order to reduce its acetic acid production. Thirty-fiveC. zemplininastrains, which were isolated from different geographic regions, were molecularly characterized, and their fermentation performances were determined. Five genetically different strains were selected for mixed fermentations withS. cerevisiae. Two types of inoculation were carried out: coinoculation and sequential inoculation. A balance between the two species was generally observed for the first 6 days, after which the levels ofC. zemplininastarted to decrease. Relevant differences were observed concerning the consumption of sugars, the ethanol and glycerol content, and acetic acid production, depending on which strain was used and which type of inoculation was performed. Sequential inoculation led to the reduction of about half of the acetic acid content compared to the pureS. cerevisiaefermentation, but the ethanol and glycerol amounts were also low. A coinoculation with selected combinations ofS. cerevisiaeandC. zemplininaresulted in a decrease of ∼0.3 g of acetic acid/liter, while maintaining high ethanol and glycerol levels. This study demonstrates that mixedS. cerevisiaeandC. zemplininafermentation could be applied in sweet wine fermentation to reduce the production of acetic acid, connected to theS. cerevisiaeosmotic stress response.

L’éthanol est un composé à usages très variés allant de la chimie à l’agroalimentaire. Cependant, la croissance actuelle du marché se fait essentiellement autour de l’utilisation de l’éthanol en tant que carburant. L’objectif de ce projet est d’intensifier la production d’éthanol à partir du sirop basse pureté, produit de la seconde cristallisation des jus d’extraction de betterave, afin de diminuer les consommations en énergie et en eau pour la production d’éthanol. Pour ce faire, en partenariat avec l’UNGDA et l’ADEME, nous avons mené des travaux de recherche sur les fermentations à haute densité afin d’obtenir des vins à teneur plus élevée en éthanol. A l’issu d’un état de l’art et de quatre visites dans des ateliers de production, une stratégie de recherche en trois points a été établie. Le premier point a consisté en la recherche d’une composition de milieu de fermentation permettant d’augmenter la concentration finale en éthanol. Le second point a eu pour but de déterminer si les besoins en nutriments se limitaient uniquement à la phase de croissance ou au contraire si l’apport de ces nutriments était bénéfique tout au long de la fermentation. Le dernier point a quant à lui utilisé l’ensemble des résultats obtenus pour définir une conduite de procédé, permettant d’obtenir la concentration finale en éthanol la plus élevée possible. Ces résultats montrent qu’il est possible de réaliser des fermentations haute densité à partir de sirop basse pureté et d’obtenir 15,2 % (v/v) d’éthanol en fin de fermentation. L’application de ces travaux dans les ateliers de production permettrait d’économiser par litre d’éthanol pur, entre 20 et 30 % d’énergie pour la distillation, entre 35 et 49 % d’eau pour la réalisation des milieux de fermentation à partir de SBP et de diminuer de 23 à 38 % le volume de déchet produit après distillation. Ethanol is a compound with a wide usage range from chemistry to food. However, the current market growth mainly concerns the use of ethanol as fuel. The objective of this project was to intensify ethanol production from low purity syrup 2, which is a substrate from sugar beet, in order to reduce the consumptions of energy and water for its production. To do this, in partnership with UNGDA and ADEME, we have conducted research on high-gravity fermentations in order to increase the ethanol concentration at the end of the fermentation. With the coming of a state of the art and four visits in production facilities, a three points research strategy has been established. The first point consisted of fermentation medium composition finding in order to increase the final ethanol concentration at the end of the fermentation. The second point was to determine if the nutrients requirements were limited only during the growth phase or, on the contrary, if nutriments were beneficial throughout the fermentation. The latter point was to use the overall results to define a fermentation process, to obtain a final ethanol concentration as high as possible. These results show that it is possible to achieve high gravity fermentation from low purity syrup and reach a final ethanol concentration of 15.2 % (v/v). The application of this work in production facilities could save per liter of pure ethanol between 20 and 30% energy for distillation, between 35 and 49 % water for the production of fermentation media from SBP and decrease from 23 to 38 % of the volume of waste produced after distillation.

Mestrado em Biotecnologia - Biotecnologia Alimentar
O objetivo do presente trabalho passou pelo estudo da adaptação de S. cerevisiae à pressão, usando ciclos consecutivos de fermentação sob pressão em níveis sub-letais. Assim, este trabalho foi divido em duas partes: numa primeira parte, foram aplicadas pressões sub-letais (entre 15-50 MPa) durante o processo fermentativo para determinar as pressões a serem utilizadas na fase posterior; na segunda parte, as culturas de S. cerevisiae realizaram fermentação sob pressão ao longo de quatro ciclos consecutivos de fermentação de modo a desencadear um mecanismo de adaptação à pressão. Neste contexto, foram testadas três pressões (15 MPa, 25 MPa e 35 MPa) e duas temperaturas (30 ºC e temperatura ambiente). De modo a monitorizar os processos, foram determinadas as concentrações de açúcares (glucose, frutose e maltose), etanol e ácidos orgânicos (cítrico, málico, succínico e acético). Para além disso, foram realizadas análises microbiológicas para determinar a viabilidade celular e concentração de biomassa. Após cada ciclo a 15 e 25 MPa, tanto o crescimento celular como a produção de etanol mostraram tendência para aumentar, sugerindo a adaptação da S. cerevisiae a estes níveis de pressão. Na verdade, no final do 4º ciclo sob ambas as pressões, a produção de etanol foi superior à observada à pressão atmosférica (8.75 g.L-1 e 10.69 g.L-1 a 15 e 25 MPa, respetivamente, comparando com 8.02 g.L-1 à pressão atmosférica). No entanto, quando a pressão aumenta para 35 MPa, o crescimento celular e a produção de bioetanol diminuíram, sendo mínimas após os 4 ciclos de fermentação consecutivos. De um modo geral, estes resultados sugerem que a adaptação a condições sub-letais de pressão (15 e 25 MPa) pode melhorar a produção de bioetanol pela S. cerevisiae, podendo esta técnica ser utilizada para aumentar rendimentos e produtividades da fermentação alcoólica
The objective of the present work was to study the adaptation of S. cerevisiae to the pressure, using consecutive cycles of fermentation under pressure at sublethal levels. Thus, this work was divided in two parts: in the first part, sublethal pressures (between 15-50 MPa) were applied during the fermentation process to determine the pressures to be used in the later phase; in the second part, S. cerevisiae cultures underwent fermentation under pressure over four consecutive fermentation cycles to trigger a pressure adaptation mechanism. In this context, three pressures (15 MPa, 25 MPa and 35 MPa) and two temperatures (30 ° C and ambient temperature) were tested. In order to monitor the processes, the concentrations of sugars (glucose, fructose and maltose), ethanol and organic acids (citric, malic, succinic and acetic) were determined. In addition, microbiological analyses were performed to determine cell viability and biomass concentration. After each cycle at 15 and 25 MPa, both cell growth and ethanol production showed a tendency to increase, suggesting the adaptation of S. cerevisiae to these pressure levels. In fact, at the end of the 4th cycle under both pressures, the ethanol production was higher than that observed at atmospheric pressure (8.75 g.L-1 and 10.69 g.L-1 at 15 and 25 MPa, respectively, comparing with 8.02 g.L-1 at pressure atmospheric). However, when the pressure increases to 35 MPa, cell growth and bioethanol production decreased, being minimal after the 4 consecutive fermentation cycles. In general, these results suggest that adaptation to sublethal pressure conditions (15 and 25 MPa) can improve bioethanol production by S. cerevisiae, and this technique can be used to increase yields and yields of alcoholic fermentation.

The fuel ethanol industry is now making use of a very efficient process where virtually all sugar substrates are converted to ethanol. Nevertheless, some metabolic by-products excreted from Saccharomyces cerevisiae tend to reduce the ethanol yield. Of such, glycerol is the major one, accounting for about 5-10% relative to the amount of ethanol produced.
Glycerol plays an important role in maintaining the redox balance within the cells by oxidizing the cytosolic NADH under anaerobic conditions. It is also believed that it acts as an osmoprotectant and would be favourably produced in high osmotic pressure conditions.
In order to mitigate the production of glycerol, various aeration strategies were investigated in a single-stage continuous fermentation system. Oxygen dissolved in the fermentation medium put the yeast in aerobiosis, acted as an oxidizing agent and hence minimised the specific glycerol production by 36% as compared to a completely anaerobic fermentation.
This has hardly been reproduced in a more industrially relevant system using a multi-stage continuous fermentation process. Indeed, oscillations in the concentrations of the various metabolites over time made difficult the assessment of significant changes. Nevertheless, these findings open the door to further investigations in order to understand the effect of oxygen in continuous fermentations using very high gravity feeds, such as in the fuel ethanol industry.

Efforts are made to change from 1st to 2nd generation bioethanol production, using lignocellulosics as raw materials rather than using raw materials that alternatively can be used as food sources. An issue with lignocellulosics is that a harsh pretreatment step is required in the process of converting them into fermentable sugars. In this step, inhibitory compounds such as furan aldehydes and carboxylic acids are formed, leading to suboptimal fermentation rates. Another issue is that lignocellulosics may contain a large portion of pentoses, which cannot be fermented simultaneously with glucose by Saccharomyces cerevisiae. In this thesis, high local cell density has been investigated as a means of overcoming these two issues. Encapsulation of yeast in semi-permeable alginate-chitosan capsules increased the tolerance towards furan aldehydes, but not towards carboxylic acids. The selective tolerance can be explained by differences in the concentration of compounds radially through the cell pellet inside the capsule. For inhibitors, gradients will only be formed if the compounds are readily convertible, like the furan aldehydes. Conversion of inhibitors by cells close to the membrane leads to decreased concentrations radially through the cell pellet. Thus, cells closer to the core experience subinhibitory levels of inhibitors and can ferment sugars. Carbohydrate gradients also give rise to nutrient limitations, which in turn trigger a stress response in the yeast, as was observed on mRNA and protein level. The stress response is believed to increase the robustness of the yeast and lead to improved tolerance towards additional stress. Glucose and xylose co-consumption by a recombinant strain, CEN.PK XXX, was also improved by encapsulation. Differences in affinity of the sugar transporters normally result in that glucose is taken up preferentially to xylose. However, when encapsulated, cells in different parts of the capsule experienced high and low glucose concentrations simultaneously. Xylose and glucose could thus be taken up concurrently. This improved the co-utilisation of the sugars by the system and led to 50% higher xylose consumption and 15% higher final ethanol titres. A protective effect by the capsule membrane itself could not be shown. Hence, the interest in flocculation was triggered, as a more convenient way to keep the cells together. To investigate whether flocculation increases the tolerance, like encapsulation, recombinant flocculating yeast strains were constructed and compared with the non-flocculating parental strain. Experiments showed that strong flocculation did not increase the tolerance towards carboxylic acids. However, the tolerance towards a spruce hydrolysate and especially against furfural was indeed increased. The results of this thesis show that high local cell density yeast cultures have the potential to aid against two of the major problems for 2nd generation bioethanol production: inhibitors and simultaneous hexose and pentose utilisation.

Akademisk avhandling som för avläggande av teknologie doktorsexamen vid Chalmers tekniska högskola försvaras vid offentlig disputation den 19 februari 2014,klockan 13.30 i KA-salen, Kemigården 4, Göteborg.

Estudou-se o processo descontínuo alimentado de fermentação alcoólica, utilizando-se mosto de melaço de cana-de-açúcar e Saccharomyces cerevisiae na forma de fermento prensado. Foram analisados a influência da vazão de alimentação exponencialmente decrescente e do tempo de enchimento do fermentador no comportamento do sistema, considerando os seguintes parâmetros: 1. produtividade em etanol e em células, 2. rendimento em etanol e 3. razão de crescimento celular.
The fed-batch ethanol fermentation of sugar-cane blackstrap molasses by the action of Saccharomyces cerevisiae (pressed yeast) was studied. The influence of exponencialy decreasing feeding rates and of the fermentor filling up time on the system behavior was analysed considering the following parameters: 1. ethanol and cell productivities, 2. ethanol yield and 3. cell growth ratio.

O Brasil é o segundo maior produtor e um dos maiores exportadores de etanol no mundo e tal biocombustível tem grande impacto na economia do país. A expectativa é de grande demanda por tal produto, quer pelo crescente consumo interno, como também em decorrência do fim do protecionismo nos Estados Unidos. Portanto, o Brasil deverá produzir mais etanol e a um custo mais reduzido para manter a competitividade frente aos combustíveis fósseis. Dentre as inovações tecnológicas estaria a fermentação com alto teor alcoólico. Contudo, um dos fatores limitantes para a implantação desta tecnologia é a ausência de leveduras apropriadas para tolerar as condições severas impostas por este tipo de fermentação, onde múltiplos estresses são impostos simultaneamente às leveduras. Assim, este trabalho se propôs a selecionar, da biodiversidade de leveduras encontradas nas destilarias brasileiras, linhagens de Saccharomyces cerevisiae com capacidade de conduzir fermentações com alto teor alcoólico e em condições de reciclo celular. A estratégia de seleção consistiu na busca de linhagens com tolerâncias múltiplas, frentes aos estresses etanólico, osmótico, ácido e térmico. Para tal, um total de 525 linhagens, obtidas de diferentes destilarias, foram submetidas a uma seleção para destacar linhagens com múltipla tolerância. Cerca de metade destas linhagens foram submetidas a uma seleção prévia avaliando-se o crescimento (D.O.570nm, durante 24 horas a 30ºC) em meio constituído de mosto misto (melaço e caldo de cana) com 25% de ART, selecionando 200 linhagens. Estas, acrescidas de mais 249 não avaliadas no meio anterior, foram igualmente submetidas a processo seletivo em meio contendo múltiplos estresses (etanólico, osmótico, ácido e térmico). Tal meio foi desenvolvido após avaliações de 26 combinações com os diferentes estresses acima mencionados e com diferentes intensidades. O objetivo foi buscar um meio que melhor discriminasse as tolerâncias das leveduras referencias: as linhagens de Saccharomyces cerevisiae PE-2 e de panificação, com e sem capacidade de implantação no processo industrial, respectivamente. A tolerância foi avaliada pela formação de biomassa (D.O.570nm, durante 24 horas a 30ºC). Assim, tal meio seletivo permitiu a seleção de 34 linhagens com perfis de tolerância igual ou superior ao da linhagem PE-2. Estas linhagens foram, a seguir, avaliadas quanto à viabilidade celular e ao crescimento em fermentações de mosto misto com teores crescentes de açúcares, ao longo de 10 reciclos a 30oC, atingindo teores de etanol de 15 a 16% (v/v). As 10 linhagens com os melhores desempenhos foram submetidas à avaliação final em fermentações simulando condições industriais, em reciclos fermentativos a 32ºC empregando-se mosto misto com teores crescentes de açúcares, permitindo aumentos nos teores de etanol de 11 a 15% (v/v) ao longo dos reciclos. Para esta avaliação final os seguintes parâmetros foram estimados: rendimento em etanol, formação de biomassa e glicerol, teores de açúcares residuais, viabilidade celular, e teores celulares dos carboidratos de reserva (glicogênio e trealose). Pelo menos 4 linhagens mostraram atributos fermentativos superiores ao da linhagem referência (PE-2), permitindo concluir que linhagens capazes de conduzirem a fermentação com alto teor de etanol podem ser obtidas da biodiversidade encontrada no ambiente das destilarias.
Brazil is the second largest ethanol producer and one of the leading ethanol exporter in the world, and this biofuel has great impact on the country economy. Huge demand is expected for this product, not only to supply the growing domestic consumption but due to the end of the United States market protectionism. In view of this, Brazil should produce more ethanol and at a lower cost to maintain competitiveness in relation to fossil fuels. One of the technological approaches which emerges is the high ethanol content fermentation. However, one of the limiting factors for this technology is the absence of proper strains to face the very harsh fermentation condition, where several stresses are simultaneously imposed to the fermenting yeast. This work aimed at selecting Saccharomyces cerevisiae strains from the biodiversity of yeasts found in Brazilian distilleries to conduct high ethanol fermentation with cell reuse. The selection strategy was to search for multiple tolerant strains to ethanol, acid, osmotic and thermal stresses. For that, a total of 525 strains, which were obtained from several distilleries, were subjected to a selection in order to highlight multi-tolerant strains. About half of these strains were subjected to a pre-screening procedure to evaluate growth (O.D.570nm, for 24 hours at 30ºC) in medium containing molasses and sugarcane juice (25% TRS), and 200 strains were selected. These 200 strains, together with 249 strains not previously evaluated, were screened in a medium imposing multiple stresses (ethanol, acid, osmotic and thermal). This medium was chosen after assessments of 26 different medium formulations with the above mentioned stresses and with different intensities. The purpose of that was to find a medium which best discriminate the tolerance of the reference yeasts: PE-2 and bakery Saccharomyces cerevisiae strains, with and without ability to persist in the industrial process, respectively. The strain tolerance was evaluated by biomass formation (O.D.570nm, for 24 hours at 30ºC). By this mean 34 strains were selected displaying similar or superior performance in comparison with PE-2 strain. These strains were then assessed for cell viability and growth in cell reuse fermentations (10 cycles), using cane juice/molasses substrates with increasing sugar content, at 30ºC, reaching 15-16% ethanol (v/v). The 10 strains with the best performances were subjected to final evaluation in fermentations simulating the industrial process with cell reuse, at 32ºC, using the same substrate with increasing sugar content, which allowed rises in ethanol content from 11 to 15% (v/v) over the cycles. For this final evaluation, the following parameters were determined: ethanol yield, biomass and glycerol formation, residual sugar levels, cell viability and storage carbohydrate levels (trehalose and glycogen). At least four strains showed superior fermentative attributes to reference strain (PE-2), leading to the conclusion that strains able to conduct high ethanol content fermentations can be obtained from the natural biodiversity found in Brazilian distilleries.

Biofuels production and utilisation can reduce the emission of greenhouse gases, dependence on fossil fuels and also improve energy security. Ethanol is the most important biofuel in the transportation sector; however, its production from lignocelluloses faces some challenges. Conventionally, lignocellulosic hydrolysis and fermentation has mostly been performed by separate hydrolysis and fermentation (SHF) or simultaneous saccharification and fermentation (SSF). SHF results in product inhibition during enzymatic hydrolysis and increased contamination risk. During SSF, suboptimal conditions are used and the fermenting organism cannot be reused. Bacterial contamination is another major concern in ethanol production, which usually results in low ethanol yield. In these studies, the above-mentioned challenges have been addressed. A novel method for lignocellulosic ethanol production ‘Simultaneous saccharification filtration and fermentation (SSFF)’ was developed. It circumvents the disadvantages of SSF and SHF; specifically, it uses a membrane for filtration and allows both the hydrolysis and fermentation to be carried out at different optimum conditions. SSFF also offers the possibility of cell reuse for several cultivations. The method was initially applied to pretreated spruce, with a flocculating strain of yeast Saccharomyces cerevisiae. SSFF was further developed and applied to pretreated wheat straw, a xylose rich lignocellulosic material, using encapsulated xylose fermenting strain of S. cerevisiae. High solids loading of 12% suspended solids (SS) was used to combat bacterial contamination and improve ethanol yield. Oil palm empty fruit bunch (OPEFB) was pretreated with fungal and phosphoric acid in order to improve its ethanol yield. An evaluation of biofuel production in Nigeria was also carried out. SSFF resulted in ethanol yield of 85% of the theoretical yield from pretreated spruce with the flocculating strain. Combination of SSFF with encapsulated xylose fermenting strain facilitated simultaneous glucose and xylose utilisation when applied to pretreated wheat straw; this resulted in complete glucose consumption and 80% xylose utilisation and consequently, 90% ethanol yield of the theoretical level. High solids loading of 12% SS of pretreated birch resulted in 47.2 g/L ethanol concentration and kept bacterial infection under control; only 2.9 g/L of lactic acid was produced at the end of fermentation, which lasted for 160 h while high lactic acid concentrations of 42.6 g/L and 35.5 g/L were produced from 10% SS and 8% SS, respectively. Phosphoric acid pretreatment as well as combination of fungal and phosphoric pretreatment improved the ethanol yield of raw OPEFB from 15% to 89% and 63% of the theoretical value, respectively. In conclusion, these studies show that SSFF can potentially replace the conventional methods of lignocellulosic ethanol production and that high solids loading can be used to suppress bacterial infections during ethanol productions, as well as that phosphoric acid pretreatment can improve ethanol yield from lignocellulosic biomass.

Thesis for the degree of Doctor of Philosophy at the University of Borås to be publicly defended on 31 October 2014, 10.00 a. m. in room E310, University of Borås, Allégatan 1, Borås.

Foram estudados os processos descohtínuo e descontínuo alimentado de fermentação alcoólica, utilizando-se mosto de melaço de cana de açúcar e Saccharomyces cerevisiae na forma de fermento prensado. Foram analisados a influência da vazão de alimentação exponencialmente decrescente, do tempo de enchimento e do nível de inóculo no comportamento do sistema, considerando os seguintes parâmetros: - produtividades em etanol e em células - rendimento em etanol - fator de conversão de substrato em células. Para o processo descontínuo alimentado, o valor máximo atingido para a produtividade em etanol foi de 16,9 g/L.h. Os resultados obtidos de rendimentos e produtividades em etanol foram semelhantes para os processos descontínuo e descontínuo alimentado (com tempo de enchimento de 3 h e constante de tempo de 1,6 h-1).
The batch and fed-batch fermentations of sugar -cane blackstrap molasses by the action of Saccharomyces cerevisiae(pressed yeast) were studied. The influence of exponentially decreasing feeding rates, fermentar filling-up time and levei of inocullum on the behavior was analysed considering the following parameters: - ethanol and cell productivities - ethanol yield - yield yeast. At fed-batch fermentation, the maximum ethanol productivity obtained was 16.9 g/L.h. The results of ethanol productivities and yield achieved for batch and fedbatch (with fermentar filling-up time of 3 h and time constant of 1.6 h-1