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.

Dissolved carbon dioxide driven repeated batch fermentation has been performed under four glucose concentrations: ~150, ~200, ~250 and ~300 g glucose l-1, with three dissolved carbon dioxide (DCO2) control conditions: without DCO2 control, with DCO2 control at 750 and 1000 mg l-1 levels. No residual glucose was observed under all performed fermentation conditions, and the repeated batch fermentation system could be operated by a computer as self-cycling system. The collected fermentation results presented that, under the same feeding concentration, ethanol concentration in the presence of DCO2 control was significantly lower than that in the absence of DCO2 control; and a higher biomass concentration in the presence of control was observed in this comparison as well. A higher biomass concentration resulted in a shorter fermentation time, which contributed to a higher ethanol production rate. The highest final ethanol concentration was observed as 113.5 g l-1 at 1000 mg DCO2 l-1 control level under ~300 g glucose l-1 condition, where the lowest ethanol production rate of 1.18 g l-1 h-1 was observed. The highest ethanol production rate was 4.57 g l-1 h-1 and its corresponding ethanol concentration was 66.7 g ethanol l-1 at 1000 mg l-1 DCO¬2 control level under ~200 g glucose l-1 condition. For all fermentation conditions, the viabilities of yeast at the end of fermentation were maintained at near 90% where their corresponding final ethanol concentrations were lower than 100 g l-1. As soon as the final ethanol concentration at the end of each cycle was greater than 110 g l-1, its corresponding viability decreased to ~70%. The ethanol conversion efficiency was maintained at ~90% and ~65% in the absence and presence of DCO2 control, respectively. Based on the changing of biomass concentration profiles in the stabilized cycles, two cell growth phases could be identified in the absence of DCO2 control, and only one cell growth phase was noticeable in the presence of DCO2 control cases. Meanwhile, a sudden decline of DCO2 readings at the end of fermentation was constantly observed in both of in the absence and in the presence of DCO2 control cases, which resulted in developing two control algorithms to determine self-cycling time. Comparison of carbon balance analysis between in the absence and in the presence of DCO2 control suggested that the availability of DCO2 control might alter the metabolic flow during fermentation; and the figure of ethanol concentration against fermentation time illustrated that the changing of DCO2 control level did not affect fermentation results, significantly. Moreover, comparisons of ethanol production rate between different processes and different initial glucose concentrations concluded that the ethanol production rate in the presence of DCO2 control was generally higher than that in the absence of DCO2 control under the same glucose concentration; and the ethanol production rate was decreased with the increasing of glucose concentration under the same DCO2 control condition. The experiment results were scaled up to 106 L as a sample analysis in production scale, which suggested that the fermentation with ~200 g glucose l-1 feeding concentration in the absence of DCO2 controlled would provide best profits in the all fermentation conditions.

Fatty acid components on yeast plasma membrane were critical in maintaining proper cell activity during bioethanol fermentation. The alteration of fatty acid composition on yeast plasma membrane was recognized as an adaptive response to several environmental stress including osmotic pressure, ethanol inhibition and nutrients limit. These stresses were exacerbated under very-high-gravity condition in which excessive fermentable sugar was provided in feedstock. Controlling redox potential was proved beneficial in improving yeast performance under very-high-gravity condition. Fatty acid synthesis and desaturation pathways involved dissolved oxygen as well as balance between NAD+/NADH and NADP+/NADPH which could be influenced by the regulation of redox potential in media. In this study, fatty acid composition profiles under different glucose concentrations and different redox potential control level were examined. Its connection with yeast cell growth, ethanol productivity and other metabolites’ concentrations were studied as well to reveal any causal correlation between redox potential control, membrane fatty acid composition and yeast activity. Two glucose concentrations used in this study were 200 g/L and 300 g/L which represented normal and very high gravity respectively in bioethanol fermentation. In 300 g/L fermentation, three redox conditions were adopted while two different redox conditions were used in 200 g/L fermentation. Biomass concentration, ethanol productivity and fatty acid composition were observed to be affected by both gravity and ORP control strategy. Final biomass concentrations were 4.302 g/L in 200 g/L glucose with no ORP control condition and 7.658 in 200 g/L glucose with ORP controlled at -100 mV condition. In 300 g/L glucose fermentation, final biomass concentrations were 3.400 g/L for no ORP control, 4.953 g/L for -150 mV ORP control and 5.260 for -100 mV ORP control. Ethanol productivities were 2.574 g/Lh for 200 g/L glucose without ORP control and 3.780 g/Lh for 200 g/L glucose with -100 mV ORP control. In 300 g/L glucose fermentation, ethanol productivity decreased to 1.584 g/Lh when no ORP control was imposed. ORP control at -150 mV could improve the ethanol productivity to 1.693 g/Lh while -100 mV ORP control was able to further enhance the ethanol productivity to 1.829 g/Lh. Fatty acid composition was observed to shift to more saturated components when no ORP control was applied. Such trend of saturation was increased by higher gravity condition. ORP control was shown to change this tendency to saturation and help restore fatty acid components on plasma membrane to a more balanced distribution.

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.

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.

The objective of this study is to evaluate and compare, both technically and economically, various glucose feeding concentrations and different redox potential settings on ethanol production under very-high-gravity (VHG) conditions. Laboratory data were collected for process modeling and two process models were created by two individual process simulators. The first one is a simplified model created and evaluated by Superpro Designer. The second one is an accurate model created by Aspen Plus and evaluated by Aspen Icarus Process Evaluator (Aspen IPE). The simulation results of the two models were also compared. Results showed that glucose feeding concentration at 250±3.95 g/L to the fermentor resulted in the lowest unit production cost (1.479 $/kg ethanol in the Superpro model, 0.764 $/kg ethanol in the Aspen Plus model), with redox potential control effects accounted. Controlling redox potential at -150 mV increased the ethanol yield under VHG fermentation conditions while no significant influences were observed when glucose feeding concentration was less than 250 g/L. Results of product sales analysis indicated that for an ethanol plant with a production rate of 85~130 million kg ethanol/year, only maintaining the glucose feeding concentration to the fermentor at around 250 g/L resulted in the shortest payout period of 5.33 years in average,, with or without redox potential control. If 300±6.42 g/L glucose feeding concentration to the fermentor is applied, it is essential to have the redox potential only controlled at -150 mV in the fermentor to limit the process payout period within 6 years. In addition, fermentation processes with glucose feeding concentration at around 200 g/L to the fermentor were estimated to be unprofitable under all studied conditions. For environmental concerns, two disposal alternatives were presented for CO2 produced during fermentation process rather than emission into atmosphere. One is to sell CO2 as byproduct, which brought 1.52 million $/year income for an ethanol plant with a capacity of 100 million kg ethanol/year. Another option is to capture and transport CO2 to deep injection sites for geological underground storage, which is already a safe and mature technology in North America, and also applicable to many other sites around the world. This would roughly add 4.78 million dollars processing cost annually in the studied scenario. Deep injection of captured CO2 from ethanol plants prevents emission of CO2 into the atmosphere, thus makes it environmental friendly.

The objective of this study is to evaluate and compare, both technically and economically, various glucose feeding concentrations and different redox potential settings on ethanol production under very-high-gravity (VHG) conditions. Laboratory data were collected for process modeling and two process models were created by two individual process simulators. The first one is a simplified model created and evaluated by Superpro Designer. The second one is an accurate model created by Aspen Plus and evaluated by Aspen Icarus Process Evaluator (Aspen IPE). The simulation results of the two models were also compared. Results showed that glucose feeding concentration at 250±3.95 g/L to the fermentor resulted in the lowest unit production cost (1.479 $/kg ethanol in the Superpro model, 0.764 $/kg ethanol in the Aspen Plus model), with redox potential control effects accounted. Controlling redox potential at -150 mV increased the ethanol yield under VHG fermentation conditions while no significant influences were observed when glucose feeding concentration was less than 250 g/L. Results of product sales analysis indicated that for an ethanol plant with a production rate of 85~130 million kg ethanol/year, only maintaining the glucose feeding concentration to the fermentor at around 250 g/L resulted in the shortest payout period of 5.33 years in average,, with or without redox potential control. If 300±6.42 g/L glucose feeding concentration to the fermentor is applied, it is essential to have the redox potential only controlled at -150 mV in the fermentor to limit the process payout period within 6 years. In addition, fermentation processes with glucose feeding concentration at around 200 g/L to the fermentor were estimated to be unprofitable under all studied conditions. For environmental concerns, two disposal alternatives were presented for CO2 produced during fermentation process rather than emission into atmosphere. One is to sell CO2 as byproduct, which brought 1.52 million $/year income for an ethanol plant with a capacity of 100 million kg ethanol/year. Another option is to capture and transport CO2 to deep injection sites for geological underground storage, which is already a safe and mature technology in North America, and also applicable to many other sites around the world. This would roughly add 4.78 million dollars processing cost annually in the studied scenario. Deep injection of captured CO2 from ethanol plants prevents emission of CO2 into the atmosphere, thus makes it environmental friendly.

Department of Food Science and Technology / MSCPNT