Otimização da produção de etanol a 42°C utilizando uma co-cultura de Saccharomyces cerevisiae e Pichia kudriavzevii em batelada alimentada
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Data
2014-06-06
Autores
Gallardo, Jéssica Carolina Medina
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Universidade Estadual Paulista (Unesp)
Resumo
O processo industrial de produção de etanol combustível é realizado sob condições não estéreis, portanto diferentes espécies de leveduras provenientes da matéria-prima e/ou do próprio ambiente podem ser encontradas no processo de fermentação. Geralmente, a temperatura nas dornas industriais é mantida ao redor de 32°C a 35°C, no entanto, devido ao curto tempo de fermentação, o resfriamento nem sempre é suficientemente eficaz na remoção de calor, atingindo temperaturas de até 40°C. Assim, uma levedura capaz de fermentar a temperaturas acima daquelas toleradas por Saccharomyces cerevisiae, levedura utilizada como inóculo nas fermentações industriais, pode ser útil na produção de etanol durante períodos de elevação de temperatura, especialmente em países de clima tropical como o Brasil. Issatchenkia orientalis é uma levedura não-Saccharomyces capaz de converter glicose e frutose em etanol a temperaturas tão altas quanto 42°C, é encontrada em processos industriais de fabricação de pães e bebidas. O presente trabalho visou a produção de etanol a partir de melaço de cana-de-açúcar a 42°C utilizando uma cultura mista das leveduras S. cerevisiae e P. kudriavzevii . Para atingir este objetivo, inicialmente, padronizaram-se as condições de fermentação em batelada alimentada a 42°C. Em ciclos únicos de fermentação, o inóculo misto de S. cerevisiae e P. kudriavzevii (24 g/L, proporção inicial 1:3, m/v) produziu 78,76±2,08 g/L de etanol com uma produtividade de 9,85±0,29 g/L/h, rendimento teórico igual a 91,97% e (Y(P/S)) de 0,47±0,01 g/g após 8 h de fermentação. A seguir foi estudada a fermentação em batelada alimentada com reciclo de células e revitalização das células entre os ciclos de fermentação. A suplementação do meio de revitalização com um coquetel de sais, não teve efeito sobre o acúmulo da biomassa, a viabilidade e a produção de etanol. Por outro lado, quando uma fonte de nitrogênio orgânico (lisado celular) foi adicionado ao meio de revitalização observou-se um aumento significativo no acúmulo de biomassa, porém sem melhoras no rendimento do processo. Já que as duas condições de revitalização estudadas não levaram a resultados relevantes em relação à produção de etanol e ao aumento da viabilidade, decidiu-se realizar experimentos de reciclo sem revitalização das células entre os ciclos. Estes experimentos confirmaram que é possível reciclar as células da cultura mista durante 5 ciclos consecutivos a 40°C e 42°C sem perdas significativas na produção de etanol, (YP/S) e viabilidade. Por fim, foi comprovada a tolerância da cultura mista a um coquetel de inibidores presentes nos hidrolisados de bagaço de cana e confirmou-se a resistência de P. kudriavzevii aos ácidos lático e acético e ao etanol a temperaturas elevadas (40°C). Estes resultados representam uma inovação tecnológica para o setor sucroalcooleiro, uma vez que a utilização da co-cultura de S. cerevisiae e P. kudriavzevii aumentaram a produção de etanol em condições de estresse. Entretanto, ensaios em escala piloto são necessários para validar este processo utilizando volumes maiores de meio.
The industrial ethanol production is carried out under non-sterile conditions, therefore different yeast species from both feedstock and environment can be found in the fermentation. Generally, the temperature in the industrial reactors is maintained between 32°C and 35°C. However, as the fermentation time is relatively short, cooling is not always sufficiently effective in removing heat, reaching temperatures up to 40°C. Thus, a yeast capable of fermenting at temperatures above those tolerated by Saccharomyces cerevisiae, the yeast commonly used as inoculum in industrial fermentations, may be useful for ethanol production during periods of high temperatures, especially in tropical countries such as Brazil. Pichia kudriavzevii is a non-Saccharomyces yeast capable of converting glucose and fructose into ethanol at temperatures as high as 42°C, which could be found in industrial processes of bread and beverages manufacture. The present work aimed the ethanol production from sugarcane molasses at 42°C using a mixed culture of the yeasts S. cerevisiae and P. kudriavzevii. In order to achieve this goal, the first part of the work was focused on the optimization of the best conditions for a fed-batch fermentation at 42°C. The mixed inoculum of S. cerevisiae and P. kudriavzevii (24 g/L, initial ratio 1:3 w/v) produced 78.76±2.08 g/L of ethanol with a productivity of 9.85±0.29 g/L/h, which is equivalent to a theoretical yield of 91.97% and Y(P/S) of 0.47±0.01 g/g after 8h of fermentation. Fed-batch fermentations were also study with cell recycle and revitalization of cells between the fermentation cycles. The supplementation of revitalization medium with a cocktail of salts had no effect on biomass yields, viability and ethanol production. On the other hand, when an organic nitrogen source (cell lysate) was added to the revitalization medium a significant increase in biomass production was observed, nevertheless no improvement of ethanol productivity was achieved. Since the two revitalization conditions did not lead to better results regarding ethanol production and cell viability, the cell recycle process was performed without cell revitalization step between cycles. The data obtained showed that it is possible to recycle the cells of the mixed culture for five consecutive cycles at both 40°C and 42°C without significant losses in ethanol production (YP/S) and viability. Finally, the mixed culture showed high tolerance to a cocktail of inhibitors usually present in sugarcane bagasse hydrolysates. Moreover, P. kudriavzevii showed high tolerance to acetic and lactic acid at high temperatures (40°C). These results represent a technological innovation for the alcohol industry, since the use of the mixed culture of S. cerevisiae and P. kudriavzevii increased the ethanol production under severe conditions. However, pilot scale tests are necessary to validate this process using larger reactors.
The industrial ethanol production is carried out under non-sterile conditions, therefore different yeast species from both feedstock and environment can be found in the fermentation. Generally, the temperature in the industrial reactors is maintained between 32°C and 35°C. However, as the fermentation time is relatively short, cooling is not always sufficiently effective in removing heat, reaching temperatures up to 40°C. Thus, a yeast capable of fermenting at temperatures above those tolerated by Saccharomyces cerevisiae, the yeast commonly used as inoculum in industrial fermentations, may be useful for ethanol production during periods of high temperatures, especially in tropical countries such as Brazil. Pichia kudriavzevii is a non-Saccharomyces yeast capable of converting glucose and fructose into ethanol at temperatures as high as 42°C, which could be found in industrial processes of bread and beverages manufacture. The present work aimed the ethanol production from sugarcane molasses at 42°C using a mixed culture of the yeasts S. cerevisiae and P. kudriavzevii. In order to achieve this goal, the first part of the work was focused on the optimization of the best conditions for a fed-batch fermentation at 42°C. The mixed inoculum of S. cerevisiae and P. kudriavzevii (24 g/L, initial ratio 1:3 w/v) produced 78.76±2.08 g/L of ethanol with a productivity of 9.85±0.29 g/L/h, which is equivalent to a theoretical yield of 91.97% and Y(P/S) of 0.47±0.01 g/g after 8h of fermentation. Fed-batch fermentations were also study with cell recycle and revitalization of cells between the fermentation cycles. The supplementation of revitalization medium with a cocktail of salts had no effect on biomass yields, viability and ethanol production. On the other hand, when an organic nitrogen source (cell lysate) was added to the revitalization medium a significant increase in biomass production was observed, nevertheless no improvement of ethanol productivity was achieved. Since the two revitalization conditions did not lead to better results regarding ethanol production and cell viability, the cell recycle process was performed without cell revitalization step between cycles. The data obtained showed that it is possible to recycle the cells of the mixed culture for five consecutive cycles at both 40°C and 42°C without significant losses in ethanol production (YP/S) and viability. Finally, the mixed culture showed high tolerance to a cocktail of inhibitors usually present in sugarcane bagasse hydrolysates. Moreover, P. kudriavzevii showed high tolerance to acetic and lactic acid at high temperatures (40°C). These results represent a technological innovation for the alcohol industry, since the use of the mixed culture of S. cerevisiae and P. kudriavzevii increased the ethanol production under severe conditions. However, pilot scale tests are necessary to validate this process using larger reactors.
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Biotecnologia, Etanol, Melaço, Levedos, Altas temperaturas