Vanda Renata Reis

Fermentative and growth performances of Dekkera bruxellensis in different batch systems and the effect of initial low cell counts in co‐cultures with Saccharomyces cerevisiae

Dekkera bruxellensis is a multifaceted yeast present in the fermentative processes used for alcoholic beverage and fuel alcohol production – in the latter, normally regarded as a contaminant. We evaluated the fermentation and growth performance of a strain isolated from water in an alcohol‐producing unit, in batch systems with/without cell recycling in pure and co‐cultures with Saccharomyces cerevisiae. The ethanol resistance and aeration dependence for ethanol/acid production were verified. Ethanol had an effect on the growth of D. bruxellensis in that it lowered or inhibited growth depending on the concentration. Acid production was verified in agitated cultures either with glucose or sucrose, but more ethanol was produced with glucose in agitated cultures. Regardless of the batch system, low sugar consumption and alcohol production and expressive growth were found with D. bruxellensis. Despite a similar ethanol yield compared to S. cerevisiae in the batch system without cell recycling, ethanol productivity was approximately four times lower. However, with cell recycling, ethanol yield was almost half that of S. cerevisiae. At initial low cell counts of D. bruxellensis (10 and 1000 cells/ml) in co‐cultures with S. cerevisiae, a decrease in fermentative efficiency and a substantial growth throughout the fermentative cycles were displayed by D. bruxellensis. Due to the peculiarity of cell repitching in Brazilian fermentation processes, D. bruxellensis is able to establish itself in the process, even when present in low numbers initially, substantially impairing bioethanol production due to the low ethanol productivity, in spite of comparable ethanol yields. Copyright

Characteristics of Saccharomyces cerevisiae yeasts exhibiting rough colonies and pseudohyphal morphology with respect to alcoholic fermentation

Among the native yeasts found in alcoholic fermentation, rough colonies associated with pseudohyphal morphology belonging to the species Saccharomyces cerevisiae are very common and undesirable during the process. The aim of this work was to perform morphological and physiological characterisations of S. cerevisiae strains that exhibited rough and smooth colonies in an attempt to identify alternatives that could contribute to the management of rough colony yeasts in alcoholic fermentation. Characterisation tests for invasiveness in Agar medium, killer activity, flocculation and fermentative capacity were performed on 22 strains (11 rough and 11 smooth colonies). The effects of acid treatment at different pH values on the growth of two strains (“52” - rough and “PE-02” - smooth) as well as batch fermentation tests with cell recycling and acid treatment of the cells were also evaluated. Invasiveness in YPD Agar medium occurred at low frequency; ten of eleven rough yeasts exhibited flocculation; none of the strains showed killer activity; and the rough strains presented lower and slower fermentative capacities compared to the smooth strains in a 48-h cycle in a batch system with sugar cane juice. The growth of the rough strain was severely affected by the acid treatment at pH values of 1.0 and 1.5; however, the growth of the smooth strain was not affected. The fermentative efficiency in mixed fermentation (smooth and rough strains in the same cell mass proportion) did not differ from the efficiency obtained with the smooth strain alone, most likely because the acid treatment was conducted at pH 1.5 in a batch cell-recycle test. A fermentative efficiency as low as 60% was observed with the rough colony alone.

Effects of feedstock and co-culture of Lactobacillus fermentum and wild Saccharomyces cerevisiae strain during fuel ethanol fermentation by the industrial yeast strain PE-2

Even though contamination by bacteria and wild yeasts are frequently observed during fuel ethanol fermentation, our knowledge regarding the effects of both contaminants together is very limited, especially considering that the must composition can vary from exclusively sugarcane juice to a mixture of molasses and juice, affecting the microbial development. Here we studied the effects of the feedstock (sugarcane juice and molasses) and the co-culture of Lactobacillus fermentum and a wild Saccharomyces cerevisiae strain (rough colony and pseudohyphae) in single and multiple-batch fermentation trials with an industrial strain of S. cerevisiae (PE-2) as starter yeast. The results indicate that in multiple-cycle batch system, the feedstock had a minor impact on the fermentation than in single-cycle batch system, however the rough yeast contamination was more harmful than the bacterial contamination in multiple-cycle batch fermentation. The inoculation of both contaminants did not potentiate the detrimental effect in any substrate. The residual sugar concentration in the fermented broth had a higher concentration of fructose than glucose for all fermentations, but in the presence of the rough yeast, the discrepancy between fructose and glucose concentrations were markedly higher, especially in molasses. The biggest problem associated with incomplete fermentation seemed to be the lower consumption rate of sugar and the reduced fructose preference of the rough yeast rather than the lower invertase activity. Lower ethanol production, acetate production and higher residual sugar concentration are characteristics strongly associated with the rough yeast strain and they were not potentiated with the inoculation of L. fermentum.

Bioethanol strains of Saccharomyces cerevisiae characterised by microsatellite and stress resistance

Strains of Saccharomyces cerevisiae may display characteristics that are typical of rough-type colonies, made up of cells clustered in pseudohyphal structures and comprised of daughter buds that do not separate from the mother cell post-mitosis. These strains are known to occur frequently in fermentation tanks with significant lower ethanol yield when compared to fermentations carried out by smooth strains of S. cerevisiae that are composed of dispersed cells. In an attempt to delineate genetic and phenotypic differences underlying the two phenotypes, this study analysed 10 microsatellite loci of 22 S. cerevisiae strains as well as stress resistance towards high concentrations of ethanol and glucose, low pH and cell sedimentation rates. The results obtained from the phenotypic tests by Principal-Component Analysis revealed that unlike the smooth colonies, the rough colonies of S. cerevisiae exhibit an enhanced resistance to stressful conditions resulting from the presence of excessive glucose and ethanol and high sedimentation rate. The microsatellite analysis was not successful to distinguish between the colony phenotypes as phenotypic assays. The relevant industrial strain PE-2 was observed in close genetic proximity to rough-colony although it does not display this colony morphology. A unique genetic pattern specific to a particular phenotype remains elusive.

CONTROLE DE MICRO-ORGANISMOS CONTAMINANTES DA PRODUÇÃO DE ETANOL COMBUSTÍVEL ATRAVÉS DE TRATAMENTO COMBINADO DE BAIXO pH, ADIÇÃO DE ETANOL E METABISSULFITO DE POTÁSSIO

Resumo Uma das principais preocupações na indústria sucro-alcooleira é combater os microorganismos contaminantes, representados pelas bactérias e leveduras que se instalam no processo e causam queda no rendimento. O objetivo desse trabalho foi propor modificações no tratamento ácido do fermento, realizado entre os ciclos fermentativos na indústria, para o controle do crescimento de linhagens rugosas de Saccharomyces cerevisiae e da bactéria Lactobacillus fermentum, em comparação com a levedura S. cerevisiae do processo (PE-2). Foram avaliados a adição de etanol à solução ácida (pH 2,0), pH 1,5 e um tratamento com metabissulfito de potássio (200 mg/L, pH 4,5) sobre o número de UFC das leveduras e bactéria. Não houve um tratamento único capaz de controlar todas as linhagens rugosas testadas. Para a linhagem rugosa 52, a adição de 13% de etanol ao tratamento ácido (pH 2,0), foi a melhor opção de controle. Para a levedura 07, a sugestão foi de abaixamento do pH do tratamento ácido de 2,0 para 1,5 para o controle do crescimento da levedura rugosa. A levedura PE-2 foi pouco afetada pelos tratamentos acima indicados. A bactéria L. fermentum teve seu crescimento afetado em todas as condições. Nenhum dos tratamentos utilizados teve efeito sobre a linhagem rugosa 36. Abstract One of the main concerns in the sugar-alcohol industry is to combat contaminating microorganisms, represented by bacteria and yeasts that settle in the process and cause a drop in yield. The objective of this work was to evaluate the effect of folic acid on the growth of strains, saccharomyces cerevisiae and bacteria, Lactobacillus fermentum, 2). (PH 2.0), pH 1.5 and a treatment with potassium metabisulfite (200 mg / L, pH 4.5) on the number of CFUs of yeasts and bacteria. There was no single treatment capable of controlling all of the roughened lines tested. For the rough lineage 52, a 13% addition of ethanol to the acid treatment (pH 2.0) was a better control option. For yeast 07, a suggestion for lowering the treatment pH from 2.0 to 1.5 for the control of rough yeast growth. The PE-2 yeast for the subsequent treatments indicated. The L. fermentum bacterium had its growth affected in all conditions. None of the treatments used had an effect on the rough lineage 36.

Potassium metabisulphite as a potential biocide against Dekkera bruxellensis in fuel ethanol fermentations

Dekkera bruxellensis is an important contaminant yeast of fuel ethanol fermentations in Brazil, whose system applies cell repitching between the fermentative cycles. This work evaluated the addition of potassium metabisulphite (PMB) on yeast growth and fermentative yields in pure and co‐cultures of Saccharomyces cerevisiae and D. bruxellensis in two situations: addition to the acidic solution in which the cells are treated between the fermentative cycles or to the fermentation medium. In the range of 200–400 mg l−1, PMB was effective to control the growth of D. bruxellensis depending on the culture medium and strain. When added to the acidic solution (250 mg l−1), a significant effect was observed in mixed cultures, because the inactivation of SO2 by S. cerevisiae most likely protected D. bruxellensis from being damaged by PMB. The physiological response of S. cerevisiae to the presence of PMB may explain the significant decrease in alcohol production. When added to the fermentation medium, PMB resulted in the control but not the death of D. bruxellensis, with less intensive effect on the fermentative efficiency. In co‐culture with the addition of PMB, the fermentative efficiency was significantly lower than in the absence of PMB.