Metabolites excreted in alcoholic fermentation as possible substrates for the growth of the genus Lactobacillus The characteristics of the Brazilian industrial process to produce bioethanol make the distilleries susceptible to the presence of contaminating microorganisms that cause in the fall of production yield. Among the stages of the process, we have the alcoholic fermentation, which consists in the metabolization of sugars by the yeast strain selected from the Saccharomyces cerevisiae species producing the ethanol. At this stage contamination occurs by yeasts of the genus Saccharomyces, yeasts not Saccharomyces and bacteria. The most commonly found bacteria are those belonging to the group of lactic bacteria (LAB), which, because they use different routes to metabolize sugars, are classified as obligate heterofermentative, obligate homofermentative and facultative heterofermentative. Among the genera of this group, Lactobacillus are the most common because of their ability to tolerate high concentrations of ethanol and sugars, high temperatures and low pH. The species L. fermentum and L. plantarum have been reported in several studies as among the most frequent species contaminating this environment. Lactobacillus contaminants are in constant interaction with the yeast strain which consequently has its fermentative efficiency reduced. This work aimed to analyze the metabolites produced during industrial alcoholic fermentation and that can be used by the contaminating bacteria, thus obtaining conditions to be competitive and persistent in the process. For this, two strains were used, isolated from distillery and identified as L. fermentum (I3a) with obligate heterofermentative metabolism and L. plantarum (I4a) with facultative heterofermentative metabolism, presenting a homofermentative metabolism under the conditions studied. Both strains were submitted to growth in the presence of glycerol, malate and pyruvate, which are metabolites produced and excreted by yeast and mannitol produced and excreted by the obligate heterofermentative bacterium. It was observed that the metabolite mannitol is an efficient source of carbon for both strains providing growth even without the presence of sugars. In addition, the combination of glucose, fructose, mannitol and malate was able to increase strains growth. However, the presence of pyruvate presented growth stimulus for the heterofermentative strain. In relation to consumption, the strains were able to metabolize mannitol, malate and pyruvate, however, they did not present glycerol consumption. Thus, both strains are benefited by yeast metabolism and the heterofermentative can reabsorb mannitol when the fermentable sugars are depleted and to make the metabolite available for homofermentative use.
The stress imposed by ethanol to Saccharomyces cerevisiae cells are one of the most challenging limiting factors in industrial fuel ethanol production. Consequently, the toxicity and tolerance to high ethanol concentrations has been the subject of extensive research, allowing the identification of several genes important for increasing the tolerance to this stress factor. However, most studies were performed with well-characterized laboratory strains, and how the results obtained with these strains work in industrial strains remains unknown. In the present work, we have tested three different strategies known to increase ethanol tolerance by laboratory strains in an industrial fuel–ethanol producing strain: the overexpression of the TRP1 or MSN2 genes, or the overexpression of a truncated version of the MSN2 gene. Our results show that the industrial CAT-1 strain tolerates up to 14% ethanol, and indeed the three strategies increased its tolerance to ethanol. When these strains were subjected to fermentations with high sugar content and cell recycle, simulating the industrial conditions used in Brazilian distilleries, only the strain with overexpression of the truncated MSN2 gene showed improved fermentation performance, allowing the production of 16% ethanol from 33% of total reducing sugars present in sugarcane molasses. Our results highlight the importance of testing genetic modifications in industrial yeast strains under industrial conditions in order to improve the production of industrial fuel ethanol by S. cerevisiae.
A suplementação de fontes nitrogenadas pode contribuir para maior tolerância de linhagens de S. cerevisiae frente às diversas condições deletérias, como as que são enfrentadas no processo brasileiro de produção de etanol. O presente estudo avaliou a influência da suplementação de aminoácidos sob o crescimento e a viabilidade celular da linhagem industrial CAT-1 em condições de estresse etanólico e osmótico (meio YNB com 10 e 12% v/v de etanol e mosto de melaço com 15, 20, 25 e 30% de ART). A suplementação de aminoácidos também foi avaliada em fermentações de melaço e xarope de cana-de-açúcar, empregando reciclo (reaproveitamento) de células. Os resultados revelaram que a suplementação com aminoácidos ocasionou efeitos distintos no comportamento fisiológico da levedura de acordo com o meio/mosto fornecido. A suplementação com histidina favoreceu a linhagem CAT-1 para maior crescimento e viabilidade em mostos provenientes tanto de melaço quanto de xarope. Os resultados revelaram que a suplementação de 200 mg.L-1 de nitrogênio amínico proveniente de aminoácidos, adicionada aos diferentes mostos, pode favorecer ou depreciar o crescimento e viabilidade da linhagem CAT-1 em fermentações simulando as condições industriais brasileiras. A suplementação com histidina se mostrou a mais promissora para o aumento da tolerância da linhagem industrial CAT-1.
The stress imposed by ethanol to Saccharomyces cerevisiae cells are one of the most challenging limiting factors in industrial fuel-ethanol production. Consequently, the toxicity and tolerance to high ethanol concentrations has been the subject of extensive research, allowing the identification of several genes important for increasing the tolerance to this stress factor. However, most studies were performed with well characterized laboratory strains, and how the results obtained with these strains work in industrial strains remains unknown. In the present work we have tested three different strategies known to increase ethanol tolerance by laboratory strains in an industrial fuel-ethanol producing strain: overexpression of the TRP1 or MSN2 genes, or overexpression of a truncated version of the MSN2 gene. Our results show that the industrial CAT-1 strain tolerates up to 14% ethanol, and indeed the three strategies increased its tolerance to ethanol. When these strains were subjected to fermentations with high sugar content and cell-recycle, simulating the industrial conditions used in Brazilian distilleries, only the strain with overexpression of the truncated MSN2 gene showed improved fermentation performance, allowing the production of 16% ethanol from 33% of total reducing sugars present in sugarcane molasses. Our results highlight the importance of testing genetic modifications in industrial yeast strains under industrial conditions in order to improve the production of industrial fuel ethanol by S. cerevisiae.
Interação entre leveduras e bactérias láticas no contexto da fermentação alcoólica brasileira: fisiologia, proteômica e metabolômica / Mariane Soares Raposo. --versão revisada de acordo com a Resolução CoPGr 6018 de 2011. --Piracicaba, 2023. 122 p. Tese (Doutorado) --
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