Participation of CWI, HOG and Calcineurin pathways in the tolerance of Saccharomyces cerevisiae to low pH by inorganic acid.

AIMS: The present work aimed at identifying the metabolic response to acid stress and the mechanisms that lead to cell tolerance and adaptation. METHODS AND RESULTS: Two strategies were used: screening deletion mutants for cell growth at neutral and acid pH compared to wild type and measurement by qPCR of the expression of yeast genes involved in different pathways. CONCLUSIONS: The results complement our previous findings and showed that the Cell Wall Integrity pathway is the main mechanism for cell tolerance to acid pH, and this damage triggers the protein kinase C (PKC) pathway mainly via the Wsc1p membrane sensor. In addition, cell wall injury might mimic the effects of high osmotic shock and activates the High Osmolarity Glycerol pathway, which amplifies the signal in the upper part of PKC pathway and leads to the activation of Ca(2+) channels by SLT2 overexpression and this Ca(2+) influx further activates calcineurin. Together, these mechanisms induce the expression of genes involved in cell cycle regulation and cell wall regeneration. SIGNIFICANCE AND IMPACT OF THE STUDY: These interactions are responsible for long-term adaptation of yeast cells to the acidic environment, and the results could drive future work on the genetic modification of yeast strains for high tolerance to the stresses of the bioethanol fermentation process.

Physiological and molecular analysis of the stress response of Saccharomyces cerevisiae imposed by strong inorganic acid with implication to industrial fermentations.

AIMS: This work aimed to identify the molecular mechanism that allows yeast cells to survive at low pH environments such as those of bioethanol fermentation. METHODS AND RESULTS: The industrial strain JP1 cells grown at pH 2 was evaluated by microarray analysis showing that most of the genes induced at low pH were part of the general stress response (GSR). Further, an acid-tolerant yeast mutant was isolated by adaptive selection that was prone to grow at low pH in inorganic but weak organic acid. It showed higher viability under acid-temperature synergistic treatment. However, it was deficient in some physiological aspects that are associated with defects in protein kinase A (PKA) pathway. Microarray analysis showed the induction of genes involved in inhibition of RNA and protein synthesis. CONCLUSIONS: The results point out that low pH activates GSR, mainly heat shock response, that is important for long-term cell survival and suggest that a fine regulatory PKA-dependent mechanism that might affect cell cycle in order to acquire tolerance to acid environment. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings might guide the construction of a high-fermentative stress-tolerant industrial yeast strain that can be used in complex industrial fermentation processes.

Chromosome instability in industrial strains of Saccharomyces cerevisiae batch cultivated under laboratory conditions.

Industrial ethanol fermentation is a complex microbiological process to which yeast cells must adapt for survival. One of the mechanisms for adaptation is thought to involve chromosome rearrangements. We found that changes in chromosome banding patterns measured by pulsed-field gel electrophoresis can also be produced in laboratory media under simulated industrial conditions. Based on analysis of their generational variation, we found that these chromosome changes were specific to the genetic backgrounds of the initial strains. We conclude that chromosome rearrangements could be one of the factors involved in yeast cell adaptation to the industrial environment.

Detection and identification of wild yeast contaminants of the industrial fuel ethanol fermentation process.

Monitoring for wild yeast contaminants is an essential component of the management of the industrial fuel ethanol manufacturing process. Here we describe the isolation and molecular identification of 24 yeast species present in bioethanol distilleries in northeast Brazil that use sugar cane juice or cane molasses as feeding substrate. Most of the yeast species could be identified readily from their unique amplification-specific polymerase chain reaction (PCR) fingerprint. Yeast of the species Dekkera bruxellensis, Candida tropicalis, Pichia galeiformis, as well as a species of Candida that belongs to the C. intermedia clade, were found to be involved in acute contamination episodes; the remaining 20 species were classified as adventitious. Additional physiologic data confirmed that the presence of these major contaminants cause decreased bioethanol yield. We conclude that PCR fingerprinting can be used in an industrial setting to monitor yeast population dynamics to early identify the presence of the most important contaminant yeasts.

Identification of Dekkera bruxellensis as a major contaminant yeast in continuous fuel ethanol fermentation.

AIMS: To identify and characterize the main contaminant yeast species detected in fuel-ethanol production plants in Northeast region of Brazil by using molecular methods. METHODS AND RESULTS: Total DNA from yeast colonies isolated from the fermentation must of industrial alcohol plants was submitted to PCR fingerprinting, D1/D2 28S rDNA sequencing and species-specific PCR analysis. The most frequent non-Saccharomyces cerevisiae isolates were identified as belonging to the species Dekkera bruxellensis, and several genetic strains could be discriminated among the isolates. The yeast population dynamics was followed on a daily basis during a whole crop harvesting period in a particular industry, showing the potential of D. bruxellensis to grow faster than S. cerevisiae in industrial conditions, causing recurrent and severe contamination episodes. CONCLUSIONS: The results showed that D. bruxellensis is one of the most important contaminant yeasts in distilleries producing fuel-ethanol from crude sugar cane juice, specially in continuous fermentation systems. SIGNIFICANCE AND IMPACT OF THE STUDY: Severe contamination of the industrial fermentation process by Dekkera yeasts has a negative impact on ethanol yield and productivity. Therefore, early detection of D. bruxellensis in industrial musts may avoid operational problems in alcohol-producing plants.

Contaminant yeast detection in industrial ethanol fermentation must by rDNA-PCR.

AIMS: The present work focuses on the possibility to use conserved primers that amplify yeast ITS1-5.8S-ITS2 ribosomal DNA locus (rDNA) to detect the presence of non-Saccharomyces cerevisiae yeast in fermentation must of bioethanol fermentation process. METHODS AND RESULTS: Total DNA was extracted from pure or mixed yeast cultures containing different cell concentrations and different contaminant/fermenting yeast concentrations and submitted to PCR. Upon improvement of detection limits and DNA extraction protocol, must samples of distillery were checked for the presence of contaminant yeast. Contaminant rDNA bands were detected only in industrial samples during contamination episodes, but not in noncontaminated must. CONCLUSIONS: The method described here could detect the presence of contaminant yeast from industrial must in eight hours after sampling. SIGNIFICANCE AND IMPACT OF THE STUDY: The improved procedure may help to avoid severe contamination episodes at fermentation industries by decreasing the detection time from 5 days to 8 h and possible quantification of contaminant yeasts that can impose economical loss to the process.

Chromosome instability in industrial strains of Saccharomyces cerevisiae batch cultivated under laboratory conditions

Industrial ethanol fermentation is a complex microbiological process to which yeast cells must adapt for survival. One of the mechanisms for adaptation is thought to involve chromosome rearrangements. We found that changes in chromosome banding patterns measured by pulsed-field gel electrophoresis can also be produced in laboratory media under simulated industrial conditions. Based on analysis of their generational variation, we found that these chromosome changes were specific to the genetic backgrounds of the initial strains. We conclude that chromosome rearrangements could be one of the factors involved in yeast cell adaptation to the industrial environment.