Glycolytic flux regulation in Saccharomyces cerevisiae during anaerobic growth and starvation

The physiology of S. cerevisiae under anaerobic growth conditions is of interest not least during implementation and development of industrial yeast-catalysed ethanol fermentations in order to maintain a productive yeast population. During growth in industrial fermentations the concentration of lactic acid can often reach considerable concentrations as a result of contamination by lactic acid bacteria. The yeast also often faces complex nutritional conditions including nutrient limitation and/or starvation. The effects of lactic or benzoic acid on glycolytic rate and product yields were studied on cells growing under carbon or nitrogen limiting conditions in anaerobic chemostat cultures (D=0.1 h-1). It was shown that during growth under nitrogen limiting conditions compared to growth under carbon limiting conditions and/or in presence of lactic acid or benzoic acid, the flow of glucose was directed to a larger extent from glycogen and biomass formation towards ethanol production. The specific ethanol production rate (mmol/gh) was also stimulated; however, the effect of lactic acid was not as large as that of benzoic acid or as the effect of growth under nitrogen limitation compared to growth under carbon limitation. High glycolytic rates/specific ethanol production rates were obtained by the weak organic acids and/or nitrogen limitation. Based on comparisons of exhibited glycolytic rate with levels of allosteric effectors of the glycolytic enzymes during the different growth conditions it was suggested that a concerted action of decreased ATP levels and increased fructose-6-phosphate levels may account for at least a part of the increase glycolytic rate during these growth conditions. Studies on the carbon or nitrogen starvation response of anaerobically growing cells were also performed. Carbon starvation of anaerobic exponentially growing cells induced an almost complete loss of fermentative capacity and viability. Nitrogen starvation reduced fermentative capacity by 70-95% depending on strain. The levels of glycolytic enzymes were not altered neither after carbon nor nitrogen starvation, however, a depletion of ATP was seen immediately upon carbon starvation but not nitrogen starvation. Growth into stationary-phase prior to carbon starvation enabled the cells to retain most of their fermentative capacity after carbon starvation. Reduction in protein synthesis prior to starvation of anaerobic exponential-phase cells could also to some extent alleviate the effects of carbon starvation. Further studies on cells grown in chemostat (D=0.1h-1) under different growth conditions that were subsequently exposed to carbon or nitrogen starvation showed that there was a positive relationship between cellular content of endogenous glycogen before carbon starvation and fermentative capacity after carbon starvation. A positive relationship after carbon starvation between the intracellular ATP levels (0-1.5 mM) and the fermentative capacity in cells grown in anaerobic chemostat cultures was also found. From these results it is suggested that anaerobically grown cells not containing any endogenous carbohydrate reserves face a lack of energy upon rapid carbon starvation which renders necessary adjustments to the starvation conditions impossible to perform, thereby reducing viability and fermentative capacity of the cell upon carbon starvation

Starvation Response of Saccharomyces cerevisiae Grown in Anaerobic Nitrogen- or Carbon-Limited Chemostat Cultures

Anaerobic starvation conditions are frequent in industrial fermentation and can affect the performance of the cells. In this study, the anaerobic carbon or nitrogen starvation response of Saccharomyces cerevisiae was investigated for cells grown in anaerobic carbon or nitrogen-limited chemostat cultures at a dilution rate of 0.1 h(−1) at pH 3.25 or 5. Lactic or benzoic acid was present in the growth medium at different concentrations, resulting in 16 different growth conditions. At steady state, cells were harvested and then starved for either carbon or nitrogen for 24 h under anaerobic conditions. We measured fermentative capacity, glucose uptake capacity, intracellular ATP content, and reserve carbohydrates and found that the carbon, but not the nitrogen, starvation response was dependent upon the previous growth conditions. All cells subjected to nitrogen starvation retained a large portion of their initial fermentative capacity, independently of previous growth conditions. However, nitrogen-limited cells that were starved for carbon lost almost all their fermentative capacity, while carbon-limited cells managed to preserve a larger portion of their fermentative capacity during carbon starvation. There was a positive correlation between the amount of glycogen before carbon starvation and the fermentative capacity and ATP content of the cells after carbon starvation. Fermentative capacity and glucose uptake capacity were not correlated under any of the conditions tested. Thus, the successful adaptation to sudden carbon starvation requires energy and, under anaerobic conditions, fermentable endogenous resources. In an industrial setting, carbon starvation in anaerobic fermentations should be avoided to maintain a productive yeast population

Characterisation of Yeasts isolated from deep igneous rock aquifers of the fennoscandian shield

The diversity of prokaryotes in the groundwater deep below the surface of the Baltic Sea at the Aspo Hard Rock Laboratory (HRL) in southeast Sweden is well documented. In addition, there is some evidence that eukaryotes, too, are present in the deep groundwater at this site, although their origins are uncertain. To extend the knowledge of eukaryotic life in this environment, five yeast, three yeastlike, and 17 mold strains were isolated from Aspo HRL groundwater between 201 and 444 m below sea level. Phenotypic testing and phylogenetic analysis of 18S rDNA sequences of the five yeast isolates revealed their relationships to Rhodotorula minuta and Cryptococcus spp. Scanning and transmission electron microscopy demonstrated that the strains possessed morphological characteristics typical for yeast, although they were relatively small, with an average length of 3 mum. Enumeration through direct counting and most probable number methods showed low numbers of fungi, between 0.01 and 1 cells mL(-1), at some sites. Five of the strains were characterized physiologically to determine whether they were adapted to life in the deep biosphere. These studies revealed that the strains grew within a pH range of 4-10, between temperatures of 4degreesC and 25-30degreesC, and in NaCl concentrations from 0 to 70 g L-1. These growth parameters suggest a degree of adaptation to the groundwater at Aspo HRL. Despite the fact that these eukaryotic microorganisms may be transient members of the deep biosphere microbial community, many of the observations of this study suggest that they are capable of growing in this extreme environment

Carbon Starvation Can Induce Energy Deprivation and Loss of Fermentative Capacity in Saccharomyces cerevisiae

Seven different strains of Saccharomyces cerevisiae were tested for the ability to maintain their fermentative capacity during 24 h of carbon or nitrogen starvation. Starvation was imposed by transferring cells, exponentially growing in anaerobic batch cultures, to a defined growth medium lacking either a carbon or a nitrogen source. After 24 h of starvation, fermentative capacity was determined by addition of glucose and measurement of the resulting ethanol production rate. The results showed that 24 h of nitrogen starvation reduced the fermentative capacity by 70 to 95%, depending on the strain. Carbon starvation, on the other hand, provoked an almost complete loss of fermentative capacity in all of the strains tested. The absence of ethanol production following carbon starvation occurred even though the cells possessed a substantial glucose transport capacity. In fact, similar uptake capacities were recorded irrespective of whether the cells had been subjected to carbon or nitrogen starvation. Instead, the loss of fermentative capacity observed in carbon-starved cells was almost surely a result of energy deprivation. Carbon starvation drastically reduced the ATP content of the cells to values well below 0.1 μmol/g, while nitrogen-starved cells still contained approximately 6 μmol/g after 24 h of treatment. Addition of a small amount of glucose (0.1 g/liter at a cell density of 1.0 g/liter) at the initiation of starvation or use of stationary-phase instead of log-phase cells enabled the cells to preserve their fermentative capacity also during carbon starvation. The prerequisites for successful adaptation to starvation conditions are probably gradual nutrient depletion and access to energy during the adaptation period