Kinetic analysis of a Saccharomyces cerevisiae strain adapted for improved growth on glycerol: Implications for the development of yeast bioprocesses on glycerol.

Glycerol is an agro-industrial residue generated in high amounts during the biodiesel production. The growing production of biodiesel is creating a worldwide glycerol surplus. Therefore, replacing sugar-based feedstock in bioprocesses by glycerol could be potentially attractive. Saccharomyces cerevisiae is one of the most commonly used microorganisms in the agri-food industry and therefore currently produced in large quantities from sugar-based feedstock. Unfortunately, growth of S. cerevisiae strains on glycerol is very low with reported µmax around 0.01 h(-1). This study demonstrates that successive growth of the S. cerevisiae CBS 8066, CEN.PK 113-7 D and Ethanol Red on glycerol as sole carbon source considerably improved the µmax from 0.01 up to 0.2 h(-1). The "adapted strain" CBS 8066-FL20 was kinetically characterized during aerobic and oxygen-limited cultivation in bioreactor and the results discussed in terms of their implication for developing glycerol-based S. cerevisiae bioprocesses.

Effect of controlled oxygen limitation on Candida shehatae physiology for ethanol production from xylose and glucose.

Carbon distribution and kinetics of Candida shehatae were studied in fed-batch fermentation with xylose or glucose (separately) as the carbon source in mineral medium. The fermentations were carried out in two phases, an aerobic phase dedicated to growth followed by an oxygen limitation phase dedicated to ethanol production. Oxygen limitation was quantified with an average specific oxygen uptake rate (OUR) varying between 0.30 and 2.48 mmolO(2) g dry cell weight (DCW)(-1) h(-1), the maximum value before the aerobic shift. The relations among respiration, growth, ethanol production and polyol production were investigated. It appeared that ethanol was produced to provide energy, and polyols (arabitol, ribitol, glycerol and xylitol) were produced to reoxidize NADH from assimilatory reactions and from the co-factor imbalance of the two-first enzymatic steps of xylose uptake. Hence, to manage carbon flux to ethanol production, oxygen limitation was a major controlled parameter; an oxygen limitation corresponding to an average specific OUR of 1.19 mmolO(2) g DCW(-1) h(-1) allowed maximization of the ethanol yield over xylose (0.327 g g(-1)), the average productivity (2.2 g l(-1) h(-1)) and the ethanol final titer (48.81 g l(-1)). For glucose fermentation, the ethanol yield over glucose was the highest (0.411 g g(-1)) when the specific OUR was low, corresponding to an average specific OUR of 0.30 mmolO(2) g DCW(-1) h(-1), whereas the average ethanol productivity and ethanol final titer reached the maximum values of 1.81 g l(-1) h(-1) and 54.19 g l(-1) when the specific OUR was the highest.

Fermentative production of L-glycerol 3-phosphate utilizing a Saccharomyces cerevisiae strain with an engineered glycerol biosynthetic pathway.

Interest in L-glycerol 3-phosphate (L-G3P) production via microbial fermentation is due to the compound's potential to replace the unstable substrate dihydroxyacetone phosphate (DHAP) in one-pot enzymatic carbohydrate syntheses. A Saccharomyces cerevisiae strain with deletions in both genes encoding specific L-G3Pases (GPP1 and GPP2) and multicopy overexpression of L-glycerol 3-phosphate dehydrogenase (GPD1) was studied via small-scale (100 mL) batch fermentations under quasi-anaerobic conditions. Intracellular accumulation of L-G3P reached extremely high levels (roughly 200 mM) but thereafter declined. Extracellular L-G3P was also detected and its concentration continuously increased throughout the fermentation, such that most of the total L-G3P was found outside the cells as fermentation concluded. Moreover, in spite of the complete elimination of specific L-G3Pase activity, the strain showed considerable glycerol formation suggesting unspecific dephosphorylation as a mechanism to relieve cells of intracellular L-G3P accumulation. Up-scaling the process employed fed-batch fermentation with repeated glucose feeding, plus an aerobic growth phase followed by an anaerobic product accumulation phase. This produced a final product titer of about 325 mg total L-G3P per liter of fermentation broth.

Very high ethanol productivity in an innovative continuous two-stage bioreactor with cell recycle.

The performance of an innovative two-stage continuous bioreactor with cell recycle-potentially capable of giving very high ethanol productivity-was investigated. The first stage was dedicated to cell growth, whereas the second stage was dedicated to ethanol production. A high cell density was obtained by an ultrafiltration module coupled to the outlet of the second reactor. A recycle loop from the second stage to the first one was tested to improve cell viability and activity. Cultivations of Saccharomyces cerevisiae in mineral medium on glucose were performed at 30 degrees Celsius and pH 4. At steady state, total biomass concentrations of 59 and 157 gDCW l(-1) and ethanol concentrations of 31 and 65 g l(-1) were obtained in the first and second stage, respectively. The residual glucose concentration was 73 g l(-1) in the first stage and close to zero in the second stage. The present study shows that a very high ethanol productivity (up to 41 g l(-1) h(-1)) can indeed be obtained with complete conversion of the glucose and with a high ethanol titre (8.3 degrees GL) in the two-stage system.

Kinetic analysis of a trehalase-overexpressing strain grown on trehalose: a new tool for respiro-fermentative transition studies in Saccharomyces cerevisiae.

AIMS: The aim was to demonstrate the use of a trehalase-overexpressing Saccharomyces cerevisiae strain grown on trehalose as a valuable tool in the studies of respiro-fermentative transition at a reduced scale. METHODS AND RESULTS: A trehalase-overexpressing strain was cultivated in synthetic medium on trehalose under aerobic conditions. This strain grew at a maximum specific growth rate of 0.16 h(-1) and showed a pure oxidative metabolism. Glucose pulse experiments were carried out in this system in order to quantify the short-term Crabtree effect. These data were then compared with glucose pulse experiments carried out in the conventional way with the wild-type strain in glucose-limited chemostats. Glucose-pulse experiments in aerobic batch cultures grown on trehalose led to a metabolic respiro-fermentative transition similar to the one observed in glucose-limited chemostats. CONCLUSIONS: This cultivation system allowed us to quantitatively mimic at the flask scale the Crabtree effect observed in conventional chemostat studies. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is of primary interest in S. cerevisiae studies in which: (i) the implementation of oxidative growth is required (as with studies of the Crabtree effect and heterologous protein production); (ii) small-scale culture systems are required (e.g. high-throughput mutant screening and isotopic labelling experiments).

Synergistic temperature and ethanol effect on Saccharomyces cerevisiae dynamic behaviour in ethanol bio-fuel production.

The impact of ethanol and temperature on the dynamic behaviour of Saccharomyces cerevisiae in ethanol biofuel production was studied using an isothermal fed-batch process at five different temperatures. Fermentation parameters and kinetics were quantified. The best performances were found at 30 and 33 degrees C around 120 g l(-1) ethanol produced in 30 h with a slight benefit for growth at 30 degrees C and for ethanol production at 33 degrees C. Glycerol formation, enhanced with increasing temperatures, was coupled with growth for all fermentations; whereas, a decoupling phenomenon occurred at 36 and 39 degrees C pointing out a possible role of glycerol in yeast thermal protection.

Aeration strategy: a need for very high ethanol performance in Saccharomyces cerevisiae fed-batch process.

In order to identify an optimal aeration strategy for intensifying bio-fuel ethanol production in fermentation processes where growth and production have to be managed simultaneously, we quantified the effect of aeration conditions--oxygen limited vs non limited culture (micro-aerobic vs aerobic culture)--on the dynamic behaviour of Saccharomyces cerevisiae cultivated in very high ethanol performance fed-batch cultures. Fermentation parameters and kinetics were established within a range of ethanol concentrations (up to 147 g l(-1)), which very few studies have addressed. Higher ethanol titres (147 vs 131 g l(-1) in 45 h) and average productivity (3.3 vs 2.6 g l(-1) h(-1)) were obtained in cultures without oxygen limitation. Compared to micro-aerobic culture, full aeration led to a 23% increase in the viable cell mass as a result of the concomitant increase in growth rate and yield, with lower ethanol inhibition. The second beneficial effect of aeration was better management of by-product production, with production of glycerol, the main by-product, being strongly reduced from 12 to 4 g l(-1). We demonstrate that aeration strategy is as much a determining factor as vitamin feeding (Alfenore et al. 2002) in very high ethanol performance (147 g l(-1) in 45 h) in order to achieve a highly competitive dynamic process.

Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process.

Several bottlenecks in the alcoholic fermentation process must be overcome to reach a very high and competitive performance of bioethanol production by the yeast Saccharomyces cerevisiae. In this paper, a nutritional strategy is described that allowed S. cerevisiae to produce a final ethanol titre of 19% (v/v) ethanol in 45 h in a fed-batch culture at 30 degrees C. This performance was achieved by implementing exponential feeding of vitamins throughout the fermentation process. In comparison to an initial addition of a vitamin cocktail, an increase in the amount of vitamins and an exponential vitamin feeding strategy improved the final ethanol titre from 126 g l(-1) to 135 g l(-1) and 147 g l(-1), respectively. A maximum instantaneous productivity of 9.5 g l(-1) h(-1) was reached in the best fermentation. These performances resulted from improvements in growth, the specific ethanol production rate, and the concentration of viable cells in response to the nutritional strategy.