Modelling the growth and ethanol production of Brettanomyces bruxellensis at different glucose concentrations.

AIM: To study the effect of glucose concentrations on the growth by Brettanomyces bruxellensis yeast strain in batch experiments and develop a mathematical model for kinetic behaviour analysis of yeast growing in batch culture. METHODS AND RESULTS: A Matlab algorithm was developed for the estimation of model parameters. Glucose fermentation by B. bruxellensis was studied by varying its concentration (5, 9.3, 13.8, 16.5, 17.6 and 21.4%). The increase in substrate concentration up to a certain limit was accompanied by an increase in ethanol and biomass production; at a substrate concentration of 50-138 g l(-1), the ethanol and biomass production were 24, 59 and 6.3, 11.4 g l(-1), respectively. However, an increase in glucose concentration to 165 g l(-1) led to a drastic decrease in product formation and substrate utilization. CONCLUSIONS: The model successfully simulated the batch kinetic observed in all cases. The confidence intervals were also estimated at each phase at a 0.95 probability level in a t-Student distribution for f degrees of freedom. The maximum ethanol and biomass yields were obtained with an initial glucose concentration of 138 g l(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: These experiments illustrate the importance of using a mathematical model applied to kinetic behaviour on glucose concentration by B. bruxellensis.

Modelling the growth dynamics of interacting mixed cultures: a case of amensalism.

In this work, a specific membrane bioreactor was used to perform co-cultures of two Saccharomyces cerevisiae yeast strains: a killer strain and a sensitive strain. Biomass could be segregated into four groups: viable killer yeasts, dead killer yeasts, viable sensitive yeasts and dead sensitive yeasts. An existing mathematical model describing the population dynamics in the mixed killer/sensitive cultures was confronted with the new experimental data. As it gave poor accuracy, some improvements were proposed and tested. In particular, a lag phase before the beginning of the lethal interaction between the two strains was introduced, in correspondence to the experimental observations.

Metabolic transition step from ethanol consumption to sugar/ethanol consumption by Saccharomyces cerevisiae.

The metabolic pathway shift between only ethanol consumption to both sugar/ethanol consumption was measured by on-line analysis of respiratory quotient of a Saccharomyces cerevisiae. The experiments were carried out in a fed-batch culture under aerobic conditions. During the transition phase, respiratory quotient (RQ) profile shows that sugar can be metabolized through the fermentative pathway even to values of RQ lower than 1.

Characterization of the metabolic shift of Saccharomyces bayanus var. uvarum by continuous aerobic culture.

Saccharomyces bayanus, being of interest for wine-making, is not as well known as S. cerevisiae and, due to many changes in the yeast classification, accurate data concerning its metabolic activity are difficult to find. In order to produce this yeast as an active dry yeast to be used as a starter in wine-making, its sensitivity to glucose was determined as the objective of our work. Using the pulse technique in continuous culture, it was found that growth in a synthetic medium was not limited by vitamins or mineral salts. We determined the critical dilution rate of a continuous culture and performed an aerobic continuous culture, measuring the respiratory quotient on-line in order to observe the metabolic shift from respiratory to fermentative metabolism. The S. bayanus var. uvarum strain studied was Crabtree-positive (glucose-sensitive) but had a weaker respiratory capacity than S. cerevisiae since the dilution rate of the metabolic shift was only 0.15 h(-1). These new data provide essential information for the biomass production of this yeast strain for wine-making.

Brettanomyces bruxellensis: effect of oxygen on growth and acetic acid production.

The influence of the oxygen supply on the growth, acetic acid and ethanol production by Brettanomyces bruxellensis in a glucose medium was investigated with different air flow rates in the range 0-300 l h(-1 ) x (0-0.5 vvm). This study shows that growth of this yeast is stimulated by moderate aeration. The optimal oxygen supply for cellular synthesis was an oxygen transfer rate (OTR) of 43 mg O(2) l(-1) x h(-1). In this case, there was an air flow rate of 60 l h(-1) (0.1 vvm). Above this value, the maximum biomass concentration decreased. Ethanol and acetic acid production was also dependent on the level of aeration: the higher the oxygen supply, the greater the acetic acid production and the lower the ethanol production. At the highest aeration rates, we observed a strong inhibition of the ethanol yield. Over 180 l h(-1) x (0.3 vvm, OTR =105 mg O(2) l(-1) x h(-1)), glucose consumption was inhibited and a high concentration of acetic acid (6.0 g x l(-1)) was produced. The ratio of "ethanol + acetic acid" produced per mole of consumed glucose using carbon balance calculations was analyzed. It was shown that this ratio remained constant in all cases. This makes it possible to establish a stoichiometric equation between oxygen supply and metabolite production.

Nutritional requirements of Brettanomyces bruxellensis: growth and physiology in batch and chemostat cultures.

The nutritional requirements of Brettanomyces bruxellensis have been investigated. Batch culture and chemostat pulse techniques were used to identify growth-limiting nutrients. The study included determination of the essential components of the culture medium and quantification of the effects of the components. Among the components tested, ammonium sulfate and yeast extract had a significant effect on glucose consumption, growth, and ethanol production. However, if the ammonium sulfate concentration is above 2 g/L, an inhibitory effect on B. bruxellensis growth is observed. The yeast extract appears to be the most important and significant component for growth. The maximum amount of synthesized biomass is proportional to the concentration of yeast extract added to the culture broth (in the tested range). Magnesium and phosphate ions are probably not essential for B. bruxellensis. These ions appear to be supplied in sufficient amounts by the yeast extract in the culture medium. Brettanomyces bruxellensis appears to have very low nutritional requirements for growth.

Stoichiometry of diauxic growth of a xylanase-producing Bacillus strain.

In this work, the establishment of material balances and stoichiometry of the growth of Bacillus sp. was undertaken. This strain produces high quantities of a xylanase suitable for use as bleach boost agent in chlorine-free bleaching sequences of paper pulp. As carbon dioxide plays an important role as a growth factor, bacterial growth in two fermentations, one fed with air and another fed with carbon-dioxide-enriched air, were compared. For this purpose, a method permitting the determination of the consumption of the two carbon sources, xylan and peptone, was proposed. The material balances revealed that in both cases, the bacteria first use peptone as their carbon source, and then xylan in the second part of the growth phase. The aerated culture showed diauxic growth on these two substrates, whereas carbon-dioxide-enriched air caused disappearance of the metabolic adaptation phase, and rendered biomass production more economic. The fermentation fed with air needed 30% more xylan than the fermentation fed with carbon-dioxide-enriched air for the same quantity of biomass produced.

Oxygen effect on lactose catabolism by a Leuconostoc mesenteroides strain: modeling of general O2-dependent stoichiometry.

Lactose metabolism of a Leuconostoc mesenteroides strain was studied in batch cultures at a pH of 6.5 and 30 degrees C in 10 L of a modified MRS (De Man, Rogosa, Sharp) broth. The end products of this heterolactic bacterium were D-lactate, acetate, ethanol, and carbon dioxide. To test the effect of oxygen on their synthesis, the medium was sparged with different gases: nitrogen, air, and pure oxygen. When oxygen was available, oxygen uptake occurred, which caused a modification in acetate and ethanol production but not in lactate or carbon dioxide production; acetate plus ethanol together were produced in constant amounts, which were independent of the level of aeration. The influence of oxygen on end-product formation could be summed up by the general equation: lactose + x O(2) --> 2 D-lactate + (x + 0.1) acetate + (2 - x) ethanol + 2 CO(2). Maximal oxygen uptake (x = 2) was reached under a 120 L/h flow rate of pure oxygen. In addition, this equation provided useful information on the possible pathway of galactose catabolism by a heterofermentative microorganism.

Influence of pH, malic acid and glucose concentrations on malic acid consumption by Saccharomyces cerevisiae.

Malic acid consumption by Saccharomyces cerevisiae was studied in a synthetic medium. The extent of malic acid degradation is affected by its initial concentration, the extent and the rate of deacidification increased with initial malate concentration up to 10 milligrams. For malic acid consumption, an optimal pH range of 3-3.5 was found, confirming that non-dissociated organic acids enter S. cerevisiae cells by simple diffusion. A full factorial design has been employed to describe a statistical model of the effect of sugar and malic acid on the quantity of malate degraded (milligrams) by a given amount of biomass (milligrams). The results indicated that the initial malic acid concentration is very important for the ratio of malate consumption to quantity of biomass. The yeast was found to be most efficient at higher levels of malate.

Alcoholic fermentation: modelling based on sole substrate and product measurement.

Optimal automatic bioreactor control requires a mathematical model adapted to the potency of reliable sensors. A new relationship describing the kinetic behavior of alcoholic fermentation is discussed. By analogy with chemical kinetics, the biological rate of substrate consumption is related to substrate and product concentration by the following equation: r(s) = kS(alpha)P(beta) Using the well known yield relation between product and substrate, it is possible to describe in both batch and continuous cultures the ethanol and sugar concentrations versus time. This pattern has been successfully tested on several fermentations performed by yeasts (S. cerevisiae, S. bayanus, and S. cerevisiae sake) and a bacterium (Z. mobilis). This simple relationship is proposed as a tool for process control alcoholic fermentation.