Nowadays, yeast biomass propagation of wine, distiller’s and brewer’s yeasts are usually produced in baker’s yeast plants. The procedure is designed as a multistage-based fermentation, previously defined for the production of baker’s yeast (Chen and Chiger, 1985; Reed and Nagodawithana, 1991) using supplemented molasses as growth media. The first stage (F1) is initiated with a flask culture containing molasses, which is inoculated with the selected yeast strain. Production cultures may be periodically renewed from the stock cultures maintained under more stringent control procedures in a central quality control laboratory. Then, the initial culture is used to inoculate the first fermentor, and cells grow in various transient stages during the batch (F2-F4) and fed-batch (F5-F6) phases of the process. In a sequence of consecutive fermentations, the yeast biomass grown in small fermentors is used to inoculate larger tanks (Reed, 1982; Chen and Chiger, 1985; Reed and Nagodawithana, 1991; Degre, 1993).

In the initial batch phase (F2), cells are exposed to the high sugars concentration present in molasses. All the other nutrients are also present in the fermentor, and pH must be adjusted to 4.5-5.0 after sterilisation to be then monitored during batch fermentation. Once the batch phase has started, the only controllable parameters are temperature and aeration. Yeast propagation typically involves continuous aeration or oxygenation, but a relatively short aeration period has been suggested to suffice (Maemura et al., 1998). However the presence of O2 from the beginning of the process allows yeast cells to synthesise lipids, thereby revitalising the sterol-deficient cell population and ensuring that fermentation can proceed efficiently. Besides, those propagation experiments carried out in non-oxygenated media considerably reduce yeast growth and increase internal oxidative stress (Boulton, 2000; Perez-Torrado et al., 2009).

During batch fermentation (F2-F4), a growth lag phase takes place in which cells synthesise the enzymes involved in gluconeogenesis and the glyoxylate cycle (Haarasilta and Oura, 1975). During the subsequent exponential phase, a very small amount of glucose is oxidised in the mitochondria, but when the sugar concentration drops below a strain-specific level or the specific growth rate in aerobic cultures exceeds a critical value (pcrit), a mixed respiro — fermentative metabolism occurs. This phenomenon has been described as the "Crabtree effect" (De Deken, 1966; Pronk et al., 1996) and was originally considered a consequence of the catabolite repression and limited respiratory capacity of S. cerevisiae (Postma et al., 1989; Alexander and Jeffries, 1990).It has also been suggested that there is no limitation in the respiratory capacity, as can be deduced from the increased respiratory capacity displayed by a PGK-overproducing mutant, indicating that the activity of respiration itself is not saturated and suggesting that it is not the main cause triggering ethanol production and inducing the long-term Crabtree effect (Van der Aar et al., 1990). However, more recent works have showed that Crabtree effect is derived from the limited mitochondrial capacity to absorb the NADH produced in the glycolysis (Vemuri et al., 2007).

Alcoholic fermentation leads to a suboptimal biomass concentration because the ATP yield is much lower than the yield obtained during respiratory carbohydrate degradation (Verduyn, 1991; Rizzi et al., 1997). However, pre-adaptation to large amounts of glucose during the batch phase is necessary to ensure the produced biomass’ optimal fermentative capacity by accumulating several necessary reserve metabolites to be used in the fed — batch phase (Dombek and Ingram, 1987; Rizzi et al., 1997; Perez-Torrado et al., 2009). In addition, prolonged growth in aerobic, glucose-limited chemostat cultures of S. cerevisiae, avoiding the batch phase, causes a partial loss of glycolytic capacity (Jansen et al., 2005). The presence of O2 during the process also allows yeast to oxidise alcoholic fermentation — produced ethanol when sucrose is exhausted, which triggers the metabolism to change from fermentation to respiration, and eliminates ethanol from the media. When ethanol is exhausted, the fed-batch phase starts (F5-F6). In the transition to the respiratory phase, an increase in the cAMP levels triggers the breakdown of storage carbohydrates and an increased influx of glucose into the glycolytic pathway. The resulting increase in the NAD+/NADH ratio stimulates respiration in combination with a drop in the ATP level, which is consumed mainly during biomass formation (Perez-Torrado, 2004; Xu and Tsurugi, 2006; Perez-Torrado et al., 2009). In some industrial wine yeast production plants, fed-batch phases are initiated without consuming ethanol from the growth media, which considerably reduces the biomass yield.

Optimisation of biomass productivity requires an increase in both the specific growth rate and the biomass yield during the fed-batch phase to the highest values possible under sugar-limited cultivation. Generally, the growth rate profile during fed-batch cultivation is controlled primarily by the carbohydrate feedstock feed rate (Beudeker et al., 1990). The control of optimum dissolved oxygen during the fed-batch phase is also essential to obtain a high biomass yield, and important studies have been done to optimise aeration control (Blanco et al., 2008). Therefore sugar-limited cultivation in the presence of O2 allows the full respiratory growth of S. cerevisiae, achieving much higher biomass yields than during the batch phase (Postma et al., 1989). If the only objective is to maximise the biomass concentration starting with a sufficiently concentrated inoculum from the batch phase, it is necessary to grow cells at a rate as close to the critical growth rate as possible (pcrit), which depends exclusively on the yeast strain (Valentinotti et al., 2002), avoiding ethanol and acetate formation. Many of the parameters that have an impact on yeast’s metabolic activities have to be controlled (Miskiewicz and Borowiak, 2005). The pH and temperature are important parameters to be controlled during this phase: maintaining pH constantly at around 4.5 by adjusting the pH automatically with acid/base solutions, and maintaining temperature at 30°C. Properly designed final fed-batch fermentations should also permit yeast cells maturation. This can be accomplished by stopping the feeding of nutrients at the end of fermentation, but allowing slight aeration to continue for an hour (Oura et al., 1974). During this period, the substrate is completely assimilated and allows ripened cells to become more stable and avoids autolysis.

Many research efforts have focused on optimising fed-batch processes for baker’s yeast production with different aims (productivity, yeast quality, or energy saving) and most have been commonly done under laboratory conditions (Van Hoek et al., 1998; Van Hoek et al., 2000; Jansen et al., 2005; Henes and Sonnleitner, 2007; Cheng et al., 2008), but rarely under pilot plant conditions (Di Serio et al., 2001; Lei et al., 2001; Gibson et al., 2007; Gibson et al.,

2008) . They have all been designed to mainly analyse the fed-batch phase without considering the whole process. The first published study on the complete industrial process was the simulation of wine yeast biomass propagation by performing batch and fed-batch phases in only one bioreactor (Perez-Torrado et al., 2005). This simplification of the process enabled the study of yeast physiology from a molecular point of view with a bench-top design (Fig. 1), whose results display a good correlation with those obtained from pilot plants and this set of parameters for further investigation.

Fig. 1. Diagram of the different stages in the industrial yeast biomass propagation process.

The parameters employed throughout the process (sucrose and ethanol production / consumption, dissolved O2, cell density and feed rate) have been adapted from Gomez — Pastor et al., 2010b. The lower panel shows representative cellular states, along with the most relevant metabolites, proteins and gene expressions throughout biomass propagation.