Yeasts have been used by humans to produce foods for thousands of years. Bread, wine, sake and beer are made with the essential contribution of yeasts, especially from the species
Nowadays, modern industries require very large amounts of selected yeasts to obtain high quality reproducible products and to ensure fast, complete fermentations. Around 0.4 million metric tonnes of yeast biomass, including 0.2 million tonnes baker's yeast alone, are produced each year worldwide. Efficient and profitable factory-scale processes have been developed to produce yeast biomass. The standard process was empirically optimised to obtain the highest yield by increasing biomass production and decreasing costs. However in recent years, several molecular and physiological studies have revealed that yeast undergoes diverse stressful situations along the biomass production process which can seriously affect its fermentative capacity and technological performance.
In this chapter, we review the yeast biomass production process, including substrates, growth configuration, yield optimisation and the particularities of brewing, baker- or wine-yeasts production. We summarise the new studies that describe the process from a molecular viewpoint to reveal yeast responses to different stressful situations. Finally, we highlight the key points to be optimised in order to obtain not only high yields, but also the best biomass fermentative efficiency, and we provide future directions in the field.
2. Molasses: A suitable substrate
Beet or cane molasses are the main substrate used in yeast production plants. These materials were selected for two main reasons: first, yeasts grow very well using the sugars present in the molasses and second, they are economically interesting since they are a waste product coming from sugar refineries without any other application. Usually, molasses contain between 65% and 75% of sugars, mainly sucrose (Hongisto and Laakso, 1978); but the composition is highly variable depending on the sucrose-refining procedure and on the weather conditions of that particular year. Sucrose is extracellularly hydrolysed by yeasts in two monosaccharides, glucose and fructose, which are transported to and incorporated into the yeast metabolism as carbon sources. However, molasses are deficient in other essential elements for yeast growth. One of them is nitrogen since its molasses content is very poor (less than 3%). Yeasts can use some of the amino acids present in molasses, but addition of nitrogen sources is needed, generally in the form of ammonium salts or urea. Magnesium and phosphate elements are also supplemented in salt forms. Finally, three vitamins (biotin, thiamine and pantothenic acid), required for fast growth, must be supplemented since their content in molasses is also very low (Oura, 1974; Woehrer and Roehr, 1981). Another negative aspect of molasses being used as a substrate to produce yeasts is the presence of different toxics that can affect yeast growth. Variable amounts of herbicides, insecticides, fungicides, fertilizers and heavy metals applied to beet or cane crops can be found in molasses and in different stocks. Moreover bactericides, which are added during sugar production in refinery plants, can be found (Reed and Nagodawithana, 1988). All these toxics can decrease yeast performance by inhibiting growth (Pérez-Torrado, 2004). In fact, a common practice in yeast plants is to mix different stocks to dilute potential toxics.
The effects of molasses composition on yeast growth have been recently analysed at molecular level by determining the transcriptional profile of yeast growing in beet molasses and by comparing it to complete synthetic media (Shima et al., 2005). The results revealed that yeast displays clear gene expression responses when grown in industrial media because of the induction of
In the last years, the price of molasses has increased because of their use in other industrial applications such as animal feeding or bioethanol production (Arshad et al., 2008; Kopsahelis et al. 2009; Xandé et al., 2010), thus rendering the evaluation of new substrates for yeast biomass propagation a trend topic for biomass producers’ research. New assayed substrates include molasses mixtures with corn steep liquor (20:80), different agricultural waste products (Vu and Kim, 2009) and other possibilities as date juice (Beiroti and Hosseini, 2007) or agricultural waste sources, also called wood molasses, that can be substrate only for yeast species capable of using xylose as a carbon source.
3. Scaling up: Bach and fed-bach
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; Pérez-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 (μcrit), 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
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; Pérez-Torrado et al., 2009). In addition, prolonged growth in aerobic, glucose-limited chemostat cultures of
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
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 (Pérez-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.
The parameters employed throughout the process (sucrose and ethanol production / consumption, dissolved O2, cell density and feed rate) have been adapted from Gómez-Pastor et al., 2010b. The lower panel shows representative cellular states, along with the most relevant metabolites, proteins and gene expressions throughout biomass propagation.
4. Desiccation of wine yeasts
In contrast to baker’s and brewer’s yeast, seasonal wine production requires the development of highly stable dry yeast products. At the end of biomass propagation, wine yeast cells are recovered and dehydrated to obtain ADY (Chen and Chiger, 1985; Degre, 1993; Gonzalez et al., 2005). After the maturation step, yeast cells are separated from fermented media by centrifugation, and are subjected to washing separations to reduce non-yeast solids, a necessary step because they affect the proper rehydration process of ADY for must fermentation. The separation process yields a slightly coloured yeast cream containing up to 22% yeast solids. After this step, the yeast cream can be stored at 4C after adjusting the pH to 3.5 to avoid microbial contaminations. The cream yeast is further dehydrated to 30-35% solids by means of rotary vacuum filters or filter presses. The filtered yeast is usually mixed with emulsifiers prior to its extrusion into yeast strands. The yeast cake is extruded through a perforated plate, while particles are loaded into the dryer and dehydrated to obtain a product with very low residual moisture. Although several types of dryers exist (roto-louvre, belt dryers, spray dryers), the one most commonly used in industry is the fluidized-bed dryer. In this dryer, heated air is blown from the bottom through yeast particles at velocities which keep them in suspension. Air is treated to reduce its water content and to ensure that the yeast temperature does not exceed 35C or 41C during drying. Drying times may vary from 15 to 60 min depending on the mass volume and the used conditions. Finally, ADY with less than 8% residual moisture is vacuum-packaged or placed in an inert atmosphere, such as nitrogen and CO2, to reduce oxidation. Depending on the strain, loss of viability is estimated at between 10% and 25% per year at 20C. For this reason, manufacturers recommend storing ADY at 4C in a dry atmosphere for a maximum 3-year period.
In order to produce an ADY product with acceptable fermentative activity and storage stability, several factors must be taken into account. The drying temperature and rate can be critical for yeast resistance to dehydration and rehydration (Beney et al., 2000; Beney et al., 2001; Laroche and Gervais, 2003). Some studies have shown that cell death during desiccation is strongly related to membrane integrity loss, leading to cell lysis during rehydration (Beney and Gervais, 2001; Laroche et al., 2001; Simonin et al. 2007; Dupont et al., 2010). A gradual dehydration kinetics, which allows a slow water efflux through the plasmatic membrane and homogenous desiccation, followed by a progressive rehydration during the starter preparation, have been related with high cell viability (Gervais et al., 1992; Gervais and Marechal, 1994¸ Dupont et al., 2010). The amount of cell constituents leaked during rehydration can also be reduced by adding emulsifiers, such as sorbitan monostearate (Chen and Chiger, 1985). Moreover, biomass propagation conditions have a major influence on yeast resistance to dehydration-rehydration. Several cultivation factors can affect cell resistance to desiccation, such as the substrate, growth phase and ion availability (Trofimova et al., 2010).
5. Yeast stress along biomass production
Several classic studies have evaluated the energy, kinetic and yield parameters of the yeast biomass production process (Reed, 1982; Chen and Chiger, 1985; Reed and Nagodawithana, 1991; Degre, 1993). However, the biochemical and molecular aspects of yeast adaptation to adverse industrial growth conditions have been poorly characterised. In recent years, a substantial effort has been made to gain insight into yeast responses during the process. It was believed that industrial conditions were optimised to obtain the best performing yeast cells, but now we know that yeast cells endure several stressful situations that induce multiple intracellular changes and challenge their technological fitness (Attfield, 1997; Pretorius, 1997; Pérez-Torrado et al., 2005). With wine yeast, moreover, the biomass is concentrated and dehydrated at the end of the process to obtain ADY yeasts that can be stored for long periods of time (Degre, 1993). Subsequently in a period of several hours during maturation and final drying processing, cells undergo nutrient limitation and a complex mixture of different stresses (thermic, osmotic, oxidative, etc.) (Garre et al., 2010). As a result, these dynamic environmental injuries seriously affect biomass yield, fermentative capacity, vitality, and cell viability (Attfield, 1997; Pretorius, 1997; Pérez-Torrado et al., 2005; Pérez-Torrado et al., 2009).
Eukaryotic cells have developed molecular mechanisms to sense stressful situations, transfer information to the nucleus and adapt to new conditions (Hohmann and Mager, 1997; Estruch, 2000; Hohmann, 2002). Protective molecules are rapidly synthesised in stressful situations and transcriptional factors are activated, thus changing the transcriptional profile of cells. Many stress response genes are induced under several adverse conditions through sequence element STRE (stress-responsive element), which targets the main transcriptional factors Msn2p and Msn4p (Kobayashi and McEntee, 1993; Martinez-Pastor et al., 1996). This pathway, also known as the “general stress response pathway”, increases the expression of many different genes, including the well-studied
Given the antiquity of yeast fermentation processes, these microorganisms have evolved in natural stressing environments, which have favoured the selection of “domesticated” yeast that displays high stress resistance (Jamieson, 1998). Studies of brewing yeast under industrial fermentations have demonstrated the suitability of the marker gene expression as a tool to study yeast stress responses in industrial processes (Higgins et al., 2003a). Monitoring stress-related marker genes, such as
As mentioned earlier, an oxygen supply is necessary to generate yeast biomass and to ensure optimal physiological conditions for effective fermentation (Chen and Chiger, 1985; Reed and Nagodawithana, 1991; Hulse, 2008). Oxygen is required for lipid synthesis, which is necessary to maintain plasma membrane integrity and function, and consequently for both cell replication and the biosynthesis of sterols and unsaturated fatty acids. Despite its potential toxicity, eliminating oxygen in the first part of the batch phase diminishes biomass yield (Boulton et al., 2000; Pérez-Torrado et al., 2009) and avoids the expression of those genes related to oxidative stress response, such as
During an industrial-scale propagation of wine and brewing yeasts, catalase and Mn superoxide dismutase activities increase as propagation proceeds (Martin et al., 2003; Gómez-Pastor et al., 2010a), indicating the importance of oxidative stress response throughout the process, whereas Sod1p (Cu/Zn superoxide dismutase) transiently accumulates at the end of the batch phase when ethanol is consumed (Gómez-Pastor et al., 2010a). A study of different types of oxidative damage during wine yeast biomass propagation has revealed that lipid peroxidation considerably increases during the metabolic transition from fermentation to respiration, which decreases to basal levels during the fed-batch phase (Gómez-Pastor et al., 2010a). Besides, the protein carbonylation analysis, one of the most important oxidative damages (Stadtman and Levine, 2000), has revealed different protein oxidation patterns during biomass propagation, which reach maximum global carbonylation levels at the end of the batch phase (Gómez-Pastor et al., 2010a). As protein oxidation causes the loss of catalytic or structural integrity, further research into the specific oxidised proteins during biomass production should be done to correlate the detriment in fermentative capacity with specific damaged proteins. In addition, reduced glutathione, an important antioxidant molecule, varies during the process as is lowers during the metabolic transition, while oxidised glutathione increases. Then, reduced glutathione increases constantly in different stages of the process (Gibson et al., 2006; Gómez-Pastor et al., 2010a). Whether glutathione is directly affected by O2 during biomass propagation remains unknown and requires further investigation.
The fed-bath phase is characterised by the accumulation of other important antioxidant molecules, such as trehalose and thioredoxin (Trx2p) (Pérez-Torrado, 2004; Gómez-Pastor, 2010), although the mRNA levels for the
Proteomic studies have also been carried out to gain a better understanding of the fluctuations in the stress-related gene mRNA levels during biomass propagation and to correlate glycolytic enzyme activities with their corresponding protein levels. However, the proteomic data available from industrial processes are very limited and usually centre on bioethanol production (Cot et al., 2007; Cheng et al., 2008) or wine and beer fermentations (Trabalzini et al., 2003; Zuzuarregui et al., 2006; Salvadó et al., 2008; Rossignol et al., 2009). Recent proteomic studies performed by 2D-gel electrophoresis during wine yeast biomass propagation have revealed that several glycolytic enzyme isoforms increase during biomass production. This is probably due to the post-translational modifications after oxidative stress exposure (Gómez-Pastor et al., 2010b; Costa et al., 2002). Trabalzini et al. (2003) suggested that some specific isoforms of glycolytic/gluconeogenic pathway enzymes in wine strains of
The industrial yeast biomass dehydration process also involves damaging environmental changes. As the biomass is being concentrated, water molecules are removed and temperature increases, all of which affect the viability and vitality of cells (Matthews and Webb, 1991). Dehydration is known to cause both cell growth arrest and severe damage to membranes and proteins (Potts, 2001; Singh et al., 2005). Removal of water molecules causes protein denaturalisation, aggregation, and loss of activity in an irreversible manner (Prestrelski et al., 1993). Additionally at the membrane level, desiccation is associated with an increased package of polar groups of phospholipids, and with the formation of endovesicles leading to cell lysis during rehydration (Crowe et al., 1992; Simonin et al., 2007). Yeasts have several strategies to maintain membrane fluidity (Beney and Gervais, 2001). One of them is to accumulate ergosterol, this being the predominant sterol in
Recent phenomic and transcriptomic analyses during the desiccation of a laboratory strain have indicated that this process represents a complex stress involving changes in about 12% of the yeast genome (Ratnakumar et al., 2011). Under these conditions, the induction of 71 genes grouped into the “environmental stress response” category was observed, suggesting a role of the general stress transcription factors Msn2p and Msn4p in the desiccation stress response. Furthermore, the phenomic screen looking for genes that are beneficial to desiccation tolerance has identified several of the transcriptional regulators or protein kinases involved in oxidative (
The accumulation of some metabolites has been related to yeasts’ resistance to drying and subsequent rehydration. One of them is the amino acid proline. This amino acid exhibits multiple functions
Interestingly, wine yeasts accumulate large amounts of disaccharide trehalose, usually in the 12-20% range of cell dry weight (Degre, 1993) although higher percentages have been detected in industrial stocks (Garre et al., 2010). Trehalose content has been proposed as one of the most important factors to affect dehydration survival. Baker’s yeasts with 5% of trehalose are 3 times more sensitive to desiccation than those cells accumulating 20% of trehalose (Cerrutti et al., 2000). The main function of this metabolite is to act as a protective molecule in stress response. This effect can be achieved in two ways: by protecting membrane integrity through the union with phospholipids (reviewed in Crowe et al., 1992); by preserving the native conformation of proteins and preventing the aggregation of partially denatured proteins (Singer and Lindquist, 1998a). The indispensability of this metabolite to survive dehydration is a controversial subject. Some studies have suggested that its presence is essential and needed in both sides of the membrane to confer suitable protection (Eleuterio et al., 1993; Sales et al, 2000). However, these results are argued alongside the
In the last few decades, the yeast biomass production industry has contributed with many advanced approaches to traditional technological tools with a view to studying the physiology, biochemistry and gene expression of yeast cells during biomass growth and processing. This has provided a picture of the determinant factors for the commercial product’s high yield and fermentative fitness. Cell adaptation to adverse industrial conditions is a key element for good progress to be made in biomass propagation and desiccation, and towards the characterisation of specific stress responses during industrial processes to clearly indicate the main injuries affecting cell survival and growth. One major aspect of relevance in the complex pattern of molecular responses displayed by yeast cells is oxidative stress response, a network of mechanisms ensuring cellular redox balance by minimising structural damages under oxidant insults. Different components of this machinery have been identified as being involved in cellular adaptation to industrial growth and dehydration, including redox protein thioredoxin, redox buffer glutathione and several detoxifying enzymes such as catalase and superoxide dismutase, plus protective molecules like trehalose which play a relevant role in dehydration.
7. Future prospects
In spite of the sound knowledge available on molecular responses to exogenous oxidants, the endogenous origin of oxidative stress in yeast biomass production, given the metabolic transitions required for growth under the described multistage-based fermentation conditions and desiccation, makes it challenging to search for the specific targets undergoing oxidative damage during both biomass propagation and desiccation, and to correlate this damage with physiologically detrimental effects. Based on the currently global data available and the use of potent analytical and genetic manipulation tools, further research has to be conducted to (i) define specific oxidised proteins and to know how this oxidation affects fermentative efficiency, (ii) identify new key elements in stress response, which can be manipulated to improve it and can be also used as markers to select suitable strains for biomass production, (iii) analyse the effects of potential beneficial additives, such as antioxidants, on yeast cells’ ability to adapt to stress, and then yeast biomass’ yield and fermentative fitness in industrial production processes.
This work has been supported by grants AGL 2008-00060 from the Spanish Ministry of Education and Science (MEC). E.G. was a fellow of the FPI program of the Spanish Ministry of Education and Science, R.G-P was a predoctoral fellow of the I3P program from the CSIC (Spanish National Research Council).