Sodium is the major cation that accumulated in roots and stems as salinity increased (Meneguzzo et al., 2000). It is evident that salt tolerance is associated with low uptake of Na+(Santa-Maria and Epstein, 2001), partial exclusion (Colmer et al., 1995) and compartmentalization of salt in the cell and within the plant (Ashraf, 1994). The preferential accumulation in roots over shoots may be interpreted as a mechanism of tolerance in at least two ways.

First, maintenance of a substantial potential for osmotic water uptake into the roots and second, restricting the spread of Na+ to shoots (Renault et al., 2001). High Na+ levels in the external medium greatly reduce the physicochemical activity of dissolved calcium and may thus displace Ca2+ from the plasma membrane of root cells. In turn, displacement of Ca2+ from root membranes by Na+ affects Na/K uptake selectivity in favor of sodium. A low Ca2+ concentration under saline conditions may severely affect the functions of membranes as barriers to ion loss from cells (Boursier and Lauchli, 1990). Various organic and inorganic solutes such as K+, Na+, Cl-, proline, and glycinebetaine have been reported to contribute to such osmotic adjustment (Saneoka et al., 2001). Salinity inhibits the accumulation of K+ and Ca2+ in roots and stems. The negative effect of NaCl on the allocation of K+, Ca2+, and Mg2+ to the leaf tissues may contribute to their deficiency and the accompanying metabolic perturbations. The altered ion and water relations have a severe impact on the photosynthetic performance of the plant (Netondo et al., 2004). Many plants accumulate high levels of free proline in response to osmotic stress. This amino acid is widely believed to function as a protector or stabilizer of enzymes or membrane structures that are sensitive to dehydration or ionically induced damage. The salt stress caused increases in proline levels. Several investigations have shown that, besides other solutes, the level of free amino acids, especially proline, increases during adaptation to various environmental stresses. Plant salt tolerance has been generally studied in relation to regulatory mechanisms of ionic and osmotic homeostasis (Ashraf and Harris, 2004). In addition to ionic and osmotic components, salt stress, like other abiotic stress, also leads to oxidative stress through an increase in Reactive Oxygen Species (ROS), such as superoxide (02-), hydrogen peroxide (H2O2) and hydroxyl radicals (OH) (Mittler, 2002). It has been reported that most abiotic stress including NaCI salt stress impose injury in plants by osmotic stress, ionic stress and generating reactive oxygen species (Shalata and Tal, 1998). During oxidative stress, the excess production of Reactive Oxygen Species (ROS) causes membrane damage that eventually leads to cell death. For protection against ROS, plants contain antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and Glutathione Reductase (GR) or as well as a wide array of non­enzymatic antioxidants (Blokhina et al., 2003). SOD is the major 02- scavenger and its enzymatic action results in H202 and O2 formation. The H202 produced is then scavenged by CAT and several classes of peroxidases. CAT, which is found in peroxisomes, cytosol and mitochondria, dismutates H202 to H20 and O2 (McKersie and Leshem, 1994). Sorghum is a salt tolerant plant therefore it seems that it uses some of the above mechanisms for its adaptation to salt and drought stress.

The biomass fractions of a tree are the stump (including roots), stem, branches and foliage (needles and leaves). Broadleaved trees and conifers have different fractions of these aboveground components (Johansson 1999a, b). For birches, the mean aboveground fractions are: stem, 75 %; branches, 18 %; and leaves, 7 %. For conifers, the mean values are 63 %, 23 % and 14 % respectively (Johansson, 1999b, c). The percentage represented by needles is higher in young than old conifers, Fig. 12.

The effect of repeated harvesting on biomass production and sprouting of downy birches growing in central and northern Finland has been studied by Hytonen and Issakainen

(2001) . Different harvesting cycles of 1, 2, 4, 8, 12 and 16 years were examined. The main results were that downy birch is not suitable for biomass production using short rotations. Most of the stumps, 87 %, did not sprout in the one year rotations, but 8-year rotations produced the same number of sprouting stumps as the longer rotations.

Reim (1929) reported that European aspen growing along the borders of farmland may produce large numbers of suckers when cultivation ceases. In a study of repeated short rotations of aspen, the number of suckers per hectare decreased with every additional rotation (Perala, 1979). The study included rotations of four or eight years and, in both cases, the number of suckers decreased over the three rotations studied.

3. Conclusions

The management methods presented here rely on the land owner having extensive and detailed knowledge of biological processes. The changes in growth of individual species and mixed stands must be known. Some of the methods are based on optimal rotation periods and adequate management of the stand, including cleaning and thinning at the correct time. Severe competition could drastically decrease tree growth. Besides the need for the site to be suitable for tree cultivation, the skill of the owners is important. The most important factor, however, is the enthusiasm and curiosity of the owner; without this, most of the methods will not produce the yields suggested in the present study.

Table 3 lists possible future management models for trees established on farmland and forest land. When operating on a small-scale, there are many alternatives and the owner can be more flexible than is possible in large-scale operations. As the possible rotation periods range from 5 to 40 years it is important to have stands of different ages to ensure a continuous supply. Efficient management of such small areas would make it possible to produce a certain amount of biomass for personal use or to sell to neighbors or local heating plants..

Figures for potential energy supply from different stand types and management options allow us to make comparisons and select appropriate ways to use available land.

Most of the methods are cheap, need a short time to establish and involve relatively straightforward management. The raw materials produced can be used to generate energy for the landowner or can be sold.

Sebastian Delgado, Rafael Villarroel, Enrique Gonzalez and Miriam Morales

Department of Chemical Engineering, Faculty of Chemistry, University of La Laguna,

Spain

1. Introduction

It is widely known that many regions in the world have scarce water resources. In these areas the groundwater aquifers are also found to be in a critical condition as a result of over­exploitation. That is why, in such regions, the reuse of wastewater is a common practice and the competent authorities undertake multiple courses of action to encourage its reuse. Legislation implementing the reclaimed wastewater reuse is likewise very demanding in terms of quality and health and safety, which has resulted in the application of new technologies for water treatment and purification. Among the new emerging technologies appears the use of micro and ultrafiltration membranes as highly efficient systems, which are economically feasible for obtaining high quality recycled water.

Over the last two decades the technology of membrane bioreactors (MBRs) has reached a significant market share in wastewater treatment and it is expected to grow at a compound annual growth rate (CAGR) of 13.2%, higher than that of other advanced technologies and other membrane processes, increasing its market value from $ 337 million in 2010 to 627 million in 2015 (BCC, 2011). Aerobic MBRs represent an important technical option for wastewater reuse, being very compact and efficient systems for separating suspended and colloidal matter, which are able to achieve the highest effluent quality standards for disinfection and clarification. The main limitation for their widespread application is their high energy demand — between 0.45 and 0.65 kWh m-3 for the highest optimum operation from a demonstration plant, according to recent studies (Garces et al., 2007; Tao et al., 2009).

The advantages of this process over the conventional activated sludge process are widely known (Judd, 2010), among these one of the most cited is the reduction in sludge production which results from operation at high solid retention time (SRT). However, its consequences for the structure and metabolism of the microbial suspensions need to be studied in detail. Generally, we would expect that microorganisms subjected to severe substrate limitation should preferentially meet their maintenance energy requirements instead of producing additional biomass (Wei et al., 2003). This substrate limitation imposed on an MBR, by operating at low food-to-microorganism ratios (F/M), should modify the activity and characteristics of the sludge and could be the key factor for determining the process performance, particularly the membrane filtration (Trussell et al., 2006).

Biokinetic models are widely used to design activated sludge process. Knowledge of biokinetics parameters allows modelling of the process including the substrate biodegradation rate and biomass growth. At low growth conditions, as is demanded in MBRs, other processes apart from microbial growth have to be taken into consideration. These have been recognized as the maintenance energy requirement, endogenous respiration and subsequent cryptic growth (Van Loosdrecht & Hence, 1999). Macroscopically they cannot be perceived, but, from a practical point of view, the global process can be described by Pirt’s equation (Pirt, 1965).

Although there are several experiences with membrane bioreactors working without biomass purge (Rosenberger et al., 2002a; Pollice et al., 2004; Laera et al., 2005), none of these authors apply any kinetics models to describe process performance. Furthermore, these results were obtained in similar conditions, by treating raw municipal wastewater with a high substrate concentration, and it is interesting to compare this behaviour with an MBR treating wastewater with a low organic load. Additionally, not enough is known about the morphology and extracellular polymeric substance (EPS) production for total sludge retention and low F/M ratios.

The aim of this chapter is to summarize the current status of membrane bioreactor technology for wastewater treatment (Section 2.1). The advantages against the conventional activated sludge process and technological challenges are assessed (Section 2.2). Some design and operation trends, based on full-scale experience, are reviewed (Section 2.3). To discuss both fundamental aspects, biotreatment and filtration, some experimental results are presented. Special attention was given to the microbial growth modelling (Section 4.1.1), biomass characterisation (Sections 4.1.2 to 4.1.5) and membrane fouling mechanisms (Section 4.2). Some of these results have at the same time been compared with biomass from a conventional activated sludge process (CAS) operated in parallel.

1.1.1 Chemical and laboratory material and equipments

For dry decomposition there were used: nitric acid (65%, p. p., Lachema Neratovice CZ and Suprapur Merck, Germany), demineralised water (quality degree 1 according to EN ISO 3696 for the calibration of ICP-OES). Water calibration solutions with one element (Analytika, Ltd., CZ) were used for the calibration of F-AAS: Ca (1.000 ± 0.002 g L-1) in 2% HCl, Cu (1.000 ± 0.002 g L-1) in 2% HNO3, Fe (1.000 ± 0.002 g L-1) in 2% HNO3, K (1.000 ± 0.002 g L-1) in 2% HNO3, Mg (1.000 ± 0.002 g L-1) in 2% HCl, Mn (1.000 ± 0.002 g L-1) in 2% HNO3 and Zn (1.000 ± 0.002 g L-1) in 2% HNO3. For the testing of the dry decomposition method, a certified reference material NIST 8436 (Durum Wheat Flour) and internal reference material (IRM) from International Plant Analytical Exchange (IPE), RM Sample 3, Wheat 684, Quarterly Report 2000.3 were used. For the calibration of AAS and testing of the method of modified dry decomposition the following materials were used: calibration solutions with one element (Analytika, Ltd., CZ) 1.000 ± 0.002 g L-1 in 2% HNO3 for elements Cu, Pb and Zn, while cadmium was dissolved in 2% HCl. Muffle oven (LM 112.10, MLW, Germany), heating plate ALTEC JRT 350 with temperature graduation per 10 °C and ultrasonic bath Elma Transonic T660/H were used for the dry decomposition of samples. Analyses of the metals were performed by atomic spectrometer VARIAN SpectrAA 110 (VARIAN A. G., Australia) with the possibility emission spectra and Varian SpectrAA 280Z atomic absorption spectrometer furnished with GTA 120 electrothermic atomizer. Laboratory hammer mills LM3100 and LM120, falling number bath FN 1500, Glutomatic 2200 and Gluten Index centrifuge 2015 made by Perten Instruments AB (Sweden) were used for the determination of Falling number, gluten content and gluten index. For protein determination there were used: nitric acid (HNO3, 65%, p. a., Lachema Neratovice, CZ), automatic nitrogen analyzer Kjeltec system. A laboratory mill LM3100, lactic acid and bromphenol blue were used for the determination of sedimentation index (Zeleny sedimentation test).

1.1.2 Soil analysis

Soil pH was measured in suspension using 1:2.5 (w/v) ratio of soil and 0.2 M KCl at 20 ± 1°C by WTW pH 340i set. Available forms of nutrients (Ca, K, Mg and P) were determined using the Mehlich 3 soil extraction procedure (Mehlich, 1984; Zbiral, 2000) and organic nitrogen by the Kjeldahl method (Bremner, 1960).

This event extended from 17 to 21 October and was characterised by a variable low level flow pattern, which had a short SALLJ episode and a changing meteorological scenario, with transient perturbations of short duration.

3.1.2 Meteorological environment and SALLJ features

On 17 October, the 1000 hPa height shows the dominance of a post-frontal high pressure system over central Argentina (Figure 12). The surface front is located over central South America. On the south-western region of Argentina, the 500/1000 hPa depths show a baroclinic zone associated with a new frontal system.

During the following day, the anticyclone moved to the Atlantic Ocean, centred about 40° W and 35° S. Behind the baroclinic zone, a low pressure system located near 65° W and 47° S, developed. A thickness through oriented from the NW to the SE, is observed over the Pacific Ocean associated to an upper air through. The low level flow over north-eastern Argentina was from the north. On 19 October, the surface low pressure region had a fast displacement towards the SE. On the other hand, an anticyclonic system moved eastward covering the southern region of Argentina. North of 30° S, central South America showed relatively lower pressures. By 20 October, the thickness through axis was over Los Andes Mountains and then moved eastward. The low pressure system on central-northern Argentina displaced to the east and accordingly, the flow near the surface turned and blew from the east over Buenos Aires. On 21 October, a low pressure system developed and evolved in agreement with the displacement of the pattern at upper levels. It is located around 40° S and 50° W. Argentina was under the influence of an extended anticyclone. The near surface flow was from the south.

Figure 13 illustrates the 850 hPa flow and SALLJ features. On 17 October the low level flow associated to the post-frontal anticyclone centred over Buenos Aires is clearly shown. A very weak SALLJ is evident in the 850-700 layer, between Los Andes and the west of an anticyclone. The smaller wind intensities are observed over the biomass burning source regions. By 18 October, the low level flow strengthened and organized in a northerly current due to the approach from the southwest of the new cold front and the presence of the anticyclone now centred at 45° W and 35° S over the Atlantic Ocean. The 850 hPa winds did not satisfy the Bonner criteria. The north-western edge of the cold front is located near 35° S and 65° W. On 19 October the SALLJ spanned from central Bolivia to Paraguay and northern Argentina. The wind was from the north. Buenos Aires was behind the cold front. Another region with low level jet occurrence is over the Atlantic Ocean centred at 15° S. South of 30° S, the flow turned counter clockwise and acquired a north-western orientation ahead of the cold front. On 20 October, a SALLJ occurred, with its southern edge near 30° S. The front remained stationary over central Argentina. A low pressure system developed in the central region of Argentina whereas the exit region of the SALLJ was on southern Brazil. During the next day, there is a clear evidence of a strengthening and rapid displacement of the cold front that is oriented NW to SE. The low-level flow was from the south up to 20° S.

The sorption of metal ions by biomass occurs via functional groups on its surface by one or more mechanisms. All the sorbents derived from different by-products of agriculture share a common network of lignin and cellulose, and differ for the presence of functional groups which characterize each single biomass. As said before, identification of the functional groups is crucial for understanding the mechanism that governs the sorption process. Indeed, each functional group presents its own coordinating abilities toward the different metal ions. These coordinating abilities can be rationalized in term of the hard/soft character both of the binding group and of the metal ion. In order to highlight the importance of each different binding group in the mechanism of metal ion adsorption, the percent incidence drawn out from 1997 to nowadays literature is presented in Fig. 1.

Fig. 1. Incidence of the different binding groups on biomass surface involved in metal ion complexation.

Potentiometric titrations, chemical treatments of the sorbent, alkaline and alkaline-earth metal ion release and spectroscopic techniques are the procedures widely followed to reveal the binding groups. A brief survey of these methods is presented in the next sections.

The microbiological investigation on crude and treated wastewaters showed a sharp decrease in indicator organisms after heat treatment (Table 1).

Aeromonas spp are spread in aquatic environments, what may explain the presence of such organism in the crude effluent. Nevertheless, some species such as A. hydropila and A. salmonicida may be responsible for lethal infections in fish, bringing considerable economic losses to aquaculture (Maluping et al., 2005; Vieira, 2003) and some others have been described as emergent pathogens for humans (Vieira, 2003). So, the presence of this microorganism in the crude wastewater claims for periodic control in aquaculture, slaughter and processing of tilapia fish, as a way of avoiding financial injury to the fish industry and to consumers.

The presence of Salmonella enterica subsp. enterica serotype Typhi was detected in the wastewater, which represents a potential risk to public health and reveals deficient sanitary conditions during manipulation in the industry, since man is the natural reservoir of this serotype. This bacterium may be transmitted by water and foods contaminated with human feces, causing a serious infectious disease (Franco & Landgraf, 1996).

detergents used during cleaning operations or from protein degradation. Although this element may be essential to most living organisms, in high concentrations it may cause the proliferation of aquatic plants in water bodies and effluents. Oils and greases in wastewaters may originate from industrial kitchens, mechanic repairs garages, boilers and other equipments, as well as from raw material. They can easily be oxidized and so exhale bad odors in the environment. COD is an indirect measure of organic compounds concentration in wastewaters and so, reflects its pollutant load (Giordano, 2004; Liu, 2007).

The physicochemical data found for crude tilapia fish processing wastewater (Table 2) indicate the need for previous treatments for a safe discharge, according to Brazilian legislation. On the other hand, the presence of such organic matter in the wastewater was important to ensure the growth of R. gelatinosus with the resulting production of cells and oxycarotenoids.

The physicochemical characteristics of wastewaters presented herein differ from others previously reported. This happens because the particular characteristics of each industrial effluent derive from crude matter composition, season of the year, water supply, reuse procedures, factory installations and industrial processing techniques, among others (Liu, 2007). For settled and unsettled wastewater from sardine processing industry, for example, pH values from 6.2 to 6.3; 63,000 mg L-1 COD and 10.88 mg L-1 TN were described (Azad et al., 2001; 2003). For white fish filleting plants, Arvanitoyannis & Kassaveti (2008) reported the generation of wastewater with 50 kg COD and Prasertsan et al. (1993) reported 5.3 to 8.3 pH; 5,950 to 157,080 mg L-1 COD; 19.30 to 82.22 g L-1 TS and 666 to 32,182 mg L-1 OG for effluents from different seafood processing plants. Concentrations around 4,300 mg L-1 COD, 800 mg L-1 OG and 6.2 to 7.0 pH also were reported for wastewater from fish processing operations by Giordano (2004).

Changes in tilapia fish wastewater physicochemical parameters after biomass recuperation comprised removals of 82% in COD, 48% in OG and 22% in TN and a decrease in pH to 7.9, rendering it suitable for discharge in the environment, according to Brazilian laws. So, the biomass production process itself worked as a biological treatment for the reduction of pollution in tilapia fish industry wastewater.

Mean cell mass production and productivity achieved with the biological treatment were 0.18 g L-1 and 0.0634 g L-1 day-1, respectively. Prasertsan et al. (1993) credit the low cell production to the anaerobiosis/light cultivation conditions, in which the synthesis of oxycarotenoids is intensified. Other authors found higher cell mass concentrations when growing phototrophic organisms in industry wastewaters but, in those cases, initial organic matter and inoculum levels were higher than the ones used in this study and/or nutritional supplementation was adopted (Azad et a!., 2001, 2003; Prasertsan et al., 1997). In this study, we opted to maintain the original wastewater composition and to use a low inoculum level in an attempt to minimize costs and render the biomass production process feasible for the industry.

The microbiological investigation on Rubrivivax gelatinosus biomass indicated low counts on total coliforms (20.27 NMP g-1), fecal coliforms (< 1.0 NMP g-1) and molds and yeasts (1.2 x 103 UFC g-1) and the absence of pathogenic organisms. This way, the product showed to be in agreement with Brazilian microbiological standards required for feed ingredients, which ensures its safe utilization.

Mean proximate composition of biomass and amino acid profile in the product are presented in Tables 3 and 4, respectively. As a typical feature of single cell proteins, the values indicate the high level of proteins in the biomass, which denotes its use in animal diets as a nutritional ingredient. Moreover, it also contained considerable amounts of all amino acids considered essential for animals, what reinforces the suggestion of its use in the supplementation of animal feeds in order to supply deficiencies that may cause, for instance, delay in protein utilization and reduction of growth, weight gain, feed conversion and immunity (Cyrino et al., 2004). In view of these findings, the bacterial biomass presents a potential for use as a nutritional ingredient for feeds.

Oxycarotenoids content in the biomass was found to be 3.03 mg g-1 dry biomass, which conferred a dark red color to the power product (L = 22.42; C = 14.22; h = 25.48). This is in agreement with Prasertsan et al. (1997), who found concentrations of 2.13 to 3.90 mg of carotenoids per gram of dry biomass of Rhodocyclus gelatinosus produced in tuna processing wastewater.

The main photosynthetic pigments produced by Rubrivivax gelatinosus are bacteriochlorophyll a and carotenoids from alternative spirilloxanthin series, which contains spheroidene, hydroxyspheroidene and spirilloxanthin as the major representants (Holt et al.,

2000) . The blend among these pigments gives the bacterial cultures a reddish color (Ponsano et al., 2002a, 2003a, 2008) that remains in the dry biomass, since sensorial and nutritional properties of lyophilized products remain intact after drying process (Pereda et al., 2005). Considering that these pigments are oxycarotenoids and so have the ability to deposit in animal tissues, this feature of the biomass suggests its application as a pigmenting ingredient for the rearing of different animals.

The use of natural or synthetic oxycarotenoids for the rearing of animals is reported by many authors. Salmonids, for instance, are noble fish natural from cold waters in North Hemisphere, but that are being commercially farmed in many parts of the world. According to Baker & Gunther (2004), in wild salmon, the natural carotenoid astaxanthin provides a majority of the color expected from this flesh. Nevertheless, for farmed salmonids, the same effect may be achieved by the use of pigmenting additives in rations. They may also be used for the raising of ornamental fish to increase skin color and beauty. For the raising of red Cyprinus carpio (Kawari), for instance, Gouveia et al. (2003) relate the utilization of carotenoids produced by micro algae Chlorella vulgaris.

For poultry products, the pigmentation varies according to market demand. In Mexico, Belgium, Italy, Peru and some regions in Brazil, for instance, the use of pigmenting ingredients in poultry production is a common practice since people prefer strong colors for broilers carcasses and egg yolks (Gouveia et al., 1996; Toyomizu et al., 2001). People often associate strong colors of a food item to safety and health and so look for strongly pigmented products. Taking it into account, Ponsano et al. (2002b, 2004a, b) added Rhodocyclus gelatinosus biomass produced in poultry slaughterhouse wastewater in broilers rations and found an increase in the color of breast meat. Polonio et al. (2010) used different concentrations of the same product in hens rations and found an improvement in yolks color, with no deleterious effects on birds performance. In the sensorial test, these authors identified the concentration of the biomass that, when used together with corn xanthophylls, provides a desired golden orange color to the yolks. Yet, Garcia et al. (2002) found an increase in yolks color, with no influence in the performance and eggs characteristics, when canthaxantin was used in hens diets.

Besides the pigmenting feature of oxycarotenoids, they are also known to exert benefits on animal health and welfare due to antioxidant properties. According to Baker & Gunther (2004), evidences suggest that the carry-over of these pigments into the human food chain could be beneficial to human health too. In humans, the consumption of oxycarotenoids is associated to aging prevention and to the decrease of the risk of diseases related to the accumulation of free radicals (Bhosale, 2004; Bhosale; Bernstein, 2005). So, for further studies on the properties of Rubrivivax gelatinosus biomass, the antioxidant ability of its carotenoids will be considered.

2. Conclusion

pigments but also for having an elevated nutritional value. Moreover, we showed that the biomass production process worked as a biological treatment for the reduction of pollution in the industrial wastewater, requiring simple and feasible methods that can be operated in the industry, so minimizing byproducts and still rendering profits from the biomass commercialization.

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; Perez-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; Perez — Torrado et al., 2005; Perez-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 HSP12 and GSY2 genes involved in protein folding and glycogen metabolism, respectively (Boy-Marcote et al., 1998; Estruch,

2000) . Furthermore, yeast cells have been seen to respond specifically to certain stresses. During thermal stress, transcriptional factor Hsf1p activates the transcription of genes, such as STI1, which code for those proteins that counteract protein denaturation and aggregation (Lindquist and Craig, 1988; Sorger, 1991). Aerobic growth during biomass propagation and pro-oxidants also generate reactive oxygen species (ROS), leading to several types of oxidative damage to cells (Gomez-Pastor et al., 2010a). To neutralise the harmful effects of oxidative stress, proteins are generated, and they participate in two major functions: antioxidants (such as GSH1, TRX2, CUP1, and CTT1) to reduce proteins and eliminate ROS damage, and metabolic enzymes (such as PMG1 and TDH2) that redirect metabolic fluxes to synthesise NADPH by slowing down catabolic pathways like glycolysis (Godon et al., 1998). Another well-known specific stress response is the high-osmolarity glycerol response pathway (Brewster et al., 1993), which induces the genes involved in glycerol synthesis (GPD1, GPP2) and methylglyoxal detoxification (GLO1). Intracellular accumulation of glycerol counteracts hyperosmotic pressure to avoid water loss (Hohmann, 2002). There are other stress response pathways that remain poorly understood, such as those involved in the adaptation to nutrient starvation. Large groups of well-known stress response genes and other genes with unknown functions, such as YPG1, are induced after exposure to one kind of stress, and are also involved in the protective mechanism against other different stresses, a phenomenon known as cross-protection (Coote et al., 1991; Piper, 1995; Trollmo et al., 1988; Varela et al., 1992; Bauer and Pretorius, 2000). The molecular responses of laboratory S. cerevisiae strains to different stresses have been thoroughly studied, and a large body of knowledge is available (Gasch and Werner-Washburne, 2002; Hohmann and Mager, 2003). In addition, several approaches for the characterisation of stress responses under industrial conditions have been carried out for wine and lager yeasts (Perez-Torrado et al., 2005; Gibson et al., 2007), and some correlations have been found between stress resistance of several yeast strains and their suitability for industrial processes (Beudeker et al., 1990; Ivorra et al., 1999; Aranda et al., 2002; Perez-Torrado et al., 2002; Zuzuarregui et al., 2005; Perez-Torrado et al., 2009; Gomez-Pastor et al., 2010a). For these reasons, the study of stress responses under industrial conditions has become an important research field to improve our knowledge of not only complex industrial processes, but of yeast capabilities.

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 HSP12, GPD1, STI1, GSY2 and TRX2, during bench-top growth trials of wine yeast biomass propagation have demonstrated that osmotic (GPD1) and oxidative stresses (TRX2) are the main adverse conditions that S. cerevisiae senses during this process (Perez-Torrado et al., 2005). Afterwards, a genome-wide expression analysis of the same process established stress-critical time points throughout the process based on the profiles of different oxidative stress response genes (Gomez-Pastor et al., 2010b). Three relevant stressful points have been defined during biomass propagation: the first during the metabolic transition from fermentation to respiration in the batch phase; the second critical point is the end of the batch phase when previously produced ethanol is completely consumed; the third interesting point is the end of the fed-batch phase, after a long period under respiratory metabolism. Among these set points, metabolic transition during the batch phase is the most relevant as several genes relating to cell stress, especially those related to oxidative stress (TRX2, GRX2 and PRX1), protein degradation, aerobic respiration and NADPH production, are induced while ribosomal proteins are dramatically repressed (Gomez-Pastor et al., 2010b). Similar results have been observed in a genome-wide expression analysis during biomass propagation of brewer’s yeasts, which also displays a strong induction of the genes involved in ergosterol biosynthesis and oxidative stress protection in initial industrial lager fermentation stages (Higgins et al., 2003b; reviewed in Gibson et al., 2007; Gibson et al., 2008). However, while osmotic stress plays a role in initial biomass propagation stages as a result of the large amount of sugar in molasses, oxidative stress takes place throughout the process as a result of aeration (reviewed in Gibson et al., 2007).

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; Perez-Torrado et al., 2009) and avoids the expression of those genes related to oxidative stress response, such as TRX2 and GRE2, which significantly increases oxidative cellular damage, such as lipid peroxidation, when the bioreactor is re­oxygenated to oxidise ethanol (Perez-Torrado et al., 2009). Clarkson et al. (1991) demonstrated that cellular antioxidant defences, such as Cu/Zn superoxide dismutase, Mn superoxide dismutase and catalase activities of brewing yeast strains, also change rapidly after adding or removing O2 from fermentation.

During an industrial-scale propagation of wine and brewing yeasts, catalase and Mn superoxide dismutase activities increase as propagation proceeds (Martin et al., 2003; Gomez-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 (Gomez-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 (Gomez-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 (Gomez-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; Gomez-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) (Perez-Torrado, 2004; Gomez-Pastor, 2010), although the mRNA levels for the TRX2 gene significantly increase during the batch phase metabolic transition (Perez-Torrado et al, 2009). On the other hand, glycogen, a secondary long-term energy storage molecule which has been related to adaptation to the respiratory metabolism (Francois and Parrou, 2001), also accumulates at the end of the fed- batch phase (Perez-Torrado, 2004). Studies using different dilution rates during the continuous cultivation of baker’s yeast have shown that the accumulation of trehalose and glycogen has a negatively effect as it increases dilution rates, which is also detrimental for fermentative capacity and cellular responses to heat stress during dehydration (Ertugay and Hamaci, 1997; Garre et al., 2009). Despite a high biomass yield and the accumulation of several beneficial metabolites obtained during the fed-batch phase, S. cerevisiae dramatically diminished fermentative capacity after prolonged glucose-limited aerobic cultivation due to several glycolytic enzymes’ diminished activity (Jansen et al., 2005).

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; Salvado 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 (Gomez-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 S. cerevisiae are involved in the physiological adaptation to different fermentation stresses. There have also been reports of the differential stress regulations of several proteins (Arg1p, Sti1p and Pdc1p) among different industrial strains possibly having important industrial implications for strain improvement and protection (Caesar et al., 2007). It is interesting to note that biomass propagation experiments using a trx2 deletion strain have shown a low number of several glycolytic enzyme isoforms and, consequently, an increase in oxidative cellular damage, such as lipid peroxidation and global protein carbonylation (Gomez-Pastor, 2010). During the metabolic transition in the batch phase, several proteins relating to oxidative stress are expressed (Prx1p, Ahp1p, Ilv5p, Pdi1p, Sod1p and Trr1p), which directly correlates with their mRNA levels observed for this growth stage (Gomez-Pastor et al., 2010b). This scenario indicates adaptation to the new condition. In contrast, the genes coding for most of the heat shock proteins, chaperons (Mge1p, Hsp60p, Ssb1p and Ssc1p) and proteins related to ATP metabolism are specifically induced during the metabolic transition, but their protein levels decline throughout the process. The proteins with the highest expression levels at the end of the biomass propagation include Tdh1p, which codifies for glyceraldehyde-3-phosphate dehydrogenase, and Bmh1p and Bmh2p, homologues to the mammalian 14-3-3 proteins involved in global protein regulation at the post-translational level (Bruckmann et al., 2007). The expression of these proteins at the end of biomass propagation is important as they control the translation of several glycolytic proteins (Fba1p, Eno1p, Tpi1p, Pck1p, Tdh1p, Tdh3p and Gpm1p), as well as the levels of those proteins involved in amino acid biosynthesis and heat shock proteins translation (Bruckmann et al., 2007). This may explain the lack of correlation between the transcriptomic and the proteomic analyses for glycolytic enzymes during biomass propagation. Under oxidative stress, some glycolytic proteins (Tdh3p, Pdc1p, Ad1p and Eno1p) have been described to be specifically modified by oxidation (Le Moan et al.,

2006) . This oxidation process could explain the loss of fermentative capacity observed in some commercial wine yeast industrial strains at the end of the biomass propagation process (Gomez-Pastor et al., 2010a, b). Regarding this hypothesis, it is worth noting that the overexpression of the TRX2 gene in industrial yeasts significantly increases the obtained biomass’ fermentative capacity by improving the oxidative stress response during propagation, and by decreasing lipid and protein oxidation (Perez-Torrado et al., 2009; Gomez-Pastor et al., 2010a, c). Figure 1 summarizes the different stresses affecting yeast cells during the biomass propagation process, especially those encountered during the batch phase, and shows the different cellular states with the most relevant metabolites, genes and proteins expressed in each propagation stage.

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 S. cerevisiae. Sterols have been proposed to maintain the lateral heterogeneity of the protein and lipid distribution in the plasma membrane because of the putative role they play in inducing microdomains, the so-called lipid rafts (Simons and Ikonen, 1997). Ergosterol synthesis has been related with yeast stress tolerance (Swan and Watson, 1998), and its beneficial role in the different processing steps of industrial yeast has been documented. Its synthesis during biomass production is critical to ensure suitable yeast ethanol tolerance in its later application in wine fermentation (Zuzuarregui et al., 2005). Moreover, the addition of oleic acid and ergosterol during wine fermentation mitigates oxidative stress by reducing not only the intracellular content of reactive oxygen species, but oxidative damage to membranes and proteins, and enhancing cell viability (Landolfo et al., 2010). Recently, experiments with a erg6A mutant strain, deficient in the ergosterol biosynthetic pathway and which accumulates mainly zymosterol and cholesta-5,7,24-trienol instead of ergosterol, have shown that the nature of sterols affects yeast survival during dehydration, and that resistance to dehydration-rehydration cycles can be restored with ergosterol supplementation during the anaerobic growth of the erg6A mutant (Dupont et al., 2010). 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 (ATF1, SKN7) and osmotic (HAL9, MSN1, MSN2, MSN4, HOG1, PBS2, SSK2) stress responses. Although studies with lab strains generate interesting information about the desiccation process, an analysis of stress marker genes during dehydration in ADY production has revealed that inductions of gene expressions in wine yeast T73 are generally moderate, although statistically significant, in some steps, such as hot air drying and final product (Garret et al., 2010). One such example is the induction of osmotic stress marker GPD1 due to water loss. However, despite the yeast biomass losing approximately 95% of water content during this dehydration process, GPD1 induction is not as important as previously observed in lab yeast strains under osmotic stress (Perez-Torrado et al., 2002). These data are in agreement with the robustness of industrial yeasts strains compared to laboratory strains (Querol et al, 2003), and also with the well-known relevance of biomass propagation conditions to confer resistance to subsequent suboptimal conditions (Bisson et al., 2007). One interesting aspect in the same study carried out by Garre and coworkers (2010) is that the highest induction is displayed by oxidative stress marker GSH1 that codes for y-glutamilcysteine synthetase activity. This observation is supported by: i) significant inductions of the other genes involved in oxidative stress response, such as TRR1 and GRX5, ii) rise in the cellular lipid peroxidation level, iii) increased intracellular glutathione accumulation, and iv) a peak of its oxidized form GSSG during the first minutes of drying. In addition, a genomic analysis of an oenological-dried yeast strain has shown a strong induction of the other genes related with oxidative stress response, such as CTT1, SOD1, SOD2, GTT1 and GTT2 (Rossignol et al., 2006). Currently, free radical damage is emerging as one of the most important injuries during dehydration. Several studies with laboratory yeast strains have shown considerable ROS accumulation during dehydration that results in protein denaturation, nucleic acid damage and lipid peroxidation (Espindola et al., 2003; Pereira et al., 2003; Franga et al., 2005, 2007). Antioxidant systems appear to be interesting targets affecting yeast’s desiccation tolerance. Several examples using lab strains have been shown. Overexpression of antioxidant enzymes genes, such as SOD1 and SOD2, increases yeast survival after dehydration (Pereira et al., 2003), whereas a mutant without cytosolic catalase activity is more sensitive to water loss (Franga et al., 2005). Glutathione seems to play a significant role in the maintenance of intracellular redox balance because glutathione-deficient mutant strains are much more oxidised after dehydration than the wild-type strain, and they show high viability loss (Espindola et al., 2003). Furthermore, addition of glutathione to gsh1 cells restores survival rates to control strain levels. Remarkably, the overexpression of the TRX2 gene in wine yeast has proved a successful strategy to improve fermentative capacity and to produce lower levels of oxidative cellular damage after dry biomass production than its parental strain (Perez-Torrado et al., 2009; Gomez-Pastor et al., 2010a).

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 in vitro: it enhances the stability of proteins, DNA and membranes, inhibits protein aggregation, and acts as a ROS scavenger; but its functions in vivo, particularly as a stress protectant, are poorly understood. Although S. cerevisiae cells do not accumulate this amino acid in response to stresses, it has been recently shown with laboratory strains that proline-accumulating mutants are more tolerant than wild-type cells to freezing, desiccation, oxidative, or ethanol stress (reviewed in Takagi, 2008; Kaino and Takagi, 2009). Self-cloning has been used to construct the baker’s yeasts that accumulate proline by carrying the disruption of the PUT1 gene involved in the degradation pathway, and expressing a mutant PRO1 gene that encodes a less sensitive y-glutamate kinase to feedback inhibition in order to enhance biosynthetic activity. The engineered yeast strain shows enhanced freeze tolerance in doughs (Kaino et al., 2008). A recent transcriptomic analysis of air-dried cells has suggested activated transport and metabolic processes to increase the intracellular concentration of proline during yeast desiccation (Ratnakumar et al., 2011).

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 tpsl mutant’s dehydration resistance, which is unable to synthesise trehalose, as other authors have indicated (Ratnakumar and Tunnacliffe, 2006). On the other hand, dehydration tolerance conferred by trehalose seems to be also related to its ability to protect cellular components from oxidative injuries (Benaroudj et al., 2001; Oku et al., 2003; Herdeiro et al., 2006; da Costa Morato et al., 2008; Trevisol et al., 2011). The addition of external trehalose during dehydration reduces intracellular oxidation and lipid peroxidationand increases the number of viable cells after dehydration (Pereira et al., 2003). Moreover, the compensatory trehalose accumulation observed in hsp12A mutants confers a higher desiccation tolerance than the parent wild-type cells, which is the result of increased protection by mutant cells against reactive oxygen species (Shamrock and Lindsey, 2008). Some studies have proved the applicability of this metabolite to improve industrial yeast tolerance to dehydration. A clear and simple example is that of Elutherio and co-workers

(1997) , where the trehalose accumulation induced by osmotic stress in the species Saccharomyces uvarum var. carlsbergensis before dehydration is enough to achieve survivals of up to 60% after drying, whereas the stationary cells presenting low trehalose levels are unable to survive. The construction of trehalose-overaccumulating strains by removing

degradative activities emerges as a useful strategy for industrial yeasts (Kim et al., 1996). Studies done with laboratory strains have shown that the deletion of genes ATH1 and NTH1, respectively encoding acid and neutral trehalase activity, improve yeast cells viability after dehydration, which is provoked by hyperosmotic stress (Garre et al., 2009). Similar approaches using baker’s yeast have also been successful, and defective mutants in neutral or acid trehalase activities exhibit higher tolerance levels to dry conditions than the parent strain, as well as increased gassing power of frozen dough (Shima et al., 1999).

6. Conclusions

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.

Rheological properties are of crucial importance due to their effect on hydrodynamic conditions near the membrane. The rheological behavior of microbial suspensions has been described in the literature as non-Newtonian pseudoplastic fluids (Rosenberger et al., 2002b). When air is dispersed in a solid-liquid suspension a change can be seen in its rheological behavior due to the change in suspension structure: with increasing shear, the structure opens and biological aggregates are reorganized resulting in a decrease in viscosity. In addition, it is accepted that the microbial suspensions have a thixotropic nature, which means that the viscosity decreases with shear rate when samples are subject to shear stress. Rheology can be described by the Bingham model, the Ostwald model and the Herschel-Bulkley model represented by Eq. (3)-(5):

(3)

( dv

^ — ml dr

In these models is the apparent viscosity, dv/dr is the shear rate and To, m and n are the model parameters. From the models we may deduce that the apparent viscosity can be described as a shear rate function.

Figure 8 shows one example of apparent viscosity reduction with the shear intensity. It decreases down to 75% when the shear varied from 13 to 130 s-1. Additionally, plotting is shown according to the Bingham, Ostwald and Herschel-Bulkley models. In general, both the Ostwald model as well as the Herschel-Bulkley model fits quite well into the experimental data, while the Ostwald was selected because of its simplicity. From the equation of the curve (Figure 8) the parameter values for Ostwald model can be obtained:

n = 0.41
m = 122 mPa s

where n is the flow behavior index and m is the consistency index.

Furthermore, as shown in Figure 8, apparent viscosity (pa)limit can be perceived for higher values (> 130 s-1 ). It does not decrease substantially with an increasing velocity gradient. Therefore, the effect of particle concentration on the viscosity can be evaluated by fitting the (pa)limit to the sludge concentration, measured as MLSS concentration (Figure 9). As expected, microbial suspension viscosity also increased with the MLSS concentration. This behaviour is commonly accepted in the literature (e. g. Pollice et al., 2007).

Therefore, the following equation (Eq. (6)) can estimate the limit apparent viscosity as a function of the MLSS concentration.

ua = 1.1-10-6-SSLM17

r alimrt

Metabolism is the sum of cellular chemical and physical activities. It involves chemical changes to reactants and the release of products using well-established pathways regulated at many levels. Knowledge of such regulation in yeasts is crucial for exploitation of yeast cell physiology in biotechnology (Talaro & Talaro, 2001). At controlled cultivation conditions oleaginous red yeasts could be a good source (producer) of lipidic primary metabolites as neutral lipids, phospholipids and fatty acids and ergosterol, which is integrate part of yeast biomembranes.

Secondary metabolism is a term for pathways of metabolism that are not absolutely required for the survival of the organism. Examples of the products include antibiotics and pigments. The induction of secondary metabolism is linked to particular environmental conditions or developmental stages. When nutrients are depleted, microorganisms start producing an array of secondary metabolites in order to promote survival (Mann, 1990). Filamentous fungi and yeasts show a relatively low degree of cellular differentiation, but still they express a complex metabolism resulting in the production of a broad range of secondary metabolites and extracellular enzymes. This very high metabolic diversity has been actively exploited for many years. In terms of biotechnological application fungi and yeast have the advantage of being relatively easy to grow in fermenters and they are therefore well-suited for large-scale industrial production. Biomass enriched by suitable mixture of primary and secondary metabolites can be used too, mainly in feed and food applications (Mann, 1990, Walker 1998).

In general, biosynthesis of individual metabolites is governed by the levels and activities of enzymes employed to the total carbon flux through the metabolic system. Efficiency of that flow depends on the cooperation of individual pathways engaged in this process and which pathway is suppressed or activated varies with the growth medium composition, cultivation conditions, microbial species and their developmental stage. Because overall yield of metabolites is directly related to the total biomass yield, to keep both high growth rates and high flow carbon efficiency to carotenoids by optimal cultivation conditions is essential in order to achieve the maximal metabolite productivity (Certik et al., 2009).