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 non­yeast 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 4°C 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 35°C or 41 °C 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 20°C. For this reason, manufacturers recommend storing ADY at 4°C in a dry atmosphere for a maximum

2- 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).

1.5 Biological process

1.5.1 Maintenance kinetics

Biomass concentration in the bioreactor is one of the most critical parameters in capital and operational costs of the process. It is known that increasing the biomass concentration reduces the bioreactor size and therefore, capital costs. However, high sludge concentration impacts on aeration efficiency (because of high viscosity) increasing membrane fouling propensity and, probably, membrane clogging (filling of the channels between the membranes with sludge solids). Therefore, a more frequent cleaning and higher aeration rate is necessary to maintain membrane permeability, which increments the operational costs. Therefore, fundamental knowledge of biomass development processes involved in the biological treatment of a MBR is required.

Figure 4 shows the typical trend of biomass evolution, expressed as total (MLSS) and volatile suspended solids (MLVSS), during the start-up and steady-state of an MBR operated without biomass purge. Biomass is developed from the microorganisms coming with the feed wastewater as the bioreactor had not been inoculated. During the initial period, biomass increased rapidly and then slower with increasing biomass concentration in the mixed liquor.

The first concern is the MLVSS/MLSS ratio, which remained within the range between 71 and 78%. It is important to note that, despite operating in conditions of total sludge
retention, this ratio remains constant throughout the experiment, indicating no significant accumulation of inorganic matter in the sludge. This may be due to the fact that a small fraction of inorganic suspended solids in the feed (5-15%) is dissolved during the process and, therefore, does not accumulate in the sludge and leaves the system with the permeate.

The second concern is the stabilisation value of the biomass concentration (MLSS and MLVSS), which is expected to depend on the hydraulic retention time (HRT) and COD removal, resulted in a stationary value of utilisation rate (U). Figure 5 shows the evolution of U with operation time where it can be observed that the system evolved until reaching a nearly constant value (0.083 ± 0.004 kg COD/kg MLVSS d). A symmetrical trend can also be observed for data obtained in a previously reported research (Delgado et al., 2010) in an MBR treating biological effluent from a WWTP. In that case, the MBR was inoculated and the initial biomass evolution was characterised by a lysis process. Afterwards, a stationary vale for U was reached (0.067 ± 0.004 kg COD/kg MLVSS d) independently of the fixed HRT value.

It is thought that the maintenance concept introduced by Pirt (1965) could be the reason for the equilibrium reached in the MBRs operated without biomass purge. Then, the utilisation rate can be described by the Pirt equation (1).

U = + km s (1)

where rs is the substrate removal rate, rx is the biomass growth rate, Y is the true sludge yield, km, S is the maintenance coefficient and X is the biomass concentration.

At very low growth rates (i. e. steady-state conditions), rx can be neglected:

U * kms (2)

Therefore, the stationary value of the utilisation rate is identical to the maintenance coefficient, which suggests that, in these substrate-limited conditions, microorganisms tend to minimize their energy requirements using the available substrate to satisfy their maintenance functions. For the presented data the best fitting parameter was km, s = 0.0035 kg COD/kg MLVSS h.

1.5.2 Microbial activity: Specific endogenous oxygen uptake rate

The measurement of the oxygen demanded by the microorganisms is a parameter frequently used for assessing aerobic activity of microbial suspensions (Vanrolleghen et al., 1995). In this sense, Pollice et al. (2004) reported that the specific endogenous respiration rates are closely related to the organic loading rates (F/M). Table 4 shows specific endogenous oxygen uptake rates (SOURe) of sludge samples at steady-state conditions and other values reported in the literature. The SOURe is considerably lower than the typical values, which confirms the maintenance energy requirement reached.

4.1.3 Sludge morphology

According to the literature, flocculant ability tends to be reduced when organic substrate is lacking (e. g. Wilen et a., 2000). In an MBR operated under substrate-limited conditions these conditions of stress are imposed and therefore a floc distribution characterised by a greater number of small flocs is expected. In addition, particle size distribution plays an important role in the formation of the cake on the membrane surface. A cake made with small particles has higher specific resistance and, therefore, is less permeable than the cake formed by larger particles (Defrance et al., 2000). As a consequence, it is crucial to analyze the effect of the several substrate-limited conditions imposed over the particle size of the flocs and the presence of small

Sludge morphology was analysed by optical microscope observations and by particle distribution measurements. In Figure 6 particle size distribution of a sludge sample at steady-state conditions is shown. Also, samples from a conventional activated sludge process (CAS) which treated the same influent were investigated and compared with the MBR sample. Figure 6 shows aggregates with bimodal distribution in CAS biomass, where 50 % of the particles have a size higher than 70 pm. In contrast, uniform and medium-sized flocs were observed in the MBR sludge, where 40 % of the particles were within the 15 to 50 pm range. Granulometric differences, which are a result of biomass separation by the membrane, are well documented in the literature (e. g. Cicek et al., 1999) and are attributable to effective particle retention by the membrane and high shear stress conditions due to air sparging for membrane fouling mitigation. Also, the low quantity of small non-floculating flocs (< 10 pm) could be due to the presence of higher organisms, which have traditionally been considered as predators that consume dispersed bacteria.

Alternatively, microscopic analysis of mixed liquor samples from the MBR is shown in Figure 7. The observations can be summarized into two main issues: firstly the absence of filamentous microorganisms, which can be linked to the process conditions, including high

dissolved oxygen and low readily biodegradable substrate concentrations (Martins et al., 2004). Secondly, as a result of the low organic loading conditions, higher organisms were also expected. In this sense, a significant quantity of worms (type Aeolosoma hemprichi) developed. Similar results were reported by Zhang (2000) where a high worm density resulted in a low sludge yield (0.10-0.15 kg MLSS/ kg COD). Worms are considered as predators with a great potential on sludge reduction and more attention has been paid to their effectiveness in wastewater treatment recently (Wei et al., 2003).

As already stated, to operate an MBR under substrate-limited conditions enhances the presence of worms that may lead to a substantial sludge reduction and improve biomass characteristics by removing small non-floculating flocs.

The genus Rhodotorula includes three active species; Rhodotorula glutinis, Rhodotorula minuta and Rhodotorula mucilaginosa (formerly known as Rhodotorula rubra) (Hoog et al., 2001). Colonies are rapid growing, smooth, glistening or dull, sometimes roughened, soft and mucoid (Figures 1 — 3). They are cream to pink, coral red, orange or yellow in color. Blastoconidia that are unicellular, and globose to elongate in shape are observed. These blastoconidia may be encapsulated. Pseudohyphae are absent or rudimentary. Hyphae are absent. Rhodotorula glutinis often called "pink yeast" is a free living, non-fermenting, unicellular yeast found commonly in nature. Rhodotorula is well known for its characteristic carotenoids "torulene, torularhodin and P-carotene. Rhodotorula glutinis is also reported to accumulate considerable amount of lipids (Perier et al., 1995).

The genus Sporobolomyces contains about 20 species. The most common one is Sporobolomyces roseus and Sporobolomyces salmonicolor (Hoog et al., 2001). Sporobolomyces colonies grow rapidly and mature in about 5 days. The optimal growth temperature is 25-30°C. The colonies are smooth, often wrinkled, and glistening to dull. The bright red to orange color of the colonies is typical and may resemble Rhodotorula spp. Sporobolomyces produces yeast-like cells, pseudohyphae, true hyphae, and ballistoconidia. The yeast-like cells (blastoconidia, 2­12 x 3-35 pm) are the most common type of conidia and are oval to elongate in shape. Pseudohyphae and true hyphae are often abundant and well-developed. Ballistoconidia are one-celled, usually reniform (kidney-shaped), and are forcibly discharged from denticles located on ovoid to elongate vegetative cells (Figures 4, 5) .

Among yeasts, Rhodotorula species is one of main carotenoid-forming microorganisms with predominant synthesis of P-carotene, torulene and torularhodin (Davoli et al., 2004; Libkind and van Broock, 2006; Maldonade et al., 2008). Cystofilobasidium (Figure 6) and Dioszegia were also found to synthesize these three pigments. Some of yeast carotenoids are modified with oxygen-containing functional groups. For example, astaxanthin is almost exclusively formed by Phaffia rhodozyma (Xanthophyllomonas dendrorhous; Frengova & Beshkova, 2009).

Nevertheless, although there are many strategies for stimulation of carotene biosynthetic machinery in yeasts, attention is still focused on unexplored yeast’s habitats for selection of hyper-producing strains what is the important step towards the design and optimization of biotechnological process for pigment formation (Libkind & van Broock, 2006; Maldonade et al., 2008).

Studies on a number of fungi, including Neurospora crassa, Blakeslea trispora, Mucor hiemalis, Mucor circinelloides and Phycomyces blakesleeanus (oleaginous fungi with carotene-rich oil) have been published over the last twenty years (Dufosse, 2006). Fungal carotenoid content is relatively simple with dominat levels of |3-carotene. Recent work with dimorphic fungal mutants M. circinelloides and Blakeslea trispora (Cerda-Olmedo, 2001) showed that these strains could be useful in a biotechnological production of carotenoids in usual fermentors. In order to study yeast physiology under different conditions, it is important to know so called "reference parameters" which these yeasts possess under optimal condition. Red or carotenogenic yeasts are well known producers of valuable carotenoids. On agar plates they form characteristic yellow, orange and red coloured colonies. Red yeast can be of ellipsoidal or spherical shape (Figures 1 — 6). Under optimal conditions (28 °C, 100 rpm, permanent lightening) they are able to grow up in 5 to 7 days. The growth curve of Rhodotorula glutinis CCY 20-2-26 as well as other studied red yeast exhibited similarly typical two-phase character with prolonged stationary phase (Figures 7, 8) probably due to the ability of the yeast cells to utilize lipid storages formed during growth as additional energy source (Marova et al., 2010). The production of carotenoids during growth fluctuated and some local maxima and minima were observed. The maximum of beta-carotene production was obtained in all strains in stationary phase after about 80 hours of cultivation.

Comparison of presented growth curves led to some partial conclusions about growth of red yeasts (Marova et al., 2010). All tested strains reached stationary phase after about 50 hours of cultivation. All strains also exhibited prolonged stationary phase with at minimum one, more often with several growth maxima. First growth maximum was observed in all strains after about 80 hours of growth. In strains followed for longer time than 100 hours additional growth maximum was observed after 105 — 140 hours. Carotenogenic yeasts probably utilize some endogenous substrates accumulated at the beginning of stationary phase. Growth maxima are mostly accompanied with carotenoid production maxima mainly in first 90 hours of cultivation. Cultivation in production media in presence of some stress factors or using waste substrates is recommended to carry out to first production maximum (about 80 — 90 hours) to eliminate potential growth inhibiton caused by nutrient starvation or toxic effect of stress. Longer cultivation can be also complicated by higher ratio of dead and living cells and in semi-large-scale and large-scale experiments also with higher production costs.

The capacity of a given biomass to absorb toxic metal ions has been traditionally quantified using either Langmuir, Freundlich, Langmuir-Freundlich isotherms, or different alternative models. These isotherms were developed under chemical assumptions that are not generally met in biosorption processes.

The main reason for their extended use is that they describe satisfactorily experimental data. They can be used for predictions, although they do not take into account external parameters, such as the pH or ionic strength. Langmuir equation

is the simplest and the one used by the most of authors. In this equation, qeq is the amount of metal ion sorbed at equilibrium, Ceq the equilibrium concentration of metal ion in solution and Ъ is the Langmuir constant related to the energy of sorption, which reflects quantitavely the affinity between the biomass and the metal ion. The parameter qmax represents the maximum capacity of the biomass to absorb a given metal ion and it is usually determined by fitting the isotherm experimental data to the equation model. The qmax values are quite almost expressed as milligrams of sorbed metal ion respect to the weight in grams of dry sorbent.

The qmax values reported in an our recent paper (Nurchi & Villaescusa, 2008), based on the survey of last ten years of literature, lie in the ranges 2.81-285.7 mg/ g for Cd2+, 11.7-32.00 mg/ g for Cu2+, 8.45-73.76 mg/ g for Pb2+, 1.78-35 mg/ g for Zn2+, 7.9-19.56 mg/ g for Ni2+, 17.2-126.9 mg/g for Cr(VI), and 3.08 mg/g for Cr3+. These quantities look more similar when expressed in molar concentrations (0.025-2.5 mmol/g for Cd2+, 0.185-0.50 mmol/g for Cu2+, 0.04-0.36 mmol/g for Pb2+, 0.027-0.53 mmol/g for Zn2+, 0.13-0.34 mmol/g for Ni2+, 0.33-2.44 mmol/g for Cr(VI), and 0.06 mmol/g for Cr3+) and the maximum quantity of metal ion sorbed by a gram of sorbent is of the order of 0.5 mmoles (values five times higher are found for Cd2+ and Cr(VI), which could be considered a reasonable result if we consider the large variability in materials and experimental conditions (particle size, pH, temperature, etc.).

In order to better characterize the behavior of a given sorbent, the use of chemical (mmol/g) instead of technical (mg/ g) units has to be recommended whenever comparisons have to be made. The results obtained in this way actually contain information on the number of coordinating sites, which can be of great utility to make provisional forecasts of the binding capacity of different metal ions, without restraints due to their atomic mass.

In literature different variables (particle size, temperature, pH, exchange and so on), and different kinetics and thermodynamic models (Langmuir, Freundlich, …) are taken into account. In the following sections 5 and 6 we will discuss the effect of temperature and pH on the sorption process. In order to design sorption processes, it is important to predict the rate at which a pollutant is removed from an aqueous solution. The rate constant and reaction order must be determined experimentally. It is usually necessary to carry out experimental studies varying several parameters such as metal ion and sorbent concentration, agitation speed, particle size, and temperature. Fitting the experimental results allows determining the kinetic mechanism, e. g. film diffusion, kinetic sorption, diffusion sorption or a combination of these processes. The kinetic models most used in biosorption studies were widely discussed in an intersting review by Ho et al., 2000.

For the microbiological characterization by biomass, total and fecal coliforms, molds and yeasts, coagulase-positive staphylococci, Aeromonas spp and Salmonella spp were investigated according to methodologies described by Vanderzant & Splittstoesser (1992). For the proximate composition of biomass, the concentrations of moisture, lipids, proteins and ash were determined according to Association of Official Analytical Chemists (1995). Amino acid determinations were carried out before and after acid hydrolysis (5 mg of extract) with a mixture containing 6 mol L-1 of HCl and 5% phenol/water (0.08 mL) for 72 h at 110°C. Samples were dried, diluted with citrate buffer pH 2.2 and filtered in a GV Millex Unity (Millipore). Amino acids analyses were performed by cation-exchange chromatography using a Shimadzu LC-10A/C-47A, sodium eluents and post-column derivatization with o — phthaldialdehyde. Identification and quantification were accomplished by the comparison of retention time and area of each amino acid with a standard containing 16 amino acids (100 nmol mL-1), respectively (Fountoulakis & Lahm, 1998).

The biomass color attributes L (lightness), C (chroma) and h (hue) were obtained from the average of three consecutive pulses launched from the optical chamber of the MiniScan XE Plus (Hunter Lab) using illuminant D65 and 2o observer, after calibration with black and white standards (Commission Internationale de l’Eclairage, 1986).

For the determination of oxycarotenoids, an adaptation of Valduga (2005) methodology was used. Pigments were extracted from biomass with dimetilsulfoxide at 55°C/30 min and alternated cycles of ultrasound at 40 kHz (Unique/USC 1800A) and shaking (Phoenix/P-56). Next, a mixture containing acetone: methanol (7:3, v/ v) was added, tubes were centrifuged at 3.400 g and 5°C/10 min and the supernatant was transferred to a 50 mL volumetric flask. Successive extractions were performed until no color remained in cells or solvent. Final dilutions were made up with methanol and the quantification of oxycarotenoids was accomplished at 448 nm (Hitachi U-1000/U-1100). Total carotenoids were estimated according to Davies (1976) using the absorption coefficient of carotenoids suggested by Liaaen-Jensen & Jensen (1971).

1.2 Drought

The agroclimatic and production-system environments of the SAT regions are very diverse. The inherent water constraints that limit crop production are variable. However, it is quite possible to broadly characterize and classify the drought patterns of a given environment using long-term water-balance modeling and geographic information system (GIS) tools (Chauhan et al., 2000). The assessment of the moisture-availability patterns of the target environments is critical for the development of best adapted crop genotypes to target environments and to identify iso-environments of drought patterns. As mentioned earlier, SAT environments are often characterized by a relatively short growing season in a generally dry semi-arid climate, with high average temperatures and potential evaporation rates. Soils are moderate to heavy, with low to moderate levels of available water content to the plants. In addition, the dry season at this location is generally rain-free, with a high mean air temperature and vapour-pressure deficits. This season provides an ideal screening environment to expose plants to controlled drought-stress treatments by regulating the timing and quantity of irrigation (Bidinger et al., 1987; Johansen et al., 1994).

Drought stress is a major limiting factor at the initial phase of plant growth and establishment. The usual effects of drought on the development of a plant are a lowered production of biomass and/or a change in the distribution of this biomass among the different organs. In addition, plant productivity under drought stress is strongly related to the processes of dry matter partitioning and temporal biomass distribution (Kage et al., 2004). Reduction of biomass due to water stress is common in both cereals and legumes, although genotypic variation does exist. In general, cereals biomass production is less affected by drought than legumes.

The types of drought occurrence is usually categorized as early, intermittent and terminal depending on the growth phase of the plant when the water deficit becomes acute. For example, long duration pigeonpea, a crop usually sown at the first onset of south Asian monsoon rains, experiences all the three types of drought.

At the early seedling stages of the crop, lack of water can adversely affect seedling growth and occasionally kill seedlings and reduce the plant population. Similar lack of water for a period of time at the later stages can affect leaf area expansion and subsequently the root and shoot growth causing intermittent set backs and relief. However at later stages once the rains cease the plants during their reproductive growth phases tend to rely on the constantly receding soil moisture leading to increasing levels of terminal drought stress affecting largely the reproductive plant parts. This may reduce the number of pod/spikelet bearing

sites or the number of seeds formed in a pod/spike or the size of the developing seeds. On the other hand, pearl millet, sorghum, groundnut and pigeonpea sown in the rainy season experience intermittent drought while chickpea that is primariely grown postrainy experiences the terminal drought (Fig 2).

Monthly rainfall from 2006 to 2010 of Quetta and Tomagh is presented in Table 1. Total annual rainfall at Quetta ranged from 105.8 to 247 mm while at Tomagh the total annual rainfall ranged from 214 to 462.6 mm. The dry matter forage production of different sites and years is presented in Table 2. The initial dry matter forage production during 2007 was 80, 60 and 184 kg/ha, respectively at Mastung, Ziarat and Loralai. Each year there were increasing trend of dry forage production and during 2010 the dry matter forage production was recorded 230, 485 and 864 kg/ha at Mastung, Zirata and Loralai, respectively (Table 2). Rainfall and its distribution during winter and spring, 2007 was comparatively better than 2006. The community degraded rangelands showed recovery potential at all sites. At Mastung the dominated range vegetation is Artemisia and Haloxylon species while at Loralai and Tomagh site perennial grasses (Cymbopogon jwarancusa, Chrysopogon aucheri) are dominated. The range recovery depends on the distribution of rainfall and management practices. The Loralai and Tomagh sites have better recovery potential of range vegetation due to occurrence of both winter and monsoon rains (Fig. 4).

Arid rangelands of Balochistan characterized by highly unpredictable and variable rainfall events, behave as non-equilibrium system. This means that both climatic and grazing factors are important in any range management and improvement interventions. There are no universally accepted grazing strategies due to specific conditions of rangelands. However, resting, restricted grazing has proved for the recovery of natural range vegetation and forage improvement in many arid and semi-arid regions. The range vegetation of Balochistan has low reproductive potential due to the adaptive strategies of the plants for survival under extreme climatic conditions. The recovery potential is also very site specific like in case of Loralai, the grasses were heavily grazed but have shown good recovery potential under favourable conditions. The optimal growth time of grasses in Balochistan is from March to June, may be extended up to October in case of monsoon rains. Therefore, resting of vegetation during this time period is very essential for recovery and forage improvement. However, if the objectives were for seed production and re-generation than at least two to three years rest period must be provided (Ahmad et al., 2010; Ahmad et al., 2007; Ahmad et al., 2000 a, b,c). Accumulated dead material of perennial grasses can decline both productivity and nutritive value (Ahmad et al., 2009; Bano et al., 2009 ) therefore, a rotation grazing may yield better results than long term protection. Enhanced growth rate of grasses in response to grazing, fire and disturbance under favourable environments have been observed (Chapin & McNaughton, 1989).

Many rangeland areas in Balochistan still have potential of natural recovery if properly grazed. As a result of protection from grazing, it is evident from the results that the community rangelands are resilient and have potential of biological recovery subject to rainfall distribution and management practices. Range productivity is greatly influenced by fluctuations in rainfall, grazing pressure and nutrients (Olson & Richard, 1989; Scoones, 1995). Above ground net primary production can be used as an integrative attribute of eco­system function (McNaughton et al., 1989). Above ground net primary production is an important variable in natural resource management because it determines forage availability for both wild and domestic herbivores. Oesterheld et al., (1992) found a strong connection between stocking density and above ground primary production for South American Rangelands. The rate of biological recovery might be slow as expected in the arid and semiarid climatic zones. The rate of vegetation recovery is also related with the rainfall distribution during the optimal growing period rather than total rainfall distribution. Strong vegetation recovery response has been reported even under desert conditions with mean annual rainfall as 60-80 mm under deep and permeable soils (Le Houerou, 1992a). From Morocco to Iran the perennial ground cover and primary productivity were enhanced by a factor of 2-5 and in most cases, 3-4 within a few years either by total or partial protection (Le Houerou, 1992a). In West Asia and North Africa range exclosures from 11 countries showed that productivity in exclosures enhanced averaged by 2.8 times than the adjacent grazed areas (Le Houerou, 1998). However, very long-term protection may not yield better results due to accumulation of dead old material that may reduce the new fresh growth. Controlled grazing may produce similar or better results than exclosures in some cases (Le Houerou, 2000). The recruitment rate of grasses may not be achieved within two to three- year protection. The changes in species composition are very slow processes in arid and semiarid areas (West et al., 1984). Limited spring season rainfall (the optimal time of seedling recruitment) in Balochistan is the main factor for low seedling recruitment even under complete protection from grazing. According to long-term meteorological data analysis in Balochistan, it is observed that above-normal rainfall amounts that promoted spring seedling emergence occur with about 10% and less than 10% probability (Keatinge and Rees, 1988).

2.5.1 Carotenoid and ergosterol enriched biomass

Red yeasts are used predominantly as carotenoid producers and, thus, carotenoid-enriched biomass is the most frequently produced. The growing scientific evidence that carotenoid pigments may have potential benefits in human and animal health has increased commercial attention on the search for alternative natural sources. Comparative success in microbial pigment production has led to a flourishing interest in the development of fermentation processes and has enabled several processes to attain commercial production levels. An important aspect of the fermentation process is the development of a suitable culture medium to obtain the maximum amount of desired product. In recent years, cheap raw materials and by-products of agro-industrial origin have been proposed as low-cost alternative carbohydrate sources for microbial metabolite production, with the view also of minimizing environmental and energetic problems related to residues and effluent disposal.

During the produt recovery process, the biomass is isolated and transformed into a form suitable for isolating carotene, which can be further isolated from the biomass with appropriate solvent, suitably purified and concentrated. Using whole biomass as final product, isolation of metabolites is not necessary and other cell active components can be utilized. Nevertheless, cell disruption is recommended for better bioavailability of the most of lipid-soluble substance (Frengova & Beshkova, 2009). Several types of microbes have been reported to produce carotenoids and carotenoid-rich biomass; but only a few of them have been exploited commercially (Bhosale, 2004).

Among the few astaxanthin producing microorganisms, Phaffia rhodozyma (Xanthophyllomyces dendrorhous) is one of the best candidate for commercial production of pigment as well as enriched biomass. Therefore, many academic laboratories and several companies have developed processes which could reach an industrial level. Phaffia/ Xanthophyllomyces has some advantageous properties that make it attractive for commercial astaxanthin production: (i) it synthesizes natural form astaxanthin (3S,3’S configuration) as a principal carotenoid, (ii) it does not require light for its growth and pigmentation, and (iii) it can utilize many types of carbon and nitrogen sources (Lukacs et al, 2006; Dufosse, 2006). Studies on physiological regulation of astaxanthin in flasks cultivations was verified in bioreactors and the ataxanthin amount reached 8.1 mg/L (Dufosse, 2006). Enhanced production of the pigment was achieved during fed-batch fermentation with regulated additions of glucose and optimized fermentation condition finally yielded up to 20 mg astaxanthin/L (Certik et al., 2009). High carbon/nitrogen ratio induced amout of astaxanthin and C/N-regulated fed-batch fermentation of P. rhodozyma led to 16 mg astaxanthin/L. Thus, this strain can be considered as a potential producer of astaxanthin. In addition, to avoid isolation of astaxanthin from cells, two-stage batch fermentation technique was used (Fang & Wang, 2002), where Bacillus circulans with a high cell wall lytic activity was added to the fermentation tank after the accumulation of astaxanthin in P. rhodozyma was completed. Astaxanthin is the principal colorant in crustaceans, salmonids and flamingos. There is current interest in using P. rhodozyma biomass in aquaculture to impart desired red pigmentation in farmed salmon and shrimps.

Biotechnological production of |3-carotene by several strains of the yeast Rhodotorula is currently used industrially. This yeast is convenient for large-scale fermentation because of its unicellular nature and high growth rate. Because Rhodotorula glutinis synthesizes P — carotene, torulene and torularhodin, the rate of production of the individual carotenoid depends upon the incubation conditions. Specially prepared mutants of Rhodotorula not only rapidly increased formation of torulene or thorularhodin, but amount of P-carotene reached the level of 70 mg/L (Sakaki et al., 2000). Better strategy than isolation of individual pigments seems to be use of the whole enriched biomass to feed and food industry.

In our recent work exogenous stress factors were used to obtain higher production of carotenoids in R. glutinis CCY 20-2-26 strain. Physical and chemical stress factors were applied as single and in combination. Adaptation to stress was used in inoculum II. Short­term UV irradiation of the production medium led to minimal changes in biomass production. The production of carotenoids in R. glutinis cells was stimulated in all samples of exponentially growing cells when compared with control cultivation. In stationary phase, the production of carotenoids was induced only by 35-min irradiation. Ergosterol production exhibited very similar changes as P-carotene production both under temperature and UV stress. Our results are in good agreement with recent findings of the effect of weak white light irradiation on carotenoid production by a mutant of R. glutinis (Sakaki et al., 2000).

Using chemical stress, the influence of osmotic (2-10 % NaCl) stress, oxidative (2-10 mM H2O2) stress and combined effects of these stress factors on the morphology, growth and production of biomass, carotenoids and ergosterol by R. glutinis CCY 20-2-26 cells were studied (Marova et al., 2010). First, R. glutinis cells were exposed to higher concentration of stress factors added into the production medium. Further, low concentrations of NaCl and H2O2 were added to the inoculum medium or to both inoculum and production media. Exposition of red yeast cells to all tested stress factors resulted in higher production of carotenoids as well as ergosterol, while biomass production was changed only slightly. Under high stress 2-3 times increase of P-carotene was observed. The addition of low salt or peroxide concentration into the inoculation media led to about 2-fold increase of carotenoid production. In Erlenmeyer flasks the best effect on the carotenoid and ergosterol production (3- to 4-fold increase) was exhibited by the combined stress: the addition of low amount of NaCl (2 mM) into the inoculum medium, followed by the addition of H2O2 (5 mM) into the production medium. The production of ergosterol in most cases increased simultaneously with the production of carotenoids.

Cultivation of R. glutinis carried out in a 2-litre laboratory fermentor was as follows: under optimal conditions about 37 g/L of yeast biomass were obtained containing approx. 26.30 mg/L of total carotenoids and 7.8 mg/L of ergosterol. After preincubation with a mild stress factor, the yield of biomass as well as the production of carotenoids and ergosterol substantially increased. The best production of enriched biomass was obtained in the presence of peroxide in the inoculation medium (52.7 g/L of biomass enriched with 34 mg/L of carotenoids) and also in combined salt/peroxide and salt/salt stress (about 30-50 g/L of biomass enriched with 15-54 mg/L of total carotenoids and about 13-70 mg/L of ergosterol). Rhodotorula glutinis CCY 20-2-26 strain could be a suitable candidate for biotechnological applications in the area of carotenoid rich biomass production. Preliminary cultivation in a 2-litre laboratory fermentor after preincubation with stress factors in well — ballanced experiments led to the yield of about 40-50 g per litre of biomass enriched by 20-40 mg of P-carotene+lycopene sum (approximately 30-50 mg of total carotenoids per litre) and about 70 mg of ergosterol per litre. Addition of simple cheap stress factor substantially increased metabolite production without biomass loss. Therefore, this strain takes advantage of the utilization of the whole biomass (complete nutrition source), which is efficiently enriched for carotenoids (provitamin A, antioxidants) and also ergosterol (provitamin D). Such a product could serve as an additional natural source of significant nutrition factors in feed and food industry (Marova et al, 2010).

Our further work was focused on possiblity to use carotenogenic yeasts cultivated on alternative nutrition sources combined with stress factors (Marova et al., 2011). Both physiological and nutrition stress can be used for enhanced pigment production. Three red yeast strains (Sporobolomyces roseus, Rhodotorula glutinis, Rhodotorula mucilaginosa) were studied in a comparative screening study. To increase the yield of these pigments at improved biomass production, combined effect of medium with modified carbon and nitrogen sources (waste materials — whey, potato extract) and peroxide and salt stress was tested. The production of carotene-enriched biomass was carried out in flasks as well as in laboratory fermentor. The best production of biomass was obtained in inorganic medium with yeast extract. In optimal conditions tested strains differ only slightly in biomass production. Nevertheless, all strains were able to use most of waste substrates. Biomass and pigment production was more different according to substrate type. It was observed that addition of non-processed or processed whey or potato extract to media can increase beta — carotene production, while biomass production changed relatively slightly (Marova et al,

2010) .

In Rhodotorula glutinis addition of whey substrate into production medium led to 3.5x increased production of beta-carotene without substantial changes in biomass. Non- processed whey or potato extract added to production media led to about 3x increase of beta-carotene production accompanied by biomass loss. The highest yield was reached after addition of lyophillized non-processed whey to INO II as well as to production media. Also potato extract added into INO II led to increased beta-carotene production while biomass yield was lower. Sporobolomyces roseus exhibited significant changes in biomass:carotene ratio dependent on whey substrate addition. Substantial biomass decrease in presence of lyophilized whey in INO II (under 5 g/L) was accompanied by very high beta-carotene yield (2.54 — 2.75 mg/ g d. w.). Potato extract addition into production medium led to about 11-times increase of P-carotene production, while production of biomass was lower than in control. Preincubation of S. roseus cells with potato extract and following cultivation in production medium with 5% hydrogen peroxide led to about 20-times higher P-carotene production as in control, in this cultivation conditions biomass decreased only slightly. In general, total production of biomass by S. roseus was about 2-x lower as in R. glutinis. So, this is the reason why S. roseus CCY 19-4-8 cells is less suitable to enriched biomass production. Rhodotorula mucilaginosa CCY 20-7-31 seems to be relatively poor producer of carotenoids when compared with the other two strains. Production of biomass in this strain was more similar to R. glutinis (about 8 g/L). However, addition of potato extract into INO II combined with salt stress in production medium enabled to reach the highest biomass as well as P-carotene production observed in this strain yet (1.56 mg/ g d. w.). It seems that this strain needs for optimal pigment/biomass production some additional nutrition factors which are no present in simple (but cheap) inorganic medium, but can be obtained from different waste substrates (also cheap).

In laboratory fermentor better producers of enriched biomass were both Rhodotorula strains. In experiments with Rhodotorula glutinis the production of yeast biomass in a laboratory fermentor was in most types of cultivation more than 30 grams per litre (about 3-times higher yield than in Erlenmeyer flasks; Table 1). The balance of cultivation in a fermentor in optimum conditions is as follows: we obtained about 37.1 g/l of biomass containing 17.19 mg per litre of P-carotene (see Table 1). The production of P-carotene was induced in most types of media combinations. High total yield of P-carotene was obtained in whey production medium (44.56 g/L of biomass; 45.68 mg of P-carotene per litre of culture). The highest total yield of P-carotene was obtained using combined whey/whey medium (51.22 mg/L); this cultivation was accompanied also with relatively high biomass production (34.60 mg/L). In experiments with Sporobolomyces roseus CCY 19-4-8 substantially higher production of biomass was obtained in fermentor when compared with cultivation in flasks. Mainly in whey medium about 3-times biomass increase (about 12 g/L) was reached and production of beta-carotene was mostly higher than in R. glutinis. Because of low biomass production, total yields were in S. roseus mostly lower than in R. glutinis cells. Yeast strain Rhodotorula mucilaginosa CCY 20-7-31 exhibited in most cases similar biomass production characteristics as R. glutninis, while pigment production was substantially lower (see Table 4). As the only substrate suitable for P-carotene production was found potato extract in INO II combined with 5% salt in production medium. Under these conditions 55.91 mg/L of P- carotene was produced in 30.12 g of cells per litre of medium (Marova et al, 2011).

The aim of all preliminary experiments carried out in laboratory fermentor was to obtain basic information about potential biotechnological use of the tested strains to the industrial production of Р-carotene/ergosterol enriched biomass. The results of both Rhodotorula strains are very promising. The yield of R. glutinis CCY 20-2-26 biomass (37 — 44.5 g/L) produced in minimal cultivation medium was similar to the maximal biomass yield obtained in fed-batch cultivation of Phaffia rhodozyma (36 g/L), which is widely used as an industrial producer of astaxanthin (Lukacs et al., 2006). The maximal production of total carotenoids by used P. rhodozyma mutant strain was 40 mg/L, which is also similar to the yields obtained in R. glutinis CCY 20-2-26 cells grown in whey medium. The highest yields of pigments were obtained in Rhodotorula glutinis CCY 20-2-26 cells cultivated on whey medium (cca 45 g per liter of biomass enriched by 46 mg/L of beta-carotene) and in Rhodotorula mucilaginosa CCY 20-7-31 grown on potato medium and 5% salt (cca 30 g per liter of biomass enriched by 56 mg/L of beta-carotene). Such dried carotenoid-enriched red yeast biomass could be directly used in feed industry as nutrition supplement (Marova et al., 2011).

An alternative for utilization of some natural substrates for production of carotenoids by Rhodotorula species is the method of cocultivation. A widespread natural substrate is milk whey containing lactose as a carbon source. Carotenoid synthesis by lactose-negative yeasts (R. glutinis, R. rubra strains) in whey ultrafiltrate can be accomplished: by enzymatic hydrolysis of lactose to assimilable carbon sources (glucose, galactose) thus providing the method of co-cultivation with lactose-positive yeasts (Kluyveromyces lactis), producers of galactosidase or by creating conditions under which lactose is transformed into carbon sources (glucose, galactose, lactic acid) easily assimilated by the yeast when they were grown in association with homofermentative lactic acid bacteria or yogurt starter culture (Frengova & Beshkova, 2009). The maximum carotenoid yields for the microbial associations [R. rubra + K. lactis; R. glutinis + Lactobacillus helveticus; R. rubra + L. casei; R. rubra + (L. bulgaricus + Streptococcus thermophillus)] were as follows: 10.20, 8.10, 12.12, 13.09 mg/l, respectively. These yields are about five times higher than that of a lactose-positive strain R. lactosa cultivated in whey reported in literature (Frengova et al., 2004). R. glutinis — Debaryomyces castellii co-cultures was produced (5.4 mg carotenoids/l) about three times the amount of total carotenoids formed by the red yeast cultured alone in low hydrolyzed corn syrup (Buzzini, 2001) The author concluded that oligosaccharides and dextrins of syrup could be utilized for pigment production by R. glutinis after hydrolysis to maltose and glucose by the extracellular amylolytic enzymes produced by D. castellii DBVPC 3503 in co­cultures.

As mentioned above, waste substrates and alternative nutrition sources were used to production of astaxanthin-enriched biomas sof Xanthophyllomonas dendrorhous sources (Lukacs et al, 2006; Dufosse, 2006). Batch culture kinetics of this yeast revealed reduction in biomass with glucose and lower intracellular carotenoid content with fructose. Figures were different when compared to sucrose. In contrast, specific growth rate constant stayed between 0.094 — 0.098 h-1, irrespective of the carbon sources employed. Although the uptake rate of glucose was found to be 2.9-fold faster than that of fructose, sucrose was found to be a more suitable carbon source for the production of carotenoids by the studied strain. When sugar cane molasses was used, both the specific growth rate constant and the intracellular carotenoid content decreased by 27 and 17%, respectively. Compared with the batch culture using 28 g/L sugar cane molasses, fed-batch culture with the same strain resulted in a 1.45- fold higher cell yield together with a similar level of carotenoid content in X. dendrorhous SKKU 0107 (Park et al, 2008).

Phaffia rhodozyma NRRL Y-17268 cells were proliferated in xylose-containing media made from Eucalyptus wood. Wood samples were subjected to acid hydrolysis under mild operational conditions, and hydrolysates were neutralized with lime. Neutralized hydrolysates were treated with charcoal for removing inhibitors and then supplemented with nutrients to obtain culture media useful for proliferation of the red yeast P. rhodozyma. Biomass was highly pigmented and volumetric carotenoid concentrations up to 5.8 mg carotenoids/L (with 4.6 mg astaxanthin/L) were reached. Further experiments in batch fermentors using concentrated hydrolysates (initial xylose concentrations within 16.6 and 40.8 g/L) led to good biomass concentrations (up to 23.2 g cells/L) with increased pigment concentration (up to 12.9 mg total carotenoids/L, with 10.4 mg astaxanthin/L) and high volumetric rates of carotenoid production (up to 0.079 mg/L/h (Parajo et al., 1998).

In the future, other types of waste materials (for instance from winemarket) are intended to be tested as carbon sources for carotenogenesis in red yeasts (Table 2). Moreover application of an environmental stress in combination with waste materials can lead to overproduction of carotenoids and lipids and decrease cost of their production. Such strategies could result into production of yeast biomass rich not only in carotenoids and other provitamins, but also in other nutrition components (proteins, PUFA, metal ions etc.) that originate both from yeast cells and from cultivation substrates. This is the way to production of complex food additives based on naturally enriched yeast biomass.

Ikuo Takeda

Shimane University Japan

1. Introduction

Phosphorus (P) is an essential element in plant nutrients, because many biochemical processes such as photosynthesis, respiration, and energy transfer depend on inorganic P or its organic derivatives. However, P is difficult for plants to obtain from the rhizosphere and P deficiency is one of the major limitations on crop production. This is because soluble P in soil, the primary P source for plants, is extremely low concentration (Condron et al., 2005) and significant portions of P in the soil are various organic complexes and unavailable (Raghothama, 2005). On a worldwide scale, land covering 5.7 billion hectares is estimated to be deficient in P for optimal crop production (Batjes, 1997). Since the soluble P in the soil is easily taken up by plants and microorganisms, continuous application of P fertilizer is necessary for crop production.

The global demand for P has increased 10-fold since the beginning of the 20th century (Cordell et al. 2009) and approximately 80% of the demand is for agricultural fertilizers (Steen, 1998). Thus, more P will be required as the world’s population increases. However, there is concern that world P resources will be depleted in the next 50-100 years, because the reserves of high-grade phosphate rock are limited (Runge-Metzger, 1995; Steen, 1998; Smil, 2000; Stewart et al., 2005). Therefore, the recovery of P is essential for sustaining food production.

Figure 1 shows a conceptual illustration of the global P cycle, which is completed by P flux from the ocean to the land, and is intimately linked to global ocean circulation. The P derived from weathering or fertilizer application on the land is washed down in rivers and enters the ocean food chain. In deep ocean water (about 2,000-3,000 m in depth), the P concentration is considerably higher than that at the surface because dead fish and plankton fall on the ocean floor. However, the P-rich water is too deep for humans to exploit. In the deep ocean, the water flows from the Atlantic Ocean to the Pacific Ocean via the Antarctic and the Indian Ocean, while the surface water flows in the opposite direction. This movement is very slow; about 2000 years is required to complete this circulation. In some areas of the Pacific Ocean, the flow rises from the bottom to the surface, but this is rare phenomenon. Because the occurrence of this rising flow depends on a complex combination of sea currents, winds, and geographical features. Consequently, these selected areas are abundant in plankton and fish. However, the P flux from ocean to land occurs only via

fishery and seabirds’ droppings (guano), unlike nitrogen that can be released into the atmosphere via denitrification. In addition, the seabed is gradually transformed into land by the geological movement of the Earth’s crust, but this occurs on a much longer time scale than do human activities. Therefore, the global P cycle is extremely limited.

Despite the limited nature of the P cycle, repeated applications of fertilizers and organic matter builds up nutrients in the soil. Strong relationships between the level of P monitored by soil tests and the amount of P lost in runoff have been reported (Pote et al., 1996; Sharpley, 1995). Thus, excessive application of P fertilizers contributes to eutrophication, which is sometimes responsible for the lack of clean water resources. From this viewpoint, the recovery of P is also essential.

The behavior of P in nature has been affected by iron (Fe) oxides since ancient time (Bjerrum & Canfield, 2002). In natural water bodies such as canals, swamps, and ponds with low oxygen groundwater seeps and circumneutral conditions, the accumulation of soft, reddish — brown sediment is often observed (Fig. 2). The essential compounds in this sediment are biogenic Fe oxides produced by microaerobic Fe-oxidizing bacteria (Emerson et al., 1999; Emerson & Weiss, 2004; James & Ferris, 2004) and this ferric substance in the sediment can adsorb P in a similar manner to abiotic P adsorbents of ferric compounds (Boujelben et al., 2008; Persson et al., 1996; Seida & Nakano, 2002; Zeng et al., 2004). Therefore, biogenic Fe oxides in nature are considered as one of the P resources. However, they have not yet been recognized as such, although they have been used for ferrous Fe removal in water treatment facilities (Pacini et al., 2005; Katsoyiannis & Zouboulis, 2004; Sogaard et al., 2001). This is because biogenic Fe oxides in natural water bodies are easily dispersed by water turbulence. In addition, it is difficult to collect only the Fe oxides as a P resource, because they usually accumulate only a few centimetres, and anaerobic and malodorous mud exists underneath (see Fig. 3). Moreover, the mud deposits that have existed for a long time may accumulate harmful substances such as heavy metals.

A new method for the recovery of P from natural water bodies using Fe-oxidizing bacteria and woody biomass as a carrier has been proposed (Fig. 3). A woody carrier is immersed in water in which Fe-oxidizing bacteria are abundant and then removed several weeks later. In this chapter, this method was tested in an agricultural area, dominated by rice paddy fields, located in the eastern part of Shimane Prefecture, Japan. As the woody carrier, sawdust from the Japanese cedar and Japanese cypress were used. Since the accumulation of biogenic Fe oxides was observed throughout the year at several locations, the water quality at these points was monitored. In addition, heavy metals on the immersed carrier were also measured, because biogenic Fe oxides have the potential to also adsorb heavy metals such as arsenic (As), cadmium (Cd), chromium (Cr), mercury (Hg), lead (Pb), zinc (Zn), and nickel

(Ni).

Statistical analysis showed significant effects of intercrop fertilization and interaction between intercrop fertilization and straw fertilization on the nitrate content in potato tubers (table 13). The highest concentration of nitrates was recorded in tubers harvested from control object. Intercrop fertilization caused a significant decrease of nitrate content in potato tubers. The lowest their concentration was noted in potato tubers fertilized with white clover, a mixture of white clover and Italian ryegrass and phacelia both plowed down in the autumn, and left till spring in the form of mulch. The nitrates content in potato tubers fertilized with Italian ryegrass, did not differ significantly from the concentrations observed in potato tubers fertilized with farmyard manure. An interaction has been noted which shows that the lowest content of nitrates was recorded in tubers fertilized with white clover and phacelia both plowed down in the autumn, and left till spring in the form of mulch, and the lowest on control object.

3.3.7 Glycoalkaloids content in potato tubers

The content of glycoalkaloids in potato tubers was significantly modified for examined factors and their interaction (table 14). Intercrop fertilization caused a significant decrease of

glycoalkaloids in potato tubers in comparison with its concentrations observed in the potato from control object. The lowest content of glycoalkaloids was noted in potato tubers fertilized with white clover, a mixture of white clover with Italian ryegrass, phacelia both plowed down in the autumn, and left till spring in the form of mulch. The concentration of glycoalkaloids in potato tubers fertilized with Italian ryegrass did not differ significantly from that recorded in the potatoes fertilized with farmyard manure. Straw fertilization also significantly modified the content of glycoalkaloids in potato tubers. At the sub-block with

straw the concentration of glycoalkaloids in potato tubers was significantly lower than that recorded in the tubers of the sub-block without straw. Investigated the interaction of factors that were characterized it shows that the lowest content of glycoalkaloids in potatoes fertilized with white clover, white clover with straw, and phacelia both plowed down in the autumn, and left till spring in the form of mulch in combination without straw and with straw, and the highest potato tubers collected from the control object.