In Sweden research on short rotations using Salix began in the end of 1900. Today 10,000­15,000 hectares of short rotation Salix stands have been established and are actively managed using advanced technology. The management is based on small-scale plots, where the farmer owns the stand and manages it. Harvesting is undertaken using machinery owned by entrepreneurs and the harvested material is sold to be used for district heating. Common rotation periods are 4-5 years with 5-6 repeated rotations; a plantation lasts a total of 20-30 years before a new one must be established. The plantations must be fertilized and in some cases treated with herbicides. Pathogens (fungi and insects) damaging the leaves and shoots will cause a reduction in growth. As the seedlings represent attractive wildlife habitat, the plantations must be fenced.

Numerous dynamic crop models have been developed for simulating crop growth in function of environmental factors (soil characteristics, climate) and of agricultural practices. Some of these models can be used for predicting crop biomass and yields and crop quality before harvest. For example the Geographic Information System (GIS) was successfully used to predict water-limited biomass production potential of various agro climatic zones of the world (Fig 3). It is very clear that the biomass producing potential of

SAT is between 300 to 600 g dry matter M-2 Y-1 that corresponds well with the observed annual productivities.

The advent of remote sensing technology supported by Geographic Information System (GIS) has opened new vistas of improving agricultural statistics systems all over the world. The applications of Remote Sensing (RS) in the field of agriculture are wide and varied, ranging from crop discrimination, inventory, assessment and parameter retrieval, on one hand, to assessing long term changes and short-term characterization of the crop environment. The use of remote sensing for crop acreage and yield estimation has been well demonstrated through various studies all over the world, and has gained importance in recent years as a means of achieving these estimates possibly in a faster mode and at a cheaper cost (Murthy et al., 1996). An integrated methodology for providing area and yield estimation and yield forecasting models with small area estimates at the block level using satellite data has been developed (Singh and Goyal, 2000; Singh et al. 2002).

The remote sensing use for drought prediction can benefit from climate variability predictions. Recent research on crop-water relations has increasingly been directed towards the application of locally acquired knowledge to answering the questions raised on larger scales. However, the application of the local results to larger scales is often questionable. Crop simulation models, when run with input data from a specific field/ site, produce a point output. The scope of applicability of these simulation models can be extended to a broader scale by providing spatially varying inputs (soil, weather, crop management) and combining their capabilities with a Geographic Information System (GIS). The main purpose of interfacing models and GIS is to carry out spatial and temporal analysis simultaneously as region-scale crop behavior has a spatial dimension and simulation models produce a temporal output. The GIS can help in spatially visualizing the results as well as their interpretation by spatial analysis of model results.

Plants were treated with eight different brassinosteroids (24-epibrassinolide; 24- epicastasterone; 4154 compound and five androstane and pregnane analogues of brassinosteroids marked KR1, KR2, KR3, KR4 and KR5) in 49-59 DC (growth phase referred to a decimal code for the growth stages of cereals — from visible awns to complete inflorescence emergence) (Zadoks et al., 1974). All brassinosteroids were applied in the form of 1 nmol L-1 of efficient compound in the water solution by spraying on all aerial biomass. Each of the tested brassinosterids was applied in four parallel replicates (4 x 10 m2 field plots). Untreated plants were cultivated as well in tetraplicates as the control variant. Applied brassinosteroids (Fig. 1) were synthesized by the Institute of Organic Chemistry and Biochemistry of the Academy of Sciences of the Czech Republic. 24-epibrassinolide (24- epiBL) and 24-epicastasterone (24-epiCS) are naturally occurring plant phytohormones, compound 4154 is a synthetic brassinosteroid registered in the Czech Republic (Registration Nr. 294343, conferred on 4 Oct 2004) and the EU (Nr. 1401278, conferred on 28 Sep. 2005). Compounds KR1 — KR5 are synthetic permanently studied brassinolide analogues, which do not occur naturally in plants and will be published after finishing the synthesis of similar structures and protecting of these compounds by a patent (Vlasankova et al., 2009).

In pot experiment plants were treated with three brassinosteroids (24-epibrassinolide, 24- epicasterone, and 4154) in two different growth stages in four parallel replicates in each brassinosteroid. Plants or experimental pots were divided before the application of brassinosteroids into four groups that differed with growth stage in the date of treatment and number of brassinosteroids applications (Table 3).

The evolution and spatial extent of the smoke plume is studied through the behaviour of the AOT500. Figure 9 shows the modelled AOT500 and the horizontal flow at 1400 m above the surface, at selected days during the analyzed period. On 23 August the smoke plume showed a relative maximum close to the emission sources, centred near 10° S and 60° W, with values higher than 2. The smoke plume had its greater longitudinal extension between

the Equator and 10° S. This feature is related with the dominance of the easterlies in that region. An outflow zone from South America towards the west is observed between 5° S and 10° S. To the south, at higher latitudes, the smoke plume had an important branch oriented from the NW to the SE reaching the latitude 20° S, with AOT values higher than 0.5. These features are due to the transport patterns at low and middle atmospheric levels, which were dominated by the flow at the western branch of the high-pressure system and the channelling effect of the Andes barrier.

On 24 August, the region with higher AOT values near the sources increased. The smoke plume had a greater latitudinal extent over Argentina, reaching 35° S. The core of the low — level jet was associated with a relative minimum. Contrarily, a relative maximum over northern Argentina appears west of the jet core. On 25 August the southern edge of the plume continues to travel towards higher latitudes and presents a shape associated with the anticyclonic circulation and the barrier effect of the Andes. Optical depths ranging from 0.3 to 0.5 cover NE of Argentina and Uruguay. An outflow region from South America towards the west is observed between 5° S and 15° S. On 26 August, Buenos Aires had AOT values between 0.5 and 0.75. The smoke plume has N-S orientation from latitudes near 15° S to 30° S. The greater values are observed near the sources, over central Brazil and Bolivia. On its southernmost extreme the plume shows a curvature associated with the high-pressure system centred over the Atlantic Ocean, near 32° S and 35° W. Cordoba is affected by aerosol optical thicknesses ranging from 0.75 to 1, which are higher than those at Buenos Aires.

On 27 August the smoke plume reached latitudes higher than 40° S. The AOT over Buenos Aires ranged from 0.75 to 1. On 28 August, the cold front succeeded in crossing the Andes and reached Argentina and afterwards, the plume started to be displaced towards the east but was still over Buenos Aires due to its pre-frontal location. During the next day, the smoke plume displaced towards the northeast, owing to the fast movement of the cold front, and reached southern Brazil. On 30 August the plume had clearly the shape of the frontal zone and reached Sao Paulo. During the next day the surface cold front was stationary over Sao Paulo. There is a region associated to the postfrontal anticyclone with a low-level recirculation of the aerosols towards the west of the plume centre. This occurs at the north­western edge of the frontal region, where the forced convection is weaker.

Techniques that can handle large numbers of genotypes include: germination or plant survival in high salinity, leaf injury as measured by membrane damage (leakage of ions from leaf discs), premature loss of chlorophyll (using a hand-held meter), or damage to the photosynthetic apparatus (using chlorophyll fluorescence). These methods can identify genotypes able to germinate, or survive, in very high salinities (over 200 mM NaCl), but do not discriminate between genotypes in their ability to tolerate the low or moderate salinities typical of many saline fields (50-100 mM NaCl). A major limitation to the use of injury or survival to identify salt-tolerant germplasm arises when the cause of injury is not known.

1.1.1.1 Screening methods based on physiological mechanisms

Because of the complex nature of salinity tolerance, as well as the difficulties in maintaining long-term growth experiments, trait-based selection criteria are recommended for screening techniques (Noble and Rogers, 1992). Traits used for screening germplasm for salinity tolerance have included Na+ exclusion, K+/Na+ discrimination (Asch et al., 2000) and Cl — exclusion (Rogers and Noble, 1992). The relationship between salinity tolerance and K+/Na+ discrimination was also considered, because K+/Na+ rather than Na+ alone has been used as an index of salinity tolerance for cultivar comparisons in wheat (Chhipa and Lal, 1995) and rice (Zhu et al., 2001). One of the mechanism of salinity tolerance that could be considered was tissue tolerance of high internal Na+ concentrations. Tissue tolerance cannot be measured directly, and is difficult to quantify. Yet it is clearly important; overexpression of vacuolar Na+/H+ antiporter that sequesters Na+ in vacuoles improved the salinity tolerance of Arabidopsis, tomato and brassica (Aharon et al., 2003).

1.2 Breeding

Breeding programs for new varieties of sweet sorghum suited to semi arid tropics, temperate areas with rainy summer, Mediterranean areas with dry summer and soil salinity, are under development (Cosentino, 1996).

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

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

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

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

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

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

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

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

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

Table 3 lists operating conditions. Permeate flux was incremented from 20 to 35 l/(hm2) in successive experimental runs. In order to maintain a constant HRT independent from the imposed permeated flux in each run, a peristaltic pump extracted from the permeate tank the flow rate necessary to maintain the required HRT and the excess of permeate was returned to the bioreactor (see Figure 1). Chemical cleaning of the membrane with sodium hypochloride (250 mg/l) was performed at the end of each experimental run.

Air was supplied through the bottom providing oxygen and stirring. The dissolved oxygen concentration was always above 1.5 mg/l in the reactor operated at 23 ± 2 °С.

1.4 Analytical methods

Dissolved oxygen (DO) was measured using a WTW 340i. Chemical oxygen demand (COD), ammonium-nitrogen (N-NH+), total suspended solids (TSS), mixed liquor suspended solids (MLSS), mixed liquor volatile suspended solids (MLVSS) were determined in conformity with the Standard Methods (American Public Health Association, 1992). Nitrite-nitrogen (N — NO2-) and Nitrate-nitrogen (N-NO3-) were measured by spectrophotometric methods with a HACH DR 2000. Microbial floc size was measured by Coulter LS100 (Coulter, UK). Proteins were determined as bovine albumin equivalent using the protein kit assay TP0300 supplied by Sigma, following the Lowry method (Lowry et al., 1951). Polysaccharides were measured as glucose equivalent by the Dubois’ method (Dubois et al., 1956).

The oxygen uptake rate was measured by following the dissolved concentration with a membrane oxygen electrode in a medium without substrate (SOURe, endogenous). The sludge rheological properties were determined by using the concentric cylinder rotational viscosimeter Visco Star plus (FungiLab, Spain). The width of the annular gap was 1.0 mm. Measurements were done at 25 °C.

1.1 Characterization of red (carotenogenic) yeasts

1.1.1 Taxonomy

Yeasts belong to the kingdom Fungi (Mycota) — a large group of eukaryotic organisms that includes microorganisms such as yeasts and moulds. Some species grow as single-celled yeasts that reproduce by budding or binary fission. Dimorphic fungi can switch between a yeast phase and a hyphal phase in response to environmental conditions. The fungal cell wall is composed of glucans and chitin. Another characteristic shared with plants includes a biosynthetic pathway for producing terpenes that uses mevalonic acid and pyrophosphate as chemical building blocks (Keller et al., 2005). Fungi produce several secondary metabolites that are similar or identical in structure to those made by plants. Fungi have a worldwide distribution, and grow in a wide range of habitats, including extreme environments such as deserts or areas with high salt concentrations or ionizing radiation, as well as in deep sea sediments. Some can survive the intense UV and cosmic radiation. Around 100,000 species of fungi have been formally described by taxonomists, but the global biodiversity of the fungus kingdom is not fully understood. There is no unique generally accepted system at the higher taxonomic levels and there are frequent name changes at every level, from species upwards. Fungal species can also have multiple scientific names depending on their life cycle and mode (sexual or asexual) of reproduction. The 2007 classification of Kingdom Fungi is the result of a large-scale collaborative research. It recognizes seven phyla, two of which—the Ascomycota and the Basidiomycota—are contained within a branch representing subkingdom Dikarya (Hibbett, 2007).

The Ascomycota constitute the largest taxonomic group within the Eumycota. These fungi form meiotic spores called ascospores, which are enclosed in a special sac-like structure called an ascus. This phylum includes single-celled yeasts (e. g., of the genera Saccharomyces, Kluyveromyces, Pichia, and Candida), and many filamentous fungi living as saprotrophs, parasites, and mutualistic symbionts.

Some yeast species accumulate carotenoid pigments, such as P-carotene, torulene, and thorularodin which cause their yellow, orange and red colours and are therefore called red yeasts. Carotenogenic yeasts are a diverse group of unrelated organisms (mostly Basidiomycota) and the majority of the known species are distributed in four taxonomic groups: the Sporidiobolales and Erythrobasidium clade of the class Urediniomycetes, and Cystofilobasidiales and Tremellales of the class Hymenomycetes (Libkind et al., 2005). Along with the most known producer Phaffia rhodozyma, there is evidence of the capacity for carotene formation by other well-known pigmented yeasts of the genus Rhodotorula (order Sporidiobolales). The composition and amount of the carotenoid pigments in numerous natural isolates of the genera Rhodotorula/ Rhodosporium and Sporobolomyces/Sporidiobolus were studied in detail (Yurkov et al., 2008).

At this time the number of red yeasts species Rhodotorula, Rhodosporidium, Sporidiobolus, Sporobolomyces, Cystofilobasidium, Kockovaella and Phaffia are known as producers of carotene pigments. Many of these strains belong to oleaginous yeasts, some of them can effectively remove heavy metals from industrial effluents and detoxify certain pollutants. Studies with yeast mutants or carotenoid biosynthesis inhibitors have shown that carotenoid-deficient yeast strains are sensitive to free oxygen radicals or oxidizing environment, and that this sensitivity can be relieved by the addition of exogenous carotenoids (Davoli et al., 2004). The major yeast pigments are |3-carotene, y-carotene, torulene, torularhodin and astaxanthin (Dufosse, 2006).

Nickel (atomic weight 58.69) is a ductile, malleable, silver-white metal (density 8.91 g/ mL). It presents the oxidation states -1, 0, +1, +2, +3, +4. More than 70% of nickel produced annually is devoted to the production of alloys; nickel is used in a variety of electrolytic procedures, in the manufacture of batteries and in welding procedures, as a catalyst in large scale processes, and in the glass and ceramics industry. In addition to 8.5 million tons per year of nickel in the atmosphere due to natural sources, 43 million tons are released by anthropogenic activities. Population exposed at soluble nickel concentration < 1 gg m-3 has no respiratory cancer risk, which is related to exposure to concentrations greater than 1 mg m-3 (workers in nickel industries). Dermal sensitivity to nickel is presented by 10-20 % of female and 1 % of male population. The nickel content in surface water ranges from 2 to 20 gg/L. The limit for nickel in drinking water is 0.07 mg/L (WHO, 2008).

1.1.3 Zinc

Zinc (atomic weight 65.41) is a soft, bluish-white metal (density 7.14 g/ mL). It presents the oxidation states 0, +2. Zinc and its products are widely used in alloy production, as anticorrosion coatings of steel and iron, in electrical devices, in rubber and tire industries, in paints, in pesticides and as chemical reagents in a number of applications. Zinc is the second most abundant trace metal in the human body: it appears in the active site of a variety of enzymes and many of the metabolic consequences of its deficiency are related to a diminished activity of zinc metallo-enzymes. Zinc is relatively nontoxic, even if daily doses greater than 100 mg during several months may lead to different disorders. Zinc imparts an undesirable astringent taste to water. Water containing zinc at concentrations in the range 3­5 mg/L also tends to appear opalescent and develops a greasy film when boiled. This feature allows the high zinc limit 3 mg/L in drinking water (WHO, 2008).

The bacterial inoculum was added, at 1% (v/v), to 100 L of treated wastewater. Cultivation was accomplished in anaerobiosis inside 100 L glass reactors at 32 + 2oC and 2,000 + 500 lux for seven days.

For the biomass recuperation, the culture was filtered at 0.2 pm, 1.5 m3 h-1 and 4.5 bar (Frings), giving origin to a concentrate containing the cells and a permeate. The concentrate was centrifuged at 3,400 g for 30 min at 5°C (Incibras Spin VI) and the resulting slime was frozen at — 40oC and lyophilized (Liobras L 101) for 48 h. Hand grinding was performed to obtain the power biomass. Procedures were repeated six times.

1.2 Process analyses

Cell mass concentration was determined from 20 mL of concentrate, after successive centrifugation (900 g/15 min) and washing cycles followed by drying at 80°C until it gets constant weight.

For productivity determination, it was considered the mean production of dry biomass per liter per day.

TN, OG, COD and pH determinations in permeate were accomplished as previously described for crude wastewater (APHA, AWWA and WPCF, 2005).