Beet or cane molasses are the main substrate used in yeast production plants. These materials were selected for two main reasons: first, yeasts grow very well using the sugars present in the molasses and second, they are economically interesting since they are a waste product coming from sugar refineries without any other application. Usually, molasses contain between 65% and 75% of sugars, mainly sucrose (Hongisto and Laakso, 1978); but the composition is highly variable depending on the sucrose-refining procedure and on the weather conditions of that particular year. Sucrose is extracellularly hydrolysed by yeasts in two monosaccharides, glucose and fructose, which are transported to and incorporated into the yeast metabolism as carbon sources. However, molasses are deficient in other essential elements for yeast growth. One of them is nitrogen since its molasses content is very poor (less than 3%). Yeasts can use some of the amino acids present in molasses, but addition of nitrogen sources is needed, generally in the form of ammonium salts or urea. Magnesium and phosphate elements are also supplemented in salt forms. Finally, three vitamins (biotin, thiamine and pantothenic acid), required for fast growth, must be supplemented since their content in molasses is also very low (Oura, 1974; Woehrer and Roehr, 1981). Another negative aspect of molasses being used as a substrate to produce yeasts is the presence of different toxics that can affect yeast growth. Variable amounts of herbicides, insecticides, fungicides, fertilizers and heavy metals applied to beet or cane crops can be found in molasses and in different stocks. Moreover bactericides, which are added during sugar production in refinery plants, can be found (Reed and Nagodawithana, 1988). All these toxics can decrease yeast performance by inhibiting growth (Perez-Torrado, 2004). In fact, a common practice in yeast plants is to mix different stocks to dilute potential toxics.

The effects of molasses composition on yeast growth have been recently analysed at molecular level by determining the transcriptional profile of yeast growing in beet molasses and by comparing it to complete synthetic media (Shima et al., 2005). The results revealed that yeast displays clear gene expression responses when grown in industrial media because of the induction of FDH1 and FDH2 genes to detoxify formate and the SUL1 expression as a response to low sulphate levels. Thus it can be concluded that molasses are far from being an optimal substrate for yeast growth. Another interesting conclusion drawn is that molecular approaches can be especially suited to gain insight into the yeast biomass production process.

In the last years, the price of molasses has increased because of their use in other industrial applications such as animal feeding or bioethanol production (Arshad et al., 2008; Kopsahelis et al. 2009; Xande et al., 2010), thus rendering the evaluation of new substrates for yeast biomass propagation a trend topic for biomass producers’ research. New assayed substrates include molasses mixtures with corn steep liquor (20:80), different agricultural waste products (Vu and Kim, 2009) and other possibilities as date juice (Beiroti and Hosseini, 2007) or agricultural waste sources, also called wood molasses, that can be substrate only for yeast species capable of using xylose as a carbon source.

1.2 Experimental setup

The experimental unit consisted of a cylindrical 220 l submerged membrane bioreactor (MBR) equipped with a submerged hollow-fibre membrane of 0.03 pm rated pore diameter and 0.93 m2 filtering surface area (ZeeWeed ZW10) supplied by GE Water & Process Technologies (Figure 3). The effluent (permeate) was extracted from the top header of the module under slight vacuum (transmembrane pressure lower than 0.12 bar). Fouling was controlled by coarse bubbling of air flow and by intermittent filtration of the permeate. The pilot plant (ZW10) was located in the wastewater treatment plant (WWTP) in Santa Cruz de Tenerife (Canary Islands, Spain).

1.3 Feedwater characteristics

The reactor was fed with screened (2.5 mm) municipal wastewater. The average feed concentrations are given in Table 2. The feedwater was characterized by a high biodegradable organic fraction (BOD5/COD = 0.52-0.67). Also, suspended solids in the water had a high organic fraction (VSS/TSS = 0.85-0.95).

Ivana Marova1, Milan Certik2 and Emilia Breierova3 1Brno University of Technology, Faculty of Chemistry, Centre for MaterialsResearch, Purkynova 118, 612 00 Brno, 2Slovak Technical University, Faculty of Chemical and Food Technology, Bratislava, 3Institute of Chemistry, Slovak Academy of Sciences, Bratislava, 1Czech Republic 2,3Slovak Republic

1. Introduction

Yeasts are easily grown unicellular eukaryotes. They are ubiquitous microorganisms, occuring in soil, fresh and marine water, animals, on plants and also in foods. The environment presents for yeast a source of nutrients and forms space for their growth and metabolism. On the other hand, yeast cells are continuously exposed to a myriad of changes in environmental conditions. These conditions determine the metabolic activity, growth and survival of yeasts. Basic knowledge of the effect of environmental factors on yeast is important for understanding the ecology and biodiversity of yeasts as well as for control the yeast physiology in order to enhance the exploitation of yeasts or to inhibit or stop their harmful and deleterious activity.

The overproduction of some metabolites as part of cell stress response can be of interest to the biotechnology. For instance carotenogenic yeasts are well known producers of biotechnologically significant carotenoid pigments — astaxanthin, P-carotene, torulen, torularhodin and under stress conditions this carotenoid accumulation was reported to be increased. Knowledge of molecular mechanism of the carotenoid production stimulation can then lead to improvement of such biotechnological process. Red yeasts are able to accumulate not only carotenoids, but also ergosterol, unsaturated fatty acids, Coenzyme Q10 and other, which can contribute to the biomass enrichment. The use of this stressed biomass in feed industry could have positive effect not only in animal and fish feeds because of high content of physiologically active substances, but it could influence nutritional value and organoleptic properties of final products for human nutrition.

Yeast biomass, mainly in the form of Saccharomyces cerevisiae, represents the largest bulk production of any single-celled microorganism throughout the world. In addition to use of

live yeast biomass for the leavening of bread dough, many other applications of yeast cells and yeast cell extracts have emerged. Most yeast biomass for industrial use is derived from Saccharomyces cerevisiae, but other yeasts have specific uses and may be grown on a range of substrates unavailable to S. cerevisiae. Some yeast strains are usable to industrial single-cell protein production from lignocellulose materials, methanol, n-alkanes, starch, oils and also other cheap carbon sources. Except compresses baker’s yeasts for baking, brewing, winemaking and distilling also other whole-cell yeast products are industrially used as animal feed, human and animal probiotics, as biosorbents for heavy metal sequestration and, also as nutritional trace element sources. Yeasts are rich sources of proteins, nucleic acids, vitamins and minerals but mostly with negligible levels of triglycerides.

Pigmented yeasts are used as feed and food colorants and, come of them, also as single cell oil producers. This chapter will be focused on controlled production of biomass and some interesting lipid metabolites of several non-traditional non-Saccharomyces yeast species. Growing interest in yeast applications in various fields coupled with significance of carotenoids, sterols and other provitamins in health and dietary requirements has encouraged "hunting" for more suitable sources of these compounds.

Mercury (atomic weight 200.59) is a heavy, liquid at room temperature, silvery colored metal (density 13.53 g/mL). It presents the three oxidation states 0, +1, +2. The most modern uses are in batteries and cells. The Castner-Kellner process, that produces chlorine and sodium hydroxide, requires mercury in the entire process. It is furthermore used in thermometers, thermostats, switches, vacuum pumps, fluorescent and energy-saving lights, tooth fillings and electrical components. Many compounds of mercury have been used as medicines since many ages. However, in recent years, as awareness about the toxicity of mercury has increased amongst people, most of the medicines have become obsolete. Mercurochrome (used in cuts and wounds) and Thimerosal (as an dental amalgamation) are the compounds that are no more used in many countries. Mascara, an ingredient of cosmetics, contains some amounts of Thimerosal. During the past ten years mercury consumption has shown a strong upward trend. The major proportion can be accounted for by the chloro-alkali industry, from which mercury is released into the environment. Most of it finds its way to watercourses exposing aquatic ecosystems where mercury accumulates. The use of seed-dressings containing mercury is decreasing, although this use of mercurial’s is still considerable, and in view of findings in other countries elevated mercury levels in seed-eating birds and their predators must be expected. Many states in the US are now very strict against the use of mercury in cosmetics and medicines. Mercury in the form of gaseous vapors is used in mercury vapor lamps, neon signs and fluorescent lamps.

Biological properties of mercury are very important and include these characteristics: inhaled mercury is more dangerous than ingested mercury; human workers and handlers of mercury may become contaminated and mercury-diseased; elemental and inorganic

mercury can be transformed to the extremely toxic methyl-mercury (CH3Hg+) by some microbes; mercury accumulates in living organisms, cells, tissues, organs and organisms; mercury can damage immune cells and tissues, and organs such as brain, heart, kidneys, lungs; mercury can be concentrated in the environment and then magnified upwards along the food chain (bioaccumulation and bio-magnification); all compounds of mercury, except those not soluble in water, are to be considered poisonous regardless of the manner of inhalation or ingestion. Mercury limit in drinking water is 0.006 mg/L (WHO, 2008).

Rubrivivax gelatinosus previously isolated from poultry slaughterhouse wastewater and characterized by morphological and biochemical tests was used in this experiment. The cells were maintained in Pfennig medium containing (per liter): 0.5 g KH2PO4; 0.4 g MgSO4.7 H2O; 0.4 g NaCl; 0.4 g NHCl; 0.05 g CaCl2.2H2O; 1.0 g sodium acetate, 0.2 g yeast extract; 0.005 g ferric citrate; 10.0 mL trace elements solution (FeSO4.7H2O 200 mg; ZnSO4.7H2O 10 mg; MnCl2.4H2O 3 mg; H3BO3 30 mg; CoCl2.6H2O 20 mg; CuCl2.2H2O 1 mg; NiCl2.6H2O 2 mg; Na2MoO4. 2H2O 3 mg); 20.0 g bacteriological agar; 10.0 ml biotin sol. (0.0015% ) and 10.0 ml thiamine-HCl sol. (0.005%). The pH was adjusted to 7.0 before autoclaving at 121oC for 15 min.

For the initial inoculum preparation, cells were grown in Pfennig liquid medium with the same pH and composition described above but bacteriological agar, under anaerobiosis (fully filled screw-crap tubes), 32 ± 2°C and 1,400 ± 200 lux for approximately 3 days, until a slight red color arose.

For the final inoculum, an aliquot from initial inoculum was transferred at 1% (v/v) to the same medium and incubation was carried out under the same conditions described before, until optical density at 600 nm reached 0.5 (Ponsano et al., 2003a).

L. Krishnamurthy, M. Zaman-Allah, R. Purushothaman,

M. Irshad Ahmed and V. Vadez

International Crops Research Institute for the Semi-Arid Tropics (ICRISAT),

Patancheru 502 324, Andhra Pradesh

India

1. Introduction

1.1 Prevalence of abiotic stresses in SAT agriculture

The semi-arid tropics (SAT) include parts of 48 countries in the developing world: in most of India, locations in south east Asia, a swathe across sub-Saharan Africa, much of southern and eastern Africa, and a few locations in Latin America (Fig 1). Semi-arid tropical regions are characterized by unpredictable weather, long dry seasons, inconsistent rainfall, and soils that are poor in nutrients. Sorghum, millet, cowpea, chickpea, pigeonpea and groundnut are the vital crops that feed the poor people living in the SAT.

Environmental stresses represent the most limiting factors for agricultural productivity. Apart from biotic stresses caused by plant pathogens, there are a number of abiotic stresses such as extremes temperatures, drought, salinity and radiation which all have detrimental effects on plant growth and yield, especially when several occur together (Mittler 2006).

salinization, as a result of sparse and seasonal rainfall and mismanagement of the natural resource base for agriculture (Evans, 1998). Expansion of irrigation does not seem feasible in many countries in Asia, the Middle East, and North Africa, where most of the available and easily accessible water resources have been already utilized. Furthermore, irrigated soils are affected by salinity with significant subsequent yield losses. Desertification may be aggravated by both extensive farming due to demographic pressure and the regional climatic changes. Hence, there is a need for the breeding programs to assign high priority for the development of crops with tolerance to both drought and salinity stress. The genetically complex control of these stresses in the plant genome may be facilitated through the manipulation of specific genes governing the component characteristics needed to achieve tolerance to salt or drought in plant crops.

Twelve parallel transects of 35 meter each were established at each site at a distance of 15 m apart each other. Three transects were used at each site for determination of forage production. Biomass estimates were made during the months of May/June (at optimal vegetation growth) to document the range productivity. At each transect four 1 x 5 m2 subplots were established on alternate site of the transect line. The vegetation inside the 1 x 5 m2 subplot was clipped at ground level, separated into leaves and wood, and oven dried. The dry matter forage production was converted into kg/ha. Descriptive analysis was used for calculation of dry forage production. Monthly, rainfall data were recorded from a rain gauge installed at Ziarat:Tomagh site while the rainfall data from Quetta site is used because due to non-availability of meteorological data of Mastung site.

There have been several reports on the enhancement of volumetric production (mg/l) as well as cellular accumulation (mg/ g) of microbial carotenoid upon supplementation of metal ions (copper, zinc, ferrous, calcium, cobalt, alluminium) in yeasts and molds (Bhosale, 2004; Buzzini et al., 2005). Trace elements have been shown to exert a selective influence on the carotenoid profile in red yeasts. It may be explained by hypothesizing a possible activation or inhibition mechanism by selected metal ions on specific carotenogenic enzymes, in particular, on specific desaturases involved in carotenoid biosynthesis (Buzzini et al., 2005). The other explanation is based on observations that presence of heavy metals results in formation of various active oxygen radicals what, in a turn, induces generation of protective carotenoid metabolites that reduce negative behaviour of free radicals. Such strategy has been applied in several pigment-forming microorganisms to increase the yield of microbial pigments (Breierova et al., 2008; Rapta et al., 2005).

In order to achieve rapid carotenoid overproduction, various stimulants can be added to the culture broth. One group of such enhancers is based on intermediates of the tricarboxylic acid cycle which play an important role in metabolic reactions under aerobic conditions, forming a carbon skeleton for carotenoid and lipid biosynthesis in microbes. Because pigment increase is paralleled by decreased protein synthesis, restriction of protein synthesis is an important way how to shift carbon flow to carotenoid synthesis (Flores — Cotera & Sanchez, 2001). It was also proposed that high respiratory and tricarboxylic acid cycle activity is associated with production of large quantities of reactive species and these are known to enhance carotenoid production (An, 2001). It should be emphasized that the degree of stimulation was dependent on the time of addition of the citric acid cycle intermediate to the culture medium. Some fungi showed that addition of organic acids to media elevated P-carotene content and concomitantly decrease y-carotene level with complete disappearance of lycopene (Bhosale, 2004).

Chemical substances capable of inhibiting biosynthetic pathways have been applied to characterize metabolic pathways and elucidate reaction mechanisms. In general, compounds that inhibit biosynthesis can act through various mechanisms, such as inhibiting the active site directly by an allosteric effect (reversible or otherwise), altering the regulation of gene expression and blocking essential biochemical pathways or the availability of cofactors, among other possibilities. From this view, number of chemical compounds including terpenes, ionones, amines, alkaloids, antibiotics, pyridine, imidazole and methylheptenone have been studied for their effect on carotene synthesis (Bhosale, 2004). In order to obtain commercially interesting carotenoid profiles, the effect of supplementation with diphenylamine (DPA) and nicotine in the culture media of Rhodotorula rubra and Rhodotorula glutinis was investigated. DPA blocks the sequence of desaturation reactions by inhibiting phytoene synthase, leading to an accumulation of phytoene together with other saturated carotenoids and nicotine inhibits lycopene cyclase, and consequently the cyclization reactions (Squina & Mercadante, 2005). Cultivation of Xanthophyllomyces dendrorhous in the presence of diphenylamine and nicotine at 4°C was reported to trigger interconversion of P — carotene to astaxanthin (Ducrey Sanpietro & Kula, 1998).

The addition of solvents such as ethanol, methanol, isopropanol, and ethylene glycol to the culture medium also stimulate microbial carotenogenesis. It should be noted that while ethanol supplementation (2%, v/v) stimulated P-carotene and torulene formation in Rhodotorula glutinis, torularhodin formation was suppressed (Bhosale, 2004). It was proposed that ethanol-mediated inhibition of torulene oxidation must be accompanied by an increase in P-carotene content suggesting a shift in the metabolic pathway to favor ring closure. Detailed studies revealed that ethanol activates oxidative metabolism with induction of HMG-CoA reductase, which in turn enhances carotenoid production. However, stimulation of carotenoid accumulation by ethanol or H2O2 was more effective if stress factors were employed to the medium in exponential growth phase than from the beginning of cultivation (Marova et al, 2004).

Mutagenesis

Mutagenesis is an alternative to classical strain improvement in the optimization of carotenoid production. Mutagenic treatment with N-methyl-N-nitro-N-nitrosoguanidine (NTG), UV light, antimycin, ethyl-methane sulfonate, y-irradiation, high hydrostatic pressure have been used successfully to isolate various strains with enhanced carotenoid- producing activity. UV mutant R. gracilis has shown 1.8 times higher carotenoid synthesizing activity than that of the parent strain and the relative share of P-carotene in the total carotenoids was 60%. The yellow colored mutant 32 was also obtained by UV mutagenesis of the pink yeast R. glutinis and produced a large quantity of total carotenoids (2.9 mg/ g dry cells), which was 24-fold higher accumulation of total carotenoids compared with the wild-type. Mutant 32 produced 120-fold more beta-carotene (2.05 mg/ g dry cells) than the parent culture in a much shorter time (36 h), which was 82% (w/w) of the total carotenoid content. Later, after the treatments of five repeated cycles by high hydrostatic pressure of 300 MPa, the mutant R. glutinis RG6p was obtained, beta-carotene production of which reached 10.01 mg/l, increased by 57.89% compared with 6.34 mg/l from parent strain (Frengova & Beshkova, 2009).

A fivefold increase in beta-carotene accumulation was reported for yellow mutant P. rhodozyma 2-171-1 which was obtained after ethyl-methane sulfonate mutagenesis of dark red strain P. rhodozyma. This mutant is likely to be blocked in the oxidase step and therefore unable to perform the conversion of beta-carotene to echinenone and latter to astaxanthin. The UV-mutant P. rhodozyma PG 104 produced 46-fold more P-carotene (92% of total carotenoids) than the parent culture (2% of total carotenoids) and maximum beta-carotene yields were 1.08 mg/ g dry cells and 9.95 mg/l. Using NTG mutagenesis two different strains of carotenoid accumulating X. dendrourhous mutants JH1 and JH2 were also isolated. Astaxanthin-overproducing mutant JH1 produced 4.03 mg astaxanthin/ g dry cells, and this value was about 15-fold higher than that of wild-type. Mutant JH2 produced 0.27 mg beta — carotene/ g dry cells, and this was fourfolds increase from that of wild-type and the mutant

X. dendrourhous JH1 produced maximum astaxanthin concentration of 36.06 mg/l and 5.7 mg/g dry cells under optimized cultivation conditions (Kim et al., 2005).

To isolate a carotenoid-hyperproducing yeast, P. rhodozyma 2A2 N was treated by low-dose gamma irradiation below 10 kGy and mutant 3A4-8 was obtained. It produced 3.3 mg carotenoids/ g dry cells, 50% higher carotenoid content than that of the unirradiated strain (antimycin NTG-induced mutant 2A2 N). Gamma irradiation produces oxygen radicals generated by radiolysis of water and could induce mutation of P. rhodozyma through a chromosomal rearrangement. A primary function of carotenoids in P. rhodozyma is to protect cells against singlet oxygen and these compounds have been demonstrated to quench singlet oxygen. Oxygen radicals have been known to cause changes in the molecular properties of proteins as well as enzyme activities. Thus, oxygen radicals generated by gamma irradiation might modify the pathway in astaxanthin biosynthesis of P. rhodozyma and cause an increase in carotenoid production of the mutant 3A4-8 isolated by gamma irradiation (Frengova & Beshkova, 2009).

In studies on heterogeneous material, requiring long equilibration times, it is hard to perform reliable calorimetric measurements. Thus, only carrying out experiments at variable temperature can give information on how this parameter affects the sorption of metal ions. From the limited extent of studies at variable temperature, only controversial conclusions can be reached. Most studies have been carried out at a fixed room temperature (20 or 25 °C). Some studies point out a low temperature influence or, at least, in a limited temperature range, giving evidence that ion exchange is the mechanisms responsible for the sorption process. Nevertheless, Kapoor and Viraraghavan, 1997, remarked that biosorption reactions are normally exothermic, which indicates that sorbent capacity increases with decreasing temperature. Conversely, Romero-Gonzalez et al., 2005, found that the sorption capacity of Agave lechuguilla leaves for Cr(VI) sorption increased on increasing the temperature from 10 to 40 °C, justifying this endothermicity with Cr(VI) reduction to Cr(III). Malkoc and Nuhoglu, 2007, confirmed the endothermicity of Cr(VI) sorption on tea factory waste, metal uptake increasing as temperature increas from 25 °C to 60 °C. The favorable temperature effect was attributed to a swelling effect within the internal structure of the sorbent enabling the large metal ions Cr(VI) to penetrate further.

Statistical analysis showed a significant effect of examined factors and their interaction on total protein content in potato tubers (table 11). Intercrop fertilization significantly increased the concentration of total protein in potato tubers in relation to its content recorded in potatoes harvested from control object. Indeed, the highest concentration of total protein were characterized by potato tubers fertilized with white clover and with phacelia both plowed down in the autumn and left till spring in the form of mulch. The content of total protein in potato tubers fertilized with a mixture of white clover with Italian ryegrass did not differ significantly from that observed in potato tubers fertilized with farmyard manure. However, fertilization of potato with Italian ryegrass caused a significant decrease in total protein content in potato tubers in comparison with farmyard manure fertilization. Straw fertilization also significantly modified the concentration of total protein in potato tubers. On objects with straw total protein content in potato tubers was significantly higher on objects without straw. An interaction has been noted, which shows that the highest concentration of total protein was characterized by a potato 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, whereas the lowest potato tubers collected from the control object without intercrop fertilization.