1.1 Role in soil

Earthworms have a critical influence on soil structure, forming aggregates and improving the physical conditions for plant growth and nutrient uptake. They also improve soil fertility by accelerating decomposition of plant littre and soil organic matter. Earthworms are the most important invertebrates in this initial stage of the recycling of organic matter in various types of soils. Curry & Byrne (1992) demonstrated that the decomposition rate of straw which was accessible to the earthworms was increased by 26-47% compared with straw from which they were excluded. Organic matter that passes through the earthworm gut and is digested in their casts is broken down into much finer particles, so that a greater surface area of the organic matter is exposed to microbial decomposition. Martin (1991) reported that casts of the tropical earthworms had much less coarse organic matter than the surrounding soil, indicating that the larger particles of organic matter were fragmented during passage through the earthworm gut. Earthworm species, such as Lumbricus terrestris, are responsible for a large proportion of the overall fragmentation and incorporation of littre in many woodlands and farmland of the temperate zone, which resulted in the formation of mulls. As a result, the surface littre and organic layers are mixed thoroughly with the mineral soil (Scheu & Wolters, 1991).

The numbers of earthworm burrows have been counted between 50 and 200 burrows m-2 on horizontal surfaces (Edwards et al., 1990). Earthworms not only improve soil aeration by their burrowing activity, but they also influence the porosity of soils. Earthworm burrows was found to increase the soil-air volume from 8% to 30% of the total soil volume (Wollny, 1890). In one soil, earthworm burrows comprise a total volume of 5 litres m-3 of soil, making a small but significant contribution to soil aeration (Kretzschmar, 1978). Water infiltration was from 4 to 10 times faster in soils with earthworms than in soils without earthworms (Carter et al., 1982). They bring large amounts of soil from deeper layers to the surface and deposit as casts on the surface. The amounts which turned over in this way greatly differ with habitats and geographical regions, ranging from 2 to 268 tons ha-1 (Beauge, 1912; Roy, 1957). The importance of this turnover, which was discussed first by Darwin (2009), can be seen by comparing the profile of a stratified mor soil (with few earthworms) with that of a well-mixed mull soil. Blanchart (1992) reported in a formation of aggregates that under natural conditions with or without earthworms, large aggregates (>2 mm) comprised only 12.9% of soil with no earthworms, whereas in soil with worms, large aggregates comprised 60.6% of soil after 30 months in the field. Devliegher & Verstraete (1997) introduced the concepts of nutrient enrichment process and gut associated process. They noted that earthworms are performing these two different functions that may have contrasting their effects on soil microbiology, chemistry and plant growth. Earthworms, such as L. terrestris, incorporate and mix surface organic matter with soil and increase biological activity and nutrient availability. However, they also assimilate nutrients from soil and organic matter as these materials pass through their gut.

The fouling rate, measured as the slope of transmembrane pressure against filtration time, has been used in many works as a fouling quantification parameter in systems operated under constant permeate flux. Experimentally, it has been found that rf depends exponentially on permeate flux (Figure 12). Therefore, a threshold flux value may be identified (32 l h-1 m-2) above which the fouling increases at an unacceptable rate.

4.3 Physico-chemical and microbiological quality of the permeate

The physical and chemical quality of the permeate was assessed by the analysis of turbidity, COD and nitrogen compounds.

The permeate had an average turbidity value of 0.59 NTU, indicating a total retention of suspended solids and macro-colloidal matter. In addition, the low turbidity of the permeate registered during the whole experimental period showed that the membrane maintained its integrity.

The organic matter content was determined by measuring the COD in feed wastewater, in the permeate and in the liquid phase of the suspension. Soluble COD (CODS) was obtained by filtering through a filter paper of 0.45 pm pore diameter. Figure 13 shows the COD of feedwater (COD feed), the soluble COD of feedwater (CODs feed), the COD of the permeate (CODp) and soluble COD of the liquid phase (CODs reactor) versus operating time. Typical fluctuations of feed wastewater can be seem in a real treatment plant. These oscillations lessened considerably in the permeate and in the liquid phase.

As it is shown in Figure 13, there is a significant difference between the total and soluble COD of feed due to the presence of suspended solids. It was estimated that approximately 68% of the COD of the feed is in a particulate form. If the soluble COD of feed is compared with the soluble COD of the CODs liquid phase (CODs reactor) a removal efficiency close to 86% can be obtained, mainly due to biological degradation and only 6% is due to the membrane separation process. It should be noted that the BOD5 was not analyzed because, through frequent and trustworthy analysis of the same water, the BOD5/COD ratio was

confirmed to be approximately constant and equal to 0.75, so the COD analysis may be considered sufficient to determine the biodegradation produced.

Also, the evolution of the ammonium nitrogen concentration in feed wastewater (N-NH4 feed) and the nitrogen compounds of the permeate ((N-NH4+)p, (N-NO2-)p, (N-NO3-)p) were measured during the experimental period (Figure 14). As can be seen, the concentrations of nitrogen-nitrate in the permeate (N-NO3-)p were in the range of 15-45 mg/l, while nitrite and ammonia were completely removed. This is interpreted as a total oxidation of ammonium to nitrate.

As shown in Table 5, no bacterial contamination indicators, bacterial pathogens or parasites were detected in the permeate. This is attributed to the ultrafiltration membrane which has a pore diameter smaller than the size of bacteria and parasitic microorganisms, so that the membrane is an effective barrier. However, Table 5 shows the presence of viral indicators. Here, results indicate a great degree of removal (99.8% and 95.3% for somatic coliphages and F-RNA bacteriophages, respectively).

Permeate microbial results proved that MBR systems are able to produce permeate of high microbial quality to be used in several applications such as land irrigation, agricultural activities etc., in accordance with local standards.

5. Conclusions

(>95%) and constant COD removal efficiency (80-98%) was achieved, regardless of the influent fluctuations. Microbial analysis of permeate showed the absence of bacterial indicators of contamination and parasitical microorganisms. At the same time, the membrane presented over 98% efficiency in the elimination of viral indicators.

Particularly interesting is the possibility of operating at maintenance energy level of the biomass, which significantly reduces sludge production. At these maintenance conditions, a minimal value for the carbon substrate utilization rate (0.07-0.1 kg COD kg-1 MLVSS d-1) was found and the system was operated successfully at permeate flux between 30 and 32 l h-1m-2 and low physical cleaning frequency. As a result of carbon substrate limited conditions, EPSs were minimized and higher organisms appeared.

Biomass development at maintenance conditions can be well described by the kinetic model based on Pirt’s equation.

Although there are many practical experiences for MBR design and operation, there are still some aspects that are not completely understood. Without any doubt, the most cited is membrane fouling. The complexity of this phenomenon is linked to the presence of particles and macromolecules with very different sizes and the biological nature of the microbial suspensions which results in a very heterogenic system. Meanwhile, the dynamic behaviour of the filtration process adds a particular complication to fouling mechanisms. Therefore, further investigation is required so as to ascertain which component in the suspension is the primary cause of membrane fouling.

6. Acknowledgements

This work has been funded by the N. R.C. (MEC project CTM2006-12226). The authors also want to express their gratitude to the MEC for a doctoral scholarship, to GE ZENON, to CANARAGUA and to BALTEN for their support and finally to the Water Analysis Laboratory of the ULL Chemical Engineering Department for analytical advice.

7. Nomenclature

CAS Conventional activated sludge process

COD Chemical oxygen demand, mg O2 /l

EPS Extracellular polymeric substance

F/M Feed to microorganisms ratio, kg COD/kg MLSS d

HRT Hydraulic retention time, h

iMBR Immersed membrane bioreactor

J Permeate flux, l/h m2

MLSS Mixed liquor total suspended solids, mg/l

MLVSS Mixed liquor volatile suspended solids, mg/l

NH4-N Ammonium nitrogen concentration, mg/l

NO2-N Nitrite nitrogen concentration, mg/l

NO3-N Nitrate nitrogen concentration, mg/l

SADm Specific membrane aeration demand, Nm3/h m2

SOURe Specific oxygen uptake rate in endogenous conditions, kg O2/kg MLVSS d

SRT Sludge retention time, days

TMP Transmembrane pressure

U Utilisation rate, kg COD/kg MLVSS d

Ergosterol, one of the most important components in fungal membranes, is involved in numerous biological functions, such as membrane fluidity regulation, activity and distribution of integral proteins and control of the cellular cycle. Ergosterol pathway is fungal-specific; plasma membranes of other organisms are composed predominantly of other types of sterol. However, the pathway is not universally present in fungi; for example, Pneumocystis carinii plasma membranes lack ergosterol. In S. cerevisiae, some steps in the pathway are dispensible while others are essential for viability (Tan et al., 2003).

Biosynthesis of ergosterol similarly to carotenoids and other isoprenoid compounds (e. g. ubiquinone), is derived from acetyl-CoA in a three-stage synthehtic process (Metzler, 2003). Stage one is the synthesis of isopenthenyl pyrophosphate (IPP), an activated isoprene unit that is the key building block of ergosterol. This step is identical with mevalonate pathway (Figure 9). Stage two is the condensation of six molecules of IPP to form squalene. In the stage three, squalene cyclizes in an astounding reaction and the tetracyclic product is subsequently converted into ergosterol. In the ergosterol pathway, steps prior to squalene formation are important for pathway regulation and early intermediates are metabolized to produce other essential cellular components (Tan et al, 2003). It should be noted that isoprenoid pathway is of great importance in secondary metabolism. Combination of C5 IPP units to squalene exemplifies a fundamental mechanism for the assembly of carbon skeletons in biomolecules. A remarkable array of compounds is formed from IPP, the basic C5 building block. Several molecules contain isporenoid side chains, for example Coenzyme Q10 has a side chain made ud of 10 isporene units.

Vegetal biomaterial can be viewed as a natural ion-exchange material that primarily contains weak acidic and basic groups on its surface. One of the common procedures to investigate whether ion-exchange is the mechanism responsible for metal sorption is to determine the concentration of alkaline and alkaline-earth metal ions or protons (when the sorbent is pretreated with acid) released from the sorbent to the solution after metal uptake. The determination of the concentration of ions released into the solution (M: Na+, K+, Ca2+, Mg2+, H+) allows the balance of the concentration of the absorbed toxic metal ion (M*), through a charge balance, not explicitly reported in equation (2).

R-M + M* 5 R-M* + M (2)

On the solid material the appearance of the sorbed metals, associated with the disappearance of alkaline and alkaline-earth metal ions, can be followed by Scanning Electron Microscopy (SEM) coupled with energy dispersive X-ray analysis (EDAX). This technique greatly contributes to indicate that ion exchange takes place between alkaline and alkaline-earth metal ions on the sorbent and the toxic metal ions in the solution.

1.2 Spectroscopic analysis

Useful information on the role of functional groups on metal sorption can be reached by non-destructive spectroscopic methods, observing the modifications induced by the metal on the spectra of the pure adsorbent.

Statistical analysis showed a significant influence of examined factors and their interaction on the commercial yield of potato tubers (table 3). The highest yields were obtained from objects fertilized white clover, a mixture of white clover and Italian ryegrass and phacelia both plowed in the autumn, and left till spring in the form of mulch. Only on object fertilized with Italian ryegrass and on control object marketable yield of potato tubers was significantly lower than that recorded in farmyard manure. Straw fertilization also significantly differentiate commercial yield of potato tubers. At the sub-block with straw marketable yield of potato tubers was significantly higher than obtained in the sub-block without straw. An interaction has been noted, which shows that indeed the highest marketable yield was obtained from the object fertilized with a mixture of white clover with Italian ryegrass and white clover with straw, and the smallest from the control object without organic fertilization.

Using a mixture of species in forest management has been common in Europe for the last three centuries. Hegre and Langhammer (1967) and Stewart et al. (2000) have presented overviews of the importance of mixed stands and their management in different countries worldwide.

In Finland and Norway, a forest stand is defined as being mixed if 20 % of its basal area is made up of broadleaved species, with conifers comprising the dominant species (Frivold, 1982). In Sweden, the proportion is 30 % and in Italy 10 % of the basal area. The Swedish definition of a mixed broadleaved and coniferous stand is "a type of stand in which the total percentage of broadleaved species is 30-70 % of the growing stock" (Anon., 2010). In Nordic countries mixed stands are the most frequent type of stand.

Mixed stands mostly establish spontaneously i. e. a planted or naturally regenerated conifer stand is mixed with naturally regenerated broadleaves. Areas of clear felling that are moist are readily colonized by broadleaves, which can establish from seeds, sprouts or suckers. The number of stems can amount to 5000 to 50,000 per hectare. However there is a conflict between broadleaf cover preventing frost damage to young spruce trees and the strong competition between broadleaves and conifer seedlings. In older stands, both species become established, competition is stabilized and the risk of frost damage declines (Johansson, 2003).

Mostly, Nordic forestry is focused on the management of stands for the production of softwood. A large number of young broadleaves are likely to compete with the conifer seedlings in such stands. In the past, the broadleaves were cut or treated with herbicides. Nowadays, with increasing interest in the supply of biomass for bioenergy production, other management systems have been introduced.

When managing mixed forest stands, a stratified mixture of shade-tolerant, late-successional species in the lower stratum and early successional species in the upper stratum is recommended (Assmann, 1970; Kelty, 1992). Mixed stands may contain alder, aspen or birch and Norway spruce (Johansson, 2003), (Fig. 4). The management of mixed stands is often based on stands which have not been cleaned at the correct time. The spontaneous establishment of broadleaved trees takes up to10 years.

Plant biomass is an important factor in the study of functional plant biology and growth analysis, and it is the basis for the calculation of net primary production and growth rate. The conventional means of determining shoot dry weight (SDW) is the measurement of oven-dried samples. In this method, tissue is harvested and dried, and then shoot dry weight is measured at the end of the experiment. For the measurement of biomass of a large number of plants, this method is time consuming and labor intensive. Also, since this method is destructive, it is impossible to take several measurements on the same plant at different time points. With the establishment of advanced technology facilities for high throughput plant phenotyping, the problem of estimating plant biomass of individual plants is becoming increasingly important. There are several technologies that can help to assess the effect of abiotic stresses like drought and soil salinity on plant growth while assisting in predicting crop yield under various environmental conditions.

In coffee conilon seedlings (Coffea canephora) with shading levels of 30%, 50%, 75% and full light, in the region of Alegre-ES/Brazil, it was found that the stem diameter was not influenced by the environment, but the height, the fresh and dry weight, volume and leaf area were greater where shading was 70% (Braun et al., 2007). But in coffee seedlings (Coffea arabica L.), Paiva et al. (2003) reported that of the with shading levels of 30%, 50% and 90%, 50% was most favorable, resulting in greater height, number of leaves and leaf area, consequently, greater vegetative growth.

Mezalira et al. (2009) when evaluating the effect of substrate, harvest period and environment of fig (Ficus carica L.) rooting in plots without cover, plots under low tunnel cover with plastic film (150 p) and plots under a low tunnel with monofilament screen (50% shading) in Dois Vizinhos-PR/Brazil, observed the greatest root production in plots with the use of low tunnel with monofilament screen and the lowest in full sun.

In Alegre-ES/Brazil, studies of germination and seedling production of guava (Psidium guajava L.) in full sun, environments covered with one, two and three screens showed that full sun and one screen promoted higher germination, rate of emergence, number of leafs, plant height and stem diameters, revealing that seedlings tend to develop less with increased levels of shading (Lopes & Freitas, 2009).

Araujo et al. (2006) evaluated the effects of three pots and three environmental conditions (greenhouse tunnel, nursery with a monofilament screen with 50% shading and natural environment) on the development of papaya (Carica papaya L.) cv. Sunrise Solo and concluded that the natural environment was most adequate for development of the seedlings at 45 days after sowing.

Fresh mass of the aerial portion (g)

Greenhouse Monofilament Aluminized coconut palm screen screen

polyethylene bag 5.50 Ac * 7.88 Ab 10.77 Aa 5.63 Ac

polystyrene trays______ 0.39 Ba___________ 0.46__________ Ba 0.48______ Ba 0.65 Ba

Dry mass of the aerial portion (g)

polyethylene bag 0.77 Ac 1.01 Ab 1.23 Aa 0.68 Ac

polystyrene trays______ 0.07 Ba___________ 0.08__________ Ba 0.09______ Ba 0.10 Ba

Fresh mass of the root portion (g)

polyethylene bag 2.67 Ac 3.71 Ab 4.57 Aa 1.57 Ad

polystyrene trays______ 0.55 Ba___________ 0.54__________ Ba 0.53______ Ba 0.43_ Ba

Dry mass of root portion (g)

polyethylene bag 0.25 Ab 0.32 Aa 0.30 Aa 0.12 Ac

polystyrene trays______ 0.05 Ba___________ 0.05__________ Ba 0.05______ Ba 0.04 Ba

* Means followed by same uppercase letters in the columns and same lowercase letters in the rows do not differ by the Tukey test at 5%;

Adapted from Costa et al. (2009a).

Table 6. Review of the analyses of mean aerial fresh mass (AFM), aerial dry mass (ADM), fresh root (RFM) and dry mass of root (RDM) in grams for the container (R) within environments (A); environments (A) inside the container (R) for the yellow passion fruit.

Costa et al. (2009a) when evaluating the production of papaya seedlings (Carica papaya L., cv ‘Sunrise Solo’) in a greenhouse with low density polyethylene film, nursery with black monofilament screen, nursery with aluminized screen and nursery with native coconut palm, using different substrates and containers in Aquidauana-MS/Brazil, observed that the best growth environment was the nursery with aluminized screen for leaf fresh weight, dry weight and fresh weight of the root system (Tables 4 and 5). The same treatments in the same region were applied on the development of passion fruit seedlings (Passiflora edulis Sims. f. flavicarpa Deg.) by Costa et al. (2009b), who found that the black monofilament screen environment provided good conditions for seedlings development. The environment with the aluminized screen also favored seedling growth (Tables 6 and 7).

Initial growth of licuri seedling (Syagrus coronata (Mart.) Becc.), at luminosity levels of 30% (monofilament screen) and 100% (full sun) in the municipality of Feira de Santana — BA/Brazil showed greatest plant growth when subjected to 30% light intensity (Chapman et al., 2006).

Martelleto et al. (2008) studied the effect of the plastic covered greenhouse, shaded greenhouse with an additional monofilament screen (30%, over the plastic), shading with only the monofilament screen (30%) and the natural environment in development of papaya cv. Baixinho de Santa Amalia (‘Solo’), and concluded that growth is favored, both in terms of plant height and trunk diameter, foliage (number of leafs/plant) and leaf area inside the greenhouse without the additional monofilament screen (Tables 8 and 9).

Seedlings of tamarind (Tamarindus indica), in Lavras-MG/Brazil, were more vigorous when cultivated in the natural environment when compared to those produced in the greenhouse and nursery with black monofilament screen providing 50% shading (Mendonga et al.,

2008) .

In Flores da Cunha-RS/Brazil, grape yields (cv. Moscato Giallo), with and without plastic cover over the crop rows, was higher in the covered environment, with greater stability of production, but did not affect the relationship between shell and pulp mass of the berries. The film increased the daily temperature at the plant canopy, not affecting relative humidity, but decreasing the photosynthetic active radiation and wind speed (Chavarria et al., 2009).

Medina et al. (2002) found a better photosynthetic performance of citrus seedlings of the orange ‘Pera’ (Citrus sinensis Osbeck) and Rangpur lime (Citrus limonia Osbeck) in the greenhouse with the use of the termorrefletora screen applying 50% of shading (aluminized screen) below the polyethylene film, in comparison with the greenhouse without the screen. According to these authors, as well as increasing photosynthesis, the screen reduced the photosynthetically active radiation and leaf temperature. These effects were not only beneficial for the maintenance of proper stomatal aperture for gas exchange, but also for better functioning of the photochemical system under adverse conditions.

With the objective of evaluating biomass of passion fruit seedlings in function of environmental conditions and substrates with percentages of organic compound in Aquidauana-MS/Brazil, Sassaqui et al. (2008) conducted an experiment in six environmental conditions: greenhouse with a height of 2.5 m; nursery with black monofilament screen with 50% shading and height of 2.5 m; nursery with aluminized screen with 50% shading and height of 2.5 m; nursery covered with native coconut palm with height of 1.8 m; plastic greenhouse with height of 4.0, zenithal opening and mobile aluminized screen beneath the film at a height of 3.5 m. The authors concluded that the polyethylene film and aluminized screen together promoted better environmental conditions for the accumulation of biomass.

Because of the biological role of the carotenoids as vitamin A precursors in humans and animals and owing to their antioxidant properties and suspected activity in preventing some forms of cancer as well, carotenoid pigments represent a group of most valuable molecules for industrial applications of red yeasts. The pharmaceutical, chemical, feed and food industries have shown increased interest in the use of carotenoids, mainly as provitamin A, but also as natural food and feed colorants. Accordingly, the red yeast P. rhodozyma is currently used for the production of astaxanthin, an important carotenoid pigment that can be exploite in aquaculture to give an appealing pink color to the fresh of farmed salmonid fish, and it also helps to impart a desirable golden color to the egg yolk and fresh of poultry. Salmon farming is an industry that is growing and gradually replacing the world’s wild salmon fisheries. The most expensive ingredient in salmonid feeds is astaxanthin, and though the actual revenues are privately held, it has been estimated that the market for astaxanthin in >US $100 milion per year (Frengova & Beshkova, 2009). Similarly to Xanthophyllomonas, also other red yeast strains could be used for industrial puropses to pruduction of carotenoids — beta-carotene, torulene, lycopene, as well as further lipid metabolites produced in cells. In many works mostly Rhodotorula glutinis sems to be perspective strain. Combined enrichment of Rhodotorula biomass by provitamin A (carotenes) and provitamin D (ergosterol) could be used in food and feed supplements (Marova et al., 2010), aditional enrichment by Coenzyme Q10 is suitable product for cosmetics and could be used also in food and feed (Dimitrova et al., 2010). Formulas based on selenium-enriched red yeast biomass with enhanced carotenoid content could be used as nutrition suplement too (Breierova et al, 2008). There is also posibility to use oleaginous red yeasts to single cell oil production; in this case production of other lipid metabolites could be reduced and the main flow of acetylCoA will be directed to fatty acid and lipid biosynthesis (Dai et al., 2007).

One limitation impacting the industrial utility of P. rhodozyma/ X. dendrourhous or Rhodotorula species has been hindered absorption of carotenoids, due to the yeast’s thick cell wall. Because of presence of other specific biologically active compounds as well as high level of nutritionally sigificant yeast cell components (proteins, unsaturated fat, vitamins…) the best strategy is to disrupt cells and to use the whole biomass without isolation of individual compounds. The biotechnology industry has developed different means of active compounds liberation by the yeast including optimization of drying conditions, mechanical breakage, microwave treatment and enzyme treatment, as described below (Frengova & Beshkova, 2009).

When disrupted cells P. rhodozyma, without cell walls are added to the diets of animals, astaxanthin is readily absorbed from the gut; it effectively colors the fresh of penreared salmonids, and also helps impart a desirable golden color to the egg yolk and fresh of poultry. Astaxanthin in yeast (X. dendrorhous) prepared by spray drying and Xat-roller milling was well absorbed by laying hens and was successfully used as a pigmentation agent in animals (An, 2005). Specifically, when spray-dried and milled yeast was supplied in the feed (40 mg astaxanthin/kg feed), astaxanthin was successfully absorbed (1,500 ng/ ml blood and 1,100 ng/ g skin) by laying hens. Extrusion temperature did not affect utilization of dietary astaxanthin or rainbow trout fresh color significantly, but cell wall disruption of red yeast cells was critical to optimize carotenoid utilization. Increasing the degree of enzymatic cell wall disruption increased fresh astaxanthin concentrations from 2.2 to 6.7 mg/kg, redness values from 5.5 to 10.7, yellowness values from 11.7 to 16.7 and astaxanthin retentions in the muscle from 3.7 to 17.4%. A formulation of P. rhodozyma cells blended with ethoxyquin, lecithin and oil prior to drying also increased astaxanthin deposition in salmonid fish fresh and rainbow trout fresh when supplied in feed as an additive. Absorption and accumulation of biological astaxanthin were higher thah those of chemical astaxanthin, probably because of the high contents of lipids in the yeast (17%). Lipid peroxide formation in skin was significantly decreased by astaxanthin. The peroxide production in chickens fed chemical astaxanthin was markedly lowered compared to biological astaxanthin (Frengova & Beshkova, 2009) .

The levels of serum transaminase activities and of lipid peroxides in fish fed oxidized oil were significantly higher that those of the control fish fed non-oxidized oil. However, the supply of freeze-dried red yeast preparation considerably decreased both enzyme activities and lipid peroxides level. Furthermore, the serum lipid (triglycerides, total cholesterol and phospholipids) concentrations were also significantly decreased. Especially, the serum triglyceride level of fish fed the red yeast was as low as that of the control. Recently was found that Zn2+ ions induced changes in yeasts (R. glutinis and R. rubra) leading to more efficient scavenging and antioxidant capacities compared with Ni2+ ions, and antioxidants (carotenoids) present in yeast’s walls showed higher ability to scavenge free radicals than those from inside the cells (Rapta et al., 2005). Later, the in vivo antioxidant and protective effects of astaxanthin isolated from X. dendrorhous against ethanol-induced gastric mucosal injury were established in animal models, especially rats (Kim et al., 2005). Oral administration of astaxanthin showed significant protection against ethanol-induced gastric lesion and inhibited elevation of the lipid peroxide levels in gastric mucosa. A histologic examination clearly indicated that the acute gastric mucosal lesion induced by ethanol nearly disappeared after pretreatment with astaxanthin (Frengova & Beshkova, 2009).

Chemopreventive and anticarcinogenic effects of carotenoids by Rhodotorula on the development of preneoplastic lesions during N-nitrosodiethylamine (DEN)-induced hepatocarcinogenesis in female Wistar strain rats were also studied (Bhosale et al., 2002). Spray-dried yeast R. glutinis (containing carotenoid pigments torulene, torularhodin and beta-carotene in proportion 58:33:2) showed significant effect on the prevention of liver tumor development. However, R. glutinis effects were relatively more significant in groups where R. glutinis was administered after DEN treatment, suggesting that R. glutinis is quite effective in the prevention of liver tumor development especially when administered after DEN treatment, indicating possible protective effects at the promotional stages.

3. Conclusions

Yeast is, due to its physiological properties, widely used in the food, feed, chemical and pharmaceutical industries for production of various valuable compounds. Red yeast is well known producer of carotenoids which are significant because of their activity as vitamin A precursors, colorants, antioxidants and possible tumor-inhibiting agents. Biological sources of carotenoids receive major focus nowadays because of the stringent rules and regulations applied to chemically synthesized/purified pigments. Compared with the extraction from vegetables, the microbial production of carotenoids is of paramount interest, mainly because of the problems of seasonal and geographic variability in the production and marketing of several of the colorants of plant origin. Moreover, red yeast is a rich source of other specific compounds — ergosterol, Coenzyme Q10, as well as unsaturated fatty acids, fats, proteins and vitamins and can be incorporated in feeds to enhance the nutritional value of yeast biomass. One limitation impacting the industrial utility of carotenogenic yeast has been complicated liberation and bioavailability of carotenoids and other active compounds, due to the yeast’s thick cell wall. The biotechnological industry has developed different means of pigment liberation by the yeast including optimization of drying conditions, mechanical breakage, microwave treatment and enzyme treatment.

The other very important limitation involved in the practical exploitation of yeasts is the high cost of microbial production. The production cost could be reduced by increasing yields of product, as well as using less expensive substrates. There is a need to improve fermentation strategies. Biomass and metabolites production by red yeast is highly variable and can be influenced by cultivation conditions (light, temperature, pH, aeration etc.). Different approaches for improving the production properties of the yeast strains, such as environmental stress, mutagenesis or genetic modification, have been studied and optimized. The other possibility for production cost reduction is using various low-cost materials as carbon or nitrogen source. The potential of several waste materials (whey, potato mass, apple mass and various cereals) as substrates for carotenoid and ergosterol production by some yeast strains belonging to the genus Rhodotorula and Sporobolomyces were succesfully examined. Mild nutrition stress cause by several waste substrates was found to be the suitable induction factor for higher carotenogenesis and ergosterol production in red yeasts.

Environmental stress was reported to induce carotenoid, ergosterol and lipid production as part of red yeast stress response. Under stress cells posses altered phenotype biotechnologically significant and/or undesirable in a dose-dependent manner. Phenotypic profiling of the environmental stress responses demonstrates genetic susceptibility of yeast to environmental stress. Low concentrations of oxidative and osmotic stress, which can under specific conditions induce carotenogenesis, have no significant effect on yeast growth. Red yeast cultivated under osmotic and oxidative stress or on various waste substrates shows no significant differences in cell morphology when compared with yeast cultivated in conventional glucose medium under optimal conditions. Thus, low environmental stress can be used for induction of carotenogenesis and use of non-toxic stress factors (salt, metals) can enable utilization o whole cell biomass to industrial use. Simple and cheap stress factor in relatively low concentration can substantially enhance biotechnologically significant metabolite production.

Growing interest in pigment and other metabolite applications in various fields coupled with their significance in health and dietary requirements has encouraged "hunting" for more suitable sources of these compounds. Due to restrictions, there is no possibility to apply carotenoids prepared by chemical synthesis for food, pharmaceutical and medical purposes. However, the success of microbial pigments, metabolites and single cell oils depends upon their acceptability in the market, regulatory approval, and the size of the capital investment required to bring the product to market. Therefore, the focus of biotechnology on highly valuable yeast biomass requires knowledge how microorganisms control and regulate the biosynthetic machinery in order to obtain metabolites and enriched biomass in high yield and at low price. From this view, attempts have been directed at the development and improvement of biotechnological processes for the utilization of red yeasts on an industrial scale. Current successes using mutation methods and molecular engineering techniques carried out over recent years have not only answered some fundamental questions related to pigment formation but has also enabled the construction of new microbial varieties that can synthesize unusual carotene metabolites. Elucidation of these mechanisms represents a challenging and potentially rewarding subject for the further research and may finally allow us to move from empirical technology to predictable carotenoid and/or isoprenoid metabolite design. Thus, the manipulation and regulation of red yeast metabolism open a large number of possibilities for academic research, demonstrates the enormous potential in its application and creates new economic competitiveness and market of microbial lipid compounds.

Figure 14 shows an example of an X-ray fluorescence spectrum of the immersed carrier. Fe was the main species detected, although silicon (Si), calcium (Ca), aluminum (Al), P, sulfur (SO4), potassium (K), chlorine (Cl) were also present. Heavy metals were not detected on most of the carriers, but traces of Pb and Zn were detected in some samples (Table 2). However, they were well below regulation levels set out in the Fertilizers Regulation Act (Ministry of Agriculture, Forestry and Fisheries, 2007) and the Guidelines against Heavy Metal Accumulation in Arable Soil (Environment Agency, 1984). This was probably because the study site was in a rural area that had not been contaminated by heavy metals and also because the immersion period was too short for these metals to accumulate.

Fig. 14. X-ray fluorescence spectrum of immersed carrier