The most common type of young stands in Nordic countries is mixed birch and Norway spruce, Fig. 5. Many reports describe how to manage birch and Norway spruce. In Finland, Norway and Sweden the management of mixed stands is common (Mielikanen, 1985; Braathe, 1988; Tham, 1988; Mard, 1997; Klang and Eko, 1999). Frivold and Groven (1996) discussed the importance of managing mixed stands for future high timber quality. The competition between the taller birches and Norway spruce may adversely affect spruce growth. Therefore the birches must be carefully managed with respect to both numbers of stems removed and controlling competition. A common recommendation is to leave 500­1000 stems ha-1 when the birches are 10-15 years old. A Finnish study of a mixed stand of birches (downy and silver) and Norway spruce examined the influence of competition (Valkonen and Valsta, 2001). A reduction of 7-15 % by volume production was reduced by 7-15 % in mixed stands with 1000 birches ha-1 compared to pure spruce stands.

Below an experiment in mixed stands of birch and Norway spruce is described (Johansson, 2000b). The experiment was started in 1983 and was based on trials established at eight localities in central and southern Sweden. The experimental stands were 20-30 years old. They were dense, 1520-20,280 stems ha-1, and self regenerated.

The experiment included three thinning regimes:

• Thinning of the birch overstory to create a shelter of 500 stems ha-1.

• Total removal of the birch trees

• Only Norway spruces

At the first cutting, to create the shelter and the pure Norway stands, 1520 to 20,280 birch stems ha-1 with a mean diameter of 5.2 cm were removed. After 5 years, 373 to 507 birch stems ha-1 with a mean diameter of 15.7 cm were recorded.

Data collected five years after the experiment started are presented in Table 2. The competition by the birch shelter did not influence the growth of Norway spruce. As shown in the table, the mean diameter of the Norway spruce trees was almost the same in the shelter as in the pure stands, 7.6 and 7.0 cm respectively.

Norwegian experiment on farmland that involved planted European aspen and Norway spruce. Aspens and Norway spruces were planted each at a density of 2000 stems ha-1. The aspens were thinned 30 years later and 580 stems ha-1 were retained. Recommendations based on the study stated that planting densities of 2000 Norway spruce and 1000 aspen ha-1 would avoid strong competition by the aspens.

5.1.5 Mixed stands of alder and Norway spruce

Naturally established mixed stands of alder are common on wet or moist sites, (Fig. 7). Few studies have examined mixed stands of alder and Norway spruce; those which do exist are based on stands that were not managed correctly during the first ten years after establishment (Lines, 1982; Johansson, 1999d).

These two methods are being used at Julich Plant Phenotyping Centre (Germany) to investigate root/shoot systems growing in sand or soil, with respect to their structures, transport routes and the translocation dynamics of recently fixed photoassimilates labelled with the short lived radioactive carbon isotope 11C. Quantitative MRI and PET data will help not only to study the differences between species, but also in phenotyping of cultivars or plant lines in which growth pattern, water relations or translocation properties are important traits with respect to plant performance (Jahnke et al. 2009). Therefore, MRI-PET combination can provide new insights into structure-function relationships of intact plants. It also allows monitoring of dynamic changes in plant properties, which has not been possible to assess systematically until now to understand plant performance such as resource use efficiency or biomass production.

Jaromlr Lachman1, Milan Kroutil1 and Ladislav Kohout2

1Department of Chemistry, Faculty of Agrobiology, Food and Natural Resources,

University of Life Sciences in Prague, 2Department of Steroid Chemistry, Institute of Organic Chemistry and Biochemistry,

Academy of Sciences of the Czech Republic, Prague

Czech Republic

1. Introduction

Brassinosteroids (BRs) are plant natural polyhydroxysteroids supporting the plant growth; their structure resembles animal steroid hormones (Bajguz, 2010). In plants, steroid hormones serve as endogenous signaling molecules. Brassinosteroids act as positive growth regulators or as compounds responsible for plant stress tolerance. Phytoecdysteroids probably show an antifeedant activity (Kamlar et al., 2010). Brassinosteroids were classified as essential plant hormones nearly thirty years after the discovery of brassinolide (the first brassinosteroid) by Groove et al. (1979) in the rape (Brassica napus L.) pollen. Presence of brassinosteroids was demonstrated in many plant species including higher and lower plants and at the same time they were detected in parts of plants, e. g. pollen, seeds, leaves, stems, roots and flowers (Sakurai et al., 1999). Up to date it was characterized 70 compounds belonging to the class of brassinosteroids, among them 65 in free form and 5 conjugated (Zulo & Adam, 2002, Bajguz & Tretyn, 2003).

Brassinosteroids are phytohormones with pleiotropic effects. They influence growth, seed germination, cell elongation, photomorphogenesis and senescence (Upreti & Murti, 2004). In relation to the growth and growth regulators, the typical effect of brassinosteroids is coincidental elicitation of cell prolongation and division (Worley & Mitchell, 1971). Investigations confirm the ability of brassinosteroids quantitatively affect plant morphogenesis; this leads to the enhancement of number and growth productive lateral shoots and branches and thereby also to the enhancement of number of spikes, pods etc. (Sakurai et al., 1999). Brassinosteroids help to overcome stresses provoked by low and high temperature, drought, salt, infection, pesticides and heavy metals (Takematsu et al., 1986; Cutler, 1991; Kulaeva et al., 1991; Schilling et al., 1991; Hathout, 1996; Bajguz 2000; Anuradha & Rao, 2001; Krishna, 2003; Janeczko et al., 2005; Cao et al., 2005; Sharma & Bhardwaj, 2007 a, b; Kagale et al., 2007; Ali & Abdel-Fattah, 2006, Ali et al., 2007, 2008, Kroutil et al., 2010 a, b). Heavy metals give rise to antioxidant stress and brassinosteroids can

it effectively reduce and induce enhancing of antioxidants under heavy metal stress (Hayat et al., 2007a). In term of the affecting of the uptake of minerals after treatment with brassinosteroids an increase of the content of minerals in aerial plant biomass was demonstrated (Nafie & El-Khallal, 2000) as well as the BRs ability to decrease uptake of heavy metals and accumulation of radioactive elements (Cs, Sr) by plants (Bajguz, 2000; Khripach et al., 1999).

In term of the affecting of the uptake of minerals after treatment with brassinosteroids an increase of the content of minerals in aerial plant biomass was demonstrated (Nafie & El — Khallal, 2000). Brassinosteroids can affect quality of plant products. Treatment with brassinosteroids at anthesis increased the starch content in rice kernels (Fujii & Saka, 2001); at tillering it increased the content of fatty acids in barley ectoplasts and the change of their rate (Khripach et al., 1999).

The aim of this work was to evaluate the ability of brassinosteroids to affect the quality parameters of spring wheat grain: change of the content of minerals in grain and the yield increase of spring wheat cultivated in rational-intensity conditions after brassinosteroids treatment. Another goal of this study was to evaluate the ability of brassinosteroids to lessen the uptake and accumulation of heavy metals (Cd, Pb, Zn, Cu) in spring wheat plants cultivated on contaminated soil of a polluted burdened region in the Czech Republic. Content of heavy metals was investigated in biomass, grains and straw of treated and control plants.

The occurrence of SALLJ in the 2002 was then determined and the pattern found was in agreement with previous studies for other years. Figure 1 shows the percent relative frequencies of SALLJ obtained for each month.

The low level flow was present all through the 2002 year, though presenting variability in its strength, frequency and location mainly related to the different synoptic conditions, and the greater scale climatological scenario. The higher frequencies of occurrence of SALLJ are observed in October and the lower in July. As previously mentioned, the aim of the present study is to relate the low-level jet east of the Andes with the dispersion of biomass burning products in South America. Figure 2 presents the number of fire spots in South America for each month in 2002. The important increase from August to October — namely the biomass burning season — is clearly evident. In consequence, we will restrict the further analysis to the events in those months.

Fig. 2. Number of fires in South America per month during 2002.

The main characteristics of the mean low-level flow are depicted in the composite fields, obtained averaging the days that comprise the SALLJ events for the burning season months (Figure 3). August and October were characterized by a northerly oriented flow, when mainly the northeast of Argentina was under its influence. In September the mean pattern was more north-westerly oriented with an outflow towards the Atlantic Ocean, over passing the southern region of Brazil. August shows the southernmost penetration, greatest horizontal wind speed gradient and vertical wind speed shear. During this month, the events are less frequent but much stronger. In the opposite, in October, there is a higher recurrence of generally weaker events. The mean low level north-westerly flow organizes at about 15° S and extends southward reaching 30-35° S.

The associated circulation patterns in conjunction with the occurrence of biomass burning caused the transport of aerosols and gases towards different regions with diverse impacts. Figure 4 shows the composites of the modelled vertically integrated aerosol optical thickness at 500 nm (AOT500) and the flow pattern for the SALLJ events. The mean plume

and flow are very well reproduced and the higher aerosol concentrations are directly related to the greater emissions during September and October.

The temporal behaviour of the AOT at the AERONET site in Buenos Aires is depicted in Figure 5 for the sub-samples SALLJ and NO-SALLJ along with the comparison with the CATT-BRAMS predicted values. The model is able to capture the evolution of the aerosol concentration. The underestimation of the values is linked to the comparison of point measurements and the model results resolution.

The relationship between the Angstrom coefficient and the AOT is frequently used to get more information about the aerosol characteristics. The greater aerosol load observed during the SALLJ events is clearly associated to higher Angstrom coefficients in agreement with the literature (Figure 6).

Fig. 6. Variation of the Angstrom coefficient (440-870nm) with the AOT 500nm obtained from the measurements at the Buenos Aires AERONET site (dots, diamond: SALLJ, square: NO-SALLJ) from August to October 2002.

About 7% of the world’s total land area is affected by salt, as is a similar percentage of its arable land (Ghassemi et al., 1995). Salinity is often accompanied by other soil properties, such as sodicity and alkalinity, which exert their own specific effects on plant growth. There are three ways in which salinity stress of crops could be reduced; 1- Farm management practices; 2- Screening; 3- Breeding which will be discussed in the followings:

1.1 Farm management practices

All irrigation waters contain some dissolved salts. Thus, soil salinization may be expected by crop irrigation. Removal of salts from the root zone may be the most effective way to eliminate the effects of salinity. However, it is expensive and requires good drainage system. It is not always possible to carry out this operation; thereby a number of other different ways could be considered such as:

a. Soil Reclamation; in a case Na ions are the major cause of soil salinity, it may be replaced with Ca ions by adding of gypsum (calcium sulfate) to the soil.

b. Reduction of the salt from seed germination zone; Seed germination and seedling establishment are the most sensitive stages to salinity. A number of approaches have been used. 1) Removal of surface soil (Qureshi et al., 2003). 2) Pre-sowing irrigation with good quality water (Goyal et al., 1999). 3) Planting seed on the ridge shoulders rather than on the ridge top of the furrow. 4) Planting in a pre-flooded field with good quality water (Goyal et al., 1999).

c. Reducing soil salinity by adding mulch, organic matter or deep tillage to the soil.

Rocio Gomez-Pastor12, Roberto Perez-Torrado2, Elena Garre1 and Emilia Matallana12

1Departamento de Bioquimica y Biologia Molecular, Universitat de Valencia. 2Departamento de Biotecnologia, Instituto de Agroqumica y Tecnologia de Alimentos,

Spain

1. Introduction

Yeasts have been used by humans to produce foods for thousands of years. Bread, wine, sake and beer are made with the essential contribution of yeasts, especially from the species Saccharomyces cerevisiae. The first references to humans using yeasts were found in Caucasian and Mesopotamian regions and date back to approximately 7000 BC. However, it was not until 1845 when Louis Pasteur discovered that yeasts were microorganisms capable of fermenting sugar to produce CO2 and ethanol. Ancient practices were based on the natural presence of this unicellular eukaryote, which spontaneously starts the fermentation of sugars. As industrialisation increased the manufacture of fermented products, the demand of yeast grew exponentially. At the end of the 19th century, addition of exogenous yeast biomass to produce bread and beer started to become a common practice. Wineries were more reluctant to alter traditional practices, and started using exogenous yeast inocula in the 1950’s, especially in countries with less wine tradition (USA, South Africa, Australia and New Zealand). In the 1960’s, yeast biomass-producing plants contributed to the technology of producing large amounts of active dry yeast (ADY), and its use rapidly spread to European countries (Reed and Nagodawithana, 1988).

Nowadays, modern industries require very large amounts of selected yeasts to obtain high quality reproducible products and to ensure fast, complete fermentations. Around 0.4 million metric tonnes of yeast biomass, including 0.2 million tonnes baker’s yeast alone, are produced each year worldwide. Efficient and profitable factory-scale processes have been developed to produce yeast biomass. The standard process was empirically optimised to obtain the highest yield by increasing biomass production and decreasing costs. However in recent years, several molecular and physiological studies have revealed that yeast undergoes diverse stressful situations along the biomass production process which can seriously affect its fermentative capacity and technological performance.

In this chapter, we review the yeast biomass production process, including substrates, growth configuration, yield optimisation and the particularities of brewing, baker — or wine — yeasts production. We summarise the new studies that describe the process from a molecular viewpoint to reveal yeast responses to different stressful situations. Finally, we

highlight the key points to be optimised in order to obtain not only high yields, but also the best biomass fermentative efficiency, and we provide future directions in the field.

As a consequence of being a relatively new technology, limited information on the life of membranes is available. However, analysis of the oldest plants evidence that membrane life can reach, or even exceed, 10 years (Verrech et al., 2010).

Recently, Ayala et al. (2011) has reported the effect of operating parameters on the permeability and integrity of cartridges taken from full-scale MBRs. Regarding permeability, a correlation of permeability loss and operation time was found, indicating that the membrane permeability reaches non-operative value after seven years of operation. The authors also suggested a significant effect of inorganic scaling on permeability loss. The correct functioning during membrane cartridge life, determined by the strength of the welding at its perimeter, appears to be related to the total volume of water permeated and the total mass of oxidant (NaOCl) used during chemical cleanings.

Changes of metal composition of plants treated with brassinosteroids were reported in quite a few experiments. Most of these experiments are related to the ability of brassinosteroids to decrease the intake of heavy metals with plants. 24-epiBL at the concentration of 10-8 mol L-1 in combination with heavy metals blocked metal accumulation in algal cells (Bajguz, 2000) and treatment of Brassica juncea plants with 24- epiBL detoxified the stress generated by NaCl and/or NiCl2 and significantly improved growth, the level of pigments and photosynthetic parameters (Ali et al., 2008b). After foliar application of brassinolide on tomato plants an increase in metals (P, K, Ca and Mg) in aerial parts of plants has been recorded (Nafie & El-Khallal, 2000). Our three-year results showed that after the brassinosteroids treatment of spring wheat some changes of the metals content were determined. However, these changes differed among the experimental years. Brassinosteroids application primarily affected content of K, Mg, Zn and Fe in grain. However, it did not affect Cu content. Brassinosteroids stimulate morphogenesis of plants which causes an increase in leaf area, number of leaves, dry and fresh mass of stems and roots and number of tillers and productive branches. Due to these effects on physiological processes in plants, an increase in the yield and quality of crops production has been observed (Sakurai et al., 1999). Yield increase depends on variety, climatic conditions, soil, application of fertilizers and also on frequency and dates of brassinosteroids application (Khripach et al., 2000, 2003; Janeczko et al., 2010). Different preparations (mixtures of natural 24-epibrassinolide and its synthetic isomers) especially used under unfavourable cultivation conditions cause an increase in yield of crops such as rice, maize, wheat, cotton, tobacco, vegetables and fruit. Exogenous brassinosteroids such as 24-epibrassinolide influences brassinosteroid balance in seedlings of wheat after soaking seeds, drenching or spraying plants and content of endogenous brassinosteroids brassinolide and castasterone varies with leaf insertion and plant age (Janeczko & Swaczynowa, 2010). The relative effects of brassinosteroids may be low, when the conditions under which plants are growing are generally favourable (Khripach et al., 2000). Treatment of barley cultivated in light-textured clay podzolic soil with brassinosteroids in a combination with nitrogen-phosphorus-potassium fertilizer (dose 60 kg N ha-1) increased grain yield by 360 kg ha-1; content of total protein in grain was not affected. However, in our experiments, where NPK fertilization at a dose of 60 kg N ha-1 was applied, no significant increase of grain yield per hectare has been proved. However, the application of brassinosteroids could reduce the negative effect of the stress factors on the yield and dry matter in wheat (Hnilicka et al., 2007, Bajguz, 2009). In a greenhouse experiment with exogenously applied 24-epibrassinolide on two hexaploid wheat (Triticum aestivum L.) cultivars, S-24 (salt tolerant) and MH-97 (moderately salt sensitive), the application of 24-epibrassinolide increased plant biomass and leaf areal per plant of both cultivars under non-saline conditions. However, under saline conditions improvement in growth due to foliar application of 24-epibrassinolide was observed only in salt tolerant cultivar (Shahbaz et al., 2008). Drought stress and high temperature were found to have a negative effect on the amount of dry matter in the above-ground wheat biomass and the yield of grain and straw. Our results regarding the total protein content are in agreement with the results of experiments with wheat after exogenous plant treatment with 24-epibrassinolide, where as well no difference in soluble protein content between control and treated plants after brassinosteroid treatment was determined (Janeczko et al., 2010). In our experiments, no difference was recorded between treated plants and control plants in other qualitative parameters such as gluten content, sedimentation index and bulk density, which are affected more likely by varietals properties, or in Falling number, which is dependent on the harvest date and weather course during the harvest period.

Enhanced resistance of brassinosteroid-treated plants to extreme temperature, salt, pathogens and environmental stresses (heavy metals) was reported by Krishna (2003). The present study revealed the effect of brassinossteroids treatment on the accumulation of Cd, Cu, Pb and Zn contents in aerial wheat biomass or grains. The obtained results are in agreement with the results of Bajguz (2000), who observed that 24-epiBL at the concentration of 10-8 mol L-1 in combination with heavy metals blocked metal accumulation in algal cells. At metal concentrations of 10-6 — 10-4 mol L-1, a combination with 24-epiBL appeared to have a stronger stimulatory effect on a number of cells than a single metal (a stronger inhibitory effect). The inhibitory effect on metal accumulation of 24-epiBL mixed with different heavy metals was arranged in the following order: zinc > cadmium > lead > copper. Our results obtained for spring wheat as an important crop confirm and are complementary to the results of Sharma & Bhardwaj (2007a, b), which describe the effects of 24-epiBL on plant growth, heavy metals uptake in the plants of Brassica juncea L. under heavy metal (Zn, Cu, Mn, Co and Ni) stress. 24-epiBL after the pre-germination treatment blocked copper metal uptake and accumulation in the plants. Likewise results of Anuradha & Rao (2007), obtained in a study on radish (Raphanus sativus L.) after the treatment with 24-epiBL and 28-homobrassinolide clearly indicated the inhibitory influence of brassinosteroids on the cadmium toxicity. Brassinosteroids supplementation alleviated the toxic effect of cadmium and increased the percentage of seed germination and seedling growth. Treatment with brassinosteroids regulates and enhances the activities of antioxidant enzymes ascorbate peroxidase, glutathione reductase, catalase, peroxidase and superoxide dismutase (Sharma, I. et al., 2010) and in drought stressed plants proline and protein content (Behnamnia et al., 2009). The application of brassinosteroids at low concentrations at a certain stage of development reduced significantly the metal absorption in barley, tomatoes and sugar beet. Our results indicate that for the decrease of heavy metals content in plants after the brassinosteroids application the growth stage of spring wheat is very important (Figs. 7 and 8).

The present study shows that the content of heavy metals in wheat plants is reduced variously in different growth stages. The plants of the second group and the third group contained in biomass at the growth stage 73-75 DC lower Pb content as compared to control

plants and the plants of the first group, which was treated with brassinosteroids last at the growth stage 29 — 31 DC. Also in the plants of the second group and the third group at the growth stage 73 — 75 DC lower Cd and Zn contents were determined (with the exception of brassinosteroid 4154 in the third group). The treatment of wheat plants with brassinosteroids 24-epiBL, 24-epiCS and 4154 at the plant growth stage 29-31 DC did not significantly influence content of the heavy metals in aerial plant biomass at the growth stage 47 — 49 DC. In the straw at the growth stage 90-92 DC, lower Pb and Zn contents were subsequently determined only in the plants treated with 24-epiBL and 24-epiCS (Zn also with the application of 4154 in the second group). Lower Cd content was determined only in the variant treated two times with 24-epiBL, which was considered as a highly active brassinosteroid. Lower Pb content was found in the grains of plants of the second group (treated two times in the stages 29-31 DC and 59-60 DC) and the third group (treated once in the stages 59-60 DC).

In terms of the content of heavy metals related to the number and growth stage of brassinosteroids applications, the most effective variants of treatment leading to decrease of

metal content proved either double treatments in the growth stages 29 — 31 DC and 59 — 60 DC (plants of the second group) or one treatment only in the stage 59 — 60 DC (plants of the third group).

Brassinosteroids are able to manage plant water economy during a drought period by decreasing plant activity with a simultaneous conservation of the whole plant for more favourable conditions. Brassinosteroid-treated plants are then able to overcome the drought period in a much better condition than non-treated plants (Sasse, 1999). Their increase in net photosynthetic rate due to brassinosteroids application has already been observed in wheat, tomato and cucumber under normal condition and environment stresses (Ogweno et al., 2008; Shabaz et al., 2008; Xia et al., 2009; Yuan et al., 2010, Hola, 2010). Nowadays biological effects not only naturally occurring brassinosteroids, but also their androstane and pregnane analogues are widely synthesised and their biological effects studied (Hnilickova et al., 2010) as well as their miscellaneous metabolic pathways in plants involving dehydrogenation, demethylation, epimerization, esterification, glycosylation, hydroxylation, side-chain cleavage and sulfonation (Bajguz, 2007). Because brassinosteroids control several
important agronomic traits (Kang & Guo, 2010) such as flowering time, plant architecture, seed yield and stress tolerance, the genetic manipulation of brassinosteroids biosynthesis, conversion or perception offers a unique possibility of both changing plant metabolism and protecting plants from environmental stresses confirming the value of further research on brassinosteroids to improve productivity and quality of agricultural crops (Divi & Krishna,

2009) or their possible use for phytoremediation application (Barbafieri & Tassi, 2010).

2. Conclusion

From the perspective of minimal heavy metals content in biomass and grains related to the number of treatments and growth stage the most effective options of application of brassinolide treatment are those, which lead to a reduction in heavy metals in biomass: either dual treatment in growth stages DC 29-31 and DC 59-60 or single treatment only in the DC 59-60. Favourable is effective reduction of the content of heavy metals in the biomass of plants in grain milk stage (DC 73-75). After treatment of plants with brassinosteroids, when the plants are harvested for ensilage, the content of toxic metals was effectively reduced. Thus, treatment of plants with brassinosteroids can effectively reduce the content of heavy metals in plants (Cd and Pb) or harvested grain (Pb) of wheat and reduce the input of these contaminants into the food chain either cereal or meat products from the food industry. From the point of view of final effect on the content of the heavy metals in plant biomass and grains, the most suitable variant appears to be the single treatment in the growth stage 59-60 DC, which is economically preferable and its final effect does not differ remarkably from double treatments. Likewise lead content in grains decreased in the plants of the second group by 70-74% and of the third group by 48-70%. Thus, treatment of plants with brassinosteroids effectively decreased content of cadmium and lead in wheat plants (biomass) and content of lead in harvested grain and diminished in such way the input of these contaminants into the food chain.

Changes in the minerals content differed according to used brassinosteroid (variant) and investigated year; however unambiguous tendencies of changes or effects were not recorded. In comparison with control plants in the year 2005 the content of minerals in grain of treated plants did not differed significantly. In the year 2006 an increase of K after treatment with 24-epiBL, 4154 and KR1 compounds and a decrease of Zn content after treatment with 24-epiCS and KR1 compounds were recorded. In the year 2007 a decrease of Mg, Mn and Fe content was determined.

Similarly grain quality was not affected by the treatment with brassinosteroids in the investigated years. Content of proteins and gluten in the grains of treated and untreated plants was not significantly different. Similar results were obtained in the sedimentation index and bulk density. Falling number values differed depending on the date of harvest and year of cultivation; in comparison with control plants no difference was recorded. The hypothesis presented is that utilisation of brassinosteroids for plant treatment in the methods of agricultural management with a normal (rational) level of agricultural engineering is not effective. However, by contrast, their application could represent a high economic gain in all cases where the conditions for the cultivation of cereals are not quite ideal, e. g. under conditions of action of different environmental plant stressors, especially with cultivation on soils contaminated with heavy toxic metals or in different arrangements of agricultural engineering. The brassinosteroids-induced enhancement of photosynthetic capacity and regulation of antioxidant enzymes or growth could be under stress factors such saline conditions cultivar specific.

Copper (atomic weight 63.54) is ductile, lustrous, reddish metal (density 8.92 g/ mL). The main application of copper is in electrical industry (transformers, generators, and transmission of electricity). Pollution derives from copper mining, brass manufacture, electroplating industries and from the use of its compounds in agriculture. Copper is known as one of the highest mammalian toxic compounds; inhalation of copper containing sprays is linked with an increase in lung cancer among exposed workers. Copper sulphate is widely used as an algaecide in water supply reservoirs affected by blooms of blue-green algae.

The maximum acceptable copper in drinking water is 2 mg/L (WHO, 2008).

1.1.2 Lead

Lead (atomic weight 207.19) is a bluish-grey, soft, dense metal (density 11.34 g/ mL). The oxidation states are 0, +2, +4. Lead is extremely resistant to corrosion and is a poor conductor of electricity. Large quantities of lead, both as the metal and as the dioxide, are used in storage batteries. Lead is also used in cable covering, as ammunition, as electrodes, in solder and as roofing material. The metal is used as shielding from radiation, e. g. in x-ray rooms and nuclear reactors. Lead oxide is also used in the manufacture of fine crystal glass. Historically, lead was used in plumbing. Tetraethyl lead was used as an anti-knock agent in petrol, and as an additive in paints. These uses have been reduced recently because of environmental concerns about cumulative lead poisoning. Although lead is one of the most useful of all the metals, used since antiquity because of its wide distribution and its easiness to be extracted and to work with, it is also the metal that has the most damaging effects on human health. Environmental contamination by lead probably dates back to Bronze Age. It can enter the human body through the uptake of food (65%), water (20%) and air (15%). Human activities, such as fuel combustion, industrial processes and solid waste combustion contribute to the rise of lead concentrations in the environment. Lead interferes with a variety of body processes and is toxic to many organs and tissues including heart, bones, intestines, kidneys, and reproductive and nervous systems. It interferes with the development of the nervous system and is therefore particularly toxic to children, causing potentially permanent learning and behavior disorders. Occupational exposure is a common cause of lead poisoning in adults. Lead can reach water through the corrosion of pipelines in water transportation systems. WHO Guidelines limit for lead in drinking water is 0.01 mg/L (WHO, 2008).

1.1 Wastewater characterization and treatment

Tilapia fish processing wastewater used in the experiment was donated by Tilapia do Brasil Inc. (Buritama City, SP, Brazil) and was made up of effluents from killing, scaling, gutting, cleaning, skinning, filleting and freezing operations, and also from cleaning operations, which were gathered and roughly filtered (grating), averaging 10,000 L h-1.

Crude wastewater was analyzed for turbidity, total solids (TS), pH, total nitrogen (TN) and oils and greases (OG), according to standard methods (American Public Health Association, American Water Works Association, Water Pollution Control Federation [APHA, AWWA and WPCF], 2005). Chemical Oxygen Demand (COD) was determined by chemical digestion (HR digestion solution for COD 0-1500 ppm; DRB200; DR2800; Hach), based on the protocol developed by Jirka & Carter (1975).

Before being used as a substrate for the bacterial growth, the wastewater was filtered in a 50 pm mesh fast filter (Gardena 1731; 3,000 L h-1) for the withdrawal of gross particles and heat treated (Incomar LTLT tank) at 65oC/30 min to eliminate pathogenic agents and repress the level of competing microorganisms. After that, wastewater was cooled to room temperature and so it was ready to receive the bacterial inoculum.

Microbiological analyses of crude and heat treated wastewater comprised mesophilic aerobic bacteria, total and fecal coliforms, molds and yeasts, Aeromonas spp and Salmonella spp, and were performed according to standard methodology (APHA, AWWA and WPCF 2005).