Biomass Alteration of Earthworm in the Organic Waste-Contaminated Soil

Young-Eun Na1, Hea-Son Bang1, Soon-Il Kim2 and Young-Joon Ahn2 1National Academy of Agricultural Science and Technology, Rural Development Administration, Suwon 441-707, 2WCU Biomodulation Major, Department of Agricultural Biotechnology,

Seoul National University, Seoul 151-921, Republic of Korea

1. Introduction

Earthworm populations show a considerable amount of variability in time and space, with mean densities and biomass ranging from less than 10 individuals and 1 g m-2 to more than 1,000 individuals and 200 g m-2 under favourable conditions. Earthworms have been considered to play a great role in soil-formation processes and in monitoring soil structure and fertility (Lavelle & Spain, 2001) because they may increase the mineralisation and humification of organic matter by food consumption, respiration and gut passage (Edwards & Fletcher, 1988; Lavelle & Spain, 2001) and may indirectly stimulate microbial mass and activity as well as the mobilisation of nutrients by increasing the surface area of organic compounds and by their casting activity (Emmerling & Paulsch, 2001). However, within particular climatic zones, earthworm assemblages, with fairly characteristic species richness, composition, abundance and biomass, can often be recognised in broadly different habitat types, such as coniferous forest, deciduous woodland, grassland and arable land (Curry, 1998).

Agriculture is facing a challenge to develop strategies for sustainability that can conserve non­renewable natural resources, such as soil, and enhance the use of renewable resources, such as organic wastes. It has been estimated that 357,861 tons of organic sludge daily were produced in South Korea in 2009 (Anon., 2009). The production and use of organic compounds have also risen rapidly over the last four decades. Organic compounds which are released either through direct discharge into the sewer system, or indirectly through run-off from roads and other surfaces are found in sewage sludge (Halsall et al., 1993). As a suitable bioindicator of chemical contamination in soil, earthworms are easy, fast and economical merits to handle. Especially, analysis of their tissues may also provide an excellent index of bioavailability of heavey metals in soils (Helmke et al., 1979; Pearson et al., 2000).

Although the acute earthworm toxicity test developed by Edwards (1984) has been widely used and an internationally accepted protocol was also used for assaying the chemical toxicity of contaminants in soils (Organisation for Economic Cooperation and Development [OECD], 1984), the chronic toxicity test to detect subtle effects of contaminants on them by long-term exposure has not been fully achieved (Venables et al., 1992). Based upon these tests, lots of information on heavy metal uptake, toxicity and accumulation by various

earthworm species have been produced. Therefore, earthworms could fill the gap by being used as potential biomarkers of ecotoxicity to various chemicals, including organic contaminants.

This chapter is particularly focused on the hazardous effects on composition, numbers and biomass of Megascolecid and Moniligastrid earthworms, which are dominant groups in South Korea, of 8 consecutive yearly applications of three levels of four different organic sludges and pig manure compost as a positive reference using field lysimeters and microcosms.

Analysis of the liquid phase. Extracellular polymeric substances

Extracellular polymeric substances (EPS) can be differentiated into two main types: bound EPS, which form the structure of the floc, and soluble EPS (often named soluble microbial products), which are soluble or colloidal form in the liquid medium. Recent studies have shown that the soluble and colloidal fraction plays an important role in membrane fouling (Drews, 2010). Their principle components are also generally recognised as proteins and polysaccharides (Sponza, 2002).


Fig. 10. Average soluble EPS concentration of feedwater, liquid-phase and permeate.

Figure 10 compares the average concentrations of proteins and polysaccharides in the feed wastewater, in the liquid-phase and in the permeate. A significant reduction in EPS can be observed in the liquid-phase in relation to feed (82% for proteins and 51% for polysaccharides), as a result of biological metabolism. On the other hand, the separation through the membrane of the polysaccharides is 31% and for the protein it is 28%, both remaining constant throughout the experimental test. These membrane retention values are similar to those found in the literature (Rosenberger et al., 2006).

A low concentration was unexpected in the liquid-phase, as the common trend is to suppose EPS accumulation resulting from polymer retention by the membrane (Masse et al., 2006). As a consequence specific microorganisms may be assumed to develop, which can degrade polysaccharides and proteins with a slow degradation rate.

4.2 Membrane performance

The isoprenoid pathway

Isoprenoids occur in all eukaryotes. Despite the astonishing diversity of isprenoid molecules that are produced, there is a great deal of similarity in the mechanisms by which different species synthesize them. In fact, the initial phase of isoprenoid synthesis (the synthesis of isopentenyl pyrophosphate) appears to be identical in all of the species in which this process has been investigated. Thus, some early steps of isporenoid pathway could be used for genetic modification.

Starting with the simple compounds acetyl-CoA, glyceraldehyde-3-phopsphate, and pyruvate, which arise via the central pathawys of metabolism, the key intermediate isopentenyl diphosphate is formed by two independent routes. It is then converted by bacteria, fungi, plants and animals into thousands of different naturally occuring products. In fungi, carotenoids are derived by sequnce reactions via the mevalonate biosynthetic pathway. The main product 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) is finaly reduced to the mevalonic acid. This two-step reduction of HMG-CoA to mevalonate is highly controlled and is also a major control factor of sterol synthesis (Metzler, 2003). From prenyl diphosphates of different chain lengths, specific routes branch off into various terpenoid end products (Figure 9).

Potentiometric titrations

Potentiometric titrations measure the acid-base properties of the sorbent and the ionic exchange properties with regard to H+ and OH — ions. The presence of acid and basic sites determines the sorbent amphoteric properties and, depending on the pH, the functional groups can be either protonated or deprotonated. Active site concentrations are generally determined by acid-base potentiometric titration of the adsorbent and related modeling. Acidity constants found in the literature can be considered as mean values, which are representative of the class of the functional groups. Potentiometric titrations can also be used to determine the pH at the point zero charge (pHpzc) of biomass. pHpzc is the pH at which the sorbent surface charge takes a zero value as the charge of the positive surface sites is equal to that of the negative ones.

The knowledge of pHpzc allows one to hypothesize on the ionization of functional groups and their interaction with metal species in solution; at solution pH’s higher than pHpzc the sorbent surface is negatively charged and could interact with metal positive species while at pHs lower than pHpzc the solid surface is positively charged and could interact with negative species. Carboxylic groups were found to be the most involved, in the majority of cases, where potentiometric titration was used to elucidate the functional groups on biomass responsible for metal ions sorption. This fact is in part expected on the basis of their easiest deprotonation in the 2 — 6 pH range which is the most suitable for metal sorption.

The Influence of Intercrops Biomass and Barley Straw on Yield and Quality of Edible Potato Tubers

Anna Plaza, Feliks Ceglarek, Danuta Buraczynska and Milena Anna Krolikowska

University of Natural Sciences and Humanities in Siedlce


1. Introduction

Potatoes destined for direct consumption should be distinguished by a high trade yield with the best qualities. (Leszynski, 2002; Boliglowa and Glen, 2003; Plaza and Ceglarek, 2009). In most European countries schemes for the verifiability of the potato product are introduced. The aim is to obtain good quality of potatoes, ensuring the reduction of harmful substances to human health and the natural environment (Spiertz et al., 1996). The beneficial effects of organic fertilization is noted here (Leszczynski, 2002; Boliglowa and Glen, 2003; Makaraviciute, 2003; Plaza et. al., 2009).

Farmyard manure is a basic manure applied in potato cultivation (Batalin et. al., 1968; Kalembasa and Symanowicz, 1985; Rozrtopowicz, 1989). For many years its amount covered the demand, but now the situation has negatively affected due to the decline in livestock, especially cattle. Decreasing amount of farmyard manure, low profitability and the rationale for a system of integrated agriculture, tend to seek alternative, energy-efficient sources of biomass. As a result, a significant role is being attributed to green manures (Grzeskiewicz i Trawczynski, 1997; Zaj^c, 1997; Ceglarek et. al., 1998; Karlsson-Strese et. al., 1998; Plaza i in.,

2009) .

Green fertilizers were mentioned many times in literature. Batalin et. al. (1968), Roztropowicz (1989), Gruczek (1994), Dzienia and Szarek (2000) emphasize that the advantage of using this type of fertilization is high labor and energy saving in relation to its amount spent on works related to the application of farmyard manure. Estler (1991), Stopes et. al. (1995), Spiertz et. al. (1996), Karlsson-Strese et. al. (1998) and Songin (1998) show that the intercrops introduction into the cultivation is not only the production of biomass. They are also a kind of absorbent material to prevent leaching of nutrients into the deeper layers of soil and groundwater, which is important in protecting the agricultural environment. From manuscripts connected with green fertilizers it is clear that among catch crops, undersown crops seem to be the cheapest source of organic matter because it does not require any additional costs associated with the cultivation and preparation of the soil before sowing, which is particularly troublesome in the cultivation of stubble crops (Ceglarek et. al., 1998). Seed cost is also low. As undersown the legumes are recommended to cultivate. The Renaissance intercrops from legumes is linked to the multilateral noticing

them, valuable, but not fully used advantages of agronomic and biological properties. The rediscovery of these plants is associated with current global trends in agricultural techniques, aiming towards the promotion of proecological and ecological agriculture (Stopes et. al., 1995; Spiertz et. al., 1996; Karlsson-Strese et. al., 1998; Duer, 1999). White clover is distinguished by a high capacity of fixing atmospheric nitrogen, and a wide range of crops to allow its existence in a very different soil conditions have long been interested for researchers across Europe (Frye et. al., 1988). In Poland, there is little experimental data determining the suitability of this species to cultivation as undersown, designed for plowing, as a green manure in integrated potato cultivation. Researches of many authors (Batalin et. al., 1968; Gromadzinski and Sypniewski, 1971; Zaj^c, 1997; Ceglarek et. al., 1998) show that undersown legumes are quite unreliable in yielding. More similar are legume mixtures with grasses (Gromadzinski and Sypniewski, 1971; Bowley et. al., 1984; Ceglarek et al,. 1998; Witkowicz, 1998; Plaza et. al., 2009). Reliable in yielding also are grasses grown in pure sowing. As a fast-growing plants and easily shading the soil interact with the position by weed reduction (Szymona et. al., 1983/1984; Sadowski, 1992; Karlsson-Strese et. al., 1998; Majda and Pawlowski, 1998; Kuraszkiewicz and Palys, 2002).

An alternative source of biomass can also be stubble crops, which were mentioned in literature many times (Sadowski, 1992; Roztropowicz, 1989; Boliglowa and Dzienia, 1996; Grzeskiewicz and Trawczynski, 1997; Dzienia and Szarek, 2000). Recently, there has been an interest of the possibility of entering non-legume plants with a short growing season. It is recommended to sow fast-growing species, with good ability of shading, and not able to produce too large, aboveground woody mass. The most common are: white mustard, oil radish and phacelia (Allson and Amstrong, 1991; Boliglowa and Dzienia, 1997; Grzeskiewicz and Trawczynski, 1997; Gutmanski et. al., 1998). Among non-legume plants cultivated in stubble crop phacelia is distinguished by rapid growth, it produces a soft aboveground mass, easily frozen in winter. Is a phytosanitary plant. In Poland, previously carried out researches on fertilizing position of phacelia only in sugar beet cultivation (Nowakowski et. al., 1997; Gutmanski et. al., 1999), still there is no experimental data evaluating its usefulness in the fertilization of potatoes.

Intercrops can be plowed down in autumn or left till spring in the form of mulch. The beneficial effects of intercrops plants left till spring in the form of mulch is to: protect the soil against wind and water erosion, gathering water from rainfall, slowing the process of mineralization of organic matter and prevent from nutrients leaching into the soil, reducing the cost of cultivation by eliminating plowing (Hoyt et. al., 1986; Gutmanski et. al., 1999). It should be noted that the green fertilizers left till spring in the form of mulch causes a slight decrease in yield, but the improvement of the quality characteristics of the fertilized plants compared to fertilization applied in the traditional form.

Another substitute source of biomass can also be the straw left on the field after harvest of cereals (Szymankiewicz, 1993; Gruczek, 1994; Snieg and Piramowicz, 1995; Dzienia and Szarek, 2000), especially used in combination with green fertilizers. Its addition to the legume biomass, not only does not reduce nitrogen losses, but also extends the period of green fertilizers acting (Nowak, 1982). In the case of non-legume plants effect of combined application of these forms of fertilization is not always positive (Dzienia, 1989; Sadowski,

1992) . In Poland, there is little on this experimental data. Thus emerges the need for research aimed at comparing the impact of intercrops biomass, stubble crops both plowed down in autumn and left till spring in the form of mulch in combinations with straw or without

straw, farmyard manure fertilization on yielding and chemical composition of edible potato tubers.

Legumes. Chickpea

stress was not allowed to occur with an optimum irrigation regime the shoot biomass productivity was near 5 t ha-1 with a seed yield of 2t ha-1. However, under the normal receding soil moisture condition, the shoot biomass productivity ranged across years from 1.8 to 3.8 and the seed yield from 0.7 to 1.6 t ha-1 (Krishnamurthy et al. 2010).

Chickpea breeding program at ICRISAT has placed high emphasis on development of early and extra early maturing varieties so that these can escape terminal drought. The early maturing crop, however, cannot accumulate enough total plant biomass due to reduced total photosynthetic period compared to the relatively longer duration varieties.

Terminal drought reduces both shoot biomass and yield in chickpea. For example the average shoot biomass reduction of 40 cultivated chickpea genotypes due to terminal drought was 44 to 61 % across two years whereas the grain yield reductions were 35 to 66% (Krishnamurthy et al. 1999). Similarly the average shoot biomass reduction of 216 (mini core) chickpea germplasm accessions due to terminal drought was 31 to 63 % across 3 years whereas the grain yield reductions were only 26 to 61% (Krishnamurthy et al. 2010). The relatively less reduction in grain yield under drought was due to an increased partitioning under the progressively built terminal drought stress.


Groundnut (Arachis hypogaea L.) is an important rainy-season crop in most of the production systems in the semi-arid tropical regions of south Asia and sub-Saharan Africa, where it is grown under varying agroecologies, either as a sole crop or intercropped with sorghum and pigeonpea. Groundnut yields are generally low and unstable under rain-fed conditions, due to unreliable rainfall patterns. Severity of drought stress depends on the stages of crop development and the duration of stress period (Wright and Nageswara Rao, 1994). Improvement of transpiration efficiency (TE) is seen as a promising strategy to improve shoot biomass and pod yield productivity under episodes of intermittent drought. Efforts were made to identify simple and easily measurable traits that are closely associated with TE such as SCMR (Nageswara Rao et al., 2001; Sheshshayee et al., 2006), SLA (Nageswara Rao and Wright, 1994; Wright et al., 1994) and carbon isotope discrimination (Hubick et al., 1986; Farquhar et al., 1988; Wright et al., 1994). Recent works have demonstrated that root dry weight and SLA were important traits related to WUE under long term drought and considered useful as selection criteria for high WUE under long term drought (Songsri et al.,

2009) .

Groundnut pod yield productivity is more adversely affected by various seasonal droughts than the shoot biomass production. For example, in a field trial where the drought intensity and the timing is managed by withholding irrigation and providing a part by line source irrigation it was established that the drought occurring between emergence to peg initiation was rather beneficial, producing greater yields than the control. However the drought occurrence between the phases of start of flowering to start of seed growth had lead to a reduction of 13 to 49% in shoot biomass and 18 to 78% in pod yield. The drought stress from the start of seed growth to maturity (terminal drought) had caused a reduction of 16 to 73% for the shoot biomass and 24 to 95 % for the seed yield (Nageswara Rao et al. 1985).


Pigeonpea (Cajanus cajan (L.) Millspaugh) is a deep-rooted and drought-tolerant leguminous food crop grown in several countries, particularly in India and India accounts for about 80% of the total world pigeonpea production. It is grown mainly by resource poor farmers in

India south east Africa and, to a varying extent, throughout the tropics, usually under rain — fed conditions.

Pigeonpea can be exposed to intermittent drought stress during dry periods of the rainy season and to terminal-drought stress in the post-rainy season. Over the last two decades, shorter-duration pigeonpea (SDP) genotypes have been developed, with some genotypes capable of reaching maturity within 90 days (Nam et al., 1993). However, the developed short-duration genotypes are usually sensitive to intermittent drought. Considerable variation in tolerance to intermittent drought has been observed in short-duration pigeonpea lines and variation in sensitivity in relation to timing of drought stress has been established (Lopez et al. 1996). As in other crops, responses to intermittent drought stress have been shown to depend on the growth stage at which the stress occurs (Nageswara Rao et al. 1985). For example Nam et al. 1993 has shown that the drought incidences at flowering cause a large reduction in productivity than drought at preflowering stage or at pod fill stage. The shoot biomass reduction was 26 to 33% across years whereas the yield reduction was 30 to 48% (Nam et al. 1993).

Effects of Protected Environments on Plant Biometrics Parameters

Edilson Costa1, Paulo Ademar Martins Leal2 and Carolina de Arruda Queiroz3

1Professor Ph. D., Agricultural Engineer, State University of Mato Grosso do Sul-UEMS, Unit of Aquidauana 2Professor Ph. D., Agricultural Engineer, University of Campinas,

College of Agricultural Engineering 3MSc in progress, Agronomist, Graduate Program in Agronomy,

Crop Area, UEMS / Aquidauana-MS


1. Introduction

There is a high correlation between the type of greenhouse used for crop production with the system used for its production, especially with the type of container and substrate used. The same protected environment may present different responses in plant biometric parameters depending on the container volume and also the chemical and physical characteristics of a particular substrate. This relationship is expressed in greater or lesser accumulation of plant biomass.

Besides of the substrate and container type, other studies seek to improve the crop yield potentials and cropping systems associated with environmental control techniques, such as cooling and/or heating systems, use of CO2 for atmospheric enrichment, color screens systems and automated control of the atmospheric parameters.

Protected environments for crop production are generally constructed of low density polyethylene film (greenhouses), and shading screens, such as monofilament screens and aluminized thermal reflective screens (are widely used. In these types of environments growing in containers is preferred because it allows for better management of both water and nutrients (Grassi Filho & Santos 2004).

Changes in the microclimate inside the greenhouses caused by the use of polyethylene result in modification of the influence of air temperature, relative humidity and solar radiation on plant growth and development, and these are dependent on the intensity, duration and quality of solar radiation (Beckmann et al., 2006; Scaranari et al., 2008). These changes affect the plants physiology (Chavarria et al., 2009), and minimize the incidence of fungal diseases and therefore application of pesticides (Chavarria et al., 2007). In vineyards, where only the rows were covered with polyethylene film, Cardoso et al. (2008) found a reduction in evaporative demand.

According to Sganzerla (1987), the advantages that the greenhouses can provide to the protected plants are numerous, as long as these facilities are correctly used. Among these

advantages some can be highlighted including harvesting crops of the season, higher product quality, early crop maturity, seedling production, better control of diseases and pests, conservation of raw materials and water, planting of selected varieties and considerable increase in production.

Despite the numerous advantages, greenhouses present poor thermal behavior since during the day elevated temperatures are observed and are difficultly avoided by natural ventilation, and at night temperatures often fall below the critical temperatures for the crops (Da Silva et al., 2000). For circumvent problems with high temperatures in greenhouses many producers use evaporative cooling systems, forcing air through a porous medium with a fan (pad-fan) or intermittent misting systems. These applications improve the thermal conditions and relative humidity during the hottest periods of the day.

Important aspects should be taken into consideration in the use of protected environments, such as knowing the different protection structures and their configurations and orientations, knowing the physiological responses of the crop to be cultivated within of the environment and knowing the energy and mass balance for the crop and its environment. This set of knowledge can aid in proper crop and environment management and obtain answers of the appropriate technology to be applied to the cropping system (Costa, 2004). The parameters of leaf growth, area and mass characterize the plant biomass, so that it can be used to determine changes in carbohydrate assimilation by the plant during a season of the year (Butler et al., 2002), where the leaf area measures the plant biomass accumulation potential and leaf dry mass allows for determination of the capacity of the plant to increase its dry weight through photosynthesis.

Microclimate environmental modifications of the greenhouse and screen, i. e., the plastic covers for vegetative production, has promoted a positive impact on crops, increasing fruit yield, leaf area and quality of products produced (Buriol et al. 1997, Segovia et al., 1997).

The microclimatic effects of the protected environment influence the emergence, initial growth and development of fruit trees, vegetables, ornamental plants and forests. The objective of this study was to perform a literature review of authors who have researched comparisons between different environmental conditions and their correlation with plant performance.

Production of red yeast biomass with accumulated metals

Heavy metals are natural components of the Earth’s crust. As trace elements, some heavy metals (e. g., copper, selenium, zinc) are essential to maintain the metabolism of the human body. However, at higher concentrations they can lead to poisoning. A special case of antioxidant/prooxidant behavior of carotenoids emerge in the presence of metals (e. g. metal-induced lipid peroxidation). In this case metal ions (Fe2+ or Cu2+) react with hydroperoxides, via a Fenton-type reaction, to initiate free radical chain processes. There are several studies which indicate that P-carotene offers protection against metal-induced lipid oxidation. Presence of carotenoid in the reaction system not only decreases the free radical concentration, but also the reduction of Fe3+ to Fe2+ by carotenoids may occur. Recently free radical scavenging and antioxidant activities of metabolites produced by carotenogenic yeasts of Rhodotorula sp. and Sporobolomyces sp. grown under heavy metal presence were studied using various EPR experiments (Rapta et al., 2005). Since carotenogenic yeast differ each to other in resistance against the heavy metals due to their individual protective system, quenching properties and antioxidant activities of carotenoids yeasts were modulated by metal ions variously. Thus, activated biosynthesis of carotenoides by yeasts exposed to heavy metal presence could be in part explained by their scavenger characters (Rapta et al., 2005) as a protection against the harmful effect of the environment.

Several divalent cations (Ba, Fe, Mg, Ca, Zn and Co) have been demonstrated to act as stimulants for growth of R. glutinis. Trace elements have been shown to exert a selective influence on the carotenoid profile in R. graminis — Al3+ and Zn2+ had a stimulatory effect on beta-carotene synthesis, while Zn2+ and Mn2+ had a inhibitory effect on torulene and torularhodin synthesis (Buzzini et al, 2005). The observed effect of trace elements on the biosynthesis of specific carotenoids in red yeasts 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. In a recent study, calcium, zink and ferrous salts were shown to have a stimulatory effect on volumetric production as well as cellular accumulation of carotenoids from the yeast R. glutinis (Bhosale & Gadre, 2001). Divalent cation salts increased the total carotenoid content (mg/L) about two times. It can be assumed that this positive response was due to a stimulatory effect of cations on carotenoid-synthesizing enzymes, or to the generation of active oxygen radicalcals in the culture broth. In contrast, the addition of manganese salt in the presence of generators of oxygen radicals had an inhibitory effect on carotenoid formation in X. dendrorhous since manganese acts as a scavenger; however, this effect could be concentration dependent as manganese is also known to act as a cofactor for enzymes involved in carotenoid biosynthesis and thus enhances carotenoid accumulation at certain concentrations (Frengova & Beshkova, 2009).

Astaxanthin content was decreased significantly at >1 mg/L FeCl3 and growth of P. rhodozyma was poor at an FeCl3 concentration of <0.1-1.0 mg/L (An et al., 2001). Carotenoid production decreased in yeast with increasing Mn2+ concentration (0-10 mg/l) when succinate was used as the sole C source, but not when growth took place in the presence of glucose. The week oxygen radical scavengers Zn2+ and Cu2+ had no effect on carotenoid production by P. rhodozyma, whereas Cu2+ below 3.2 |iM increased the astaxanthin content of cells P. rhodozyma but at the expense of a slightly decreased growth. In yeast, there are at least two intracellular enzyme systems requiring copper: cytochrome-c — oxidase and superoxide dismutase. These enzymes are probably related to the increased astaxanthin production seen in concentrations of Cu2+ below 3.2 |iM. Copper deficit decreases the activity of antioxidant enzyme Cu, Zn-superoxide dismutase, as reported previously and may induce oxidative stress and astaxanthin synthesis because of diminished antioxidant defences. In contrast, iron below 1 |iM decreased both the growth and astaxanthin content of cells P. rhodozyma (Flores-Cotera & Sanchez, 2001).

Selenium (Se) is a key trace element required in small amounts in humans and animals for the function of a number of Se-dependent enzymes; however, this element can also be toxic in larger doses. Se is incorporated into proteins to provide selenoproteins, which are important antioxidant enzymes; other selenoproteins participate in the regulation of thyroid function and play a role in the immune system (Wang & Xu, 2008). Organically bound Se is considered as more bioavailable and suitable for dietary application than sodium selenite or podium selenate, the two inorganic forms of Se commonly used in the feed industry. Yeasts

naturally incorporate Se into the biomass where it is stored as selenomethionine. The organic form of Se produced in yeasts is of the similar type as that obtained from food. Recently preparation of antioxidant formula based on carotenoid forming yeasts Rhodotorula glutinis and Sporobolomyces roseus that also efficiently accumulated selenium from the growth medium was reported (Breierova et al., 2008).

In the presence of Se, carotenogenic yeast strains produced less carotene pigments. The results obtained indicate that the most dramatic change was observed in the significantly lowered levels of P-carotene, while torularhodin and torulene contents decreased to a lesser extent (Breierova et al., 2008). Previously, it has been shown that Cd, Ni, and Zn induce the opposite effect and stimulate production of P-carotene. It was found that direct incorporation of Se into yeast cells during cultivation in Se-rich medium can not be used for preparation Se-enriched yeast biomass. Instead, cultivation of the yeasts and a subsequent treatment with sodium selenite during 24h should be applied. A non-lethal and simultaneously maximum tolerated concentration of Se was determined based on the growth curves of the individual strains. A 60-ppm concentration was used with all strains, and the distribution of Se in the cells, on the surface of cells, and in the exopolymers was analyzed. The maximum Se sorption was observed with the cells of species Rhodotorula glutinis CCY 20-2-26 (17 mg/ g dry weight), while its exopolymers accumulated only 7% of the total adsorbed Se. The remaining Se was sorbed onto the fibrillar part of the cell wall and into the cells. Similarly, two other studied strains, CCY 19-6-4 and CCY 20-2-33, sorbed Se primarily into cells (63-74%) and the fibrillar part of cell wall (2-22%), whereas exopolymers bound only 12-32% of the total sorbed amount. The yeasts with high content of the carotenoid pigments and selenium may be used for the preparation of a new type of antioxidant formula that could be directly applied for various human and animal diets. Such a formula can only be produced by separate processes of the cultivation of red yeasts and a subsequent sorption of selenium into the cells (Breierova et al., 2008).

In general, 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, in agreement with previous studies reporting activation or inhibition by metal ions in microbial desaturases (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 pigment­forming microorganisms to increase the yield of microbial pigments (Rapta et al., 2005; Breierova et al., 2008).

Immersion test

The immersion test was conducted in an agricultural drainage canal (at point 1 in Fig. 4) where reddish-brown sediment accumulated, and sheathed bacteria (Leptothrix spp.) were found to be abundant (Fig. 6). The test was performed during the irrigation period for paddy fields (from May to September 2009) and the non-irrigation period (from October 2009 to April 2010), because the canal is mainly fed by drainage water from paddy fields via surface outlets and underdrains, and the water quality is affected by the paddy field irrigation.

In this test, the woody carrier was placed in a container of non-woven bag and lowered to the bottom of the canal. The carrier in the container was removed from the water after immersion for 4 weeks. The Fe collected on the immersed carrier was analyzed by the 1,10- phenanthroline method (Stucki & Anderson, 1981), and the P adsorbed on the Fe oxides was analyzed by the Bray-2 method (Byrnside & Sturgis, 1958). The Bray-2 P is a portion of the soil P and is one of the indexes of available P for plant uptake. In this study, the adsorbed P is expressed as g/kg instead of the conventional expression of Bray-2 P (mg P2O5/100 g dry material). In addition, the water samples were collected at weekly intervals and the water quality of D-Fe and PO4-P was analyzed by the above-mentioned methods.

1.2 Elemental analysis

Elemental analysis of the immersed carrier was carried out by X-ray fluorescence spectrometry system (Shimadzu, EDX-720) at a voltage of 50 kV and a current of 1 mA.

Savoriness of potato tubers

Statistical analysis revealed significant effects of intercrop fertilization and interaction between intercrop fertilization and straw fertilization on savoriness of potato tubers (table17). Intercrop fertilization improved the savoriness of potato tubers in comparison with savoriness of potato tubers harvested from control object. The best savoriness had potato tubers fertilized with white clover, the mixture of white clover with Italian ryegrass, and phacelia both plowed down in the autumn, and left till spring in the form of mulch. Savoriness of potato tubers fertilized with Italian ryegrass did not significantly differ from savoriness of potato tubers fertilized with farmyard manure. There has been an interaction which shows that the best savoriness had potato tubers fertilized with white clover in combination without straw and with straw, and the worst potato tubers from control object.

3. Discussion

Shortage of farmyard manure due to the decline in farm animal stocks, low profitability and the rationale for integrated production tend to look for alternative and efficient ways of potato fertilization. The most important here are green fertilizers from undersown crops and stubble crops and straw left on field after cereal harvest. Selection of underplant crops as alternative sources of biomass, dictated the results of Batalina et al. (1968) and Ceglarka (1982). Batalin et al. (1968) initiated studies to evaluate the fertilizer value of underplant crops legumes, and Ceglarek (1982) conducted a thorough research on the determination of yield and chemical composition of crop residues of underplant crops. However Gutmanski et al. (1998) have evaluated the value of fertilizer of oil radish, white mustard and phacelia used in sugar beet cultivation, which became the motivation for taking this type of research in potato cultivation. Under the conditions of this experiment, from the group of underplant crops yielding on the highest level was Italian ryegrass and a mixture of white clover with Italian ryegrass. The high biomass production of grasses also show results of Gromadzinski and Sypniewski (1971), Zajerc and Witkowicz (1996), Ceglarka et al. (1998), Witkowicz (1998) and Kuraszewicza and Palys (2002). In own researches, phacelia grown in stubble intercrop yielded at a similar level as white clover cultivated as an intercrop. This is consistent with the results of Gromadzinski and Sypniewski (1971), Witkowicz (1998), Trawczynski and Grzeskiewicz (1997) and Nowakowski et. al. (1997), Ceglarek and Plaza, 2000). In the experiment the addition of straw to the intercrops caused a significant increase of the amount of dry matter and macronutrients.

Nowak (1982) indicates a predominance of green manure on the farmyard manure. This follows from the fact that the nutrients contained in green manure are generally more easily absorbed than the components of farmyard manure, due to rapid decomposition of organic matter. In this experiment, among intercrops the highest value of fertilizing showed undersown: a mixture of white clover with Italian ryegrass and white clover. Batalin et. al. (1968) the highest yields of potato tubers received after plowing the undersown of red clover and serradella, and Ceglarek et al. (1998) after plowing the mixtures of legume with Italian ryegrass. These differences are due to different rates of mineralization used forms of fertilization and the fact that the introduction into the soil with a mixture of larger amounts of biomass and macronutrients. According to Nowak (1982), during the decomposition of legumes may occur high losses of nitrogen. Depending on the temperature, humidity and time of decomposition, nitrogen losses could amount up to 50%. To prevent it, to the decomposing mass of legumes material rich in carbon should be added, such as grasses, in order to increase the C:N. In this experiment yields of potato tubers fertilized with Italian ryegrass were significantly smaller than in farmyard manure. However, in this case, tuber yields were significantly higher than those obtained on control object, without intercrop fertilization. The increase of tuber yield after plowing down the grass also found Sadowski

(1992) , Spiertz et al. (1996), Duer and Jonczyk (1998) and Reust et al. (1999), but yields were lower than on the farmyard manure. This is because the introduction into the soil a large amount of biomass, with a low content of macronutrients (Sadowski 1992; Duer and Jonczyk 1998). In addition, grasses have a wide ratio C:N. In this case, the less nitrogen mineralization, which is used primarily by soil microorganisms. In own research, the value of stubble crop fertilizer from phacelia plowed down in autumn and left till spring in the form of mulch equal the fertilizer value of farmyard manure. This is understandable because of non-legume stubble crops biomass of this plant was notable for its high content of macronutrients. This is confirmed by results of Dzienia (1989), Trawczynski and Grzeskiewicz (1997) and Nowakowski, et al. (1997) and Rozylo (2002).

In potato fertilization of stubble crops can also be used in the form of mulch. However, thus fertilizing the position, with the exception of phacelia, in terms of fertilizer could not match with farmyard manure. This is confirmed by research of Boliglowa and Dzienia (1996) and Dzieni and Szarka (2000) on potato fertilization by mulch from white mustard. In the system of integrated agriculture can recommend this method of fertilization, especially with phacelia mulch, while significantly reducing of costs. The beneficial effects of intercrops plants left on the field in the form of mulch slows the mineralization of organic matter, does not allow for leaching of nitrogen, stored water from the autumn-winter rainfalls, improves soil structure and enriches it in organic matter (Hoyt et al. 1986; Frye et al., 1988; Dzienia and Boligowa, 1993; Gutmanski et al., 1999).

In that experiment fertilization with spring barley straw gave a lower effect than farmyard manure fertilization. This is consistent with the results of Sadowski (1992), Szymankiewicz

(1993) , Snieg and Piramowicza (1995) and Ceglarek et al. (1998). However, its use combined with intercrop undersown of white clover and stubble crop left till spring in the form of mulch clearly strengthened its fertilising value. Potato tubers yields of after fertilization of these forms were comparable, in the case of white clover yields higher than those recorded on farmyard manure. Also Ceglarek et al. (1998) recommend the combined use of legumes as undersown.

Intercrops fertilization with straw affects not only for the amount of received yieldss, but also on quality, so reciprocal arrangement of the components involved in potato tubers (Roztropowicz, 1989; Grzeskiewicz and Trawczynski, 1997; Boliglowa and Glen 2003). The dry matter content and starch in potato tubers depends on the genetic factor, the distribution of rainfall and temperatures during the growing season and on agronomic factors, mainly from fertilizer (Rostropowicz, 1989; Grzeskiewicz and Trawczynski 1997; Ceglarek et al., 1998; Dzienia and Szarek, 2000; Leszczynski 2002; Plaza and Ceglarek 2009; Makaraviciute 2003). In own studies, intercrop fertilization stimulated the content and dry matter yield of potato tubers and starch content and yield. The highest concentration of dry matter were characterized potatoes fertilized with mixture of white clover with Italian ryegrass and with phacelia plowed down in the autumn and left till spring in the form of mulch, and starch — potatoes fertilized with Italian ryegrass and phacelia plowed down and left till spring in the form of mulch. Research of Ceglarek et al. (1998) showed that potatoes fertilized with legume mixtures with Italian ryegrass include the most dry matter and Italian ryegrass fertilized include the most starch. Boliglowa and Glen (2003) have not indicated significant differences between the starch content in potatoes fertilized with farmyard manure, and white mustard both plowed down in the autumn, as left till spring in the form of mulch. a Different view present Mazur and Julkowski (1982) claiming that potato fertilization with legumes works better on the percentage starch content than with farmyard manure fertilization. In own studies, potato fertilization with stubble intercrop in the form of mulch increased the concentration of dry matter and starch in potato tubers as compared to that of intercrops plowed down in autumn. A similar relationship, but in sugar beet cultivation proved Gutmanski et al. (1998). However Dzienia and Szarek (2000) and Boliglowa and Glen (2003) found no significant differences between the starch content in potato tubers fertilized with farmyard manure, and white mustard both plowed down in the autumn, and left till spring in the form of mulch. Under the conditions of this experiment straw fertilization increased starch content in potato tubers, and in studies Glen et al. (2002) did not decrease significantly the concentration of this component. Consumption potato tubers should contain about 0.3% reducing sugar, and 1% of total sugars. With increased content of total sugars, potatoes taste sweet (Gluska 2000; Leszczynski, 2000, 2002). In own studies, fertilization of potato with intercrop and straw caused a significant decrease in reducing sugars and total sugars in potato tubers as compared to the control object, without intercrop fertilization. Also, according to Leszczynski (2002) and Makaraviciute (2003) organic fertilizers reduce the concentration of sugars in potato tubers. However, the studies of Mondy and Munshi (1990) showed that enrichment of soil in substance abounds in nitrogen reduces the starch content and increases the sugar content in potato tubers. In own studies, potato fertilization with white clover did not result in significant differences in the amount of reducing sugars and total sugars as compared to farmyard manure fertilization.

In light of these studies used forms of organic fertilization stimulated the concentration of vitamin C in potato tubers. The highest concentrations of vitamin C were characterized in potatoes fertilized with white clover and phacelia both plowed down in the autumn, and left till spring in the form of mulch in combination without the straw and with straw. Also, the findings of other authors (Garwood et al. 1991; Weber and Putz 1999; Leszczynski 2002; Sawicka and Kus 2002; Hamouz et al. 2005, 2007; Plaza and Ceglarek 2009) indicate a positive correlation between organic fertilization and vitamin C content in potato tubers.

In own researches, intercrop fertilization preferably affected on protein content in potato tubers. Also in the researches of Mazur and Julkowskiego (1982), Sawicka (1991), Leszczynski (2002) and Sawicka and Kus (2002) saw an increase in concentration of true protein in potato tubers cultivated in organic fertilizers. Most preferably, the discussed feature influenced white clover fertilization, also phacelia both plowed down in the autumn, and left till spring in the form of mulch in combination, without straw and with straw. A similar relationship has proved Wiater (2002). Potatoes cultivation in the position fertilized with legume plants and phacelia plants take larger amounts of nitrogen from soil than potatoes cultivated in position fertilized with green fertilizers. Nitrogen contained in the biomass of white clover and phacelia, is gradually mineralization is evenly shared to the potato crop, leading to total conversion of protein nitrogen. In own stuies, the lowest nitrate content was reported in potato tubers fertilized with white clover and phacelia both plowed down in the autumn and left till spring in the form of mulch. Only after Italian ryegrass applying nitrate content in potato tubers did not differ significantly from that recorded in potatoes fertilized with farmyard manure. The above relationship is explained by the fact that the biomass of white clover, or phacelia outside the higher content of nitrogen contained a few fibers which ensured its rapid degradation. Thanks to this all nutrients, including nitrogen available to potatoes plant are evenly distributed, allowing the total conversion of mineral nitrogen in protein nitrogen. This is consistent with the results of Dzienia et al. (2004) and Boliglowy and Glen (2003), who showed that potato tubers fertilized with white mustard and rye straw contained significantly less nitrates than potatoes fertilized with farmyard manure. According to Leszczynski (2002) use of farmyard manure, whose chemical composition is not controlled, may increase for example nitrogen and other components content in the plant. However Boliglowa and Glen (2003) showed that the nitrate content in potato tubers fertilized with white mustard developed at a similar level as in the potatoes fertilized with farmyard manure. In own studies the highest concentration of nitrates reported in potato tubers from the control object, only with mineral fertilization. This is due to the fact that mineral fertilizers, especially nitrogen increased the content of nitrogen compounds, mainly non-protein, including free amino acids, amines, ammonium nitrogen and nitrate nitrogen and reduces the share of protein in general (Wiater, 2002).

In this experiment the lowest concentration of glycoalkaloids in potatoes fertilized with white clover, a mixture of white clover and Italian ryegrass and phacelia both plowed down in the autumn, and left till spring in the form of mulch. According to Rudella et al. (2005) intercrop cultivation with a favorable ratio of carbon to nitrogen regenerates the soil environment, increases the humus content, the number of microorganisms, enzymes and other biologically active compounds in the soil, which inhibits the accumulation of harmful substances in potato tubers. In the experiment only after the applying of Italian ryegrass the concentration of glycoalkaloids in potato tubers was at the similar level as in the potato fertilized with farmyard manure. However, in this case the content of glycoalkaloids in tubers was significantly lower than that in potatoes cultivated without intercrop fertilization. Leszczynski (2002) shows that organic fertilizers reduce the harmful substances content in potato tubers by enriching the soil with organic substance which inhibits the synthesis process of glycoalkaloids. In own studies, straw fertilization also significantly differentiate the content of glycoalkaloids in potato tubers. On objects with straw the content of glycoalkaloids in potato tubers was significantly lower than on objects without straw. This is consistent with the results of research of Plaza et al. (2010). In this experiment the highest concentration of glycoalkaloids in potato tubers has been harvested from the control object, only with mineral fertilization. Also, the studies of Mondy and Munshi (1990), Hamouz et al. (2007), Kolodziejczyk et al. (2007) and Rytel et al. (2008) mineral fertilization increased the content of glycoalkaloids (solanine and chakoniny) in potato tubers. However, it should be noted that the potato in comparison with other crops have little ability to accumulate harmful substances for human. Moreover, the use of green manure and straw greatly reduces their concentration in comparison to traditional farmyard manure.

[1] In our case bioprocess parameter E was not considered-

[2] Bioprocess parameters and values of their levels are indicated in Table 1.

[3] Means in the same column followed by same letter do not differ by the Tukey test (P <0.05).

Adapted from Costa & Leal (2008)

Table 1. Leaf area (LA), leaf fresh mass (LFM) and leaf dry mass (LDM) for the strawberry cultivar Tudla, during August-October (ASO) and November to March (NDJFM).

Cultivars of chicory (Cichorium endivia L.), AF-254 and Marina, produced under a natural environment and within a low tunnel constructed of white polypropylene in the region of Ponta Grossa-PR/Brazil, presented greater head mass in the low tunnel and a greater number of leaves in the natural environment. The AF-254 cultivar was more productive but more susceptible to tipburn in the protected environment (SA & Reghin, 2008).

Cunha et al. (2005) evaluated the radiation balance and yield of sweet pepper, hybrid Elisa, in a protected environment (a non-acclimatized greenhouse oriented in the NNW-SSE direction, covered with low density polyethylene film) and in a field located in Botucatu — SP/Brazil. The authors observed that plants in the protected environment present not only greater plant height and total dry matter during of total cycle, but also a greater leaf area index. However this environment showed less net energy for growth and development of the crop.

Interactions between greenhouse environments, substrates types and different cucumber hybrids were evaluated by Costa et al. (2010) and verified different behavior of the substrates in the different environments studied, noting that the seedling growth was affected by the environments and the substrates. Response of cucumber hybrids in terms of seedlings dry biomass depended on the substrate and the growing environment. The substrate "soil and coconut fiber" increased biomass accumulation in the greenhouse and nursery with black the monofilament screen. The substrate "soil and organic compost" showed greater aerial biomass in the nursery with the aluminized screen. Hybrid ‘Safira’ accumulated more root biomass in the substrate "soil and coconut fiber" and when using the screens. The hybrid ‘Nikkei’ accumulated higher root biomass in the nursery with the aluminized screen and in the substrate "soil and coconut fiber" and did not differ from the substrate "soil and saw-dust". Hybrids ‘Aladdin F1’ and ‘Nobre F1’ accumulated similar root biomass in the environments, where the ‘Aladdin F1’ had a higher accumulation of biomass in the substrates "soil and organic compound" and "soil and coconut fiber", while the hybrid ‘Noble F1’ showed greater accumulation in "soil and coconut fiber", showing no difference from "soil and saw-dust" (Tables 2 and 3).