For the determination of kefir grain mass, the gravimetric method was used. Therefore, kefir grains were separated first from the fermentation medium with plastic household sieve. Then the grains were washed with cold water and dried on filter paper to remove of bulk of adhered water. Finally, kefir grain mass was determined by weighting on Mettler-Toledo analytical balance (PG5002-S).

1.3 Taguchi’s experiment design methodology

Dr. Genichi Taguchi has defined the optimization criterion quality as a consistency in achieving the desired or targeted value and minimization of the deviation (Ranjit, 1990). This goal is connected with the performance of a series of experiments with different bioprocess parameters at different levels. The bioprocess parameter is a factor affecting the optimization criterion quality, and its value is called the ‘level’. The number of experiments and their sequence are determined by standard OA. When planning the experiments using four bioprocess parameters at four levels, we use the OA L16. Such a plan envisages the performance of 16 experiments, which is significantly less when compared to the full factorial DoE with 44 = 128 experiments.

Due to performing only a part of the envisaged experiments using the traditional full factorial DoE methodology, it is necessary to include an analysis of the results confidence. The standard statistical technique is used for this purpose, the so-called ‘analysis of variance’ (ANOVA), which recognizes the relative impact of the bioprocess parameters for the optimization criterion (in our case daily kefir grain increase mass) value.

The mathematical algorithm of the ANOVA statistical technique is based on calculation of the variance, which is an indicator of the optimization criterion quality. The ratio between the variance of the bioprocess parameter and the error variance shows whether the parameter affect on the product’s quality. The equations required for calculating the relative impact of various significant bioprocess parameters affecting the optimization criterion are presented bellow. The meanings of symbols are described in the sub-chapter "Nomenclature".

We compare variance ratio of bioprocess parameter j, Fj, to the standardized value at defined level of significance, Fm, n, which is obtained from the standard F tables (Ranjit, 1990), whereby m stands for the degree of freedom of bioprocess parameter j and n means the degree of freedom of error variance, and thus determine the bioprocess parameter impact accordingly. In the case where the variance ratio of bioprocess parameter j falls below Fm, n, the bioprocess parameter has no impact on the optimization criterion, therefore, it is pooled and ignored in the calculations. Consequently, the variance error changes, as the sum of squares and degree of freedom of the pooled bioprocess parameter are added to the error sum of squares and degree of freedom of error variance, respectively. By using the adjusted variance error, we determine new variance ratio of bioprocess parameter j and compare them again by the Fm, n. The process of pooling is sequential, which means that the parameter having the smallest impact on the optimization criterion should be pooled first, then we re­calculate the variance ratio of bioprocess parameter j and continue pooling until each bioprocess parameter meets the condition Fj > Fm, n. If the pooling process begins to perform, Taguchi recommends pooling bioprocess parameters until the degree of freedom of error variance is approximately half the total degree of freedom irrespective of significant test criterion validity Fj > Fm, n for all remaining bioprocess parameters (Taguchi, 1987). When the pooling procedure is completed, the relative impact of bioprocess parameter j and error on optimization criterion can be calculated using Eqs. (10) and (11).

Membrane permeate flux is an important design and operational parameter that impacts significantly in CAPEX and OPEX. Typical operation flux rates for various full-scale iMBRs applied to treat municipal wastewater treatment are over 19-20 l/h m2 (Judd, 2010) with a peak flux (< 6 h) in the range 37-73 l/h m2 (Asano et al., 2006).

A recent analysis of design and operation trends of the larger MBR plants in Europe (Lesjean et al., 2009), shows a broad difference between the design and operation flux. For Kubota systems, the designed maximum daily net fluxes are 14-48 l/h m2 (mean at 32 l /h m2) while for the GE Zenon modules they are 20-37 l/h m2 (mean at 29 l/h m2). However, it is interesting to note that for both systems the operation net flux is over 18 l/h m2. Further differences are the same regardless of whether this is a new plant or a retrofit, or more or less conservative designs of a specific plant. In fact, the authors indicate that the averaged trend of the design maximum net flux and operation mean flux have moderately increased by only 3 l/h m2 during the last 6 years. Given the impact of this discrepancy over CAPEX (i. e. higher membrane surface demand) and OPEX (i. e. higher membrane replacement costs) different solutions have been proposed: a plant has been designed in parallel to conventional activated sludge systems (hybrid systems), which can absorb the peak flows, or by addition of a buffer tank for flow equalisation.

In a comprehensive cost analysis of a large HF MBR plant, Verrecht et al. (2010) show the impact of both solutions on plant costs over the cycle life of the plant. While comparing a hybrid system with an MBR designed to manage maximum flow conditions, results indicate that the average energy demand for the full-flow MBR is 57% higher, as a result of under­utilization of the membrane available area and excess of membrane aeration. With regard to the adding of a buffering tank, the authors pointed out that the cost of buffering would be covered by reducing the required membrane surface area. However, this solution should increase the scale size of the plant by 10% compared to CAS treating the same flow. Therefore, the authors conclude that hybrid MBR plant is the most desirable option. Examples of some full-scale facilities with this hybrid system would be the Brescia plant with GE/Zenon in Italy, or the Sabadell plant with Kubota in Spain.

1.2 Content of Cd, Cu, Pb and Zn in aerial plant biomass (cultivation in experimental pots)

Experimental plants of the first group (A-I, B-I and C-I) and the second one (A-II, B-II and C-II) treated with BRs in the plant growth stage 29-31 DC did not differ in the growth stage 47 — 49 DC from untreated control and the plants of the third group (not treated in the stage 29 — 31 DC). The first differences in the content of investigated metals were shown in the plant growth stage 73 — 75 DC (Table 5). A distinct trend in copper content was not observed in the plant biomass. Content of lead decreased in all variants of treated plants. A decreased lead content was determined in the plants of the second group (as a whole treated two times) and the third (A-III, B-III and C-III) group (as a whole treated ones), in which the last BRs application was performed in growth stage 59-60 DC. In the first group that was treatedonly once in the stage 29-31 DC, lead content was higher than those in the other two groups. Similarly to lead content in the plants of the second group and the third group, lower cadmium and zinc contents were determined as related to the contents of the first group and in control plants (with the exception of plants treated with 4154 in the third group, where the lower Zn content was not determined). After the harvest of plants in the growth stage 90-92 DC (Table 5), a lower copper content in the first group and the third group (with the exception of plants treated with 4154 in the third group) was determined in plant straw. Likewise in the growth stage 73-75 DC, lower zinc content was determined in all plants of the second group and in the plants of the third group treated with 24-epiBL and 24-epiCS.

growth stage 47-49 growth stage 73-75growth stage 90-92 DC DC DC

(plants) (plants) (straw)

growth stage 47-49 DCgrowth stage 73-75 DCgrowth stage 90-92 DC (plants) (plants) (straw)

Fig. 3. Content of zinc in aerial biomass of plants (mg kg-1 DM)

No significant difference of the cadmium content in the growth stage 90-92 DC was found, with the exception of plants treated with 4154 in the first group and 24-epiBL in the second group. Lower lead content was determined in the plants of the second group and the third group treated with 24-epiBL and 24-epiCS. Copper content was affected more likely
according to the actual and individual status of plants, however, in some cases these physiological processes could be affected by brassinosteroids treatment (Fig. 2). Zinc content in aerial biomass decreased during plant growth (Fig. 3). A significant decrease of cadmium content was determined after the applications of brassinosteroids in the growth stage 73-75 DC in the plants of the second group and the third group (Fig. 4). Lead was accumulated in the plant biomass of the control group during the all vegetation period (Fig. 5). Lower lead

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growth stage 47-49 DC growth stage 73-75 DC growth stage 90-92 DC

(plants) (plants) (straw)

growth stage 47-49 DC growth stage 73-75 DC growth stage 90-92 DC (plants) (plants) (straw)

Fig. 5. Content of lead in aerial biomass of plants (mg kg-1 DM)

content in the stage 90-92 DC was found only in the plants of the second group and the third group that were treated with 24-epiBL and 24-epiCS. No significant difference was found in the plants of the first group that were treated in the growth stage 29-31 DC and in the treatment with brassinosteroid 4154.

1.3 Content of Cd, Cu, Pb and Zn in grains (cultivation in experimental pots)

After the application of BRs, lead content in grains decreased in the second and the third group (Table 5). While copper content significantly decreased in the plants of the third group following 24-epiCS treatment, the decrease of copper content was not statistically significant in other variants. Effect of brassinosteroids on the content of metals in grains of control plants is shown in Fig. 6.

The element aluminium (atomic weight 26.98) is a silver white metal (density 2.7 g/mL). In its inorganic compound it presents only two oxidation states: 0, +3. Aluminium is the most abundant metal in the Earth’s crust, and the third most abundant element, after oxygen and silicon. Because of its extremely low redox-potential potential in nature, it is found combined in over 270 different minerals as oxides or silicates. Aluminium is remarkable for low density and for its ability to resist corrosion due to the phenomenon of passivation. Structural components made from aluminium and its alloys are vital to the aerospace industry and are very important in transportation and building. Aluminium compounds are widely used in the paper industry, in the dye production, in the textile industry, in processed food, and as a component of many cosmetic and pharmaceutical preparations. Soluble aluminium salts have demonstrated toxic effects in elevated concentrations. Its toxicity can be traced to deposition in bone and the central nervous system. Because aluminium competes with calcium for absorption, increased amounts of dietary aluminium may contribute to osteopenia (reduced skeletal mineralization). In very high doses, aluminium can cause neurotoxicity. In a smaller amount it can give in susceptible people contact dermatitis, digestive disorders, vomiting or other symptoms upon contact or ingestion.

Owing to limitations in the animal data as a model for humans and the uncertainty surrounding the human data, a health-based WHO guideline value cannot be derived; however, practicable levels based on optimization of the coagulation process in drinking — water plants using aluminium-based coagulants are derived: 0.1 mg/L or less in large water treatment facilities, and 0.2 mg/L or less in small facilities (World Health Organization [WHO], 2008).

Sorghum is an important food cereal in many parts of worldwide. According to the U. S. National Sorghum Producers Association (2006), approximately 50% of the world production of sorghum grain is used as human food. Sorghum grain is a staple diet in Africa, the Middle East, Asia and Central America where its processed grain may be consumed in many forms including porridge, steam-cooked product, tortillas, baked goods, or as a beverage (CGIAR, 2009). China and India account for almost all of the food use of sorghum in Asia, in other parts of the world, sorghum grain is used mainly as an animal feed. It has the distinct advantage (compared to other major cereals) of being drought-resistant and many subsistence farmers in these regions cultivate sorghum as a staple food crop for consumption at home (Murty and Kumar, 1995). Therefore sorghum acts as a principal source of energy, protein, vitamins and minerals for millions of the poorest people living in these regions (Klopfenstein and Hoseney, 1995). The improvement of sorghum nutrient availability is critical for food security. Cereal scientists and sorghum food processors are thus faced with the challenge of identifying the factors that adversely affect, and developing processing procedures that improve sorghum protein digestibility. Most parts of the sorghum plant are used as animal feed. Growing sorghum may be grazed, or the aerial parts of the plant may be ensiled or dried and fed as stover or silage for ruminant animals. Whole sorghum grain is cracked, ground, or steam flaked and fed to poultry, swine, dairy and beef cattle as a source of energy. Crop residues are a major animal feed resource in many crop-livestock farming systems. They are very useful in ameliorating the problem of inadequacy of feeds for ruminant livestock during the dry season. Although useful as dry season feeds, crop residues, particularly those of cereal origin, are low in protein and energy content (Agyemang et al., 1998). The stover of sorghum also is used as fodder for animals. The nutrient composition of sorghum grain is presented in Table 8.

Table 8. Proximate analysis of S. bicolor grain (dry matter basis) (Quotation from OECD, 2010)

The earthworm fauna of South Korea is dominated by the family Megascolecidae and identified 101 species, with 12 species in Lumbricidae, 9 species in Moniligasteridae and 80 species in Megascolecidae (Fig. 1) (Hong, 2000, 2005; Hong et al., 2001). In general, earthworms are classified into three types based upon life style and burrowing habit (Bouche, 1972). The epigeal forms (e. g., Lumbricus rubellus and Eisenia fetida) hardly burrow in soil at all, but inhabit decaying organic matters on the surface, including manure or compost heaps. The endogenous species (e. g., Allolobophora chlorotica and Allolobophora caliginosa) produce shallow branching burrows in the organo-mineral layers of the soil. Lastly, the anectic forms (e. g., L. terrestris and Allolobophora longa) are deep burrowing species, producing channels to a depth of one meter or more. Megascolecidae species identified in Korean ecosystem come under anectic forms. Occurrence of earthworms in agroecosystem appeared the most individuals of Amynthas agrestis, Amynthas heteropodus and Amynthas koreanus (Hong & Kim, 2007).

1.2 Biomonitor for biological hazard assessment on soil contamination

Concerns about contamination of soil and detrimental effects of contaminants on the living environment have resulted in a strong and growing interest in soil organisms among environmental scientists and legislators. Legislation in many countries has recently focused on the need of sensitive organisms from the soil environment for environmental monitoring. Many toxic materials have been accumulated along with food webs. The decomposer levels are frequently the first to be affected since the organic matter and the soil are the ultimate sink for most contaminants. Ecologically, earthworms are near the bottom of the terrestrial tropic levels. The effects of contaminants on earthworms which were kept in soil in the laboratory have been studied (Edwards & Thompson, 1973). These tests tended to produce consistent and reproducible results because 10 individuals of E. fetida were used and these worms were an intimate contact with pesticides. van Hook (1974) demonstrated that earthworms could serve as useful biological indicators of contamination because of the fairly consistent relationships between the concentrations of various contaminants and mortality of earthworm. The basic requirements of finding a species easy to rear and genetically homogeneous could be fulfilled by using representatives of the species, although there have been arguments for the use of Eisenia andrei or a genetically controlled single strain of the E. fetida complex (Bouche, 1992). Callahan et al. (1994) have suggested that E. fetida may be a representative of the species, Allolobophora tuberculata, Eudrilus eugeniae and Perionyx excavatus based upon the concentration-response relationship for 62 chemicals when applying the Weibull function. Habitational earthworms, including E. fetida, are useful as biological indicator species in the ecological sense or a more useful biomonitor species. It has been proposed that A. heteropodus could be adopted as a bioindicator in agroecosystem because of dominant species in South Korea (Kim et al., 2009).

Sarfraz Ahmad and Muhammad Islam

Arid Zone Research Centre, Quetta,

Pakistan

1. Introduction

Pakistan has total land area of 88 million hectare (ha) and about 65% of this is rangelands. Five different types of range ecological zones (Sub-alpine and temperate, Sub-tropical humid, Sub-tropical sub-humid, Tropical arid and semi-arid deserts plains, and Mediterranean) have been described in Pakistan (Khan & Mohammad, 1987). These rangelands are the major feed source of about 97 million heads of livestock. Precipitation varies from 125 mm to over 1500 mm per annum. About 60 to 70% of monsoon rains received during the months of July to September while the winter rains occur from December to February (Khan, 1987).

Balochistan has a total area of 34 million ha of which only 4% (1.47 m ha) is under cultivation while 60% of the cultivated area is rainfed (Khan, 1987). Approximately, 93 % of this province (Fig. 1) is characterized as rangelands (FAO, 1983) Arid and semi-arid areas are falling within the rainfall zones of 50-200 mm and 250-400 mm, respectively (Kidd et al.,

1988) . Rainfall patterns are unpredictable with great variations. Like other arid and semiarid rangelands of the world, Balochistan ranges also provide a diversity of uses, including forage for livestock, wildlife habitat, medicinal plants, water storage and distribution, energy, minerals, fuel wood, recreational activity, wilderness and natural beauty.

Livestock rearing is the main activity of the inhabitants of Balochistan. Sheep and goats are the main livestock of the province. About 87% of the people in Balochistan directly or indirectly drive their livelihood from livestock rearing (Heymell, 1989). About 20 million sheep and goats population have been reported in Balochistan (GOB, 1996 ). Rangelands are the major feed source of these animals and approximately 90% of total feed requirements of sheep and goats were being met from rangelands (FAO, 1983). Overgrazing, drought, erosion, and human induced stresses caused severe degradation of rangelands in Balochistan (Islam et al., 2008; Hussain & Durrani, 2007). The degradation of rangelands includes changes in composition of desirable plant species, a decrease in rangeland diversity and productivity, reduction of perennial plant cover, and soil erosion (Milton et al., 1994).

In Balochistan, the mixed grass-shrub steppe is more common than single plant communities. The range vegetation types in Balochistan changes from south to north along the rainfall distribution. In South, shrub species Haloxylon species and Artemisia species while in north perennial grass species Cymbopogon jwarancusa and Chrysopogon aucheri are dominant. The fragile ranges of Balochistan are degrading very rapidly due to heavy

grazing pressure, aridity, and human disturbances. However, still many of these ranges have potential for improvement by using grazing management practices, natural recovery of vegetation and artificial re-vegetation at suitable sites coupled with better water harvesting and conservation practices.

Natural re-vegetation practices particularly grazing management may restore vigor and accelerate the spread of desirable species (Vallentine, 1980). Grazing management alone may not accelerate the succession towards desirable species in arid and semiarid rangelands due to limited precipitation where artificial re-vegetation would involve the establishment of adapted species either by seed or transplanting seedlings (Roundy & Call, 1988). Restoration and rehabilitation are the two main procedures for regeneration of a depleted rangeland. Restoration or biological recovery means to bring the ecosystem to their pristine situation and rehabilitation or artificial recovery is the artificial establishment of a new type of vegetation different from the pristine native vegetation (Le Houerou, 2000). Biological or artificial recovery may include increase in biomass, plant cover, organic matter, soil micro and macro-organisms, better water intake and turnover, lower evaporation and runoff. Biological recovery may be obtained by protecting the target area from human and livestock intrusion. The purpose of rehabilitation of rangelands may be diverse like forage production, timber production, landscaping, wind breaks, sand dune fixation, and erosion control (Le Houerou, 2000).

A major concern of arid and semiarid ranges is the progressive reduction of secondary productivity and diversity (West, 1993) and how to manage these changes (Walker, 1993). The management and improvement of arid and semi-arid ranges is always a challenging job. Different theoretical models of rangelands have been developed and few are also being tested in different rangeland ecosystems of the world. However, the arid rangeland ecosystem of Balochistan is very dynamic where major climatic and agricultural changes are occurring. Hence many range management projects were carried out with little success.

Therefore, there is a need to re-look into research, policy and management issues for better productivity of rangelands and livestock.

The isoprenoid pathway in yeasts is important not only for sterol biosynthesis but also for the production of non-sterol molecules, deriving from farnesyl diphosphate (FPP), implicated in N-glycosylation and biosynthesis of heme and ubiquinones. FPP formed from mevalonate in a reaction catalyzed by FPP synthase (Erg20p). In order to investigate the regulation of Erg20p in Saccharomyces cerevisiae, a two-hybrid screen was used for its searching and five interacting proteins were identified. Subsequently it was showed that Yta7p is a membrane-associated protein localized both to the nucleus and to the endoplasmic reticulum. Deletion of Yta7 affected the enzymatic activity of cis- prenyltransferase (the enzyme that utilizes FPP for dolichol biosynthesis) and the cellular levels of isoprenoid compounds. Additionally, it rendered cells hypersensitive to lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) that acts upstream of FPP synthase in the isoprenoid pathway. While HMGR is encoded by two genes, HMG1 and HMG2, only HMG2 overexpression was able to restore growth of the yta7- cells in the presence of lovastatin. Moreover, the expression level of the S. cerevisiae YTA7 gene was altered upon impairment of the isoprenoid pathway not only by lovastatin but also by zaragozic acid, an inhibitor of squalene synthase (Kuranda et al., 2009).

All enzymes involved in carotenoid biosynthesis are membrane-associated or integrated into membranes. Moreover, carotenoid biosynthesis requires the interaction of multiple gene products. At present more than 150 genes, encoding 24 different crt enzymes involved in carotenogenic branch of isoprenoid pathway, have been isolated from bacteria, plants, algae and fungi. The availability of a large number of carotenogenic genes makes it possible to modify and engineer the carotenoid biosynthetic pathways in microorganisms. A number of genetically modified microbes, e. g. Candida utilis, Escherichia coli, Saccharomyces cerevisiae, Zymomonas mobilis, etc. have been studied for carotenoid production (Wang et al. 2000; Schmidt-Dannert, 2000; Lee & Schmidt-Dannert, 2002; Sandmann 2001). However, lack of sufficient precursors (such as IDP, DMADP and GGDP) and limited carotenoid storage capability is the main task how to exploate these organisms as commercial carotenoid producers. Therefore, effort has been focuced on increasing the isoprenoid central flux and levels of carotenoid precursors. For example, overexpression of the IDP isomerase (idi — catalyzes the isomerization of IDP to DMAP) together with an archaebacterial multifunctional GGDP synthase (gps — converts IDP and DMADP directly to GGDP) resulted in a 50-fold increase of astaxanthin production in E. coli (Wang et al., 2000).

By combination of genes from different organisms with different carotenoid biosynthetic branches, novel carotenoids not found in any other pathway can be synthesized. Most Mucor species accumulate |3-carotene as the main carotenoid. The crtW and crtZ astaxanthin biosynthesis genes from Agrobacterium aurantiacum were placed under the control of Mucor circinelloides expression signals. Transformants that exhibited altered carotene production were isolated and analyzed. Studies revealed the presence of new carotenoid compounds and intermediates among the transformants (Papp et al., 2006). Fusarium sporotrichioides was genetically modified for lycopen production by redirecting of the isoprenoid pathway toward the synthesis of carotenoids and introducing genes from the bacterium Erwinia uredovora (Leathers et al, 2004). Carotenoid biosynthetic pathway of astaxanthin producers of Phaffia/Xanthophyllomyces strains has also been engineered and several genes, such as phytoene desaturase, isopentenyl diphosphate isomerase and epoxide hydrolase were isolated and expressed in E. coli (Verdoes et al., 2003; Lukacz, 2006).

FTIR is one of the most used techniques. Infrared Spectroscopy belongs to the group of molecular vibrational spectroscopies which are molecule-specific, and give direct information about the functional groups, their kind, interactions and orientations. Its sampling requirements allow the gain of information from solids, and in particular from solid surfaces. Even if historically IR has been mostly used for qualitative analysis, to obtain structural information, nowadays instrumental evolution makes non-destructive and quantitative analysis possible, with significant accuracy and precision. The shift of the bands and the changes in signal intensity allow the identification of the functional groups involved in metal sorption. Using this technique, carbonyl, carboxylic, aromatic, amine, and hydroxyl groups has been found to be involved in metal uptake by different biosorbents.

1.2.1 Diffuse reflectance infrared fourier transform spectroscopy (DRIFTS)

DRIFTS occurs when light strikes on the surface of a material and is partially reflected and transmitted. The light that penetrates the material may be absorbed or reflected out again. The diffuse reflectance (radiation reflected from an absorbing material) is thus composed of surface-reflected and bulk re-emitted components, and contains information relative to the structure and composition of the sample. Even if DRIFTS has been not of large use, it has found interesting applications on verifying the enhancement of cadmium sorption capacity by juniper wood when carbonyl groups were substituted by sulfonic groups and on determining that Cr3+, Cu2+ and Zn2+ were sorbed onto the organic polymeric fraction of olive mill wastewater by ion exchange between alkaline and alkaline-earth metal ions and protons bound to carboxylic groups.

2.1.2 The dry matter content in potato tubers

The dry matter content in potato tubers was significantly differentiated by the intercrop fertilization, straw fertilization and their interaction (table 4). The highest concentration of

dry matter characterized potato tubers fertilized with white clover, a mixture of white clover with Italian ryegrass and phacelia both plowed down in the autumn, as left till spring in the form of mulch.

The dry matter content in potato tubers fertilized with Italian ryegrass was significantly lower than in potatoes fertilized with farmyard manure. On control object, without organic fertilization dry matter content in potato tubers was significantly lower. Straw fertilization also significantly modified dry matter content in potato tubers. At the sub-block with straw potatoes distinguished by a higher concentration of dry matter than the tubers at sub-block without straw. From the interaction of researched factors showed that the highest content of dry matter was noted in potato tubers fertilized with white clover with straw, a mixture of white clover with Italian ryegrass in combinations without straw and with straw, phacelia in combination with straw, and phacelia used in the form of mulch with a straw or without the straw, and the lowest in potato tubers harvested from control object without organic fertilization.