Microbial-enhanced Selenium and Iron Biofortification of Wheat (Triticum aestivum L.) - Applications in Phytoremediation and Biofortification

Selenium (Se) is an essential trace element for humans and other mammals. Most dietary Se is derived from crops. To develop a Se biofortification strategy for wheat, the effect of selenate fertilization and bacterial inoculation on Se uptake and plant growth was investigated. YAM2, a bacterium with 99% similarity to Bacillus pichinotyi, showed many plant growth promoting characteristics. Inoculation with YAM2 enhanced wheat growth, both in the presence and absence of selenate: YAM2-inoculated plants showed significantly higher dry weight, shoot length and spike length compared to un-inoculated plants. Selenate also stimulated wheat growth; Un-inoculated Se-treated plants showed a significantly higher dry weight and shoot length compared to control plants without Se. Bacterial inoculation significantly enhanced Se concentration in wheat kernels (167%) and stems (252%), as well as iron (Fe) levels in kernels (70%) and stems (147%), compared to un-inoculated plants. Inoculated Se-treated plants showed a significant increase in acid phosphatase activity, which may have contributed to the enhanced growth. In conclusion; Inoculation with Bacillus sp. YAM2 is a promising Se biofortification strategy for wheat and potentially other crops.     

Effect of selenium on distribution of macro- and micro-elements to different tissues during wheat ontogeny

Selenium (Se) is essential for health of humans, animals, and plants. Especially wheat is a major source of Se in the terrestrial food chain. In this study, an element analysis was optimized and the content of Ca, Mg, K, S, P, Fe, Se, Mn, Cu, Zn, and Mo in leaves, roots, and seeds were measured during growth of wheat (Triticum aestivum L. cv. Manu) in Hoagland nutrient solution with 5 and 15 μM Na₂SeO₄. Se was transported to all investigated tissues and accumulated in the seeds in proportion to used amounts. The supplementation of Se, independently of concentration, weakly modified the micro- and macro-elements content in the seedlings. In the flag-leaf stage, an increase of the Mo and S content in the shoots and the S and Cu content in the roots was found. Moreover, in the generative phase, a decrease in Ca and Fe in the roots was registered. Increased Se in the nutrient solution strongly stimulated the Se accumulation in the seeds.     

Selenium uptake by plants as a function of soil type,organic matter content and pH

In pots containing sandy soils at two levels (pH 5 and 7) to which 0.5 mg Se L^-1 soil had been added, an increase in the proportion of clay soil or peat soil led to a decrease in the uptake of Se by spring wheat grain (Triticum aestivum L., var. Drabant) and winter rape plants (Brassica napus L., var. Emil). The effect was most pronounced for the smallest additions of clay and peat soils. Differences in Se uptake between the two pH levels were greatest in treatments where the additions of clay and peat soils were small. At the high pH, an increase in clay content from 7% to 39% resulted in a decrease in Se uptake of 79% for wheat and 70% for rape. At the low pH, the uptake decreased by 72% and 77%, respectively. At the higher pH, an increase in the content of organic matter from 1.4% to 39% resulted in decreases in Se uptake of 88% for wheat grain and 69% for rape. At the low pH, Se uptake decreased by 63% and 48%, respectively. Adding peat soil to clay soil had little effect on Se uptake. Among the limed, unmixed clay, sand and peat soils to which Se had not been added, uptake was highest from the sandy soil, i.e. 8.3 ng Se/g wheat grain and 42 ng Se/g rape. The lowest uptake rates were obtained in the clay soil, i.e. 3.0 ng Se/g for wheat grain and 9.0 ng Se/g for rape.     

Characterisation of endophytic Bacillus thuringiensis strains isolated from wheat plants as biocontrol agents against wheat flag smut

To explore effective and ecofriendly means of controlling wheat flag smut (WFS) using biocontrol agents, three endophytic strains (58-2-1, 37-1 and YC-1) of Bacillus sp. were isolated from winter wheat plants in China and identified as Bacillus thuringiensis based on their 16S rDNA sequences as well as phenotypic characteristics. Four morphological (leaf length, root length, dry weight and tiller numbers) and one physiological [root vigour (RV)] parameters of wheat plants treated by strains 58-2-1 and 37-1 were significantly enhanced compared to the control. The soluble sugar contents in the roots of wheat samples treated by the two strains were significantly lower than the control. The resistance of wheat varieties to WFS was investigated by inoculation tests. Of the 12 wheat varieties tested, 6 (Yunhan-618, Bainongaikang-58, Kaimai-20, Zhengmai-9023, 04-zhong-36 and Yanzhan-4110) were identified as WFS-highly resistant (HR), 3 (Pumai-9, Jinboshi-1 and Yunong-202) WFS-moderately resistant (MR), 1 (Yubao-1) WFS-susceptible (S) and 2 (Yumai-012 and Yunong-416) WFS-highly susceptible (HS) varieties. The Urocystis tritici-induced yield loss on the S/HS varieties was significantly higher than that on the HR/MR ones. The strains 58-2-1 and 37-1 had control efficacies of 6.7–100% (av.54.8%) and 33.3–100% (av. 66.5%) on 9 and 7 out of 12 varieties, respectively. The strains 58-2-1 and 37-1 had enhanced yields of 10.2–54.9% (av. 32.9%) and 2.8–43.4% (av. 24.8%) on 10 and 8 out of 12 varieties, respectively. This is the first report on endophytic B. thuringiensis strains isolated from wheat plants with the abilities to suppress WFS and to enhance yields on multiple wheat varieties.     

Production of thermostable pectinase and xylanase for their potential application in bleaching of kraft pulp

A very high level of alkalophilic and thermostable pectinase and xylanase has been produced from newly isolated strains of Bacillus subtilis and Bacillus pumilus respectively. Enzyme production for pectinase was carried out under SSF using combinations of cheap agricultural residues while xylanase was produced under submerged fermentation using wheat bran as substrate to minimize the cost of production of these enzymes Among the various substrates tested, the highest yield of pectinase production was observed by using combination of WB + CW (6592 U/g of dry substrate) supplemented with 4% yeast extract when incubated at 37 °C for 72 h using deionized water of pH 7.0 as moistening agent. The biobleaching effect of these cellulase free enzymes on kraft pulp was determined. Both xylanase and pectinase showed stability over a broad range of pH from 6 to 10 and temperature from 55 to 70 °C. The bleaching efficiency of the pectinase and xylanase on kraft pulp was maximum after 150 min at 60 °C using enzyme dosage of 5 IU/ml of each enzyme at 10% pulp consistency with about 16% reduction in kappa number and 84% reduction in permanganate number. Enzyme treated pulp when subjected to CDED₁D₂ steps, 25% reduction in chlorine consumption and upto 19% reduction in consumption of chlorine dioxide was observed for obtaining the same %ISO brightness. Also an increase of 22 and 84% in whiteness and fluorescence respectively and a decrease of approximately 19% in the yellowness of the biotreated pulp were observed by pretreatment of the pulp with our enzymatic mixture.     

Isolation of polyacrylamide-degrading microorganisms from soil

Two polyacrylamide-degrading bacterial strains, No. 2 and No. 11, were isolated from soil, and identified asBacillus sphaericus No. 2 andAcinetobacter sp. No. 11, respectively. Both strains grew on medium containing polyacrylamide as sole carbon and nitrogen sources.B. sphaericus No. 2 andA. sp. No. 11 reduced by 16% and 19% of the initial polyacrylamide concentration, respectively. Optimal pH and temperature in growth ofAcinetobacter sp. No. 11 were 8.0 and 37°C, respectively. After 14-day cultivation ofA. sp. No. 11, the average molecular weight of polyacrylamide has been shifted from 2.3×10⁶ to 0.5×10⁶.     

Chromium-resistant bacteria and cyanobacteria: impact on Cr(VI) reduction potential and plant growth

Two chromium-resistant bacterial strains, Bacillus cereus S-6 and Ochrobactrum intermedium CrT-1, and two cyanobacterial strains, Oscillatoria sp. and Synechocystis sp., were used in this study. At initial chromate concentrations of 300 and 600 μg K₂CrO₄ mL⁻¹, and an inoculum size of 9.6×10⁷ cells mL⁻¹, B. cereus S-6 completely reduced Cr(VI), while O. intermedium CrT-1 reduced Cr(VI) by 98% and 70%, respectively after 96 h. At 100 μg K₂CrO₄ mL⁻¹, Synechocystis sp. MK(S) and Oscillatoria sp. BJ2 reduced 62.1% and 39.9% of Cr(VI), respectively, at 30°C and pH 8. Application of hexavalent chromate salts adversely affected wheat seedling growth and anatomical characters. However, bacterial inoculation alleviated the toxic effects, as reflected by significant improvements in growth as well as anatomical parameters. Cyanobacterial strains also led to some enhancement of various growth parameters in wheat seedlings.

     

Insect Diversity in Phytoremediation and Bioaccumulation of Se

A variety of plant species are being considered for the phytoremediation of selenium (Se) contaminated soils in agricultural regions of central California. Use of this plant-based technology may also attract a wide range of insects to these Se-accumulating plants. The first field study surveyed the diversity of insects attracted to tall fescue, birdsfoot trefoil, kenaf, and Indian mustard. Over 7500 specimens were collected by a sweep net collection technique for one complete growing season. Most of the 84 families identified were associated with beneficial insects, although pestiferous insects, for example, thrips, aphids, lygus, and leafhoppers, were also found. In the second study the bioaccumulation of Se in the cabbage looper [Trichoplusia ni (Hübner)] was investigated on Indian mustard grown in Se-rich water culture solution. Neonate larvae were transferred to plants and fed on Se-treated and no Se treated plants (controls) for 14 days, respectively. Pupae were collected from each treatment and incubated until adult insects emerged. Almost 50% fewer pupae were collected from Se-treated plants compared with “controls”, resulting in fewer adult insects. Selenium concentrations were as high as 3173 μg Se kg^-1 DW in adult insects hatched from Se-treated plants compared with <5 μg Se kg^-1 DW in insects from “controls”. Based on both studies, we concluded that insect diversity should be determined and insects monitored for bioaccumulation of Se on phytoremediation sites in agricultural regions.     

Root morphology, root-hair development and rhizosheath formation on perennial grass seedlings is influenced by soil acidity

Cadmium (Cd) exerts a detrimental effect on the metabolism of plants, whereas selenium (Se) may protect them against various stressors through its antioxidative activity. In this in vitro study we investigated the impact of Se (2 µM Na₂SeO₄) on the growth, nutrient (P, S, K, Ca, Mg, B, Mn, Fe and Zn) concentrations and cell integrity of rape (Brassica napus oleifera) and two wheat (Triticum aestivum) genotypes subjected to Cd stress (600 µM CdCl₂). Rape accumulated both Cd and Se more than did wheat. In all plants, Cd markedly reduced the biomass, enhanced lipid peroxidation and diminished plasmalemma fluidity. A drop in the K uptake and the reduced plasmalemma permeability diminished the K efflux from the leaf cells. In contrast, Cd elevated S concomitantly with Zn, indicating an activity of detoxifying SH groups and SOD isoenzymes. When added alone, Se promoted the growth of all plants, it enhanced the accumulation of S, but the impact on other nutrients remained minor. In Cd-stressed plants, Se tended to counterbalance the Cd-induced changes in nutrients, it also reduced the lipid peroxidation and exerted positive effects on the cell membrane stability. The Cd stress and the protective role of Se were most evident in rape. The Finnish wheat genotype was less tolerant to Cd than the Polish one.     

Selenium Modifies the Effect of Short-Term Chilling Stress on Cucumber Plants

The objective of this study was to investigate the effect of selenium (Se) supply (0, control; 2.5, 5, 10, or 20 μM) on cucumber (Cucumis sativus L.) cv. Polan F1 plants grown under short-term low temperature stress. About 14–16 day-old seedlings, grown at an optimal temperature (25/20°C; day/night), were exposed to short-term chilling stress with a day/night temperature of 10°C/5°C for 24 h, for a further 24 h at 20°C/15°C, and then transferred to 25/20°C (re-warming) for 7 days. Se did not affect the fresh weight (FW) of plants at a concentration of 2.5–10 μM, but in the presence of 20 μM Se, the biomass of shoots significantly decreased. The contents of chlorophylls and carotenoids witnessed no significant change after Se supplementation. Compared with the control, the Se-treated plants showed an increase of proline content in leaves, once after chilling and again after 7 days of re-warming. However, proline levels were much higher immediately after chilling than after re-warming. The malondialdehyde (MDA) content in the root of plants treated with 2.5–10 μM Se decreased directly after stress. This was in comparison with the plants grown without Se, whereas it increased in roots and leaves of plants exposed to 20 μM Se. Seven days later, the MDA level in the root of plants grown in the presence of Se was still lower than those of plants not treated with Se and generally witnessed no significant change in leaves. Although Se at concentrations of 2.5–10 μM modified the physiological response of cucumber to short-term chilling stress, causing an increase in proline content in leaves and diminishing lipid peroxidation in roots, the resistance of plants to low temperature was not clearly enhanced, as concluded on the basis of FW and photosynthetic pigments accumulation.     

Potato peel as a solid state substrate for thermostable α-amylase production by thermophilic Bacillus isolates

Summary Potato peel was found to be a superior substrate for solid state fermentation, compared to wheat bran, for the production of α-amylase by two thermophilic isolates of Bacillus licheniformis and Bacillus subtilis. Under optimal conditions, B. licheniformis produced 270 units/ml and 175 units/ml of α-amylase on potato peel and wheat bran, respectively, while the corresponding values for B. subtilis were 600 units/ml and 265 units/ml. The enzyme from B.␣licheniformis was optimally active at 90 °C and pH 9.0, while that from B. subtilis at 60 °C and pH 7.0. The nature of the experimental data permitted excellent polynomial fits, on the basis of which, two master equations, corresponding to the isolated strains, were derived for estimation of enzyme activity for any set of values of temperature, particle size, moisture, and incubation time within the indicated ranges.     

Characterizing Se transfer in the soil-crop systems under field condition

Aims

A comparison was performed between plant species to study Se accumulation and translocation in the crops under field condition.

Methods

Wheat, rice and canola were sampled with respective cultivated soils from the Yangtze River Delta area. The concentrations of total Se and bio-available Se and a number of parameters (N, P, S, Fe, Ca, Mg, Al, K, Mn, pH and organic carbon) were analyzed, and the net translocation coefficients of Se in the soil-crop systems were calculated.

Results

The concentrations of Se in plants significantly differed between crop species, in spite of concentrations of total Se and bio-available Se and related parameters in the soils showing no significant difference among the wheat, rice and canola sampled sites. With regard to the seeds, wheat exhibited significantly higher Se concentration than rice and canola; whereas for the straw and root, wheat showed lower Se concentration than canola and rice. The net translocation coefficients of Se in different soil-crop systems exhibited different patterns, suggesting that the difference is mainly caused by a discrepancy in Se translocation from straw to grain.

Conclusions

Wheat has a weaker capability to accumulate Se compared with rice and canola, but a significantly stronger capability to transport Se from its straw to seed. These differences might be related to the influence of S on Se differences, comparing the biochemical behavior and transport of S and Se in plants of different plant species. Selenium follows sulfur during accumulation in wheat and rice because both elements are accumulated in plant tissues mainly in the form of amino acids; whereas in canola, the influence of S on Se accumulation is not as obvious as in wheat and rice because the seeds contain more non-amino acid organic S compounds.

     

Bacterial Reduction of Cr(VI) and Fe(III) in In Vitro Conditions

ABSTRACT Chemical reduction of Cr(VI) can be a strategy to detoxify toxic metals in oxidized states, whereas reduction of Fe(III) could enhance the availability of Fe in the form of Fe(II) to boost plant growth. However, it creates another problem of chemical sludge disposal. Hence, microbial conversion of Cr(VI) to Cr(III) and Fe(III) to Fe(II) is preferred over the chemical method. Out of 11 bacterial strains isolated from the rhizospheric zone of Typha latifolia growing on fly ash dump sites, four isolates were selected for the reduction of Cr(VI) and Fe(III) and were identified as Micrococcus roseus NBRFT2 (MTCC 9018), Bacillus endophyticus NBRFT4 (MTCC 9021), Paenibacillus macerans NBRFT5 (MTCC 8912), and Bacillus pumilus NBRFT9 (MTCC 8913). These strains were individually tested for survival at different concentrations of Cr(VI) and Fe(III), pH, and temperature, and then, their ability for reduction of both metals was evaluated at optimum pH 8.0 and temperature 35°C. The results indicated that NBRFT5 was able to reduce the maximum amount, 99% Cr(VI) and 98% Fe(III). Other strains also reduced these metals to different levels, but less than NBRFT5. Hence, these strains may be used for decontamination of metal-contaminated sites, particularly with Cr(VI) and Fe(III) through the reduction process.     

Synergistic biodegradation of pentachlorophenol by <Emphasis Type="Italic">Bacillus cereus</Emphasis> (DQ002384), <Emphasis Type="Italic">Serratia marcescens</Emphasis> (AY927692) and <Emphasis Type="Italic">Serratia marcescens</Emphasis> (DQ002385)

The consortium of Bacillus cereus (DQ002384), Serratia marcescens (AY927692) and Serratia marcescens (DQ002385) were used for pentachlorophenol (PCP) degradation. The consortia showed better overall removal efficiencies than single strains by utilization of PCP as a carbon and energy source confirmed by pH dependent dye indicator bromocresol purple (BCP) in mineral salt media (MSM). Mixed culture was found to degrade up to 93% of PCP (300 mg/l) as compared to single strains (62.75–90.33%), at optimized conditions (30 ± 1°C, pH 7 ± 0.2, 120 rpm) at 168 h incubation. PCP degradation was also recorded at 20°C (62.75%) and 37°C (83.33%); pH 6 (70%) and pH 9 (75.16%); 50 rpm (73.33%) and 200 rpm (91.63%). The simultaneous release of chloride ion up to 90.8 mg/l emphasized the bacterial dechlorination in the medium. GC–MS analysis revealed the formation of low molecular weight compound, i.e., 6-chlorohydroxyquinol, 2,3,4,6-tetrachlorophenol and tetrachlorohydroquinone, from degraded sample as compared to control.     

Selenium accumulation by sequentially grown wheat and rice as influenced by gypsum application in a seleniferous soil

A field experiment was conducted for 2 years on an alkaline calcareous seleniferous soil to study the effect of different levels of gypsum (0.2 – 3.2 t ha⁻¹) applied to wheat only in the first year on Se accumulation by wheat (Triticum aestivum L.) – rice (Oryza sativa L.) cropping sequence. With gypsum application, grain yield of both rice and wheat crops increased by 0.4 – 0.5 t ha⁻¹; the increase in straw yield was 0.4 – 1.1 t ha⁻¹. Significant reduction in Se accumulation by wheat was observed with gypsum application up to 0.8 t ha⁻¹ and its residual effect was evident on the following crops for 2 years. Reduction in Se accumulation varied from 53 to 64% in wheat grain, 46 to 49% in wheat straw, 35 to 63% in rice grain and 36 to 51% in rice straw with an application of gypsum at 0.8 t ha⁻¹. A corresponding increase in S concentration was observed. In the gypsum-treated plots, the ratio of S:Se increased by 6 – 8 times in wheat and 3 – 6 times in rice. Reduction in Se accumulation by crop plants through gypsum application may help in lowering the risk of Se over-exposure of animals and humans that depend on diet materials grown on high selenium soils. This revised version was published online in June 2006 with corrections to the Cover Date.     

Construction, characterization and expression of full length cDNA clone of sheep YAP1 gene

RT-PCR, 5′RACE, 3′RACE were used to clone sheep full length cDNA sequence of YAP1 (Yes-associated protein 1), eukaryotic expression plasmid and a mutant that cannot be phosphorylated at Ser42 was successfully constructed. The amino acid sequence analysis revealed that sheep YAP1 gene encoded water-soluble protein and its relative molecular weight and isoelectric point was 44,079.0 Da and 4.91, respectively. Sub-cellular localization of YAP1 was in the nucleus, it is hydrophilic non-transmembrane and non-secreted protein. YAP1 protein contained 33 phosphorylation sites, seven glycosylation sites and two WW domains. The secondary structure of YAP1 was mainly composed of random coil, while the tertiary structure of domain area showed a forniciform helix structure. YAP1 gene was expressed in different tissues, the highest expression was in kidney and the lowest was in hypothalamus. The CDS of sheep YAP1was amplified by RT-PCR from healthy sheep longissimus dorsi muscle, cloned into pMD19-T simple vector by T/A ligation. YAP1 coding region was further sub-cloned into pEGFP-C1 vector by T4 Ligase to construct a eukaryotic expression plasmid and then make the eukaryotic expression vector as the template to construct the phosphorylation site mutant. PCR, restriction enzyme and sequencing were used to confirm the recombinant plasmid. The sheep full-length YAP1 cDNA sequence is 1712 in length encoding 403 amino acids. It was confirmed that the sheep YAP1 CDS was correctly inserted into eukaryotic expression vector and serine had been mutated to alanine by PCR, restriction digestion and sequencing. The result showed that the recombinant plasmid pEGFP-C1-YAP1 and pEGFP-C1-YAP1 S42A was constructed correctly, this will help for further studies on the YAP1 protein expression and its biological activities.     

Production of highly active fungal milk-clotting enzyme by solid-state fermentation

Abstract

Cheese production is projected to reach 20 million metric tons by 2020, of which 33% is being produced using calf rennet (EC 3.4.23.4). There is shortage of calf rennet, and use of plant and microbial rennets, hydrolyze milk proteins non-specifically resulting in low curd yields. This study reports fungal enzymes obtained from cost effective medium, with minimal down streaming, whose activity is comparable with calf and Mucor rennet. Of the fifteen fungi that were screened, Mucor thermohyalospora (MTCC 1384) and Rhizopus azygosporus (MTCC 10195) exhibited the highest milk-clotting activity (MCA) of 18,383?±?486?U/ml and 16,373?± 558?U/ml, respectively. Optimization exhibited a 33% increase in enzyme production (30?g wheat bran containing 6% defatted soy meal at 30?°C, pH 7) for M. thermohyalospora. The enzyme was active from pH 5–10 and temperature 45–55?°C. Rhizopus azygosporus exhibited 31% increase in enzyme production (30?g wheat bran containing 4% defatted soy meal at 30?°C, pH 6) and the enzyme was active from pH 6–9 at 50?°C. Curd yields prepared from fungal enzyme extract decreased (5–9%), when compared with calf rennet and Mucor rennet. This study describes the potential of fungal enzymes, hitherto unreported, as a viable alternative to calf rennet     

Selenium in cereals: improving the efficiency of agronomic biofortification in the UK

Wheat, despite its relatively low selenium (Se) concentration in the UK, is still an important dietary Se source and its biofortification by use of Se fertiliser may be an efficient means to increase the relatively low Se status of the population. We need to know more about the fate of Se applied to the soil and how to ensure the efficiency of Se application, and the three studies reported in this issue of Plant and Soil are timely and informative. Selenium in soil, both globally and locally, is notoriously variable; however, the soils in these studies yielded wheat grain Se concentrations in the narrow range of 16–44 ng/g. The low plant Se levels reported here are not surprising, given that selenite is the dominant Se form in these soils. A regression equation (which used total and extractable Se and extractable S as variables) explained a high proportion of the variance in grain Se concentration. Sulphur application (a common practice on UK wheat growing soils) had variable effects on grain Se concentration, depending on soil S status, pH and possibly other factors. A fertiliser methodology study investigated ways to optimise Se application for the purpose of biofortification. It was calculated that an application of a modest 10 g Se/ha as selenate would increase the grain Se concentration of UK wheat from around 30 ng/g to 300 ng/g. The national Se fertiliser program in Finland shows that this increase would have a large effect on population Se status. However, Se recovery in grain at this application rate is only 14%, and it can be argued that large-scale agronomic biofortification of cereals with Se would be somewhat wasteful of a relatively scarce trace element. Selenium’s effects and interactions in soil, plants, animals and humans are complex and often surprising and will keep researchers busy well into the future.     

Frementation of biowaste to H2 by Bacillus licheniformis

Completely damaged wheat grains, unfit for human consumption, were fermented to H₂ by Bacillus licheniformis strain JK1. Batch-culture fermentation of wheat slurries [6% (w/v) total solids and 5.8% (w/v) organic solids (OS)] evolved 225, 205 and 203 l of biogas-H, a mixture of H₂, CO₂ and H₂S, per kg OS at pH 6, 7 and 8, respectively. H₂ constituted 25% to 41% of the total biogas-H evolved. In single-stage continuous culture, H₂ generated/kg OS reduced was 70 l at pH 6 and 74 l at pH 7 and 8.V.C. Kalia, S.R. Jain, and A. Kumar are and A.P. Joshi was with the Centre for Biochemical Technology, Mall Road, University Campus, Delhi-110007, India; A.P. Joshi is now with the Chemical Engineering Division, National Chemical Laboratory, Pune-410008, India.