Sharad P. Kale

Monitoring of organochlorine pesticide residues in the Indian marine environment

Organochlorine pesticide residues in sediment and fish samples collected from the east and west coasts of India are presented. HCH isomers and DDT and its metabolites are the predominantly identified compounds in most of the samples. Despite the higher quantity of consumption, HCH and DDT levels in fish in India were lower than those in temperate countries suggesting a lower accumulation in tropical fish, which could be due to rapid volatilization and degradation of these insecticides in the tropical environment. The predominance of alpha- and beta-HCH reflect the use of technical grade HCH in India. The high temperature in the tropics also enhances the elimination rate of chemicals in fish, as the biological half-lives of semivolatile compounds such as DDT are short at high temperature.

Performance of electron acceptors in catholyte of a two-chambered microbial fuel cell using anion exchange membrane

The performance of the cathodic electron acceptors (CEA) used in the two-chambered microbial fuel cell (MFC) was in the following order: potassium permanganate (1.11V; 116.2 mW/m(2))>potassium persulfate (1.10 V; 101.7 mW/m(2))>potassium dichromate, K(2)Cr(2)O(7) (0.76 V; 45.9 mW/m(2))>potassium ferricyanide (0.78 V; 40.6 mW/m(2)). Different operational parameters were considered to find out the performance of the MFC like initial pH in aqueous solutions, concentrations of the electron acceptors, phosphate buffer and aeration. Potassium persulfate was found to be more suitable out of the four electron acceptors which had a higher open circuit potential (OCP) but sustained the voltage for a much longer period than permanganate. Chemical oxygen demand (COD) reduction of 59% was achieved using 10mM persulfate in a batch process. RALEX™ AEM-PES, an anion exchange membrane (AEM), performed better in terms of power density and OCP in comparison to Nafion®117 Cation Exchange Membrane (CEM).

Enhanced tolerance and remediation of anthracene by transgenic tobacco plants expressing a fungal glutathione transferase gene

Plants can be used for remediation of polyaromatic hydrocarbons, which are known to be a major concern for human health. Metabolism of xenobiotic compounds in plants occurs in three phases and glutathione transferases (GST) mediate phase II of xenobiotic transformation. Plants, although have GSTs, they are not very efficient for degradation of exogenous recalcitrant xenobiotics including polyaromatic hydrocarbons. Hence, heterologous expression of efficient GSTs in plants may improve their remediation and degradation potential of xenobiotics. In the present study, we investigated the potential of transgenic tobacco plants expressing a Trichoderma virens GST for tolerance, remediation and degradation of anthracene-a recalcitrant polyaromatic hydrocarbon. Transgenic plants with fungal GST showed enhanced tolerance to anthracene compared to control plants. Remediation of (14)C uniformly labeled anthracene from solutions and soil by transgenic tobacco plants was higher compared to wild-type plants. Transgenic plants (T(0) and T(1)) degraded anthracene to naphthalene derivatives, while no such degradation was observed in wild-type plants. The present work has shown that in planta expression of a fungal GST in tobacco imparted enhanced tolerance as well as higher remediation potential of anthracene compared to wild-type plants.

Bioremediation of DDT in soil by genetically improved strains of soil fungus Fusarium solani.

Bioremediation of DDT in soil by genetically improved recombinants of the soil fungus Fusarium solani was studied. The parent strains were isolated from soil enriched with DDD or DDE (immediate anaerobic and aerobic degradation products of DDT), as further degradation of these products are slow processes compared to the parent compound. These naturally occurring strains isolated from soil, however, are poor degraders of DDT and differed in their capability to degrade its metabolites such as DDD, DDE, DDOH and DBP and other organochlorine pesticides viz. kelthane and lindane. Synergistic effect was shown by some of these strains, when grown together in the medium containing DDD and kelthane under mixed culture condition. No synergism in DDE degradation was observed with the strains isolated from enriched soil. DDD-induced proteins extracted from individual culture filtrate (exo-enzyme) when subjected to SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) showed complementary polypeptide bands in these strains i.e., each strain produced distinct DDD degrading polypeptide bands and the recombinant or hybrid strains produced all of the bands of the two parents and degraded DDD better than the parental strains. Recombinant hybrid strains with improved dehalogenase activity were raised by parasexual hybridisation of two such complementary isolates viz. isolate 1(P-1) and 4(P-2) showing highest complementation and are compatible for hyphal fusion inducing heterokaryosis. These strains are genetically characterised as Kel+BenRDBP-Lin- and Kel-BenrDBP+Lin+ respectively.Recombinants with mixed genotype, i.e., Kel+BenRDBP+Lin+ showing superior degradation quality for DDT were selected for bioremediation study. Recombination was confirmed by polypeptide band analysis of DDD induced exo-proteins from culture filtrate usingSDS-Polyacrylamide Gel Electrophoresis (PAGE) and RAPD (Random Amplified Polymorphic DNA) of genomic DNA using PCR (Polymerase Chain Reaction) technique. SDS-PAGE showed combination of DDD induced polypeptide bands characteristic of both the parents in the recombinants or the hybrids. PCR study showed the parent specific bands in the recombinant strains confirming gene transformation.

Overlapping and distinct functions of two Trichoderma virens MAP kinases in cell-wall integrity, antagonistic properties and repression of conidiation.

We have studied the functions of the Trichoderma virens TmkB, a homologue of the yeast cell-wall integrity MAP kinase Slt2, using gene knockout. The functions of TmkB were compared to those of the pathogenicity MAP kinase homologue (TmkA). Like the tmkA loss-of-function mutants, tmkB mutants exhibited reduced radial growth and constitutive conidiation in dark as well as in liquid shake cultures. The tmkB mutants, in contrast to tmkA mutants, had cell-wall integrity defects, as shown by autolysis of the mycelia and increased sensitivity to cell-wall degrading enzymes. Interestingly, the tmkB mutants were not autolytic on the synthetic Vogels minimal medium. The tmkB mutants had attenuated ability to overgrow the plant pathogen Sclerotium rolfsii, while retaining the ability to overgrow Rhizoctonia solani and Pythium spp., a phenotype also exhibited by the tmkA mutants. This first functional analysis of a cell-wall integrity MAPK in Trichoderma spp., a group of economically important fungi, shows the importance of this signaling pathway in biocontrol. Common phenotypes of the TmkA and TmkB pathways suggest that the two MAPKs may share some substrates, perhaps subunits of key transcription factors, thus dependent on two phosphorylation events for their activity.

Studies on degradation of 14C-DDT in the marine environment.

Degradation of 14C-DDT was studied in a marine ecosystem for 60 days and in marine sediments under moist and flooded conditions using a continuous flow system for a period of 130 days. 14C-DDT residues were recovered in sediments of the marine ecosystem at uniform level of 60-65% of the applied 14C-activity throughout the incubation period. DDD was a major metabolite in sediments while DDMU was a major metabolite in clams. Clams brought about substantial degradation of DDT. However, 14C-residues recovered form clams are not suggestive of significant bioaccumulation. In the continuous flow experiment, under both moist and flooded conditions, DDT underwent degradation and about 22% of the applied 14C-activity was recovered as volatiles under both conditions. In sediments, extractable 14C-residues accounted for about 30 and 19% under moist and flooded conditions, respectively. DDT was the major compound in extractable residues as identified by TLC-autoradiographic procedures. More bound residues were formed under flooded than under moist conditions.

Isolation and characterization of microalgae for biodiesel production from Nisargruna biogas plant effluent.

Increasing energy demand and depleting fossil fuel sources have intensified the focus on biofuel production. Microalgae have emerged as a desirable source for biofuel production because of high biomass and lipid production from waste water source. In this study, five microalgae were isolated from effluents of Nisargruna biogas plants. These isolates were identified based on morphology and partial 18S and 23S rRNA gene sequences. Growth and lipid accumulation potential of these microalgae were investigated. One isolate, Chlorella sp. KMN3, accumulated high biomass (1.59 ± 0.05 g L(-1)) with moderate lipid content (20%), while another isolate Monoraphidium sp. KMN5 showed moderate biomass accumulation of 0.65 ± 0.05 g L(-1) with a very high (35%) lipid content. The fatty acid methyl esters mainly composed of C-16:0, C-18:0, C-18:1 and C-18:2. This observation makes these microalgae immensely potential candidate for biodiesel production using the effluent of a biogas plant as feed stock.

Studies on degradation of 14C-chlorpyrifos in the marine environment

Degradation of 14C-chlorpyrifos was studied in a marine ecosystem for 60 days and in marine sediment under moist and flooded conditions using a continuous flow system allowing a total 14C-mass balance for a period of 40 days. In the marine ecosystem, 14C-chlorpyrifos underwent rapid degradation and very little (1-2%) 14C-residues of the applied activity were detected after two months in sediments. Clams were major component of the ecosystem and played a significant role in degradation of the insecticide. In the continuous flow system chlorpyrifos did not undergo substantial mineralization. Volatilization accounted for 0.8-1% loss during first ten days of application. The amounts of extractable 14C-activity were higher in flooded sediments than in moist sediment. More bound residues were formed under moist conditions. TCP (3,5,6-trichloro-2-pyridinol) was the major degradation product formed under both moist and flooded conditions, its formation being higher in the latter conditions. These studies underline the role of clams in degradation of chlorpyrifos and lack of microbial degradation. In absence of clams, chlorpyrifos underwent abiotic degradation in marine sediment with formation of bound residues.

Biomimetic synthesis of nanocrystalline silver sol using cysteine: stability aspects and antibacterial activities

The study reports the development of a simple, environmentally benign green chemical route to produce stable silver nanoparticle (Ag-np) sols with excellent antibacterial properties under ambient conditions. The method involves the room temperature reduction of AgNO3 by cysteine (aq) and requires no additional capping/stabilizing agent. It essentially mimics the redox reaction that takes place during incubation of the cell-free extract from Trichoderma asperellum in the presence of AgNO3 (aq) (P. Mukherjee, M. Roy, B. P. Mandal, G. K. Dey, P. K. Mukherjee, J. Ghatak, A. K. Tyagi and S. P. Kale, Nanotechnology, 2008, 19, 075103), wherein cysteine, a biomolecule present in the fungal extract, acts as a potential reducing agent. Additionally, cysteine acts as a capping molecule in the present case. Formation of Ag-nps was evidenced from UV-Vis, TEM, XRD and EDS studies. The stability of Ag sols has been shown to depend strongly on the concentration of cysteine relative to that of AgNO3. Sols obtained by reacting 0.1 mM of cysteine with 1 mM of AgNO3 remained stable for more than one month at 24 °C. The role of cysteine as capping molecule and the possible modes of its linkages with Ag-nps was studied by FT-IR, XPS and Raman spectroscopy. Bonding of Ag with either or all the three, thiolate, amino and carboxylate groups of the cysteine molecule via stable PH configuration is believed to have resulted in the stabilization of the Ag-nps. Antibacterial activity of the cysteine capped Ag sol was studied along with that of the Ag sol obtained by fungal route. Both the sols exhibited excellent and comparable efficacies as bactericidal agents against gram negative bacteria E. coli BW (25113), with one of the lowest minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values published so far.

Synthesis of uniform gold nanoparticles using non-pathogenic bio-control agent: Evolution of morphology from nano-spheres to triangular nanoprisms

Green synthesis of gold nanospheres with uniform diameter and triangular nanoprisms with optically flat surface was carried out using a non-pathogenic bio-control agent Trichoderma asperellum for reduction of HAuCl(4). Kinetics of the reaction was monitored by UV-Vis absorption spectroscopy. No additional capping/complexing agent was used for stabilizing the gold nanoparticles. Evolution of morphology from pseudospherical nanoparticles to triangular nanoprisms was studied by transmission electron microscopy (TEM). It revealed that three or more pseudospheres fused to form nanoprisms of different shapes and sizes. Slow rate of reduction of HAuCl(4) by constituents of cell-free fungal extract was instrumental in producing such exotic morphologies. Isolation of gold nanotriangles from the reacting masses was achieved by differential centrifugation.