Haresh Keharia

Production, partial purification and characterization of organic solvent tolerant lipase from Burkholderia multivorans V2 and its application for ester synthesis

Burkholderia multivorans V2 (BMV2) isolated from soil was found to produce an extracellular solvent tolerant lipase (6.477 U/mL). This lipase exhibited maximum stability in n-hexane retaining about 97.8% activity for 24h. After performing statistical optimization of medium components for lipase production, a 2.2-fold (14 U/mL) enhancement in the lipase production was observed. The crude lipase from BMV2 was partially purified by ultrafiltration and gel permeation chromatography with 24.64-fold purification. The K(m) and V(max) values for partially purified BMV2 lipase were found to be 1.56 mM and 5.62 micromoles/mg min. The metal ions Ca(2+), Mg(2+) and Mn(2+) had stimulatory effect on lipase activity, whereas Cu(2+), Fe(2+) and Zn(2+) strongly inhibited the lipase activity. EDTA and PMSF at 10mM concentration strongly inhibited the lipase activity. Non-ionic and anionic surfactants stimulated the lipase activity. BMV2 lipase was proved to be efficient in synthesis of ethyl butyrate ester under non-aqueous environment.

Decolorization screening of synthetic dyes by anaerobic methanogenic sludge using a batch decolorization assay

The nonspecific ability of anaerobic sludge bacteria obtained from cattle dung slurry was investigated for 17 different dyes in a batch assay system using sealed serum vials. Experiments using Reactive Violet 5 (RV 5) showed that sludge bacteria could effectively decolorize solutions having dye concentrations up to 1000 mg l−1 with a decolorization efficiency of above 75% during 48 h of incubation. Headspace gas composition of anaerobic batch systems for varying dye concentration revealed that lower concentrations of RV 5 (upto 500 mg l−1) were found to be stimulatory to the methanogenic activity of sludge bacteria. However at higher dye concentrations, the headspace gas composition was found to be similar to batch assay controls without dye, indicating that dye at higher concentrations was inhibitory to methanogenic bacteria of sludge. The optimum inoculum and incubation temperature for maximum decolorization of RV 5 was found to be 9.0 g l−1(in terms of total solids) and 37°C, respectively. Of sixteen other dyes tested, nine (Reactive Black 5, Reactive Blue 31, Reactive Blue 28, Reactive Red HE8B, Reactive Yellow, Reactive Golden Yellow, Mordant Orange, Novatic Olive R S/D & Navilan Yellow GL) were decolorized with more than 88% efficiency; three (Orange II, Navy Blue HER & Novatic Blue BC S/D) were decolorized with about 50–65% efficiency, whereas other three dyes (Procion Orange H2R, Procion Brilliant Blue HGR & Novatic Blue BC S/D) were decolorized with less than 40% efficiency. Though Ranocid Fast Blue was decolorized with about 92.5% efficiency, this was merely due to sorption, whereas the other dyes were decolorized due to biotransformation.

The suitability of Jatropha seed press cake as a biosorbent for removal of hexavalent chromium from aqueous solutions.

ABSTRACT Jatropha seed press cake (JPC), a biodeisel waste, was investigated for its use as biosorbent for Cr(VI) removal from wastewater. The acid-pretreated biomass exhibited 1.9-fold higher biosorption efficiency for Cr(VI). The Cr(VI) biosorption efficiency was found to increase with decrease in pH of aqueous medium. The adsorption capacity of biosorbent for Cr(VI) increased with increasing concentration of Cr(VI). The biosorption of Cr(VI) by acid-treated JPC followed a pseudo-second-order kinetics. The results of equilibrium studies showed that the biosorption process fitted the Langmuir isotherm model, with a maximum adsorption capacity of 22.727 mg of Cr(VI)/g of biosorbent at 30°C. The activation energy was found to be 27.114 kJ/mol, suggesting that the adsorption process was mainly a physical process. The important thermodynamic parameters of adsorption (ΔG, ΔH, andΔS) were determined, which indicated that the Cr(VI) sorption by JPC is a spontaneous and endothermic process.

Production of indole-3-acetic-acid (IAA) by the white rot fungus Pleurotus ostreatus under submerged condition of Jatropha seedcake.

The synthesis of phytohormone, indole-3-acetic acid (IAA), is not only confined to flowering plants but bacteria (especially plant growth-promoting rhizobacteria and few pathogens), yeasts, and other fungi are also known to produce this hormone and in many cases even at higher levels than plants. Three white rot fungi, Trametes versicolor, Pleurotus ostreatus, and Phanerochaete chryosporium, were examined for their ability to produce IAA when incubated with L-tryptophan. The maximum IAA production (473.55 ± 3.32 μg ml−1) was observed upon 18 d of incubation at 37°C using a medium containing 2% (w/v) Jatropha seedcake as substrate, with pH adjusted to 7.0. The IAA produced by P. ostreatus was further confirmed and characterized by thin layer chromatography and Gas Chromatograph-Turbomass Mass Spectrometer. The biological activity of IAA obtained from the culture supernatant of P. ostreatus was determined using wheat coleoptile bioassay.

Hexavalent chromium sorption by biomass of chromium tolerant Pythium sp.

The removal of Cr(VI) from aqueous solutions by live and pretreated fungal biomass of Pythium sp was investigated in a batch mode. The influence of biomass dose, solution pH, initial metal ion concentration, temperature and pretreatment of biomass on biosorption efficiency was studied. The acid pretreated biomass adsorbed 1.7 times more hexavalent chromium in comparison to untreated biomass. The chromium removal rate increased with decrease in pH and increase in Cr(VI) concentration, biomass dose and temperature. The adsorption data was described well by Freundlich isotherm model. Evaluation of biosorption mechanism using infrared spectroscopy showed the involvement of positively charged amino groups in Cr(VI) biosorption. The biosorption of Cr(VI) by Pythium sp. followed second order kinetics, the biosorption process was found to be spontaneous and endothermic with high affinity of biomass for Cr(VI). (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Ester synthesis using Candida rugosa lipase immobilized on magnetic nanoparticles

Abstract Magnetic nanoparticles were synthesized by co-precipitation under hydrothermal conditions. The average diameter of the magnetic nanoparticles was found to be in the range of 15 ± 5 nm with an average surface area of 112.15 m2 g−1. Immobilization of lipase on magnetite nanoparticles was confirmed by FTIR, differential scanning calorimetry and thermal gravimetric analysis. The activation energy of the free enzyme was 1.9-fold higher than that of the immobilized lipase for hydrolytic reactions. Additionally, the lower KM and higher Vmax values of the immobilized enzyme for hydrolysis of 4-nitrophenyl palmitate indicated an increased efficiency of the immobilized lipase. The immobilized lipase exhibited higher esterification efficiency compared with free lipase for synthesis of ethyl isovalerate. It also exhibited fairly good reusability, with about 8.5% reduction in esterification efficiency for ethyl isovalerate synthesis over ten cycles of reuse.

Identification and characterization of novel surfactins produced by fungal antagonist Bacillus amyloliquefaciens 6B

The broad‐spectrum fungal antagonist, Bacillus amyloliquefaciens 6B (BA6B), isolated from the Jakhao coast of Kutch, India, was investigated for its antifungal metabolites using mass spectrometry. The cyclic lipopeptides harvested from the cell‐free fermentation broth of BA6B by acid precipitation and subsequently dissolved in methanol were subjected to liquid chromatography coupled with electrospray ionization mass spectrometry (LC–ESI–MS/MS) for their identification and sequence determination. The 26 types of surfactin variants were identified from the methanolic extract by LC–ESI–MS/MS analysis. Among 26 surfactin species, several new cyclic as well as acyclic surfactin variants based on the variation in the β‐hydroxy fatty acid (β‐OH FA) chain length and/or in amino acid positions 4, 5, 6, and 7 were identified. The mass spectrometric analysis of crude extract also enabled the identification of 11 unique molecular mass ions with minimum two or maximum four types of isobaric peptide variants.

Transformation of textile dyes by white-rot fungus Trametes versicolor.

We have investigated transformation of eight industrial dyes by a whiterot fungus, Trametes versicolor. The fungus was found to decolorize Reactive Golden Yellow R, Procion Red, Reactive Violet 5, Reactive Blue 28, and Ponceau Red 4R at an initial dye concentration of 80 ppm within 72 h of incubation, whereas it took 5 d to completely decolorize Reactive Black 5 (40 ppm). However, it did not significantly decolorize Reactive Red 152 and Novatic Blue BC S/D. During decolorization in liquid medium, laccase and manganese-independent peroxidase (MiP) activities were detected in culture filtrate of T. versicolor. Dye-decolorizing activity of the culture was found to be associated with H2O2-dependent activity of the culture filtrate. Furthermore, dye-decolorizing activity of the culture filtrate was not influenced by Mn2+ or veratryl alcohol, thus suggesting a role of extracellular MiP in decolorization of synthetic dyes by T. versicolor.

Toxicity analysis of azo Red BS and Methyl Red dye solutions on earthworm (Pheretima phosthuma), micro-organisms, and plants

Abstract Azo dyes containing effluent from various textile industries adversely affects water resources, soil fertility, aquatic organisms as well as animals. Pure cultures of bacteria or bacterial consortia have been successfully applied for the biodegradation of toxic dye effluents. In this view, toxicity analysis of Red BS and Methyl Red dyes and their biologically decolorized solutions were studied on earthworm (Pheretima phosthuma), plants, and micro-organisms. Different types of morphological symptoms were observed upon exposure of dye solutions on earthworm. Mortality rate in terms of LD50 value was determined for both the dye solutions. The LD50 of untreated Red BS and Methyl Red dye solution was 120.22 and 218.77 mg l−l, respectively. Alteration in the protein content was observed in various organs, head, clitella, and abdomen of earthworms on exposure of dye solutions and the presence of proteins under stress condition was studied using Sodium dodecyl sulphate-Polyacrylamide Gel Electrophoresis....

Eco-Friendly Phyto-Synthesis of Silver Nanoparticles Using Jatropha Seedcake Extract

Phytosynthesis of metal nanoparticles is gaining importance due to their biocompatibility, low toxicity and eco-friendly nature. In the present study, an aqueous extract of Jatropha seedcake (JSC) is assessed as a reducing and stabilizing agent for the synthesis of silver nanoparticles (AgNPs). The maximum reduction of silver ions occur when 1 mM AgNO3 solution is treated with 0.1 volume fraction of JSC extract in boiling water bath for 10 min. The synthesis of AgNPs is monitored by the excitation of surface plasmon resonance using UV-vis spectrophotometry. The AgNPs are found to be mono-dispersed, spherical with average particle size of 10.48 ± 74 nm when analyzed by transmission electron microscopy. The selected area electron diffraction (SAED) ring pattern indicated the polycrystalline nature of the AgNPs. The x-ray diffraction data further confirm the presence of characteristic (111), (200), (220), (311) and (222) diffraction planes of face centered cubic structure, and the calculated lattice parameter comes out to be 4.083 A. FTIR analysis reveals the involvement of proteins and phenols in reduction and stabilization of nanoparticles. The synthesized AgNPs have significant antibacterial action on both the Gram positive and negative bacteria.