A bio-relevant supramolecular Co(ii)-complex for selective fluorescence sensing of µM range inorganic As(iii) in aqueous medium and its intracellular tracking in bacterial systems.

A novel Co(ii)-complex {i.e. [Co(n-BuM)(DPA)(H2O)2]·H2O} [n-BuMH2 = n-butylmalonic acid and DPA = 2,2'-dipyridylamine] was synthesized. The supramolecular feature, i.e. a metal coordinated and free guest water mediated hydrogen-bonding interaction assisted supramolecular ππ assembly, has been observed in the crystal structure of the Co(ii)-complex in the solid state. The role of different water molecules (metal coordinated and free guest water molecules) had also been scrutinized via theoretical studies. The fluorescent nature of the aqueous solution of the Co(ii)-complex has been utilized for selective µM range toxic inorganic As(iii)-sensing in aqueous medium. The Co(ii)-probe is very specific towards toxic As(OH)3 even in the presence of several ions and other arsenic sources like inorganic As(v)-oxoanions and organic arsenic species like cacodylic acid. The bio-relevant nature of the fluorescent probe of the Co(ii)-complex has also been examined. The luminous Co(ii)-probe has been employed for the intracellular tracking of As(iii) in bacterial systems including As(iii)-resistant bacteria Bacillus aryabhattai and As(iii) non-resistant bacteria Bacillus subtilis.

Molecular Study of Indigenous Bacterial Community Composition on Exposure to Soil Arsenic Concentration Gradient.

Community structure of bacteria present in arsenic contaminated agricultural soil was studied with qPCR (quantitative PCR) and DGGE (Denaturing Gradient Gel Electrophoresis) as an indicator of extreme stresses. Copy number of six common bacterial taxa (Acidobacteria, Actinobacteria, α-, ß- and γ-Proteobacteria, Firmicutes) was calculated using group specific primers of 16S rDNA. It revealed that soil contaminated with low concentration of arsenic was dominated by both Actinobacteria and Proteobacteria but a shift towards Proteobacteria was observed with increasing arsenic concentration, and number of Actinobacteria eventually decreases. PCA (Principle Component Analysis) plot of bacterial community composition indicated a distinct resemblance among high arsenic content samples, while low arsenic content samples remained separated from others. Cluster analysis of soil parameters identifies three clusters, each of them was related to the arsenic content. Further, cluster analysis of 16S rDNA based DGGE fingerprint markedly distributed the soil bacterial populations into low (< 10 ppm) and high (> 10 ppm) arsenic content subgroups. Following analysis of diversity indices shows significant variation in bacterial community structure. MDS (Multi Dimensional Scaling) plot revealed distinction in the distribution of each sample denoting variation in bacterial diversity. Phylogenetic sequence analysis of fragments excised from DGGE gel revealed the presence of γ-Proteobacteria group across the study sites. Collectively, our experiments indicated that gradient of arsenic contamination affected the shape of the soil bacterial population by significant structural shift.

A simple one pot purification of bacterial amylase from fermented broth based on affinity toward starch-functionalized magnetic nanoparticle.

Surface-functionalized adsorbant particles in combination with magnetic separation techniques have received considerable attention in recent years. Selective manipulation on such magnetic nanoparticles permits separation with high affinity in the presence of other suspended solids. Amylase is used extensively in food and allied industries. Purification of amylase from bacterial sources is a matter of concern because most of the industrial need for amylase is met by microbial sources. Here we report a simple, cost-effective, one-pot purification technique for bacterial amylase directly from fermented broth of Bacillus megaterium utilizing starch-coated superparamagnetic iron oxide nanoparticles (SPION). SPION was prepared by co-precipitation method and then functionalized by starch coating. The synthesized nanoparticles were characterized by transmission electron microscopy (TEM), a superconducting quantum interference device (SQUID, zeta potential, and ultraviolet-visible (UV-vis) and Fourier-transform infrared (FTIR) spectroscopy. The starch-coated nanoparticles efficiently purified amylase from bacterial fermented broth with 93.22% recovery and 12.57-fold purification. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the molecular mass of the purified amylase was 67 kD, and native gel showed the retention of amylase activity even after purification. Optimum pH and temperature of the purified amylase were 7 and 50°C, respectively, and it was stable over a range of 20°C to 50°C. Hence, an improved one-pot bacterial amylase purification method was developed using starch-coated SPION.

SPION-mediated soil DNA extraction and comparative analysis with conventional and commercial kit-based protocol.

Direct isolation of soil DNA comes as an emerging technology to understand the microbial diversity of a particular environment circumventing the dependency on culturable methods. Soil DNA isolation is tough due to the presence of various organic components present in soil which interfere in extraction procedure. Here, we report a novel direct soil DNA extraction protocol utilizing bare superparamagnetic iron oxide nanoparticles and its comparison with conventional and commercial kit-based soil DNA extraction methods. The quality, quantity and feasibility of the recovered DNA from all the three methods towards various molecular techniques were checked. Our magnetic nanoparticle-based soil DNA extraction successfully yields pure DNA without any RNA or protein contamination as revealed by the nanodrop spectrophotometer and agarose gel electrophoretic study. Different methods of soil DNA extraction were evaluated on the basis of PCR, denaturing gradient gel electrophoresis and real-time PCR. Soil DNA extracted using conventional method fails to carry out critical molecular biology techniques where as magnetic nanoparticle-based soil DNA extraction gave good results which is comparable to commercial kit. This comparative study suggests that protocol described in this report is novel, less time consuming, cost effective with fewer handling steps and yields high quantity, good quality DNA from soil.