Abstract
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Abstract
Bacterial community within the arsenic (As) contaminated groundwater of West Bengal, India was investigated using both culture -independent and -dependent approaches to elucidate microbial role in As mobilization. Analysis of 16S rRNA gene from groundwater metagenome revealed that the bacterial community was mainly composed of α-Proteobacteria (56%) and Firmicutes (29%), along with β-Proteobacteria, Verrucomicrobia, Sphingobacteria and unclassified members as minor groups.
Denaturing Gradient Gel Electrophoresis (DGGE) analysis of metagenome derived 16S rRNA gene showed a stable community composition over a period of three years. In culture dependent approach, 64 bacterial strains were isolated, identified and characterized to investigate their metabolic potential with respect to interaction with As.
16S rRNA gene analysis revealed that arsenite (As3+) oxidizing bacterial isolates were related to genera Achromobacter, Alcaligenes, Hydrogenophaga and Pseudoxanthomonas while arsenate (As5+) reducing strains showed their affiliation with genera Agrobacterium, Brevundimonas, Ochrobactrum and Rhizobium. More than 66%
and 34% isolates were able to produce siderophore and phosphatase, respectively. Among these bacteria, 10 strains were selected for detail metabolic and molecular characterization. Along with As-resistance and -transformation (oxidation/reduction) abilities, these strains were found to be endowed with wide respiratory diversity in terms of utilizing varieties of electron -donors and -acceptors (including arsenic) under aerobic and anaerobic conditions, respectively. Genes involved in As -transformation [As3+
oxidase (aoxB) and As5+ reductase (arsC)] and -resistance [efflux pumps, arsB, acr3(1) and acr3(2)] have been detected within these strains. A detail characterization of As3+
oxidation process by As3+ oxidizing strain Achromobacter KAs3-5 was carried out. A prominent role of As5+ reducing bacteria (possessing cytosolic As5+ reductase) over As3+
oxidizing organism was noted. Microcosm experiments with As transforming strains (Ochrobactrum sp. and Rhizobium sp.) showed release of considerable As into aqueous phase from its sediment bound form. Total groundwater bacterial community also showed As release in to aqueous phase when incubated with the same As bearing sediments.
Addition of carbon source accelerated the As release process. During real time PCR based expression study, concomitant up regulation of arsC, arsB, acr3p, siderophore receptor (tonB), nitrate reductase (ntrf) and sulfate reductase (dsr) genes indicated their possible role in release of sediment bound As. Overall analysis from this study enlightens the involvement of siderophore and cytosolic As5+ reductase based process in mobilization of As into groundwater of Bengal Delta Plain (BDP).
Keywords: Arsenic; bacteria; As3+ oxidation; As5+ reduction; As mobilization;
microcosm; real-time PCR; aoxB; arsC; arsB; acr3; tonB.
