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Abstract
Anthropogenic nitrate contamination is a serious problem in many natural environments. Nitrate removal by microbial action is dependent on molybdenum (Mo), which is required by nitrate reductase in denitrification and dissimilatory nitrate reduction to ammonium. The contaminated area at Oak Ridge Reservation (ORR) is an extreme acidic environment contaminated by nitrate, various metals, etc. In this study, it was demonstrated that Fe and Al mineral formation that occurs as the pH of acidic synthetic groundwater is increased, decreases soluble Mo to low picomolar concentrations, a process proposed to mimic environmental diffusion of acidic contaminated groundwater. Analysis of ORR sediments revealed recalcitrant Mo in the contaminated core that co-occurred with Fe and Al, consistent with Mo scavenging by Fe/Al precipitates. ORR strain Bacillus strain EB106-08-02-XG196 isolated from contaminated sediment is more tolerant to multiple metals (Cd, Ni, Cu, Co, Mn and U) but also exhibits more robust growth compared to control strains under low molybdate concentrations (< 1 nM), including Pseudomonas fluorescens N2E2 from a pristine ORR environment. Molybdate is taken up by the molybdate binding protein, ModA, of the molybdate ATP-binding cassette transporter. ModA of XG196 is phylogenetically distinct from those of other characterized ModA proteins. Isothermal titration calorimetry analysis showed XG196 ModA molybdate binding constant (KD) is 2.2 nM, about one order of magnitude lower than those of P. fluorescens N2E2 (27.0 nM) and E. coli K12 (25.0 nM). XG196 ModA also showed higher affinity for molybdate than for tungstate (KD 11 nM), whereas the ModA proteins from P. fluorescens N2E2 and E. coli K12 ModA had similar affinities for the two oxyanions. This high molybdate affinity coupled with resistance to multiple metals gives strain XG196 a competitive advantage in Mo-limited environments contaminated with high concentrations of metals and nitrate, as found at ORR. Nitrous oxide (N2O), a greenhouse gas, can be produced in ORR by nitrification, denitrification, chemodenitrification. Effects of ORR environment-related metals (Mo, Cu, Cd, Co, Ni, U and metal mix) on N2O emission were demonstrated by ORR strains Rhodanobacter R12 and Acidovorax 3H11 in this study.