The effects of metals on environmental denitrification
Vaccaro, Brian Jeffrey
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Denitrification, microbial reduction of nitrate to dinitrogen, completes the global nitrogen cycle, returning bioavailable nitrogen to the atmospheric reservoir of inert dinitrogen. The work presented focuses on the effects of molybdenum, copper, zinc and aluminum on denitrifying metabolism in Pseudomonas stutzeri strain RCH2 in the context of contaminated groundwater at the Oak Ridge Reservation (ORR). High concentrations of nitrate, aluminum and other contaminants (including copper and zinc) have leached into ORR groundwater from uranium processing waste, however molybdenum concentrations are extremely low. Molybdenum is the catalytic metal used for nitrate reduction, the first step of denitrification, and results are presented on the effects of molybdenum limitation on denitrifying metabolism using the RB-TnSeq genome-wide fitness assay. New or clarified protein functions are proposed: molybdate transport by an uncharacterized transporter, nitric oxide sensing by NnrS, nitrate chemotaxis by a CheR homolog, identification of the functional paralog in several cases, and others. RB-TnSeq was combined with regulon analysis and construction of individual mutant strains to study genes and proteins involved in resistance to toxic concentrations of copper and zinc in strain RCH2. Data is presented suggesting that three proteins (CorB, CorC and Psest_3226) not known to transport zinc and one (an Mrp antiporter subunit) not known to transport copper do function in this capacity in strain RCH2. Functional transporter paralogs are identified, and conclusions are drawn concerning transporter classification. Denitrification targets of copper and zinc toxicity are also assessed. Finally, the potential for aluminum to cause molybdenum limitation in acid contaminated environments such as ORR is explored. In aerobic environments, molybdenum exists as molybdate, which can adsorb to aluminum or ferric iron hydroxide minerals. This phenomenon can explain the particularly low concentrations of molybdenum in contaminated groundwater at ORR. Aluminum- and iron-based molybdenum depletion is demonstrated in the low concentration regime where molybdenum limitation occurs, and aluminum-based molybdenum limitation of P. stutzeri growth is demonstrated. Implications of molybdenum depletion via neutralization of acidic environments, such as acid mine drainage sites, is discussed. Specifically, eutrophication in coastal regions could be exacerbated by extending the lifetime of nitrate in freshwater affected by these processes.