Nitrate-Utilizing Microorganisms Resistant to Multiple Metals from the Heavily Contaminated Oak Ridge Reservation

Contamination of environments with nitrate generated by industrial processes and the use of nitrogen-containing fertilizers is a growing worldwide problem. While nitrate can be removed from contaminated areas by microbial denitrification, nitrate frequently occurs with other contaminants, such as heavy metals, that have the potential to impede the process. Herein nitrate-reducing microorganisms were enriched and isolated from both groundwater and sediments at the Oak Ridge Reservation (ORR) using concentrations of nitrate and metals (Al, Mn, Fe, Co, Ni, Cu, Cd, and U) similar to those observed in a contaminated environment at ORR. Seven new metal-resistant, nitrate-reducing strains were characterized and their distribution across both non-contaminated and contaminated areas at ORR was examined. While the seven strains have varying pH ranges for growth, carbon source preferences and degrees of resistance to individual and combinations of metals, all were able to reduce nitrate at similar rates both in the presence and absence of the mixture of metals found in the contaminated ORR environment. Four strains were indentified in groundwater samples at different ORR locations by exact 16S RNA sequence variant analysis and all four were found in both non-contaminated and contaminated areas. By using environmentally-relevant metal concentrations, we successfully isolated multiple organisms from both ORR non-contaminated and contaminated environments that are capable of reducing nitrate in the presence of extreme mixed-metal contamination. Importance Nitrate contamination is a global issue that affects groundwater quality. In some cases, co-contamination of groundwater with nitrate and mixtures of heavy metals could decrease microbial-mediated nitrate removal thereby increasing the duration of nitrate contamination. Herein, we used metal and nitrate concentrations that are present in a contaminated site at the Oak Ridge Reservation to isolate seven metal-resistant strains. All were able to reduce nitrate in the presence of high concentrations of a mixture of heavy metals. Four of seven strains were located from pristine as well as from contaminated sites at the Oak Ridge Reservation. Further study of these nitrate-reducing strains will uncover mechanisms of resistance to multiple metals that will increase our understanding of the impact of nitrate and metal contamination on groundwater microbial communities.