Denitrification in the Uranium and Nitrate-Contaminated Terrestrial Subsurface

Nitrate (NO3-) and uranium (U) are priority co-contaminants at U.S. Department of Energy (DOE) managed nuclear legacy waste sites, where nitric acid was extensively used to process uranium waste. This combination of a low pH and mixed metal contamination in a subsurface environment is also representative of legacy nuclear waste sites worldwide. The subsurface at DOE's Oak Ridge Integrated Field Scale Research Challenge (OR-IFRC) site is heavily contaminated with NO3-, radionuclides, heavy metals, and halogenated organics. NO3- concentrations in the near source zone (adjacent to the former S-3 ponds) reaches extraordinarily high concentrations (in the range of 10-1000 mM). Extensive research and field scale experiments have focused on ways of removing nitrate and recognize microbially-mediated denitrification as the most significant process in bioremediation and natural attenuation strategies. However, high levels of contamination decreases diversity of cultivable and non-cultivable bacterial populations in OR-IFRC groundwater, and low pH can inhibit denitrification activity. Denitrification is a microbially mediated dissimilatory reduction of nitrate to produce gaseous end products (N2O, N2). Denitrification is mediated by a group of facultative anaerobes including bacteria, fungi and archaea which display a wide range in phylogenetic affiliation and metabolic capabilities. Though nitrate respiring microorganisms have been studied extensively in soils and aquatic environments, the mechanisms controlling in situ metabolism of NO3- reduction remain poorly understood in terrestrial aquifers. The relationships between environmental factors (e.g. geochemistry, contaminant, pH), denitrifying community composition, and denitrification rates are intertwined and complex. Hence, the main objective of this dissertation was characterization of the microbial community mediating denitrification and understand their mechanisms and controls in a radionuclide contaminated terrestrial subsurface. The objectives of Chapter 1 were to extensively characterize microbial diversity and composition in acidic to circumneutral subsurface groundwater samples (pH 3.1-7.1) using a polyphasic approach. Multivariate analyses with geochemical and contaminant variables, and microbial community indices, showed the groundwater pH had the strongest effect of any variable on these communities. Our pyrosequencing survey of microbial diversity across the watershed has provided an initial insight into the large-scale distribution patterns of microbes in this unique environment, with greater variability in physical and geochemical attributes. The community was strongly dominated (>80%) by Proteobacteria, most of which fell into the Gamma-, Beta- and Alpha-Proteobacteria, and community composition was driven primarily by differences in diversity of the proteobacteria. This study also confirms that Gammaproteobacteria as the most dominant taxa with correlation to low pH, with Rhodanobacter sp. as the predominant genus. Furthermore, the data indicates that: (1) the diversity of microbial communities, more specifically that of Gammaproteobacteria is affected by the measured geochemical variables more notably pH, NO3-, N2 and TOC; (2) the diversity of Alphaproteobacteria and Deltaproteobacteria were higher in low contaminant wells; (3) pH appeared to be a strong predictor of relative lineage abundance with samples with low pH levels (pH <4.5) clustering separately from those with moderate pH values and (4) bacterial assemblages identified included genera related to known nitrate, U(VI), sulfate and Fe(III) reducers and fermenters. Bacterial SSU rRNA gene copy numbers did not change significantly between circumneutral pH groundwater samples and low pH samples. In Chapter 2, we report for the first time fungal communities characterized in a uranium and nitrate contaminated subsurface environment and also the potential contribution of fungi to contaminant transformation (nitrate attenuation). The abundance, distribution, and diversity of fungi in subsurface groundwater samples were determined using quantitative and semi quantitative molecular techniques, including quantitative PCR of eukaryotic small-subunit rRNA genes and pyrosequencing of fungal internal transcribed spacer (ITS) regions. Potential bacterial and fungal denitrification was assessed in sediment-groundwater slurries amended with antimicrobial compounds and in fungal pure cultures isolated from the subsurface. Our results demonstrate that subsurface fungal communities are dominated by members of the phylum Ascomycota, and a pronounced shift in fungal community composition occurs across the groundwater pH gradient at the field site, with lower diversity observed under acidic (pH<4.5) conditions. Fungal isolates recovered from subsurface sediments, including cultures of the genus Coniochaeta, which were detected in abundance in pyrosequence libraries of site groundwater samples, were shown to reduce nitrate to nitrous oxide. Denitrifying fungal isolates recovered from the site were classified and found to be distributed broadly within the phylum Ascomycota and within a single genus of the Basidiomycota. Potential denitrification rate assays with sediment-groundwater slurries showed the potential for subsurface fungi to reduce nitrate to nitrous oxide under in situ acidic pH conditions. A concerted field-scale effort was undertaken to test the hypothesis that the microbial denitrification is stimulated by pH adjustment due to the alleviation of low pH stress and/or metal toxicity at the OR-IFRC site (Chapter 3). Sediment and groundwater samples were collected over a year, and microbial enumeration by DAPI counts and MPNs, SSU rRNA gene amplicon pyrosequencing, potential metabolic rate measurements for denitrification and oxygen consumption and geochemical analysis were used to evaluate the shifts in community composition as a function of pH manipulation. The potential denitrification rates measured for contaminated sediments prior to pH manipulation ranged from 0.39 nmol gdry wt-1 day-1 to 8.70 nmol N2O-N gdry wt-1 day-1; and denitrification optima was observed at pH 5.5 with maximum potential rates of 156.9 nmol N2O-N gwet wt-1 day-1. Addition of base resulted in an increase of groundwater pH from 3.5 to 5.5 in the injection well (FW128). However, little or no change in pH was observed in down-gradient wells. Similar trends were observed in total bacterial and denitrifier counts such that total bacterial numbers decreased with change in pH and over time. Potential rates were variable but generally decreased with time in those wells where the pH substantially increased after base addition. Shifts in community composition were observed, and addition of base resulted in a strong selection of a limited number of microbial groups, predominantly of phylum Proteobacteria. The results from this study demonstrate that the denitrifying community is sensitive to perturbation and responds slowly to pH elevation. A relatively rapid pH increase may act as a stressor that inhibits microbial activity over the short term of this study.