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. / A Dissertation submitted to the Department of Earth, Ocean, and Atmospheric Sciences in partial fulfillment of the Doctor of Philosophy. / Spring Semester 2016. / April 4, 2016. / Denitrification, low pH, Radionuclinde, Subsurface Bacteria, Subsurface Fungi, Terrestrial Subsurface / Includes bibliographical references. / Jeffrey P. Chanton, Professor Directing Dissertation; Christopher Coutts, University Representative; Marcus Huettel, Committee Member; Amy Baco-Taylor, Committee Member; Christopher W. Schadt, Committee Member; Stefan J. Green, Committee Member; Joel E. Kostka, Committee Member.

Surficial sediments and sediment cores were collected from two distinct depositional regimes of the York River subestuary of Chesapeake Bay to document seasonal inputs, spatial variability, and longer-term (>40 years) fate of total organic carbon (TOC), lipid biomarker compounds and polycyclic aromatic hydrocarbons (PAHs). These regimes included biological mixing in the lower York and episodic mixing at the mid river site. Compounds were selected to represent a range of chemical reactivities, biological and anthropogenic sources, and modes of entry to the environment. The depositional environments were characterized with a suite of analytical tools: x-radiographs, Eh, 210Pb and 137Cs, total organic carbon, total nitrogen, and their stable isotopes. Each compound class was quantified in extractable and "bound" phases. Episodic mixing at the mid-river site resulted in stronger oxidizing conditions that promoted enhanced degradation of labile organic matter (e.g. diatoms) vs. refractory material (e.g. higher plants) in extractable sedimentary phases from sediments <5 yrs old. However, while apparent rate constants for bulk organic matter and total lipid were higher in older sediments (<40 years) under physically mixed conditions, degradation rates of fatty acid and sterol biomarkers were similar at both study sites. PAHs and lipid biomarkers isolated from "bound" phases were better preserved over time than corresponding "extractable" compounds. However, stabilization in the bound phase was not the same among compound classes. Differences in compound class fate were a function of inherent compound class reactivity (fatty acids > sterols and PAHs) rather than source or depositional regime. While compounds in bound phases may be formed over time during organic matter diagenesis, organic compounds did not increase in bound residues over time regardless of depositional regime, suggesting that bound phase compounds are formed within the source organism or very rapidly upon cell death and/or deposition to the sediments. The fate of organic carbon in coastal sediments is dependent upon the source and reactivity of organic carbon, the depositional regime, and its association with the underlying sediment/macromolecular matrix. Models of coastal carbon dynamics that consider these parameters and how they change will yield more accurate forecasts of coastal biogeochemical cycling.

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Understanding the distribution, abundances and metabolic activities of microbial life in the subsurface is fundamental to our understanding of the role microbes play in many areas of inquiry such as terrestrial biogeochemical cycling and the search for extraterrestrial life. The deep terrestrial subsurface is known to harbor microbial life at depths of up to several kilometers where, in some cases, organisms live independently from the photosphere and atmosphere. Ancient fracture fluids trapped within the crystalline basement of the Canadian Precambrian Shield have been shown to be preserved on geologic timescales (millions to billions of years). Significant challenges exist when probing the deep terrestrial subsurface including the low biomass abundance, heterogeneous distribution of biomass, and the potential for matrix effects during sampling and analysis. This Master’s thesis project has two main parts. The first study utilizes phospholipid fatty acid (PLFA) analysis to determine the extent of mineral matrices on the effectiveness of PLFA extraction and analysis from deep terrestrial subsurface samples. This was done by creating a bacterial dilution series of known concentration to inoculate one of two mineral matrices, granite or bentonite. This study revealed the presence of significant influence of mineral matrices on PLFA extraction and demonstrated the unreliability of PLFA-based biomass conversion factors with respect to complex microbial communities. The second study in this thesis combine PLFA analysis with stable carbon isotope analysis to characterize microbial communities associated with fracture fluids with mean residence times of ~1.4 Ga from boreholes located ~2.4km below the surface in Kidd Creek Mine, in Timmins, Ontario. Characterizing communities in subsurface systems has large implications for the search for life on other planets and moons, acting as an analogue environment. Large volumes of water from two boreholes, 12261 and 12299, were passively filtered for 6-12 months to collect microbial biomass. Borehole adjacent biofilms were also collected along with mine service water, which served as a control. All samples had significant biomass associated with them but were distinct in PLFA fingerprint and δ13C – PLFA signatures indicating the presence of three distinct microbial communities living in association with the fracture fluids and gases. These results have implications for the potential existence of ancient deep subsurface communities that have survived geologic time in isolation, in particular with relation to the subsurface of Mars, as well as give us insight into life on the early Earth. / Thesis / Master of Science (MSc)

Between 40 % and 50 % of the Earth’s primary production occurs in marine environments, primarily by phytoplankton. The trace metal micronutrients iron, zinc and cobalt are known to exert a significant biological control on phytoplankton productivity by serving as essential active centres in enzymatic processes such as inorganic carbon, nitrogen and phosphorus acquisition. The distributions and biogeochemistries of iron, zinc and cobalt therefore, have the potential to impact upon the global carbon cycle and hence climate. This research involves investigations into the biogeochemical cycling of iron, zinc and cobalt in the Atlantic Ocean. Iron measurements were conducted during October and November 2009 to determine the distribution and biogeochemistry of iron in the upper water column of the Atlantic Ocean along an Atlantic Meridional Transect (AMT-19). In addition, deck board incubation experiments were performed to establish the role of iron in controlling rates of di-nitrogen (N2) fixation in the North Atlantic. The distribution patterns and biogeochemistries of iron, zinc and cobalt in the South Atlantic at 40° S were determined during the UK GEOTRACES Section GA10 cruises of October 2010 and December 2011 to January 2012. Iron distributions in North Atlantic surface waters were primarily controlled by the transport and deposition of atmospheric dust particles. In the North Atlantic, elevated surface dissolved iron concentrations (0.50 - 1.65 nM) were associated with wet and dry deposition of Saharan dust between 4 and 29° N. To the south of 4° N, surface dissolved iron concentrations were markedly reduced (0.14 nM) indicating that high precipitation rates in the Inter-Tropical Convergence Zone (4 - 10° N) formed a barrier to the large-scale transport of Saharan dust particles, thus iron, to the South Atlantic. Here, the low surface dissolved iron concentrations were balanced by a total dissolvable iron flux out of the surface mixed layer (3.2 µmol m-2 y-1) that was comparable to atmospheric input estimates. Nitrogen fixation rates in the North Atlantic were highest (0.3 – 1.1 nmol L-1 d-1) where surface dissolved iron concentrations were elevated (1.02 nM) and decreased with increasing latitude as iron decreased. Hence, iron variability in the North Atlantic was sufficient to influence nitrogen fixation over a large spatial scale. In the South Atlantic Ocean at 40° S, the vertical and horizontal distributions of dissolved zinc and cobalt showed distinct gradients associated with the water masses present. Zinc concentrations ranged from 15 pM in open ocean surface waters to 8 nM in Antarctic Bottom Waters, whilst cobalt ranged from 2 pM to 80 pM in intermediate waters and was scavenged in deeper waters. Growth limiting mixed layer zinc concentrations resulted from the lack of a direct return path for zinc to the South Atlantic pycnocline with Sub-Antarctic Mode Water. Low zinc in this return path was identified by a linear correlation between zinc and soluble reactive phosphorus that showed a kink at ~ 500 m, much deeper than that observed in other oceanographic regimes. A seasonal study in the Southeast Atlantic revealed that the depletion of zinc over spring-summer periods resulted in an increase in the nutritional importance of cobalt and a shift towards phytoplankton with a cellular preference for cobalt over zinc and/or the ability to co-substitute these two trace metals at the molecular level. These key findings demonstrate the physico-chemical and biological influences that interact to control the distributions and biogeochemistries of iron, zinc and cobalt across diverse oceanographic regimes of the Atlantic Ocean, provide the first examination of zinc and cobalt biogeochemistries along the productive 40° S parallel and highlight the need for additional research in this region.

The flux of carbon in to and out of the atmosphere exerts a fundamental control over the Earth's climate. The oceans contain almost two orders of magnitude more carbon than the atmosphere, and consequently, small fluctuations within the oceanic carbon reservoir can have very significant effects on air-sea CO₂ exchange, and the climate of the planet. Pelagic carbonates represent a major long-term flux of carbon from the surface ocean to deep-sea sediments. Within sediments, the biologically produced carbonates act as a longterm carbon store, but also as chemical recorders of past surface ocean conditions. Counterintuitively, despite the production and sedimentation of carbonate acting as a CO₂ sink, over periods shorter than the mixing-time of the ocean, the pH change associated with calcium carbonate precipitation enriches the surface waters in CO₂ and elevates the equilibrium value of gaseous exchange with the atmosphere. Coccolithophores, ubiquitous marine photosynthetic plankton, produce calcium carbonate plates, coccoliths, which account for around one third of all marine calcium carbonate production. Sedimentary coccoliths therefore represent a valuable repository of surface ocean geochemical data, as well as a very significant carbon-cycle flux. This thesis examines how the mass of calcium carbonate produced by coccolithophores has changed in response to rising levels of atmospheric CO₂. A -40% increase in average coccolith mass over the last 230 years, paralleling anthropogenic CO₂ release, is demonstrated within a high-accumulation rate North Atlantic sediment core. Additionally, a flow-cytometry method is presented, which enables the automatic separation of coccoliths from clay particles in sedimentary samples, representing the first step in a coccolith cleaning procedure, which should ultimately enable down-core measurements of coccolith trace-element/calcium ratios. Complementing this work I describe results from continuous dissolution analysis of cultured coccoliths which allows a first-order evaluation of trace-element partitioning into coccoliths produced by the species Coccoliths pelagicus, and present a conceptual methodology to allow the determination of single-species coccolith chemical data.

Phytoplankton across the majority of the world’s oceans are thought to be limited by the availability of either nitrate or iron (Fe). However, the spatial resolution of experiments confirming this is low. Two thesis chapters present the results of bottle enrichment experiments at high spatial resolution across (i) the South Subtropical Convergence (SSTC) in the South Atlantic, and (ii) the Scotia Sea-Drake Passage sector of the Southern Ocean. These studies have added detail to the boundaries of limiting nutrients in these regions. Patterns of Fast Repetition Rate fluorometry (FRRf) derived parameters, physiological regulation of these parameters including influences of community structure, and the environmental controls driving them are analysed. Given its role as an essential micronutrient, there has been much effort in constraining potential sources of bioavailable Fe to the ocean, with one such source receiving recent interest: erupted ash from volcanoes. Bottle-scale ash-incubation experiments alongside conventional iron additions and laboratory ash-leaching experiments were conducted, the results of which suggest phytoplankton would respond strongly to ash deposition in the High Nitrate, Low Chlorophyll (HNLC) areas of the Southern Ocean. Particularly notable was the evidence these experiments provided for potential (co-)limitation of phytoplankton in these waters by the micronutrient manganese. The first three chapters of this thesis highlight a number of biogeochemical implications of trace metal stress, particularly that of Fe stress. Therefore, the ability to map the oceanographic extent of Fe-stressed regions using remote sensing would represent a particularly useful advance in marine biogeochemistry. Theoretically it could be possible to map Fe stress from space using satellite images of chlorophyll fluorescence, yet there are important uncertainties that need to be addressed before this can be carried out. In particular, a better understanding of the midday non-photochemical quenching driven reductions in chlorophyll fluorescence occurring at the time satellite images are captured is required. Analysis of over 200 non-photochemical quenching experiments collected over three research cruises, has allowed us to explore non-photochemical quenching and its relevance for using sunlight induced chlorophyll fluorescence to assess broad patterns of Fe stress. Our results have confirmed that satellite fluorescence quantum yields have the potential to reveal broad regions of Fe stress, however a dynamic non-photochemical quenching correction derived from our experiments and analysis was necessary to achieve this.

Dry deposition of nitrate, estimated from a box model based on NO$\rm\sb{x}$ emissions and rain chemistry monitoring data over the contiguous 48 states, accounts for about half of the total US NO$\rm\sb{x}$ emissions, a deposition flux twice that of measured wet deposition. Thus, total atmospheric nitrate deposition is roughly three times wet only deposition. Ten subregions of wet only nitrate depositions were delineated by EOF analysis from the entire U.S.A., in which each has a narrow range of annual deposition flux and exhibits unique seasonal variation. The study was based on statistical analysis of chemical concentrations measured for more than 10 years in weekly rainfall samples of the National Atmospheric Deposition Program, NADP, and more than 20 years of river water samples of the U.S. Geological Survey, USGS. NO$\rm\sb{x}$ emissions appear to regulate the annual average total deposition fluxes while in the subregions rainfall characterizes the seasonal and shorter term variations in wet only depositions. Atmospheric wet and dry deposition ("acid rain") appears to be the principal source of nitrogen in twelve northern Florida watersheds that range from Pensacola to Gainesville (Escambia to Alachua Counties). River fluxes of total dissolved nitrogen average close to the atmospheric deposition fluxes of nitrate and ammonium ions. Factor analysis was applied to the data sets to resolve principal components: (1) in atmospheric data, that distinguish air pollution nitrate and sulfate from sea salt sodium and chloride, and (2) in surface water data, that distinguish ground water calcium, magnesium, and silica from meteoric water nitrate and sulfate. River concentration ratios N/P in the watersheds are high, averaging twice the Redfield mole ratio N/P = 16 for aquatic plant nutrients. The results indicate that excess dissolved nitrogen could be / temporarily recycled in the watersheds but not retained, so that it could eventually flow to the coastal zone where N may be a limiting nutrient for marine plants. Hydrologic conditions, which exhibit variations on seasonal and longer time scales, play an important role in the transport of nutrients and other species in the rivers. / Source: Dissertation Abstracts International, Volume: 57-04, Section: B, page: 2448. / Major Professor: John W. Winchester. / Thesis (Ph.D.)--The Florida State University, 1996.