Acid mine drainage (AMD) is a major issue for the mining industry and a global environmental concern. It is facilitated heavily by microbially catalyzed sulfur oxidation/ disproportionation reactions involving reduced sulfide components in mine wastes that result in the release of harmful acidity and metals. The microbial processes catalyzing sulfide mineral leaching of mine waste rock resulting in AMD have been previously studied. However, the initiation of AMD processes in mining wastewaters has not been well studied. Post extraction, highly contaminated, mining wastewater is stored in retention (tailings) impoundments on site and treated to prevent impacts such as toxicity, contamination, and acidity prior to discharge to the receiving environment. Tailings, the waste stream generated through extraction of sulfide ores contains a variety of aqueous sulfur oxidation intermediate compounds (SOIs), such as polysulfides, elemental sulfur, and sulfur oxyanions, of the form SxOy2- termed “thiosalts” by the industry, which can also generate AMD. These wastewaters typically initially start off with circumneutral pH, but if thiosalts are present in high enough concentrations, microbial processes can cause net-acid generation leading to AMD. The microbial ecology and functionality of endemic tailings impoundment microbial assemblages in the “pre-net acid generating phase” (PNAG) of circumneutral mine wastewaters, as well as the associated sulfur species and reactions are not well understood. Thus, early-stage indicators that would offer mines proactive monitoring tools for improved tailings impoundment management are currently lacking. In collaboration with our mining industry partners, who provided tailings impoundment water samples, this dissertation tackles these limitations. This dissertation aimed to identify important microbes and the SOI important to the initiation of AMD in PNAG tailings impoundment wastewaters and to determine potential markers (microbial and/or geochemical) associated with these initiating AMD processes that would inform the development of monitoring tools in mine water management. The objectives of this doctoral research were to constrain the S biogeochemistry of the PNAG phase by characterizing both expression levels of sulfur oxidation genes and sulfur speciation under experimental conditions designed to assess the roles of microbial community, SOI geochemistry and pH. Specifically three well constrained laboratory experiments determined: (1) gene expression by a pure culture of Acidithiobacillus thiooxidans and sulfur speciation with either thiosulfate or elemental sulfur added as the starting SOI substrate (Chapter 3); (2) parent mining wastewater communities and associated sulfur oxidizing bacteria (SoxB) enrichments collected seasonally at two mines and grown at either pH 7-5 or pH 5-3 genetically (16S amplicon) (Chapter 4); and (3) geochemical sulfur pathways of three unique mine wastewater SoxB enrichment communities for six distinct simulated mine wastewater thiosulfate and/or tetrathionate treatments (Chapter 5). Results presented in Chapter 3 expand the understanding of the reactions and enzymes involved in S0 and S2O32- metabolism by a pure strain sulfur oxidizing bacteria A. thiooxidans ATCC 19377 by developing the first models integrating gene expression, solution sulfur speciation, electron microscopy and spectroscopy. These novel results reveal that A. thiooxidans S2O32- metabolism involves the conversion of S2O32- to SO42-, S0 and S4O62-, mediated by the sulfur oxidase complex (Sox), tetrathionate hydrolase (TetH), sulfide quinone reductase (Sqr) and heterodisulfate reductase (Hdr) proteins. These same proteins, with the addition of rhodanese (Rhd), were identified to convert S0 to SO32-, S2O32- and polythionates in the A. thiooxidans S0 metabolism model. The results of this chapter advance understanding by revealing (1) the important role specifically of TetH in S2O32- metabolism; (2) Hdr proteins, rather than Sdo proteins, are likely associated with S0 oxidation; (3) that formation of intracellular S2O32- is a critical step in S0 metabolism, and (4) that recycling of internally generated SO32- occurs, through comproportionating reactions that result in S2O32-. Results summarized in Chapter 4, identify that pH is the most important factor influencing which sulfur oxidizing bacteria (SoxB) occur irrespective of total S concentration of SOI substrate provided in enrichment experiments for two mines of different parent wastewater geochemistry. Mine 1 exhibited a lower total S and reactive soluble sulfur compounds (oxidation state < +VI) concentrations, and greater parent wastewater microbial community diversity with more unique sequences relative to Mine 2. All experimental SoxB enrichment experiment microbial communities evidenced a shift in dominance from primarily Alphaproteobacteria (28% - 77%) at circumneutral pH to Gammaproteobacteria (>80%) under moderately acidic pH values. A further pH dependent shift was observed at the genus level, from Halothiobacillus spp. dominating the circumneutral pH SoxB enrichments to Thiomonas spp. dominating the mildly acidic SoxB enrichments. These results provide some of the first putative biological indicators to improve prediction and management of sulfur processes and AMD onset within mining wastewaters. Chapter 5 results importantly assess the influences of SOI chemistry and SoxB consortia identity on SOI cycling. Results identify that SOI substrate, whether S2O32- , S4O62-, or S2O32- + S4O62-, was a more important determinant of microbial sulfur outcomes than the relative abundances of Halothiobacillus spp. and Thiomonas spp.. Further, three pH dependent phases of microbial sulfur processing were identified: Phase 1, pH > 5, dis/comproportionation reactions and acid consuming reactions were prevalent alongside oxidation, with S2O32- identified as an important indicator; Phase 2, pH 5 – 3, further dis/comproportionation occurred, with S0 emerging as an important indicator signalling the progression of the system towards net acid generation with S4O62- or S2O32- as the major S species present and; Phase 3, pH < 3, i.e. full AMD conditions, with dominant oxidation reactions resulting in SO42- and acid generation. Collectively, these results identify specific SOI species important at different pH stages in AMD initiation and development across different microbial SoxB, providing new indicators that may serve as signals associated with predictive tool development. The integration of the novel results of this thesis revealed some of the first biologically informed possible indicators (bacterial and geochemical) that will enable improved management through proactive monitoring tools for AMD initiation in mining wastewaters. / Dissertation / Doctor of Science (PhD)

As oil production increases in Alberta’s Athabasca Oil Sands Region (AOSR), optimization of tailings management processes will be integral to the successful reclamation of tailings-based environments. Syncrude Canada Ltd. has established an innovative dry-storage method for their wastes known as composite tailings (CT) that supports mine closure objectives by providing a base for terrestrial reclamation landscapes. Syncrude’s Sandhill Reclamation Fen is the first instrumented research wetland of its kind to be developed in the AOSR and it overlays a sand-capped composite tailings deposit in a retired open-pit mine site. This stratified sulfur-rich environment is highly anthropogenically altered and consists of three distinct zones: a constructed wetland, a 10m layer of sand, and 40m of CT. As oil sands tailings systems are becoming globally significant sulfur reservoirs due to their size, sulfur content, and diverse microbial communities, understanding the mechanisms behind H2S generation in novel tailings structures will help inform our understanding of sulfur-rich environments. This study is the first to characterize the sulfur biogeochemistry in each zone of the Sandhill Reclamation Fen deposit in an effort to establish the potential for microbial sulfur cycling and explore the mechanisms controlling H2S generation. Porewater ΣH2S(aq) was detected at all depths, increasing with depth from the surface of the wetland (<1.1 μM) and peaking in the sand cap (549 μM). Across all sampling trips, ΣH2S(aq) concentrations were consistently highest in the sand cap, with sampling-associated H2S gas concentrations in the wells reaching 104-180 ppm. Abundance of dissolved sulfate (0.14-6.97 mM) did not correlate to the distribution of ΣH2S, and dissolved organic carbon (21.47-127.72 mg/L) only positively correlated with the observed maxima of ΣH2S in the sand-cap. Identical sodium and chloride distributions in the sand and CT supported the model of upward migration of CT-derived porewater and fines into the sand cap. Functional metabolic enrichments established the ability of endemic microbial communities from all depths of the deposit to oxidize and reduce sulfur. Experimental microcosms demonstrated 1) the dependence of ΣH2S generation on the presence of fine particles; 2) stimulation of endemic microbial sulfur reduction through amendment with labile carbon and 3) increased generation of ΣH2S in the presence of thiosulfate over sulfate. Field and experimental results indicated that the bioaccessibility of recalcitrant organic carbon in the deposit likely controls rates of ΣH2S generation at depth. While the mechanisms relating CT-derived fines to ΣH2S in the sand cap are still unconstrained, the sand layer is clearly a bioreactive mixing-zone supporting optimal conditions for ΣH2S accumulation. These findings inform our understanding of biogeochemical sulfur cycling in novel oil sands reclamation deposits and will advise on-going optimization of tailings-based landscape management practices. / Thesis / Master of Science (MSc)

This thesis presents a multi-proxy paleoenvironmental reconstruction from the sedimentary archives of Lilandsvatnet, a small arctic lake on Vestvågøy, in the Lofoten Islands, Norway. Lofoten has a rich history of human settlements existing throughout the Holocene. The catchment of Lilandsvatnet was the location of a prominent Viking chieftain farm that existed throughout the Iron Age, and the sedimentary archive contains a strong signal of prehistoric and historic human settlements and land-use practices. Paleoenvironmental reconstructions in this thesis show evidence for Holocene environmental variability in response to both natural and anthropogenic forcing. Cryptotephra deposits from Icelandic eruptions further contrain sediment chronology in the study, allowing reconstructions of subtle changes in the landscape with excellent chronological control during the late Holocene period of settlement. Additionally, I attempt to improve existing methods for crypto-tephrochronology through the development of new techniques.

Natural ecosystems are strongly affected by changes in resource supply (bottom-up forces) and by changes in upper trophic levels (top-down forces). The extent to which these processes impact a system depends largely on the responses of organisms at middle trophic levels. In seagrass beds, a group of mid-level consumers known as mesograzers form a critical link in the chain of impact, connecting seagrass and epiphytic algae with predatory fishes and crustaceans. I observed dramatic seasonal and interannual changes in mesograzer abundance and species composition in eelgrass (Zostera marina) beds of lower Chesapeake Bay, Virginia, and endeavored to explain the top-down and bottom-up causes and consequences of those changes with field studies and controlled experiments. A field cage experiment showed that grazing, predation and nutrient enrichment all had strong effects on the eelgrass community, but that the effects of each factor varied for different community components (Chapter 1). A second experiment delved deeper into the predation dynamic by manipulating the diversity of both predators and mesograzers in macroalgal mesocosms. Increasing predator diversity increased the strength of predation, but increasing mesograzer diversity conferred resistance to some types of predation (Chapter 2). to assess the influence of top-down and bottom-up forces in a more natural context, I analyzed the long-term changes in biotic and abiotic components of an eelgrass bed at the Goodwin Islands National Estuarine Research Reserve. I found that abiotic processes had strong effects on both consumer and resource abundance, and could therefore initiate either top-down or bottom-up control of eelgrass community structure (Chapter 3). to examine this top-down and bottom-up control in more detail I explicitly compared the ecological relationships seen in the field to those observed in mesocosm experiments. Mesocosm experiments tended to find a greater influence of top-down effects and a lesser influence of bottom-up effects, relative to field observations (Chapter 4). Finally, I took a snapshot of the eelgrass food web itself by examining the gut contents and stable carbon and nitrogen isotopic ratios of predators, mesograzers, and plants. I found that direct grazing on eelgrass does occur, but that microalgae and detritus provide the main trophic support for the epifaunal community (Chapter 5). Overall, my results suggest that both top-down and bottom-up forces control eelgrass community structure via mesograzers, but that top-down control in the field is more subtle and more intimately tied with bottom-up control than has been indicated by some manipulative experiments.

Lakes are a significant source of the powerful greenhouse gas methane (CH4) globally. Methaneis produced through microbial processes in anoxic sediments. Methane emission from lakes ishighly variable in space and time. Consequently, is it difficult to predict the methane production rate and at present time it cannot be predicted from sediment characteristics. Therefore, methane production in the sediment of contrasting Swedish lakes was investigated, in order to find out if methane production rate can be related to sediment characteristics, and if a predictive model that recently was developed for Brazilian reservoirs is applicable to Swedish lakes. For this, sediment cores were collected from six lakes, differing in their sediment characteristics and geographical position as well as one river. The sediment cores were sliced into one centimetre thick layers. The different layers were incubated and methane production rate was measured. The sediment layers were also analysed for water content, median grain size, total nitrogen and carbon content as well as age. The influence of sediment age and C:N ratio as predictors for methane production were tested with a mixed linear model and a non-linear model. Both models showed that age had a significant effect on methane production rate (p &lt; 0.001). The C:N ratio also had a statistically significant effect on methane production rate only shown with the non-linear model, however this effect was weak. Applying the recently published predictive model for methane production rate in Brazilian reservoir sediments to this data from the Swedish lakes, provided a good prediction of methane production rate in the nutrient-rich Swedish lakes, however it overestimated the methane production rate of the humic-rich boreal lakes and sediment older than 50 years. In summary, a model using age as predicting factor was developed fitting all the studied Swedish lakes. In addition, the predictive model developed in Brazilian reservoirs for the methane production rate was valid only for the studied nutrient-rich Swedish lakes and the studied oligotrophic Swedish lakes.

Forest fragmentation is a ubiquitous consequence of anthropogenic land-use change, yet its effects on ecosystem processes and biogenic carbon (C) cycling remain unclear, especially belowground. Forest edges, or the boundary of forest and non-forest land cover, experience altered environmental conditions that affect soil biogeochemical cycling and microbial communities. Urbanization can further alter forest soil dynamics and may interact with perturbations at the forest edge in complex, nonlinear ways. Though soils comprise over 40 % of the global forest C sink, the net effects of interacting global change drivers (e.g., rising temperatures, fragmentation, urbanization) are largely unknown, introducing large uncertainties into estimates of soil C fluxes and our understanding of soil ecology. I co-designed and implemented an observational field campaign of forest edges along an urbanization gradient in Massachusetts to characterize soil C cycling and its drivers from the forest edge to the interior. I use field measurements of soil temperature, moisture and C efflux as CO2, or soil respiration, to find diverging trends in soil C losses at urban and rural forest edges. I find that urban soil respiration rates are less sensitive to rising temperatures than rural soils and that urban edges are even less sensitive than their interior counterparts. I then perform a holistic characterization of soil properties and microbial activity to explore the effects of multiple, simultaneous environmental perturbations on forest edge soils. I report that soil C content does not reflect diverging trends in soil C efflux between rural and urban sites and, instead, is generally lower at the forest edge, suggesting a decoupling between edge soil C pools and fluxes. I also report that soil properties often mediated by human activity, such as pH, temperature, and trace element concentrations, broadly predict soil C dynamics from edge to interior across the urbanization gradient. Finally, I conduct a meta-analysis of published studies on forest edge soil C cycling and its drivers and interpret the findings through a lens of broader global change. I demonstrate that soil conditions converge at the forest edge across the globe, where soils are hotter, drier, and less acidic than the forest interior. I find limited investigation of soil C fluxes and substantial variability in edge soil C stocks, and I conclude that forest edges are not direct analogs for global change experiments. My dissertation demonstrates that soil C cycling is significantly altered by both forest fragmentation and urbanization. I highlight the need for further study both in situ at the forest edge and through multi-factor manipulation experiments to improve our understanding of an increasingly fragmented and urbanized forest landscape.

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