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Our vision is to create a program dedicated to accelerating innovation that improves the quality, efficiency, and resilience of human dominated landscapes, including our cities, farms, and industrial lands. Humans dramatically alter and manipulate the global landscape for food and fiber production, mineral extraction, urban development, waste disposal and many other purposes. Impacts to essential ecosystem functions and values range from local (e.g. mining and land development) to global (e.g. carbon emissions) with a clear need for development of appropriate management systems for their mitigation. By using a systems approach that interfaces environmental scientists and ecologists with relevant disciplines, this proposed signature area within Global Systems Science (GSS) will build upon existing group strengths in soil remediation, water quality, hydrology, urban soils, land reclamation, agroecosystem management, forest ecology, wetland restoration, soil-waste management and integrated modeling across multiple spatial and temporal scales to develop a more holistic approach to landscape management. We will also...

Microorganisms are absolutely critical to myriad aspects of the human existence. As a field of study, microbiology could and should serve a greater role on our campus, as it has key connections with many of the Destination and Strategic Growth Areas. We propose the development of a broad concept area in microbiology that will serve as a nexus, as it is applied to solve critical global challenges related to food, energy, water and health, by bridging across multiple disciplines at Virginia Tech (VT). There is increasing recognition of microbes as a driving force in natural and managed environments, biological processes, and ecological structure. Conversely, the importance of culture and individual behavior in affecting microbial communities has also become apparent....

The development of viable solutions to the global threat of antimicrobial resistance requires a transdisciplinary approach that simultaneously considers the clinical, biological, social, economic, and environmental drivers responsible for this emerging threat. The vision of the Antimicrobial Resistance Mitigation (ARM) group is to build upon and leverage the present strengths of Virginia Tech in ARM research and education using a multifaceted systems approach. Such a framework will empower our group to recognize the interconnectedness and interdependent nature of this threat and enable the delineation, development, and testing of resilient approaches for its mitigation. We seek to develop innovative and sustainable approaches that radically advance detection, characterization, and prevention of antimicrobial resistance emergence and dissemination in human-dominated and natural settings...

Water scarcity is mainly caused by overwhelming human consumption and contamination, from production of water-thirsty meats and vegetables, biofuel crop production, industrial uses, and rapid urbanization. The scale of water scarcity makes it an interconnected global issue and efforts to minimize the gap between water supply and demand are critical...

Plant establishment is a key component of land reclamation and restoration, but ultimately full recovery of other beneficial ecosystem services that were provided prior to disturbance is desired for successful restoration. Many of these ecosystem services, primarily related to nutrient cycling and other biogeochemical processes, are regulated by microorganisms. However, the factors that promote the return of beneficial soil microorganisms – and the ecological functions they perform – to restored ecosystems are not well understood. Our previous work in the Powell River Project has discovered that the type of vegetation used to reforest reclaimed mine soils may not only control the resulting plant community, but also the trajectory of the soil microbial community and the processes they mediate related to important ecosystem services. In this work, we experimentally tested the hypothesis that carbon substrate diversity, provided by plant root exudates, is an important mechanism in shaping soil microbial communities as ecosystems recover. In a controlled greenhouse experiment, we analyzed microbial community structure and diversity of dissolved organic carbon substrates in experimental PRP soil mesocosms planted with one of five plant diversity treatments: chicory, clover, foxtail, rye, or an even mix of all four. Results indicate that plant species has significant effects on soil dissolved organic carbon substrate quantity and quality, as well as soil microbial community structure, with clover and chicory resulting in the most distinct changes. In addition, among the non-leguminous plant species, patterns of carbon substrate and microbial diversity tracked each other, suggesting an important interaction between the two.

This file contains supplementary figures and tables for Wepking et al.

A significant amount of the research to date at the Powell River Project (PRP) has been focused on reforestation, with the assumption that tree growth will ultimately lead to the re-establishment of a fully functioning forest ecosystem. Soil microorganisms are a critical component of this system because they mediate many of the ecosystem services for which forests are valued including carbon sequestration, soil formation, nutrient retention, watershed protection, and groundwater purification. We are characterizing the response of soil microbial communities to land reclamation approaches in the PRP to provide critical information about the restoration of the microbial component of the forest ecosystem. The objectives of this project are threefold: 1) characterize the recovery of soil microorganisms over time; 2) determine if alternate reclamation practices affect microbial diversity and community structure; and 3) compare restored microbial communities to un-mined forest soils to identify potential indicators that ‘healthy’ microbial communities are returning to reclaimed soils. We have identified a variety of reclamation plots within the PRP that represent a range of ages between 5 and 30 years to look at the effects of time. We have also sampled two other sets of to determine effects of reclamation practices: one where soils were amended with biosolids and another that was planted with pines as opposed to the standard hardwood mix. We are using genomic sequencing to fully characterize bacterial and fungal organisms present in soil samples from each plot to determine microbial diversity and community structure. Preliminary results suggest that bacterial communities recover quickly, becoming indistinguishable from communities in undisturbed soils within 10 to 30 years. In addition, certain taxa such as Bacteroidetes, Verrucomicrobia, and Gemmatimonadetes appear to respond to age since reforestation and may contain taxa that can be used to gauge restoration progress.

Nicholas B Norgard, Brian T BadgleyUniversity at Buffalo, School of Pharmacy and Pharmaceutical Sciences, Buffalo, NY, USAAbstract: Eptifibatide is a glycoprotein IIb/IIIa receptor antagonist used to reduce the incidence of ischemic events in patients with acute coronary syndromes and those undergoing percutaneous coronary intervention. A minority of patients given eptifibatide develop acute, profound thrombocytopenia (

Opportunistic premise (i.e., building) plumbing pathogens (OPPPs) and antibiotic resistant bacteria are emerging microbial concerns in drinking water. OPPPs, such as Legionella pneumophila, are the leading cause of drinking water disease in many developed countries. Contributing factors include the relative success in controlling fecal pathogens, the presence of complex building plumbing systems that create habitats for OPPPs, and the relative resistance of OPPPs to disinfectants, and aging populations that are susceptible to infection. Concurrently, drinking water is increasingly being scrutinized as a potential environment that is conducive to horizontal gene transfer of antibiotic resistance genes (ARGs), selection pressure for enhanced survival of resistant bacteria, and a route of transmission of antibiotic resistant pathogens. While maintaining a disinfectant residual is an established approach to controlling OPPPs in premise plumbing, some studies have indicated that co-resistance and cross-resistance to disinfectants can increase the relative abundances of resistant bacteria and ARGs. Thus, there may be trade-offs to controlling both OPPPs and antibiotic resistance in premise plumbing that call for controlled study aimed at optimizing residual disinfection application for this purpose. A critical review of the scientific literature in Chapter 2 revealed that premise plumbing is a biologically and chemically complex environment, in which the choice of pipe material has cascading effects on water chemistry and the corresponding premise plumbing microbiome. This, in turn, has broad implications for the control of OPPPs, which need to be elucidated through controlled experiments in which worst case premise plumbing conditions are held constant (e.g., warm temperature), while other variables are manipulated. Chapter 3 introduces the convectively-mixed pipe reactors (CMPRs) as a novel low-cost, small footprint approach to replicably conduct such experiments. The CMPRs were demonstrated to effectively simulate key chemical and biological phenomena that occur in distal reaches of premise plumbing. In Chapter 4, the CMPRs were leveraged to study the interactive effects of four disinfectants (chlorine, monochloramine, chlorine dioxide, and copper-silver ionization) and three pipe materials (PVC copper, and iron). The CMPRs were inoculated with two antibiotic-resistant OPPPs: Pseudomonas aeruginosa and Acinetobacter baumannii. It was found that pipe-material (PVC or PVC combined with iron or copper) profoundly impacted the water chemistry in a manner that dictated disinfection efficacy. In Chapter 5, we applied shotgun metagenomic shotgun sequencing to evaluate effects of the combination of pipe material and disinfectant type on the wider microbial community, especially their ability to select for or reduce ARGs. In Chapter 6, we used CMPRs and metagenomic sequencing in a study comparing Dutch drinking water practices to our prior testing in an American system. Dutch drinking water is of interest because of lack of historical use of disinfectants was hypothesized to result in a microbial community that is relatively depleted of ARGs or mobile genetic elements, which can enhance spread of ARGs as disinfectants are applied. Generally, it was found that OPPPs required higher doses of disinfectants for inactivation than the general microbial community, sometimes concentrations approaching the regulatory limits in the US (e.g., 4 mg/L of total chlorine). Even successful reductions were modest, typically ~1-log, and failed to eliminate either P. aeruginosa or A. baumannii. Moreover P. aeruginosa, A. baumannii, and non-tuberculous mycobacteria varied substantially in their preference for pipe material and susceptibility to disinfectants. We found that disinfectants tended to increase the relative abundance of OPPPs, ARGs, and mobile genetic elements. Disinfectants were sometimes associated with net increases in levels of these pathogens and genes when applied at low levels (e.g., 0.1 mg/L of monochloramine), which effectively acted to reduce competition from less resistant and non-pathogenic taxa. When a low dose of monochloramine was applied to PVC CMPRs in the US, we estimated from metagenomic sequencing data that this water contained roughly 100,000 cells per milliliter of taxa known to contain pathogenic members. The Dutch drinking water exhibited more diverse microbial communities and lower relative abundances of taxa containing pathogens. ARGs were two times proportionally more abundant in CMPRs operated in the US without disinfectant than in the corresponding CMPRs operated in the Netherlands. The findings of this dissertation can help to optimize the application of in-building disinfectant addition for addressing concerns related both to OPPPs and antibiotic resistance. The studies herein highlight the necessity of developing comprehensive OPPP and antibiotic resistance control strategies that emphasize not just disinfectant dose, but other key control parameters such as contact time, hydraulics, and temperature. The functional diversity of OPPPs, antibiotic resistant bacteria, and the background premise plumbing microbiome further necessitates broad, holistic programs for monitoring and control. Doctor of Philosophy Efforts to provide safe drinking water face two emerging threats: the rise of pathogens that thrive in the plumbing environment that delivers water to the tap and the rise of antibiotic resistance. In the US and many other parts of the world, opportunistic pathogens are the predominant agents responsible for disease spread by tap water. Opportunistic pathogens tend to infect aged or immunocompromised individuals (hence, 'opportunistic') and grow well in in-building plumbing. Globally, antibiotic resistance is on the rise and becoming a fundamental threat to modern medicine. Pathogenic bacteria become resistant to antibiotics used to treat infections when they acquire antibiotic resistance genes (ARGs), which can happen either by mutation or from other resistant bacteria sharing ARGs. Overuse or misuse of antibiotics can impose selection pressure that stimulates horizontal gene transfer and enhance survival of bacteria that are resistant. Prior studies have suggested that under some circumstances, disinfectants used to control pathogens in drinking water can also select for antibiotic resistant bacteria. Thus, the overarching goal of this research was to optimize the type and dose of disinfectant used, depending on building-level factors such as pipe material, for effectively controlling proliferation of both opportunistic pathogens and antibiotic resistance. This dissertation largely focuses on in-building plumbing systems, which are home to potentially tens of thousands of bacterial cells per milliliter of water or per square centimeter of internal pipe surfaces. These bacteria interact not only with each other and other microbes, but also with features of the plumbing environment, such as the water chemistry or the pipe materials. Building plumbing systems are highly intricate ecosystems that can undermine the effectiveness of disinfectants provided by utilities. One major contribution of this research is the development of the convectively-mixed pipe reactors (CMPRs) as a simple and easy-to-use test system that recreates combinations of features of interest encountered in in-building plumbing. We applied the CMPRs to study two common residual disinfectants (chlorine and monochloramine) supplied by water utilities, and two other disinfectants (chlorine dioxide and copper-silver ionization) which are commonly dosed by building operators, especially in hospitals and other buildings housing individuals susceptible to infection. These four disinfectants were applied to CMPRs consisting of PVC, copper, and iron pipe. Chemical, culture, and DNA methods were used to understand how these disinfectants affected the microbes and their ecology. We then took the opportunity to set up CMPRs in the Netherlands, where there has been no historical exposure to chlorine because their water quality regulations emphasize limiting nutrients in the water and elevating the hot water line temperatures as means to control microbial growth. The CMPRs effectively produced worst-case plumbing scenarios, where opportunistic pathogens were especially difficult to control through residual disinfection. Dosed disinfectants tended to be no longer measurable in the water after five hours. The CMPRs also showed that the disinfectant most effective for one pathogen could be the least effective for another. If doses were applied near regulatory limits, the concentrations of pathogens and antibiotic resistance genes decreased. However, opportunistic pathogens tended to survive better than background populations of bacteria. Bacteria carrying ARGs also survived some disinfectant conditions better as well. Thus, if doses were applied at levels that could inactivate some microbes, but not the opportunistic pathogens, pathogen abundances sometimes increased. These results were largely confirmed in the experiment with Dutch drinking water. Here, chlorine appeared to be more problematic than monochloramine in terms of enriching pathogens and antibiotic resistance. We also noted that Dutch waters garnered more diverse microbial communities, with fewer DNA markers for pathogens and antibiotic resistance. In general, this research takes a key step towards optimizing application of residual disinfectants for control of both opportunistic pathogens and antibiotic resistance. Because disinfectants can have negative impacts on drinking water microbial communities when supplied insufficiently, it is important that the other features of in-building plumbing, such as the selection of pipe material or the hydraulics, facilitate disinfectants reaching all portions of plumbing and at the necessary concentrations. It is recommended that the selection process for disinfectant type and dose considers the plumbing materials and other conditions such that disinfection can be aimed towards controlling multiple opportunistic pathogens, which can vary in their susceptibility, and antibiotic resistance.

Antimicrobial resistance (AMR) threatens our modern standard of living with the potential return to a pre-antibiotic condition where deadly infections are no longer treatable. Wastewater treatment plants (WWTPs) are vital components in water sanitation infrastructure and are now also being recognized as valuable monitoring points for antibiotics, antibiotic resistant bacteria (ARB), and antibiotic resistance genes (ARGs) disposed of or excreted by human populations. Hospital waste water is of special interest as a potential focused monitoring point and in general research is needed to establish the benefits of both on-site and community-scale wastewater treatment as important barriers to the disseminators of ARGs into the environment. The research aims described herein examine these components of wastewater treatment and how they relate to AMR indicators identified through metagenomic sequencing. Through monitoring of local WWTPs, it was found that AMR indicators shifted over time and in relation to human behavior that changed due to the COVID-19 pandemic. Hospital wastewater did not measurably impact the microbiome during simulated activated sludge wastewater treatment according to broad-scale metagenomic ARG profiling; however, some clinically-relevant ARGs escaped treatment. Lastly, a study of a transect of WWTPs indicated impacts on the abundance of certain ARGs in downstream riverine receiving environments. Nonetheless, there appeared to be a number of other factors at play, and upstream and downstream resistomes tended to remain similar, calling for further research to delineate impacts of various wastewaters and treatments on ARGs in affected aquatic environments. Doctor of Philosophy Antimicrobial resistance (AMR) occurs when bacteria, viruses, and fungi are able to survive in the presence of antibiotics because they carry antibiotic resistance genes (ARGs) encoded in their DNA. AMR is a major public health concern as it makes it so that antibiotics are no longer effective against potentially deadly infections. Wastewater treatment plants (WWTPs) are being discovered as a hub of opportunity for monitoring potential AMR problems in a community. WWTPs receive sewage from homes and various industries. This sewage contains rich information for researchers to examine in terms of which antibiotics, bacteria, and ARGs are circulating in the community. This makes it possible to find out which antibiotics are being consumed in the community and which ARGs might be prevalent. The purpose of this research was to better understand both how WWTPs can be used as monitoring points for AMR and how they can be improved to help reduce ARGs emitted to rivers and streams where treated water is discharged. It was found that the types of ARGs prevalent in wastewater changed over time, especially during the COVID-19 pandemic as people worked from home and changed habits regarding doctors' visits, which impacted antibiotic use. Hospital sewage was studied as a useful indicator of pathogens and ARGs that are challenging a community and also the antibiotics being used. This research explored what happened to ARGs during the treatment of domestic (i.e., from people's homes) wastewater along with hospital wastewater and found that hospital wastewater introduced some ARGs that are typically found in clinical settings, but did not negatively impact the overall wastewater treatment process. Finally, the impact that WWTPs have on rivers to which treated water is discharged was explored. The results indicated that certain ARGs were elevated downstream of the WWTPs. However, when examining all ARGs together, no major shifts due to the treated wastewater were apparent.

Since penicillin was discovered in 1928, antibiotic usage in human and veterinary medicine and prevalence of antibiotic resistant bacteria (ARB), has been increasing. While antibiotics and antibiotic resistance genes (ARGs) naturally occur in soils, increasing abundances of ARGs correlate with increased antibiotic usage in agricultural settings. When livestock are treated with antibiotics, the antibiotic compounds, ARB, and ARGs can enter soil via manure excreted onto pastures or applied to other fields as fertilizer, thereby spreading antimicrobial resistance (AMR) in the environment. In addition to human health implications, increased AMR has negative impacts on ecosystem services such as carbon and nitrogen cycling. While many studies have researched antibiotic persistence in agricultural systems and their impacts on soil microbial communities, there are still significant knowledge gaps around the long-term effects of antibiotic exposure in soils, how those impacts differ among soils, and how elements of AMR may differentially transport through soil. To address these knowledge gaps, our objectives were to 1) examine the impact of multi-year repeated additions of manure from cattle administered antibiotics on soil microbial communities, 2) determine the interactive effects of soil moisture and type on soil microbial communities exposed to antibiotics and manure, and 3) differentiate between vertical transport of AMR in the form of viable ARB or ARGs in extracellular plasmids. Our results demonstrate that soil bacterial community structures were consistently altered by 3-year additions of manure from cattle administered antibiotics compared to soil amended with antibiotic-free manure. Furthermore, ARG abundances were higher in soils with manure additions compared to soil without manure, although this was true regardless of whether the cattle were administered antibiotics, suggesting that manure and antibiotic impacts on soil microbial communities can persist over multi-year of repeated manure applications. Additionally, in microcosms, effects of manure from cattle administered antibiotics on ARG abundances, microbial community structures, respiration, and nitrogen pools in soil were seen across multiple soil types and moisture contents, suggesting environmental conditions can alter how manure and antibiotics impact microbial community structure and nutrient cycling. Finally, ARB flowed readily through saturated soil, but were also detectable in the top 5 cm of soil columns. However, ARGs on extracellular plasmids did not flow through soil columns and were not detected in soil, indicating that extracellular DNA does not persist or transport through the soil to any meaningful degree. Overall, these results indicate a nuanced approach is required to mitigate the environmental spread of AMR. Soil management strategies for addressing the AMR crisis should consider the broader context of manure management, as high ARG abundances can come from application of manure from antibiotic-free cattle, and soil microbial communities in individual environments may have varied responses to manure antibiotic exposure. Furthermore, the transport of AMR through soil is complex and dynamic, as elements of AMR may transport differently through soil and require separate consideration in modeling and management. Future AMR management practices that consider diverse factors that affect persistence and spread of AMR in the environment can help protect livestock productivity and maintain the efficacy of antibiotics to protect human and animal health. Doctor of Philosophy Antibiotics are an important tool used to fight infections in humans, pets, and livestock. As antibiotics are used more frequently, the bacteria they target are more likely to develop resistance to the antibiotics, leading to increasing cases of infections that are harder to treat and higher risk. Antibiotic resistance can persist and spread in multiple forms, including the antibiotic compounds themselves, as antibiotic resistant bacteria (ARB), or as the genetic material that encodes for antibiotic resistance genes (ARGs). In agricultural systems, when livestock are treated with antibiotics they can excrete the antibiotics, along with ARB and ARGs, in the manure, which is then applied to land as fertilizer. In addition to the associated health risks, the spread of antibiotic resistance impacts microscopic bacteria and fungi in the soil, which are important for recycling nutrients for plants and maintaining ecosystem health. The overall goal of this dissertation was to gain a better understanding of how manure from cattle given antibiotics impacts these bacteria and fungi when manure is applied to the soil. The specific objectives were to 1) look impacts after long-term (multiple years) of manure addition, 2) examine how bacteria and fungi might respond differently to antibiotics in soils of different type or with different amounts of water, and 3) determine if ARGs that exist as free genetic material outside of living bacteria can be moved through the soil with flowing water in the same way as living bacteria. Results showed that while the composition of bacterial and fungal communities in the soil vary from year to year, adding manure with and without antibiotics had both caused different and consistent changes on the composition of bacterial communities. There were also higher concentrations of ARGs in soil that had manure added, however antibiotics in the manure did not cause ARGs to increase further, suggesting that even antibiotic-free manure can impact the spread of antibiotic resistance. Experimental work also demonstrated that the soil type and water content of soil can alter how bacteria and fungi respond to antibiotics in manure. The composition of bacterial and fungal communities, their activity rates, and the amount of nitrogen – an important plant nutrient with availability that is strongly affected by microbial activity – all differed with soil type and water content. Thus, while antibiotic resistance antibiotic resistance can cause measurable changes in soil across a range of environmental conditions, it is also likely to persist and spread in different ways in different environments. Finally, when water containing elements of AMR was added to soil, ARB were shown to both move through the soil easily and remain near the top of soil. In contrast, ARGs contained on genetic material outside of living cells did not move through the soil and were broken down within a few days, suggesting that antibiotic resistance likely spreads through living bacteria more than genes outside of cells. Overall, this work highlights the complexity of understanding the role of environmental transmission in the antibiotic resistance crisis and demonstrates the need for nuanced management approaches that take specific environments and conditions into account.

Fungi play important roles in various environments. Some of them infect plants and cause economically important diseases. However, many fungal pathogens cause similar symptoms or are even spread asymptomatically, making it difficult to identify them morphologically. Therefore, culture-independent, sequence-based diagnostic methods that can detect and identify fungi independently of the symptoms that they cause are desirable. Whole genome metagenomic sequencing has the potential to enable rapid diagnosis of plant diseases without culturing pathogens and designing pathogen-specific probes. In my study, the MinION nanopore sequencer, a portable single‐molecule sequencing platform developed by Oxford Nanopore Technologies, was employed to detect the fungus Calonectria pseudonaviculata (Cps), the causal agent of the devastating boxwood blight disease of the popular ornamental boxwood (Buxus spp.). Various DNA extraction methods and computational tools were compared. Detection was sensitive with an extremely low false positive rate for most methods. Therefore, metagenomic sequencing is a promising technology that could be implemented in routine diagnostics of fungal diseases. Other fungi may play important roles in the atmosphere because of their ice nucleation activity (INA). INA is the capacity of some particles to induce ice formation above the temperature that pure water freezes (-38°C). Importantly, INPs affect the ratio of ice crystals to liquid droplets in clouds, which in turn affects Earth's radiation balance and the intensity and frequency of precipitation. A few fungal species can produce ice nucleating particles (INPs) that cause ice formation at temperatures ≥ –10°C and they may be present in clouds. Two such fungal genera are Fusarium and Mortierella but little is known about their INPs and the genetic basis of their INA. In my study, F. avenaceum and M. alpina were examined in detail. INPs of both species were characterized and it was found that strains within both species varied in regards to the strength of INA. Whole genome sequencing and comparative genomic studies were then performed to identify putative INA genes. Differential expression analyses at different growth temperatures were also performed. INP properties of the two species shared similarities, both appearing to consist of secreted aggregates larger than 30 kDa. Low temperatures induced INA in both species. Lists of candidate INA genes were identified based on their presence in the strains with the strongest INA and/or induction of their expression at low temperatures and because they either encode secreted proteins or enzymes that produce other molecules known to have INA in other organisms. These genes can now be characterized further to help identify the fungal INA genes in both species. This can be expected to help increase our understanding of the role of fungal INA in the atmosphere. Doctor of Philosophy Fungi are important to life on Earth and play roles in the environments that surround us. On the one hand, fungi can make plants sick and some plant diseases may even cause economic losses to farmers. If the cause of a disease can be identified accurately in an early stage before symptoms develop, disease transmission may be prevented and plants may be protected from disease. However, it is a challenge to find out which fungus causes which disease since symptoms of different fungal diseases look very similar. Typically, we have to wait for plants to become very sick or we have to isolate the fungus that causes a disease to identity it, which may be time-consuming and not lead to precise identification. DNA sequencing technologies have the potential to lead to more sensitive, faster, and more accurate disease diagnosis and, therefore, may help prevent disease outbreaks. In my study, the MinION nanopore sequencer, a small portable device, was used to detect the fungus causing boxwood blight on boxwood. By loading the DNA of unhealthy boxwood on the device, the boxwood blight pathogen was identified within a very short time. Thus, this method is a promising diagnostic method that may be applied to detect other plant fungal diseases as well. On the other hand, fungi may affect Earth's climate by affecting how many water droplets in clouds are frozen, which in turn affects Earth's temperature and how often and how much it rains and snows. Fungi may affect the freezing of water droplets in clouds since some of them have ice nucleation activity (INA), which is the capacity to catalyze ice formation at a higher temperature than the temperature at which pure water freezes (-38°C), and they may be present in clouds. So far, INA has only been found in a few fungi, including the species Fusarium avenaceum and Mortierella alpina, but the mechanism of their INA is poorly understood. In my study, multiple F. avenaceum and M. alpina strains were examined in detail. Two approaches were used. First, strains in each species were compared with each other to find out how strong their INA is. Once it was found that they differed in their strength of INA, their genomes were sequenced and compared to find genes present in the most active strains and missing from the least active strains since it is these genes that may contribute to INA. It was also found that both fungal species had stronger INA when they were grown at lower temperatures. Therefore, the expression of their genes between higher and lower temperatures was compared to find the genes that were more highly expressed at lower temperatures since it is these genes that may cause INA. Based on previous studies, fungal INPs may either consist of secreted proteins or be the products of biosynthetic gene clusters. Therefore, the list of potential genes was reduced by looking for genes encoding either secreted proteins or biosynthetic gene clusters. The list of these potential INA genes will make it easier to identify the INA genes in F. avenaceum and M. alpina and determine the role of fungi in affecting the weather and climate on Earth.

In terrestrial ecosystems, microorganisms are the dominant drivers of virtually all ecosystem processes, particularly cycling of carbon (C), nitrogen (N), and phosphorus (P). These microbial functions are critical for promoting ecosystem services that support human well-being, such as provisioning of clean drinking water, nitrogen retention, and carbon storage. In forests of the Appalachian region of the eastern US, these ecosystem services are threatened by multiple anthropogenic influences, including present and past land use activities (e.g., logging, conversion to agriculture) and climate change (e.g., intensifying droughts). However, despite the central importance of microbial communities in promoting ecosystem functions, impacts of land management and climate change on soil microorganisms remain poorly understood in the region. This dissertation seeks to address the following questions: 1) How does a new forest management practice, Rhododendron understory removal, influence the ecosystem functions of soil microbial communities? 2) Do historical land management activities have long-term legacy effects on the structure and ecosystem functions of soil microbial communities? And 3) Does historical land use influence responses of soil microbial communities to intensifying drought? In chapter 2, I show that experimental Rhododendron understory removal increased soil C and N availability, thereby promoting increased total microbial biomass. This increased microbial biomass resulted in elevated production of microbial extracellular enzymes, which increased rates of C and N cycling in soils following Rhododendron removal. In chapter 3, I examined soils across several historically disturbed and adjacent undisturbed reference forests and show that historical management activities, e.g., logging, conversion to agriculture, have long-term effects on soil microbial communities 4-8 decades after management activities occurred. These effects included increased bacterial diversity, increased relative abundance of r-selected bacterial taxa, and increased abundance of arbuscular mycorrhizal fungi. In chapter 4, I show that key soil biogeochemical processes, i.e., C mineralization, N mineralization, and nitrification, exhibit generally higher rates in historically disturbed forests relative to adjacent reference forests. Further, I attributed these changes in ecosystem process rates to changes in key aspects of microbial communities, including microbial biomass, extracellular enzyme activities, and bacterial r- vs K-selection. In chapter 5, I conducted a drought-rewetting experiment and show wide-ranging effects of experimental drought on soil microbial communities, including altered diversity, community composition, and shifts in the relative abundances of several specific taxa. Further, drought responses were particularly evident in soils from historically disturbed forests, indicating influences of land management on responses of soil communities to climate change. Finally, in chapter 6, I show that the experimental drought also influenced several ecosystem-scale properties of soils, including increased soil N pools and increased respiratory C loss. Overall, my dissertation reveals wide-ranging effects of anthropogenic activities on soil microorganisms and shows that microbial communities will influence forest responses to global change at the ecosystem scale. Doctor of Philosophy Forest ecosystems of the southern Appalachian region provide numerous services that support human well-being, including provisioning of clean drinking water and the retention of nutrients and carbon (C). These ecosystem services are dependent on several processes that occur in soil, including the C and nitrogen (N) cycles. These cycles, in turn, are carried out primarily by microorganisms (bacteria and fungi) that live in soil. The ecosystem services provided by these forests are being threatened in the Appalachian region by a variety of land use activities such as logging and conversion of forests to agriculture and are also being threatened by climate change. However, despite the critical role of microorganisms in supporting ecosystem services, effects of land use activities and climate change on soil microorganisms are largely unknown in the Appalachian region. The goal of this dissertation is to answer the following questions: 1) How does Rhododendron understory removal, a new land management practice, influence soil microbial communities? 2) What are the long-term effects of historical land use activities, such as logging and conversion to agriculture, on soil microbial communities? And 3) In what ways will intensifying droughts influence soil microorganisms in Appalachian forests? I used a variety of approaches to answer these questions, including experiments and observational approaches. In chapter 2, I show that experimental Rhododendron understory removal increased the overall size of microbial communities (i.e., more microbial biomass) due to greater availability of soil resources (i.e., C and N). These larger microbial communities produced more enzymes involved in C and N metabolism, thereby increasing rates of C and N cycling in soils. In chapter 3, I surveyed soils from several forests that were disturbed by humans ~40-80 years previously (i.e., logged, converted to agriculture) and showed that these historical human activities have many long-term effects on soil bacteria and fungi. Specifically, in historically disturbed soils, I observed higher diversity of bacteria, higher abundance of rapidly growing (i.e., r-selected) bacteria and higher abundance of some groups of fungi that associate with plant roots and aid in acquiring nutrients for plants (i.e., mycorrhizal fungi). In chapter 4, I show that these historically disturbed soils also had altered rates of C and N cycling and that these altered cycling rates were associated with changes in several properties of microbial communities, including microbial enzymes, microbial biomass, and growth rates of bacteria (i.e., r-selection). In chapter 5, I conducted an experimental drought in these soils and show that drought has wide-ranging effects on many aspects of microbial communities, including effects on diversity, species composition, and abundances of many specific microbial taxonomic groups. Further, responses of microbial communities were larger in soils from historically disturbed forests, showing that past management will influence microbial responses to future climate change. In chapter 6, I show that many aspects of the soil ecosystems as a whole were also impacted by the experimental drought. In particular, soils exposed to drought released more CO2 over the course of the experiment and had higher N concentrations than control soils. Overall, my dissertation identifies many influences of land management and climate change on soil microbial communities and shows that these microorganisms will influence forest responses to global change at the ecosystem scale.

Stormwater bioretention cells (BRCs) are a variety of green stormwater infrastructure with the potential to restore pre-urban water balance, provided they can be tailored to infiltrate and evapotranspire (i.e., harvest) urban runoff in proportions consistent with pre-urban hydrologic conditions. This paper evaluates their capacity to do so, focusing on evapotranspirative harvest, which is relatively understudied, and the capacity of CSR (Competitve, Stress-tolerant, and Ruderal) functional type to serve as an overarching framework characterizing the water use strategy of BRC plants. The goal is to determine if harvest (and therefore the ratio of urban runoff infiltrated to harvested; the I:H ratio) might be fine-tuned to meet pre-urban values in BRCs through informed manipulation of plant community composition. This study focuses on 3 critical plant water use traits, the turgor loss point, the point of incipient water stress, and maximum stomatal conductance. A global plant traits meta-analysis identified degree of plant competitiveness and stress tolerance as significant determinants of all three water use traits, with stem type (woody vs herbaceous) also being significant, but only for turgor loss point. Based on these results, six water use scenarios appropriate for plants with different CSR type/stem type combinations were developed. BRC plants spanning the range of CSR types necessary to actionize these scenarios were determined to be available in eight major climate zones of the coterminous US, suggesting that regulating plant water use in BRCs using CSR is likely feasible. Hydraulic simulations (Hydrus 1D) were conducted for each scenario in all eight climate zones and revealed significant differences in evapotranspirative harvest and I:H ratios in simulated BRCs. Competitive woody plants had the highest evapotranspiration and lowest I:H ratios; 1.5-1.8 times more evapotranspiration and a 1.6-2 times lower I:H ratio than stress tolerant herbaceous plants, on average, across climate zones. Despite these significant differences, no simulated BRC in any climate zone was capable of reproducing pre-urban I:H ratios, regardless of plant type. More water was infiltrated than harvested in all scenarios and climates with the inverse being true for all pre-urban conditions. This suggests that absent additional sources of harvest (e.g., use of BRC water for nonpotable purposes such as toilet flushing and outdoor irrigation, or adoption of novel BRC designs that promote lateral exfiltration, stimulating "extra" evapotranspiration from nearby landscapes), BRCs will be unable to restore pre-urban water balance on their own. If true, then using BRCs in combination with other green technologies (particularly those biased towards harvest), may be the best path forward for balancing urban water budgets. Master of Science Stormwater bioretention cells (BRCs) are a variety of green infrastructure designed to manage urban stormwater flows that can dramatically reduce the amount of stormwater that is rapidly (and unnaturally) conveyed from paved surfaces to ecosystems. Their ability to recreate natural flow conditions is dependent on them balancing rates of infiltration – slowly filtering water down to the water table – and evapotranspiration – letting plants capture and transpire water. This paper evaluates the extent to which different plant functional types (competitive, stress tolerant, and ruderal (weedy)) can be used to regulate this balance, bringing urban hydrologic conditions closer to pre-urban ones. Competitiveness and stress tolerance were found to significantly influence plant water use traits, as was plant stem type (woody vs herbaceous) to a lesser extent (i.e., managing water budgets using CSR functional type is theoretically possible). Published BRC vegetation guidelines in 8 major US climate zones were found to include both competitive and stress tolerant species (i.e., the range of functional types required to regulate BRC water balance exists, suggesting it is feasible). Finally, hydraulic simulations conducted under six plant water use scenarios (reflecting different CSR types and stem types) revealed significant differences in the ratio of water infiltrated to evapotranspired by BRCs (i.e., changing plant functional types can meaningfully influence BRC water balance). This said, the magnitude of this effect may be insufficient to return urban catchments to a pre-urban state. All BRCs infiltrated too much water in our simulations suggesting that absent additional sources of harvest (for instance., use of BRC water for nonpotable purposes such as toilet flushing or outdoor irrigation), BRCs will be unable to restore pre-urban water balance on their own. If true, then using BRCs in combination with other green technologies (particularly those biased towards harvest), may be the best path forward for balancing urban water budgets.

Widespread use of antibiotics in livestock production can result in the dissemination of bacteria carrying antibiotic resistance genes (ARGs) to the broader environment. Within agroecosystems, ARGs can pose a risk to livestock handlers, farmers, and ultimately consumers. The overall goals of this dissertation are to examine the presence of resistance (antibiotic, metal) in agricultural soils and evaluate the most critical potential points of best management control of antibiotic resistance spread along the agricultural production chain. The relative impacts of agricultural practices, manure management, native soil microbiota, and type of crop grown and harvested on the agricultural resistome are multi-dimensional and cannot be captured via a single analytical technique or by focusing on one specific point in the agricultural process. Culture-, molecular "indicator"-, and next-generation sequencing- based methods were employed to characterize antibiotic resistance via taxonomic and functional profiles on the broader manure, soil, and vegetable surface microbial communities through 16S rRNA amplicon sequencing and shotgun metagenomics. Although antibiotic concentrations dissipated in the soil after 28 days after amendment application, antibiotic resistance presence was recoverable throughout the entire 120d growing season in the compost and manure amendments, the amended soil, and on vegetable surfaces. The addition of organic fertilizers increased antibiotic resistance presence compared to background levels. Further, metals and metal resistance were also measured in the amended soils and were found to be at greater levels in the inorganically fertilized soils compared to the manures and compost amended soils. Analysis of the widespread agroecosystem microbial community composition and broader metagenome has characterized varying genera profiles in the soil and on the vegetable surfaces and specific ARG and mobile genetic element (plasmid) co-occurrences. These co-occurrences highlight which ARGs may be most critical for future antibiotic resistance dissemination research. It is imperative to employ multiple methods when measuring agricultural resistance, as one method alone may miss significant patterns and lead to different best management recommendations. Linking the livestock manure, soil, and vegetable surface-associated ARBs, ARGs, resistomes, and microbiomes will help identify critical control points for mitigation of agricultural dissemination of antibiotic resistance to the environment and food production. Doctor of Philosophy By 2050, it is estimated that antibiotic resistant infections will be the leading cause of death worldwide. It is important to consider human, animal, and environmental health when researching antibiotic resistance, which is known as a "One Health" approach. In this dissertation work, I focus on the environmental side of antibiotic resistance in our agricultural systems. Agriculture is a known source of antibiotic resistance due to its use of antibiotics in livestock as a treatment for illness, and in some instances, as a growth promoter. Over one growing season, I measured antibiotic resistance in an agricultural setting using many techniques. First, I analyzed the effects of inorganic (chemical) versus organic (manure and compost) fertilization on antibiotic resistance in the soil. I measured antibiotic resistance by growing antibiotic resistant bacteria, quantifying specific antibiotic resistant genes (ARGs) using DNA amplification, and quantifying all the ARGs in the soil using a next-generation sequencing (NGS) technique called shotgun metagenomics. I found that adding manure to the soil increases ARGs compared to background soil levels, and that composting in an effective management strategy in decreasing ARGs in the soil over time. Second, I analyzed the same effects of fertilization on metal resistance in the soil. I was able to use the same NGS dataset to measure metal resistance genes (MRGs). I found that adding inorganic chemical fertilizer increases MRGs in the agricultural soils compared to the organic (manure or compost) fertilizer. Additionally, I studied the microbes that live in the agricultural soils using another kind of NGS data specific for microbial identification. I found that although there were small differences between the microbial populations in the soil when fertilizers were added, they returned to similar composition over the growing season. Lastly, I measured antibiotic resistance and microbes throughout the entire agricultural system. I picked the point of fertilization (manure management), soil, and the lettuce surface to evaluate if antibiotic resistance spreads from the farm to the vegetable that ends up on a consumer's plate. I found that at each point antibiotic resistance is present, but at different levels. Composting reduces ARGs compared to raw manure. Agricultural soils may act as a natural buffer to antibiotic resistance. Lettuce plants grown in compost fertilized soils have less ARGs than lettuce plants grown in manure. There are many agricultural management practices that effectively reduce antibiotic resistance and using all of them plus many measurement methods will ultimately help farmers and consumers reduce antibiotic resistance in our agricultural systems.

Anthropogenic freshwater salinization, which is caused by numerous human activities including agriculture, urbanization, and deicing, impacts an estimated 37% of the contiguous drainage area in the United States. High salt concentrations in brackish and marine environments (~1,500 – 60,000 µS cm-1) influence aquatic bacteria. Less is known about the effects of freshwater salt concentrations (≤ 1,500 µS cm-1) on bacterial ecology, despite the pervasiveness of freshwater salinization. Bacteria perform many fundamental ecosystem processes (e.g. biogeochemical cycling) and serve as indicators of human health risk from exposure to waterborne pathogens. Thus, to understand how salt pollution affects freshwater ecosystems, there is a critical need to understand how freshwater salinization is impacting bacterial ecology. Using a series of controlled mesocosm experiments, my objectives were to determine how (1) survival of fecal indicator bacteria (FIB), (2) the diversity of native freshwater bacterial communities, and (3) bacterial respiration and nutrient uptake rates responded across a freshwater salinity gradient of different salt profiles. Survival rates (t90) of Escherichia coli, the EPA recommended freshwater FIB, increased by over 200% as salinity increased from 30 to 1,500 µS cm-1. Survival rates were also significantly higher in water with elevated Mg2+ relative to other base cations, suggesting that different salt sources and ion profiles can have varied effects in FIB survival. Thus, freshwater salinization could cause accumulating concentrations of FIB even without increased loading, increasing the risk of bacterial impairment. Diversity of native bacterial communities also varied across a freshwater salinity gradient, with a general increase in species richness as salinity reached 1,500 µS cm-1. Community variability (β-diversity) was greatest at intermediate salinities of 125 – 350 µS cm-1 and decreased towards the upper and lower extremes (30 and 1,500 µS cm-1, respectively). These diversity patterns suggest that osmotic stress is an environmental filter, but filtering strength is lowest at intermediate salinities causing a change from more deterministic to more stochastic assembly mechanisms. Different salt types also produced distinct bacterial community structures. Lastly, bacterial respiration doubled as salinity increased to 350 – 800 µS cm-1, revealing a subsidy-stress response of bacterial respiration across a freshwater salinity gradient. Corresponding changes in nitrogen and phosphorus uptake increased N:P ratios in ambient water, especially in mesocosms with elevated Ca2+, which could affect nutrient limitation in salinized streams enriched with Ca2+. Bacterial community structure based on Bray-Curtis dissimilarity was not correlated to pairwise changes in respiration rates but was linked to net nitrogen and phosphorus uptake after five days. Collectively, these results establish that freshwater salinization alters bacterial ecology at the individual population, whole community, and ecosystem process scales. Further, different salt types (e.g., CaCl2, MgCl2, NaCl, KCl, sea salt) had varying effects on bacteria at all levels and should be considered when predicting the effects of salinization on freshwater ecosystems. Developing more nuanced salt management plans that consider not only amount, but different types, of salts in freshwaters could help improve our ability to predict human health risk from waterborne pathogens and mitigate/ reduce salinity-induced impacts to freshwater ecosystem processes and services. Doctor of Philosophy Humans rely on streams, rivers, and lakes for many services including transportation, recreation, food, and clean drinking water. Despite our reliance on freshwater ecosystems, human activity has significantly degraded freshwater resources worldwide. Recently, salt pollution caused by human activity on land, known as freshwater salinization, has emerged as a widespread water quality issue. Numerous human activities including agriculture, urbanization, resource extraction, and deicing have increased freshwater salt concentrations in 37% of the United States' contiguous drainage area. Large changes in salinity (i.e. from freshwater to oceanic salinities) are known to affect bacteria that perform many important ecological functions, such as nutrient cycling and water purification, while the effects of smaller changes in salinity more typical within the freshwater range are unknown. I used controlled laboratory experiments to determine how freshwater salinization affects (1) survival rates of Escherichia coli, (2) diversity of native bacterial communities, and (3) bacterial nutrient cycling. My results revealed that freshwater salinization can significantly increase how long E. coli survive in freshwater. E. coli are used to detect the presence of waterborne pathogens and reduce human health risk. Thus, freshwater salinization might reduce the reliability of E. coli as an indicator of waterborne pathogens as well as increase concentrations of bacterial that are potentially harmful to human health in freshwater. Additionally, freshwater salinization affected bacterial diversity by altering the ways in which bacterial communities form. In general, the number of bacterial species present increased as salinity reached the upper freshwater limit, but communities were most variable at intermediate freshwater salt concentrations. These diversity patterns suggest that different salt concentrations can either cause or reduce stress in bacteria, resulting in significantly different bacterial communities. Lastly, moderate increases in freshwater salt concentrations doubled bacterial respiration and nutrient uptake rates. Bacterial respiration influences how energy flows through ecosystems, and freshwater salinization could potentially alter this process. Different salt types also had different effects of bacterial ecology. Collectively, my results establish that freshwater salinization impacts bacteria at the individual, community, and ecosystem levels.

Plants synthesize a diverse array of phytochemicals in response to interactions with herbivores, pathogens, and commensal microbes. These phytochemicals may simultaneously enhance crop defense and quality, representing a potential pest management strategy. However, plant chemical responses to different types and levels of biotic interactions remain unclear, particularly in fruit tissues, and the feasibility of inducing these defenses through elicitor application in field environments also requires further examination. Thus, apples were used to 1) examine the impact of distinct communities of biotic interactions among plants, insects, and microbes on fruit phenolic chemistry, and 2) examine the impact of the phytohormones jasmonic acid (JA), salicylic acid (SA), and melatonin (M) on fruit phenolic chemistry and resistance against pests and pathogens. Ultimately, phenolic defenses were induced by fungal damage primarily in ripe pulp tissues, where there was also a positive relationship between fungal endophyte and phenolic diversity, supporting a broad hypothesis that chemical diversity may increase with biotic diversity. Additionally, two compounds were upregulated in response to fungal damage: chlorogenic acid and an unidentified benzoic acid. Elicitor applications did not affect phenolic chemistry, but the combined application of JA-SA analogues had some chemical or physical effect, as this treatment reduced emergence of the insect Rhagoletis pomonella. Thus, fruit induced defenses may be tissue-specific and subject to temporal, environmental, or genotypic variation. Overall, these chapters examined the relationship between biotic interactions and induced fruit chemistry, with the goal of improving understanding of plant-microbe-insect interactions and incorporating these interactions into more sustainable agricultural practices. Master of Science Plants may produce a diverse array of defensive phytochemical compounds in response to interactions with herbivores, pathogens, and the microorganisms that reside within plant tissues. These phytochemicals may simultaneously improve crop defenses and quality, representing a potential agricultural management strategy. However, plant chemical responses to different types and levels of biotic interactions are not well-understood, particularly in fruit tissues, and the feasibility of activating these defenses in fruits through the application of phytohormones that regulate defense pathways as a potential management strategy also requires further examination. Thus, apples were used to 1) examine the impact of distinct communities of biotic interactions among plants, insects, and microbes on fruit chemistry, focusing on phenolics, an important class of phytochemical compounds, and 2) examine the impact of the defense-activating phytohormones jasmonic acid (JA), salicylic acid (SA), and melatonin (M) on fruit phenolic chemistry and resistance against pests and pathogens. Ultimately, phenolic defenses were activated by fungal damage primarily in ripe pulp tissues, where there was also a positive relationship between fungal endophyte and phenolic diversity, supporting a broad hypothesis that chemical diversity may increase with biotic diversity. Additionally, two compounds were produced in response to fungal damage: chlorogenic acid and an unidentified benzoic acid. Phytohormone applications did not affect phenolic chemistry, but the application of the combined JA-SA analogues had some chemical or physical effect, as this treatment reduced emergence of the insect Rhagoletis pomonella. Overall, the phytochemical defenses activated by biotic interactions in fruits may occur primarily in certain tissue types, and may also vary due to environmental conditions, time of year, or plant species. These chapters examined the relationship between fruit chemistry and biotic interactions with the goal of improving understanding of plant-microbe-insect interactions and incorporating these interactions into more sustainable agricultural practices.

Soil microorganisms are primarily limited by carbon (C) availability. The majority of C entering belowground food webs comes directly from local flora. Plant derived labile C compounds affect microbial community structure and function, which in turn drive ecosystem function. Research has focused on dissolved organic C (DOC) from litter leachates and root exudates. These compounds are often readily assimilable by soil microorganisms and are precursors for stable soil organic matter formation. Due to diffusion limitation DOC rarely travels far beyond its origin, meaning most soil microorganisms are unable to access these compounds unless they are located near the C source. However, recent studies have illuminated the importance of volatile organic compounds (VOCs) in soil ecosystems. VOCs are produced in abundance and, as vapors, they are able to travel through soil more rapidly than DOC. This dissertation aims to investigate the importance of VOCs commonly produced during the decomposition of leaf litter. We used three separate microcosm experiments to answer the following questions. 1) How do abundant VOCs affect microbial activity in soil? 2) How do VOCs affect nitrogen (N) transformations and the microbes associated with N transformations? 3) How do VOCs affect microbial community composition? 4) Are VOCs from decomposing litter incorporated into soil C pools? In chapter 2, we show that methanol and acetone – common litter derived VOCs – increase microbial activity and labile soil C, while also decreasing available nitrate, and ammonia oxidizing archaea. Interestingly, this decrease in nitrifiers did not affect nitrification rate after VOC addition was ceased. In chapter 3, we demonstrate that soil microbial taxa respond differently to DOC and VOCs at different soil moisture levels. Specifically, DOC primarily affected taxa abundance in wetter soils, while the insoluble VOC α-pinene had the largest impact at lower moisture levels, and methanol affected abundance at all moisture levels. Finally, in chapter 4, we demonstrate that VOCs from decomposing leaf litter altered soil bacterial and fungal communities, and VOC derived C entered all measured soil organic matter pools without direct contact between decomposing litters and the soil. This work demonstrates the importance of VOCs on soil microbial communities and ecosystem function. The VOC induced increase in microbial activity, and the effects of VOCs at low moisture levels suggest that VOCs may function in the bulk soil in a manner similar to DOC in rhizosphere soil. Additionally, the incorporation of VOC-C into soil organic matter pools identifies a hitherto unrecognized mechanism for soil organic matter formation. Doctor of Philosophy Soil microorganisms live in an environment where their access to carbon containing compounds limits their growth. In these belowground environments most of the carbon flows from aboveground plant matter through soil microbes into the organisms that consume those microbes. The carbon from plants not only feeds the soil microbes but also changes the type of microbes and how those microbes process important chemicals in the environment – e.g., carbon and nitrogen. Previously, research has focused on carbon compounds that are able to dissolve in water. Often, these compounds originate from liquids that plants release from their roots, or dissolve like tea when leaves are soaked in water. Soil microorganisms can often use these dissolved carbon compounds and directly incorporate them into their biomass. Additionally, these compounds can be stored in soil - sequestering that carbon in the soil, potentially long term. However, dissolved compounds are unable to move very quickly through soil, and the soil microorganisms that live far from the source of these compounds do not have access to them. However, recent studies have found that another form of carbon, volatile organic compounds, are also produced in abundance in the soil environment. These compounds can travel through the air in the soil, as well as in the soil water. When in the air, VOCs travel very quickly and can also travel farther than dissolved compounds. This dissertation aims to investigate the importance of volatile organic compounds that are produced during the decomposition of leaves. We carried out three experiments using small volumes of soil under controlled conditions in the laboratory. We aimed to answer the following questions. 1) How do abundant volatile organic compounds affect microbial activity in soil? 2) How do volatile organic compounds affect microbial processing of nitrogen containing compounds, and the populations of microorganisms that process those compounds? 3) How do volatile organic compounds affect the composition of microorganism in the soil? 4) Are volatile organic compounds from decomposing leaves able to be stabilized in the soil. In chapter 1, we show that methanol and acetone – common volatile compounds produced during the decomposition of leaves– increase microbial activity, and microbial available carbon in soil. Methanol and acetone also decreased available nitrate (an important N containing compound) and a group of organisms that produce nitrate called ammonia oxidizing archaea. Interestingly, once we stopped adding methanol and acetone to the soil the production of nitrate did not differ, meaning that the nitrate producing community was able to recover from the reduction in ammonia oxidizing archaea. In chapter 2, we demonstrated that soil microbial taxa respond differently to dissolved carbon and volatile organic compounds across a gradient of soil moisture. Specifically, dissolved carbon primarily affected taxa abundance in wetter soils, while the insoluble volatile α-pinene had the largest impact at lower moisture levels, and the volatile compound methanol affected abundance of microbial taxa at all moisture levels. Finally, in chapter 3, we demonstrate that volatile organic compounds produced during the decomposition of leaves altered the composition of both bacterial and fungal communities in the soil. Also, and possibly most interestingly, carbon from those volatile organic compounds was stored in all of the pools of carbon that we measured. Together these chapters demonstrate the importance of volatile organic compounds on soil microbial communities and ecosystem function. Since volatile organic compounds induced an increase in microbial activity we are able to infer that soil microorganisms are using these compounds; paired with our observation that volatile organic compounds affected microbial taxa at lower moisture levels than the dissolved compounds did, we can infer that volatile compounds may function as a carbon source in parts of the soil that do not have access to dissolved carbon. Additionally, the incorporation of carbon from volatile organic compounds into soil identified a hitherto unrecognized mechanism for soil carbon sequestration.