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Global climate change represents one of the greatest threats to biodiversity. Phenomena such as rising surface water temperature, increased UV radiation, and ocean acidification have brought negative impacts to ecosystems and their inhabitants. Sensitive to various abiotic factors, microbial eukaryotic communities in aquatic systems are particularly being affected by these environmental changes. Specifically, warming temperature not only can directly affect plankton through limiting growth and inhibiting physiological processes, but can also indirectly impact these organisms by altering light and nutrient availability via loss of sea ice and changes in thermal stratification in various environments. Mixotrophic chrysophytes are an important lineage of protists that often dominate phytoplanktonic blooms in both freshwater and marine systems. Studies have shown mixotrophic organisms’ nutrient-acquiring strategies are influenced by abiotic environmental factors. Temperature in particular, is known to alter growth rate and bacterivory. In response to rising temperature, mixotrophs can either become more phototrophic or more heterotrophic, depending on species, resulting in changes of their role in aquatic food webs and potentially leading to shifts in overall community composition and structure. The objective of this research is to investigate the influence of different environmental factors on primary production and heterotrophic ingestion in marine and freshwater chrysophytes, providing an understanding on how climate change may alter physiological response and survival, with indicative changes in community structures and food webs. The influence of irradiance, nutrient concentrations, and temperature on mixotrophic responses of the Arctic marine chrysophyte Dinobryon faculiferum was investigated, where our results demonstrated an increase in heterotrophic ingestion in response to rising temperature. We also found bacterivory contributes a major proportion of D. faculiferum’s carbon budget in comparison to primary production, which is different from previous studies on Dinobryon species that appeared to be more reliant on phototrophy. Conversely, the freshwater chrysophyte Chrysolepidomonas dendrolepidota, exhibited the opposite temperature effect. The freshwater species was more reliant on primary production and ingested less as temperature increased. Such varying responses showcased diverse nutrient strategies on the mixotrophic spectrum, suggesting generalization of mixotrophic mode in predictive models should be approached with caution. Additional work was done to gain insight on the biogeography of C. dendrolepidota, of which little is known about its distribution. The presence of C. dendrolepidota was not detected through metadata analysis, nor was it detected across several waterbodies sampled in this study. Our results suggested possible rare distribution and endemism of C. dendrolepidota. / Biology

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.

Molecular biology techniques were used in this research to assess the changes that occur in aquatic microbial communities when exposed to chemical wastes and the response of genetically engineered microorganisms (GEMs) in irradiated soils. Different molecular and microbial approaches to monitoring both types of microbial community changes can provide information about those systems at less complex and more sensitive levels than can be achieved with more traditional methods. Traditionally, aquatic microbial communities are evaluated by taxonomic identification and enumeration. Changes in species richness (diversity of taxa) and density of representative taxa were evaluated by DNA-DNA hybridization of prokaryotic communities at stations above and below points of industrial effluent release. Preliminary studies in 1990 indicated a positive correlation (0.95) between traditional identification and enumeration and DNA-DNA hybridization. This correlation was approaching significance but limited by a small sampling size. Study sites in 1991 did not have high correlation (0.04 and 0.36). Low correlation values were the result of methodological difficulties in the hybridization process rather than actual disparities between the two approaches. Graphically, the trends in protozoan species richness and densities of represented taxa were reflected in the DNA-DNA hybridization over time. Improvements in a molecular method include the use of radioisotopes for membrane bound hybridizations or liquid hybridizations (COT curves). In irradiated clay and loam microcosms, genetically-engineered Erwinia carotovora and wildtype declined significantly (p ≤ 0.05) over a 60 day period. Interestingly, in the clay soil, both the GEM and wildtype remained at decreased but detectable levels (using the MPN technique) after 60 days. In the loam soil, the GEM declined below levels of detection while the wildtype persisted and displayed a highly variable growth die-off pattern before declining in numbers. No significant (p ≥ 0.05) differences between the GEM and wildtype were observed in the clay soil. In the loam soil, wildtype survived at significantly (p ≤ 0.05) greater densities than the GEM over 60 days. Differences in soil characteristics as well as the type of GEM used resulted in novel patterns of persistence. An alternative to the MPN method for determining low densities of organisms is the polymerase chain reaction (PCR) in conjunction with DNA probes and hybridization techniques. For environmental probes to be used, the DNA sequence or a portion of the sequence must be determined to serve as a template for PCR amplification. Both ends of a probe specific for 63 serologically distinct strains of E. carotovora were sequenced in the initial phase of the PCR amplification and detection process. This research employs several molecular and microbial methods for the determination of changes in microbial community structure and function in response to chemical or irradiation perturbations. / Master of Science

Gastrointestinal tract (GIT) microorganisms play important roles in animal health, including providing energy and vitamins, improving the host immune response and preventing pathogenic microorganisms from colonizing. Prebiotic feed supplementation offers an alternative to antimicrobial growth promoters by stimulating key populations of the GIT bacteria that can ferment these non-digestible compounds, producing various short chain fatty acids used by the animal. The objective of this study was to quantify the effects of a proprietary Saccharomyces cerevisiae fermentation product (XPC, Diamond V Mills, Inc., Cedar Rapids, IA) inclusion in nursery diets on the microbial diversity and growth performance of pigs before, during and after an oral challenge with Salmonella. Pigs (n= 40) were weaned at 21 d of age, blocked by body weight (BW) and assigned in a 2Ã 2 factorial arrangement consisting of diet (control or 0.2% XPC) and inoculation (broth or Salmonella). Diet had no effect on pig growth performance prior to inoculation; however, consumption of XPC altered the composition of the gastrointestinal microbial community resulting in increased growth performance prior to inoculation. After Salmonella infection, XPC altered the composition of the gastrointestinal microbial community resulting in increased (P < 0.05) populations of Bacteroidetes and Lactobacillus. Infection with Salmonella and treatment of the piglets with ceftiofur-HCl resulted in alterations to the species richness and abundance of key members of the GIT community. The addition of XPC to the diets of weaning pigs results in greater compensatory gains after infection with Salmonella and an increase in beneficial bacteria within the GIT. / Master of Science

Microbial community composition plays a vital role in soil biogeochemical cycling. Information that explains the biogeography of microorganisms is consequently necessary for predicting the timing and magnitude of important ecosystem services mediated by soil biota, such as decomposition and nutrient cycling. Theory developed to explain patterns in plant and animal distributions such as the prevalent relationship between ecosystem productivity and diversity may be successfully extended to microbial systems and accelerate an emerging ecological understanding of the "unseen majority." These considerations suggest a need to define the important mechanisms which affect microbial biogeography as well as the sensitivity of community structure/function to changing climatic or environmental conditions. To this end, my dissertation covers three data chapters in which I have 1) examined patterns in bacterial biogeography using gradients of environmental severity and productivity to identify changes in community diversity (e.g. taxonomic richness) and structure (e.g. similarity); 2) detected potential bacterial ecotypes associated with distinct soil habitats such as those of high alkalinity or electrical conductivity and; 3) measured environmental controls over the function (e.g. primary production, exoenzyme activity) of soil organisms in an environment of severe environmental limitations. Sampling was performed in the polar desert of Antarctica's McMurdo Dry Valleys, a model ecosystem which hosts microbially-dominated soil foodwebs and displays heterogeneously distributed soil properties across the landscape. Results for Chapter 2 indicate differential effects of resource availability and geochemical severity on bacterial communities, with a significant productivity-diversity relationship that plateaus near the highest observed concentrations of the limiting resource organic carbon (0.30mg C/g soil). Geochemical severity (e.g. pH, electrical conductivity) primarily affected bacterial community similarity and successfully explained the divergent structure of a subset of samples. 16S rRNA amplicon pyrosequencing further revealed in Chapter 3 the identity of specific phyla that preferentially exist within certain habitats (i.e. Acidobacteria in alkaline soils, Nitrospira in mesic soils) suggesting the presence of niche specialists and spatial heterogeneity of taxa-specific functions (i.e. nitrite oxidation). Additionally, environmental parameters had different explanatory power towards predicting bacterial richness at varying taxonomic scales, from 57% of phylum-level richness with pH to 91% of order- and genus-level richness with moisture. Finally, Chapter 4 details a simultaneous sampling of soil communities and their associated ecosystem functions (primary productivity, enzymatic decomposition) and indicates that the overall organic substrate diversity may be greater in mesic soils where bacterial diversity is also highest, thus a potentially unforeseen driver of community dynamics. I also quantified annual rates of soil production which range between 0.7 - 18.1g C/m2/yr from the more arid to productive soils, respectively. In conclusion, the extension of biogeographical theory for macroorganisms has proven successful and both environmental severity and resource availability have obvious (although different) effects on the diversity and composition of soil microbial communities. / Ph. D.

Antibiotic resistance is ubiquitous due to high usage of antibiotics and the capability of bacteria to transfer genes both horizontally and vertically. While this has dire implications for human health, the potential to disturb microbial communities and ecosystem functions they regulate is under appreciated. Antibiotics are commonly used in the livestock sector, accounting for 80% of antibiotic use domestically. This dissertation addresses three facets of this problem. Chapter 2 is a nation-wide survey of antibiotic resistance at dairy operations, aimed at understanding how ecosystem function is affected in situ. Chapter 3 describes a field-experiment, seeking to determine whether antibiotics have effects beyond soil through impacts on plant-microbe-soil feedbacks, thus potentially altering terrestrial ecosystem function. Chapter 4 investigates how rising global temperature interacts with antibiotic exposure through a microcosm-incubation experiment. These multiple stressors (i.e. temperature and antibiotics) could alter microbial community composition or physiology with repercussions on function. Additionally, chapter 4 seeks to determine whether microbes acclimate to continued antibiotic exposure. In chapter 2 I present evidence that increased antibiotic resistant gene (ARG) abundance with exposure to antibiotics and manure, and a correlation between ARGs and microbial stress. This increase in microbial stress results in elevated soil carbon loss. Chapter 3 shows that antibiotic exposure can change plant function – presumably through impacts on rhizospheric microbial community composition. Plants assimilate more nitrogen, but more carbon is lost from the system overall seemingly due to plant-soil-microbe tradeoffs. Chapter 4 shows a temporally dependent temperature–antibiotic interactive effect. Initially, pirlimycin increased microbial respiration at high temperatures, however this diminishes with time. Additional studies of microbial respiration at a range of temperatures show that microbial acclimation to antibiotic exposure may be taking place. However, interactive effects of high temperature and antibiotics appear to inhibit active microbial biomass production. Possible explanations to both of these patterns are the underlying differences in microbial community composition, specifically the fungal:bacterial. My results show that antibiotics not only lead to increased ARG abundance, but also have wide ranging effects on communities and ecosystem processes that are likely to be compounded in the face of global change. / Ph. D. / Antibiotic resistance is becoming ubiquitous. While implications for human health are dire, underappreciated are the potential effects on environmental microbes, given that microbes are drivers of ecosystem function. Antibiotics are commonly used in livestock production, accounting for 80% of antibiotic use domestically, with a substantial proportion of the administered antibiotics passing through livestock while still functional. Therefore understanding how antibiotics may be impacting livestock-associated soils is critical. This dissertation is divided into three data-driven chapters, each addressing a facet of this question. In chapter 2 I show that antibiotic exposure can increase microbial stress and decrease microbial efficiency. This reduction in microbial efficiency results in increased soil carbon loss. In chapter 3 I show that antibiotic exposure can change carbon and nitrogen cycling in plants, presumably through impacts on root-associated microbial composition. Plants assimilated more nitrogen, but more carbon was lost from the system overall, when soil was exposed to manure from cattle administered the antibiotic pirlimycin. Chapter 4 describes an interactive effect between temperature and antibiotic exposure, however, this effect appears to diminish with time. The pirlimycin treatment increased microbial respiration at high temperatures, however this effect was not observed in the second year of v the field portion of this study. Additional experimentation showed some evidence of microbial acclimation to multiple stressors. However other evidence described within this chapter paints a different picture, as interactive effects of high temperature and antibiotics appeared to inhibit active microbial biomass production. Possible explanations to both of these patterns are the underlying differences in microbial community composition, specifically broad differences in the ratio of fungi to bacteria. Therefore antibiotics not only lead to reduced microbial efficiency, but also have wide ranging effects on communities and ecosystem processes that are likely to be compounded in the face of global change.