Thesis advisor: Heather Craig Olins / Microbes play a critical role in the Earth’s ecosystems, and freshwater microbial communities are underappreciated players in biogeochemical cycles. Vernal pools are ecologically important habitats that are particularly sensitive to global warming. Microbial communities in vernal pools and other freshwater wetlands are both critical to supporting life on Earth as well as incredibly vulnerable to climate change. This thesis describes for the first time microbial community composition in freshwater wetlands in Newton, Massachusetts. Beta diversity analysis reveals that sites host distinct microbial communities, something not always seen at these spatial scales. Sediment samples from the Bare Pond vernal pool were dominated by Acidobacteria, Actinobacteria, Alphaproteobacteria, and Chloroflexi. Analysis of surface vs. subsurface sediment samples reveal taxonomic patterns that cross multiple sites. These findings are a first step towards better understanding ecologically important microbial activity in these local sites, and freshwater wetlands more broadly. / Thesis (BS) — Boston College, 2020. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biology.

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The diversity of microorganisms and the influence of their enzymatic activities in soil are critical to the maintenance of good soil health. Changes in these parameters may be the earliest predictors of soil quality changes, potentially indicating anthropogenic influences. The goal of this study was to investigate the soil microbial diversity and function of grasslands along an urbanization gradient. Soil samples were collected in the Potchefstroom municipal area, South Africa, at specific sites. Sampling sites were described as urban, suburban and rural - according to the V-I-S (Vegetation-Impervious surface-Soil) model of Ridd (1995). Soil samples were collected over a warmer, wet season (May) and a colder, dry season (August) over two years (2007 and 2008). Collected soil samples were characterised using certain physical and chemical parameters. Plant species composition and abundance were determined at each site, along with basic site data (soil compaction, percentage ground cover, percentage bare ground, percentage organic material present). The Shannon-Weaver diversity index was used to calculate biodiversity values for all the investigated sites regarding collected plant species composition. The microbial component of the soil was quantified and characterized using culture-dependent and culture-independent techniques. Culture-dependent techniques included the investigation of the aerobic heterotrophic bacteria and fungi. Organisms were plated out on different media, and the bacterial component was broadly grouped using morphology. Dominant organisms were identified by sequencing of PCR amplified 16S ribosomal DNA fragments. Shannon-Weaver index for bacterial diversity was determined for each of the sites. Denaturing gradient gel electrophoresis (DGGE) profiling of selected bacterial communities were also conducted. Microbial community function was determined using enzyme assays of five major groups of enzymes, namely (i) dehydrogenase; (ii) β-glucosidase; (iii) acid phosphatase, (iv) alkaline phosphatase and (v) urease. Plant species results were then brought into context with microbiological diversity and functionality results using multivariate statistics. Physical and chemical parameters of the collected soil samples revealed patterns present along the urbanization gradient. The pH values were mostly higher in the sub-urban and urban sites than in the rural sites. Electrical conductivity values were generally highest in the sub-urban sites. Plant species composition revealed trends along the urbanization gradient. Ordinations clearly grouped the plant species into rural, sub-urban and urban groups regarding plant species composition. Rural sites had the highest number of plant species. Shannon-Weaver values regarding the plant diversity supported the plant species composition data indicating higher plant diversity in the rural areas, followed by the sub-urban and the urban areas. Plant structural data indicated that forbs were most numerous in the rural sites, and less so in the urban sites. Higher average aerobic heterotrophic bacterial levels were present in the urban soil samples. The bacterial levels were lower in the sub-urban and rural soil samples. Subsequent identification of the dominant bacteria in the soil samples revealed organisms of the genus Bacillus dominated the aerobic heterotrophic bacterial communities in the soil samples. Bacillus species dominated the soil samples along the urbanization gradient. Shannon-Weaver indices based on culture-dependent methods indicated that urban sites had the highest biodiversity. These results could have been exaggerated, because of an overestimation of the number of bacterial morphotypes present in samples. Fungal levels were higher in the soil from samples collected at the rural samples sites. The culture-independent method (DGGE) was not optimized and inconclusive results were obtained. Enzyme assays revealed that potential dehydrogenase, β-glucosidase and urease activity followed a trend along the urbanization gradient, with urban samples registering the highest values and rural sites the lowest. Enzymes involved in carbohydrate catabolism (β-glucosidase and dehydrogenase) registered significantly higher potential activity in urban sites than the sub-urban and rural sites. The results could indicate that urban sites have the potential to lose carbon at higher rates than the rural sites. This aspect may need further investigation. Higher potential urease activity could indicate higher N-cycling in the urban soil environment. Ordination results for soil-, plant- and microbial diversity as well as microbial functionality indicated certain trends along the urbanization gradient. Plant species composition and structure data indicated that urbanization has a definite effect on the plant communities in the urban ecosystem. Results regarding aerobic heterotrophic bacteria populations and potential enzyme activity of the dehydrogenase, β-glucosidase (both active in the carbon cycle) and urease (active in the nitrogen cycle) illustrated clear trends along the urbanization gradient. In conclusion, results indicated that urbanization has an effect on plant species composition, and the population and function of aerobic heterotrophic bacteria and the fungal population. Furthermore, this study demonstrated the potential of using microbial diversity and activity as tools to investigate carbon utilization and storage along an urban-rural gradient. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2011

The diversity of microorganisms and the influence of their enzymatic activities in soil are critical to the maintenance of good soil health. Changes in these parameters may be the earliest predictors of soil quality changes, potentially indicating anthropogenic influences. The goal of this study was to investigate the soil microbial diversity and function of grasslands along an urbanization gradient. Soil samples were collected in the Potchefstroom municipal area, South Africa, at specific sites. Sampling sites were described as urban, suburban and rural - according to the V-I-S (Vegetation-Impervious surface-Soil) model of Ridd (1995). Soil samples were collected over a warmer, wet season (May) and a colder, dry season (August) over two years (2007 and 2008). Collected soil samples were characterised using certain physical and chemical parameters. Plant species composition and abundance were determined at each site, along with basic site data (soil compaction, percentage ground cover, percentage bare ground, percentage organic material present). The Shannon-Weaver diversity index was used to calculate biodiversity values for all the investigated sites regarding collected plant species composition. The microbial component of the soil was quantified and characterized using culture-dependent and culture-independent techniques. Culture-dependent techniques included the investigation of the aerobic heterotrophic bacteria and fungi. Organisms were plated out on different media, and the bacterial component was broadly grouped using morphology. Dominant organisms were identified by sequencing of PCR amplified 16S ribosomal DNA fragments. Shannon-Weaver index for bacterial diversity was determined for each of the sites. Denaturing gradient gel electrophoresis (DGGE) profiling of selected bacterial communities were also conducted. Microbial community function was determined using enzyme assays of five major groups of enzymes, namely (i) dehydrogenase; (ii) β-glucosidase; (iii) acid phosphatase, (iv) alkaline phosphatase and (v) urease. Plant species results were then brought into context with microbiological diversity and functionality results using multivariate statistics. Physical and chemical parameters of the collected soil samples revealed patterns present along the urbanization gradient. The pH values were mostly higher in the sub-urban and urban sites than in the rural sites. Electrical conductivity values were generally highest in the sub-urban sites. Plant species composition revealed trends along the urbanization gradient. Ordinations clearly grouped the plant species into rural, sub-urban and urban groups regarding plant species composition. Rural sites had the highest number of plant species. Shannon-Weaver values regarding the plant diversity supported the plant species composition data indicating higher plant diversity in the rural areas, followed by the sub-urban and the urban areas. Plant structural data indicated that forbs were most numerous in the rural sites, and less so in the urban sites. Higher average aerobic heterotrophic bacterial levels were present in the urban soil samples. The bacterial levels were lower in the sub-urban and rural soil samples. Subsequent identification of the dominant bacteria in the soil samples revealed organisms of the genus Bacillus dominated the aerobic heterotrophic bacterial communities in the soil samples. Bacillus species dominated the soil samples along the urbanization gradient. Shannon-Weaver indices based on culture-dependent methods indicated that urban sites had the highest biodiversity. These results could have been exaggerated, because of an overestimation of the number of bacterial morphotypes present in samples. Fungal levels were higher in the soil from samples collected at the rural samples sites. The culture-independent method (DGGE) was not optimized and inconclusive results were obtained. Enzyme assays revealed that potential dehydrogenase, β-glucosidase and urease activity followed a trend along the urbanization gradient, with urban samples registering the highest values and rural sites the lowest. Enzymes involved in carbohydrate catabolism (β-glucosidase and dehydrogenase) registered significantly higher potential activity in urban sites than the sub-urban and rural sites. The results could indicate that urban sites have the potential to lose carbon at higher rates than the rural sites. This aspect may need further investigation. Higher potential urease activity could indicate higher N-cycling in the urban soil environment. Ordination results for soil-, plant- and microbial diversity as well as microbial functionality indicated certain trends along the urbanization gradient. Plant species composition and structure data indicated that urbanization has a definite effect on the plant communities in the urban ecosystem. Results regarding aerobic heterotrophic bacteria populations and potential enzyme activity of the dehydrogenase, β-glucosidase (both active in the carbon cycle) and urease (active in the nitrogen cycle) illustrated clear trends along the urbanization gradient. In conclusion, results indicated that urbanization has an effect on plant species composition, and the population and function of aerobic heterotrophic bacteria and the fungal population. Furthermore, this study demonstrated the potential of using microbial diversity and activity as tools to investigate carbon utilization and storage along an urban-rural gradient. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2011

In the Central Valley of Costa Rica in the Department of Heredia, I investigated the soil chemical properties and microbial communities under four native shade tree species in a coffee agroforestry system. In the second year of the study, Effective Microorganisms, a microbial inoculant, was applied to examine its effect on soil chemistry. The shade tree species included in this study were Anonna muricata L., Diphysa americana Mill., Persea americana Mill., and Quercus spp. L. Plots measured 20 by 30 meters and were replicated three times for each shade tree species except for Quercus spp., which only had two replications. Twelve composite soil samples were collected from each plot in 2008 and again in 2009, and twelve composite foliar samples were taken from the coffee plants in each plot in 2008. The results of this study indicated that the species of native shade tree had a significant effect on soil ammonium-N, nitrate-N, total dissolved nitrogen and magnesium. Sun or shade position had a significant effect on dissolved organic nitrogen and dissolved organic carbon. The species of native shade tree also had a significant effect on the composition of soil microbial communities. PLFA analysis revealed a significant difference in soil fungi abundance in soil samples from Annona plots relative to those from Persea plots. Effective microorganisms in combination with the tree species, as well as in combination with species and sun or shade position, had a significant interaction effect on soil ammonium-N, with the EM-treated plots showing higher concentrations of soil ammonium-N. There was a significant positive correlation between soil pH and foliar calcium, as well as soil dissolved organic nitrogen and foliar %N, at p< 0.01. This study suggests that Quercus spp. is a tree species that may help to regulate the cycling of nitrogen in the coffee agroecosystem. Annona muricata appears to inhibit the action of some fungal species and may reduce the occurrence of fungal pathogens in the soil, although the present study did not explore this issue. Although Diphysa americana is a legume, it does not appear to increase the amount of soil nitrogen in the vicinity of the coffee plants themselves. All four tree species in this study improve coffee soils by increasing soil concentrations of dissolved organic nitrogen and dissolved organic carbon. Coffee yield data and long term observations on the health of the coffee plants would clarify whether one of these species is particularly beneficial, from an agronomic perspective, for the productivity of this coffee agroecosystem.

Proper management of soil organic matter (SOM) contributes to increasing plant productivity and reducing dependency on mineral fertilizers. Organic matter is widely regarded as a vital component of a healthy soil as it plays an important role in soil physical, chemical and biological fertility. Plant residues are the primary source of SOM. Therefore, proper SOM management requires a better understanding of plant residue decomposition kinetics in order to synchronize nutrient release during decomposition and plant uptake and prevent nutrient losses. In natural and managed ecosystems, residues are added frequently to soil, in the form of dead roots and litter fall of plant species with different C/N ratios. However, in most studies on residue decomposition, residues with different C/N ratios are added once and the effect of the presence of plants on residue decomposition is rarely investigated. In this project, four experiments were carried out with different objectives in order to close these knowledge gaps. The aim of the first experiment was to investigate the effect of frequent wheat residue addition on C mineralization and N dynamics. The experiment consisted of five treatments with different frequency of residue addition (2% w/w of wheat residues in total): once (100%W), every 16 days (25%), every 8 days (12.5%) or every 4 days (6.25%) and noresidue addition (control) with four replicates. The results showed that increasing frequency of low-N wheat residue addition increased C mineralization. Compared to 100%W, cumulative respiration per g residue at the end of the incubation (day 80) was increased by 57, 82 and 92% at 25%W, 12.5%W and 6.25%W, respectively. Despite large increases in cumulative respiration, frequent residue addition did not affect inorganic N or available N concentrations, microbial biomass C and N or soil pH. It is concluded that experiments with single residue additions may underestimate residue decomposition rates in the field because with several additions, soil microbes respire more of the added C (and possibly native soil C) per unit biomass but that this does not change their N requirements or the microbial community composition. In the second experiment, the effect of mixing of high and low C/N residues at different times during incubation was investigated. There were 4 addition times; 25% of a total of 2% (w/w) residue was added either as wheat (high C/N) or lupin (low C/N) residue. Wheat residue was added to lupin residues on days 16 (LW-16), 32 (LW-32) or 48 (LW-48). Additional treatments were 100%L (added 25% of lupin residues on days 0, 16, 32 and 48) and 100%W (added 25% of wheat residues on days 0, 16, 32 and 48) and 0% (the control) with four replicates. Adding high C/N residues into decomposing low C/N ratio residue strongly decreased the respiration rate compared to the addition of low C/N residues, and lowered the availability of inorganic N, but significantly increased soil pH and altered microbial community composition. By the end of the incubation on day 64, the cumulative respiration of LW-16, LW-32 and LW-48 was similar and approximately 30% lower than in the treatment with only lupin residue addition. The third experiment studied the effect of spatial separation of high and low C/N residues on decomposition and N mineralization. Each microcosm consisted of two PVC caps of 70 mm diameter and 20 mm height with the open end facing each other separated by a 30μm mesh. The caps were filled with soil mixed with either low or high C/N residue with three replicates. Contact of high and low C/N residues led to an increase in the decomposition rate of the high C/N residues at the interface whereas it decreased it in the low C/N residues. The results showed that N and soluble C compounds moved from the easily decomposable residues into the surrounding soil, thereby enhancing microbial activity, increasing inorganic N and significantly changing soil pH in the layer 0-5 mm from the interface compared to the 5-10 mm layer of the high C/N residues, whereas the movement of soluble C and N to high C/N residues decreased the decomposition of the low C/N residues. The final experiment investigated the effect of living plants on decomposition of high and low C/N residues. Wheat was grown in pots with a 30 μm mesh at the bottom. After a root mat had formed (>50% root coverage), a PVC cap with soil with high and low C/N residues (2% w/w) was placed against the mesh. The presence of plant roots significantly increased the respiration rate, N immobilization and increased the soil pH in the 0-5 mm layer in the first 4 days compared to the 5-10 mm layer. This enhanced microbial activity (and probably microbial biomass) can be explained by root exudates. The microbial community composition of plant treatments differed significantly from treatments without plants and the effect was greater in the immediate vicinity of the roots. / Thesis (M.Ag.Sc.) -- University of Adelaide, School of Earth and Environmental Sciences, 2009

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The rapid spread of antibiotic resistance is a major global health concern, jeopardizing the successful treatment of bacterial infections. Natural environments are potential hotspots for the emergence and spread of antibiotic resistance genes (ARGs). Among these potential hotspots, aquatic ecosystems are of particular concern, as they receive wastewater containing antibiotic-resistant bacteria and ARGs originating from both human and animal sources. Several key questions remain to be addressed. What is the fate of ARGs in receiving water bodies? What are implications of environmental ARGs for human health? How does wastewater discharge impacts aquatic microbial communities with regard to the overall ecosystem well-being? The objective of this work was to investigate the impact of wastewater, seasonal variations, and the riverine compartment on the prevalence of selected ARGs and the composition of natural microbial communities in a near-pristine river, and to specifically assess the effect of antibiotics on riverine microbial communities. Quantitative real-time PCR was used to monitor the abundance of three indicator ARGs (sul1 and sul2, conferring resistance against sulfonamide antibiotics, and intI1, a marker for anthropogenic pollution) upstream and downstream from a wastewater treatment plant (WWTP). Furthermore, the impact of WWTP effluent on the riverine microbial community was examined through 16S rRNA amplicon sequencing. Wastewater was the main source of all three target genes and significantly altered the microbial community in the river. The surface water compartment served as a dissemination route for ARGs, with increased prevalence even 13 km downstream of the WWTP, particularly during the summer season when the proportion of wastewater in the river was high. Notably, riverbed biofilms served as a local reservoir for ARGs only at the discharge point, with little abundance of target genes further downstream. The sulfonamide antibiotic sulfamethoxazole (SMX) was persistent in both near-pristine and wastewater-impacted river water when introduced at a concentration of 12.5 µg/L, but had neglectable effects on the microbial community diversity. Interestingly, concentrations as high as 100 µg/L SMX induced a short-term increase in microbial activity in both surface water and biofilm compartment, as revealed by bulk and nanoscale measurements. Altogether, this work underscores the fundamental role of wastewater treatment in combating the environmental dissemination of antibiotic resistance.:Summary 1 Zusammenfassung 5 1 Introduction 9 1.1 Rundown of the global antibiotic resistance crisis 9 1.1.1 History of antibiotics 9 1.1.2 Emergence of antibiotic resistance 9 1.1.3 Integrons as vehicles for antibiotic resistance 10 1.1.4 Risks related to environmental antibiotic resistance 12 1.2 Fate of antibiotic resistance genes in the aquatic environment 14 1.2.1 Genetic indicators for antibiotic resistance 14 1.2.2 River surface water compartment as dissemination route for antibiotic resistance 15 1.2.3 River biofilm compartment as reservoir for antibiotic resistance 17 1.3 Impact of antibiotics on aquatic microbial communities 18 1.4 Fate and effect of sulfamethoxazole in surface waters 20 2 Scope of the thesis 22 3 Main findings and scientific implications 24 3.1 Fate of antibiotic resistance genes after wastewater discharge into a near-pristine river 24 3.1.1 Wastewater is the primary source for aquatic antibiotic resistance 24 3.1.2 Drought increases the antibiotic resistance prevalence in surface waters 25 3.1.3 Riverbed biofilms serve as local reservoirs for antibiotic resistance genes 26 3.2 Anthropogenic pollution is the key driver for microbial community alteration 26 3.3 Sulfamethoxazole increases the microbial activity of aquatic microbial communities 27 4 Conclusions and future perspective 29 5 References 31 6 Publications 43 6.1 Publication 1 43 6.2 Publication 2 56 6.3 Publication 3 69   7 Appendix 94 7.1 Declaration of independent work 94 7.2 List of publications and conference contributions 95 7.2.1 Publications 95 7.2.2 Conference contributions 96 7.3 Contribution of Co-authors 97 7.4 Curriculum vitae 101 7.5 Acknowledgements 104 7.6 Supplementary Material 105 7.6.1 Supplementary Material for Publication 1 105 7.6.2 Supplementary Material for Publication 2 118 7.6.3 Supplementary Material for Publication 3 125

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