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Increasing socio-economic growth and development of South Africa’s freshwater systems require continuous augmentation of water sources to meet the growing water requirements of communities and industries. Anthropogenic disturbances have caused the water quality of many freshwater systems to drastically deteriorate due to constant disposal of domestic, industrial, and agricultural waste into surface waters. Government agencies make use of biomonitoring programmes to effectively manage the countries’ freshwater resources. These programmes use a variety of biological indicators (e.g., macroinvertebrates, fish, diatoms and algal species) and physico-chemical variables to determine the state of the environment. However, attempts to use microbial community structures as bioindicators of anthropogenic perturbations are greatly neglected. This study used molecular techniques (PCR-DGGE and 454-pyrosequencing) and multivariate analysis to develop a robust monitoring technique to determine the impacts of environmental disturbances on bacterial community compositions in river systems in the North West Province. Significant contributions made by this project included the establishment of a bacterial diversity framework for South African freshwater systems that are impacted by a variety of anthropogenic activities (e.g., urban and informal settlements, agriculture and mining). Furthermore, case studies demonstrated the prevalence of specific taxa at polluted sites, as well as positive and negative associations between taxa and environmental variables and pollutants. Finally, biogeochemical cycles could be partially matched to bacterial community structures in river systems. The first part of the project included a pilot study that investigated bacterial structures in a segment of the Vaal River in response to environmental parameters using molecular techniques and multivariate analysis. The most important observations made during this study included the generation of a larger bacterial diversity dataset by pyrosequencing compared to PCR-DGGE. In addition, metagenomic and multivariate analyses provided clues about potential biogeochemical roles of different taxa. The second and third part of the project included two case studies that investigated bacterial communities in the Mooi River and Wonderfonteinspruit in response to environmental activities. Both these systems are impacted by a variety of external sources such as urban and informal settlements, agriculture, and mining. The results demonstrated that perturbations nearby the Mooi River and Wonderfonteinspruit caused the overall water quality to deteriorate which in turn had a profound impact on bacterial community composition. Bacterial community structures at reference/control sites (Muiskraal and Turffontein dolomitic eye) had overall high species diversity (richness and evenness), whereas polluted sites showed lower species diversity and were dominated by the Beta- and Gammaproteobacteria, Bacteroidetes, and Verrucomicrobia. In addition, various potential pathogens (e.g. Eschirichia/Shigella, Legionella, Staphylococcus, Streptococcus etc.) were identified at impacted sites. Multivariate analysis suggested that bacterial communities and certain taxa (Malikia, Algoriphagus, Rhodobacter, Brevundimonas and Sphingopyxis) at polluted sites were mainly impacted by temperature, pH, nutrient levels, and heavy metals. Finally, the proportion of nitrogen and sulphur bacteria corresponded well with the nitrogen and sulphur levels measured in the Wonderfonteinspruit. Based on these results, it was concluded that bacterial community structures might provide a good indicator of anthropogenic disturbances in freshwater systems and may be incorporated into biomonitoring programs. / PhD (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Increasing socio-economic growth and development of South Africa’s freshwater systems require continuous augmentation of water sources to meet the growing water requirements of communities and industries. Anthropogenic disturbances have caused the water quality of many freshwater systems to drastically deteriorate due to constant disposal of domestic, industrial, and agricultural waste into surface waters. Government agencies make use of biomonitoring programmes to effectively manage the countries’ freshwater resources. These programmes use a variety of biological indicators (e.g., macroinvertebrates, fish, diatoms and algal species) and physico-chemical variables to determine the state of the environment. However, attempts to use microbial community structures as bioindicators of anthropogenic perturbations are greatly neglected. This study used molecular techniques (PCR-DGGE and 454-pyrosequencing) and multivariate analysis to develop a robust monitoring technique to determine the impacts of environmental disturbances on bacterial community compositions in river systems in the North West Province. Significant contributions made by this project included the establishment of a bacterial diversity framework for South African freshwater systems that are impacted by a variety of anthropogenic activities (e.g., urban and informal settlements, agriculture and mining). Furthermore, case studies demonstrated the prevalence of specific taxa at polluted sites, as well as positive and negative associations between taxa and environmental variables and pollutants. Finally, biogeochemical cycles could be partially matched to bacterial community structures in river systems. The first part of the project included a pilot study that investigated bacterial structures in a segment of the Vaal River in response to environmental parameters using molecular techniques and multivariate analysis. The most important observations made during this study included the generation of a larger bacterial diversity dataset by pyrosequencing compared to PCR-DGGE. In addition, metagenomic and multivariate analyses provided clues about potential biogeochemical roles of different taxa. The second and third part of the project included two case studies that investigated bacterial communities in the Mooi River and Wonderfonteinspruit in response to environmental activities. Both these systems are impacted by a variety of external sources such as urban and informal settlements, agriculture, and mining. The results demonstrated that perturbations nearby the Mooi River and Wonderfonteinspruit caused the overall water quality to deteriorate which in turn had a profound impact on bacterial community composition. Bacterial community structures at reference/control sites (Muiskraal and Turffontein dolomitic eye) had overall high species diversity (richness and evenness), whereas polluted sites showed lower species diversity and were dominated by the Beta- and Gammaproteobacteria, Bacteroidetes, and Verrucomicrobia. In addition, various potential pathogens (e.g. Eschirichia/Shigella, Legionella, Staphylococcus, Streptococcus etc.) were identified at impacted sites. Multivariate analysis suggested that bacterial communities and certain taxa (Malikia, Algoriphagus, Rhodobacter, Brevundimonas and Sphingopyxis) at polluted sites were mainly impacted by temperature, pH, nutrient levels, and heavy metals. Finally, the proportion of nitrogen and sulphur bacteria corresponded well with the nitrogen and sulphur levels measured in the Wonderfonteinspruit. Based on these results, it was concluded that bacterial community structures might provide a good indicator of anthropogenic disturbances in freshwater systems and may be incorporated into biomonitoring programs. / PhD (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

<p>Rising atmospheric CO2 concentration, currently about 390 ppm, causes climate change and is expected to reach 500 ppm or higher this century due to human activities. Soils are the largest terrestrial pool of carbon, and changes in soil carbon storage due to plant and microbial activities could affect atmospheric CO2 levels. This dissertation studies soil carbon and microbial responses to an experimental preindustrial-to-future CO2 gradient (250-515 ppm) in a grassland ecosystem. Two contrasting soil types are studied in the gradient, providing insight on how natural ecosystem variation modifies CO2 effects.</p><p>Although total soil organic carbon (SOC) did not change with CO2 treatment after four growing seasons, fast-cycling SOC pools did respond to CO2, particularly in the black clay soil. Microbial biomass increased 18% and microbial activity increased 30% across the CO2 gradient in the black clay, but neither factor changed with CO2 in the sandy loam. Similarly a one-year laboratory soil incubation showed that a fast-cycling SOC pool increased 75% across the CO2 gradient in the black clay. Size fractionation of SOC showed that coarse POM-C, the youngest and most labile fraction, increased four-fold across the CO2 gradient in the black clay, while it increased 50% across the gradient in the sandy loam. CO2 enrichment in this grassland increased the fast-cycling soil organic carbon pool as in other elevated CO2 studies, but only in the black clay soil.</p><p>CO2 also induced changes in microbial community composition, and we explored the functional consequences in a microcosm experiment. Soil collected in the third growing season of CO2 treatment was used to inoculate Indiangrass seedlings grown in the lab. The elevated CO2 soil inoculum had higher microbial biomass C/N (C/N = 21) than the subambient CO2 soil inoculum (C/N = 16), suggesting a difference in community composition. Mean plant height in elevated CO2 soil inoculum (475 ppm) was 57% greater than in subambient CO2 soil inoculum (300 ppm), but the difference was not statistically significant. Similarly, total leaf N from plants in elevated CO2 soil was 28% greater on average than in subambient CO2 soil, but not significantly different. CO2-induced microbial effects on plant growth were either negligible or occurred at finer microbial taxonomic levels, making them difficult to resolve at the whole-community level.</p><p>Soil fungi decompose soil organic matter, and studying fungal community responses to CO2 could improve our understanding of soil carbon responses. We studied fungal communities in the CO2 gradient using Sanger sequencing and pyrosequencing of rDNA. As in our soil C study, fungal community responses to CO2 were mostly linear, and occurred mostly in the black clay soil. Fungal species richness increased linearly with CO2 treatment in the black clay. The relative abundance of Chytridiomycota (chytrids) increased linearly with CO2 in the black clay, while the relative abundance of Glomeromycota (arbuscular mycorrhizal fungi) increased linearly with CO2 in the sandy loam. Increased labile C availability at elevated CO2 and/or decreased inorganic N may explain the increase in fungal species richness and Chytridiomycota abundance in the black clay, while increased P limitation may explain the stimulation of Glomeromycota at elevated CO2 in the sandy loam. Across both soils, fungal species richness increased linearly with soil respiration, an index of decomposition rate (p = 0.01, R2 = 0.46). Adding fungal species may have improved decomposition efficiency, but it is also possible that species richness and decomposition increased due to another factor such as C quantity. Soil type strongly structured both fungal community and arbuscular mycorrhizal fungal community composition.</p><p>Together, these studies suggest that soil C and fungal community responses to CO2 were mostly linear, and were most apparent in the black clay soil. Soil type strongly influenced fungal community composition as well as which phyla responded to CO2. Therefore, soil type could be a useful addition to predictions of soil carbon and microbial responses to future CO2 levels.</p> / Dissertation

The fern genus Pteridium comprises a number of closely related species distributed throughout the world. Collectively they are called bracken ferns and have historically been treated as a single species, Pteridium aquilinum. Bracken is notorious as a toxic weed that colonizes open fields and poisons livestock. Bracken is also easily cultured and has become one of the most intensively studied ferns. Bracken has been used as a model system for the study of the fern life cycle, fern gametophyte development, the pheromonal mechanism of sex determination, toxicology, invasion ecology, and climate change. This dissertation places bracken within a global evolutionary perspective and establishes bracken as an emerging system for evolutionary genomics in ferns. Bracken samples from around the world were examined for chloroplast DNA variation to infer historical phylogenetic and biogeographic evolutionary events. New high-throughput DNA sequencing technologies and bioinformatic approaches were used to determine the complete chloroplast genome sequence in bracken, to identify novel RNA editing sites in chloroplast transcripts, and to identify gene sequences that are expressed in the gametophyte stage of the fern life cycle. These data represent an important genomic resource in ferns and were examined within a functional and evolutionary perspective. Several novel approaches and analyses were developed in the course of this research. Results presented in this dissertation provide novel insights into fern biology and land plant evolution.

Carrion decomposition is an essential ecosystem function as it is an important component of nutrient cycling. Carrion decomposition has primarily been attributed to insect consumption, with little attention given to microbial communities or their potential interactions with insects. The first objective was to use passive insect-trapping methods to assess primary colonizer communities on swine carcasses between two treatments: 1) carrion with access to insects and 2) carrion excluded from insect access for five days using exclusion cages. Despite similarities between succession patterns within each treatment, carcasses initially exposed to insects had significantly fewer insect taxa. Therefore, collections of adult insect communities associated with carrion are promising as an indication of whether or not there has been a delay in insect colonization of a resource. There has yet to be a study documenting bacterial communities during carrion decomposition. The second objective was to describe bacterial community succession and composition during decomposition in the presence and absence of naturally occurring insects. Total genomic DNA was used to identify bacterial community composition via a modified bacterial tagged encoded FLX amplicon pyrosequencing. I obtained 378,904 sequences and documented distinct bacterial community successional trajectories associated with insect access and exclusion carcasses. By the fifth day of decomposition, Proteus was the dominant (72%) bacterial genus on exclusion carcasses while Psychrobacillus (58%) and Ignatzschineria (18%) were dominant bacterial genera on insect carcasses. These data are the first to document bacterial community composition and succession on carrion. My final objective was to assess microbial community function in response to carrion insect colonization using metabolic profiling. I characterized microbial community metabolic function in the presence and absence of the primary necrophagous insects. I documented significant microbial community metabolic profile changes during active decomposition of carcasses. Mean carcass microbial community metabolic function with insect access continuously decreased over decomposition during both field seasons. Thus demonstrating microbial metabolic activity may have discriminatory power to differentiate early and late stages of decomposition. Overall, my data contributes to an understudied area of microbial research important to organic matter decomposition, forensic entomology, and microbial-insect ecological interactions.

From acid seeps and deep-sea thermal vents to glacial ice and hypersaline lakes, extreme environments contain relatively simplified communities consisting of extremophiles that have evolved to survive and thrive under adverse abiotic conditions. In more neutral environments, microorganisms use dormancy as a common life history strategy to weather temporal fluctuations of resources or stresses until more 'optimal' conditions are present. It is unclear if dormancy is an essential survival mechanism for microorganisms in extreme environments; however, recent studies suggest that extreme environments may create stable conditions for extremophiles to the extent that dormancy is of less ecological importance. Using lake salinity levels as measurements of "extreme," we evaluated the dormancy of bacterial and archaeal phyla and lake chemistry in five hypersaline and five freshwater lakes across the western United States. Dormancy was calculated using targeted metagenomics to analyze 16S rDNA and rRNA tag sequences. It was hypothesized that bacteria and archaea in hypersaline lake communities would exhibit lower levels dormancy than bacterial and archaeal communities in geologically similar freshwater lake controls. It was also hypothesized that microbial dormancy would decrease as the dominant extreme environmental variable increased in the lakes. As hypothesized, overall dormancy decreased at least 2-fold in hypersaline compared to freshwater lakes for both bacteria and archaea. Of the predominant phyla and subclasses, Firmicutes, Bacteroidetes, and Gammaproteobacteria each demonstrated at least a seven-fold decrease in dormancy in hypersaline lakes compared to freshwater lakes. Specifically, species within the genus Clostridium were responsible for 85% of the dormancy observed in the phylum Firmicutes. Also as hypothesized, microbial dormancy decreased as salinity increased in the lakes. Lower dormancy in hypersaline lakes correlated with increasing salinity while lower dormancy in freshwater lakes correlated with increasing total phosphorus levels. These results suggest that dormancy is a less common life history strategy for microorganisms in extreme environments; it is proposed that this is due to the relatively stable environment in hypersaline lakes and the reduced number of available microbial niches. These results also suggest that the dominant extreme stress (i.e., salinity) may override other driving factors in an environment to ultimately determine microbial community composition, diversity and richness.

Multiple mechanisms control bacterial community structure but two in particular, the "legacy" of past environmental conditions, and the "priming" of bacteria to respond to seasonal or reoccurring fluctuations in resources, have the potential to determine both bacterial communities, as well as, temporal shifts in active bacterial taxa. To begin to evaluate the legacy effects of resources on microbial communities, we added four limiting resources annually (i.e., water only; C-mannitol + water; N-NH4NO3 + water; and C, N + water) and measured shifts in bacterial community composition after seven years in a cold desert ecosystem in the McMurdo Dry Valleys, Antarctica. Further, to investigate the ecological significance of priming, we conducted a series of stable isotope probing experiments (i.e., 18O-DNA SIP with 18O-labeled water, 13C-DNA SIP with 13C-labeled mannitol, 15N-DNA with 15N- NH4NO3, and a combined C and N SIP) and characterized the responding (i.e., isotopically labeled) and seed bank (i.e., unlabeled) bacterial communities. We performed each of the SIPs in soil microcosms corresponding to a single resource manipulation (e.g., 13C-labeled mannitol in C addition soils). We hypothesized that all long-term additions of nutrients and water will lead to a distinct bacterial community—a legacy effect due to the nutrient and water impoverished state of Antarctica soils. We also hypothesized that the stronger the legacy effects demonstrated by a specific community the more adapted or primed bacterial species will be to take advantage of the resource and respond. As hypothesized, resource additions created distinct bacterial legacy but to different degrees among the treatments. The extent of the resource legacy effects was greatest in the CN, intermediate in water and N, and lowest in C communities relative to the control communities, suggesting that C induced changes in communities were intensified by tandem N additions and that water alone created a more distinct legacy than water and C additions combined. Contrary to our hypothesis, the stronger the legacy effects, the less adapted or primed the community was to take advantage of resource additions. For example, the CN treatment that induced the greatest effect on bacterial communities had the lowest number of species (20.9%) in common between the responding and seed bank communities. This inverse relationship may be due to only two species (i.e., Arthrobacter, Actinobacteria and Massilia, Betaproteobacteria) really being primed to take advantage of CN and these species constituting over 75% of the seed bank community. Water, N, and C additions had similar levels of priming with 38.4%, 41.4%, and 36.3% of the responding species being present in the seed bank community, respectively. But of these three treatments, only the priming with water resulted in a unique responding community, suggesting that water, a universal bacterial resource, was enough to prime bacteria. Furthermore, water generates the most diverse responding community of all the resources with stemming from all of the fourteen dominant phyla. We did find patterns of ecological coherence among the responders, especially in the major responders (i.e., responders that increased in relative recovery by at least ten-fold). These responders were predominantly found in only three phyla (i.e., Actinobacteria, Bacteriodetes, and Gammaproteobacteria) regardless of resource addition. Alternatively minor responders (i.e., responders that increased in relative recovery at least two-fold) were contained in fourteen different phyla with specific taxa stimulated by CN (i.e., Betaproteobacteria) and N and water (i.e., Deltaproteobacteria). Further, resource additions elicited responses from 37% of bacterial species with species specializing on a specific resource (e.g., Chloroflexi) or being a generalist (e.g., Planctomycetes and Gammaproteobacteria). Our results offer the first direct links between legacy and priming effects on bacterial community composition and demonstrate that these mechanisms are not always complimentary leading to the formation of similar communities but may both be essential to maintain the high levels of bacterial diversity. Further, all resources produced elicited responders that were either specialists of generalists demonstrating that even bacteria in the extreme environment of Antarctica respond to pulses of resources.

In aquatic environments the availability of nitrogen (N) generally limits primary production. N2-fixing prokaryotes (diazotrophs) can convert N2 gas into ammonium and provide significant input of N into the oceans. Cyanobacteria are thought to be the main N2-fixers but diazotrophs also include a wide range of heterotrophic bacteria. However, their activity and regulation in the water column is largely unknown. In this thesis the distribution, diversity, abundance, and activity of marine and estuarine heterotrophic diazotrophs was investigated. With molecular methods targeting the nifH gene, encoding the nitrogenase enzyme for N2 fixation, it was shown that diverse nifH genes affiliating with heterotrophic bacteria were ubiquitous in surface waters from ten marine locations world-wide and the estuarine Baltic Sea. Through enrichment cultures of Baltic Sea surface water in anaerobic N-free medium, heterotrophic N2 fixation was induced showing that there was a functional N2-fixing community present and isolates of heterotrophic diazotrophs were obtained. In Sargasso Sea surface waters, transcripts of nifH related to heterotrophic bacteria were detected indicating heterotrophic N2-fixing activity. Nitrogenase expression is thought to be highly regulated by the availability of inorganic N and the presence of oxygen. Low oxygen zones within the water column can be found in association with plankton. The presence of diazotrophs as symbionts of heterotrophic dinoflagellates was investigated and nifH genes related to heterotrophic diazotrophs rather than the cyanobacterial symbionts were found, suggesting that a symbiotic co-existence prevailed. Oxic-anoxic interfaces could also be potential sites for heterotrophic N2 fixation. The Baltic Sea contains large areas of anoxic bottom water. At the chemocline and in anoxic deep water heterotrophic diazotrophs were diverse, abundant and active. These findings extend the currently known regime of N2 fixation to also include ammonium-rich anaerobic waters. The results of this thesis suggest that heterotrophic diazotrophs are diverse and widely distributed in marine and estuarine waters and that they can also be active. However, limits in the knowledge on their physiology and factors which regulate their N2 fixation activity currently prevent an evaluation of their importance in the global marine N budget.

Master of Science / Department of Plant Pathology / Karen A. Garrett / Ari Jumpponen / Traditional fallow periods in the Bolivian highlands are being shortened in an effort to increase short-term crop yields, with potential long-term impacts on soil communities. Using 454-pyrosequencing, we characterized fungal and bacterial community responses to (1) the length of fallow period and (2) the presence of the plants Parasthrephia sp. or Baccharis sp. (both locally known as ‘thola’). Thola is widely considered by farmers as beneficial to soil health, although it is also frequently harvested as a source of fuel by farmers. Soils in one study area, Ancoraimes, had higher levels of organic matter, nitrogen and other macronutrients compared to the other study area, Umala. In our analyses, Ancoraimes soils supported more diverse fungal communities, whereas Umala had more diverse bacterial communities. Unexpectedly, the longer fallow periods were associated with lower fungal diversity in Umala and lower bacterial diversity in Ancoraimes. Fungi assigned to genera Verticillium, Didymella, and Alternaria, and bacteria assigned to genera Paenibacillus, Segetibacter, and Bacillariophyta decreased in abundance with longer fallow period. The presence of thola did not significantly affect overall soil fungal or bacterial diversity, but did increase the frequency of some genera such as Fusarium and Bradyrhizobium. Our results suggest that fallow period has a range of effects on microbial communities, and that the removal of thola from the fields impacts the dynamics of the soil microbial communities.