Thesis (MSc (Microbiology))--Stellenbosch University, 2008. / The soil environment is thought to contain a lot of the earth’s undiscovered biodiversity. The aim of this study was to understand the extent of microbial diversity in the unique ecosystem of the Western Cape’s fynbos biome. It is known that many processes give rise to this immense microbial diversity in soil. In addition the aim was to link microbial diversity with the soils physio-chemical properties as well as the plant community’s structure. Molecular methods especially automated ribosomal intergenic spacer analysis (ARISA) was used in the study. The most important property of environmental DNA intended for molecular ecology studies and other downstream applications is purity from humic acids and phenolic compounds. These compounds act as PCR inhibitors and need to be removed during the DNA extraction protocol. The fist goal in the study was to develop an effective DNA extraction protocol by using cationic locculation of humic acids. The combination of cationic flocculation with CuCl2 and the addition of PVPP and KCl resulted in a high yield of DNA, suitable for PCR amplification with bacterial and fungal specific primers. Determining the reproducibility and accuracy of ARISA and ARISA-PCR was important because these factors have an important influence on the results and effectiveness of these techniques. Primer sets for automated ribosomal intergenic spacer analysis, ITS4/ITS5, were assessed for the characterization of the fungal communities in the fynbos soil. The primer set delivered reproducible ARISA profiles for the fungal community composition with little variation observed between ARISAPCR’s. ARISA proved useful for the assessment and comparison of fungal diversity in ecological samples. The soil community composition of both fungal and bacterial groups in the Sand fynbos was characterized. Soil from 4 different Sand fynbos sites was compared to investigate diversity of eubacterial and fungal groups at the local as well as a the landscape scale. A molecular approach was used for the isolation of total soil genetic DNA. The 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified when performing bacterial ARISA from total soil community DNA (BARISA). Correspondingly, the internal transcribed spacers, ITS1, ITS2 and the 5.8S rRNA gene from the fungal rRNA operon were amplified when undertaking fungal ARISA (F-ARISA). The community structure from different samples and sites were statistically analysed. ARISA data was used to evaluate different species accumulation and estimation models for fungal and bacterial communities and to predict the total community richness. Diversity, evenness and dominance were the microbial communities were used to describe the extent of microbial iversity of the fynbos soils. The spatial ordination of the bacterial and fungal species richness and diversity was considered by determining the species area relationship and beta diversity of both communities. The correlation between the soil physio-chemical properties was determined. The plant community structure data was correlated with the fungal and the bacterial community structure. The results indicated that bacterial species numbers and diversity were continually higher at the local scale. Fungi however showed higher species turnover at the landscape scale. Bacterial community structure showed stronger links to the plant community structure whereas the fungi community structure conformed to spatial separation patterns. To further investigate the diversity of soil microbes the potential of genus specific primes was investigated. The genus Penicillium is widespread in the soil environment and the extent of its diversity and distribution is however not. For this reason Penicillium was chosen as a model organism. To expand the insight into the diversity of Penicillium species in the fynbos soil ecosystem, a rapid group specific molecular approach would be useful. Penicillium specific primers targeting the 18S rRNA ITS gene region were evaluated. Fungal specific primers ITS4 and ITS5, targeting the internal transcribed region (ITS) were used to target Penicillium specific in the soil sample. Nested PCR, using primer Pen-10 and ITS5, was then utilized to target Penicillium species specifically. The discrimination of Penicillium species was possible due to length heterogeneity of this gene region. Eight different peaks was detected in the soil sample with ARISA and eight different species could be isolated on growth media. The technique proved useful for the detection and quantification of Penicillium species in the soil.

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

In den vergangenen Jahren rückten Pflanzen-assoziierte Bakterien auf Grund ihrer Bedeutung für die Pflanzengesundheit und das ökologische Gleichgewicht zunehmend in den Fokus aktueller Forschungen. Trotz der stetig steigenden Zahl wissenschaftlicher Studien ist der Einfluss von Bewirtschaftungsmaßnahmen auf die Diversität dieser Bakteriengemeinschaften in Grünlandökosystemen ver-gleichsweise wenig untersucht. In dieser Studie haben wir neue und interessante Erkenntnisse über die Diversität von Pflanzen-assoziierten Bakterien in Grünlandökosystemen gewonnen. Sämtliche Untersuchungen dieser Arbeit wurden auf der GrassMan-Fläche in den Mittelgebirgslagen des Solling in Deutschland durchgeführt. Das GrassMan-Experiment wurde 2008 in einer Matrix von Wiesenplots schachbrett-artig auf historisch altem Grünland errichtet. Die Bewirtschaftungsintensität unterschied sich bezüglich der Häufigkeiten (einmal jährlich im Juli oder dreimal jährlich im Mai, Juli und September) und der Düngung (keine Düngung bzw. Düngung mit NPK). Außerdem wurde durch gezielten Herbizid-Einsatz gegen Monokotylen oder gegen Dikotylen ein Gradient in der Anzahl der Pflanzenarten erzeugt. Die Arbeit umfasst drei Hauptthemen. Erstens wurde der Einfluss verschiedener Bewirtschaftungsmaßnahmen auf die bakterielle Endophyten-gemeinschaft in den drei Grasarten Festuca rubra, Lolium perenne und Dactylis glomerata untersucht. Hierfür wurden im September 2010 und im April, Juli und September 2011 Pflanzenproben auf den Dikotylen-reduzierten Plots gesammelt. Die Umwelt-DNS wurde aus den Proben extrahiert und als Template für 16S PCRs eingesetzt. Die Struktur der bakteriellen Endophyten-Gemeinschaft wurde mittels DGGE-Analyse der erhaltenen PCR-Produkte untersucht. Wir konnten Unterschiede der Endophyten-Gemeinschaftsstrukturen hinsichtlich der verschiedenen Bewirtschaftungsintensitäten feststellen. Während die Düngung einen starken Effekt auf die bakterielle endophytische Diversität sowohl in F. rubra als auch in L. perenne hatte, wurden die bakteriellen Endo-phyten in D. glomerata nicht dadurch beeinflusst. Die Proben von L. perenne, die von den ungedüngten Plots stammten, bildeten zudem eindeutige Gruppen bei der Analyse der DGGE-Banden bezüglich der zwei Schnitthäufigkeiten. Somit beeinflusste auch die Mahd die bakterielle Endophyten-Gemeinschaft in den Pflanzen. Weiterhin konnten wir einen starken saisonalen Effekt auf die Struktur der endophytischen Gemeinschaft nachweisen. Da saisonale Veränderungen und die Pflanzenart die Zusammensetzung der endophytischen Bakteriengemeinschaft beeinflussten, können sich die Auswirkungen unterschiedlicher Bewirtschaf-tungsintensitäten mit der Zeit und der untersuchten Pflanzenart verändern. Dieses Ergebnis sollte bei zukünftigen Studien berücksichtigt werden. Das zweite Ziel dieser Arbeit war die Beantwortung der Frage, wie sich oberirdische Herbivorie auf die Bakteriengemeinschaft in der Rhizosphäre auswirkt. Hierfür wurde im Herbst 2010 ein Lysimeter-Experiment auf der GrassMan-Fläche errichtet. Nach einer zweiwöchigen Herbivorie durch Grashüpfer und Schnecken im Sommer 2011 wurden Bodenproben von jedem Lysimeter genommen. Um Einblicke in die Zusammensetzung der bakteriellen Gemeinschaft in der Rhizosphäre zu erhalten, wurde die Gesamt-DNS aus den Bodenproben extrahiert und als Template in 16S rDNS PCRs eingesetzt. Die Gemeinschaftsstruktur wurde mittels DGGE-Analyse bzw. Pyrosequenzierung der erhaltenen PCR Produkte untersucht. Die Herbivorie hatte keinen Einfluss auf die Anzahl der Bakterien (richness), während leichte Änderungen in der relativen Abundanz von einigen Bakteriengruppen festgestellt wurden. So war zum Beispiel die relative Abundanz einer unkultivierten Acidobacterium-Art in den Herbivorie-Lysimetern erhöht. Bestandteil des Lysimeter-Experiments war zudem die Untersuchung des Einflusses der Pflanzenartenanzahl und der verschiedenen Bewirtschaftungs-maßnahmen auf die bakterielle Gemeinschaft in der Rhizosphäre. Der Einsatz von Herbiziden und eine niedrigere Schnittfrequenz reduzierten die Artenanzahl (richness) der Bakterien in der Rhizosphäre. Die Düngung hatte keinen Einfluss auf die Anzahl der Arten. Weitere Analysen zeigten, dass eine Vielzahl von verschiedenen bakteriellen Taxa in der Rhizosphäre durch die untersuchten Maßnahmen beeinflusst wurde. So war die Abundanz der Acidobacteria in den gedüngten Plots signifikant geringer. Das Gegenteil trat bei den Actinobacteria auf. Abschließend lässt sich sagen, dass Pflanzen-assoziierte Bakterien sowohl in der Endosphäre und Rhizosphäre durch Bewirtschaftungsmaßnahmen beeinflusst werden. Die Untersuchung der Wirkung von verschiedenen Bewirtschaftungsintensitäten im Grünland und von oberirdischer Herbivorie auf Pflanzen-assoziierte Bakterien kann zu einem besseren Verständnis der multitrophischen Interaktionen zwischen Pflanzenart, Bakteriengemeinschaft und oberirdischen Herbivoren führen. Außerdem können uns die Ergebnisse dieser Arbeit helfen, die Effekte unterschiedlicher Bewirtschaftungsmaßnahmen auf Pflanzen-assoziierte Bakteriengemeinschaften und damit zusammenhängende Effekte auf das Bodenökosystem vorherzusagen.

<p>This research examines three potential mechanisms by which bacteria can adapt to different temperatures: changes in strain-level population structure, gene regulation and particle colonization. For the first two mechanisms, I utilize bacterial strains from the Vibrionaceae family due to their ease of culturability, ubiquity in coastal environments and status as a model system for marine bacteria. I first examine vibrio seasonal dynamics in temperate, coastal water and compare the thermal performance of strains that occupy different thermal environments. Our results suggest that there are tradeoffs in adaptation to specific temperatures and that thermal specialization can occur at a very fine phylogenetic scale. The observed thermal specialization over relatively short evolutionary time-scales indicates that few genes or cellular processes may limit expansion to a different thermal niche. I then compare the genomic and transcriptional changes associated with thermal adaptation in closely-related vibrio strains under heat and cold stress. The two vibrio strains have very similar genomes and overall exhibit similar transcriptional profiles in response to temperature stress but their temperature preferences are determined by differential transcriptional responses in shared genes as well as temperature-dependent regulation of unique genes. Finally, I investigate the temporal dynamics of particle-attached and free-living bacterial community in coastal seawater and find that microhabitats exert a stronger forcing on microbial communities than environmental variability, suggesting that particle-attachment could buffer the impacts of environmental changes and particle-associated communities likely respond to the presence of distinct eukaryotes rather than commonly-measured environmental parameters. Integrating these results will offer new perspectives on the mechanisms by which bacteria respond to seasonal temperature changes as well as potential adaptations to climate change-driven warming of the surface oceans.</p> / Dissertation

Nitrification is a biological oxidation of NH3 to NO2- and then to NO3-. Understanding how the nitrifier community responds to agricultural management is essential because the community composition is complex and functional distinction of subgroups occurs. Better managing nitrifiers could benefit the environment by increasing nitrogen (N) fertilizer use efficiency, decreasing NO3- leaching, and reducing NO and N2O emissions. This study examined how long-term N fertilization and tillage influenced nitrifier density, ratios, nitrification rates, and the community structure of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), and nitrite-oxidizing bacteria (NOB). The study site was a long-term (>40 years) continuous maize (Zea mays L.) experiment with three N fertilization rates (0, 168, and 336 kg ha-1) and either no-tillage (NT) or plow tillage (PT). Most Probable Number method was used to estimate the density of AOB and NOB; the shaken slurry method was used to measure potential nitrification rates; PCR-denaturing gradient gel electrophoresis (DGGE) was used to analyze nitrifier communities. Tillage, fertilization, and their interaction all significantly influenced the AOB and NOB densities, the ratio of AOB to NOB, and potential nitrification rate. Nitrifier densities and potential nitrification rates increased with increased N fertilization; NOB density increased faster than AOB density with fertilization. The influence of tillage on nitrification was different for different fertilization rates. The trends for nitrifier density and potential nitrification rate were not consistent. Nitrifier community structure was influenced by sample season, N fertilization rates, tillage, and their interaction. Different nitrifier groups had different responses to the treatments. The AOB became more diverse with increasing N input; tillage rather than N fertilizer played a dominant role affecting the AOA community; two NOB genera had different responses to N fertilization rates: Nitrobacter diversity increased with more N applied; Nitrospira was the opposite. Unique bands/members were discovered in different treatments, manifesting environmental selection. Long-term field trials were useful in better understanding how soil management influenced the relationship between nitrifier densities, nitrification rates, and community structure, which may facilitate new approaches to optimize nitrification and provide new clues to discover which environmental factors most influence the nitrifier community in agroecosystems.

<p>Although there are many examples of microbial biogeography, few microbes have been studied at high taxonomic resolution over large spatial scales. As a result, the environmental and ecological processes that drive niche partitioning, diversity, composition, and activity of microbial taxa are often poorly understood. To address this gap, I examine the most abundant phytoplankton in the global ocean, Prochlorococcus sp., a marine cyanobacterium. Using amplicon libraries of the Prochlorococcus internal transcribed spacer (ITS) region and 23S rRNA gene as markers, I demonstrate several key differences between the two major high light (HL) clades of Prochlorococcus. First, by examining ITS amplicon libraries at high taxonomic resolution it is revealed that “sub-ecotype” clades have unique, cohesive responses to environmental variables and distinct biogeographies, suggesting that presently defined ecotypes can be further partitioned into ecologically meaningful units. Whereas unique combinations of environmental traits drive the distribution of the HL-I sub-ecotype clades, the HL-II sub-ecotype clades appear ecologically coherent. Second, using 23S rRNA and rDNA libraries I show that activity (rRNA) and abundance (rDNA) are highly correlated for Prochlorococcus across all sites and operational taxonomic units (OTUs) in the surface ocean, demonstrating a tight coupling between activity and abundance. Finally, I investigate the associations between Prochlorococcus and the rest of the microbial community in the North Pacific and find region-specific trends in both strength and sign. Associations with other microbes are strongest for HL-I in the temperate region and strongest for HL-II in the sub-tropical gyre. This dissertation clarifies the relative importance of the environment, geography, community, and taxonomy in terms of their role in creating complex assemblages of Prochlorococcus and helps improve our understanding of how marine microbial communities are assembled in situ.</p> / Dissertation

Stimulating in situ microbial communities in oil reservoirs to produce natural gas is a potentially viable strategy for recovering additional fossil fuel resources following traditional recovery operations. Little is known about what geochemical parameters drive microbial population dynamics in biodegraded, methanogenic oil reservoirs. We investigated if microbial community structure was significantly impacted by the extent of crude oil biodegradation, extent of biogenic methane production, and formation water chemistry. Twenty-two oil production wells from north central Louisiana, USA, were sampled for analysis of microbial community structure and fluid geochemistry. Archaea were the dominant microbial community in the majority of the wells sampled. Methanogens, including hydrogenotrophic and methylotrophic organisms, were numerically dominant in every well, accounting for, on average, over 98% of the total Archaea present. The dominant Bacteria groups were Pseudomonas, Acinetobacter, Enterobacteriaceae, and Clostridiales, which have also been identified in other microbially-altered oil reservoirs. Comparing microbial community structure to fluid (gas, water, and oil) geochemistry revealed that the relative extent of biodegradation, salinity, and spatial location were the major drivers of microbial diversity. Archaeal relative abundance was independent of the extent of methanogenesis, but closely correlated to the extent of crude oil biodegradation; therefore, microbial community structure is likely not a good sole predictor of methanogenic activity, but may predict the extent of crude oil biodegradation. However, when the shallow, highly biodegraded, low salinity wells were excluded from the statistical analysis, no environmental parameters could explain the differences in microbial community structure. This suggests that the microbial community structure of the 5 shallow, up-dip wells was different than the 17 deeper, down-dip wells. Also, the 17 down-dip wells had statistically similar microbial communities despite significant changes in environmental parameters between oil fields. Together, this implies that no single microbial population is a reliable indicator of a reservoir's ability to degrade crude oil to methane, and that geochemistry may be a more important indicator for selecting a reservoir suitable for microbial enhancement of natural gas generation.

In wetland soils, hydrology is considered to be one of the primary factors shaping wetland function and microbial community structure, but plant-soil interactions are also important mechanisms affecting microbial nutrient transformations. The research presented here considered the interactive effect to describe how hydrology and the presence of plants alter the soil profile, the development of the bacterial community, and their associated functions. To achieve this goal, plots were established in three hydrologically-distinct regimes (Wet, Intermediate, and Dry) within a non-tidal freshwater wetland along the James River (Charles City County, Virginia). Inside each main plot, ten subplots were cleared of all aboveground plant material; five plots were left to re-grow (“Vegetated” reference), while the remaining five were weeded each week to maintain bare soil (“Clipped” treatment subplots). Manipulations were started at the beginning of the growing season, and sampling continued until the following winter. Every eight weeks, soil cores (30 cm) were collected and analyzed for a variety of soil properties (e.g., pH, OM, C:N, redox, vegetation and root biomass), microbial community structure (16S-rDNA-based T-RFLP),bacterial abundance (Acridine Orange Direct Count), and soil function (Extracellular Enzyme Activity (EEA)). A mixed-effects repeated measures analysis of variance (ANOVA) was used to better understand how each variable responded within each hydrological regime and treatment. Principal component analysis (PCA) and Partial Mantel tests were used to elucidate how saturation and vegetation influence the microbial community structure and soil enzyme function. Bacterial community properties and soil functions followed differences in soil saturation and associated physicochemical parameters (i.e., pH and redox). Correlations with wetland vegetation were primarily related to seasonal changes in plant community composition and biomass, and differences between experimental treatments were small. Evidence suggests the present plant species and the amount of above- and belowground biomass plays a more selective role shaping bacterial communities and soil function. Due to the short-term of this study and tight soil correlations, it is difficult to determine if observed differences are a product of the plant community or soil saturation, but it is clear that each is important. Based on the literature, plant effects were smaller in this wetland than might be expected. This experiment took place in a recently exposed lake basin, so plant-soil-microbe interaction may not be well established. As the wetland matures, relative importance of vegetation is expected to increase and impact bacterial composition and function. Collectively, these results demonstrate that wetlands are not a product of one separate variable, but result from various factors interlinked to shape microbial communities and soil functions.

Aquatic bacteria respond to changing environmental conditions through a variety of mechanisms including changes in abundance, shifts in community composition and variable activity states. In the tidal-freshwater James River, variation in bacterial abundance was linked to nutrient availability and autochthonous production with highest bacterial densities associated with low-nutrient, high-chlorophyll a conditions. Laboratory experiments revealed that bacterial growth rates were nutrient limited at the low-nutrient site, while co-limitation (nutrients, glucose, light) was apparent at the high nutrient site. Despite large differences in abundance, community composition was similar based on TRFLP and 16S rDNA pyrosequencing. Community similarity was lower among rRNA libraries suggesting that variable activity states are prevalent in natural communities. Rare taxa were more likely to be metabolically active and were capable of dramatic growth under microcosm conditions.

Saltwater intrusion due to global change is expected to have a detrimental effect on the biogeochemistry of tidal freshwater wetlands. Of particular concern is that fact that salinization can alter the role of these ecosystems in the global carbon cycling by causing shifts in microbial metabolism that alter greenhouse gas emissions and increase carbon mineralization rates. However, our understanding of how wetland microbial community dynamics will respond to saltwater intrusion is limited. To address this knowledge gap and increase our understanding of how microbial communities in tidal freshwater wetlands change over time (1, 3, 12, and 49 weeks) under elevated salinity conditions, an in situ soil transplant was conducted. Throughout the 49 weeks of saltwater exposure, salinity had no effect on soil quality (organic matter content and C:N ratio). In contrast, the concentration of porewater ion species (SO4-2, NO3-, and NH4+) considerably increased. The activity of hydrolytic enzymes, (ß-1,4-glucosidase and 1,4-ß-cellobiohydrolase) gradually decreased with prolonged exposure to saline conditions; by the final sampling event (49 weeks), activity was reduced by ~70% in comparison to the freshwater controls. Short term exposure to salinity (3 and 12 weeks) had a greater effect on phenol oxidase, decreasing activity by 10-20%. Saltwater exposure had an immediate (1 week) effect on potential rates of carbon mineralization; overall, carbon dioxide production doubled and methane production decreased by ~20-fold. These changes in gas production were correlated to increased salinity and to changes in the abundance of methanogens and sulfate reducing bacteria, suggesting a shift in the terminal step in organic matter degradation from methanogenesis to sulfate reduction. Principal component analysis revealed distinct changes in soil environmental conditions and carbon metabolism within weeks, but the response of the microbial community was slower (months to a year). Taken together, results from this study indicate that the response of tidal freshwater wetlands to salinization is driven by complex interactions of microbial related processes and environmental changes that are dependent on the duration of exposure. Assessing the impact of environmental perturbation on ecosystem function may be better achieved by complementary analysis of both microbial community structure and function.