Understanding the response of soil microbial communities to various environmental stresses is of current interest, because of their pivotal role in nutrient cycling, soil organic matter mineralization and influence on plant growth. Determining the affect of several biotic and abiotic factors on soil microbial communities is the overall objective of the study. The specific goals are to determine 1) the response of microbial communities to water deficit in soil and 2) how the presence of a rich biotic community determines the direction of microbial community development in cultures. Both goals are novel and unique contributions to understanding microbial ecology in soil. Dynamics in water potentials due to drying and rewetting of soil impose significant physiological challenges to soil microorganisms. To cope with these fluctuations, many microorganisms alter the chemistry and concentration of their cytoplasmic contents. The aim of this research is to understand how the microbial biomass and their cytoplasm change in response to water potential deficits under in situ soil conditions. To address this objective we characterized intracellular and extracellular metabolites in moist, dry and salt stressed soils. Our results provided the first direct evidence that microbial communities in soil in situ utilize sugars and sugar alcohols to cope with low water potential. While the cultivation and isolation of microorganisms is essential to completely explore their physiology and ecology, 99% of soil microbes resist growing in cultures. Presence of very unnatural conditions in the culture plates was considered as main reason for low cultivability. Thus, a culture-based study was conducted whereby microorganisms were grown in association with their native habitat with an objective of mimicking native conditions to promote the growth of previously uncultivated microorganisms. Moreover, the importance of biotic communities (microbe-microbe) and abiotic soil effects were assessed on bacterial growth. Our results strongly indicate that the presence of living microbial community in the vicinity of the target culture resulted in the cultivation of novel members of rare bacterial taxa from phyla Verrucomicrobia, Bacteroidetes, Proteobacteria, and Planctomycetes. These results emphasize the need to develop new culturing methods to tap the hidden microbial potential for emerging anthropogenic needs.

Presented diploma thesis focuses on a new type of lightweight polyurethane foam (PUR), which has been manufactured with the intention of it being biodegradable within currently valid norms on biodegradation of plastic materials. The future use of said polyurethane foam is as carrier for odor repellent to avoid wildlife-vehicle collisions in agriculture landscape, where, after the end of its lifespan it could be left to biodegrade in soil the environment. The examined material, PUR BIO-10, was tested for biodegradability in laboratory microcosms according to standardized method ASTM D5988-03. Biodegradability was tested in two separate soil types - forest soil and agricultural soil, which have been selected with the future use of the material in mind. According to the method biodegradability was measured as mineralization of the material by capturing evolved carbon dioxide. Two trials with different treatments were executed. In the first trial the material was introduced directly into the soil and in the second trial the material was tested in a litterbag to avoid soil contamination which was problematic for further analysis. During the first trial, after 90 days, the mineralization of polyurethane foam was 10.65 ± 2.54 % in the forest soil and 20.48 ± 9.18 % in the agricultural soil. During the...

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<p>Modern microbialites provide the opportunity to explore the influences of biology on microbialite formation and understand how biosignatures can be preserved in these structures. In this study, we used the isotopic compositions (δ¹³C) of phospholipid fatty acids (PLFAs) and their structurally-defined profiles, in conjunction with calcium carbonate isotopic compositions and imaging to evaluate microbial autotrophic and heterotrophic processes associated with freshwater microbialites from Kelly Lake, British Columbia. This was done to determine what types of metabolism may have been influencing microbialite growth and whether a biosignature of this process was preserved. In addition, PLFA profiles from a microbialite-derived pure culture were analyzed under various growth conditions to assess environmental influences on microbial PLFA composition.</p> <p>Although the majority of the δ¹³C values of Kelly Lake microbialite surface carbonates fell within the range predicted for equilibrium precipitation, samples collected from 26 m were found to have enriched δ¹³C_carb values and are likely a biosignature of autotrophy at this depth. PLFA profiles and δ¹³C_PLFAvalues also supported the predominance of autotrophy, however, they indicated that heterotrophic organisms were also present. This data suggests that autotrophic metabolisms have influenced the local geochemistry in the past, at least at 26 m, and are likely substantial contributors to microbialite growth.</p> <p>Changes in temperature, pH, NaCl concentrations, and cell densities were found to induce variations in the PLFA profiles of the <em>Exiguobacterium</em> strain RW2. The degree of PLFA unsaturation changed in each of the different culture conditions, and was predominantly adjusted through alterations in the branched monoenoic PLFAs, particularly i-17:1Δ⁵. These results highlight the difficulties associated with applying PLFA profiles as evidence for shifts in a microbial community composition, since altered growth conditions can induce intra-specific PLFA changes.</p> / Master of Science (MSc)

<p>Investigating the presence of microbial communities in the Earth's deep terrestrial subsurface and the metabolic processes taking place in these environments provides insight into the some of the ultimate limits for life on Earth, as well as the potential for microbial life to exist within the subsurface of other planetary bodies. This Master's thesis project utilized phospholipid fatty acid (PLFA) analysis, in combination with carbon isotope analyses (δ¹³C and Δ¹⁴C), to explore the presence and activity of microbial communities living within deep terrestrial subsurface fracture water systems and low permeability, deep sedimentary rocks. Deep fracture water systems, ranging from 0.9 to 3.2 km below land surface, were sampled for microbial communities via deep mine boreholes in the Witwatersrand Basin of South Africa. PLFA concentrations revealed low biomass microbial communities, ranging from 2x10¹ to 5x10⁴ cells per mL and the PLFA profiles contained indicators for environmental stressors, including high temperatures and nutrient deprivation. δ¹³C and Δ¹⁴C analyses of PLFAs and potential carbon sources (dissolved inorganic carbon (DIC), dissolved organic carbon (DOC) and methane) identified microbial utilization of methane in some systems and utilization of DIC in others. Evidence for microbial oxidation of methane and chemoautotrophy in these systems is consistent with a self-sustaining deep terrestrial subsurface biosphere that is capable of surviving independent of the photosphere. Viable microbial communities were also identified within deep (334 to 694 m depth) sedimentary rock cores sampled from the Michigan Basin, Canada. PLFA analyses revealed microbial cell densities ranging from 1-3 x 10⁵ cells/mL and identified PLFA indicators for environmental stressors. These results demonstrate the ubiquity of microbial life in the deep terrestrial subsurface and provide insight into microbial carbon sources and cycling in deep microbial systems which may persist in isolation over geologic timescales.</p> / Master of Science (MSc)

Investigation of life’s limitations on Earth provides the necessary information to constrain where life outside of Earth may be proliferating or previously existed. This Master’s thesis applied phospholipid fatty acid (PLFA) analysis in combination with organic carbon and 16S rRNA gene data to assess and characterize microbial communities through both microcosms and in situ samples of bentonite clay, which is an intended barrier component for the long-term storage of high-grade nuclear waste. Microcosm experiments were set up to test the impact of water activity in as-received, uncompacted bentonite clays using a high (0.99) and low (0.93) water activity over a one month period. Under aerobic incubation water activities of 0.93 and 0.99 had no resolvable effect between water activity levels on the growth of cells of indigenous communities of microbes in as-received uncompacted bentonite. Growth was detected under both water activities by a significant increase in total PLFA abundance. The increase in PLFA over the period of the study suggested an approximate increase in cells from 4x10^6 to 2x10^7 E.coli equivalent cells/g. The distribution of the PLFA and genetics data suggests the community is composed predominantly of gram-positive aerobic heterotrophs with lesser amounts of anaerobic bacteria and eukaryotes potentially in the form of fungi. Similar cell abundances and community structures were identified in the Tsukinuno Mine bentonite DGR analogue site which is a ~12 to 16 Ma deposit approximately 200 m below the surface. Total PLFA recovered from the core subsamples ranged from 32 pmol PLFA/g to 431 pmol PLFA/g, which corresponds to a range from 7.5x10^5 to 1.2x10^7 E.coli equivalent cells/g, across all cores. The community was composed of both aerobic and anaerobic bacteria consisting of gram-positive and gram-negative bacteria, as well as possible sulfate-reducing bacteria and eukaryotes. / Thesis / Master of Science (MSc)

Soil is an important ecosystem that supports a wide variety of organisms such as bacteria, fungi, arthropods and nematodes. This sensitive ecosystem may be influenced by various factors, including agricultural management practices. With the introduction of genetically modified (GM) glyphosate-tolerant (RoundUp ® Ready: RR) crops, herbicides such as glyphosate have been increasingly used. However, little is known about the effect of glyphosate on the biological communities in these herbicide-sprayed soils. With the intimate proximity that microorganisms and nematodes have with the roots of plants, these organisms can be used to assess changes that may occur in the soil surrounding roots of RR crops. The aim of this study was to determine microbial community structure and nematode diversity, with emphasis on that of non-parasitic nematodes, in soil samples from conventional soybean (CS) - and RR- soybean fields compared to that in adjacent natural veld (NV) areas. Samples were collected from twenty three sites at six localities that are situated within the soybean-production areas of South Africa. These sites represented fields where RR and CS soybean grew, as well as surrounding NV. All RR fields have been treated with glyphosate for no less than five years. Microbial community structures of the twenty three sites in the RR, CS and NV ecosystems were determined by phospholipid fatty acid (PLFA) analyses. Nematode diversity was determined by extracting the nematodes from soil samples and conducting a faunal analysis. Soil physical and chemical properties were determined by an independent laboratory, Eco-Analytica (North West University, Potchefstroom) according to standard procedures. Results from this study indicated differences in microbial community structure between the various localities. However, there were no significant (p ≤ 0.05) differences in microbial community structures between RR- and CS ecosystems. Soils of both RR- and CS crops were primarily dominated by bacteria. Nematode identification and faunal analysis also indicated no significant (p ≤ 0.05) differences between the different non-parasitic/beneficial nematodes that were present in soils of these two ecosystems during the time of sampling. Non-parasitic nematode communities were primarily dominated by bacterivores. A faunal analysis indicated that most of the sites contained enriched, but unstructured soil food-webs. However, four of the sites showed enriched and structured food webs due to the presence of non-parasitic nematodes with high coloniser-persister (cp) values. Relationships between non-parasitic nematode – and microbial communities showed that there was a positive relationship between nematode functional groups and their corresponding microbial prey. From the results obtained in this study, it can be concluded that the community structures of both non-parasitic nematodes and microorganisms shared similarities. These community structures showed no long-term detrimental effects of glyphosate application in the soils surrounding roots of RR soybean crops. Relationships existed between non-parasitic nematode and microbial communities in the rhizosphere of soybean crops and natural veld. For example, bacterivore nematodes had a strong positive relationship with gram-negative bacteria. Similar but weaker relationships also existed between carnivores, omnivores, plantparasitic nematodes and gram-negative bacteria. A positive relationship also existed between fungivores and fungal fatty acids. This emphasises the value of these organisms as indicators of soil health and also the impact that agricultural practices can have on soils. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2014