The first part of the study involved revival and isolation of AEFB from five depths along a sediment core. Three procedures were used to extract endospores from sediment particle matrixes to determine which yielded the highest counts of AEFB. The optimal procedure was then applied to screen samples from the five depths, followed by plating onto four different media types to achieve growth and maximize isolation. The study explored two means of diversity analysis of AEFB isolates, namely, Rep-PCR and HRMA. Rep-PCR provided a strain-level resolution of the isolates and was used for classification of isolates into OTUs.
Phylogenetic analysis was employed to determine the evolutionary relationships present between representatives of each OTU. As part of physiological profiling, a protocol involving microtiter plate assays was developed to test salinity and pH tolerance ranges of selected AEFB isolates. Lastly, metabolic diversity was evaluated by testing the substrate utilization capabilities of selected isolates using Biolog EcoPlates™.
Based on the findings of this study, the following was established:
♦ AEFB were present at all five sample depths along the Mfabeni Peatland sediment core, ranging in age from 589 to 37 906 cal years BP. This indicates that ancient sedimentary deposits do serve as a reservoir for dormant endospores. It was found that a bead-beating, buffer- mediated technique based on a modified indirect DNA extraction protocol yielded the highest CFU/g values based on preliminary testing. Extraction of endospores from different types of
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sediment could be carried out using this technique to determine if it could be applied across a wider spectrum of archival material.
♦ The decreasing trend observed in CFU/g with increasing sample age indicates that rates of survival of endospores decrease as they lose viability over time. The higher numbers of AEFB observed at the shallowest depth may indicate that AEFB populations at this depth are influenced by a close proximity to oxic conditions. The survival of AEFB at a depth of 344 cm, albeit at very low CFU/g, suggests that the accumulating layers of organic material within peatlands do serve to trap and preserve dormant endospores over extended periods of time.
♦ Most OTUs were unique to the media types upon which they were isolated, demonstrating the diverse nutritional requirements amongst the AEFB isolates. A large proportion of OTUs would not have been isolated if only a single type of media had been selected. This was an important finding since several studies use only a single media type for isolation (Fredrickson et al., 1991; Shivaji et al., 2011). It was also found that the nutritionally-limited 10% TSA and R2A media and environmentally-based Marine Agar were able to support a higher level of OTU diversity. This was not unexpected since many environmental organisms are adapted to the nutrient-limiting conditions which are often prevalent in natural environments.
♦ Rep-PCR was found to be an effective tool for providing strain-level resolution of isolates.
Fingerprinting profiles could be easily compared allowing for the differentiation of isolates into OTUs. HRMA was found to be suitable for comparing and grouping closely-related isolates and was able to distinguish isolates at a species level. The high throughput and elimination of gel electrophoresis made this a fast and simple technique to conduct. Rotor- Gene™ Software aided in the analysis of the melt curves and provided raw fluorescence data which could then be used for statistical analysis. Both techniques were found to be suitable for the analysis of diversity amongst AEFB in terms of the initial cost and throughput demands.
However, for the degree of resolution required for the current study, Rep-PCR was able to provide a higher level of discrimination and differentiation amongst AEFB isolates, allowing for a greater degree of genetic diversity to be distinguished.
♦ Genotyping revealed distinctions in diversity amongst isolates obtained from sample depths of different ages. Based on the results of Rep-PCR, 94% of OTUs were found to be unique to their depths. This supported the notion that endospores are able to remain static and immobile once deposited. This further supports the inference that the endospores revived were of an age equivalent to that of the radiocarbon-dated sample material from which they were isolated. The
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high numbers of certain OTUs at some of the depths suggest that these bacteria may have been residing in the ecosystem, as opposed to being transported there.
♦ Several aerobic endospore-forming genera were present along the sediment core. Members of some of the genera were found to be unique to specific sample depths, providing further evidence which discounts endospore migration through the core. For instance, members of the Lysinibacillus and Brevibacillus genera were only present in the younger sampling depths, whereas members of the Domibacillus genus were unique to the older depths. On the other hand, members of the Bacillus and Paenibacillus genera were present at multiple depths along the sediment core, indicating that they may be abundant in the region. Many isolates also matched species which have been previously isolated from peatlands. Some isolates from the younger sampling depths were taxonomically-similar to isolates from older depths. However, it was found that no common strains were present across the younger and older depths.
♦ The changes in diversity across different sampling depths refute the possibility of carry- through or contamination between the samples. The results support the notion that the bacteria isolated were ancient in origin.
♦ 16S rRNA gene sequencing results also revealed a proportion of isolates with low sequence similarity matches to currently-listed species. This suggests the possibility of some of these isolates being novel species or strains which require further characterization. These results suggest that by examining ecosystems which are able to remain stable over time, it is possible to isolate potentially uncharacterized bacterial species or strains.
♦ Responses of selected AEFB isolates to salinity testing revealed that a high percentage of isolates preferred lower salt concentrations. Halotolerant bacteria were also present throughout the core. Water fluctuations within peatlands have direct impacts on salinity levels, which may have an influence on the residing AEFB.
♦ pH testing revealed that a high percentage of isolates favoured near-neutral to basic pH. This could be attributed to the initial cultivation media used, which were neutral-based, thereby favouring the selection of neutrophiles. In addition, the presence of alkaline microenvironments within the peat may support the growth of alkaliphiles. A higher proportion of acidophiles may be isolated by enriching for these bacteria with the initial cultivation media used. This could allow for the bacteria, which are able to thrive in the acidic peat, to be isolated.
♦ Overall, it was determined that salinity and pH were not major factors driving the changes in diversity between isolates from different depths. This could suggest the possibility of
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relatively stable salinity and pH conditions over time within the region. For future studies, temperature tolerance ranges can be tested to determine the effect of this environmental parameter on AEFB diversity.
♦ Biolog EcoPlates™ were found to be a useful tool for determining the substrate utilization capabilities of the isolates against environmentally-relevant substrates. It provided an efficient means of producing results relating to metabolic diversity, which could then be statistically analyzed for comparison between isolates of different depths. The utilization of a diverse range of substrates by each isolate demonstrated the varied metabolic capabilities present amongst the AEFB. The most commonly utilized substrates were found to be Tween 40 and Tween 80.
The high degree of utilization of these compounds suggests that fatty acid metabolism may be prevalent amongst AEFB populations associated with the peatland environment. Substrates such as D-xylose and pyruvic acid methyl ester were also utilized to a high degree. These are substrates which are associated with decomposing plant material and thus, would be expected to be present in peat.
♦ Distinct patterns of substrate utilization were discerned between isolates from the younger and older depths. Multivariate analysis revealed, with significance, that the age of the sample from which the isolates were obtained did have an effect on the variation observed in substrate utilization patterns amongst isolates. This finding is promising since it supports the notion that past climate changes, which impacted on vegetation and conditions within the peatland, could have influenced the genetic and physiological diversity of the AEFB isolates recovered from different sections of the sediment core.
6.3. DO DORMANT AEFB MEET THE REQUIREMENTS OF A PALAEO-