Graduation date: 2003

The gastrointestinal tract is colonized by an abundant and diverse community of microorganisms which has a profound impact on the health of the host. The profiling of these microbial communities with traditional culture-based methods identifies only a fraction of microbes present with limited specificity, high labour costs and limited sample throughput. To overcome these limitations, a molecular hybridization assay was developed and characterized using the target gene chaperonin 60 (cpn60). The interspecies discriminatory ability of the hybridization assay was determined by hybridizing cpn60 gene fragments from a known species to a series of cpn60 gene fragments derived from related species with distinct but similar cpn60 sequences. Species with less than 85% cpn60 sequence identity to the probe DNA were effectively distinguished using the hybridization approach. To characterize complex microbial communities, universal PCR primers were used to amplify a fragment of 549-567 nucleotides from cpn60 (the cpn60 universal target (UT)) using template DNA extracted from the ileal contents of pigs fed diets based on corn (C), barley (B), or wheat (W), or from plasmids containing the cpn60 UT selected from a clone library generated from these contents. The intensity of hybridization signals generated using labelled probes prepared from library clones designated B1 (Bacillales-related), S1 (Streptococcus-related), C1 (Clostridiales-related), and L10 (Lactobacillales-related) and targets prepared from ileal contents of C, W, or B-fed pigs correlated closely with the number of genomes of each bacterial group as determined by quantitative PCR. Universal PCR primers were also used to amplify genomic DNA extracted from jejeunal contents of pre- and post-weaning piglets. Labelled probe DNA was prepared from S1, L10, LV (Lactobacillus vaginalis-related) and EC (E.coli) library clones. The resulting signal intensities correlated with quantitative polymerase chain reaction (qPCR) data for L10 and LV, but minimal correlation was observed for the S1 and EC groups. A cpn60- based macroarray has potential as a tool for identification and semi-quantification of shifts in colonization abundance of bacteria in complex communities, providing a similar amount of data as techniques such as denaturation gradient gel electrophoresis or terminal restriction fragment length polymorphism analysis.

The gastrointestinal tract is colonized by an abundant and diverse community of microorganisms which has a profound impact on the health of the host. The profiling of these microbial communities with traditional culture-based methods identifies only a fraction of microbes present with limited specificity, high labour costs and limited sample throughput. To overcome these limitations, a molecular hybridization assay was developed and characterized using the target gene chaperonin 60 (cpn60). The interspecies discriminatory ability of the hybridization assay was determined by hybridizing cpn60 gene fragments from a known species to a series of cpn60 gene fragments derived from related species with distinct but similar cpn60 sequences. Species with less than 85% cpn60 sequence identity to the probe DNA were effectively distinguished using the hybridization approach. To characterize complex microbial communities, universal PCR primers were used to amplify a fragment of 549-567 nucleotides from cpn60 (the cpn60 universal target (UT)) using template DNA extracted from the ileal contents of pigs fed diets based on corn (C), barley (B), or wheat (W), or from plasmids containing the cpn60 UT selected from a clone library generated from these contents. The intensity of hybridization signals generated using labelled probes prepared from library clones designated B1 (Bacillales-related), S1 (Streptococcus-related), C1 (Clostridiales-related), and L10 (Lactobacillales-related) and targets prepared from ileal contents of C, W, or B-fed pigs correlated closely with the number of genomes of each bacterial group as determined by quantitative PCR. Universal PCR primers were also used to amplify genomic DNA extracted from jejeunal contents of pre- and post-weaning piglets. Labelled probe DNA was prepared from S1, L10, LV (Lactobacillus vaginalis-related) and EC (E.coli) library clones. The resulting signal intensities correlated with quantitative polymerase chain reaction (qPCR) data for L10 and LV, but minimal correlation was observed for the S1 and EC groups. A cpn60- based macroarray has potential as a tool for identification and semi-quantification of shifts in colonization abundance of bacteria in complex communities, providing a similar amount of data as techniques such as denaturation gradient gel electrophoresis or terminal restriction fragment length polymorphism analysis.

Thesis--University of Kentucky (M.S.), 2003. / Title from document title page. Document formatted into pages; contains ix, 127 p. : ill. Includes abstract and vita. Includes bibliographical references (p. 110-126).

The biological world is complex. Communities contain a multitude of interacting species, while populations contain extensive genetic variation. How much complexity must one consider to understand patterns and processes of interest? When are species interactions and community properties shaped by evolution? Conversely, when is evolution altered by community context? I test these questions in a series of experiments with simple microbial communities. The first data chapter investigates the impact of competition on the evolution of phage resistance in bacteria. This work demonstrates that community context can dramatically alter the evolution of resistance to phage. Next I tested the impact of evolution on assembly of a three species community. I demonstrate that evolution can influence the content of a microbial community by altering the process of assembly. Finally, I investigated the evolutionary origin and maintenance of cross-feeding mutualisms. This work suggests that species interactions can enable novel evolutionary pathways, and that evolution can significantly increase the productivity of cross-feeding communities. Jointly these experiments suggest that consideration of the interplay between ecological and evolutionary forces can provide insight into the complexity of the natural world. / text

published_or_final_version / Dentistry / Master / Master of Dental Surgery

Anaerobic digestion of agricultural wastes provides an opportunity for renewable energy production while reducing emissions of greenhouse gasses such as carbon dioxide and methane from crop and livestock production. While anaerobic digestion is possible under a wide range of temperatures and reactor configurations, it does require a stable methanogenic community composed of hydrolytic and fermentative bacteria and methanogenic archaea in order to maintain robust methane production. Research focused on characterizing and optimizing the microbial community during anaerobic digestion is increasingly exploiting DNA-based methods. In addition to providing an in-depth phylogenetic survey, these techniques permit examination of dynamic changes in α- and β-diversity during the digestion process and in response to perturbations in the system. This study used universal target amplification, next generation sequencing, and quantitative PCR to characterize the Bacteria and Archaea in digestate from thermophilic batch anaerobic digesters processing different combinations wheat ethanol stillage waste and cattle manure. The results indicated that the bacterial community was composed primarily of Firmicutes, with Proteobacteria and Bacteroidetes also numerically abundant. While less phylogenetically diverse, the archaeal community showed robust populations of both hydrogenotrophic and acetoclastic methanogens. A core microbiome present across all reactors was identified and differences in the relative abundances of the bacteria within the core community suggested significant niche overlap and metabolic redundancy in the reactors. A time-course study correlating the abundances of individual Bacteria and Archaea to methane production and volatile fatty acid catabolization identified several microorganisms hypothesized to be critical to both hydrogenotrophic and acetoclastic methanogenesis. Individual Bacteria most closely related to Clostridium spp. and Acetivibrio spp. were 10-1000-fold less abundant in reactors suffering from volatile fatty acid accumulation and inhibition of methanogenesis. Additionally, failing reactors were devoid of robust populations of acetoclastic methanogens. Microorganisms identified as critical during the time-course study were targeted for isolation in vitro and a robust methanogenic consortium consisting of at least 9 bacteria and both a hydrogenotrophic and an acetoclastic methanogen was stably propagated. Addition of this bioaugmentation consortium to digesters experiencing classic symptoms of acid crisis resulted in reduced acetate accumulation and initiation of methanogenesis. One acetoclastic methanogen, most likely a novel species from the genus Methanosarcina, showed particularly robust growth in the recovered bioaugmented reactors, increasing 100-fold in the first 7 days post-treatment. A combination of Illumina shotgun and Roche 454 paired-end sequencing chemistry was used to generate a high quality draft genome for this organism. Analysis of the annotated genome revealed diverse metabolic potential with a full complement of genes for acetoclastic, hydrogenotrophic and methylotrophic methanogenesis pathways represented. Taken as a whole, this thesis provides the foundation for using microbial community characterization to inform anaerobic digester design and operation. By identifying organisms of interest, correlating their abundance to specific biochemical functions and confirming their hypothesized functions in situ, microorganisms critical for robust methane production were acquired. The logical extension of this work is to establish monitoring tools for microorganisms identified as critical to specific performance parameters, to enumerate them in real-time, and to use that data to improve reactor operation.

No description available.

Soil microbial communities can be characterized by community structure and function (community composition) across a spectrum of spatial scales, and variation in soil microbial composition has been associated with a number of environmental gradients. This study investigates the structure and function of soil microbial communities under mature, undisturbed forested sites across a range of regional climates in British Columbia and Alberta, and also examines the variation in community composition within sites. Phospholipid fatty acid analysis was used to investigate the structure of soil microbial communities and total soil microbial biomass at each site. Extra-cellular enzyme assays established the functional potential of the soil microbial community at each site. Multivariate analysis of the data showed that the soil microbial communities under different forest types did significantly separate along the regional climate gradient by both community structure and function, despite high local variation in the communities. Soil moisture content and soil organic matter concentration consistently exhibited the strongest relationship with microbial community characteristics, although the functional and structural responses to the external drivers were different. Microbial community function and structure also changed with soil depth but not with time of sampling. Microbial community function was related to the regional annual average precipitation gradient. Most of the locations exhibited unique microbial community functional profiles in their soil layers; however the enzyme activities in the samples from the driest (Ponderosa Pine) and wettest (Mountain Hemlock) locations were notably different from each other and from those of the other locations, especially in the organic layers. The moist maritime-influenced Coastal Western Hemlock (CWH) forest exhibited microbial community structural characteristics which were unique from those of the other forest locations. The higher abundance of bacteria relative to fungi in the CWH forest soils may be related to the significantly higher available nitrogen concentrations at this site.

Antarctica provides some of the most extreme environments on earth. Low temperatures, low water availability and nutrient deficiency are contributing factors to the limited colonisation of Antarctic biotopes, particularly in the continental Dry Valleys. The survival of microorganisms in this harsh continent provides the basis for the significance of this study. This study aimed to explore microbial phylotypic diversity across a 500 m altitudinal transect in the Miers Dry Valley, Ross Desert, East Antarctica. The study also attempted to infer from phylogenetic data, the possible presence of indicative phenotypes which might contribute to a functional microbial community.