Food Science and Technology Department


Date of this Version

Summer 8-10-2012


Legge, R. (2012) Analysis of microbial diversity by amplicon pyrosequencing. PhD Diss., University of Nebraska-Lincoln.


A DISSERTATION Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy, Major: Food Science and Technology, Under the Supervision of Professor Andrew Benson. Lincoln, Nebraska: August, 2012

Copyright (c) 2012 Ryan Matthew Legge


Microorganisms numerically dominate terrestrial biodiversity, and play important biochemical and geochemical roles in the environments they inhabit. To understand structure and function of complex ecosystems, it is essential to identify primary drivers of microbial diversity and community structure. Historically, the study of microbial ecology was reductive, limited to microbes able to be cultured and enumerated. Microbes meeting this criterion were thought to comprise the dominating members of the environments they were isolated from, however, estimates suggesting up to 99% of the endogenous species are uncultivable with existing methodologies; a concept that reflects experimental failure, rather than a verifiable conclusion. Therefore surveys of microbial community members relying solely on culture-based techniques will severely underestimate the extent of microbial diversity. Analytical methods for DNA sequencing have progressed over the last 30 years allowing for increasingly detailed analysis of microbial communities. Microbes can be recognized and their function can be understood at the DNA/RNA level without cultivation bias through molecular techniques which analyze content based on microbial DNA isolated from environmental samples. Using high-capacity sequencing, environmental samples can be characterized at resolution, ultimately allowing communities to be compared on the basis of their taxonomic or phylogenetic content as well as on functions the microbes carry out. In this dissertation research, two unique studies were explored. Studies focus on differences in composition of microbial communities as a phenotype in the GI tract of animals in a genetic selection experiment and as a measure of contamination risk in food production with 454-based pyrosequencing of the 16s rRNA amplicon. These two studies give credence to the applications of “-omics” techniques in addressing questions relevant to fundamental and applied biological disciplines. Ultimately, studies like these are creating paradigm shifts in how we view food production and human health as they begin to uncover the entire microbial community to unparalleled levels. Continued advancements in the technology itself and the associated bioinformatics tools will influence a broad cross-section of problems in food production, heath care, and water and land management.

Advisor: Andrew Benson