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16S rRNA-Based Tag Pyrosequencing of Complex Food and Wastewater Environments: Microbial Diversity and Dynamics

16S rRNA-Based Tag Pyrosequencing of Complex Food and Wastewater Environments: Microbial Diversity and Dynamics. (December 2010) Katherine Grace McElhany, B.S., Texas A&M University Chair of Advisory Committee: Dr. Suresh D. Pillai Environmental microbiology has traditionally been performed using culture-based methods. However, in the last few decades, the emergence of molecular methods has changed the field considerably. The latest development in this area has been the introduction of next-generation sequencing, including pyrosequencing. These technologies allow the massively parallel sequencing of millions of DNA strands and represent a major development in sequencing technologies. The purpose of this study was to use both pyrosequencing and traditional culture-based techniques to investigate the diversity and dynamics of bacterial populations within milk and untreated sewage sludge samples. Pasteurized and raw milk samples were collected from grocery stores and dairies within Texas. Milk samples were analyzed by plating, pyrosequencing, and an assay for the presence of cell-cell signaling molecules. Samples were processed, stored, and then evaluated again for spoilage microflora. The results of this study showed that raw milk had a considerably higher bacterial load, more diversity between samples, and a significantly higher concentration of pathogens than pasteurized milk. Additionally, this study provided evidence for varying spoilage microflora between raw and pasteurized milk, as well as evidence for the production of cell-cell signaling molecules by bacterial organisms involved in milk spoilage.