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



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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. Four samplings of untreated sewage sludge were collected from wastewater treatment plants in seven different municipalities across the United States. Samples were subjected to quantification of selected bacterial organisms by culture and a pyrosequencing analysis was performed on extracted community DNA. The results of this study showed that untreated sewage sludge is inhabited by a huge diversity of microorganisms and that certain municipalities may have distinct bacterial populations that are conserved over time. Additionally, this study provided some evidence for seasonal differences in several of the major bacterial phyla. Lastly, this study emphasized the challenges of comparing results obtained by culture and pyrosequencing. In conclusion, this study showed that both milk and sewage are highly diverse, dynamic environments that can contain organisms of public health concern. The use of both culture-based methods and pyrosequencing in this study proved a complementary approach, providing a more comprehensive picture of both microbial environments.