Pyrosequencing of the 16S rRNA gene to reveal bacterial pathogen diversity in biosolids Kyle Bibby, Emily Viau, Jordan Peccia* Department of Chemical Engineering, Environmental Engineering Program, Yale University, New Haven, CT 06520, USA article info Article history: Received 17 March 2010 Received in revised form 14 May 2010 Accepted 25 May 2010 Available online 9 June 2010 Keywords: Pathogen Anaerobic digestion Sludge Compost 16S abstract Given the potential for a variety of bacterial pathogens to occur in variably stabilized sewage sludge (biosolids), an understanding of pathogen diversity and abundance is necessary for accurate assessment of infective risk when these products are land applied. 16S rDNA was PCR amplified from genomic DNA extracted from municipal wastewater residuals (mesophilic- and thermophilic-phased anaerobic digestion (MAD and TPAD), composting (COM)), and agricultural soil (SOIL), and these amplicons were sequenced using massively parallel pyrosequencing technology. Resulting libraries contained an average of 30,893 16S rDNA sequences per sample with an average length of 392 bases. FASTUNIFRAC- based comparisons of population phylogenetic distance demonstrated similarities between the populations of different treatment plants performing the same stabilization method (e.g. different MAD samples), and population differences among samples from different biosolids stabilization methods (COM, MAD, and TPAD). Based on a 0.03 Jukes-Cantor distance to 80 potential bacterial pathogens, all samples contained pathogens and enrichment ranged from 0.02% to 0.1% of sequences. Most (61%) species identified were opportunistic pathogens of the genera Clostridium and Mycobacterium. As risk sciences continue to evolve to address scenarios that include multiple pathogen exposure, the analysis described here can be used to determine the diversity of pathogens in an envi- ronmental sample. This work provides guidance for prioritizing subsequent culturable and quantitative analysis, and for the first time, ensuring that potentially significant pathogens are not left out of risk estimations. ª 2010 Elsevier Ltd. All rights reserved. 1. Introduction More than 7 million dry tons of sewage sludge are produced annually in the U.S., Bastian (1997). Globally, this waste stream continues to increase as urban populations increase, municipal wastewater treatment facilities move toward biological nutrient removal, and urban areas of developing nations build sewer systems and centralized treatment works. In the U.S., sewage sludges that have been stabilized by digestion or composting are termed “biosolids” if there is a resulting beneficial use. Greater than 60% of stabilized sewage sludges are reused through application to agricultural land, Bastian (1997), Spicer (2002). While the agricultural benefits of land applying biosolids are well documented, Tenenbaum (1997), the potential pathogen content of biosolids and associated health complaints from residents living near biosolids land application sites have resulted in widespread public health concerns and community opposition to this practice, NRC (2002). The pathogen content of biosolids is likely diverse. In large municipalities, centralized wastewater treatment facilities commonly serve over 1 million residents. Although enteric pathogens are the traditional focus of biosolids management * Corresponding author. Tel.: þ1 203 432 4385; fax: þ1 203 432 4387. E-mail address: jordan.peccia@yale.edu (J. Peccia). Available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/watres water research 44 (2010) 4252 e4260 0043-1354/$ e see front matter ª 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2010.05.039