Molecular Diversity of a North Carolina Wastewater Treatment Plant as Revealed by Pyrosequencing

Abstract

We report the results of pyrosequencing of DNA collected from the activated sludge basin of a wastewater treatment plant in Charlotte, NC. Using the 454-FLX technology, we generated 378,601 sequences with an average read length of 250.4 bp. Running the 454 assembly algorithm over our sequences yielded very poor assembly, with only 0.3% of our sequences participating in assembly of significant contigs. Of the 117 contigs greater than 500 bp long that were assembled, the most common annotations were to transposases and hypothetical proteins. Comparing our sequences to known microbial genomes showed nonspecific recruitment, indicating that previously described taxa are only distantly related to the most abundant microbes in this treatment plant. A comparison of proteins generated by translating our sequence set to translations of other sequenced microbiomes shows a distinct metabolic profile for activated sludge with high counts for genes involved in metabolism of aromatic compounds and low counts for genes involved in photosynthesis. Taken together, these data document the substantial levels of microbial diversity within activated sludge and further establish the great utility of pyrosequencing for investigating diversity in complex ecosystems.Although largely invisible in the urban landscape when they are functioning well, wastewater treatment plants are integral to the municipal obligation to protect public health, aquatic ecosystems, and the quality of life. At the heart of wastewater treatment plants is a process whereby a dense microbial consortium is employed to remove organic and nutrient contaminants. The microbes used to treat wastewater are a crucial tool in environmental protection. The current use of molecular techniques that do not require the isolation and cultivation of microorganisms (1, 33), including 16S rRNA analysis (6,13,20) and fluorescent in situ hybridization (8), have greatly expanded our understanding of wastewater microbial communities. Researchers have identified many bacteria of importance to wastewater treatment, including the bacteria involved in biological phosphorus removal (5, 16, 29), nitrifiers (8, 19, 25), denitrifiers (3, 12, 17), and methanogens (18, 36). Molecular techniques have also improved our understanding of fundamental processes such as nitrification and denitrification, as well as plant upsets, such as foaming (9, 24), which can decrease treatment efficiency.In this paper, we apply recently developed pyrosequencing technology to probe the molecular diversity of the aerobic basin of a wastewater treatment plant in Charlotte, NC. In line with other studies of complex microbial communities (28, 32), we observed astounding levels of diversity. We found that substantial regions of the genomes of the most prevalent microbes in the wastewater treatment plant are poorly described by existing sequence databases. Our results demonstrate that despite recent technological advances that allow identification of microorganisms, the microbial population of wastewater treatment plan...

Bacterial assembly and temporal dynamics in activated sludge of a full-scale municipal wastewater treatment plant

“…Some bacterial classes, such as Alphaproteobacteria, Actinobacteria and Nitrospira, tended to be more persistent, whereas others (for example, Deltaproteobacteria, most sulfate-reducing bacteria in this class, which depend on the aerobic/anaerobic condition) were more transitory over the 5-year sampling period. Persistent OTUs were primarily affiliated with 12 classes, such as Alphaproteobacteria, Actinobacteria, Gammaproteobacteria, Acidimicrobia, Sphingobacteria and Anaerolineae, which are also key bacterial groups commonly found in activated sludge of different municipal WWTPs (Wagner and Loy, 2002;Sanapareddy et al, 2009;Xia et al, 2010;Zhang et al, 2012). By contrast, intermittent and transient OTUs included a larger proportion (30-35%, Supplementary Table S4) of populations from other bacterial classes, such as TM7-1, TM7-3, Synergistia, Verrucomicrobiae and Chlamydia (Supplementary Table S4).…”

MiSeq Sequencing Analysis of Bacterial Community Structures in Wastewater Treatment Plants

“…However, to our knowledge only a very limited number of studies have used Illumina MiSeq technology to examine the microbial community structures in WWTPs. Also, these early studies only focused on the microbial communities in a single wastewater treatment system [13] and did not examine the relationships between microbial community structures and environmental variables, thus it is not clear how wastewater characteristics, operational parameters, and geographic locations influence the bacterial community structures.…”

Metagenomic analysis of a stable trichloroethene-degrading microbial community

“…Metagenomic data provide a broad view of the genetic composition of a community, including information about the identity and potential metabolic capabilities of community members. Metagenomic approaches have been used to study a variety of microbial communities, including those inhabiting termite guts, human intestines, wastewater treatment plants and acid mines (Tyson et al, 2004;Gill et al, 2006;Warnecke et al, 2007;Sanapareddy et al, 2009). In the case of dechlorinating communities, metagenomic data can provide insights into the organisms that support dechlorination activity (Waller, 2009).…”