Scott Pease

Project title: An Analysis of Viral Metagenomes in Acetate-fed Anaerobic Reactors

Degree: MS | Program: Environmental Health (EH) | Project type: Thesis/Dissertation
Completed in: 2012 | Faculty advisor: John Meschke


Anaerobic digesters (ADs) are an important engineered system used to effectively treat wastewater sludge. The ecology of these digesters is dominated by the acetoclastic methanogens, Methanosaeta spp. and Methanosarcina spp. Though CRISPR sequences have been identified in the genomes of these methanogens, no phages infecting these archaea have been identified. Analysis of the virus size fraction of digester samples by transmission electron microscopy (TEM) revealed the presence of numerous virus-like particles (VLPs), consistent with phages within the family Siphoviridae, which contains phages that infect both archaea and bacteria. To further investigate these VLPs, acetate-fed anaerobic reactors were established and samples removed for characterization of the viral size fraction (VSF) by DNA sequencing. Samples from functional ADs were also processed to assess the similarity to the reactor communities. Samples were 0.2 um filtered, concentrated, placed in CsCl density gradient, and DNase treated before DNA extraction. To assess sample purity and diversity, one sample was initially amplified and sequenced. Samples were then sequenced using an Ion Torrent PGM. Sequences were quality trimmed and assembled, and then analyzed using MG-RAST. Taxonomic profiling yielded ~10% of reactor sequences having significant matches, compared to 25-32% of digester sequences, and indicated dominance by the order Caudovirales, consistent with TEM images. Reactor sequence matches to CRISPR spacers of Methanosaeta concilii suggest the presence of viruses of acetoclastic methanogens in these systems. Functional profiling indicated an abundance of DNA methylase genes, consistent with a prior study. Taxonomic and functional comparison indicated moderate differences between the hourly fed reactor and other samples. Functional comparison to viral metagenomes from other environments indicated significantly lower metabolic gene frequency in our samples. DNA analysis of the VSFs is consistent with microscopy results that phages are a robust component of the digester communities. URI