Identification of Key Organisms, Genes and Pathways in Benzene-degrading Methanogenic Cultures

Benzene is a carcinogenic hydrocarbon and a widespread groundwater contaminant. Microbial mineralization of benzene is possible, but the process is slow and not well understood under the anaerobic conditions that are often prevalent at contaminated sites. The goals of this study were to gain a better understanding of key organisms, genes and metabolic pathways associated with benzene degradation in methanogenic enrichment cultures. Conditions required for the successful growth of the cultures were also explored. Potential key organisms were identified via 16S rRNA gene cloning studies, performed using several cultures that had been enriched in parallel from the same hydrocarbon-contaminated site. In all cultures, closely related members of the Syntrophobacterales were found to have survived and thrived over nearly a decade of enrichment. These organisms, referred to as ORM2-like organisms, were phylogenetically most similar to those present in cultures enriched from other benzene-contaminated sites. To link organisms with their potential roles in anaerobic benzene biodegradation, we sequenced, assembled and analyzed the metagenome of one of the benzene-degrading methanogenic cultures. Several assemblies were created from Illumina sequencing data and a combination of these proved to be optimal. An unsupervised binning method was developed to achieve near-complete genomes of the three most abundant phylotypes, including a group of three closely-related ORM2-like species, a hydrogen-utilizing Methanoregula sp., and a previously unidentified organism in the culture, classified as a member of the Candidate Division OD1. The ORM2-like organisms were found to harbor a suite of genes linked to the degradation of the central metabolite benzoyl-CoA. Proteomic data confirmed that these genes were expressed under benzene-degrading conditions, providing strong evidence for the role of ORM2-like organisms as benzene fermenters. Activity of hydrogenotrophic and acetoclastic methanogens was confirmed; other organisms may play peripheral roles that increase the culture robustness. The absence of an easily identified putative anaerobic benzene carboxylase in the ORM2-like genomes suggests a novel mechanism for benzene activation by these organisms. The work presented here provides a valuable blueprint that could be used in identifying biomarkers of anaerobic benzene biodegradation, or in exploring the genomes of organisms from novel genera, orders and even phyla.Ph