Functional soil metagenomics: elucidation of polycyclic aromatic hydrocarbon degradation potential after 10 years of in situ bioremediation

Polycyclic aromatic hydrocarbons (PAHs) are the most ubiquitous contaminants and pose a significant risk to humans and the environment. Microbial degradation represents the major mechanism responsible for the ecological recovery of PAH-contaminated sites. Here we present a culture-independent approach to assess the microbial aerobic catabolome for PAH-degradation. To study the microbial community of a PAH-contaminated soil subjected to 10 years of in situ bioremediation, the community structure was assessed by Illumina-based deep sequencing of amplicons targeting the V5-V6 region of 16S rRNA gene. In a complementary approach, a metagenomic library was prepared in pCCFos and 425 000 clones subjected to activity-based screening for key catabolic ring-cleavage activities using 2,3-dihydroxybiphenyl as substrate. Since most of the genes encoding extradiol ring-cleavage enzymes on 672 fosmids could not be identified using primers based on currently available sequence information, 200 fosmid inserts were sequenced using the Illumina technology. To accurately annotate catabolic genes encoded by the environmental metagenome, we developed manually curated databases for catabolic key gene families involved in degradation of aromatics – AromaDeg. Using this phylogenomics-based bioinformatic tool, we successfully overcame the overwhelming level of misannotations in databases. Sequence information of the fosmid inserts revealed not only the presence of novel extradiol dioxygenase genes but also additional key genes of aromatic metabolic pathways only distantly related to previously described variants. The given detailed framework of the metagenome dedicated to PAH-metabolism will serve as base for a careful analysis of the metabolic net acting on PAHs under real environmental conditions through metatranscriptomic analysis.