Sequencing and PCR-Based Detection of Phenylacetate Decarboxylase Genes in Toluene-Producing Cultures Derived From a Superfund Site Near Baton Rouge, Louisiana

The broad goal of the research described in this thesis was to gain a better understanding of microbial communities capable of anaerobically producing toluene during bioremediation strategies aimed at stimulating reductive dechlorination at a Superfund site located near Baton Rouge, Louisiana. Full-length sequences of genes encoding enzymes putatively responsible for toluene production in a groundwater-derived enrichment culture were determined. Quantitative PCR (qPCR) methods were developed to detect two target genes: 1) a glycyl radical enzyme reportedly responsible for the conversion of phenylacetate to toluene and 2) a partial 16S rRNA gene belonging to the putative toluene-producing organism, a member of the phylum Acidobacteria related to Candidatus Koribacter versatalis strain Ellin345. The newly developed qPCR methods were applied to quantify the two target genes over time in a toluene-producing enrichment culture derived from site groundwater. qPCR analysis revealed a positive correlation between quantities of the two target genes and toluene accumulation in batch culture. qPCR analysis also revealed a high correlation between quantities of the two target genes. Collectively, experimental results support the notion that the microorganisms present at the Louisiana Superfund site responsible for toluene production are members of the phylum Acidobacteria related to Candidatus Koribacter versatalis Ellin345