Semester of Graduation

August 2022

Degree

Master of Science in Civil Engineering (MSCE)

Department

Civil and Environmental Engineering

Document Type

Thesis

Abstract

While the aromatic hydrocarbon toluene is widely regarded as a contaminant associated with discharges of crude oil and refined petroleum products (e.g., gasoline), the enzyme phenylacetate decarboxylase catalyzes the decarboxylation of phenylacetate to yield toluene. Research described in this thesis further investigated toluene biosynthesis by a microbial consortium originating from chlorinated solvent-contaminated groundwater near Baton Rouge, Louisiana. In anoxic growth media supplemented with phenylacetate as a precursor, toluene accumulation was found to be maximal at near-neutral to slightly acidic pHs (toluene accumulation greater than 1 mg/L at pH in the range of 5.6 to 7.6, but was not observed for pH ≤5.0 or ≥8.0) and low salinity (when ≤3 g/L NaCl was added). Toluene accumulation was observed with and without CO2 and H2 in the anaerobic headspace (up to 10 mol%), when L-cysteine hydrochloride was added as a reducing agent, and when L-cysteine hydrochloride was added in conjunction with the antibiotics ampicillin (≤500 mg/L), vancomycin (≤10 mg/L) or kanamycin (≤5 mg/L). Additional experiments were conducted to explore the spatial distribution and phylogenetic diversity of toluene-producing microorganisms in the northern gulf coast region of the United States. Enrichment cultures inoculated with sediment from 15 of 17 different water bodies distributed throughout Louisiana and as far east as the Florida panhandle produced toluene that accumulated to aqueous-phase concentrations over 100 mg/L. Next generation sequencing of partial 16S rRNA genes in four toluene-accumulating enrichment cultures revealed a relatively high proportion of sequences affiliated with the family Acidobacteriaceae (subdivision 1) of the phylum Acidobacteriota in all four cultures. Nearly full-length Acidobacteriota 16S rRNA gene sequences and diverse phenylacetate decarboxylase gene sequences were recovered from all of the toluene-accumulating enrichment cultures. Results of experiments presented in this thesis demonstrate that toluene-producing bacteria are widespread throughout Louisiana and the northern gulf region of the U.S. This has important implications for bioremediation strategies involving fermentable substrate addition to the subsurface, and it also points to the potential for widespread toluene production in areas where proteins may be biodegraded in anoxic environments.

Date

6-26-2022

Committee Chair

Moe, William M.

Available for download on Wednesday, June 25, 2025

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