Semester of Graduation

Spring 2018


Master of Civil Engineering (MCE)


Department of Civil and Environmental Engineering

Document Type



The broad goal of the research described in this thesis was to better understand the potential for biological toluene production to occur in groundwater at a Superfund Site located in southeast Louisiana. Previous literature reported that bacterial isolates under laboratory test conditions and undefined microbial communities in anaerobic sludge digesters and seasonally stratified lakes can produce toluene biogenically. Most of the research reported on this subject previously, however, has been on systems in Europe and little research has been reported previously on such phenomena in North America.

In the research described here, studies were conducted utilizing groundwater from a Superfund site employing an enhanced bioremediation strategy to reductively dehalogenate chlorinated solvents. When incubated at ambient room temperature (~22˚C) and provided glucose and either phenylalanine or phenylacetic acid, microbial communities originating from the groundwater displayed the ability to biogenically produce toluene. When provided with 0.8 mM (132 mg/L) phenylalanine, cultures grown in liquid media accumulated toluene to aqueous-phase concentrations 0.44 mM to 0.54 mM of toluene, 41 mg/L and 50 mg/L respectively, in less than 30 days. In experiments in which an enrichment culture derived from site groundwater was supplied with either phenylacetic acid or phenylalanine at concentrations ranging from 0-400 mg/L (0-2.42 mM and 0-2.94 mM respectively), there was a high correlation between toluene accumulated at the end of a 28-day incubation period and the concentration of phenyl-containing precursor supplied. The observed toluene yield was 0.52 moles toluene produced per mole phenylacetic acid supplied and 0.575 moles toluene produced per mole phenylalanine supplied. The toluene-producing capabilities of the enrichment cultures were maintained during propagation through four sequential transfers and after long-term (~15 months) storage at 4°C.

The finding that groundwater microbial communities in areas undergoing enhanced bioremediation to treat chlorinated solvent contamination can produce toluene at levels in excess of drinking water standards has important implications for the field of bioremediation. Further research is necessary to identify how widespread biogenic toluene production is in areas undergoing enhanced remediation. Further research is also required to identify the microbes responsible for toluene production.



Committee Chair

Moe, William