Biodegradation of aromatic hydrocarbons : microbial and isotopic studies

Diegor, Elizabeth Justa M. (2000) Biodegradation of aromatic hydrocarbons : microbial and isotopic studies. Masters thesis, Memorial University of Newfoundland.

[English] PDF (Migrated (PDF/A Conversion) from original format: (application/pdf)) - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. Download (15MB) [English] PDF - Accepted Version Available under License - The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. (Original Version)

Abstract

Aromatic hydrocarbon contamination of soil and groundwater is a widespread environmental problem. Among the compounds of interest is a range of low molecular weight aromatic hydrocarbons that includes the so-called BTEX compounds (benzene, toluene, ethylbenzene, xylenes). Aerobic biodegradation by natural populations of microorganisms represents one of the primary mechanisms by which petroleum and other hydrocarbon pollutants are eliminated in the environment. Several indicators have been utilized to evaluate this process but their measurement (e.g., of hydrocarbon concentration, bacterial count, metabolites) maybe affected by other chemical and physical processes. Stable carbon isotope analysis is one technique that has been previously used in environmental studies particularly in tracing sources of organic pollutants. Compounds have characteristic carbon isotopic compositions that can be used to pinpoint their origins. Any process in which the compounds are involved may likewise impart significant isotopic fractionation. It is shown that abiotic processes affect the ¹²C/¹³C ratio but biological transformation is known to produce the largest fractionation. -- The purpose of this study is to determine the magnitude and direction of transformation of stable carbon isotopes (¹²C, ¹³C) during microbial degradation of selected low molecular weight hydrocarbon compounds such as toulene, ethylbenzene, naphthalene, methanol and hexadecane. Coupled with this objective is the identification of the various species that make up the consortium used in the study and the metabolic pathways by which these organisms degrade the compound. The overarching goal is to examine if the isotopic fractionation associated with such pathways can be employed for monitoring in situ bioremediation. -- Replicate microbial biodegradation experiments modified from an earlier protocol were done using microbial cultures grown aerobically at room temperature. Optical density measurements during the course of the experiments were undertaken to establish microbial growth. In addition, hydrocarbon isotope analysis was conducted by periodically removing a specific headspace concentration from the culture flask and analyzing it by gas chromatography continuous flow isotope ratio mass spectrometry (GC-IRMS). -- Laboratory biodegradation studies on toluene showed increase in microbial growth from increases in optical density measurements with corresponding decreases in hydrocarbon concentrations and no significant changes in the δ¹³C values. Similar observations were obtained using a higher substrate concentration (10 μl of toluene) except for differences in incubation periods. Experiments conducted on ethylbenzene as the substrate likewise demonstrated the same effects on microbial biomass as well as in concentrations of the residual hydrocarbon. Carbon isotopic compositions also remained relatively constant during microbial growth. -- Taxonomic identification of the microcosm resolved several strains that composed the different hydrocarbon-specific cultures. These bacterial strains consisted of Gram negative rods as well as Gram positive coccL Gram negatives included strains from the genera of Pseudomonas, Stenotrophomonas, Oligella and Acidovorax while Gram positives belonged to Micrococcus, Staphylococcus, Dermacoccus and Kokuria (or Erythromyxa). -- Results of the present study were compared with other published works. Similarities and differences in the outcomes of the respective experiments indicate that the occurrence of isotopic fractionation depends on the degradative pathways utilized by the respective microbial consortia. In particular, the nature of the initial metabolic step (e.g., attack on methyl group versus scission of aromatic ring) could control the extent of carbon isotope fractionation. -- Based on the results of the present study, application of stable carbon isotope analysis in aerobic degradation of aromatic hydrocarbons, particularly the BTEX compounds, does not appear promising for assessment of natural or engineered in situ bioremediation. Future studies should look more closely into the different degradative pathways and enzyme systems used by individual microorganisms as well as mixed populations and their effects on the magnitude of isotopic fractionation. Site-specific studies are also necessary to determine the inherent presence of (these) microbial consortia and quantify the associated biological isotope fractionation.

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