Persistence, Bioremediation and Fate of Unresolved Complex Mixtures (UCMs) of Hydrocarbons in the Environment

Max Frenzel

PhD – March 2008

University of Exeter, Exeter, U.K.

When crude oil is released into the environment it becomes subject to a number of abiotic (weathering) and biotic (biodegradation) processes. These processes remove the more abundant, gas chromatographically resolvable components of the oil (such at the n-alkanes) and the remaining material appears as an unresolved complex mixture (UCM). UCMs are characteristic of weathered and heavily biodegraded crude oils and are known to persist in the environment for many years and to be toxic. Therefore UCMs should be considered as candidates for bioremediation. To date bacteria capable of degrading these persistent, toxic environmental pollutants remain unidentified. In order to culture such bacteria two alkylcyclohexyltetralins and two butylcyclohexylbutanoic acids were synthesised as model compounds. These contained structural features consistent with those agreed upon for aromatic and acidic UCM components respectively. All compounds were purified or synthesised successfully (all > 81% pure) and in good yield (> 80 mg) for use in biodegradation studies. Structures were confirmed by GC-MS, JR spectroscopy and NMR spectroscopy.

These compounds were used in enrichment culture with sediment collected from Whitley Bay, U.K. (a naturally hydrocarbon-polluted site). After 46 days incubation with the isolated consortia 86.5% of 6-cyclohexyltetralin and 75.6% of 1-(3'-methylbutyl)-7-cyclohexyltetralin (MBCHT) were biodegraded when compared with abiotic controls. Abiotic losses were minimal over the incubation period (mean loss 10% ± 10%). No biodegradation of the model naphthenic acids was observed. PCR-DGGE and 16S rRNA gene clone libraries were used to characterise the microbial communities involved. 16S rRNA gene clone libraries of the consortia able to degrade 6-cyclohexyltetralin and MBCHT were both dominated by sequences identified as Rhodococcus sp.

A UCM isolated from Tia Juana Pesado crude oil (Venezuela) was exposed for 46 days to the isolated Whitley Bay consortium able to biodegrade MBCHT. Comprehensive two-dimensional gas chromatography-time of flight-mass spectrometry (GC×GC-T0F-MS) and mass fragmentology analysis were used to study changes in the composition of the UCM over the exposure period. This revealed that biodegradation of the lower molecular weight branched alkylbenzenes (BABs), -alkylindans and tetralins (BATs), and -alkylindenes (BAIs) had occurred, however large numbers of compounds were also sorbed onto the surface of the cells. Changes in the microbial community were monitored by PCR-DGGE of the 16S rRNA gene, whilst changes in the toxicity of the oil fraction was assessed using the Microtox® test and a blue mussel (Mytilus edulis) feeding rate assay and compared to controls incubated in the absence of the consortium. Mussels exposed to the undegraded and biodegraded UCMs (7.5 mg L-1) exhibited feeding rates of 45 and 108% that of control organisms respectively, indicating that the biodegraded oil fraction was significantly less toxic than the undegraded fraction. GC×GC-T0F-MS and mass fragmentology analysis of mussel tissue extracts revealed that this decrease in toxicity may be partly explained by the loss of the BABs, BATs and BAIs from the UCM in the biodegraded sample. Using selective media a strain of Rhodococcus was isolated that was able to biodegrade 63% of MBCHT after 21 days incubation. Several putative metabolites of a minor synthetic impurity were identified indicating that the alicyclic ring of MBCHT might be the site of initial enzyme attack.

This study is the first to identify the bacteria involved in the biodegradation of cyclohexylalkyltetralins. This study has also shown that it is possible to use bacteria to reduce the toxicity of UCMs to microorganisms and invertebrates. The identification of such bacteria is an important step toward the bioremediation of UCM contaminated sites.

© 2008 by M. Frenzel. All Rights Reserved

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