| The purpose of this work was to determine whether a cultivation-independent approach could be used to monitor aerobic bioremediation of aromatic hydrocarbons. A cultivation-independent approach would be a significant improvement over standard cultivation-based methods which are time intensive and are subject to several well-known biases. Catechol 2,3-dioxygenase genes were used as a marker for monitoring aerobic bioremediation because catechol 2,3-dioxygenase enzymes are involved in the biodegradation pathways of numerous environmentally significant aromatic compounds. Polymerase chain reaction (PCR) primers specific for catechol 2,3-dioxygenase genes were designed and tested on genetically well characterized strains of bacteria. A quantitative competitive PCR method was modified for use with these primers and evaluated in the presence of potential PCR inhibitors. Toluene, non-target DNA, and soil organic matter did not inhibit PCR. Use of this technique allowed detection of 102 to 10 3 genes. The detection limit was improved to 100 to 101 genes when a gene probe was used. Microcosm studies were performed to determine which aromatic compounds enriched for dioxygenase genes detectable with this method. Naphthalene, m-xylene, and p-xylene all enriched for detectable catechol 2,3-dioxygenase genes while benzene, toluene, and o-xylene produced only transient, weakly detectable genes. In a field study catechol 2,3-dioxygenase genes were detected at a petroleum-contaminated site where an oxygen-releasing compound was being used to stimulate aerobic biodegradation. At a different petroleum-contaminated site where clay soils predominated, catechol 2,3-dioxygenase genes were generally not present. This is consistent with other data which indicates that aerobic bioremediation of aromatic compounds was occurring at the site where the oxygen-releasing compound was being used, and was not occurring at the second field site. The results of this field study demonstrate that the molecular genetic method developed provides a more accurate and rapid detection of aromatic hydrocarbon-degraders than cultivation-based assays. |
