Functional genes and gene array analysis as tools for monitoring hydrocarbon biodegradation:Dissertation

Bioremediation is considered to be an environmentally sustainable and cost-effective means for environmental clean-up. However, a comprehensive understanding of the biodegradation potential of intrinsic microbial communities and their response to decontamination measures is required for the effective management of bioremediation processes. In this thesis, the potential to use functional genes encoding hydrocarbon-degradative enzymes as indicators of aerobic hydrocarbon biodegradation was investigated. Small-scale functional gene macro- and microarrays targeting aliphatic, monoaromatic and low molecular weight polyaromatic hydrocarbon (PAH) biodegradation were developed in order to simultaneously monitor the biodegradation of mixtures of hydrocarbons. The validity of the array analysis in monitoring hydrocarbon biodegradation was evaluated in microcosm studies and field-scale bioremediation processes by comparing the hybridization signal intensities to hydrocarbon mineralization, real-time polymerase chain reaction (PCR), dot blot hybridization and both chemical and microbial monitoring data. The results obtained by real-time PCR, dot blot quantification and gene array analysis were in good agreement with hydrocarbon biodegradation. In laboratory-scale microcosms, mineralization of several hydrocarbons could be monitored simultaneously using functional gene array analysis. In the field-scale bioremediation processes, the detection and enumeration of hydrocarbon-degradative genes provided important additional information for process optimization and design. In creosote-contaminated groundwater, the functional gene array analysis demonstrated that the aerobic PAH-biodegradation potential that was present at the site, but restrained under the oxygen-limited conditions, could be successfully stimulated with air sparging and nutrient infiltration. During ex situ bioremediation of diesel oil- and lubrication oil-contaminated soil, the functional gene array analysis revealed inefficient hydrocarbon biodegradation, caused by poor aeration during composting. The functional gene array specifically detected upper and lower biodegradation pathways required for complete mineralization of hydrocarbons. Hydrocarbon-degrading bacteria representing 1 % of the microbial community could be detected without prior PCR amplification. Molecular biological monitoring methods based on functional genes provide powerful tools for the development of more efficient remediation processes. With careful assay design, the analysis can be directed to common biodegradation functions rather than specific genotypes. The parallel detection of several functional genes using functional gene array analysis is an especially promising tool for monitoring the biodegradation of mixtures of hydrocarbons