Assessment of microbial community changes and limiting factors during bioremediation of hydrocarbon-polluted soil with new miniaturized physiological methods

Due to human activities, organic pollutants are spilled to the environment where they threaten public health, often as contaminants of soil or groundwater. Living organisms are able to transform or mineralize many organic pollutants, and bioremediation techniques have been developed to remove pollutants from a contaminated site. However, fast and easy methods to document both the efficacy of bioremediation and the changes in soil microbial communities during bioremediation are not well developed. The major aim of this thesis was to develop miniaturized methods targeting the physiology of microbial communities during pollutant degradation in soil and to assess pollution-induced community changes. Furthermore, the methods should identify factors limiting efficient pollutant biodegradation. Petroleum hydrocarbons have been chosen as pollutants because these compounds are frequently spilled, readily water-soluble, partly toxic and carcinogenic, and therefore undesirable in soils and drinking water supplies. The influence of petroleum hydrocarbons on a microbial community in the vadose zone was assessed under field conditions. An artificial hydrocarbon mixture consisting of volatile and semi-volatile compounds similar to jet-fuel was emplaced in a previously uncontaminated vadose zone in nutrient-poor glacial melt water sand. The experiment included monitoring of microbial parameters and CO2 concentrations in soil gas over 3 months, in- and outside the hydrocarbon vapor plume that formed around the buried petroleum. Microbial and chemical analyses of vadose zone samples were performed on material from 9 cores drilled on 3 days at 3 distances from the buried petroleum mass to 3.3 m depth. Significantly elevated CO2 concentrations were observed after contamination. Total cell numbers as determined by fluorescence microscopy were strongly correlated with soil organic carbon and nitrogen content but varied little with contamination. Redundancy analysis (RDA) allowed direct analysis of effects of selected environmental variables or the artificial contamination on microbiological parameters. Variation in biomass and CO2 production was to 46 % explained by soil parameters and to 39.8 % by the duration of contamination. The microbial community structure was assessed by community-level physiological profile analysis using BiologTM EcoPlates. Only 35.9 % of variation in BiologTM data could be attributed to soil parameters and contamination, however, the samples with greatest exposure to hydrocarbons grouped together on RDA plots. It is concluded that at this nutrient-poor site, the microbial community was dominated by the natural heterogeneity and that the influence of petroleum hydrocarbon vapor was weak. BiologTM Eco-Plates combined with RDA were able to distinguish between samples with high and low hydrocarbon exposure. However, the method was not sensitive enough to produce consistent patterns in three replicates when extracts from subsurface soils with low