Characterization of chemoheterotrophic bacteria associated with the in situ bioremediation of a waste-oil contaminated site

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Abstract
In the course of an in situ bioremediation, different hydrologically controllable test plots were installed on the ground of a waste-oil contaminated site, and continuously injected with nutrient solution and the electron acceptors NO3−, O2, and H2O2. In a two-year period, groundwater samples obtained from different recovery wells within these field plots, in addition to subsoil samples, were monitored for several chemical and microbiological parameters. The removal of hydrocarbons observed in the water samples could not unambiguously be attributed to biodegradation, and was probably caused by groundwater treatment measures. However, chemical (gaschromatographic) and microbiological data from the subsoil samples indicated a biological degradation of pollutants. Analysis of the groundwater samples of the different test plots revealed only minor quantitative differences. With time, only a slight increase in bacterial numbers on different media, including hydrocarbon-agar, was observed. In general, chemical and microbiological analyses of groundwater samples cannot replace analyses of subsoil samples for a sufficient documentation of in situ remediation processes in subsoil. From the groundwater and subsoil samples, 3,446 pure cultures, obtained from R2A agar, were characterized morphologically and physiologically, and identified in order to study the culturable bacterial communities. Several qualitative differences in composition and diversity of the bacterial communities among the test plots were observed. More than 70 different species or taxonomic groups (most of them known as hydrocarbon degrading taxa) could be identified from the groundwater samples; these were mainly the Gram-negative genera Acinetobacter, Alcaligenes, Comamonas, Hydrogenophaga, Pseudomonas, Flavobacterium/Flexibacter/Cytophaga, and others. A high proportion of Gram-positive organisms (42.5%), belonging to Bacillus and the various genera of coryneform and nocardioform organisms, were isolated from the subsoil samples.