ASSESSMENT OF POPULATION DYNAMICS OF SPECIFIC STRAINS IN GROUNDWATER BIOAUGMENTATION EXPERIMENTS BY TWO DIFFERENT MOLECULAR TECHNIQUES


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Abstract

A set of microcosm experiments was performed to understand the behaviour of special degraders in bioaugmentation experiments. In the experiments the following chlorobenzene degraders were used: the genetically modified Pseudomonas putida F1ΔCC, and the two wild-type strains Pseudomonas putida GJ31 and Pseudomonas aeruginosa RHO1. These strains were used at an initial cell density of 105 cells mL−1 groundwater which had been spiked with 1,2-dichlorobenzene (1,2-DCB), 1,4-dichlorobenzene (1,4-DCB) and, as main contaminant, chlorobenzene (CB). The population dynamics and behaviour of the three special degraders within the groundwater microcosms were studied by single-strand conformation polymorphism (SSCP) analysis of 16S rDNA fragments amplified from directly extracted community DNA and fluorescent in situ hybridization (FISH) with species-specific probes. RHO1 disappeared after 4 days as detected by FISH in contrast to SSCP-detection where RHO1 could be found during the whole incubation time. Whereas GEM F1ΔCC and wild-type strain GJ31 survived the whole incubation for 20 days. With both methods we were able to detect all strains with high specificity among the indigenous microbial community. The data sets obtained from SSCP analysis and FISH were highly correlated. Specific band intensity within the SSCP fingerprints and the cell counts determined by FISH gave a quantitative overview about the introduced strains.

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