Introduction and PCR detection of Desulfomonile tiedjei in soil slurry microcosms

Abstract

Bioremediation is currently attracting considerable interest as a technique of enhancing the degradation of troublesome pollutants by competent microorganisms for the rehabilitation of contaminated soil. A number of authors have demonstrated the feasibility of introducing new metabolic activities into a specific microbial environment using pure bacterial strains or consortia (Oldenhuis et al., 1989; Ahring et al., 1992; Møller and Ingvorsen, 1993; Brunsbach and Reineke, 1993, 1995). The new bio-catalytic potential can be established in the inoculated site either through the growth and metabolism of the added microorganisms or by the transfer of degradative genes (Mergeay et al., 1990; Zhou and Tiedje, 1995). In this work, we explored the introduction of reductive dechlorination activity into non-sterile soil microcosms by inoculation with a pure anaerobic bacterium strain and we developed reliable molecular methodology for detection of the strain over time after inoculation. Desulfomonile tiedjei was used as model microorganism because of its well-characterised dechlorination activity. This bacterium which can dechlorinate chloroaromatics (De Weerd et al., 1990) is a fastidious sulfate-reducing anaerobe with the capacity to grow syntrophically within a methanogenic consortium (Dolfing and Tiedje, 1986). In addition to demonstrating the successful maintenance of D. tiedjei in the microcosms by following the reductive dechlorination of 3-chlorobenzoate, we also developed a molecular probe. Its use for detecting D. tiedjei in soil slurry microcosms was based on the PCR amplification of the 16S rDNA gene. This genomic fragment is widely used as a probe for detecting bacteria in natural and polluted environments as shown by the exponential increase of recent publications (Amann et al., 1990; Britschgi and Fallon, 1994; Raskin et al., 1994; Amann et al., 1995; Degrange and Bradin, 1995; Briglia et al., 1996; Hales et al., 1996; Wang et al 1996). However, to obtain DNA suitable for PCR amplification different DNA extraction and purification methods had to be tested and compared (Dijkmans et al., 1993; Volossiouk et al., 1995; Zhou et al, 1996) before developing an adequate methodology for the soil and the bacterium used throughout this study.