INTRODUCTION AND PCR DETECTION OF DESULFOMONILE-TIEDJEI IN SOIL SLURRY MICROCOSMS

The aim of this work was to test the feasibility of introducing an ana erobic microbial reductive dechlorination activity into non sterile so il slurry microcosms by inoculation with the pure anaerobic bacterial strain Desulfomonile tiedjei, which is capable of dechlorinating 3-chl orobenzoate to benzoate. To show that the bacterium was established in the microcosms we followed the expression of the reductive dechlorina tion activity and a molecular probe based on PCR amplification of the 16S rDNA gene was developed. However, the success of PCR amplification of the 16S rDNA gene depends on the DNA extraction and purification m ethodologies applied, as shown through the use of several protocols. I n this study we report a DNA extraction and purification method which generates sufficient and very clean DNA suitable for PCR amplification of the D. tiedjei 16S rDNA gene. The threshold of detection was about 5.10(3) bacteria per gram of soil slurry. Introduction of D. tiedjei in soil slurry microcosms proved successful since 3-chlorobenzoate dec hlorination activity was established with this bacterium in microcosms normally devoid of this dechlorination capacity. Indeed, the addition of D. tiedjei to microcosms supplemented with acetate plus formate as cosubstrate, at their respective concentrations of 5 and 6 mM, led to a total biotransformation of 2.5 mM of 3-chlorobenzoate within 12 day s. After complete 3-chlorobenzoate dechlorination, the 16S rDNA gene o f this bacterium was specifically detected only in the inoculated micr ocosms as shown by PCR amplification followed by restriction mapping c onfirmation.