Introduction and PCR detection of Desulfomonile tiedjei in soil slurry microcosms

The aim of this work was to test the feasibility ofintroducing an anaerobic microbial reductivedechlorination activity into non sterile soil slurrymicrocosms by inoculation with the pure anaerobicbacterial strain Desulfomonile tiedjei, which iscapable of dechlorinating 3-chlorobenzoate tobenzoate. To show that the bacterium was establishedin the microcosms we followed the expression of thereductive dechlorination activity and a molecularprobe based on PCR amplification of the 16S rDNA genewas developed. However, the success of PCRamplification of the 16S rDNA gene depends on the DNAextraction and purification methodologies applied, asshown through the use of several protocols. In thisstudy we report a DNA extraction and purificationmethod which generates sufficient and very clean DNAsuitable for PCR amplification of the D. tiedjei16S rDNA gene. The threshold of detection was about5.10³ bacteria per gram of soil slurry.Introduction of D. tiedjei in soil slurrymicrocosms proved successful since 3-chlorobenzoatedechlorination activity was established with thisbacterium in microcosms normally devoid of thisdechlorination capacity. Indeed, the addition of D. tiedjei to microcosms supplemented with acetateplus formate as cosubstrate, at their respectiveconcentrations of 5 and 6 mM, led to a totalbiotransformation of 2.5 mM of 3-chlorobenzoate within12 days. After complete 3-chlorobenzoatedechlorination, the 16S rDNA gene of this bacteriumwas specifically detected only in the inoculatedmicrocosms as shown by PCR amplification followed byrestriction mapping confirmation.