QUANTIFICATION OF DESULFITOBACTERIUM-FRAPPIERI STRAIN PCP-1 AND CLOSTRIDIUM-LIKE STRAIN-6 IN MIXED BACTERIAL-POPULATIONS BY COMPETITIVE POLYMERASE-CHAIN-REACTION

Competitive polymerase chain reaction (cPCR) was used to quantify two anaerobic, Gram-positive bacteria, Desulfitobacterium frappieri strain PCP-1 and Clostridium-like strain 6 in mixed bacterial populations. c PCR was done with specific primers targeting the respective 16S rRNA g enes of these strains and an internal standard (IS). The IS for each s train had the same primer binding sites, size and sequence as the targ et DNA, except that a unique restriction site was introduced by PCR-me diated site-directed mutagenesis. To distinguish between the IS and th e target DNA, appropriate restriction digestion was done, cPCR was per formed on genomic DNA of both strains, from which, a close value of th e number of genomes added in the PCR mixtures was obtained, cPCR was a lso carried out with DNA extracted from a soil inoculated with a known amount of strain PCP-1 cells (determined by a:plating method). The ce ll number of strain PCP-1 in the soil, determined by the cPCR, was sim ilar to the number of CFU inoculated. Evaluation of the strain PCP-1 c ell concentration was achieved in a pentachlorophenol (PCP)-degrading methanogenic consortium, from which strain PCP-1 was isolated. This co nsortium was used in a continuous flow activated sludge reactor to tre at PCP-contaminated industrial effluents. cPCR revealed that strain PC P-1 was present at a concentration of 1.8x10(5) cells/mL in the reacto r. The cell concentration of strain 6 was evaluated in a phenol-degrad ing methanogenic consortium, from which strain 6 was isolated. Strain 6 was estimated by cPCR to be 6.5x10(5) cells/mL in a 20-day-old conso rtium culture. This is the first time that these two anaerobic Gram-po sitive bacteria were quantified in mixed bacterial populations. (C) 19 98 Elsevier Science B.V.