Evolution of bacterial diversity during enrichment of PCP-degrading activated soils

The microbiota of completely mixed soil slurry was acclimated with pentachl orophenol (PCP) or with a wood preservative mixture (WPM) containing severa l pollutants such as PCP and petroleum hydrocarbons. The impact of these co mpounds on the bacterial diversity was studied by using molecular tools. PC R amplifications of the 16S ribosomal RNA gene sequences (rDNA) were carrie d out with total DNA extracted from soil slurry samples taken at different time points during the enrichment process of the PCP and WPM reactors. The composition of these PCR products, reflecting the bacterial diversity, was monitored by the single-strand-conformation polymorphism (SSCP) method. Our results showed that the complexity of the SSCP profiles in the PCP reactor decreased significantly during the enrichment process, whereas they remain ed complex in the WPM reactor. PCR-amplified 16S rDNA libraries were genera ted from each reactor. The SSCP method was used to rapidly screen several c lones of these libraries to find specific single-strand DNA migration profi les. In the PCP-activated soil, 96% of examined clones had the same SSCP pr ofile, and sequences of representative clones were related to the genus Sph ingomonas, suggesting that the enrichment with PCP resulted in a selection of little phylogenetic diversity. Four different SSCP profiles were observe d with the 68 examined clones from the WPM reactor. Representative clones o f these profiles were related to Methylocystaceae or Rhizobiaceae, to sulfu r-oxidizing symbionts, to the genus Acinetobacter, and to the genus Sphingo monas. We also cloned and sequenced PCR-amplified DNA related to the pcpB g ene, coding for the Sphingomonas PCP-4-monooxygenase and detected in both r eactors after two weeks of enrichment Of the 16 examined clones, deduced am ino acid sequences of 13 clones were highly related to the Sphingomonas sp. strain UG30 pcpB. The three remaining pcpB clones were not closely related to the three known Sphingomonas pcpB.