Cultivation‐independent in situ molecular analysis of bacteria involved in degradation of pentachlorophenol in soil

Abstract

The central aim of this study was to determine which components of an indigenous bacterial community in pristine grassland soil were capable of degrading pentachlorophenol (PCP) using two cultivation-independent, in situ, molecular techniques. The first involved polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) amplification of 16S rRNA genes from DNA and RNA, respectively, extracted from PCP-amended soil. The second involved stable isotope probing (SIP), with incubation of soil with 13C-PCP and molecular analysis of 13C-labelled RNA, derived from cells incorporating PCP or its breakdown products, after separation from 12C-RNA by ultracentrifugation. Bacterial communities were characterized by denaturing gradient gel electrophoresis (DGGE) analysis of amplification products. PCP was degraded at an approximate rate of 1.18+/-0.25 (SEM) mg kg-1 day-1 and 39% of the measurable PCP fraction was degraded after incubation for 63 days. PCP degradation was associated with significant changes in bacterial community structure, leading to the appearance of seven bands in both DNA- and RNA-based DGGE profiles, the latter providing clearer evidence of qualitative shifts in community structure. The majority of novel bands increased in relative intensity during the first 35 days and subsequently decreased in relative intensity as incubation continued. Sequence and phylogenetic analysis of six of these bands indicated most to have closest database relatives that were uncultured bacteria with sequence homologies to reported hydrocarbon degraders. No band could be detected in RNA-SIP-DGGE profiles derived from 13C-RNA fractions at day 0 but several faint bands appeared in these fractions after incubation of soil for 4 days, indicating assimilation of PCP or its degradation products. These bands increased in intensity during subsequent incubation for 21 days and decreased with further incubation. With one exception, RNA-SIP-DGGE and RNA-DGGE profiles were similar, indicating that RNA-targeted DGGE, in this case, provided a good indication of the metabolically active microbial community.