Structural and spatial analysis of the microbial communities in soil contaminated with polycyclic aromatic hydrocarbons

The microbial communities of an aged, creosote-contaminated soil (CMN) and an uncontaminated soil (PMN) from nearby were compared. Polymerase chain reaction (PCR) amplicons of small subunit ribosomal RNA-encoding genes were resolved on a denaturing gradient gel (DGGE) and transformed into clone libraries. The CMN community was less diverse than the PMN, as evidenced by 1) the finite number of DGGE bands versus the smeared profile of the PMN and 2) the finite number of unique clones versus the flat rank abundance of the PMN clones. The CMN clone library contained sequences belonging only to the Proteobacteria, whereas the PMN sequences represented numerous phylotypes. A soil-embedding technique was developed for visualizing microorganisms in soil that preserved spatial arrangements as compared to conventional slurry-based methods. 16S rRNA probes were hybridized to cells in three soils contaminated with polycyclic aromatic hydrocarbons (PAH) and the uncontaminated soil after incubation with pyrene. Spatial relationships between cells and pyrene were determined using a two-population stereological analysis. Microbial localization with respect to pyrene was found to be nonrandom for the slurry-based method and random for the aggregate method. This was likely due to the close association of cells and pyrene with soil particles in the slurried samples. Other patterns also emerged: 1) the higher the background PAH contamination, the more cells clustered with respect to pyrene, and 2) the clustering tendency increased over time for the least contaminated soil and decreased for the others. These data suggest that better-adapted populations are present in more contaminated soils. The change in the bacterial community structure of the spiked PMN was monitored by PCR-DGGE. The PMN profile became less diverse over time but never approached that of the CMN, even after an additional pyrene spike. The community did not recover its original diversity, indicating either permanent succession or an ongoing selection of certain populations due to constant contaminant availability in the soil. A group of pyrene degraders, PG3, was detected whose 16S rRNA gene copy number correlated with pyrene mineralization, suggesting that the appearance of this group following an acute exposure to PAH could be an indicator of remediation potential