• Bacteria;
  • 16S rRNA;
  • PCR;
  • Subsurface;
  • Sediment


Culture-based techniques have traditionally been the primary tools utilized for studying the microbiology of terrestrial subsurface environments. Recently, nucleic acid-based methods have been employed to further characterize the microbial diversity in subsurface sediments and rocks, but the results have not been related to individual bacteria cultivated from the same environment. Restriction fragment length profiles of 16S rRNA genes derived from bulk community DNA or bacterial isolates were compared to determine the efficacy of PCR-based methods for studying microbial diversity and phylogeny in a deep (188 m) subsurface environment. The phylogenetic relatedness between 16S rRNA genes of enrichment cultures and individual clones was also determined through DNA sequence analysis of 16S rRNA genes. Restriction fragment length profiles from PCR clone libraries accounted for 64% of recovered isolates and 55% of the estimated culturable diversity based upon their 16S rDNA RFLP signatures. DNA sequence comparisons between the 16S rDNA of the most commonly occurring isolates and clones confirmed that similar DNA sequences were contained within the RFLP groups used to categorize the isolates and clones. For 7 of 8 RFLP groups for which DNA sequences were obtained, nearest neighbor assignments corresponded at the genus level but suggested that 16S rDNA sequences from multiple genera were contained within single RFLP profiles. Phylogenetic analysis of 16S rRNA sequences supported the nearest neighbor inferences and indicated that 16S rDNA clones derived from bulk sediment were specifically related to isolates recovered on enrichment plates. This study has shown that a majority of the cultivated aerobic heterotrophic bacteria in a subsurface sediment could be described by 16S rDNA clones obtained from directly extracted DNA, but that PCR-based methods cannot account for all organisms from a given sample. Consequently, a more comprehensive assessment of microbial diversity in subsurface (and probably other) environments can be obtained by using a combination of culture- and molecular-based techniques than by using either method alone.