Microbial diversity in a hydrocarbon- and chlorinated-solvent-contaminated aquifer undergoing intrinsic bioremediation

Abstract

A culture-independent molecular phylogenetic approach was used to survey constituents of microbial communities associated with an aquifer contaminated with hydrocarbons (mainly jet fuel) and chlorinated solvents undergoing intrinsic bioremediation. Samples were obtained from three redox zones: methanogenic, methanogenic-sulfate reducing, and iron or sulfate reducing. Small-subunit rRNA genes were amplified directly from aquifer material DNA by PCR with universally conserved or Bacteria- or Archaea-specific primers and were cloned. A total of 812 clones were screened by restriction fragment length polymorphisms (RFLP), approximately 50% of which were unique. All RFLP types that occurred more than once in the libraries, as well as many of the unique types, were sequenced. A total of 104 (94 bacterial and 10 archaeal) sequence types were determined. Of the 94 bacterial sequence types, 10 have no phylogenetic association with known taxonomic divisions and are phylogenetically grouped in six novel division level groups (candidate divisions WS1 to WS6); 21 belong to four recently described candidate divisions with no cultivated representatives (OP5, OP8, OP10, and OP11); and 63 are phylogenetically associated with 10 well-recognized divisions. The physiology of two particularly abundant sequence types obtained from the methanogenic zone could be inferred from their phylogenetic association with groups of microorganisms with a consistent phenotype. One of these sequence types is associated with the genus Syntrophus; Syntrophus spp. produce energy from the anaerobic oxidation of organic acids, with the production of acetate and hydrogen. The organism represented by the other sequence type is closely related to Methanosaeta spp., which are known to be capable of energy generation only through aceticlastic methanogenesis. We hypothesize, therefore, that the terminal step of hydrocarbon degradation in the methanogenic zone of the aquifer is aceticlastic methanogenesis and that the microorganisms represented by these two sequence types occur in syntrophic association.

FIG. 1

Diagram of sampling locations and profile of clone libraries. Approximate redox zone and soil sample depths are indicated along with groundwater sampling points from sampling well ML3. The water table (∇) is approximately 5.2 m below the ground surface (m bgs).

FIG. 2

Diagrammatic radial representation of selected putative bacterial divisions. Filled wedges indicate that sequences representing these divisions were obtained in the present study. The numbers in parentheses next to the divisions indicate the number of sequences obtained from the respective divisions. Nonfilled wedges are reference divisions with no representation among the Wurtsmith clones. Wedges labeled WS indicate previously undetected candidate divisions.

FIG. 3

Evolutionary distance dendrograms of bacterial and archaeal 16S rDNA sequence types obtained from the Wurtsmith aquifer. Putative divisions are listed outside the brackets for panels b, c, d, f, and g; subdivisions are listed for panel a (Proteobacteria) and panel e (low-G+C gram-positive organisms). Six novel candidate divisions determined in this study are labeled WS1 to WS6. Reference sequences were chosen with the ARB parsimony insertion tool and database. Bacillus subtilis (D26185) and Synechococcus sp. strain PCC 6301 (X01296) were used as the outgroups for panels a, b, c, d, and f; Synechococcus sp. strain PCC 6301 and E. coli (J01695) were used as the outgroups for panel e; and Thermatoga maritima (M21774) and Aquifex pyrophilus (M83548) were used as the outgroups for panel g. Branch points supported (bootstrap values, >74%) by rate-corrected maximum likelihood, parsimony, and distance analyses are indicated by filled circles; open circles indicate branch points supported by some analyses but only marginally supported (bootstrap values, 50 to 74%) or not supported (bootstrap values, <50%) by others. Branch points without circles were not resolved (bootstrap values, <50%) as specific groups in different analyses.

FIG. 4

Proposed secondary and tertiary interactions in the region from positions 503 to 542 (E. coli numbering) of 16S rRNA sequences. Sequences in italic type represent the binding site for the universal 515F primer. Connected boxes indicate proposed tertiary interactions. Nucleotides in bold type indicate differences between the WCHB1-01 and E. coli rRNAs.