Microbial communities associated with anaerobic benzene degradation in a petroleum-contaminated aquifer

Abstract

Microbial community composition associated with benzene oxidation under in situ Fe(III)-reducing conditions in a petroleum-contaminated aquifer located in Bemidji, Minn., was investigated. Community structure associated with benzene degradation was compared to sediment communities that did not anaerobically oxidize benzene which were obtained from two adjacent Fe(III)-reducing sites and from methanogenic and uncontaminated zones. Denaturing gradient gel electrophoresis of 16S rDNA sequences amplified with bacterial or Geobacteraceae-specific primers indicated significant differences in the composition of the microbial communities at the different sites. Most notable was a selective enrichment of microorganisms in the Geobacter cluster seen in the benzene-degrading sediments. This finding was in accordance with phospholipid fatty acid analysis and most-probable-number-PCR enumeration, which indicated that members of the family Geobacteraceae were more numerous in these sediments. A benzene-oxidizing Fe(III)-reducing enrichment culture was established from benzene-degrading sediments and contained an organism closely related to the uncultivated Geobacter spp. This genus contains the only known organisms that can oxidize aromatic compounds with the reduction of Fe(III). Sequences closely related to the Fe(III) reducer Geothrix fermentans and the aerobe Variovorax paradoxus were also amplified from the benzene-degrading enrichment and were present in the benzene-degrading sediments. However, neither G. fermentans nor V. paradoxus is known to oxidize aromatic compounds with the reduction of Fe(III), and there was no apparent enrichment of these organisms in the benzene-degrading sediments. These results suggest that Geobacter spp. play an important role in the anaerobic oxidation of benzene in the Bemidji aquifer and that molecular community analysis may be a powerful tool for predicting a site's capacity for anaerobic benzene degradation.

FIG. 1

Hierarchical cluster analysis of PLFAs extracted from sediment samples from IR-1, IR-2, IR-3, and the uncontaminated site.

FIG. 2

Distribution of Geobacteraceae-indicative PLFA functional groups at sites IR-1, IR-2, and IR-3 and the uncontaminated site.

FIG. 3

DGGE profiles of bacterial 16S rDNA fragments retrieved from Bemidji sediments along the contaminant gradient. Triplicate DNA extractions were analyzed from each site. A 60 to 80% denaturant gradient range was used.

FIG. 4

DGGE analysis of 16S rDNA fragments amplified from the uncontaminated site (Uncont.) and benzene-oxidizing site IR-3 with primers that target 16S rDNAs of most bacteria in several different denaturant gradient ranges.

FIG. 5

16S rRNA phylogenetic tree of Geobacteraceae and delta proteobacterial sequences from the uncontaminated site and site IR-3 and from benzene-oxidizing enrichment cultures. Phylogenetic trees were constructed by using maximum-parsimony methods with 100 bootstrapped data sets. A total of 366 base positions were considered in the analysis. Bootstrap values of greater than 50 (of 100 trees) are shown adjacent to the nodes. The scale bar is in fixed nucleotide substitutions per 100 sequence positions. A similar tree topology was observed for trees constructed by using least-squares methods (data not shown).

FIG. 6

Phylogenetic analysis of 16S rRNA sequences from the uncontaminated and benzene-oxidizing sites and from enrichment cultures. Phylogenetic trees were constructed by using maximum-parsimony methods with 100 bootstrapped data sets. Bootstrap values (of 100 trees) that are greater than 50 are shown adjacent to the nodes. A total of 402 base positions were considered in the analysis. The scale bar is in fixed nucleotide substitutions per 100 sequence positions. A similar tree topology was observed for trees constructed by using least-squares methods (data not shown).

FIG. 7

DGGE analysis of 16S rDNA fragments amplified from the uncontaminated (uncont.) site and the benzene-oxidizing site (IR-3) by using Geobacteraceae-targeted primers.