Depth-resolved quantification of anaerobic toluene degraders and aquifer microbial community patterns in distinct redox zones of a tar oil contaminant plume

Abstract

Microbial degradation is the only sustainable component of natural attenuation in contaminated groundwater environments, yet its controls, especially in anaerobic aquifers, are still poorly understood. Hence, putative spatial correlations between specific populations of key microbial players and the occurrence of respective degradation processes remain to be unraveled. We therefore characterized microbial community distribution across a high-resolution depth profile of a tar oil-impacted aquifer where benzene, toluene, ethylbenzene, and xylene (BTEX) degradation depends mainly on sulfate reduction. We conducted depth-resolved terminal restriction fragment length polymorphism fingerprinting and quantitative PCR of bacterial 16S rRNA and benzylsuccinate synthase genes (bssA) to quantify the distribution of total microbiota and specific anaerobic toluene degraders. We show that a highly specialized degrader community of microbes related to known deltaproteobacterial iron and sulfate reducers (Geobacter and Desulfocapsa spp.), as well as clostridial fermenters (Sedimentibacter spp.), resides within the biogeochemical gradient zone underneath the highly contaminated plume core. This zone, where BTEX compounds and sulfate--an important electron acceptor--meet, also harbors a surprisingly high abundance of the yet-unidentified anaerobic toluene degraders carrying the previously detected F1-cluster bssA genes (C. Winderl, S. Schaefer, and T. Lueders, Environ. Microbiol. 9:1035-1046, 2007). Our data suggest that this biogeochemical gradient zone is a hot spot of anaerobic toluene degradation. These findings show that the distribution of specific aquifer microbiota and degradation processes in contaminated aquifers are tightly coupled, which may be of value for the assessment and prediction of natural attenuation based on intrinsic aquifer microbiota.

FIG. 1.

Depth profiles of representative aromatic contaminants (A) and reduced electron acceptors (A and B) in sediment samples (sed.) and groundwater (aq.) from the Flingern aquifer. Samples were taken in June (sed.) and September (aq.) 2005. Error bars indicate the means of duplicate measurements, and the absence of error bars indicates single measurements. Plume and redox compartments were specified in accordance with the biogeochemical data. GW, groundwater.

FIG. 2.

Depth distribution of bacterial 16S rRNA genes and bssA genes of the F1 cluster of unknown anaerobic toluene degraders (58) as measured via qPCR. Shown are the means of gene cp g⁻¹ sediment (wet weight) ± standard errors of three independent DNA extracts for each depth.

FIG. 3.

Depth-resolved 16S rRNA gene T-RFLP fingerprinting of bacterial community structures in plume compartments of the Flingern aquifer. (A) Shannon-Wiener diversity index (H′) calculated for the entire T-RFLP data set. (B) Representative T-RFLP electropherograms of selected depths. Community patterns marked by an asterisk were subsequently selected for cloning. Selected characteristic T-RFs mentioned in the text are indicated by their base pair lengths.

FIG. 4.

(A) PC ordination of the overall variance in depth-resolved bacterial community composition as analyzed by T-RFLP fingerprinting. The depths at which specific fingerprints were retrieved are indicated next to the ordination points. Communities marked by an open square were subsequently selected for cloning. Inferred PC factors 1 and 2 accounted for 21.3% and 18% of total variance, respectively. (B) Loading plot of inferred PC factors on specific T-RFs. The identities (bp) of selected T-RFs with characteristic factor loading are indicated.