No abstract

An in situ mesocosm system was designed to monitor the in situ dynamics of the microbial community in polluted aquifers. The mesocosm system consists of a permeable membrane pocket filled with aquifer material and placed within a polypropylene holder, which is inserted below groundwater level in a monitoring well. After a specific time period, the microcosm is recovered from the well and its bacterial community is analyzed. Using this system, we examined the effect of benzene, toluene, ethylbenzene, and xylene (BTEX) contamination on the response of an aquifer bacterial community by denaturing gradient gel electrophoresis analysis of PCRamplified 16S rRNA genes and PCR detection of BTEX degradation genes. Mesocosms were filled with nonsterile or sterile aquifer material derived from an uncontaminated area and positioned in a well located in either the uncontaminated area or a nearby contaminated area. In the contaminated area, the bacterial community in the microcosms rapidly evolved into a stable community identical to that in the adjacent aquifer but different from that in the uncontaminated area. At the contaminated location, bacteria with tmoA-and xylM/xylE1-like BTEX catabolic genotypes colonized the aquifer, while at the uncontaminated location only tmoA-like genotypes were detected. The communities in the mesocosms and in the aquifer adjacent to the wells in the contaminated area consisted mainly of Proteobacteria. At the uncontaminated location, Actinobacteria and Proteobacteria were found. Our results indicate that communities with long-term stability in their structures follow the contamination plume and rapidly colonize downstream areas upon contamination.

tmoA and related genes encode the alpha-subunit of the hydroxylase component of the major group (subgroup 1 of subfamily 2) of bacterial multicomponent mono-oxygenase enzyme complexes involved in aerobic benzene, toluene, ethylbenzene and xylene (BTEX) degradation. A PCR-denaturing gradient gel electrophoresis (DGGE) method was developed to assess the diversity of tmoA-like gene sequences in environmental samples using a newly designed moderately degenerate primer set suitable for that purpose. In 35 BTEX-degrading bacterial strains isolated from a hydrocarbon polluted aquifer, tmoA-like genes were only detected in two o-xylene degraders and were identical to the touA gene of Pseudomonas stutzeri OX1. The diversity of tmoA-like genes was examined in DNA extracts from contaminated and non-contaminated subsurface samples at a site containing a BTEX-contaminated groundwater plume. Differences in DGGE patterns were observed between strongly contaminated, less contaminated and non-contaminated samples and between different depths, suggesting that the diversity of tmoA-like genes was determined by environmental conditions including the contamination level. Phylogenetic analysis of the protein sequences deduced from the amplified amplicons showed that the diversity of TmoA-analogues in the environment is larger than suggested from described TmoA-analogues from cultured isolates, which was translated in the DGGE patterns. Although different positions on the DGGE gel can correspond to closely related TmoA-proteins, relationships could be noticed between the position of tmoA-like amplicons in the DGGE profile and the phylogenetic position of the deduced protein sequence.