# Distinct Bacterial Consortia Established in ETBE-Degrading Enrichments from a Polluted Aquifer
## 4 Discussion  
We used aerobic enrichment cultures with ETBE as sole source of carbon to reveal the potential for aerobic ETBE degradation at the studied aquifer, and to identify ETBE-degrading consortia. Water from two wells, one from the anoxic plume of the aquifer, and one situated upstream the polluted zone were used to establish the enrichment cultures. Distinct consortia with ETBE-biodegradation potential could establish from the two wells. Moreover, the _ethB_ gene was distributed independently of the communities' taxonomic composition. From an ecological perspective, these results highlight the importance of the so-called rare biosphere as a potential reservoir of ETBE degraders. This work has been done in a context of a large bioremediation study on a gasoline-polluted aquifer, where bacterial communities and chosen biodegradation genes were monitored in situ over time, using a high-throughput taxonomic microarray and PCR of functional genes (not shown). The microarray has been previously developed to target 19 bacterial phyla at different taxonomic levels [34; 39], and it was implemented with 68 new probes targeting uncultured bacteria from the studied aquifer as well as known ETBE and MTBE degrading isolates. The microarray has been previously validated with sequencing and qPCR. It has been shown that the microarray was semi-quantitative, enabling between-sample comparisons based on signal intensities from individual probes [33; 38; 42]. Between-probe comparisons (i.e., between-taxon comparisons) are, however, not quantitative. Therefore, we used cloning-sequencing of chosen samples to reveal the main taxa present. Though both methods bring some bias due to PCR, there was a good agreement between the two methods concerning the presence of bacterial taxa. Not surprisingly, microarray revealed some taxa that were not shown by cloning-sequencing, which is consistent with its higher sensitivity [42]. Figure 3: Phylogenetic tree based on _ethB_ sequences recovered from PW ETBE-enrichment subculture S3 (23 sequences), UW ETBE-enrichment subculture S1 (42 sequences) and PW water (45 sequences), and highly similar (\(>\)99% identity, according to blastn) _ethB_ sequences from published strains. The Neighbor-Joining tree was constructed based on phylogenetic distances calculated with Kimura-2 parameter (816 nucleotide positions included in the analysis), and bootstrapped over 1000 replicates. The collapsed parts of the tree (presented as black triangles) are labeled with the corresponding samples and numbers of clones (in brackets). Bootstrap values are marked in bold next to the branches. The scale bar shows phylogenetic distance. This study has shown that the bacterial community in the polluted well, compared to the upstream well, had a reduced diversity, which is consistent with previous studies on BTEX contaminated sites [41; 43]. It was enriched in Betaproteobacteria and in anaer-like taxa, in line with the anaerobic character of the well (Table 1). Similar results were obtained with water from other wells situated in the plume (not shown). The depletion of oxygen in BTEX-contaminated sites is a common state and the redox potential seems to be the main factor driving the composition of bacterial communities in such sites [44]. Despite the distinct initial conditions, ETBE-degrading consortia could be established from water of both aerobic unpolluted well and the anaerobic polluted well. Comparison of the original aquifer communities with their respective enrichment cultures showed that the biodegradation consortia might represent only a minor part of the original aquifer community. Deeper techniques such as next-generation sequencing could be helpful to estimate the relative abundance of the rare taxa in original aquifer samples. The failure of biostimulation approach (either oxygen or oxygen and salt addition) in further laboratory experiments with polluted water from the studied aquifer [4] confirms the scarcity of the ETBE-degrading bacteria.