# Distinct Bacterial Consortia Established in ETBE-Degrading Enrichments from a Polluted Aquifer
## 4 Discussion
In contrast, bioaugmentation using the UW ETBE-enrichment culture characterized in this study led to the complete ETBE biodegradation of polluted water under laboratory conditions at 15 \({}^{\circ}\)C, which is the mean aquifer temperature [4]. The taxonomical composition of the enrichment communities did not reflect their biodegradation potential. On the one hand, the initial ETBE-enrichment cultures from the UW had very similar taxonomical composition, while they differed in their capacity of ETBE and TBA degradation. On the other hand, enrichment cultures from UW and PW that degraded ETBE were composed by completely distinct bacterial taxa. These results highlight the high degree of functional redundancy of the aquifer and enrichment communities. The lack of convergence in bacterial community composition in contaminated aquifers or soils was also noted in other studies [44; 45], indicating that distinct consortia with the same functional properties may be present at one site. Consequently, similarities in taxonomical composition of ETBE-degrading bacterial consortia from different studies and sites can be seen only on high taxonomic levels. Alpha-, Beta- and Gammaproteobacteria, Actinobacteria, Acidobacteria and Chloroflexi are reported in this and other studies [12; 22], prompting to search candidate biodegradation bacteria within these groups. We did not aim to test individual strains from ETBE-enrichment cultures for their biodegradation potential, it is worth noting, however, that several bacterial genera that include strains with ETBE-biodegradation properties such as _Mycobacterium_, _Pseudonocardi_ and _Varivorax_ were detected in the ETBE-enrichment cultures by microarray. _Pseudonocardi_ was additionally confirmed in UW ETBE-enrichment culture by sequencing. It is not known whether the _Mesorhizobium_ and _Hydrogenophag_ spp. dominating in the UW and PW ETBE-enrichment clone libraries, respectively, were involved in the initial ETBE oxygenation step or in the degradation of other compounds occurring in the course of ETBE biodegradation, such as TBA. A slight ETBE- (and no TBA-) degradation capacity was reported once in an isolate of _Mesorhizobium_ from a bacterial enrichment culture degrading mixture of hydrocarbons and gasoline additives [46]. _Mesorhizobium_ spp. were also detected in three out of five ETBE-enrichments originating from different aquifers in the study of [PERSON] et al. [12], and once they even reached the relative abundance of 22%, but no ETBE-degrading strain was obtained in their study. ETBEdegradation was never reported in _Hydrogenophaga_, although one _Hydrogenophaga_ strain (ENV735) was shown to be able to degrade MTBE and TBA [47]. _Hydrogenophaga_ was suggested to have the main role in MTBE biodegradation in the presence of BTEX [48], while it was considered only as an accompanying microflora in a MTBE-enrichment culture from a gasoline-polluted site [49]. In our study, _Hydrogenophaga_ was enriched from the BTEX-polluted plume of the aquifer and its ability to degrade oxygenates merits further studies. The successful amplification of _ethB_ genes from both types of enrichments as well as from the polluted well confirms that the gene _ethB_ is a good candidate for an in situ marker of the ETBE biodegradation potential, as suggested in [4]. The great level of conservation observed for the _ethB_gene obtained from highly distinct bacterial communities and consortia noticed in this study further indicates that this gene may be horizontally transferred among different hosts. The natural diversity of _ethB_ may, however, remain hidden as the currently used _ethB_ primers [7] are based on only three sequences. Indeed, [PERSON] et al. [22] did not detect _ethB_, when using primers based on _A. tertiaricarbonis_ L108 sequence [50], in their ETBE-degrading cultures from polluted groundwater, despite the fact that ETBE-degradation was shown to be cytochrome P450-dependent.