# Distinct Bacterial Consortia Established in ETBE-Degrading Enrichments from a Polluted Aquifer
## 1 Introduction  
Oxygenates methyl tert-butyl ether (MTBE) and ethyl tert-butyl ether (ETBE) are widely used as enhancers of gasoline octane index, replacing previously used lead tetraethyl. These chemicals are, however, highly soluble in water and became a major pollutant in aquifers after accidental gasoline spills. Both compounds are poorly biodegradable and potentially carcinogenic. Their biodegradation often terminates with a toxic compound tert-butyl alcohol (TBA) [1; 2]. The biodegradation of ETBE, in comparison to MTBE, has been studied less [3], but is of concern because ETBE has been found as an aquifer pollutant in some cases in Europe, especially in France [4]. Few bacterial isolates have been shown to degrade ETBE so far, belonging either to Betaproteobacteria, for example, _Aquincola tertiaricarbonis_ L108, _Comamonas testosteroni_ E1 and _Rubriviox_ sp. IFP 2047, or to Actinobacteria, for example, _Mycobacterium_ sp. IFP 2009, _Rhodococcus equii_ IFP 2002, _Rhodococcus zopfi_ IFP 2005, _Rhodococcus ruber_ IFP 2001, _Rhodococcus_ sp. IFP 2014 and other strains, _Pseudonocardiia tetrahydrofuranoxydans_ K1 and _Pseudonocardiia_ sp. IFP 2050 [5; 6; 7; 8; 9; 10; 11; 12]. The betaproteobacterial degraders were also able to degrade MTBE and TBA [6; 8; 12]. In all cases, the degradation of ETBE was observed only under aerobic conditions. In situ and microcosm studies, however, indicated that ETBE-degradation may also proceed under anaerobic conditions [13; 14]. In _Rhodococcus ruber_ IFP2001, it was shown that the operon _ethABCD_ was essential for ETBE degradation [15]. The operon codes for a cytochrome P450 monooxygenase system involved in the initial oxydation of ETBE [1]. The expression of the gene _ethB_, encoding the cytochrome P450, is inducible by ETBE [16]. The _ethABCD_ operon is flanked by class II transposon sequences, indicating that it may be horizontally transferred to other bacterial species. Indeed, highly conserved (\(>\)99% identity) _eth_ genes were found in _Rhodococcus zopfi_ IFP2005 and _Mycobacterium_ sp. IFP2009 [7]. All these strains were able to grow on ETBE but accumulated TBA, showing that this operon itself is not sufficient to achieve complete ETBE mineralization. The accumulation of TBA may present a problem for in situ treatments because of its toxicity. The co-culture with microorganisms able to grow on TBA may be required for successful bioremediation of oxygenase-contaminated sites. Indeed, a complete degradation of ETBE by a bacterial consortium was obtained from a polluted aquifer, in which _Rhodococcus_ sp. degraded ETBE to TBA and _Bradyrhizobium_ sp. further degraded TBA to CO\({}_{2}\)[13]. In addition, oxygenates in aquifers are usually accompanied by BTEX and other gasoline compounds, which implies that large bacterial consortia may be required for a full depollution of contaminated sites. Mentioned and other observations suggest the use of a community-based rather than a one-strain-based approach, as the best alternative to study these processes in the environment [17]. Few studies evaluated the composition of microbial consortia presenting oxygenate degradation. They indicated that the potential for intrinsic oxygenase degradation was dependent on the composition of the indigenous aquifer community, in combination with site-specific geochemical conditions [18; 19; 20]. These studies mainly focused on MTBE, while the information on the composition of microbial communities in ETBE polluted sites and natural consortia capable for ETBE biodegradation remain scarce [11; 12; 21; 22]. In a study on bacterial enrichment cultures from five different aquifers, [PERSON] et al. [12] showed that the ETBE-degrading consortia had distinct taxonomical composition with prevailing Proteobacteria in three of them, and with Actinobacteria occurring in all of them. This confirmed the importance of the composition of the original aquifer communities, and also indicated which taxa might play a crucial role in ETBE degradation.