Identification of Aerobic ETBE‐Degrading Microorganisms in Groundwater Using Stable Isotope Probing

A limited number of microorganisms have been identified with the capability to degrade ethyl tert‐butyl ether (ETBE) in the environment. Knowledge of the identity and distribution of ETBE‐degrading microorganisms is important for the implementation of management measures such as natural attenuation and bioremediation at ETBE‐release sites. In this study, DNA‐stable isotope probing (SIP) was used to identify microorganisms able to aerobically degrade 13C‐labeled ETBE in laboratory microcosms constructed with groundwater and aquifer material from an ETBE‐release site. Microorganisms in the Class γ‐proteobacteria, Order β‐proteobacteriales, Family Burkholderiaceae, and classified as Methylibium and Leptothrix, respectively, were identified as primary ETBE degraders. Comparisons with ETBE‐responsive microorganisms (those which increased in abundance after the addition of ETBE), identified by high‐throughput sequencing of microcosms established from the same site, showed that only a small proportion of the ETBE‐responsive organisms were primary degraders as determined by SIP. ETBE degraders were taxonomically related to microorganisms able to degrade other gasoline components, but not ETBE, implying that this functionality results from acquisition of the eth gene cluster by these organisms. These ETBE degraders could also be identified at ETBE‐release sites, but at low relative abundance and generally only in those locations from which the microcosms had been established. Therefore, we recommend that molecular investigations of ETBE‐contaminated sites focus on functional genes (i.e., the eth gene cluster) rather than specific taxa.