# Distinct Bacterial Consortia Established in ETBE-Degrading Enrichments from a Polluted Aquifer
## Materials and Methods
### Enrichment Cultures and Their Biodegradation Potential  
Initial enrichment cultures and subsequent subcultures were carried out as follows (Figure S1). ETBE-enrichment samples are labeled I1 to I3 for the initial cultures and S1 to S3 for the subcultures. UW ETBE-enrichment subcultures S1-S3 were obtained from the initial UW culture I1, whereas PW ETBE-enrichment subcultures S1-S3 were obtained from the initial PW culture I3 (Figure S1). The establishment and handling of enrichment cultures was done in a laminar flow hood to maintain sterile conditions. For initial enrichment cultures, water samples (20 mL) from UW and PW were filtered (0.22 \(\upmu\)m) to discard any pollutant that could be dissolved in water. The filters were introduced to air-tight 500 mL-Schott flasks containing 150 mL mineral medium (KH\({}_{2}\)PO\({}_{4}\) 1.4 g/L; K\({}_{2}\)HPO\({}_{4}\) 1.7 g/L; MgSO\({}_{4}\) \(\times\) 7 H\({}_{2}\)O 0.5 g/L; NH\({}_{4}\)NO\({}_{3}\) 1.5 g/L; CaCl\({}_{2}\)\(\times\) 2 H\({}_{2}\)O 0.04 g/L; FeO\({}_{4}\)\(\times\) 7 H\({}_{2}\)O 1 mg/L) with 100 mg/L ETBE representing the sole source of carbon. A vitamin solution and an oligo-element solution were added as previously described [23]. The headspace in the flasks was calculated so that oxygen was in a large excess with regard to the theoretical oxygen demand of ETBE. The initial enrichment cultures were set in triplicates and incubated under agitation at 30 \({}^{\circ}\)C. The flasks were closed with a stopper and equipped with a side-arm enabling sampling in the course of incubation. Samples of 2 mL were regularly withdrawn to measure ETBE and TBA concentrations (see further). When ETBE was fully consumed, the cultures were spiked with ETBE so as to reach the initial concentration (1 to 3 times). The initial enrichment cultures were sampled (40 mL) at 67 days (replicate I3 of PW initial enrichment culture) and 253 days (the other replicates), centrifuged, and cell pellets were stored at -80 \({}^{\circ}\)C for DNA extraction.  
\begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline  & **ETBE** & **MTBE** & **TBA** & **BTEX** & **TOC \({}^{\text{a}}\)** & **Nitrates** & **Sulfrates** & **Oxygen** \\  & **(mg/L)** & **(mg/L)** & **(mg/L)** & **(mg/L)** & **(mg/L)** & **(mg/L)** & **(mg/L)** & **(mg/L)** \\ \hline
**PW** & 160.8 & 2.5 & 1.9 & 4.4 & 204 & ND \({}^{\text{b}}\) & 7 & 0.33 \\
**UW** & ND & ND & ND & ND & ND & 3 & 25 & 2.54 \\ \hline \hline \end{tabular}  
\({}^{\text{a}}\) Total organic carbon, \({}^{\text{b}}\) ND—not detected.  
\end{table}
Table 1: Chemical analysis of water from the upstream (UW) and the polluted (PW) well.  
After the complete biodegradation of ETBE in the initial enrichment cultures, triplicate enrichment subcultures were established from one flask for UW (I1) and PW (I3), by transferring 30 mL of the initial enrichment cultures into 150 mL fresh minimal medium containing 100 mg/L ETBE (Figure S1). After 1 week, 40 mL of the enrichment subcultures were centrifuged and the bacterial pellets were stored at \(-80\)\({}^{\circ}\)C until DNA extraction. After the complete ETBE biodegradation, the enrichment subcultures were spiked with ETBE so as to reach the initial concentration and the oxygenate concentrations were further monitored. This was done twice in order to confirm the biodegradation capacities of the established community.  
ETBE and TBA were quantified by flame ionization detection on a Varian 3300 gas chromatograph (Varian, Les Ulis, France) equipped with a 0.32 mm \(\times\) 25 m Porabond-Q capillary column (J&W Scientific, Chromoptic, Auxerre, France), using a two-step temperature gradient ranging from 105 \({}^{\circ}\)C to 210 \({}^{\circ}\)C at 10 \({}^{\circ}\)C/min. The column was then maintained at 210 \({}^{\circ}\)C for 20 min. Helium (1.6 mL/min) was used as the carrier gas. Samples were filtered through 0.22 \(\upmu\)m filters (Prolabo, Fontenay-sous-Bois, France) and injected without further treatment.