# Distinct Bacterial Consortia Established in ETBE-Degrading Enrichments from a Polluted Aquifer
## 3 Results
### Bacterial Communities in the ETBE-Enrichment Cultures  
The microarray analysis of the ETBE-enrichment communities originating from UW and PW wells revealed that they differed significantly from the corresponding water communities as well as from each other (Figure 1; ANOSIM, \(P<0.01\) in all cases). There was no convergence between the ETBE-enrichment communities originating from UW and PW (Figure 1). The initial enrichment cultures and the subcultures originating from the same well grouped together, showing little turnover in the course of the experiment. Also, the three initial enrichment cultures from UW grouped together  
Figure 1: Multidimensional Scaling of bacterial communities from PW water (black circles), UW water (white circles), PW ETBE-enrichment cultures (black triangles) and UW ETBE-enrichment cultures (white triangles). Water samples are labeled with month/year of sampling. ETBE-enrichment samples are labeled II-I3 for the initial cultures and S1–S3 the subcultures (see Figure S1 for more details). Samples that were used for 16S rRNA and _ethB_ gene sequencing are marked with an asterisk. The distances between samples were based on Bray-Curtis similarity matrix, which was calculated from normalized probe signals. Low 2D stress value (<0.1) indicates that the distances between samples in reduced 2-dimensional space correspond well with the actual multivariate distance between the samples. (Figure 1), despite their distinct biodegradation rates and differences in their TBA biodegradation potential (Figure S1). SIMPER analysis together with Wilcoxon rank sum tests showed which probes significantly (\(P<0.05\)) contributed to up to 50% differences in bacterial community composition between UW- and PW-enrichment cultures (Figure S2). Notably, the enrichments (initial cultures and subcultures grouped together) from PW, in comparison to the UW enrichments, had higher signals for probes targeting Betaproteobacteria belonging to _Hydrogenophaga_ (HyMa2, HydETBE1, HydETBE3), _Nitrosospira_ (Nit1B, Nit1C), _Variovorax_ (Varpar) and _Aquabacterium_ (AquacIn2), Gammaproteobacteria _Nitrosococcus_ (Nibmob) and _Eikenella/Neisseria_ (Eikcor2), and _Paenibacillus_ (Pav, Palv2; Firmicutes). In contrast, the UW enrichment cultures had higher signals for probes targeting Alphaproteobacteria from the family Rhizo157, Rhi) related to _Mesorhizobium_/_Rhizobium_/_Devosia_ (Phyllobat, MesocIn1, Davocln1), from Actinobacteria _Pseudonocardi_ (Psdonoca), _Gordonia_ (Gordcln3) and _Mycobacterium_ (Mycoba2), and from Chloroflexi (Dehacln5, Dehacln6), as compared to PW enrichments. These probes distinguishing the UW and PW enrichments also significantly contributed to the differences between the enrichments and their respective initial water samples (Figure S2). Though there were no overall differences in the community composition between the three initial ETBE enrichment cultures from UW (Figure 1), it is interesting to note that the enrichment culture I3, which showed only a slight ETBE biodegradation activity at the time of sampling, lacked signal from the probe MesocIn1 for _Mesorhizobium_ (Figure S2). There was no significant difference in bacterial community composition between the enrichments that completely degraded ETBE and those that degraded ETBE but accumulated TBA, based on ANOSIM analysis of microarray data. When individual probes were analyzed with Wilcoxon rank sum tests (\(P<0.05\)), only XAX818 (for _Xanthomonas_) gave significantly higher signals in the enrichments that showed a complete ETBE degradation, while Ralstcln2 (for _Ralstonia_/_Cupiravidus_/_Wautersia_, including certain clones from UW) gave significantly lower signals. The dominating taxa in the two ETBE-enrichment subculture communities originating from UW and PW that were able to degrade ETBE were further studied by sequencing the 16S rRNA gene.