Fig. S1. Location of the Integrated Field Research Challenge (IFRC) at Rifle, Colorado (USA) and the well layout used during the 2006 acetate-based biostimulation experiment. Cross-well mixing pumps and tubing were used to circulate acetate-amended groundwaters between the five injection wells indicated, as illustrated schematically in Fig. 4 of the main text. Well installation and well completion details as described by Williams and colleagues (2011).


Fig. S2. Removal of total aqueous selenium (gray symbols) from groundwater during a 25-day acetate injection experiment at the Rifle IFRC site in August–September 2010 configured in an identical fashion to that reported here. The 2006 experimental location is approximately 100 m to the west of that used in 2010. As with the B04 location in 2006, the background well (top) in 2010 exhibited seasonal fluctuations in total selenium over the monitoring period. As with the 2006 M16 results, pre-injection selenium concentrations (c. 125 μg l) at the first downgradient well (bottom) fell rapidly as soon as acetate (open symbols) was detected. Of note, selenium concentrations remained well below the US Environmental Protection Agency's maximum contaminant level for selenium in drinking water (50 μg l) long after acetate was exhausted from the system (data not shown).


Fig. S3. A and C. Scanning electron micrograph (SEM) images depicting representative locations (white crosses) where energy dispersive X-ray spectroscopy (EDS) data were obtained for elemental analysis. In contrast to the SEM images in Fig. 2, the accelerating voltages were much higher for the EDS analysis (20 versus 5 keV in Fig. 2), which gives rise to the loss in contrast/resolution of fine scale details.

B and D. EDS data from the regions depicted in A and C. indicating selenium-rich areas of the tubing biofilm.


Fig. S4. Photograph of enrichment cultures after c. 3 weeks using two different sources of microbial innocula: tubing biofilms (left) and Rifle site groundwater (middle). A sterile control with identical media composition is shown on the right; media contained acetate (20 mM) and selenate (10 mM) as the sole electron donor and acceptor, respectively, as described in the text.


Fig. S5. Transmission electron micrographs of the selenium-respiring enrichment cultures. The morphological qualities of the Se0 nanospheres are similar to those produced biologically and extensively characterized by Oremland and colleagues (2004). Rapid volatilization of the electron-dense minerals prevented characterization by selected area electron diffraction.


Appendix S1. Site description and supplementary experimental procedures describing selenium speciation analytical protocols and 16S-based rRNA analysis of tubing-derived biofilms and groundwater enrichment cultures.

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