In addition to the strong evidence presented by PCR analysis (amoA gene) in this study that support the presence of AOB on C.comosa roots, the evidence based on chemical analysis also supports AOB activity through conversion of ammonium to nitrite and nitrate with a simultaneous decrease in oxygen (Figs. 1 and S2). During the initial enrichment period (cycle AME 1 for three weeks; Figs. S2-S4, in SM), significant ammonium removal and nitrate production were measured in the absence of TCE-free enrichment. This compares well to previous reports of nitrification and denitrification in wetland mesocosms using 15N radiolabeled ammonium (Reddy et al., 1989); in that study, planted, therefore
oxygenated, reactors demonstrated significantly greater 15N2 in the reactor headspace. The results of our investigation are also comparable with another study where soil inocula from the rhizosphere of G. maxima were used to establish continuous cultures of ammonium oxidizers at very low NH4 + concentrations (Bollmann and Laanbroek, 2001). In the present investigation, ammonium oxidation was sustainable with the excised root samples for over 9 weeks (with periodic replenishment of growth medium,
described above), showing no significant decline in NH4 + removal (Fig. 1a), even with TCE amendments.