Anaerobic ammonium-oxidizing (anammox) bacteria perform an important step in the global nitrogen cycle: anaerobic oxidation of ammonium and reduction of nitrite to form dinitrogen gas (N(2)). Anammox organisms appear to be widely distributed in natural and artificial environments. However, their roles in groundwater ammonium attenuation remain unclear and only limited biomarker-based data confirmed their presence prior to this study. We used complementary molecular and isotope-based methods to assess anammox diversity and activity occurring at three ammonium-contaminated groundwater sites: quantitative PCR, denaturing gradient gel electrophoresis, sequencing of 16S rRNA genes, and (15)N-tracer incubations. Here we show that anammox performing organisms were abundant bacterial community members. Although all sites were dominated by Candidatus Brocadia-like sequences, the community at one site was particularly diverse, possessing four of five known genera of anammox bacteria. Isotope data showed that anammox produced up to 18 and 36% of N(2) at these sites. By combining molecular and isotopic results we have demonstrated the diversity, abundance, and activity of these autotrophic bacteria. Our results provide strong evidence for their important biogeochemical role in attenuating groundwater ammonium contamination.

Elevated ammonium concentrations in pump‐and‐treat effluent from a volatile organic compounds–contaminated municipal aquifer originate from two industrial sources: infiltration of drainage from the blending operations of a fertilizer company (FC) located in the recharge area ( of 500 to 700 parts per million [ppm] N and of 150 to 300 ppm N) and leakage from waste water treatment ponds maintained by an adjacent chemical company (CC) ( of 50 to 70 ppm N, with no ). Geochemical and isotope data are used to trace the mechanisms for the strong attenuation observed between the source areas and the municipal ground water treatment wells ( < 10 ppm N). Conservative mixing calculations demonstrate a loss of and along the flowpath relative to K+ and Cl−. Reactive loss of in these anoxic ground water is attributed to anaerobic oxidation by anammox bacteria. Lines of evidence leading to this conclusion include (1) loss of both and under anoxic conditions along the flowpath; (2) a progressive enrichment of and , indicating reactive loss of ammonium and nitrate; (3) values greater than coexisting , which precludes loss by nitrification to ; and (4) significant N2 overpressuring with increasing values. Anaerobic ammonium oxidation by anammox bacteria uses nitrate as the electron donor: . The recently discovered anammox reaction is more energetically favorable than denitrification and is now considered to play a major role in the global nitrogen cycle. It has been observed in waste water bioreactors and sea water but not previously in ground water.