Manganese-mediated dissimilatory nitrate reduction to ammonium of nitrate-dependent anaerobic methane oxidation

Nitrate-dependent anaerobic methane oxidation (n-DAMO) archaea not only partially denitrify nitrate to nitrite to feed n-DAMO bacteria or Anammox bacteria but also independently perform manganese-dependent anaerobic oxidation of methane. Despite their widespread coexistence in natural ecosystems, how nitrate and manganese simultaneously drive anaerobic methane oxidation remains unresolved. Here, we demonstrate that n-DAMO archaea converted 35.2% nitrate to ammonium with the presence of birnessite and 41.3% with the presence of nano MnO₂. Through multi-omics analysis, two potential pathways for nitrate ammonification were identified in DAMO archaea, ammonia-forming nitrite reductase (Nrf) pathway and reverse hydroxylamine:ubiquinone reductase module (reverse-HURM) pathway. Significant expression of genes involved in Nrf pathway and reverse-HURM pathway was detected after introducing birnessite or nano MnO₂ in n-DAMO culture. Consequently, a large portion of nitrate was converted into ammonium by n-DAMO archaea rather than nitrite, reducing the availability of nitrite to support n-DAMO bacteria, which led to a sharp decline in their population abundance from 6.0% to 1.69%. These findings indicate that the metabolic shift of n-DAMO archaea from partial denitrification to nitrate ammonification is triggered when nitrate and Mn(IV) act as dual electron acceptors. Given the widespread coexistence of diverse electron acceptors in natural environments, further investigation is needed to understand the in-situ physiology of n-DAMO microorganism and their contributions to methane oxidation and nitrogen cycle.