Multipathway of Nitrogen Metabolism Revealed by Genome-Centered Metatranscriptomics from Pyrite-Guided Mixotrophic Partial Denitrification/Anammox Installations

Further reducing the organic requirements is essential for the sustainable development of partial denitrification/anammox technology. Here, an innovative mixotrophic partial denitrification/anammox (MPD/A) installation fed with pyrite and few organics was realized, and the average nitrogen and phosphorus removal rates were as high as 96.24 ± 0.11% and 79.23 ± 2.06%, respectively, with a C/N ratio of 0.5. To understand the nature by which MPD/A achieves efficient nitrogen removal and organic conservation, the electron transfer-dependent nitrogen escape and energy metabolism were first elucidated using multiomics analysis. Apart from heterotrophic denitrification and anammox, the results revealed some unexpected metabolic couplings of MPD/A systems, in particular, putative nitrate-dependent organic and pyrite oxidation among nominally heterotrophic Denitratisoma (PRO3) strains, which accelerated nitrate gasification with a low-carbon supply. Additionally, Candidatus Brocadia (AMX) employed extracellular solid-state electron acceptors as terminal electron sinks for high-rate ammonium removal. AMX transported ammonium electrons to extracellular γFeO(OH) (generated from pyrite oxidation) through the transient storage of menaquinoline pools, cytoplasmic migration via multiheme cytochrome(s), and OmpA protein/nanowires-mediated electron hopping on cell surfaces. Further investigation observed that extracellular electron flux resulted in the transfer of more energy from the increased oxidation of the electron donor to the ATP, supporting nitrite-independent ammonium removal.