Quantification of enhanced nitrogen removal pathways of pyrite interaction with anammox sludge system

Fe and S could improve the anammox performance by coupling with the N cycle. However, the coexistence of Fe and S complicates the metabolic process, troubling the quantification of nitrogen removal pathways. Herein, the nitrogen removal pathways were quantificationally analyzed by 15 N stable isotope tracing and elaborate batch tests, and the enhancing mechanism was illuminated by physiological and metatranscriptome analyses in pyrite (FeS 2 )-amended anammox system. We found that anammox, denitrification and Feammox (NH 4 + oxidation coupled with Fe(III) reduction) contributed to 80.22 %, 19.53 % and 0.25 % nitrogen removal in this process. With pyrite addition, Fe, S release and the extracellular polymeric substances secretion were responsible for the enhancement of nitrite and nitrate removal via chemical, autotrophic and heterotrophic denitrification, improving nitrogen removal by 14 % and 19 %. The network analyses further confirmed that the multiple nitrogen removal pathways (i.e., anammox, Feammox and denitrification) contribute to pyrite-induced nitrogen removal enhancement in the anammox system. This work enables the in-depth understanding, future design and optimization of the pyrite-amended anammox system, providing a fundamental basis for developing Fe- and S-based anammox enhancement technologies .