Granular Sludge Coupling Nitrate/Nitrite Dependent Anaerobic Methane Oxidation with Anammox: from Proof-of-Concept to High Rate Nitrogen Removal

This work developed a novel Membrane Granular Sludge Reactor (MGSR) equipped with a gas permeable membrane module for efficient methane delivery to cultivate nitrate/nitrite dependent anaerobic methane oxidation (n-DAMO) microorganisms in granular sludge. As proof of concept, the MGSR was fed with synthetic wastewater containing nitrate and ammonium to facilitate the growth of n-DAMO microorganisms. The granular sludge of n-DAMO and Anammox was gradually developed and achieved a nitrogen removal rate of 1.08 g NO₃^--N L^-1 d^-1 and 0.81 g NH₄⁺-N L^-1 d^-1. Finally, enriched granular sludge was successfully applied for nitrogen removal from the synthetic partial nitritation effluent. The combined dominance of n-DAMO archaea, Anammox bacteria, and n-DAMO bacteria in the microbial community was confirmed by 16S rRNA amplicon sequencing. Fluorescence in situ hybridization revealed that a layered structure was formed in the granular sludge with Anammox bacteria in the outer layer and n-DAMO microorganisms in the inner layer when granules were fed with nitrite and ammonium. The high performance of nitrogen removal (16.53 kg N m^-3 d^-1) with satisfactory effluent quality (∼8 mg N L^-1) and excellent biomass retention capacity (43 g VSS L^-1) make the MGSR promising for the practical application of n-DAMO and Anammox in wastewater treatment.