Rapid formation of granules coupling n-DAMO and anammox microorganisms to remove nitrogen

Granular sludge exhibits unique features, including rapid settling velocity, high loading rate and relative insensitivity against inhibitors, thus being a favorable platform for the cultivation of slow-growing and vulnerable microorganisms, such as anaerobic ammonium oxidation (anammox) bacteria and nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) microorganisms. While anammox granules have been widely applied, little is known about how to speed up the granulation process of n-DAMO microorganisms, which grow even slower than anammox bacteria. In this study, we used mature anammox granules as biotic carriers to embed n-DAMO microorganisms, which obtained combined anammox + n-DAMO granules within 6 months. The results of whole-granule 16S rRNA gene amplicon sequencing showed the coexistence of anammox bacteria, n-DAMO bacteria and n-DAMO archaea. The microbial stratification along granule radius was further elucidated by cryosection-16S rRNA gene amplicon sequencing, showing the dominance of n-DAMO archaea and anammox bacteria at inner and outer layers, respectively. Moreover, the images of cryosection-fluorescence in situ hybridization (FISH) verified this stratification and also indicated a shift in microbial stratification. Specifically, n-DAMO bacteria and n-DAMO archaea attached to the anammox granule surface initially, which moved to the inner layer after 4-months operation. On the basis of combined anammox + n-DAMO granules, a practically useful nitrogen removal rate (1.0 kg N/m³/d) was obtained from sidestream wastewater, which provides new avenue to remove nitrogen from wastewater using methane as carbon source.