Visual evidence for anammox granules expanding their size by aggregation of anammox micro-granules.

Granular sludge is superior in sustainable wastewater treatment; however, no consensus has achieved in its formation mechanism. In this study, we provide visual and experimental evidences to reveal how the large anammox granules formed. Micro-observation of anammox granules illustrated that some special anammox granules were clearly composed of numerous micro-granules, which enveloped by transparent extracellular polymeric substances (EPS). Static culture experiment proved that anammox granules were easy to aggregate and form a larger entirety within approximately 14 days when there were no severe external disturbances (mainly hydraulic shear force). Stratified EPS extraction and selective enzymatic digestion tests further elucidated that tightly-bound EPS and extracellular proteins were the most vital constituents in maintaining the structure of anammox granules, and the minimal size of anammox micro-granules that aggregated to form large anammox granules was approximately 100-150 μm in the reactor studied herein. Our findings highlight that anammox granules could expand their size and form larger granules by the aggregation of anammox micro-granules, representing a natural but significant granule formation and enlargement mechanism. Understanding the enlargement mechanism could consummate the granulation process and help to culture large size anammox granules.