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

Abstract

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.