Uncover the secret of granule calcification and deactivation in up-flow anaerobic sludge bed (UASB) reactor with long-term exposure to high calcium.

Calcification of anaerobic granular sludge causing the decrease of microbial activity has been focused for several decades, but the mechanism of calcification and deactivation of calcified granule are still lacking. In this study, the calcification process of anaerobic granular sludge was analyzed in the UASB reactor with long-term exposure to a high calcium level of 2 gCa2+L-1. Greyish-white calcified granules with larger size and density first appeared at the bottom of sludge bed. Characterization of granular surface demonstrated that calcified granules had smaller specific surface area and pore volume with the disappearing channels in granules visualized over calcification. The coverage of calcite on granular surface with a thickness of 500-600 µm was observed by a microscope, which is responsible for the blockage of channels for substrate diffusion and transport. The evaluation of mass transfer showed that the 'effectiveness factor' η of calcified granules was higher than 1.23, proving the existence of mass transfer resistance. Furthermore, the interactions between calcium and extracellular polymeric substances (EPS) revealed the possible mechanism of calcite location at the outer layer of anaerobic granular sludge: The bulk calcite directly binds the EPS such as humic acids via charging functional groups, and bulk CO32- could react easily with Ca2+ bound to EPS according to thermodynamic analysis.