Response of methanogens in calcified anaerobic granular sludge: Effect of different calcium levels

[Display omitted] •Ca effect on performance and microbial activity of calcified AnGS was studied.•Ca mass flow combined Ca2+, pH and HCO3− validated the Ca precipitation.•The relationship between different Ca forms and functional microbes was estimated.•Hydrogenotrophic Methanobacterium aggregated at higher Ca2+ concentration. Inhibition of high calcium during anaerobic wastewater treatment has been studied in recent years, focusing on calcium precipitates in anaerobic granule but neglecting the effect of functional microbes. In this study, key factors of calcification and microbial behaviors especially methanogens of calcified anaerobic granule (AnGS) were investigated in batch assays with calcium level varying from 0 to 5 g L−1. The results showed that the COD removal efficiency and specific methane activity of calcified AnGS were restrained with calcium addition, especially high calcium (>2 g L−1), and little tolerance of calcified AnGS to Ca2+ was underlined compared with non-calcified AnGS. Analysis of calcium mass flow from solution to sludge validated the formation of calcium precipitates influenced by calcium concentration, pH and HCO3−. Besides, death of microbes in outer layer of anaerobic granules was triggered by calcium precipitation. Most importantly, aceticlastic Methanothrix genus was the dominant methanogen, and its relative abundance was correlative negatively with cumulative decrease of bulk Ca2+. Hydrogenotrophic Methanobacterium was enriched at higher calcium level, and it suggested that hydrogenotrophic methanogenesis could play a role in alleviating the inhibition of high calcium.