Anaerobic ammonium-oxidizing (anammox) is a novel wastewater treatment process, which can realize efficient nitrogen removal with low operational cost. The anammox process is usually executed at 35 degrees C by heating devices. The potential effects of overheating on anammox culture are still remained as an open question. In this study, we share experiences about behavior detection and activity recovery of damaged anammox culture after accidental overheating. When anammox culture suffered 48 degrees C for 1 h, flocculating and deflocculating occurred, resulting in the suspended culture and the settled. Fluorescence in situ Hybridization, Flow Cytometry and Non-invasive Micro-test Technique showed that the settled culture contained more anammox bacteria than the suspended, leading to its higher death probability as time course, higher bulk liquid H+ flux rate and faster activity restoration for nitrogen removal after overheating. The autofluorescence of Candidatus Brocadia fulgida varied during this process and could be considered as a characteristic fingerprint for these anammox bacteria. Experimental results also indicated the shear force produced by 150 rpm stir would contribute to the further damage of the heated bacteria. Reducing shear stress by low stirring rates and static cultivation might potentially be used as effective strategies in the repair of damaged anammox culture. (C) 2014 Elsevier B.V. All rights reserved.