The anaerobic ammonium oxidation (anammox) process has been recognized as a promising sewage treatment approach. Considering the susceptibility, it is meaningful to study the behaviors of anammox bacteria under the unfavorable conditions. Here, we found that anammox bacteria more probably tended to aggregation by the regulation of c-di-GMP against the unfavorable environmental stresses (low temperature, aerobic condition and low pH). Further using multiple protein sequence alignment, we systematically examined the functionality of thirteen genes encoding putative c-di-GMP metabolic enzymes in anammox organism Candidatus Jettenia caeni, revealing most of the predicted enzymes were predicted to be active. Particularly, ectopic expression of jcaA, a gene encoding a protein with both GGDEF domain and EAL domain, suggested that it encoded a functional enzyme capable of both synthesizing and degrading c-di-GMP, which was clearly confirmed by in vitro enzymatic assays and reverse transcription polymerase chain reaction (RT-PCR). Furthermore, the catalytic mechanism was simulated by the means of three-dimensional homology modeling and molecular docking. The identification of cdi-GMP turnover and its role in granulation for anammox organism provides a new perspective for regulation of its aggregation capability and further promotion of anammox performance in the application of wastewater treatment process. (C) 2017 Elsevier Ltd. All rights reserved.