The anaerobic ammonium oxidation (anammox) process, which is applicable at ambient temperature, is necessary to develop more versatile nitrogen removal technologies. In this study, two anammox reactors, Low-R1 and Low-R2 inoculated with activated sludge respectively in Kumamoto and Hokkaido, Japan, achieved nitrogen removal rates (NRRs) of 1.5 lcg-N/m(3)/day at 20 degrees C. The specific anammox activity (SAA) of the Low-R1 and Low-R2 sludge samples had peaks, respectively, of 2.8 +/- 0.3 mg-N/g-VSS/h at 25 degrees C and 4.2 +/- 03 mg-N/g-VSS/h at 30 degrees C and dropped over the optimum temperature. Moreover, the SAA values of the Low-R1 and Low-R2 were higher at 10-25 degrees C and 10-35 degrees C, respectively, than that of an anammox reactor inoculated with activated sludge in Kumamoto operated at 35 degrees C (Mod-R). The apparent activation energy for anammox of Low-R1, Low-R2, and Mod-R were 108 14/mol (10-25 degrees C), 73 kJ/mol (10-30 degrees C), and 89 kJ/mol (10-35 degrees C), respectively. Candidatus Kuenenia stuttgartiensis dominated in the Mod-R sludge. The Low-R1 sludge was comprised of Ca. K. stuttgartiensis, Ca. Brocadia caroliniensis and Ca. B. fulgida and uncultured anammox-like or planctomycete-like bacteria. The Low-R2 sludge was comprised of various uncultured anammox-like or planctomycete-like bacteria. As Low-R2 was constructed, enrichment of freshwater anammox bacteria at low temperature with seed sludge collected from cold regions is expected to be an effective strategy for anammox applications under a wide temperature range. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.