A co-culture system comprising an acetogenic bacterium, Sporomusa ovata DSMZ2662, and a denitrifying bacterium, Pseudomonas stutzeri JCM20778, enabled denitrification using H-2 as the sole external electron donor and CO2 as the sole external carbon source. Acetate produced by S. ovata supported the heterotrophic denitrification of P. stutzeri. A nitrogen balance study showed the reduction of nitrate to nitrogen gas without the accumulation of nitrite and nitrous oxide in the co-culture system. S. ovata did not show nitrate reduction to ammonium in the co-culture system. Significant proportions of the consumed H-2 were utilized for denitrification: 79.9 +/- 4.6% in the co-culture system containing solid-phase humin and 62.9 +/- 11.1% in the humin-free co-culture system. The higher utilization efficiency of hydrogen in the humin-containing system was attributed to the higher denitrification activity of P. stutzeri under the acetate deficient conditions. The nitrogen removal rate of the humin-containing co-culture system reached 0.19 kg NO3--N.m(-3).d(-1). Stable denitrification activity for 61 days of successive sub-culturing suggested the robustness of this co-culture system. This study provides a novel strategy for the in situ enhancement of microbial denitrification. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.