Denitrification by Pseudomonas stutzeri coupled with CO2 reduction by Sporomusa ovata with hydrogen as an electron donor assisted by solid-phase humin

• Denitrifying co-culture of Pseudomonas and Sporomusa was established.
• Denitrification was coupled with acetogenesis from CO2 with H2.
• Humin increased nitrate reducing capacity of co-culture when acetate was deficient.
• More than 60% of consumed H2 in co-culture system was used for denitrification.
• The co-culture system showed robust denitrification by successive transfer.

A co-culture system comprising an acetogenic bacterium, Sporomusa ovata DSMZ2662, and a denitrifying bacterium, Pseudomonas stutzeri JCM20778, enabled denitrification using H2 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 H2 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.