In microbial electrolysis cells (MECs), the hydrogen production yield is often limited by the occurrence of methanogenesis. To reduce methane production, an air treatment process was applied as a costeffective approach, however, the reported method of using air or oxygen sparging may cause an energy loss because of residual dissolved oxygen in the MEC solution. In this study, an air-exposed biofilm was applied to improve H 2 production in single-chamber MECs.Twelve reactors with 0.8 V applied voltage were operated in four batch conditions (three replicates for each): (a) the biofilm was aerated for 10 minutes before the medium was refilled (air speed: 0.8-1 L min À1 ); (b) the biofilm was air-exposed for 10 minutes before the medium was refilled; (c) a fresh medium was used to immediately refill after the reacted medium was discharged; (d) nitrogen gas was sparged for 10 minutes after the fresh medium was refilled (as a control treatment). It was found that the H 2 yield increased by $60% after biofilm aeration under condition (a), and the hydrogen production rate was up to 1.3 mL per mL per reactor d, while little methane was detected. In contrast, under conditions (c) and (d), the maximum production rate of methane was 0.1 mL CH 4 per mL reactor per d, while the production rate of hydrogen decreased to 0.8 mL per mL reactor per d. This work indicated that a short-term aeration treatment could substantially affect energy recovery and methanogen communities located in biofilms.