With the goal of maximizing the H2-harvesting efficiency, we designed an upflow single-chamber microbial electrolysis cell (MEC) by placing the cathode on the top of the MEC and carried out a program to track the fate of H2 and electron equivalents in batch experiments. When the initial acetate concentration was 10 mM in batch-evaluation experiments lasting 32 h, the cathodic conversion efficiency (CCE) from coulombs (i.e., electron equivalents in current from the anode to the cathode) to H2 was 98 +/- 2%, the Coulombic efficiency (CE) was 60 +/- 1%, the H2 yield was 59 +/- 2%, and methane production was negligible. However, longer batch reaction time (approximately 7 days) associated with higher initial acetate concentrations (30 or 80 mM) led to significant H2 loss due to CH4 accumulation: up to 14 +/- 1% and 16 +/- 2% of the biogas at 30 and 80 mM of acetate, respectively. Quantitative PCR proved that no acetoclastic methanogens were present, but that hydrogenotrophic methanogens (i.e., Methanobacteriales) were present on both electrodes. The hydrogenotrophic methanogens decreased the CCE by diverting H2 generated at the cathode to CH4 in the upflow single-chamber MEC. In some experiments, the CE was greater than 100%. The cause was anode-respiring bacteria oxidizing H2 and producing current which recycled H2 between the cathode and the anodes, increasing CE to over 100%, but with a concomitant decline in CCE, despite negligible CH4 formation.