Pellicles are biofilms found at the air-liquid interface and are widely distributed in natural environments. In this study, a simple pellicle detection method was established, and using this new method, the pellicle formation activities of Shewanella oneidensis MR-1 and its 42 cytochrome c mutants were analysed. The results showed that the pellicle was initiated at very early stages of incubation. Aerotaxis was the major external factor, while energy acquirement was the main internal factor for pellicle initiation. Among the 42 cytochrome c mutants, 17 mutants, including those deficient in aerobic respiration, sulfur or sulfite/ sulfate respiration, nitrite respiration, metal respiration, DMSO respiration and fumarate respiration, exhibited delayed pellicle initiation. The results suggest that S. oneidensis utilizes the electron acceptors simultaneously under anoxic conditions and that the disruption of any of these anaerobic respiration routes would retard pellicle initiation. (C) 2019 Elsevier Inc. All rights reserved.