Three different organic substrates, K-medium, sterilized activated sludge (SAS), and methanol, were examined for utility as substrates for enriching manganese-oxidizing bacteria (MnOB) in an open bioreactor. The differences in Mn(Il) oxidation performance between the substrates were investigated using three down-flow hanging sponge (DHS) reactors continuously treating artificial Mn(II)-containing water over 131 days. The results revealed that all three substrates were useful for enriching MnOB. Surprisingly, we observed only slight differences in Mn(II) removal between the substrates. The highest Mn(II) removal rate for the SAS-supplied reactor was 0.41 kg Mn.m(-3).d(-1), which was greater than that of K-medium, although the SAS performance was unstable. In contrast, the methanol-supplied reactor had more stable performance and the highest Mn(II) removal rate. We conclude that multiple genera of Comamonas, Pseudomonas, Mycobacterium, Nocardia and Hyphomicroblum play a role in Mn(II) oxidation and that their relative predominance was dependent on the substrate. Moreover, the initial inclusion of abiotic-MnO2 in the reactors promoted early MnOB enrichment.