Bioleaching is of increasing interest due to its high e ciency for improving sludge dewaterability and removing heavy metals. However, a traditional single-phase bioleaching cannot run continuously at a high-e ciency status due to a destruction of the microbial synergistic effect in a low-pH environment. So, in this study, a series of multi-compartment ba ed-ow trials were carried out to assess the effect of phase separation on sludge bioleaching by comparing the bioleaching process of a two-phase bioreactor with that of two single-phase bioreactors. Energy substrate and sludge re ux were introduced into two different compartments to form two phases, namely selection phase and leaching phase. The results show that phase separation obviously shortened the start-up duration for sludge bioleaching from 7 days in single-phase bioleaching to 4 days in two-phase bioleaching. The dewaterability improvement of bioleached sludge also was enhanced by phase separation with the relative decreases of 25.0 -33.3% for speci c resistance to ltration (SRF) and 14.2% for capillary suction time (CST), which was attributed to lower pH values, zeta potentials of closer to zero and less DOM of bioleached sludge after the twophase bioleaching. Phase separation generally increased the dissolution ratio of heavy metals with the