The seed sludge used in this experiment was a mixture of two different kinds of sludge i.e. seed sludge from previous experiment (Qiao et al., 2013a,b) and a new seed sludge obtained from a waste water treatment plant located in Sendai city, Japan. The sludge from previous experiment was long term acclimated to coffee grounds but was inhibited by over load. The new seed sludge was mixed with previous sludge to supply RO 4929097 with high activity. The coffee residues (spent coffee grounds, coffee liquid), milk waste and municipal sludge were provided by Tokyo Gas Co., Ltd., Japan. The characteristics of feedstock are presented in Table 1. Coffee grounds, coffee liquid, milk waste and municipal sludge were mixed with mass-based ratio 14.6:65.3:12.2:7.9, based on the requirement of the process design of the full scale plant and then homogenized using a high speed blender at 18.500 rpm for 30 min, then stored in a substrate tank at low temperature (4 °C) for subsequent use to reduce changes in substrate composition. The total solids of feedstock used in vesicles experiment was around 70 g/L.