Anaerobic ammonium oxidation (anammox) is a novel wastewater treatment process. The intrinsic nature of anammox bacteria makes their traditional isolation from a consortium impossible. This study presents a detailed assessment of different pretreatments for percoll density gradient centrifugation (PDGC) to purify anammox bacteria and proposes that the extraction of extracellular polymeric substances (EPS) could significantly increase the purification performance. The results demonstrate that both EPS extraction using cation exchange resin for 2 h and mild ultrasound at an optimal power of 7.2 kJ were suitable pretreatments to increase the purification efficiency in comparison to PDGC performed directly from an original consortium (optimal conditions identified as 8000 rpm, 30 min). Importantly, the implementation of EPS extraction before ultrasound was verified to have the capacity to further increase the purification efficiency up to 95%, which demonstrates the prominent effects of EPS extraction on the PDGC performance. Compared to the previous report, EPS extraction was applied instead of the first centrifugation prior to PDGC to remove large flocs, which correspondingly increased the purified amounts. To clarify the involved mechanism, the bacteria aggregating behaviors before and after EPS extraction over time were visually tracked using a particle size analyzer. The results of this study help to obtain large amounts of anammox bacteria and elucidate the anammox characteristics and communities in both natural and engineered systems. (C) 2015 Elsevier B.V. All rights reserved.