Granule formation frequently occurs in denitrifying phosphorus (P) removal systems. The differences between granules and flocculants, including P removal capacities, bio-physiochemical properties, and microorganism distribution and diversity, are however still poorly understood. Physical and biochemical characteristics of granules and sludge flocs were investigated through two laboratory-scale sequencing batch reactors (SBRs). One reactor was operated as a flocculent SBR and the other was operated as a granular SBR, using a granule diameter of 1.92 +/- 0.78 mm. Granular sludge had a higher tolerance to inhibitors (nitrite/free nitrite acids (FNAs)), and a higher percentage of P accumulating organisms (PAOs) (72% vs. 64% in flocs) than flocculent sludge. The granule crush tests and higher PAOII (unable to use nitrate as an electron acceptor) to PAOs ratios (over 72%) by fluorescent in situ hybridization showed that in both reactors, glycogen accumulating organisms (GAOs) were mainly responsible for nitrate to nitrite reduction, and PAOII further reduced nitrite to nitrogen gas in association with anoxic P uptake; GAOs wash-out weakened the mutual relationship between GAOs and PAOII to some extent, which made denitrification of nitrate to nitrite inefficient and weakened subsequent anoxic P removal. GAOs existed mainly on the surface of the granules, whereas PAOs (PAOI + PAOII) were distributed both on the surface and in the interior of the granules. Thus, GAOs had easier access to carbon sources but were at risk of suffering from exposure to FNA. (C) 2014 Elsevier B.V. All rights reserved.