The present study exposes the effect of deagglomeration using rhamnolipid on anaerobic granular biosolids (AGB) followed by ultrasonic homogenization for effective biohydrogen production. Rhamnolipid was used to remove the extracellular polymeric substance bound over the surface of AGB to increase the rate of biogranular lysis during ultrasonic homogenization. Extracellular polymeric substance (EPS) removal was achieved at an optimum rhamnolipid dosage of 0.04 g Rh/g SS. Ultrasonic homogenization (UH) of AGB demands 27016 kJ/kg TS of specific energy to achieve 16.8% and 13.9% of biogranular lysis and biosolids reduction, respectively. However, rhamnolipid-alkaline pH induced ultrasonic homogenization (RAUH) demand lesser (12607 kJ/kg TS) and achieves greater biogranular lysis (25.4%) and biosolids reduction (20.7%). RAUH significantly saves the net energy. Exponential first order kinetic analysis was done to evaluate and compare the biohydrogen production potential of RAUH with that of UH. The biohydrogen production was found to be 55.1 mL H-2/g COD and 36.7 mL H-2/g COD for RAUH and UH respectively. A higher positive net energy of 2.62 kWh/kg AGB was achieved by RAUH when compared to UH (-3.49 kWh/kg anaerobic granular biosolids). (C) 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.