Low frequency ultrasonic treatment was investigated to enhance hydrogen production by bacteria under both batch and continuous operation. Optimal ultrasonic time and intensity as examined in batch experiments were found to be 10 s and 100 W, respectively. Hydrogen production was increased by 20% while ultrasonic treatment could effectively shorten batch test reaction times. Using these optimized conditions, continuous experiments were conducted in two parallel expanded granular sludge bed (EGSB) reactors. The ultrasonic treated EGSB reactor obtained a specific hydrogen production rate of 4.38 hg VSS.d, which was 32% higher than that in the control reactor. Ethanol-type fermentation was achieved at a faster rate and with a 72.7% shorter startup time using ultrasonic treatment. Microbial activity under ultrasonic treatment was shown to resist lower pH and to maintain satisfactory hydrogen production. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.