The improvement of batch photofermentative biohydrogen production was investigated using ultrasonication pre-treatment on a combined effluent of palm oil and pulp and paper mills. The effects of the amplitude (30-90%) and ultrasonication duration (5-60 min) were investigated in terms of their influences on the biohydrogen yield and chemical oxygen demand (COD) removal. The recommended ultrasonication parameters were found at the higher ranges of amplitude and duration (A70T45). Using A70T45 ultrasonication, the production of biohydrogen at 30 degrees C could be enhanced up to 8.72 mL H-2/mL(medium), with a total COD removal of 36.9%. During pre-treatment at A70T45, an energy input of 775 J/mL was supplied to disintegrate complex compounds into simpler structures. As a result, an increase in the soluble organic matter concentration was achieved, which led to enhanced biohydrogen production. On the other hand, the lowest biohydrogen yield (4.67 mL H-2/mL(medium)) and total COD removal (28.8%) were obtained in the control without pre-treatment. The enthalpy of the photofermentation process was estimated to be 141.1 kJ/mol with a threshold temperature of 30.9 degrees C based on a modified Arrhenius approach. (C) 2016 Elsevier Ltd. All rights reserved.