Three parallel lab-scale expanded granular sludge bed(EGSB) reactors were employed to evaluate the start-up process of bio-hydrogen production from molasses wastewater.With other start-up parameters controlled the same,the effect of inoculating sludge pre-treatments was studied.Two methods of pre-treatments(pre-heating and pre-aeration) were conducted for the inoculating sludge in two reactors compared with a non-pre-treatment reactor(control reactor).The results showed that the reactors with pre-treatment performed better in the start-up process than the control one.The hydrogen-producing bacteria were efficiently enriched at the start-up period,shortening the start-up process.At the end of the start-up stage,the acidification rate of the pre-treatment reactors was 10%—30% more than the control one,and hydrogen production increased by 1.69—1.82 times.Considering cost and feasibility,the authors suggest applying the pre-aeration method to treat the inoculating sludge to get more efficient start-up in the industrial scale bio-hydrogen production plant.