BACKGROUND: The conversion of lipid-extracted microalgal biomass residues (LMBRs) into hydrogen serves the dual role in renewable energy production and sustainable development of microalgal biodiesel industry. In this work, batch experiments were performed to convert LMBRs pretreated by a thermo-alkaline method into hydrogen. In addition, repeated batch cultivation was conducted using the pretreated LMBRs as feedstocks under optimal pretreatment condition to further obtain high hydrogen production. RESULTS: The optimal pretreatment conditions for LMBRs were NaOH dosage of 8 g L-1, pretreatment time of 2.5 h and solid content of 6.7%, which resulted in 160% and 500% improvement in the hydrogen yield and hydrogen production rate, respectively. In addition, it was found that the repeated batch cultivation was a very useful method to improve the hydrogen production and hydrogen production rate. CONCLUSIONS: The thermo-alkaline pretreatment resulted in remarkable improvements in LMBR solubilization, which led to an increase in hydrogen production. Repeated batch cultivation was a useful method to adapt and enrich hydrogen-producing bacteria that could ferment LMBRs with high hydrogen yield. (C) 2010 Society of Chemical Industry