This study focuses on a scale-up procedure considering two vital parameters light energy and mixing for microalgae cultivation, taking Chlamydomonas reinhardtii as the model microorganism. Applying two stage hydrogen production protocol to 1 L flat type and 2.5 L tank type photobioreactors hydrogen production was investigated with constant light energy and mixing time. The conditions that provide the shortest transfer time to anaerobic culture (light energy; 2.96 kJ s(-1) m(-3) and mixing time; 1 min) and highest hydrogen production rate (light energy; 1.22 kJ s(-1) m(-3) and mixing time; 2.5 min) are applied to 5 L photobioreactor. The final hydrogen production for 5 L system after 192 h was measured as 195 +/- 10 mL that is comparable with the other systems is a good validation for the scale-up procedure. (C) 2012 Elsevier Ltd. All rights reserved.