The properties of eggshells (EGS) as neutralizing and immobilizing agent were investigated for hydrogen (H-2) production using crude glycerol (CG) by co-culture system. Eggshells of different sizes and concentrations were used during batch and repeated-batch fermentation. For batch and repeated-batch fermentation, the maximum H-2 production (36.53 +/- 0.53 and 41.16 +/- 0.95 mmol/L, respectively) was obtained with the EGS size of 33 mu m < x(5) < 75 lm. Hydrogen production increased with the decreasing size of EGS. Eggshells maintained the fermentation pH (6.00-6.30) and provided immobilization support as confirmed by scanning electron microscopy. As media components, the EGS concentration of 0.25% (w/v) was found to be optimum for maximum H-2 production (31.66 +/- 0.55 mmol/L) and the production profile was comparable to H-2 production (32.07 +/- 0.92 mmol/L) obtained with all media components. In scale-up study with semi-continuous bioreactor (7.5 L), almost 1.5-fold increase (in comparison to mono-culture) i.e. 312.12 mmol-H-2/L-of medium with 86.65% glycerol utilization was obtained. (C) 2016 Elsevier Ltd. All rights reserved.