Increasing consumption of fossil fuels as well as the concern over pollution and global climate change has accelerated the development of the sustainable biofuel industry. Biodiesel, bioethanol and biomethane are already commercially available as alternatives of fossil fuels and the search for a more environmentally friendly biofuel, preferentially produced from non-food raw materials and capable of fulfilling the transportation energy requirement of the world for longer duration, is ongoing. In this context, biohydrogen produced from waste biomass is an ideal option. It has higher energy content compared to fossil and biofuels of equivalent mass and produces water as the only major emission during combustion. In the present investigation, crude glycerol (CG) generated as by-product of the biodiesel production process has been used as feedstock for biohydrogen production and different supplements have been evaluated for increasing the product yield. Nano-spray dried ferric citrate particles have been found to enhance the hydrogen production by 50.45%. Hydrogen production using extremely low CG concentration of 100 mg L⁻¹ has been found to produce 22.7 mol-H₂ per kg CG; which is 2.75-fold higher than 8.25 mol-H₂ per kg CG, known for dark fermentation.