• Biodiesel wastes;
  • Hydrogen;
  • Klebsiella pneumoniae;
  • Regression analysis;
  • Uniform design


Biodiesel wastes containing glycerol were utilized by Klebsiella pneumoniae DSM 2026 to produce hydrogen. The optimization of medium components was performed using both Plackett-Burman and uniform design methods. Using the Plackett-Burman design, glycerol, yeast extract, NH4Cl, KCl and CaCl2 were found to be the most important components, which were further investigated by uniform design and second-order polynomial stepwise regression analysis. The optimized medium containing 20.4 g·L-1 glycerol, 5.7 g·L-1 KCl, 13.8 g·L-1 NH4Cl, 1.5 g·L-1 CaCl2 and 3.0 g·L-1 yeast extract resulted in 5.0-fold increased level of hydrogen (57.6 mL/50 mL medium) production compared to initial level (11.6 mL/50 mL medium) after 24 h of fermentation The optimization of fermentation condition (pH, temperature and inoculum) was also conducted. When the strain grew in the optimized medium under optimal fermentation condition in a 5-L stirred tank bioreactor for batch production, hydrogen yield and production reached 0.53 mol/mol and 117.8 mmol/L, respectively. The maximum hydrogen evolution rate was 17.8 mmol/(L·h). Furthermore, 1,3-propanediol (6.7 g·L-1) was also obtained from the liquid medium as a by-product.