Optimization of media and process parameters for bioconversion of crude glycerol to hydrogen by locally isolated klebsiella pneumoniae

Bioconversion of crude glycerol to biohydrogen is promising because the cost for capital investment and operation is cheaper, and it can help in reducing waste thus making it a clean process. However, not many microbes can metabolise glycerol efficiently under anaerobic or oxygen-limited conditions. This makes the production and yield of hydrogen produced to be low. Therefore, this study sought to isolate new indigenous bacteria that can consume crude glycerol (85%) and convert it into biohydrogen without the need for pretreatment or acclimatization. Dark fermentation approach was employed since this approach offers much advantages in terms of substrates and microbe producers used. The aim of study was achieved through isolation and screening for potential hydrogen producers followed by identification using 16S rRNA technique. Next, optimisation of medium composition and optimisation of operating parameters to obtain the optimum production and yield of hydrogen and ethanol including glycerol uptake. The study was finalised by a kinetic study for growth and substrate utilisation by kinetic model and potential hydrogen production by modified Gompertz model. The best hydrogen producer had been successfully isolated, screened and identified as Klebsiella pneumoniae strain HS11286. The 2-level fractional factorial design (28–3) for medium composition optimisation and response surface methodology study employing Box-Behnken design for operating parameters were employed to evaluate the interactive effects of several respective factors. The fermentation was conducted in 2 L reactor with a working volume of 1.8 L. Medium optimisation study gave the composition of 30 g/L glycerol, 3.5 g/L K2HPO4, 3.98g/L KH2PO4, 2.69 g/L (NH4)2SO4, 0.03 g/L CaCl2.2H20, 0.054 g/L FESO4.7H2O, 3.0 g/L yeast extract, and 0.54 g/L MgSO4.7H2O as the optimised medium composition. This optimised composition yielded 588.68±0.04 mmol H2/mol glycerolconsumed, 9345±63.64 mL of hydrogen, 97±1.41% glycerol uptake and 0.024±0.001 mmol ethanol/mol glycerolconsumed. Meanwhile, the optimum operating condition was found best at pH 6.0, temperature 32.5°C and 25% headspace with 82% desirability. This yielded 601.07±10.69 mmol H2/mol glycerolconsumed of hydrogen yield, 9935±176 mL of hydrogen, 97±1.4% of glycerol uptake and 0.045±0.002 mmol ethanol/mol glycerolconsumed. Finally, kinetic parameters for specific growth rate (μ) was at 0.106 h−1, glycerol consumption rate (Qgly) at 1.572 g/L/h, and yield coefficient Yp/x, Yp/s and Yx/s at 30758.51 mL/g cell, 479.26 mL/g substrate and 0.016 g cell/g substrate, respectively. Meanwhile, the modified Gompertz model gave a prediction of 10155 mL of hydrogen at 620 mL/h. In conclusion, Klebsiella pneumonia strain HS11286 has the potential to produce almost 10 litre of hydrogen in a short period (less than 48 h) without the need to pretreat the glycerol or to acclimatize the bacteria in crude glycerol