Glycerol Box-Behnken design a b s t r a c tEnterobacter aerogenes has a known ability to convert glycerol during a fermentative process to yield hydrogen and ethanol as the main products. A Box-Behnken design and response surface methodology were used to determine the optimal concentration of some media constituents and oxygen to maximize the yield of biohydrogen. Results indicated that the concentration of the salts studied: NH 4 NO 3 , FeSO 4 , and Na 2 HPO 4 and; the presence of oxygen in the pre-culture significantly influence the production of biohydrogen. Optimal conditions were determined to be 7.5% O 2 in the inoculum transfer step, ratio of inocula 18%, 8 g/L of Na 2 HPO 4 , 0.00625 g/L of FeSO 4 and 1.5 g/L of NH 4 NO 3 . These optimal conditions resulted in a measured yield of 0.85 mol H 2 /mol glycerol at a substrate concentration of 15 g/L and a maximum predicted yield of 0.95 mol H 2 /mol glycerol at a substrate concentration of 21 g/L. These results were obtained using lower concentrations of salts than in previous studies, corresponding to a 76% cost savings. These experimental results also demonstrated the importance of optimizing the amount of oxygen present in the biological system rather than maintaining complete anaerobic conditions.

Crude glycerol from the biodiesel manufacturing process is being produced in increasing quantities due to the expanding number of biodiesel plants. It has been previously shown that, in batch mode, semi-anaerobic fermentation of crude glycerol by Enterobacter aerogenes can produce biohydrogen and bioethanol simultaneously. The present study demonstrated the possible scaling-up of this process from small batches performed in small bottles to a 3.6-L continuous stir tank reactor (CSTR). Fresh feed rate, liquid recycling, pH, mixing speed, glycerol concentration, and waste recycling were optimized for biohydrogen and bioethanol production. Results confirmed that E. aerogenes uses small amounts of oxygen under semi-anaerobic conditions for growth before using oxygen from decomposable salts, mainly NH4NO3, under anaerobic condition to produce hydrogen and ethanol. The optimal conditions were determined to be 500 rpm, pH 6.4, 18.5 g/L crude glycerol (15 g/L glycerol) and 33% liquid recycling for a fresh feed rate of 0.44 mL/min. Using these optimized conditions, the process ran at a lower media cost than previous studies, was stable after 7 days without further inoculation and resulted in yields of 0.86 mol H2/mol glycerol and 0.75 mol ethanol/mole glycerol.

a b s t r a c tEnterobacter aerogenes have a known ability to convert glycerol (GL) in a fermentative process to yield hydrogen and ethanol as the main by-products. The concentration of some media constituents was optimized to maximize biohydrogen yield and rate of production. E. aerogenes were cultured in aerobic conditions, and then transferred into anaerobic conditions before being cultured in a minimum mineral synthetic media (MMSM) containing 15 g/L GL. The concentration of selected salts were optimized in the following ranges: 0e300 mg/L MgSO 4 , 0e14 g/L Na 2 EDTA, 0e10 mg/L CaCL 2 , 0e10 g/L Na 2 HPO 4 , and 0 e9.7 g/L KH 2 PO 4 . The results of the full factorial design indicated that the production of biohydrogen required a minimal concentration of 3.5 mg/L EDTA, 200 mg/L MgSO 4 .7H 2 O and no CaCl 2 .2H 2 O. A significant interaction between EDTA and MgSO 4 was also observed. Results from the phosphate salts optimization showed that Na 2 HPO 4 gave better results than KH 2 PO 4 . The optimal conditions determined using pure glycerol (commercial grade glycerol), were successfully applied to the fermentation of crude glycerol from biodiesel production. The results indicated promising yields of 0.79 and 0.84 mol/mol of glycerol for bioethanol and biohydrogen, respectively, and this at a faster rate than reported previously for E. aerogenes.

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