| In a search to turn the economically and environmentally non-valuable "waste" streams of biodiesel production into a profitable byproduct, a mutant strain of Enterobacter aerogenes ATCC 13048 was developed by six-tube subculturing technique. This technique is based on the principle of adaptive evolution, and involved subculturing the bacterium in a tryptic soy broth without dextrose (TSB) containing specific glycerol and ethanol concentration for six consecutive times. Then, the six consecutive subculturing was repeated in a fresh TSB of higher glycerol and ethanol concentrations. A new mutant strain, E. aerogenes S012, which could withstand a combination of 200 g/l glycerol and 30 g/l ethanol concentrations, was developed. The wild and mutant strains were used for the fermentation of pure (P-) and recovered (R-) glycerol. Taguchi and full factorial methods of design of experiments were used to screen and optimize the important process factors that influence the microbial production of ethanol. A statistically sound regression model was used to establish the mathematical relationship between the process variables and ethanol production. Temperature of 38°C, agitation speed of 200 rpm, pH of 6.3-6.6, and microaerobic condition were the optimum process conditions. Different pretreatment methods to recover glycerol from the crude glycerol and the subsequent fermentation method showed that direct acidification using 85% H3PO4 was the best. The R-glycerol contained 51% pure glycerol and 21% methanol. The wild strain, E. aerogenes ATCC 13048, produced only 12 g/l and 12.8 g/l ethanol from 20 g/l P- and R-glycerol respectively, and could not utilize higher glycerol concentrations. The mutant, E. aerogenes S012, produced ethanol amount and yield of 43 g/l and 1.12 mol/mol-glycerol from P-glycerol, respectively within 96 h. It also produced ethanol amount and yield of 26.8 g/l and 1.07 mol/mol-glycerol, respectively, from R-glycerol within the same duration. In a fermentation to estimate hydrogen production using a respirometer, the hydrogen yield and volumetric rate of 1.06 mol/mol-glycerol and 217 ml/l/h, respectively were obtained from 6% P-glycerol in 72 h by E. aerogenes S012. The result was higher from R-glycerol, which produced hydrogen yield and productivity of 1.83 mol/mol-glycerol and 326 ml/l/h, respectively. |
