Response surface methodology and optimization of the processes for bioethanol production from Calabash (Crescentia cujete) Using Cronobacter malonaticus

Aim: Response surface methodology (RSM) model was applied to optimize ethanol production from Calabash (Crescentia cujete) pulp juice using Cronobacter malonaticus. Study Design: The Calabash pulp was squeezed with muslin cloth, and vacuum filtered to clear solution before use. The clear juice was tested for reducing sugars using the Dinitrosalicylic acid (DNS) method. Twenty three (23) runs, including 3 controls, of the fermentation was conducted at varying temperatures, pH, and volumes of inoculum. The process parameters (input variables): volumes of inoculum, temperature, and pH were subjected to response surface model, using the Central Composite Design (CCD). Place and Duration of Study: This study was carried out in the Environmental Microbiology Laboratory, University of Port Harcourt for six months. Methodology: Fermentation was done in conical flasks covered with cotton wool and foil in a stationary incubator for four days (96 hours). Active stock culture of Cronobacter malonaticus was used, with inoculum developed using Marcfaland’s method. Samples were collected every 24 hours, centrifuged, filtered and analyzed for measurement of the output variables: Reducing sugar, cell density and ethanol concentration. Results: The concentration of reducing sugars from Calabash pulp was 3.2 mg/ml. Results obtained also revealed that the fermentation can take place on a wide range of temperature 28-32°C. The optimal pH range for performance of C.malonaticus for the fermentation process was pH 5.95-6.5. The optimum volume of inoculum was 10%v/v (i.e. 10 ml in 90 ml juice). The optimized process using the RSM model gave 5.08% v/v bioethanol, being the highest achieved at pH6.08 and 28oC . Conclusion: The bioethanol yield from Calabash substrate is reasonable considering that the bacterium used is not known for ethanol production. Also the concentration of reducing sugars in the substrate and the duration of fermentation could be responsible for the yield.