| Summary: | This paper evaluates the feasibility of bioethanol production from onion waste by <i>Saccharomyces cerevisiae</i> and <i>Pichia pastoris</i> and their novel co-culture through fermentation. The process parameters were optimized for each strain and their combination to observe the synergistic effect of co-fermentation. A dinitro salicylic acid (DNS) test was conducted to study the reducing sugar content of samples at different time intervals. Fourier transform infrared (FTIR) spectroscopic analysis was used to compare results for functional groups of samples before and after fermentation, and gas chromatography with flame ionization detection (GC-FID) analysis was performed to measure the bioethanol concentration obtained at different combinations of pH (5, 5.5, 6), temperature (20 °C, 30 °C, 40 °C), and time (24–110 h). The maximum bioethanol concentration was achieved through a monoculture of <i>Saccharomyces cerevisiae</i>, i.e., 30.56 g/L. The ethanol productivity was determined based on the ethanol concentration and fermentation time ratio. The energy content was determined using the obtained ethanol value and the specific energy content of ethanol, i.e., 30 kJ/g. The productivity and energy of bioethanol obtained at this maximum concentration were 0.355 g/L h and 916.8 kJ/L, respectively, after 86 h of fermentation at 30 °C and pH 5. <i>Pichia pastoris</i> produced a maximum of 21.06 g/L bioethanol concentration with bioethanol productivity and energy of 0.264 g/L h and 631.8 kJ/L, respectively, after 72 h of fermentation at 30 °C and pH 5. The coculture fermentation resulted in 22.72 g/L of bioethanol concentration with bioethanol productivity and energy of 0.264 g/L h and 681.6 kJ/L, respectively, after 86 h of fermentation at 30 °C and pH 5. The results of reducing sugars also supported the same conclusion that monoculture fermentation using <i>Saccharomyces cerevisiae</i> was the most effective for bioethanol production compared to <i>Pichia pastoris</i> and co-culture fermentation.
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