Abstract
Citrus fruits are widely consumed worldwide due to their nutritional and health benefits. However, the disposal of citrus waste poses significant environmental challenges. Orange peels (OP) are a substantial by-product of fruit processing and hold great potential as a source for bioethanol production, promoting investment in utilizing agricultural waste for biofuel purposes. OP offers a cost-effective substrate for producing value-added compounds, including bioethanol. Autoclaved-water treated OP biomass exhibited the highest release of reducing sugars (68.2%) this results supported by SEM images of that Autoclaving has definite effect on the structure of the OP particles. Among the five tested microbes, Candida tropicalis was selected as a promising bioethanol candidate due to its ethanol tolerance and ability to utilize xylose. Preliminary screening using Plackett-Burman Design (PBD) was conducted to identify six influential factors affecting the fermentation process at three levels, determining the optimum response region for bioethanol production by C. tropicalis. The significant variables were further investigated using Response Surface Methodology-Central Composite Rotatable Design (RSM-CCRD) at five levels, a novel approach in this study. The addition of cysteine and resazurin as reducing agents increased bioethanol production by 2.9 and 2.1 times, respectively, from the treated OP. Under the optimized conditions obtained from RSM-CCRD, bioethanol production reached 16.7 mg/mL per mg/ml reducing sugars. Implementing all the optimized conditions, including an initial pH of 5.75, 3% yeast extract, 2.25 g/L cysteine, 4% inoculum size, 0.6 g/L ZnSO4, 0.29 g/L MgSO4, 0.3 g/L MnSO4, and substrate treatment with active charcoal before fermentation, the bioethanol yield increased by 2.2 times after three days of fermentation using co-cultures of C. tropicalis and Kluyveromyces marxianus. The fermentation process was conducted at 30 °C and 150 rpm. Exploring OP as a low-cost renewable substrate and employing efficient microorganisms open new avenues for bioethanol production.
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