Response Surface Optimization of Bioethanol Production from Roselle (Hibiscus Sabdariffa L.) using Saccharomyces Cerevisiae
Keywords:
Bioethanol; Hibiscus sabdariffa; roselle; Saccharomyces cerevisiae ; fermentation; response surface methodology; optimization; renewable biomass.Abstract
Bioethanol production from renewable biomass has gained increasing attention due to the growing demand for alternative forms of energy and environmental issues from fossil fuels. This study investigated the production and optimization of bioethanol from zobo (Hibiscus sabdariffa L.) extract using Saccharomyces cerevisiae isolated from palm wine. The roselle extract was characterized for physicochemical properties, fermentable sugars, organic acids, and mineral composition. Fermentation was conducted using one-factor-at-a-time (OFAT) screening followed by response surface methodology (RSM) based on central composite design (CCD) to evaluate the effects of fermentation time, initial pH, substrate concentration, and temperature on ethanol production. The extract contained glucose (22.90 mg/g), fructose (16.50 mg/g), and sucrose (11.70 mg/g), with malic acid (45.65 mg/g) as the predominant organic acid. The extract had a pH of 3.56, moisture content of 10.80 g/100 g, and ash content of 9.70 g/100 g, with calcium (0.55 mg/g), potassium (0.46 mg/g), and iron (0.22 mg/g) as major mineral components. Preliminary screening identified 60 h, pH 6, substrate concentration of 60 g/L, and temperature of 40 °C as favorable conditions. RSM produced a statistically significant quadratic model (F = 17.81, p = 0.0010) with R² = 0.9765, adjusted R² = 0.9217, and adequate precision of 13.6882. The lack of fit was non-significant (p = 0.0924), with a coefficient of variation of 5.16%. Fermentation time, initial pH, and substrate concentration had significant linear effects on ethanol production, while temperature was not significant as a linear term. The pH × temperature interaction and the quadratic effects of fermentation time and pH were also significant. The model predicted optimum conditions of 60 h, pH 6, substrate concentration of 80 g/L, and temperature of 40 °C, yielding an experimental ethanol concentration of 5.10 g/L compared with predicted value of 5.31 g/L, corresponding to a deviation of 3.95%. Diagnostic plots indicated that the model assumptions were reasonably satisfied; however, the negative predicted R² (−1.1933) indicated limited predictive reliability for new observations. The findings demonstrate the potential of roselle extract as a substrate for bioethanol production and the applicability of RSM for optimizing the fermentation process.
References
1. Altınışık, S., Nigiz, F. U., Gürdal, S., Yılmaz, K., Tuncel, N. B., & Koyuncu, S. (2024). Optimization of bioethanol production from sugar beet processing by-product molasses using response surface methodology. Biomass Conversion and Biorefinery, 15(7), 9875–9888. https://doi.org/10.1007/s13399-024-05786-w
2. Apuyor, K. E., Asiagwu, A. K., Otobrise, C., Apuyor, S. E., & Apuyor, A. (2026). Biodiesel Production from Jatropha curcas (l.) Seed Oil Using a Green-Synthesized MGO Nanocatalyst and RSM Method for Optimization. Asian Journal of Applied Chemistry Research, 17(2), 32–57. https://doi.org/10.9734/ajacr/2026/v17i2389
3. Apuyor, K. E., Otobrise, C., Apuyor, S. E., Asiagwu, A. K., & Onofuevure, A. O. (2026). GC–MS analysis of Jatropha curcas seed oil and ethanol derived biodiesel produced using green-synthesized MgO nanocatalyst. FUDMA Journal of Sciences, 10(2), 55–64. https://doi.org/10.33003/fs-2026-1002-4346
4. Aquinas, N., Ramananda, B. M., & Selvaraj, S. (2024). Optimization of curdlan production and ultrasound assisted extraction processes from Priestia megaterium. Scientific Reports, 14(1). https://doi.org/10.1038/s41598-024-77880-y
5. Asiagwu, A. K. (2017). Kinetics of fermentation of local species of cashew juice using Saccharomyces cerevisiae (local extract). International Journal of Engineering Research & Technology (IJERT), 6(7), 420–426.
6. Beigbeder, J.-B., de Medeiros Dantas, J. M., & Lavoie, J.-M. (2021). Optimization of Yeast, Sugar and Nutrient Concentrations for High Ethanol Production Rate Using Industrial Sugar Beet Molasses and Response Surface Methodology. Fermentation, 7(2), 86. https://doi.org/10.3390/fermentation7020086
7. Bušić, A., Marđetko, N., Kundas, S., Morzak, G., Belskaya, H., Ivančić Šantek, M., Komes, D., Novak, S., & Šantek, B. (2018). Bioethanol Production from Renewable Raw Materials and its Separation and Purification: a Review. Food Technology and Biotechnology, 56(3).
https://doi.org/10.17113/ftb.56.03.18.5546
8. Chen, A., Si, Q., Xu, Q., Pan, C., & Chen, J. (2025). Evaluation of Stress Tolerance and Fermentation Performance in Commercial Yeast Strains for Industrial Applications. Foods, 14(1), 142.
https://doi.org/10.3390/foods14010142
9. Chen, Q., & Qi, J. (2023). How much should we trust R² and adjusted R²: Evidence from regressions in top economics journals and Monte Carlo simulations. Journal of Applied Economics, 26(1). https://doi.org/10.1080/15140326.2023.2207326
10. Dadi, M., Siwale, W., Munalula, F., Ravi, S. N., Mundike, J., Ramasamy, S., & Mulenga, C. (2025). A comprehensive review of advances in bioenergy including emerging trends and future directions. Discover Energy, 5(1). https://doi.org/10.1007/s43937-025-00095-3
11. Douradinho, R., Sica, P., Tonoli, F., Mattos, E., Oliveira, M., Pinto, A., Mota, L., Faria, T., Costa, V. F., Leite, G., Arthur, V., Coelho, S., & Baptista, A. (2023). Osmotic Stress Alleviation in Saccharomyces cerevisiae for High Ethanol Fermentations with Different Wort Substrates. Stresses, 3(4), 813–826. https://doi.org/10.3390/stresses3040055
12. Edo, G. I., Samuel, P. O., Jikah, A. N., Oloni, G. O., Ifejika, M. N., Oghenegueke, O., Ossai, S., Ajokpaoghene, M. O., Asaah, E. U., Uloho, P. O., Akpoghelie, P. O., Ugbune, U., Ezekiel, G. O., Onoharigho, F. O., Agbo, J. J., & Essaghah, A. E. A. (2023). Proximate composition and health benefit of Roselle leaf (Hibiscus sabdariffa). Insight on food and health benefits. Food Chemistry Advances, 3, 100437. https://doi.org/10.1016/j.focha.2023.100437
13. Egharevba, F., Omoregie, O., & Iyayi, E. A. (2014). Optimization of fermentation conditions for ethanol production from cassava peels. African Journal of Biotechnology, 13(12), 1345–1352.
14. Fentahun, M. (2026). Bioethanol production using sugar beet (Beta vulgaris) juice as substrate by stress-tolerant yeast strains isolated from Areke. Scientific Reports, 16(1).
https://doi.org/10.1038/s41598-026-45172-2
15. Ghosh, S., Chakraborty, S., & Ray, S. (2012). Optimization of ethanol production from sweet sorghum juice by Saccharomyces cerevisiae . Biomass and Bioenergy, 47, 233–241.
16. Hemalatha, S., & Anbuselvi, S. (2013). Physicochemical constituents of roselle (Hibiscus sabdariffa L.). Journal of Chemical and Pharmaceutical Research, 5(3), 214–216.
17. Ibrahim, Z. A., Soliman, W. S., Ahmed, O. K., & Abbas, M. T. (2026). Roselle (Hibiscus sabdariffa) stem residues as a sustainable plant-based culture medium for isolation of endophytic bacteria. Scientific Reports, 16(1), 14394. https://doi.org/10.1038/s41598-026-47923-7
18. Idowu-Adebayo, F., Toohey, M. J., Fogliano, V., & Linnemann, A. R. (2021). Enriching street-vended zobo (Hibiscus sabdariffa) drink with turmeric (Curcuma longa) to increase its health-supporting properties. Food & Function, 12(2), 761–770. https://doi.org/10.1039/d0fo02888f
19. Izquierdo-Vega, J., Arteaga-Badillo, D., Sánchez-Gutiérrez, M., Morales-González, J., Vargas-Mendoza, N., Gómez-Aldapa, C., Castro-Rosas, J., Delgado-Olivares, L., Madrigal-Bujaidar, E., & Madrigal-Santillán, E. (2020). Organic Acids from Roselle (Hibiscus sabdariffa L.)—A Brief Review of Its Pharmacological Effects. Biomedicines, 8(5), 100. https://doi.org/10.3390/biomedicines8050100
20. Jawad, A. H., Alkarkhi, A. F. M., & Norulaini, N. A. N. (2013). Production of bioethanol from Hibiscus sabdariffa L. using Saccharomyces cerevisiae . Journal of Industrial and Engineering Chemistry, 19(5), 1602–1607.
21. Juneja, A., & Kumar, D. (2024). Production of Ethanol from Plant Biomass. Handbook of Biorefinery Research and Technology: Production of Biofuels and Biochemicals, 3–32.
https://doi.org/10.1007/978-981-97-7586-6_37
22. Jung, E., Kim, Y., & Joo, N. (2013). Physicochemical properties and antimicrobial activity of roselle (Hibiscus sabdariffa L.). Journal of the Korean Society of Food Science and Nutrition, 42(6), 924–931.
23. Kant, G., Hasan, A., Yadav, P., Pandey, A., & Srivastava, S. (2025). The generational shift in biofuels: A path toward sustainable energy solutions. Biomass and Bioenergy, 196, 107757.
https://doi.org/10.1016/j.biombioe.2025.107757
24. Karuppaiya, M., Sasikumar, E., Viruthagiri, T., & Vijayagopal, V. (2010). Optimization of process conditions using response surface methodology (RSM) for ethanol production from cashew apple juice. Chemical Engineering Communications, 197(1), 1–14.
25. Li, N., Simon, J. E., & Wu, Q. (2024). Determination of anthocyanins, organic acids, and phenolic acids in hibiscus market products using LC/UV/MS. Journal of Food Science, 89(2), 1098–1113. https://doi.org/10.1111/1750-3841.16909
26. Li, Y., Lu, J., Gu, G., & Shi, Z. (2002). Mathematical modeling and optimization of fermentation processes. Journal of Biotechnology, 94(2), 179–192.
27. Mondal, P., Sadhukhan, A. K., Ganguly, A., & Gupta, P. (2021). Optimization of process parameters for bio-enzymatic and enzymatic saccharification of waste broken rice for ethanol production using response surface methodology and artificial neural network-genetic algorithm. *3 Biotech, 11*(1), 28. https://doi.org/10.1007/s13205-020-02553-2
28. Mwanyesya, O. P., Mtashobya, L. A., & Emmanuel, J. K. (2025). Value addition on bioethanol produced from different feedstocks: a review of recent outlook. Discover Applied Sciences, 7(12). https://doi.org/10.1007/s42452-025-07377-4
29. Nwogwugwu, N. U., Abu, G. O., Akaranta, O., & Chinakwe, E. C. (2019). Application of Response Surface Methodology for Optimizing the Production of Bioethanol from Calabash (Crescentia cujete) Substrate Using Saccharomyces cerevisiae . Journal of Advances in Microbiology, 1–12.
https://doi.org/10.9734/jamb/2019/v17i230139
30. Olorunfemi, O. B., Boboye, B. E., & Okonko, I. O. (2005). Isolation and characterization of yeast from palm wine. African Journal of Biotechnology, 4(10), 1120–1124.
31. Opara, C. C., & Nwahia, C. O. (2012). Production of alcoholic beverage from zobo flower juice using Saccharomyces cerevisiae isolated from palm wine. Journal of Biochemistry and Technology, 3(4), 436–437.
32. Parapouli, M., Vasileiadi, A., Afendra, A.-S., & Hatziloukas, E. (2020). Saccharomyces cerevisiae and Its Industrial Applications. AIMS Microbiology, 6(1), 1–31. https://doi.org/10.3934/microbiol.2020001
33. Phong, H. X., Klanrit, P., Dung, N. T. P., Thanonkeo, S., Yamada, M., & Thanonkeo, P. (2022). High-temperature ethanol fermentation from pineapple waste hydrolysate and gene expression analysis of thermotolerant yeast Saccharomyces cerevisiae . Scientific Reports, 12(1).
https://doi.org/10.1038/s41598-022-18212-w
34. Ramin, H., & Mir Taher, M. (2026). Model-based optimization of bioethanol production from sugar beet molasses using Zymomonas mobilis PTCC 1718: A response surface methodology approach. Chemical Engineering Journal Advances, 27, 101277. https://doi.org/10.1016/j.ceja.2026.101277
35. Rinki, Yadav, P., Sharma, A., Dahiya, P., Kashyap, A., Walia, A., Bhatt, A. K., & Bhatia, R. K. (2024). Upcycling of tetra pack waste cellulose into reducing sugars for bioethanol production using Saccharomyces cerevisiae . Biotechnology for Sustainable Materials, 1(1).
https://doi.org/10.1186/s44316-024-00003-0
36. Rozina, Ahmad, M., & Zafar, M. (2022). Biomass as Sustainable Material for Bioethanol Production. Handbook of Smart Materials, Technologies, and Devices, 453–475. https://doi.org/10.1007/978-3-030-84205-5_19
37. Sahana, G. R., Balasubramanian, B., Sebastian, J. K., Pappuswamy, M., Liu, W.-C., Meyyazhagan, A., Kamyab, H., Chelliapan, S., & Joseph, B. V. (2024). A review on ethanol tolerance mechanisms in yeast: Current knowledge in biotechnological applications and future directions. Process Biochemistry, 138, 1–13. https://doi.org/10.1016/j.procbio.2023.12.024
38. Sewsynker-Sukai, Y., & Gueguim Kana, E. B. (2022). Optimization of bioethanol production from lignocellulosic biomass using response surface methodology: A review. Biofuel Research Journal, 9(2), 1595–1610.
39. Shankar, T. J., Sokhansanj, S., & Bi, X. (2015). Optimization of bioethanol production from lignocellulosic biomass. Bioresource Technology, 189, 1–8.
40. Tadesse, T., Dase, D., Koricha, A. D., & Bacha, K. (2024). Optimization of Bioethanol Production Using Response Surface Methodology for Stress-Tolerant Wild Yeasts Isolated from Natural Forests. International Journal of Energy Research, 2024(1). https://doi.org/10.1155/2024/7086047
41. Thatiyamanee, P., Laopaiboon, P., & Laopaiboon, L. (2024). Optimizing bioethanol production from sweet sorghum stem juice under very high gravity fermentation and temperature stress conditions. Carbon Resources Conversion, 100274. https://doi.org/10.1016/j.crcon.2024.100274
42. Thuy, C. X., Pham, Van T., Nguyen, T. T. N. H., Nguyen, T. T. N., Ton, N. T. A., Tuu, T. T., & Vu, N. D. (2024). Effect of Fermentation Conditions (Dilution Ratio, Medium pH, Total Soluble Solids, and Saccharomyces cerevisiae Yeast Ratio) on the Ability to Ferment Cider from Tamarillo (Solanum betaceum) Fruit. Journal of Food Processing and Preservation, 2024(1), e8841207.
43. Zhang, X., Yang, Q., Yang, M., Guan, C., Wang, Z., Tao, L., & Tian, Y. (2025). Optimization of fermentation process of passion fruit-roselle complex fruit wine with immobilized yeast. China Brewing, 44(4), 213–220. https://doi.org/10.11882/j.issn.0254-5071.2025.04.031
Downloads
Published
Issue
Section
License
Copyright (c) 2026 Auhor(s)
This work is licensed under a Creative Commons Attribution 4.0 International License.
Articles published in the Journal of Advanced Multidisciplinary Studies (JAMS) are licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0), unless otherwise stated. Authors retain copyright of their work and grant JAMS the right of first publication.
