DOI: https://doi.org/10.9767/bcrec.20721

Optimization and Kinetic Studies of Enzymatic Saccharification of Double-Stage Ozonolysis Pretreated Oil Palm Empty Fruit Bunch for Enhanced Sugar Production

1Chemical Reaction Engineering Group (CREG), Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia (UTM), 81310 Johor Bahru, Johor, Malaysia

2Research Center for Electronics, National Research and Innovation Agency (BRIN), KST Sama’un Samadikun, Jl. Sangkuriang, Bandung, 40135, Indonesia

3Centre of Lipid Engineering Applied Research (CLEAR), Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia (UTM), 81310 Johor Bahru, Johor, Malaysia

4 Department of Bioprocess and Polymer Engineering, Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia (UTM), 81310 Johor Bahru, Malaysia

5 UTM-MPRC Institute for Oil & Gas, Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia (UTM), 81310 Johor Bahru, Johor, Malaysia

6 Energy Management Group, Faculty of Chemical and Energy Engineering, Universiti Teknologi Malaysia (UTM), 81310 Johor Bahru, Johor, Malaysia

7 Université Clermont Auvergne, CNRS, Clermont auvergne INP, Institut Pascal, 63000 Clermont-Ferrand, France

View all affiliations
Received: 30 Apr 2026; Revised: 25 Jun 2026; Accepted: 26 Jun 2026; Available online: 4 Jul 2026; Published: 30 Oct 2026.
Editor(s): Istadi Istadi
Open Access Copyright (c) 2026 by Authors, Published by BCREC Publishing Group
Creative Commons License This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.
Fulltext View|Download

Citation Format:
Cover Image
Abstract

Oil Palm Empty Fruit Bunch (OPEFB), an abundant lignocellulosic biomass in Malaysia, is a promising feedstock for producing Total Reducing Sugar (TRS), although its recalcitrant structure limits enzymatic conversion. This study proposes a novel double-stage ozonolysis pretreatment integrated with intermediate alkaline swelling to enhance cellulose accessibility and saccharification efficiency. Structural modifications were confirmed through compositional analysis, TGA, XRD, FTIR, and SEM. Enzymatic saccharification (ES) was optimized using Response Surface Methodology (RSM) based on a Face-Centered Central Composite Design (FCCCD), evaluating reaction time, biomass loading, and temperature. Analysis of Variance (ANOVA) indicated a significant model (R2 = 0.88), with optimal conditions of 44 h reaction time, 1.8 % w/v biomass loading, and 50 °C temperature, achieving a maximum TRS yield of 42.75%. The double-stage ozonolysis outperformed single-stage and alkaline pretreatments, yielding the highest cellulose enrichment (up to 79 wt%) and improved digestibility. Kinetic analysis revealed a substantial reduction in the Michaelis-Menten constant ( ) from 175.713 to 9.010 mg/mL, indicating enhanced enzyme–substrate affinity. These findings demonstrate a robust and efficient strategy for improving biomass-to-sugar conversion. Copyright © 2026 by Authors, Published by BCREC Publishing Group. This is an open access article under the CC BY-SA License (https://creativecommons.org/licenses/by-sa/4.0).

Keywords: Oil palm empty fruit bunch; Biomass pretreatment; Ozonolysis; Optimization; Enzymatic saccharification; Total reducing sugar
Funding: (FRGS) I FRGS/1/2022/TK05/UTM/02/11; (MyTIGER) 2024 I Vot Number 1U072; (UTM) Matching grant I Vot Number 05M00; MTSF I Vot Number 4J754).

Article Metrics:

Article Info
Section: 3rd International Symposium of Reaction Engineering, Catalysis and Sustainable Energy 2025 (RECASE 2025)
Language : EN
Recent articles
ZIF-67 Incorporated Sodium Alginate/Polyvinyl Alcohol Composite Beads for Efficient Adsorptive Removal of Reactive Blue 19 from Aqueous Solution Improvements in Photocatalytic Activity in Visible Light Destruction of Chlorpyrifos in a Batch Reactor Applying the Z-Scheme g-C3N4/Fe3O4/CuWO4/CuO Heterojunction, Including Fabrication, Characterization, and Recycling Efficient Deoxygenation of Palm Oil to Green Diesel Using a Metal Oxide Catalysts Supported on ZrO2-Enhanced Graphene Oxide Influence of Ni/CeO2 on Pyrolytic Conversion of FOBS to Hydrogen Formation Tailoring the Structural Evolution of Co-Supported Fibrous ZSM-5 via Hydrothermal Aging for Syngas Production in Ethanol Dry Reforming Optimization and Kinetic Studies of Enzymatic Saccharification of Double-Stage Ozonolysis Pretreated Oil Palm Empty Fruit Bunch for Enhanced Sugar Production More recent articles
  1. Bhattacharyya, S., Chakraborty, S., Dattaa, S., Drioli, E., Bhattacharjee, C. (2013). Production of total reducing sugar ( TRS ) from acid hydrolysed potato peels by sonication and its optimization. Environmental Technology, 34(9). DOI: 10.1080/09593330.2012.733965
  2. Mahardika, M., Zakiyah, A., Ulfa, S.M., Ahmad, R., Hassan, M.Z., Amelia, D., Knight, V.F., Nor, M. (2024). Recent Developments in Oil Palm Empty Fruit Bunch (OPEFB) Fiber Composite Recent Developments in Oil Palm Empty Fruit Bunch (OPEFB) Fiber. Journal of Natural Fibers, 21(1) DOI: 10.1080/15440478.2024.2309915
  3. Mankar, A.R., Pandey, A., Modak, A., Pant, K.K. (2021). Pretreatment of lignocellulosic biomass: A review on recent advances. Bioresource Technology, 334, 125235. DOI: 10.1016/j.biortech.2021.125235
  4. Malik, K., Sharma, P., Yang, Y., Zhang, P., Zhang, L., Xing, X., Yue, J., Song, Z., Nan, L., Yujun, S., El-Dalatony, M.M., Salama, E.S., Li, X. (2022). Lignocellulosic biomass for bioethanol: Insight into the advanced pretreatment and fermentation approaches. Industrial Crops and Products, 188, 115569. DOI: 10.1016/j.indcrop.2022.115569
  5. Harahap, B.M., Dewantoro, A.I., Maulid, M.R., Mardawati, E., Yarlina, V.P. (2020). Autoclave-assisted weak acid pretreatment of oil palm empty fruits bunches for fermentable sugar production. IOP Conference Series: Earth and Environmental Science, 443(1), 1–13. DOI: 10.1088/1755-1315/443/1/012080
  6. Woo, W.X., Tan, J.W., Tan, J.P., Indera Luthfi, A.A., Abdul, P.M., Abdul Manaf, S.F., Yeap, S.K. (2022). An Insight into Enzymatic Immobilization Techniques on the Saccharification of Lignocellulosic Biomass. Industrial and Engineering Chemistry Research, 61(30), 10603–10615. DOI: 10.1021/acs.iecr.2c01154
  7. Diyanilla, R., Hamidon, T.S., Suryanegara, L., Hussin, M.H. (2020). Overview of Pretreatment Methods Employed on Oil Palm Biomass in Producing Value-added Products : BioResources, 15(4), 9935–9997. DOI: 10.15376/biores.15.4.9935-9997
  8. Suhartini, S., Ainur, N., Hidayat, N., Idrus, S., Hoon, Y., Melville, L. (2023). Comparison of acid and alkaline pre-treatment on methane production from empty palm oil fruit bunches ( OPEFB ): Effect on characteristics , digester performance , and correlation of kinetic parameters. Renewable Energy, 215(July), 119009. DOI: 10.1016/j.renene.2023.119009
  9. Obeng, A.K., Premjet, D., Premjet, S. (2019). Combining Autoclaving with Mild Alkaline Solution as a Pretreatment Technique to Enhance Glucose Recovery from the Invasive Weed Chloris barbata. Biomolecules, 9(4), 120. DOI: 10.3390/biom9040120
  10. . Zhang, H., Han, L., Dong, H. (2021). An insight to pretreatment, enzyme adsorption and enzymatic hydrolysis of lignocellulosic biomass: Experimental and modeling studies. Renewable and Sustainable Energy Reviews, 140(12), 110758. DOI: 10.1016/j.rser.2021.110758
  11. . Orduña, J., Andrés, J., Vargas, M., Micali, O., Metzker, G., Gomes, E., Boscolo, M. (2019). Soaking and ozonolysis pretreatment of sugarcane straw for the production of fermentable sugars. Industrial Crops & Products, (May), 111959. DOI: 10.1016/j.indcrop.2019.111959
  12. . Shamjuddin, A., Ab Rasid, N.S., Michele Raissa, M.M., Abu Zarin, M.A., Wan Omar, W.N.N., Syahrom, A., Mohd Szali Januddi, M.A.F., Saidina Amin, N.A. (2021). Kinetic and dynamic analysis of ozonolysis pre-treatment of empty fruit bunch in a well-mixed reactor for sugar production. Energy Conversion and Management, 244(9), 114526. DOI: 10.1016/J.ENCONMAN.2021.114526
  13. . Ab Rasid, N.S., Shamjuddin, A., Abdul Rahman, A.Z., Amin, N.A.S. (2021). Recent advances in green pre-treatment methods of lignocellulosic biomass for enhanced biofuel production. Journal of Cleaner Production, 321, 129038. DOI: 10.1016/j.jclepro.2021.129038
  14. . Travaini, R., Martín-Juárez, J., Lorenzo-Hernando, A., Bolado-Rodríguez, S. (2015). Ozonolysis: An advantageous pretreatment for lignocellulosic biomass revisited. Bioresource Technology, 199, 2–12. DOI: 10.1016/J.BIORTECH.2015.08.143
  15. . Ethaib, S., Omar, R., Mustapa Kamal, S.M., Awang Biak, D.R., L. Zubaidi, S. (2020). Toward Sustainable Processes of Pretreatment Technologies of Lignocellulosic Biomass for Enzymatic Production of Biofuels and Chemicals: A Review. BioResources, 15(4), 10063–10088. DOI: 10.15376/biores.15.4.ethaib
  16. . Abdur Rasid, N.S., Shamjuddin, A., Saidina Amin, N.A. (2021). Chemical and structural changes of ozonated empty fruit bunch (EFB) in a ribbon-mixer reactor. Bulletin of Chemical Reaction Engineering & Catalysis, 16(2), 383–395. DOI: 10.9767/bcrec.16.2.10506.383-395
  17. . Arnandan, P.T., Wan Omar, W., Wan, N., Husna, Z., Sufri, M., Muhaimin, A., Akmal, D., Ahmad, J., Mba, M., Raissa, M., Shamjuddin, A., Chang, K., Aishah, N., Amin, S. (2025). Novel sequential ozonolysis-hydrolysis treatments for microcrystalline cellulose synthesis from oil palm empty fruit bunch. Biomass and Bioenergy, 199(January), 107902. DOI: 10.1016/j.biombioe.2025.107902
  18. . Mba Makam, R.M., Wan Omar, W.N.N., Thevi Arnandan, P., Abd Muhaimin, M.S., Jihat @ Ahmad, D.A., Shamjuddin, A., Hassan, Z.H., Chang, K., Saidina Amin, N.A. (2025). Sustainable carboxymethyl cellulose from ozonated empty fruit bunch for application as food coating additive. International Journal of Biological Macromolecules, 319(P4), 145660. DOI: 10.1016/j.ijbiomac.2025.145660
  19. . Mohd, N.H., Othaman, R., Mackeen, M.M., Maskat, M.Y. (2021). Effect of pretreatment and enzymatic hydrolysis for oligosaccharide production from oil palm mesocarp Fibre. Sains Malaysiana, 50(6), 1673–1683. DOI: 10.17576/jsm-2021-5006-14
  20. . Tan, H.T., Lee, K.T. (2012). Understanding the impact of ionic liquid pretreatment on biomass and enzymatic hydrolysis. Chemical Engineering Journal, 183, 448–458. DOI: 10.1016/j.cej.2011.12.086
  21. . Rizal, N.F.A.A., Ibrahim, M.F., Zakaria, M.R., Bahrin, E.K., Abd-Aziz, S., Hassan, M.A. (2018). Combination of superheated steam with laccase pretreatment together with size reduction to enhance enzymatic hydrolysis of oil palm biomass. Molecules, 23(4), 811. DOI: 10.3390/molecules23040811
  22. . Ahmad Rizal, N.F.A., Abd-aziz, S., Yee, P.L., Hassan, M.A. (2018). Pre-treatment of Oil Palm Biomass for Fermentable Sugars Production. Molecules, 23, 1–14. DOI: 10.3390/molecules23061381
  23. . Sari, F.P., Falah, F., Anita, S.H., Panji, K., Permana, R., Laksana, B., Fatriasari, W., Hermiati, E. (2021). Pretreatment of Oil Palm Empty Fruit Bunch ( OPEFB ) at Bench-Scale High Temperature-Pressure Steam Reactor for Enhancement of Enzymatic Saccharification. Int Journal of Renewable Energy Development, 10(2), 157–169. DOI: 10.14710/ijred.2021.32343
  24. . Yadav, V., Anand, A., Goyal, A. (2026). Enhanced enzymatic saccharification by statistically optimizing the delignification of bamboo biomass. Biomass and Bioenergy, 206(November 2025), 108646. DOI: 10.1016/j.biombioe.2025.108646
  25. . Brahma, D., Brahma, S., Kumar, Y. (2026). Face centered central composite design-Response surface methodology (FCC-RSM) a multivariate optimization of crystal violet dye adsorption by Cajanus cajan residues with thermodynamic, kinetic and mechanistic insights. Next Materials, 10(October 2025) DOI: 10.1016/j.nxmate.2025.101476
  26. . Hassan, Z.H., Saidina Amin, N.A., Wan Omar, W.N.N., Abd Muhaimin, M.S., Michele Raissa, M.M., Arnandan, P.T., Shamjuddin, A. (2026). Parametric Studies on Delignification using Ozonolysis Pretreatment of Oil Palm Empty Fruit Bunch for Cellulose Nano-fiber Production. Journal of Advanced Research in Micro and Nano Engineering, 42(1), 177–204. DOI: 10.37934/armne.42.1.177204
  27. . Seok, J., Lee, Y.Y., Hyun, T. (2016). Bioresource Technology A review on alkaline pretreatment technology for bioconversion of lignocellulosic biomass. Bioresource Technology, 199, 42–48. DOI: 10.1016/j.biortech.2015.08.085
  28. . Akindoyo, J.O., Ghazali, S.B., Beg, M.D.H. (2015). Structural and Thermal Properties of Ultrasound Treated Oil Palm Empty Fruit Bunch (Opefb) Fiber. Tenth TheHER International Conference, (February), 60–64
  29. . Mitbumrung, W., Rungraung, N., Muangpracha, N., Akanitkul, P., Winuprasith, T. (2022). Approaches for Extracting Nanofibrillated Cellulose from Oat Bran and Its Emulsion Capacity and Stability. Polymers 2022, Vol 14, Page 327, 14(2), 327. DOI: 10.3390/POLYM14020327
  30. . Liyana, N., Zailuddin, I. (2020). Morphology , mechanical properties , and biodegradability of all-cellulose composite films from oil palm empty fruit bunch. (July), 1–11. DOI: 10.1002/pls2.10008
  31. . Asmoro, N.W., Hidayat, C., Ariyanto, T., Millati, R. (2023). Cellulose Isolation From Oil Palm Empty Fruit Bunch Using Different Pretreatment Processes. Journal of Applied Science and Engineering, 26(11), 1513–1520. DOI: 10.6180/jase.202311_26(11).0001
  32. . Hemashini, T., Lee, C.K., Goh, C.F., Tye, Y.Y., Ismayati, M., H’ng, Y.Y., Leh, C.P. (2024). Elucidating the Correlation of Lignocellulosic Compositions and Physicochemical Alterations in Oil Palm (Elaeis guineensis) Biomass on Enzymatic Saccharification Yield. Bioenergy Research, 17, 1612–1630. DOI: 10.1007/s12155-024-10762-3
  33. . Noor Arbaain, E.N., Kamal Bahrin, E., Ibrahim, M.F., Ando, Y., Abd-Aziz, S. (2019). Biological Pretreatment of Oil Palm Empty Fruit Bunch by Schizophyllum commune ENN1 without Washing and Nutrient Addition. Processes, 7(402), 2–8. DOI: 10.3390/pr7070402
  34. . Loh, S.K., Kassim, M.A., Bukhari, N.A. (2018). Optimisation of process conditions for ethanol production from enzymatically saccharified empty fruit bunch using response surface methodology (RSM). Journal of Oil Palm Research, 30(December), 642–654. DOI: 10.21894/jopr.2018.0045
  35. . Heris, S., Fitria, A., Nurfajrin, N., Fahriya, S., Sari, P., Risanto, L., Fatriasari, W. (2020). Optimization of Microwave-Assisted Oxalic Acid Pretreatment of Oil Palm Empty Fruit Bunch for Production of Fermentable Sugars. Waste and Biomass Valorization, 11(6), 2673–2687. DOI: 10.1007/s12649-018-00566-w
  36. . Qin, C.K., Majid, N.Z., Shamjuddin, A., Amin, N.A.S., Yussuf, M.A.M., Hassan, Z.H., Hamid, N.H.A., Zainan, N.H., Jaafar, N.R. (2025). Parametric optimization on acid hydrolysis of ozone pre-Treated oil palm empty fruit bunch for enhanced glucose production. AIP Conference Proceedings, 3225(1), 1–6. DOI: 10.1063/5.0267373
  37. . Baksi, S., Sarkar, U., Villa, R., Basu, D., Sengupta, D. (2023). Conversion of biomass to biofuels through sugar platform : A review of enzymatic hydrolysis highlighting the trade-off between product and substrate inhibitions. Sustainable Energy Technologies and Assessments, 55(September 2022) DOI: 10.1016/j.seta.2022.102963
  38. . Shiva, Rodríguez-Jasso, R.M., López‑Sandin, I., Aguilar, M.A., López-Badillo, C.M., Ruiz, H.A. (2023). Intensification of enzymatic saccharification at high solid loading of pretreated agave bagasse at bioreactor scale. Journal of Environmental Chemical Engineering, 11(1), 109257. DOI: 10.1016/j.jece.2022.109257
  39. . Shah, S.R., Ishmael, U.C., Palliah, J.V., Asras, M.F.F., Ahmad, S.S.B.N.W. (2016). Optimization of the enzymatic saccharification process of empty fruit bunch pretreated with laccase enzyme. BioResources, 11(2), 5138–5154. DOI: 10.15376/biores.11.2.5138-5154
  40. . Yemis, O., Mazza, G. (2012). Optimization of furfural and 5-hydroxymethylfurfural production from wheat straw by a microwave-assisted process. Bioresource Technology. 109, 215–223. DOI: 10.1016/j.biortech.2012.01.031
  41. . Hu, Y., Priya, A., Chen, C., Liang, C., Wang, W., Wang, Q., Sze, C., Lin, K., Qi, W. (2023). Recent advances in substrate-enzyme interactions facilitating efficient biodegradation of lignocellulosic biomass : A review. International Biodeterioration & Biodegradation, 180, 105594. DOI: 10.1016/j.ibiod.2023.105594
  42. . Abd Majid, N.Z., Shamjuddin, A., Asli, U.A., Mohd Fauzi, N.A.F., Hussain, S.N., Masri, A.N., Zainan, N.H., Jaafar, N.R. (2025). Optimization of Temperature, Cellulase Concentration and pH value of Enzymatic Saccharification for Producing Sugar from Ozone Pre-treated Oil Palm Empty Fruit Bunch. Bioprocessing and Biomass Technology, 4(1), 33–37. DOI: 10.11113/bioprocessing.v4n1.73
  43. . Wang, Q., Chang, L., Wang, W., Hu, Y., Yue, J. (2023). Simultaneous saccharification of hemicellulose and cellulose of corncob in a one-pot system using catalysis of carbon based solid acid from lignosulfonate. Royal Society of Chemistry. 13, 28542–28549. DOI: 10.1039/d3ra05283d
  44. . Mutrakulcharoen, P., Pornwongthong, P. (2020). Inhibitory Effect of Inorganic Salts Residuals on Cellulase Kinetics in Biofuel Production from Lignocellulose Biomass. 2020 International Conference and Utility Exhibition on Energy, Environment and Climate Change (ICUE). DOI: 10.1109/ICUE49301.2020.9307055
  45. . Singh, S., Agarwal, M., Bhatt, A., Goyal, A., Moholkar, V.S. (2015). Ultrasound enhanced enzymatic hydrolysis of Parthenium hysterophorus : A mechanistic investigation. Bioresource Technology, 192, 636–645. DOI: 10.1016/j.biortech.2015.06.031
  46. . Brown, J., Lindstrom, J.K., Ghosh, A., Rollag, S.A., Brown, R.C. (2024). Production of sugars from lignocellulosic biomass via biochemical and thermochemical routes. Frontiers in Energy Research, 12(February), 1–34. DOI: 10.3389/fenrg.2024.1347373
  47. . Ioelovich, M. (2015). Study of kinetics of enzymatic hydrolysis of cellulose materials. ChemXpress, 8(4), 231–239. DOI: 10.13140/RG.2.1.3185.6806
  48. . Wang, K., Shen, Z., Wang, X., Li, Z., Cheng, S. (2024). Advances in enhancing the enzymatic saccharification process of lignocellulosic biomasses for bioethanol productionsti. Biomass and Bioenergy, 191, 107450. DOI: 10.1016/j.biombioe.2024.107450
  49. . Angeltveit, C.F., Várnai, A., Eijsink, V.G.H., Horn, S.J. (2024). Enhancing enzymatic saccharification yields of cellulose at high solid loadings by combining different LPMO activities. Biotechnology for Biofuels and Bioproducts, 17(1), 1–14. DOI: 10.1186/s13068-024-02485-6

Last update:

No citation recorded.

Last update:

No citation recorded.