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Shivangi Singh*,
Dr. Richa Khare,
Dr. Manoj Kumar Srivastava,
Dr. Smriti Khare,
- Research Scholar, Amity School of Applied Sciences, Amity University, Uttar Pradesh, India
- Assistant Professor, Amity School of Applied Sciences, Amity University, Uttar Pradesh, India
- Principal Scientist, ICAR -Indian Institute of Sugarcane Research, Lucknow, India
- Assistant Professor, Amity School of Applied Sciences, Amity University, Uttar Pradesh, India
Abstract
In this study, bioethanol production by simultaneous saccharification and fermentation (SSF) process was compared between fed-batch and batch mode using sugarcane bagasse (SCB) as raw 2 material, after undergoing various pre-treatments prior to hydrolysis. The effect of these various parameters (viz. initial pH, fermentation temperature, enzyme addition, yeast strain and solids loading) on high titre ethanol production was investigated. Glucan, xylan, and lignin contents were 41.95, 21.70 and 24.11 per cent respectively in untreated sugarcane bagasse. They were pre-treated with 1.8% w/v NaOH at a solid to liquid ratio of 1:6, which resulted in a glucan content of 61.01%, xylan 26.94% and a reduced lignin of 8.95% showing effective delignification and enrichment in glucan and xylan. At tested conditions, initial pH of 5.6 gave the maximum ethanol yield whereas 37℃ gave a balanced condition of cellulase activity and yeast fermentation. The temperature-tolerant Indian repository strain Kluyveromyces marxianus MTCC 242/NCYC 827 displayed a stronger ability to withstand high temperature but S. cerevisiae MTCC 174/NCIM 3187 displayed better ethanol tolerance which was selected for use in highsolids fed-batch experiments. Supplementation of the full cellulase dosage at the initial phase of the prehydrolysis process accelerated the production of ethanol compared to the multiple supplementation, even though it did not speed up the liquefaction of substrate. A subsequent fed-batch SSF showed that the ethanol productivity peaked at 0.88, 1.04 and 1.15 g/L/h at 19, 26 and 33% solids (w/v), respectively. Soils loading of 33% was used to obtain the highest ethanol obtained which was 75.57 ± 2.14 g/L after 120 hours. The maximum theoretical ethanol yield that was obtained was around 72.77% after 96 hours. The results presented here clearly indicate the potential of the alkaline pre-treatment combined with cellulase addition initially, sequential feeding of the substrates and the use of ethanol-tolerant yeast in the production of high-titre second generation bioethanol from sugarcane bagasse. The process is especially applicable to the major sugar cane growing areas in India where the amount of bagasse generated can be used for generating renewable fuel.
Keywords: Sugarcane bagasse, second-generation bioethanol, alkaline pre-treatment, SSF, fed- batch fermentation, high solids loading, Saccharomyces cerevisiae.
References
1. Sandri JP, Ordeñana J, Milessi TS, Zangirolami TC, Mussatto SI. Solid feeding and co- culture strategies for an efficient enzymatic hydrolysis and ethanol production from sugarcane bagasse. Environmental Technology & Innovation. 2023 May 1;30:103082.
2. Canilha L, Chandel AK, Suzane dos Santos Milessi T, Antunes FA, Luiz da Costa Freitas W, das Graças Almeida Felipe M, Da Silva SS. Bioconversion of sugarcane biomass into ethanol: an overview about composition, pretreatment methods, detoxification of hydrolysates, enzymatic saccharification, and ethanol fermentation. BioMed Research International. 2012;2012(1):989572.
3. Gao Y, Xu J, Yuan Z, Jiang J, Zhang Z, Li C. Ethanol production from sugarcane bagasse by fed‐batch simultaneous saccharification and fermentation at high solids loading. Energy Science & Engineering. 2018 Dec;6(6):810-8.
4. Hawaz E, Tafesse M, Tesfaye A, Kiros S, Beyene D, Kebede G, Boekhout T, Groenwald M, Theelen B, Degefe A, Degu S. Bioethanol production from sugarcane molasses by co- fermentation of Saccharomyces cerevisiae isolate TA2 and Wickerhamomyces anomalus isolate HCJ2F-19. Annals of Microbiology. 2024 Apr 3;74(1):13.
5. Saini JK, Saini R, Tewari L. Lignocellulosic agriculture wastes as biomass feedstocks for second-generation bioethanol production: concepts and recent developments. 3 Biotech. 2015 Aug;5(4):337-53.
6. Menegol D, Fontana RC, Dillon AJ, Camassola M. Second-generation ethanol production from elephant grass at high total solids. Bioresource Technology. 2016 Jul 1;211:280-90.
7. Bhutto AW, Harijan K, Qureshi K, Bazmi AA, Bahadori A. Perspectives for the production of ethanol from lignocellulosic feedstock–A case study. Journal of Cleaner Production. 2015 May 15;95:184-93.
8. Modenbach AA, Nokes SE. Enzymatic hydrolysis of biomass at high-solids loadings–a review. Biomass and bioenergy. 2013 Sep 1;56:526-44.
9. Qureshi AS, Zhang J, Bao J. High ethanol fermentation performance of the dry dilute acid pretreated corn stover by an evolutionarily adapted Saccharomyces cerevisiae strain. Bioresource technology. 2015 Aug 1;189:399-404.
10. Ye G, Zeng D, Zhang S, Fan M, Zhang H, Xie J. Ethanol production from mixtures of sugarcane bagasse and Dioscorea composita extracted residue with high solid loading. Bioresource technology. 2018 Jun 1;257:23-9. 35
11. Limayem A, Ricke SC. Lignocellulosic biomass for bioethanol production: current perspectives, potential issues and future prospects. Progress in energy and combustion science. 2012 Aug 1;38(4):449-67.
12. Gao Y, Xu J, Yuan Z, Zhang Y, Liang C, Liu Y. Ethanol production from high solids loading of alkali-pretreated sugarcane bagasse with an SSF process. BioResources. 2014 May 1;9(2):3466-79.
13. Suryawati L, Wilkins MR, Bellmer DD, Huhnke RL, Maness NO, Banat IM. Simultaneous saccharification and fermentation of Kanlow switchgrass pretreated by hydrothermolysis using Kluyveromyces marxianus IMB4. Biotechnology and bioengineering. 2008 Dec 1;101(5):894-902.
14. Carrasco J. Selection of thermotolerant yeasts for simultaneous saccharification and fermentation (SSF) of cellulose to ethanol. Applied Biochemistry and Biotechnology. 1991 Jan 1.
15. Liu Y, Xu J, Zhang Y, Yuan Z, He M, Liang C, Zhuang X, Xie J. Sequential bioethanol and biogas production from sugarcane bagasse based on high solids fed-batch SSF. Energy. 2015 Oct 1;90:1199-205.
16. Koppram R, Olsson L. Combined substrate, enzyme and yeast feed in simultaneous saccharification and fermentation allow bioethanol production from pretreated spruce biomass at high solids loadings. Biotechnology for biofuels. 2014 Apr 8;7(1):54.
17. Zhang Y, Xu JL, Yuan ZH, Liu YY, He MC. Comparison of different pretreatment methods on sugarcane bagasse and fractal-like kinetics of enzymatic hydrolysis. Adv New Renew Eng. 2013;1(2):166-9.
18. Zhang W, Lin Y, Zhang Q, Wang X, Wu D, Kong H. Optimisation of simultaneous saccharification and fermentation of wheat straw for ethanol production. Fuel. 2013 Oct 1;112:331-7.
19. Ghose TK. Measurement of cellulase activities. Pure and applied Chemistry. 1987 Jan 1;59(2):257-68.
20. Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, Crocker DL. Determination of structural carbohydrates and lignin in biomass. Lab. Anal. Proced. 2008 Apr 25;1617(1):1- 6.
21. Varga E, Klinke HB, Réczey K, Thomsen AB. High solid simultaneous saccharification and fermentation of wet oxidized corn stover to ethanol. Biotechnology and bioengineering. 2004 Dec 5;88(5):567-74.
22. Ding JC, Xu GC, Han RZ, Ni Y. Biobutanol production from corn stover hydrolysate pretreated with recycled ionic liquid by Clostridium saccharobutylicum DSM 13864. Bioresource technology. 2016 Jan 1;199:228-34.
23. Alvira P, Tomás-Pejó E, Ballesteros M, Negro MJ. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresource technology. 2010 Jul 1;101(13):4851-61.
24. Wang W, Wang Q, Tan X, Qi W, Yu Q, Zhou G, Zhuang X, Yuan Z. High conversion of sugarcane bagasse into monosaccharides based on sodium hydroxide pretreatment at low water consumption and wastewater generation. Bioresource Technology. 2016 Oct 1;218:1230-6.
25. Roche CM, Dibble CJ, Knutsen JS, Stickel JJ, Liberatore MW. Particle concentration and yield stress of biomass slurries during enzymatic hydrolysis at high‐solids loadings. Biotechnology and bioengineering. 2009 Oct 1;104(2):290-300.
26. Lin YS, Lee WC, Duan KJ, Lin YH. Ethanol production by simultaneous saccharification and fermentation in rotary drum reactor using thermotolerant Kluveromyces marxianus. Applied energy. 2013 May 1;105:389-94. 36
27. Mohapatra S, Mishra C, Behera SS, Thatoi H. Application of pretreatment, fermentation and molecular techniques for enhancing bioethanol production from grass biomass–A review. Renewable and Sustainable Energy Reviews. 2017 Oct 1;78:1007-32.
28. Gao Y, Xu J, Yuan Z, Zhang Y, Liu Y, Liang C. Optimization of fed-batch enzymatic hydrolysis from alkali-pretreated sugarcane bagasse for high-concentration sugar production. Bioresource technology. 2014 Sep 1;167:41-5.
29. Park I, Kim I, Kang K, Sohn H, Rhee I, Jin I, Jang H. Cellulose ethanol production from waste newsprint by simultaneous saccharification and fermentation using Saccharomyces cerevisiae KNU5377. Process Biochemistry. 2010 Apr 1;45(4):487-92.
30. Park I, Kim I, Kang K, Sohn H, Rhee I, Jin I, Jang H. Cellulose ethanol production from waste newsprint by simultaneous saccharification and fermentation using Saccharomyces cerevisiae KNU5377. Process Biochemistry. 2010 Apr 1;45(4):487-92.
31. Li H, Kim NJ, Jiang M, Kang JW, Chang HN. Simultaneous saccharification and fermentation of lignocellulosic residues pretreated with phosphoric acid–acetone for bioethanol production. Bioresource technology. 2009 Jul 1;100(13):3245-51.
32. Zhang T, Zhu MJ. Enhanced bioethanol production by fed-batch simultaneous saccharification and co-fermentation at high solid loading of Fenton reaction and sodium hydroxide sequentially pretreated sugarcane bagasse. Bioresource Technology. 2017 Apr 1;229:204-10.
33. Gao Y, Xu J, Zhang Y, Yu Q, Yuan Z, Liu Y. Effects of different pretreatment methods on chemical composition of sugarcane bagasse and enzymatic hydrolysis. Bioresource technology. 2013 Sep 1;144:396-400.
34. Aditiya HB, Mahlia TM, Chong WT, Nur H, Sebayang AH. Second generation bioethanol production: A critical review. Renewable and sustainable energy reviews. 2016 Dec 1;66:631-53.
35. Bellido C, González-Benito G, Coca M, Lucas S, García-Cubero MT. Influence of aeration on bioethanol production from ozonized wheat straw hydrolysates using Pichia stipitis. Bioresource technology. 2013 Apr 1;133:51-8.
36. Carrillo-Nieves D, Alanís MJ, de la Cruz Quiroz R, Ruiz HA, Iqbal HM, Parra-Saldívar R. Current status and future trends of bioethanol production from agro-industrial wastes in Mexico. Renewable and Sustainable Energy Reviews. 2019 Mar 1;102:63-74.
37. Choudhary J, Singh S, Nain L. Bioprospecting thermotolerant ethanologenic yeasts for simultaneous saccharification and fermentation from diverse environments. Journal of bioscience and bioengineering. 2017 Mar 1;123(3):342-6.
38. Cronjé Y, Farzad S, Mandegari M, Görgens JF. A critical review of multiple alternative pathways for the production of a high-value bioproduct from sugarcane mill byproducts: the case of adipic acid. Biofuel Research Journal. 2023 Sep 1;10(3):1933-47.
39. Kasavi C, Finore I, Lama L, Nicolaus B, Oliver SG, Oner ET, Kirdar B. Evaluation of industrial Saccharomyces cerevisiae strains for ethanol production from biomass. Biomass and Bioenergy. 2012 Oct 1;45:230-8.
40. Ruyters S, Mukherjee V, Verstrepen KJ, Thevelein JM, Willems KA, Lievens B. Assessing the potential of wild yeasts for bioethanol production. Journal of Industrial Microbiology and Biotechnology. 2015 Jan 1;42(1):39-48.
41. Song Y, Cho EJ, Park CS, Oh CH, Park BJ, Bae HJ. A strategy for sequential fermentation by Saccharomyces cerevisiae and Pichia stipitis in bioethanol production from hardwoods. Renewable Energy. 2019 Aug 1;139:1281-9.
42. Zeng Y, Li L, Wang Y, Dong Y, Wang M, Wang W, Gao L, Suo Y. Efficient ethanol production from non-detoxified sugarcane molasses by an engineered Wickerhamomyces anomalus. Renewable Energy. 2026 Mar 1;259:125125. 37
43. Vaz LP, Sears HB, Miranda EA, Holwerda EK, Lynd LR. Solubilization of sugarcane bagasse by mono and cocultures of thermophilic anaerobes with and without cotreatment. Bioresource Technology. 2024 Aug 1;406:130982.
44. Batalha LA, Han Q, Jameel H, Chang HM, Colodette JL, Gomes FJ. Production of fermentable sugars from sugarcane bagasse by enzymatic hydrolysis after autohydrolysis and mechanical refining. Bioresource technology. 2015 Mar 1;180:97-105.
45. Da Silva AS, Inoue H, Endo T, Yano S, Bon EP. Milling pretreatment of sugarcane bagasse and straw for enzymatic hydrolysis and ethanol fermentation. Bioresource technology. 2010 Oct 1;101(19):7402-9.
46. Wang Z, Dien BS, Rausch KD, Tumbleson ME, Singh V. Fermentation of undetoxified sugarcane bagasse hydrolyzates using a two stage hydrothermal and mechanical refining pretreatment. Bioresource technology. 2018 Aug 1;261:313-21.
Research and Reviews : A Journal of Biotechnology
| Volume | 16 | |
| 02 | ||
| Received | 18/10/2026 | |
| Accepted | 29/10/2026 | |
| Published | 09/11/2026 | |
| Publication Time | 22 Days |