Waste and Biomass Valorization

, Volume 10, Issue 11, pp 3279–3293 | Cite as

Fed-Batch Saccharification as a Strategy towards Reducing Enzyme Dosage and Enhancing Fermentable Sugar Yield from Pretreated Lignocellulo-Starch Biomass

  • M. G. Mithra
  • M. S. Sajeev
  • G. PadmajaEmail author
Original Paper



Lignocellulo-starch biomass (LCSB) comprising root and vegetable processing residues contain starch also along with cellulose and hemicellulose and hence require whole slurry saccharification using an enzyme cocktail containing starch hydrolysing enzyme as well along with cellulase and xylanase. The cost-effective production of ethanol necessitates high fermentable sugar yield (> 80 g/l) in the hydrolysate which is possible through fed-batch saccharification as it overcomes issues such as low mass transfer and high viscosity of slurry encountered on high solids loading. To our knowledge no information is available on the fed-batch saccharification of LCSBs and hence the objective of the study was to investigate the possibility of enhancing the sugar yield at low enzyme dosage through fed-batch saccharification approach.


Fed-batch saccharification of pretreated LCSBs [steam (ST), dilute sulphuric acid (DSA) and microwave-assisted DSA (MW-DSA)] in enhancing the sugar yield was investigated using a triple enzyme cocktail at two cumulative loading densities (15 g/100 ml and 20 g/100 ml) and compared with the respective batch system.


The hydrolysis yield from ST fed-batch system was very high (84–95%) for the residues followed by 83–90% yield from MW-DSA system at 15 g/100 ml cumulative substrate loading. Glucose and xylose were uniformly present in all the hydrolysates with higher levels of glucose in the steam pretreated fed-batch system SFB1 (15 g/100 ml). High phenolic retention in the hydrolysates did not affect saccharification as detoxification chemicals were supplemented.


Fed-batch saccharification enhanced the sugar yield from pretretated LCSBs and based on the hydrolysis yield, 15 g/100 ml cumulative substrate loading was better than 20 g/100 ml loading and steam pretreatment (45 min) emerged as the best. Pulsed addition of substrate with only one-time enzyme feeding at the start resulted in enzyme saving during the fed-batch saccharification.

Graphical Abstract


Lignocellulo-starch biomass Pretreatment Fed-batch saccharification Enzyme dosage Sugars Phenolics Detoxification 



The authors acknowledge the financial support received for the study from the Kerala State Council for Science, Technology & Environment (KSCSTE) through Grant No. 853/2015/KSCSTE and to the Director, ICAR-CTCRI for the facilities provided. The help extended by Dr. J. Sreekumar, Principal Scientist (Agricultural Statistics) for the statistical analysis is also thankfully acknowledged. Authors are also thankful to Dr. A. N. Jyothi, Principal Scientist and Mr. V. R. Vishnu, Senior Research Fellow, ICAR- CTCRI for the help extended for HPLC studies.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

12649_2018_373_MOESM1_ESM.docx (15 kb)
Supplementary material 1 (DOCX 14 KB)


  1. 1.
    Himmel, M.E., Ding, S.Y., Johnson, D.K., Adney, W.S., Nimlos, M.R., Brady, J.W., Foust, T.D.: Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science. 325, 804–807 (2007)CrossRefGoogle Scholar
  2. 2.
    Wyman, C.E.: Biomass ethanol: technical progress, opportunities and commercial challenges. Annu. Rev. Energ. Environ. 24, 189–226 (1999)CrossRefGoogle Scholar
  3. 3.
    Alvira, P., Tomas-Pejo, E., Ballesteros, M., Negro, M.J.: Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour. Technol. 101, 4851–4861 (2010)CrossRefGoogle Scholar
  4. 4.
    Yang, B., Wyman, C.E.: Pretreatment: the key to unlocking low cost cellulosic ethanol. Biofuels Bioprod. Bioref. 2, 26–40 (2008)CrossRefGoogle Scholar
  5. 5.
    Agbogbo, F.K., Coward-Kelly, G., Torry-Smith, M., Wenger, K.: Fermentation of glucose/xylose mixtures using Pichia stipitis. Process Biochem. 41, 2333–2336 (2006)CrossRefGoogle Scholar
  6. 6.
    Zhu, Z., Zhu, M., Wu, Z.: Pretreatment of sugarcane bagasse with NH4OH-H2O2 and ionic liquid for efficient hydrolysis and bioethanol production. Bioresour. Technol. 119, 199–207 (2012). CrossRefGoogle Scholar
  7. 7.
    Zhang, T., Zhu, M.J.: Enhancing enzymolysis and fermentation efficiency of sugarcane bagasse by synergistic pretreatment of Fenton reaction and sodium hydroxide extraction. Bioresour. Technol. 214, 769–777 (2016). CrossRefGoogle Scholar
  8. 8.
    Larsson, S., Quintana-Sáinz, A., Reimann, A., Nilvebrant, N.O., Jönsson, L.J.: Influence of ignocelluloses-derived aromatic compounds on oxygen limited growth and ethanolic fermentation by Saccharomyces cerevisiae. Appl. Biochem. Biotechnol. 84, 617–632 (2000)CrossRefGoogle Scholar
  9. 9.
    Palmqvist, E., Hahn-Hägerdal, B.: Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. Bioresour. Technol. 74, 25–33 (2000)CrossRefGoogle Scholar
  10. 10.
    Olsson, l., Hahn-Hägerdal, B.: Fermentation of lignocellulosic hydrolysates for ethanol production. Enzyme Microb. Technol. 18, 312–331 (1996)CrossRefGoogle Scholar
  11. 11.
    Parajó, J.C., Domínguez, H., Domínguez, J.M.: Biotechnological production of xylitol. Part 3: operation in culture media made from lignocellulose hydrolysates. Bioresour. Technol. 66, 25–40 (1998)CrossRefGoogle Scholar
  12. 12.
    Modig, T., Lidén, G., Taherzadeh, M.J.: Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase. Biochem. J. 363, 769–776 (2002)CrossRefGoogle Scholar
  13. 13.
    Tejirian, A., Xu, F.: Inhibition of enzymatic cellulolysis by phenolic compounds. Enzyme Microb. Technol. 48, 239–247 (2011)CrossRefGoogle Scholar
  14. 14.
    Ximenes, E.A., Kim, Y., Mosier, N.S., Dien, B.S., Ladisch, M.R.: Inhibition of cellulases by phenols. Enzyme Microb. Technol. 46, 170–176 (2010)CrossRefGoogle Scholar
  15. 15.
    Zhu, W., Houtman, C.J., Zhu, J.Y., Gleisner, R., Chen, K.F.: Quantitative predictions of bioconversion of aspen by dilute acid and SPORL pretreatments using a unified combined hydrolysis factor (CHF). Process Biochem. 47, 785–791 (2012)CrossRefGoogle Scholar
  16. 16.
    Qing, Q., Yang, B., Wyman, C.E.: Xylo-oligomers are strong inhibitors of cellulose hydrolysis by enzymes. Bioresour. Technol. 101, 9624–9630 (2010)CrossRefGoogle Scholar
  17. 17.
    Ethaib, S., Omar, R., Kamal, S.M.M., Biak, D.R.A.: Microwave-assisted pretreatment of lignocellulosic biomass: a review. J. Eng. Sci. Technol. 21, 97–109 (2015)Google Scholar
  18. 18.
    Li, H., Qu, Y., Yang, Y., Chang, S., Xu, J.: Microwave irradiation- a green and efficient way to pretreat biomass. Bioresour. Technol. 199, 34–41 (2016) Scholar
  19. 19.
    Cavka, A., Jönsson, L.J.: Detoxification of lignocellulosic hydrolysates using sodium borohydride. Bioresour. Technol. 136, 368–376 (2013)CrossRefGoogle Scholar
  20. 20.
    Eriksson, T., Börjesson, J., Tjerneld, F.: Mechanism of surfactant effect in enzymatic hydrolysis of lignocelluloses. Enzyme Microb. Technol. 31, 353–364 (2002)CrossRefGoogle Scholar
  21. 21.
    Kristensen, J.B., Börjesson, J., Bruun, M.H., Tjerneld, F., Jørgensen, H.: Use of surface active additives in enzymatic hydrolysis of wheat straw lignocelluloses. Enzyme Microb. Technol. 40, 888–895 (2007)CrossRefGoogle Scholar
  22. 22.
    Zhang, X., Qin, W., Paice, M.G., Saddler, J.N.: High consistency enzymatic hydrolysis of hardwood substrates. Bioresour. Technol. 100, 5890–5897 (2009)CrossRefGoogle Scholar
  23. 23.
    Jørgensen, H., Vibe-Pedersen, J., Larsen, J., Felby, C.: Liquefaction of lignocelluloses at high-solids concentrations. Biotechnol. Bioeng. 96, 862–870 (2007)CrossRefGoogle Scholar
  24. 24.
    Modenbach, A.A., Nokes, S.E.: The use of high-solids loadings in biomass simultaneous saccharification and fermentation of pretreated wheat straw to ethanol. Appl. Biochem. Biotechnol. 33, 67–81 (2012)Google Scholar
  25. 25.
    Zhang, Y., Lin, Y., Yuan, Z.H., Qi, W., Zhuang, X.S., He, M.C.: High solids and low enzyme loading based saccharification of agricultural biomass. BioResources. 7, 345–353 (2012)Google Scholar
  26. 26.
    Hodge, D.B., Karim, M.N., Schell, D.J., McMillan, J.D.: Soluble and insoluble solids contributions to high-solids enzymatic hydrolysis of lignocelluloses. Bioresour. Technol. 99, 8940–8948 (2008)CrossRefGoogle Scholar
  27. 27.
    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. Bioresour. Technol. 167, 41–45 (2014)CrossRefGoogle Scholar
  28. 28.
    Wanderley, M.C.A., Martin, C., Rocha, G.J.M., Gouveia, E.R.: Increase in ethanol production from sugarcane bagasse based on combined pretreatments and fed-batch enzymatic hydrolysis. Bioresour. Technol. 128, 448–453 (2013)CrossRefGoogle Scholar
  29. 29.
    Zhang, T., Zhu, M.J.: 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. Bioresour. Technol. 229, 204–210 (2017). CrossRefGoogle Scholar
  30. 30.
    Mosier, N., Hendrickson, R., Ho, N., Sedlak, M., Ladisch, M.R.: Optimization of pH controlled liquid hot water pretreatment of corn stover. Bioresour. Technol. 96, 1986–1993 (2005)CrossRefGoogle Scholar
  31. 31.
    Benjamin, Y., Cheng, H., Gorgens, J.F.: Optimization of dilute sulphuric acid pretreatment to maximize combined sugar yield from sugarcane bagasse for ethanol production. Appl. Biochem. Biotechnol. 172, 610–630 (2014)CrossRefGoogle Scholar
  32. 32.
    Saha, B.C., Nichols, N.N., Cotta, M.A.: Comparison of separate hydrolysis and fermentation versus simultaneous hydrolysis and fermentation of pretreated wheat straw to ethanol by Saccharomyces cerevisiae. J. Biobased Materials Bioener. 7, 409–414 (2013)CrossRefGoogle Scholar
  33. 33.
    Binod, P., Sindhu, R., Singhania, R.R., Vikram, S., Devi, L., Nagalakshmi, S., Kurien, N., Sukumaran, R.K., Pandey, A.: Bioethanol production from rice straw: an overview. Bioresour. Technol. 101, 4767–4774 (2010)CrossRefGoogle Scholar
  34. 34.
    Yasuda, M., Miura, A., Shiragami, T., Matsumoto, J., Kamei, I., Ishii, Y., Ohata, K.: Ethanol production from non-pretreated napier grass through a simultaneous saccharification and fermentation process followed by a pentose fermentation with Escherichia coli K011. J. Biosci.Bioeng. 114, 188–192 (2012)CrossRefGoogle Scholar
  35. 35.
    Zhu, Z.-S., Li, X.-H., Zheng, Q.-M., Zhang, Z., Yu, Y., Wang, J.-F., Liang, S.-Z., Zhu, M.-J.: Bioconversion of amixture pf paper sludge and extraction liquor from water prehydrolysis of Eucalyptus chips to ethanol using separate hydrolysis and fermentation. BioResources 6, 5012–5026 (2011)Google Scholar
  36. 36.
    Li, A., Antizar-Ladislao, B., Khraisheh, M.: Bioconversion of municipal solid waste to glucose for bio-ethanol production. Bioprocess. Biosyst. Eng. 30, 189–196 (2007)CrossRefGoogle Scholar
  37. 37.
    Lissens, G., Klinke, H., Verstraete, W., Ahring, B., Thomsen, A.B.: Wet oxidation treatment of organic household waste enriched with wheat straw for simultaneous saccharification and fermentation into ethanol. Environ. Technol. 25, 647–655 (2004)CrossRefGoogle Scholar
  38. 38.
    Tang, Y.Q., Koike, Y., Liu, K., An, M.Z., Morimura, S., Wu, X.L., Kida, K.: Ethanol production from kitchen waste using the flocculating yeast, Saccharomyces cerevisiae strain KF-7. Biomass Bioener. 32, 1037–1045 (2008)CrossRefGoogle Scholar
  39. 39.
    Zhu, M.-J., Li, P., gong, X.-F., Wang, J.-F.: A comparison of the production of ethanol between simultaneous saccharification and fermentation and separate hydrolysis and fermentation using unpretreated cassava pulp and enzyme cocktail. Biosci. Biotechnol. Biochem. 76, 671–678 (2012)CrossRefGoogle Scholar
  40. 40.
    Singh, A., Kuila, A., Adak, S., Bishai, M., Banerjee, R.: Utilization of vegetable wastes for bioenergy generation. Agric. Res. 1, 213–222 (2012)CrossRefGoogle Scholar
  41. 41.
    Mithra, M.G., Padmaja, G.: Compositional profile and ultrastructure of selected root and vegetable processing residues subjected to steam and dilute sulfuric acid pretreatment. Curr. Biotechnol. (2016); CrossRefGoogle Scholar
  42. 42.
    Mithra, M.G., Padmaja, G.: Comparative alterations in the compositional profile of selected root and vegetable peels subjected to three pretreatments for enhanced saccharification. Internat. J. Environ. Agric. Biotechnol. 2, 1732–1744 (2017)CrossRefGoogle Scholar
  43. 43.
    Mithra, M.G., Padmaja, G., Sreekumar, J.: Optimization of microwave-assisted dilute acid pretreatment for enhanced structural breakdown and enzymatic saccharification of lignocellulo-starch biomass. Curr. Microwave Chem. 4, 1–13 (2017). CrossRefGoogle Scholar
  44. 44.
    Mithra, M.G., Padmaja, G.: Strategies for enzyme saving during saccharification of pretreated lignocellulo-starch biomass: Effect of enzyme dosage and detoxification chemicals. Heliyon (2017). e00384CrossRefGoogle Scholar
  45. 45.
    Mithra, M.G., Sreekumar, J., Padmaja, G.: Binary- and triple-enzyme cocktails and their application mode affect fermentable sugar release from pretreated lignocellulo-starch biomass. Biomass Conv. Biorefin. 2017; CrossRefGoogle Scholar
  46. 46.
    Ghose, T.K.: Measurement of cellulase activities. Pure Appl. Chem. 59, 257–268 (1987)CrossRefGoogle Scholar
  47. 47.
    Tomaz, T., Roche, A.: Hydrophobic interaction, chromatography of Trichoderma reesei cellulase on polypropylene glycol-sepharose. Separation Sci. Technol. 37, 1–11 (2002)CrossRefGoogle Scholar
  48. 48.
    Nelson, N.: A photometric adaptation of the Somogyi method for determination of glucose. J. Biol. Chem. 153, 375–380 (1944)Google Scholar
  49. 49.
    Divya Nair, M.P., Padmaja, G., Moorthy, S.N.: Biodegradation of cassava starch factory residue using a combination of cellulases, xylanases and hemicellulases. Biomass Bioener. 35, 1211–1218 (2011)CrossRefGoogle Scholar
  50. 50.
    Anon.: STARGEN™002: Granular starch hydrolyzing enzyme for ethanol production. Product information (2009) published by Genencor International, a Division of Danisco, Danisco US Inc, Accessed 22 Dec 2014
  51. 51.
    Mithra, M.G., Padmaja, G.: Phenolic inhibitors of saccharification and fermentation in lignocellulo-starch prehydrolysates and comparative efficacy of detoxification treatments. J. Biomass Biofuel. 3, 1–15 (2016). CrossRefGoogle Scholar
  52. 52.
    Singleton, V.L., Rossi, A.: Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am. J. Enol. Vitic. 16, 144–158 (1965)Google Scholar
  53. 53.
    SAS.: 2010; SAS/STAT Software Version 9.3, SAS Institute Inc, CaryGoogle Scholar
  54. 54.
    Kurakake, M., Ooshima, H., Kato, J., Harano, Y.: Pretreatment of bagasse by non-ionic surfactant for the enzymatic hydrolysis. Bioresour. Technol. 49, 247–251 (1994)CrossRefGoogle Scholar
  55. 55.
    Larsson, M., Arasaratnam, V., Mattiason, B.: Effect of poly (ethylene-glycol) on Saccharomyces cerevisiae with respect to growth and ethanol production. J. Microb. Biotechnol. 3, 22–29 (1988)Google Scholar
  56. 56.
    Gupta, R., Kumar, S., Gomes, J., Kuhad, R.C.: Kinetic study of batch and fed-batch enzymatic saccharifiaction of pretreated substrate and subsequent fermentation to ethanol. Biotehnol. Biofuels. (2012). CrossRefGoogle Scholar
  57. 57.
    Hendriks, A.T.W.M., Zeeman, G.: Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresour. Technol. 100, 10–18 (2009)CrossRefGoogle Scholar
  58. 58.
    Jung, Y.H., Park, H.M., Kim, D.H., Yang, J., Kim, K.H.: Fed-batch enzymatic saccharification of high solids pretreated lignocelluloses for obtaining high titers and high yields of glucose. Appl. Biochem. Biotechnol.
  59. 59.
    Xiong, J., Ye, J., Liang, W.Z., Fan, P.M.: Influence of microwave on the ultrastructure of cellulose. J. South China Univ. Technol. 28, 84–89 (2000)Google Scholar
  60. 60.
    Hu, Z.H., Wen, Z.Y.: Enhancing enzymatic digestibility of switchgrass by microwave-assisted alkali pretreatment. Biochem. Eng. J. 38, 369–378 (2008)CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Division of Crop UtilizationICAR- Central Tuber Crops Research InstituteThiruvananthapuramIndia

Personalised recommendations