Augmented hydrolysis of acid pretreated sugarcane bagasse by PEG 6000 addition: a case study of Cellic CTec2 with recycling and reuse

  • Pratibha Baral
  • Lavika Jain
  • Akhilesh Kumar Kurmi
  • Vinod Kumar
  • Deepti AgrawalEmail author
Research Paper


In an integrated lignocellulosic biorefinery, the cost associated with the “cellulases” and “longer duration of cellulose hydrolysis” represents the two most important bottlenecks. Thus, to overcome these barriers, the present study aimed towards augmented hydrolysis of acid pretreated sugarcane bagasse within a short span of 16 h using Cellic CTec2 by addition of PEG 6000. Addition of this surfactant not only enhanced glucose release by twofold within stipulated time, but aided in recovery of Cellic CTec2 which was further recycled and reused for second round of saccharification. During first round of hydrolysis, when Cellic CTec2 was loaded at 25 mg protein/g cellulose content, it resulted in 76.24 ± 2.18% saccharification with a protein recovery of 58.4 ± 1.09%. Filtration through 50KDa PES membrane retained ~ 89% protein in 4.5-fold concentrated form and leads to simultaneous fractionation of ~ 70% glucose in the permeate. Later, the saccharification potential of recycled Cellic CTec2 was assessed for the second round of saccharification using two different approaches. Unfortified enzyme effectively hydrolysed 67% cellulose, whereas 72% glucose release was observed with Cellic CTec2 fortified with 25% fresh protein top-up. Incorporating the use of the recycled enzyme in two-stage hydrolysis could effectively reduce the Cellic CTec2 loading from 25 to 16.8 mg protein/g cellulose. Furthermore, 80% ethanol conversion efficiencies were achieved when glucose-rich permeate obtained after the first and second rounds of saccharification were evaluated using Saccharomyces cerevisiae MTCC 180.


Cellic CTec2 PEG 6000 PES membrane filter Recycling Saccharification 



This work is financially supported by the Department of Biotechnology (DBT, India) under Indo-UK Industrial Waste Challenge 2017, with grant number being GAP 3513. The authors are thankful to Dr Anjan Ray, Director CSIR-Indian Institute of Petroleum for providing necessary facilities to complete this work and his constant motivation. Mrs. Lavika Jain is grateful to Council of Scientific and Industrial Research (CSIR) New Delhi, India, for awarding her Senior Research Fellowship.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Carriquiry MA, Du X, Timilsina GA (2011) Second generation biofuels: Economics and policies. Energy Policy 39:4222–4234CrossRefGoogle Scholar
  2. 2.
    Lynd LR, Liang X, Biddy MX, Allee A, Cai H, Foust T, Himmel ME, Laser MS, Wang M, Wyman CE (2017) Cellulosic ethanol: status and innovation. Curr Opin Biotechnol 45:202–211CrossRefGoogle Scholar
  3. 3.
    Mohanram S, Amat D, Choudhary J, Arora A, Nain L (2013) Novel perspectives for evolving enzyme cocktails for lignocellulose hydrolysis in biorefineries. Sustain Chem Process 1:15CrossRefGoogle Scholar
  4. 4.
    Nguyen TY, Caib CM, Kumar R, Wyman CE (2017) Overcoming factors limiting high-solids fermentation of lignocellulosic biomass to ethanol. PNAS 114:11673–11678CrossRefGoogle Scholar
  5. 5.
    Berlin A, Gilkes N, Kurabi A, Bura R, Tu M, Kilburn D, Saddler J (2005) Weak lignin-binding enzymes, a novel approach to improve activity of cellulases for hydrolysis of lignocellulosics. Appl Biochem Biotechnol 121:163–170CrossRefGoogle Scholar
  6. 6.
    Whitehead TA, Bandi CK, Berger M, Park J, Chundawat SPS (2017) Negatively supercharging cellulases render them lignin-resistant. ACS Sustain Chem Engg 5:6247–6252CrossRefGoogle Scholar
  7. 7.
    Gomes DG, Serna-Loaiza S, Cardona CA, Gama M, Domingues L (2018) Insights into the economic viability of cellulases recycling on bioethanol production from recycled paper sludge. Bioresour Technol 267:347–355CrossRefGoogle Scholar
  8. 8.
    Rodrigues AC, Felby C, Gama M (2014) Cellulase stability, adsorption/desorption profiles and recycling during successive cycles of hydrolysis and fermentation of wheat straw. Bioresour Technol 156:163–169CrossRefGoogle Scholar
  9. 9.
    Jain L, Kurmi AK, Agrawal D (2018) Feasibility studies with lignin blocking additives in enhancing saccharification and cellulase recovery: mutant UV-8 of T. verruculosus IIPC 324 a case study. Enzyme Microb Technol 118:44–49CrossRefGoogle Scholar
  10. 10.
    Fahmy M, Sohel MI, Vaidya AA, Jack MW, Suckling ID (2019) Does sugar yield drive lignocellulosic sugar cost? Case study for enzymatic hydrolysis of softwoods with added polyethylene glycol. Proc Biochem 80:103–111CrossRefGoogle Scholar
  11. 11.
    Luo X, Liu J, Zheng P, Li M, Zhou Y, Huang L, Chen L, Shuai L (2019) Promoting enzymatic hydrolysis of lignocellulosic biomass by inexpensive soy protein. Biotechnol Biofuels 12:51CrossRefGoogle Scholar
  12. 12.
    Alhammad A, Adewale P, Kuttiraja M, Christopher LP (2018) Enhancing enzyme-aided production of fermentable sugars from poplar pulp in the presence of non-ionic surfactants. Biprocess Biosys Eng 41(8):1133–1142CrossRefGoogle Scholar
  13. 13.
    Olson DG, McBride JE, Shaw AJ, Lynd LR (2011) Recent progress in consolidated bioprocessing. Curr Opin Biotechnol 23:1–10CrossRefGoogle Scholar
  14. 14.
    Gomes D, Rodrigues AC, Domingues L, Gama M (2015) Cellulase Recycling in biorefineries- is it possible? Appl Microbiol Biotechnol 99:4131–4143CrossRefGoogle Scholar
  15. 15.
    Jørgensen H, Pinelo M (2016) Enzyme recycling in lignocellulosic biorefineries. Biofuel Bioprod Biorefin 11:150–167CrossRefGoogle Scholar
  16. 16.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  17. 17.
    Ghosh D, Dasgupta D, Agrawal D, Kaul S, Adhikari DK, Kurmi AK, Arya PK, Bangwal D, Negi MS (2015) Fuels and chemicals from lignocellulosic biomass: an integrated biorefinery approach. Energ Fuel 29:3149–3157CrossRefGoogle Scholar
  18. 18.
    Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, Crocker D (2012) Determination of structural carbohydrates and lignin in biomass. Laboratory Analytical Procedure, NREL/TP-5104-2618Google Scholar
  19. 19.
    Trivedi N, Baghel RS, Bothwell J, Gupta V, Reddy RK, Lali AM, Jha B (2016) An integrated process for the extraction of fuel and chemicals from marine macroalgal biomass. Sci Rep 6:30728CrossRefGoogle Scholar
  20. 20.
    Qi B, Luo J, Chen G, Chen X, Wan Y (2012) Application of ultrafiltration and nanofiltration for recycling cellulase and concentrating glucose from enzymatic hydrolyzate of steam exploded wheat straw. Bioresour Technol 104:466–472CrossRefGoogle Scholar
  21. 21.
    Reis L, Fontana RC, Delabona PS, Lima DJS, Camassola M, Pradella JGC, Dillon AJP (2013) Increased production of cellulases and xylanases by Penicillium echinulatum S1M29 in batch and fed-batch culture. Bioresour Technol 146:597–603CrossRefGoogle Scholar
  22. 22.
    Sun FF, Hong J, Hu J, Saddler JN, Fang X, Zhang Z, Shen S (2015) Accessory enzymes influence cellulase hydrolysis of the model substrate and the realistic lignocellulosic biomass. Enzyme Microb Technol 79–80:42–48CrossRefGoogle Scholar
  23. 23.
    Ramos LP, Silva L, Ballem AC, Pitarelo AP, Chiarello LM, Silveira MHL (2015) Enzymatic hydrolysis of steam-exploded sugarcane bagasse using high total solids and low enzyme loadings. Bioresour Technol 175:195–202CrossRefGoogle Scholar
  24. 24.
    Cannella D, Hsieh CC, Felby C, Jørgensen H (2012) Production and effect of aldonic acids during enzymatic hydrolysis of lignocellulose at high dry matter content. Biotechnol Biofuels 5:26CrossRefGoogle Scholar
  25. 25.
    Rodrigues AC, Haven MØ, Lindedam J, Felby C, Gama M (2015) Celluclast and Cellic® CTec2: saccharification/fermentation of wheat straw, solid–liquid partition and potential of enzyme recycling by alkaline washing. Enzyme Microb Technol 79–80:70–77CrossRefGoogle Scholar
  26. 26.
    Gomes D, Gama M, Domingues L (2018) Determinants on an efficient cellulase recycling process for the production of bioethanol from recycled paper sludge under high solid loadings. Biotechnol Biofuels 11:111CrossRefGoogle Scholar
  27. 27.
    Knutsen JS, Davis RH (2004) Cellulase retention and sugar removal by membrane ultrafiltration during lignocellulosic biomass hydrolysis. Appl Biochem Biotechnol 114:585–600CrossRefGoogle Scholar
  28. 28.
    Hu J, Mok YK, Saddler JN (2018) Can we reduce the cellulase enzyme loading required to achieve efficient lignocellulose deconstruction by only using the initially absorbed enzymes? ACS Sustain Chem Engg 6:6233–6239CrossRefGoogle Scholar
  29. 29.
    Haven MØ, Lindedam J, Jeppesen MD, Elleskov M, Rodrigues AC, Gama M, Jørgensen H, Felby C (2015) Continuous recycling of enzymes during production of lignocellulosic bioethanol in demonstration scale. Appl Energ 159:188–195CrossRefGoogle Scholar
  30. 30.
    Patel H, Chapla D, Shah A (2017) Bioconversion of pretreated sugarcane bagasse using enzymatic and acid followed by enzymatic hydrolysis approaches for bioethanol production. Renew Energ 109:323–331CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Biochemistry and Biotechnology Area, Materials Resource Efficiency DivisionCSIR- Indian Institute of PetroleumDehradunIndia
  2. 2.Academy of Scientific and Innovative Research (AcSIR)GhaziabadIndia
  3. 3.Bioenergy and Resource Management Centre, School of Water, Energy and EnvironmentCranfield UniversityCranfieldUK

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