Skip to main content
SpringerLink
Log in

Enzymatic hydrolysis of structurally upgraded lignocellulosic biomass with the aid of humic acid: a case study in a membrane integrated bioreactor

  • Published:
Environment, Development and Sustainability Aims and scope Submit manuscript

Abstract

Substrate accessibility by an enzyme is the rate-limiting step in cellulose digestion. The newly schematized humic acid (HA)-assisted alkali pretreatment was done to delignify and upgrade the bagasse’s characteristics for the pragmatic accessibility of the enzyme. The proposed pretreatment resulted in ~90–100% lignin recovery with distinctive properties. After delignification, the upgraded biomass harnessed as solid support to immobilize the enzyme, ultimately reducing the cost and step for designing physical support for immobilization. HA, an active ligand, with its enriched functional moieties such as carboxyl, carbonyl, hydroxyl, and amide group, bind prior to the substrate (i.e., delignified bagasse) that immobilizes enzyme and hydrolyses cellulose portion. The interaction mechanisms of pretreated bagasse with enzyme were demonstrated through molecular docking using Auto Dock software and UV (Ultraviolet) Spectrophotometric analysis. The pretreatment efficiency was analyzed as a case study in a large-scale reactor by subjecting pretreated bagasse to batch enzymatic hydrolysis with a low enzyme-loading rate of 14 FPU/g of cellulose. Cellulose conversion of 88% (i.e., 20.92 g/L of glucose) was achieved in 48 h, respectively. Subsequently, the flat-sheet cross-flow ultrafiltration (UF) membrane-based continuous recycling unit was operated at a flux of 22.5 L/m2hr toward the purification of glucose (i.e., 15.75 g/L of glucose). The performance of PES10-UF membrane module chaperoning with hydrolysis reactor for the designed hydrolysis scheme was examined in light of critical flux, pure water permeability, and irreversible fouling studies. The flow rate of 300 LPH (L/hr) and the transmembrane membrane pressure (TMP) of 1.5 bar showed high antifouling performance.

Graphical abstract

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig.10
Fig. 11

Similar content being viewed by others

References

  • Abdullah, N., Rahman, M. A., Dzarfan Othman, M. H., Jaafar, J., & Aziz, A. A. (2018). Preparation, characterizations and performance evaluations of alumina hollow fiber membrane incorporated with UiO-66 particles for humic acid removal. Journal of Membrane Science, 563(May), 162–174. https://doi.org/10.1016/j.memsci.2018.05.059

    Article  CAS  Google Scholar 

  • Abels, C., Thimm, K., Wulfhorst, H., Spiess, A. C., & Wessling, M. (2013). Membrane-based recovery of glucose from enzymatic hydrolysis of ionic liquid pretreated cellulose. Bioresource Technology, 149, 58–64. https://doi.org/10.1016/j.biortech.2013.09.012

    Article  CAS  Google Scholar 

  • Dadi, A. P., & Sasidhar Varanasi, C. A. S. (2006). Enhancement of Cellulose Saccharification Kinetics Using an Ionic Liquid Pretreatment Step. Bioresource and Bioengineering, 95(5), 904–910. https://doi.org/10.1002/bit

    Article  CAS  Google Scholar 

  • Andrić, P., Meyer, A. S., Jensen, P. A., & Dam-Johansen, K. (2010). Reactor design for minimizing product inhibition during enzymatic lignocellulose hydrolysis: I. Significance and mechanism of cellobiose and glucose inhibition on cellulolytic enzymes. Biotechnology Advances, 28(3), 308–324. Doi: https://doi.org/10.1016/j.biotechadv.2010.01.003

  • Asar, M. F., Ahmad, N., & Husain, Q. (2020). Chitosan modified Fe3O4/graphene oxide nanocomposite as a support for high yield and stable immobilization of cellulase: Its application in the saccharification of microcrystalline cellulose. Preparative Biochemistry and Biotechnology, 50(5), 460–467. https://doi.org/10.1080/10826068.2019.1706562

    Article  CAS  Google Scholar 

  • Basu, H., Saha, S., Mahadevan, I. A., Pimple, M. V., & Singhal, R. K. (2019). Humic acid coated cellulose derived from rice husk: A novel biosorbent for the removal of Ni and Cr. Journal of Water Process Engineering, 32(March), 100892. https://doi.org/10.1016/j.jwpe.2019.100892

    Article  Google Scholar 

  • Berg JM, Tymoczko JL, S. L., & Freeman, N. Y. W. H. (2002). Biochemistry. 5th edition. Section 8.4, The Michaelis-Menten Model Accounts for the Kinetic Properties of Many Enzymes. https://www.ncbi.nlm.nih.gov/books/NBK22430/

  • Britain, G., Croix, P., Sciences, F., & Louvain, D. (1986). Formation of [ “ C ] Cellulase-Humic Complexes and Their Stability, 18(3), 251–254.

  • Bu, L., Nimlos, M. R., Shirts, M. R., Ståhlberg, J., Himmel, M. E., Crowley, M. F., & Beckham, G. T. (2012). Product binding varies dramatically between processive and nonprocessive cellulase enzymes. Journal of Biological Chemistry, 287(29), 24807–24813. https://doi.org/10.1074/jbc.M112.365510

    Article  CAS  Google Scholar 

  • Chhaya, M., & S., Majumdar, G. C., & De, S. (2012). Clarifications of stevia extract using cross flow ultrafiltration and concentration by nanofiltration. Separation and Purification Technology, 89, 125–134. https://doi.org/10.1016/j.seppur.2012.01.016

    Article  CAS  Google Scholar 

  • Dixon, M., Webb, E. C., Thorne, C. I. R., & Tipton, K. F. (1981). Enzymes ( Third Edition ) Biology of Collagen, 130(1).

  • Espirito Santo, M., Rezende, C. A., Bernardinelli, O. D., Pereira, N., Curvelo, A. A. S., deAzevedo, E. R., et al. (2018). Structural and compositional changes in sugarcane bagasse subjected to hydrothermal and organosolv pretreatments and their impacts on enzymatic hydrolysis. Industrial Crops and Products, 113(January), 64–74. https://doi.org/10.1016/j.indcrop.2018.01.014

    Article  CAS  Google Scholar 

  • Gao, J., Chen, L., Zhang, J., & Yan, Z. (2014). Improved enzymatic hydrolysis of lignocellulosic biomass through pretreatment with plasma electrolysis. Bioresource Technology, 171, 469–471. https://doi.org/10.1016/j.biortech.2014.07.118

    Article  CAS  Google Scholar 

  • Gennari, A., Simon, R., Denise, N., Sperotto, D. M., Valim, C., Augusto, L., et al. (2022). International Journal of Biological Macromolecules Application of cellulosic materials as supports for single-step purification and immobilization of a recombinant β -galactosidase via cellulose-binding domain, 199(October 2021), 307–317.

  • Hsieh, C. C., Cannella, D., Jørgensen, H., Felby, C., & Thygesen, L. G. (2014). Article Previous Article Next Article Table of Contents Cellulase Inhibition by High Concentrations of Monosaccharides. Journal of Agricultural and Food Chemistry, 62, 3800–3805. https://doi.org/10.1021/jf5012962

    Article  CAS  Google Scholar 

  • Hwangbo, M., Tran, J. L., & Chu, K. H. (2019). Effective one-step saccharification of lignocellulosic biomass using magnetite-biocatalysts containing saccharifying enzymes. Science of the Total Environment, 647, 806–813. https://doi.org/10.1016/j.scitotenv.2018.08.066

    Article  CAS  Google Scholar 

  • Kaur, P., Taggar, M. S., & Kalia, A. (2021). Characterization of magnetic nanoparticle–immobilized cellulases for enzymatic saccharification of rice straw. Biomass Conversion and Biorefinery, 11(3), 955–969. https://doi.org/10.1007/s13399-020-00628-x

    Article  CAS  Google Scholar 

  • Laskowski, R. A., Gerick, F., & Thornton, J. M. (2009). The structural basis of allosteric regulation in proteins. FEBS Letters, 583(11), 1692–1698. https://doi.org/10.1016/j.febslet.2009.03.019

    Article  CAS  Google Scholar 

  • Li, Y., & Wilson, D. B. (2008). Chitin binding by Thermobifida fusca cellulase catalytic domains. Biotechnology and Bioengineering, 100(4), 644–652. https://doi.org/10.1002/bit.21808

    Article  CAS  Google Scholar 

  • Liu, W., Wang, B., Hou, Q., Chen, W., & Wu, M. (2016). Effects of fibrillation on the wood fibers’ enzymatic hydrolysis enhanced by mechanical refining. Bioresource Technology, 206, 99–103. https://doi.org/10.1016/j.biortech.2016.01.074

    Article  CAS  Google Scholar 

  • Liu, Z., Xu, F., Zu, Y., Meng, R., & Wang, W. (2016). Study on water-dispersible colloids in saline-alkali soils by atomic force microscopy and spectrometric methods. Microscopy Research and Technique, 79(6), 525–531. https://doi.org/10.1002/jemt.22662

    Article  CAS  Google Scholar 

  • Maeda, R. N., Serpa, V. I., Rocha, V. A. L., Mesquita, R. A. A., Anna, L. M. M. S., De Castro, A. M. H., et al. (2011). Enzymatic hydrolysis of pretreated sugar cane bagasse using Penicillium funiculosum and Trichoderma harzianum cellulases. Process Biochemistry, 46(5), 1196–1201. https://doi.org/10.1016/j.procbio.2011.01.022

    Article  CAS  Google Scholar 

  • Mohamad, N. R., Marzuki, N. H. C., Buang, N. A., Huyop, F., & Wahab, R. A. (2015). An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes. Biotechnology and Biotechnological Equipment, 29(2), 205–220. https://doi.org/10.1080/13102818.2015.1008192

    Article  CAS  Google Scholar 

  • Nebbioso, A., Piccolo, A., Lamshöft, M., & Spiteller, M. (2014). Molecular characterization of an end-residue of humeomics applied to a soil humic acid. RSC Advances, 4(45), 23658–23665. https://doi.org/10.1039/C4RA01619J

    Article  CAS  Google Scholar 

  • Nebbioso, A., & Piccolo, A. (2012). Analytica Chimica Acta Advances in humeomics: Enhanced structural identification of humic molecules after size fractionation of a soil humic acid. Analytica Chimica Acta, 720, 77–90. https://doi.org/10.1016/j.aca.2012.01.027

    Article  CAS  Google Scholar 

  • Nguyen, L. T., Neo, K. R. S., & Yang, K. L. (2015). Continuous hydrolysis of carboxymethyl cellulose with cellulase aggregates trapped inside membranes. Enzyme and Microbial Technology, 78, 34–39. https://doi.org/10.1016/j.enzmictec.2015.06.005

    Article  CAS  Google Scholar 

  • Nguyen, T. T. T., & Le, V. V. M. (2013). Effects of ultrasound on cellulolytic activity of cellulase complex. International Food Research Journal, 20(2), 557–563.

    CAS  Google Scholar 

  • Nitsos, C., Stoklosa, R., Karnaouri, A., Vörös, D., Lange, H., Hodge, D., et al. (2016). Isolation and characterization of organosolv and alkaline lignins from hardwood and softwood biomass. ACS Sustainable Chemistry and Engineering, 4(10), 5181–5193. https://doi.org/10.1021/acssuschemeng.6b01205

    Article  CAS  Google Scholar 

  • Pal, R. K., & Chakraborty, S. (2013). A novel mixing strategy for maximizing yields of glucose and reducing sugar in enzymatic hydrolysis of cellulose. Bioresource Technology, 148, 611–614. https://doi.org/10.1016/j.biortech.2013.09.009

    Article  CAS  Google Scholar 

  • Qi, B., Chen, X., Su, Y., & Wan, Y. (2011). Enzyme adsorption and recycling during hydrolysis of wheat straw lignocellulose. Bioresource Technology, 102(3), 2881–2889. https://doi.org/10.1016/j.biortech.2010.10.092

    Article  CAS  Google Scholar 

  • 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. Bioresource Technology, 104, 466–472. https://doi.org/10.1016/j.biortech.2011.10.049

    Article  CAS  Google Scholar 

  • Reinikainen, T., Ruohonen, L., Nevanen, T., Laaksonen, L., Kraulis, P., Jones, T. A., et al. (1992). Investigation of the function of mutated cellulose‐binding domains of Trichoderma reesei cellobiohydrolase I. Proteins: Structure, Function, and Bioinformatics, 14(4), 475–482. Doi: https://doi.org/10.1002/prot.340140408

  • Rios, G. M., Belleville, M. P., Paolucci, D., & Sanchez, J. (2004). Progress in enzymatic membrane reactors - A review. Journal of Membrane Science, 242(1–2), 189–196. https://doi.org/10.1016/j.memsci.2003.06.004

    Article  CAS  Google Scholar 

  • Robin, T., Reuveni, S., & Urbakh, M. (2018). Single-molecule theory of enzymatic inhibition. Nature Communications, 9(1), 1–9. https://doi.org/10.1038/s41467-018-02995-6

    Article  CAS  Google Scholar 

  • Safari Sinegani, A. A., Emtiazi, G., & Shariatmadari, H. (2005). Sorption and immobilization of cellulase on silicate clay minerals. Journal of Colloid and Interface Science, 290(1), 39–44. https://doi.org/10.1016/j.jcis.2005.04.030

    Article  CAS  Google Scholar 

  • Saha, K., Verma, P., Sikder, J., Chakraborty, S., & Curcio, S. (2019). Synthesis of chitosan-cellulase nanohybrid and immobilization on alginate beads for hydrolysis of ionic liquid pretreated sugarcane bagasse. Renewable Energy, 133, 66–76. https://doi.org/10.1016/j.renene.2018.10.014

    Article  CAS  Google Scholar 

  • Selvam, K., Senbagam, D., Selvankumar, T., Sudhakar, C., Kamala-Kannan, S., Senthilkumar, B., & Govarthanan, M. (2017). Cellulase enzyme: Homology modeling, binding site identification and molecular docking. Journal of Molecular Structure, 1150, 61–67. https://doi.org/10.1016/j.molstruc.2017.08.067

    Article  CAS  Google Scholar 

  • Shi, K. Y., Yin, S. D., Tao, X. X., Du, Y., He, H., Lv, Z. P., & Xu, N. (2013). Quantitative measurement of coal bio-solubilization by ultraviolet-visible spectroscopy. Energy Sources, Part a: Recovery, Utilization and Environmental Effects, 35(15), 1456–1462. https://doi.org/10.1080/15567036.2010.521800

    Article  CAS  Google Scholar 

  • Sluiter, A., Hames, B., Ruiz, R., Scarlata, C., Sluiter, J., Templeton, D., & Nrel, D. C. (2012). Determination of Structural Carbohydrates and Lignin in Biomass Determination of Structural Carbohydrates and Lignin in Biomass, (August).

  • Smilek, J., Sedláček, P., Kalina, M., & Klučáková, M. (2015). On the role of humic acids’ carboxyl groups in the binding of charged organic compounds. Chemosphere, 138, 503–510. https://doi.org/10.1016/j.chemosphere.2015.06.093

    Article  CAS  Google Scholar 

  • Soni, S. K., Batra, N., Bansal, N., & Soni, R. (2010). Bioconversion of sugarcane bagasse into second generation bioethanol after enzymatic hydrolysis with in-house produced cellulases from aspergillus sp. s4b2f. BioResources, 5(2), 741–757. Doi: https://doi.org/10.15376/biores.5.2.741-757

  • Stickel, J. J., Adhikari, B., Sievers, D. A., & Pellegrino, J. (2018). Continuous Enzymatic Hydrolysis of Lignocellulosic Biomass in a Membrane-Reactor System, 93(8), 2181–2190.

    CAS  Google Scholar 

  • Sun, R., Fang, J., & Rowlands, P. (1998). Physico-Chemical and thermal characterisation of alkali-soluble lignins from wheat straw. Polymer. https://doi.org/10.1295/polymj.30.289

    Article  Google Scholar 

  • Tang, W., Wu, X., Huang, C., Huang, C., Lai, C., & Yong, Q. (2020). Humic acid-assisted autohydrolysis of waste wheat straw to sustainably improve enzymatic hydrolysis. Bioresource Technology, 306(February), 123103. https://doi.org/10.1016/j.biortech.2020.123103

    Article  CAS  Google Scholar 

  • Tang, W., Wu, X., Huang, C., Ling, Z., Lai, C., & Yong, Q. (2021). Biotechnology for Biofuels Comprehensive understanding of the effects of metallic cations on enzymatic hydrolysis of humic acid - pretreated waste wheat straw. Biotechnology for Biofuels, 14, 1–10. https://doi.org/10.1186/s13068-021-01874-5

    Article  CAS  Google Scholar 

  • Uma Maheswari, R., Mavukkandy, M. O., Adhikari, U., Naddeo, V., Sikder, J., & Arafat, H. A. (2020). Synergistic effect of humic acid on alkali pretreatment of sugarcane bagasse for the recovery of lignin with phenomenal properties. Biomass and Bioenergy, 134(June 2019), 105486. Doi: https://doi.org/10.1016/j.biombioe.2020.105486

  • van Tilbeurgh, H., Loontiens, F. G., Engelborgs, Y., & Claeyssens, M. (1989). Studies of the cellulolytic system of Trichoderma reesei QM 9414: Binding of small ligands to the 1,4-β-glucan cellobiohydrolase II and influence of glucose on their affinity. European Journal of Biochemistry, 184(3), 553–559. https://doi.org/10.1111/j.1432-1033.1989.tb15049.x

    Article  Google Scholar 

  • Xu, C., Zhang, J., Zhang, Y., Guo, Y., Xu, H., Xu, J., & Wang, Z. (2019). Enhancement of high-solids enzymatic hydrolysis efficiency of alkali pretreated sugarcane bagasse at low cellulase dosage by fed-batch strategy based on optimized accessory enzymes and additives. Bioresource Technology, 292(June), 121993. https://doi.org/10.1016/j.biortech.2019.121993

    Article  CAS  Google Scholar 

  • Yang, F., Jin, E. S., Zhu, Y., Wu, S., Zhu, W., Jin, Y., & Song, J. (2015). A mini-review on the applications of cellulose-binding domains in lignocellulosic material utilizations. BioResources, 10(3), 6081–6094. Doi: https://doi.org/10.15376/biores.10.3.Yang

  • Yu, H., Xiao, W., Han, L., & Huang, G. (2019). Characterization of mechanical pulverization/phosphoric acid pretreatment of corn stover for enzymatic hydrolysis. Bioresource Technology, 282, 69–74. https://doi.org/10.1016/j.biortech.2019.02.104

    Article  CAS  Google Scholar 

  • Zhao, H., Jones, C. L., Baker, G. A., Xia, S., Olubajo, O., & Person, V. N. (2009). Regenerating cellulose from ionic liquids for an accelerated enzymatic hydrolysis. Journal of Biotechnology, 139(1), 47–54. https://doi.org/10.1016/j.jbiotec.2008.08.009

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The Department of Biotechnology, Government of India, has funded part of this research work under the bilateral collaboration between India and Brazil (DBT- India, and MCTI-CNPq-Brazil) vide no. DBT/In-Bz/2013-16/06. The authors are also thankful to the Department of Science and Technology, Government of India (DST) for the grants under the DST-FIST (Level-I) Program [SR/FST/ETI-405/2015(C)] with which the infrastructure for the present research was developed. All the authors are grateful to Dr. Utpal Adhikari, Assistant professor, Department of chemistry, NIT Durgapur, for the valuable guidance and suggestions for upgrading the working strategies during the implementation of the project. The authors would like to express our sincere gratitude to Prof. Hassan A. Arafat (Center for Membrane and Advanced Water Technology, Khalifa University, Abu Dhabi, United Arab Emirates) for the help extended toward improving the language of the manuscript.

Author information

Authors and Affiliations

  1. Department of Chemical Engineering, National Institute of Technology Durgapur, West Bengal, Durgapur, 713209, India

    R. Uma Maheswari, Ayan Mondal, Gopinath Halder & Jaya Sikder

  2. Pondicherry Centre for Biological Science and Educational Trust, Kottakuppam, Tamilnadu, 605104, India

    Krishnaraj Thirugnanasambantham

Authors
  1. R. Uma Maheswari
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Krishnaraj Thirugnanasambantham
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ayan Mondal
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gopinath Halder
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jaya Sikder
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Gopinath Halder or Jaya Sikder.

Ethics declarations

Conflict of interest

The authors reported no potential conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Maheswari, R.U., Thirugnanasambantham, K., Mondal, A. et al. Enzymatic hydrolysis of structurally upgraded lignocellulosic biomass with the aid of humic acid: a case study in a membrane integrated bioreactor. Environ Dev Sustain 25, 4033–4064 (2023). https://doi.org/10.1007/s10668-022-02233-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10668-022-02233-6

Keywords

Search

Navigation