Bioprocess and Biosystems Engineering

, Volume 41, Issue 8, pp 1153–1163 | Cite as

Differential reinforcement of enzymatic hydrolysis by adding chemicals and accessory proteins to high solid loading substrates with different pretreatments

  • Jian Du
  • Wenxia Song
  • Xiu Zhang
  • Jian Zhao
  • Guodong Liu
  • Yinbo QuEmail author
Research Paper


High dosage of enzyme is required to achieve effective lignocellulose hydrolysis, especially at high-solid loadings, which is a significant barrier to large-scale bioconversion of lignocellulose. Here, we screened four chemical additives and three accessory proteins for their effects on the enzymatic hydrolysis of various lignocellulosic materials. The effects were found to be highly dependent on the composition and solid loadings of substrates. For xylan-extracted lignin-rich corncob residue, the enhancing effect of PEG 6000 was most pronounced and negligibly affected by solid content, which reduced more than half of enzyme demand at 20% dry matter (DM). Lytic polysaccharide monooxygenase enhanced the hydrolysis of ammonium sulfite wheat straw pulp, and its addition reduced about half of protein demand at the solid loading of 20% DM. Supplementation of the additives in the hydrolysis of pure cellulose and complex lignocellulosic materials revealed that their effects are tightly linked to pretreatment strategies.


Lignocellulosic biomass Additives Accessory proteins Cellulose conversion High-solid enzymatic hydrolysis 



The study was supported by Heilongjiang Provincial Key Laboratory of Environmental Microbiology and Recycling of Argo-Waste in Cold Region, Heilongjiang Bayi Agricultural University, China (No. 201715).

Compliance with ethical standards

Conflict of interest

The authors declare no competing financial interest.


  1. 1.
    Himmel ME, Ding S-Y, Johnson DK, Adney WS, Nimlos MR, Brady JW, Foust TD (2007) Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 315(5813):804–807CrossRefPubMedGoogle Scholar
  2. 2.
    Paulova L, Patakova P, Branska B, Rychtera M, Melzoch K (2015) Lignocellulosic ethanol: technology design and its impact on process efficiency. Biotechnol Adv 33(6):1091–1107CrossRefPubMedGoogle Scholar
  3. 3.
    Jorgensen H, Vibe-Pedersen J, Larsen J, Felby C (2007) Liquefaction of lignocellulose at high-solids concentrations. Biotechnol Bioeng 96(5):862–870CrossRefPubMedGoogle Scholar
  4. 4.
    Du J, Zhang F, Li Y, Zhang H, Liang J, Zheng H, Huang H (2014) Enzymatic liquefaction and saccharification of pretreated corn stover at high-solids concentrations in a horizontal rotating bioreactor. Bioprocess Biosyst Eng 37(2):173–181CrossRefPubMedGoogle Scholar
  5. 5.
    Du J, Li Y, Zhang H, Zheng H, Huang H (2014) Factors to decrease the cellulose conversion of enzymatic hydrolysis of lignocellulose at high solid concentrations. Cellulose 21(4):2409–2417CrossRefGoogle Scholar
  6. 6.
    Du J, Cao Y, Liu G, Zhao J, Li X, Qu Y (2017) Identifying and overcoming the effect of mass transfer limitation on decreased yield in enzymatic hydrolysis of lignocellulose at high solid concentrations. Biores Technol 229:88–95CrossRefGoogle Scholar
  7. 7.
    Ellila S, Fonseca L, Uchima C, Cota J, Goldman GH, Saloheimo M, Sacon V, Siika-Aho M (2017) Development of a low-cost cellulase production process using Trichoderma reesei for Brazilian biorefineries. Biotechnol Biofuels 10:30CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Klein-Marcuschamer D, Oleskowicz-Popiel P, Simmons BA, Blanch HW (2012) The challenge of enzyme cost in the production of lignocellulosic biofuels. Biotechnol Bioeng 109(4):1083–1087CrossRefPubMedGoogle Scholar
  9. 9.
    Nguyen TY, Cai CM, Osman O, Kumar R, Wyman CE (2016) CELF pretreatment of corn stover boosts ethanol titers and yields from high solids SSF with low enzyme loadings. Green Chem 18(6):1581–1589CrossRefGoogle Scholar
  10. 10.
    Bhagia S, Kumar R, Wyman CE (2017) Effects of dilute acid and flowthrough pretreatments and BSA supplementation on enzymatic deconstruction of poplar by cellulase and xylanase. Carbohyd Polym 157:1940–1948CrossRefGoogle Scholar
  11. 11.
    Alkasrawi M, Eriksson T, Börjesson J, Wingren A, Galbe M, Tjerneld F, Zacchi G (2003) The effect of Tween-20 on simultaneous saccharification and fermentation of softwood to ethanol. Enzym Microb Technol 33(1):71–78CrossRefGoogle Scholar
  12. 12.
    Kristensen JB, Börjesson J, Bruun MH, Tjerneld F, Jørgensen H (2007) Use of surface active additives in enzymatic hydrolysis of wheat straw lignocellulose. Enzym Microb Technol 40(4):888–895CrossRefGoogle Scholar
  13. 13.
    Eriksson T, Karlsson J, Tjerneld F (2002) A model explaining declining rate in hydrolysis of lignocellulose substrates with cellobiohydrolase I (Cel7A) and endoglucanase I (Cel7B) of Trichoderma reesei. Appl Biochem Biotechnol 101(1):41–60CrossRefPubMedGoogle Scholar
  14. 14.
    Yang B, Wyman CE (2006) BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates. Biotechnol Bioeng 94(4):611–617CrossRefPubMedGoogle Scholar
  15. 15.
    Liu H, Zhu JY, Fu SY (2010) Effects of lignin-metal complexation on enzymatic hydrolysis of cellulose. J Agric Food Chem 58(12):7233–7238CrossRefPubMedGoogle Scholar
  16. 16.
    Li K, Wang X, Wang J, Zhang J (2015) Benefits from additives and xylanase during enzymatic hydrolysis of bamboo shoot and mature bamboo. Biores Technol 192:424–431CrossRefGoogle Scholar
  17. 17.
    Agger JW, Isaksen T, Várnai A, Vidal-Melgosa S, Willats WG, Ludwig R, Horn SJ, Eijsink VG, Westereng B (2014) Discovery of LPMO activity on hemicelluloses shows the importance of oxidative processes in plant cell wall degradation. Proc Natl Acad Sci 111(17):6287–6292CrossRefPubMedGoogle Scholar
  18. 18.
    Horn SJ, Vaaje-Kolstad G, Westereng B, Eijsink VG (2012) Novel enzymes for the degradation of cellulose. Biotechnol Biofuels 5(1):1–13CrossRefGoogle Scholar
  19. 19.
    Kang K, Wang S, Lai G, Liu G, Xing M (2013) Characterization of a novel swollenin from Penicillium oxalicum in facilitating enzymatic saccharification of cellulose. BMC Biotechnol 13(1):1CrossRefGoogle Scholar
  20. 20.
    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. Enzym Microb Technol 79:42–48CrossRefGoogle Scholar
  21. 21.
    Harris PV, Xu F, Kreel NE, Kang C, Fukuyama S (2014) New enzyme insights drive advances in commercial ethanol production. Curr Opin Chem Biol 19:162–170CrossRefPubMedGoogle Scholar
  22. 22.
    Kumar R, Wyman CE (2009) Effect of additives on the digestibility of corn stover solids following pretreatment by leading technologies. Biotechnol Bioeng 102(6):1544–1557CrossRefPubMedGoogle Scholar
  23. 23.
    Carvalho AFA, Oliva Neto PD, Silva DFD, Pastore GM (2013) Xylo-oligosaccharides from lignocellulosic materials: chemical structure, health benefits and production by chemical and enzymatic hydrolysis. Food Res Int 51(1):75–85CrossRefGoogle Scholar
  24. 24.
    Song W, Han X, Qian Y, Liu G, Yao G, Zhong Y, Qu Y (2016) Proteomic analysis of the biomass hydrolytic potentials of Penicillium oxalicum lignocellulolytic enzyme system. Biotechnol Biofuels 9:68CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, Crocker D (2008) Determination of structural carbohydrates and lignin in biomass. NREL Lab Anal Proced 1617:1–16Google Scholar
  26. 26.
    Wood TM, Bha K (1988) Methods for measuring cellulase activities. Methods Enzymol 160:87–112CrossRefGoogle Scholar
  27. 27.
    Hu Y, Xue H, Liu G, Song X, Qu Y (2015) Efficient production and evaluation of lignocellulolytic enzymes using a constitutive protein expression system in Penicillium oxalicum. J Ind Microbiol Biotechnol 42(6):877–887CrossRefPubMedGoogle Scholar
  28. 28.
    Peng S, Cao Q, Qin Y, Li X, Liu G, Qu Y (2017) An aldonolactonase AltA from Penicillium oxalicum mitigates the inhibition of β-glucosidase during lignocellulose biodegradation. Appl Microbiol Biotechnol 9(101):3627–3636CrossRefGoogle Scholar
  29. 29.
    Sipos B, Szilágyi M, Sebestyén Z, Perazzini R, Dienes D, Jakab E, Crestini C, Réczey K (2011) Mechanism of the positive effect of poly (ethylene glycol) addition in enzymatic hydrolysis of steam pretreated lignocelluloses. CR Biol 334(11):812–823CrossRefGoogle Scholar
  30. 30.
    Selig MJ, Thygesen LG, Felby C (2014) Correlating the ability of lignocellulosic polymers to constrain water with the potential to inhibit cellulose saccharification. Biotechnol Biofuels 7(1):159–159CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Roberts KM, Lavenson DM, Tozzi EJ, McCarthy MJ, Jeoh T (2011) The effects of water interactions in cellulose suspensions on mass transfer and saccharification efficiency at high solids loadings. Cellulose 18(3):759–773CrossRefGoogle Scholar
  32. 32.
    Hsieh CW, Cannella D, Jorgensen H, Felby C, Thygesen LG (2014) Cellulase inhibition by high concentrations of monosaccharides. J Agric Food Chem 62(17):3800–3805CrossRefPubMedGoogle Scholar
  33. 33.
    Hsieh CW, Cannella D, Jorgensen H, Felby C, Thygesen LG (2015) Cellobiohydrolase and endoglucanase respond differently to surfactants during the hydrolysis of cellulose. Biotechnol Biofuels 8:52CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Li J, Li S, Fan C, Yan Z (2012) The mechanism of poly (ethylene glycol) 4000 effect on enzymatic hydrolysis of lignocellulose. Colloids Surf B 89:203–210CrossRefGoogle Scholar
  35. 35.
    Srivastava R, Madamwar D, Vyas V (1987) Activation of enzymes by reversed micelles. Biotechnol Bioeng 29(7):901–902CrossRefPubMedGoogle Scholar
  36. 36.
    Pereira A, Hoeger IC, Ferrer A, Rencoret J, Del Rio JC, Kruus K, Rahikainen J, Kellock M, Gutierrez A, Rojas OJ (2017) Lignin films from spruce, eucalyptus, and wheat straw studied with electroacoustic and optical sensors: effect of composition and electrostatic screening on enzyme binding. Biomacromolecules 18(4):1322–1332CrossRefPubMedGoogle Scholar
  37. 37.
    Guo F, Shi W, Sun W, Li X, Wang F, Zhao J, Qu Y (2014) Differences in the adsorption of enzymes onto lignins from diverse types of lignocellulosic biomass and the underlying mechanism. Biotechnol Biofuels 7(1):38CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Pereira A, Hoeger IC, Ferrer A, Rencoret J, del Rio JC, Kruus K, Rahikainen J, Kellock M, Gutiérrez A, Rojas OJ (2017) Lignin films from spruce, eucalyptus, and wheat straw studied with electroacoustic and optical sensors: effect of composition and electrostatic screening on enzyme binding. Biomacromol 18(4):1322–1332CrossRefGoogle Scholar
  39. 39.
    Rahikainen JL, Martin-Sampedro R, Heikkinen H, Rovio S, Marjamaa K, Tamminen T, Rojas OJ, Kruus K (2013) Inhibitory effect of lignin during cellulose bioconversion: the effect of lignin chemistry on non-productive enzyme adsorption. Biores Technol 133:270–278CrossRefGoogle Scholar
  40. 40.
    Lu X, Zheng X, Li X, Zhao J (2016) Adsorption and mechanism of cellulase enzymes onto lignin isolated from corn stover pretreated with liquid hot water. Biotechnol Biofuels 9:118CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Roberts V, Stein V, Reiner T, Lemonidou A, Li X, Lercher JA (2011) Towards quantitative catalytic lignin depolymerization. Chem-A Eur J 17(21):5939–5948CrossRefGoogle Scholar
  42. 42.
    Wang Z, Xue J, Liu W (2012) Nitrogen fixation and chelating property of wheat ammonium sulfite pulping spent liquor. BioResources 7(1):0777–0788Google Scholar
  43. 43.
    Lou H, Zhu J, Lan TQ, Lai H, Qiu X (2013) pH-Induced lignin surface modification to reduce nonspecific cellulase binding and enhance enzymatic saccharification of lignocelluloses. ChemSusChem 6(5):919–927CrossRefPubMedGoogle Scholar
  44. 44.
    Hu J, Arantes V, Pribowo A, Gourlay K, Saddler JN (2014) Substrate factors that influence the synergistic interaction of AA9 and cellulases during the enzymatic hydrolysis of biomass. Energy Environ Sci 7(7):2308CrossRefGoogle Scholar
  45. 45.
    Harris PV, Welner D, McFarland K, Re E, Navarro Poulsen J-C, Brown K, Salbo R, Ding H, Vlasenko E, Merino S (2010) Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family. Biochemistry 49(15):3305–3316CrossRefGoogle Scholar
  46. 46.
    Quinlan RJ, Sweeney MD, Leggio LL, Otten H, Poulsen J-CN, Johansen KS, Krogh KB, Jørgensen CI, Tovborg M, Anthonsen A (2011) Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components. Proc Natl Acad Sci 108(37):15079–15084CrossRefPubMedGoogle Scholar
  47. 47.
    Rodríguez-Zúñiga UF, Cannella D, de Campos Giordano R, Giordano RdLC, Jørgensen H, Felby C (2015) Lignocellulose pretreatment technologies affect the level of enzymatic cellulose oxidation by LPMO. Green Chem 17(5):2896–2903CrossRefGoogle Scholar
  48. 48.
    Andberg M, Penttilä M, Saloheimo M (2015) Swollenin from Trichoderma reesei exhibits hydrolytic activity against cellulosic substrates with features of both endoglucanases and cellobiohydrolases. Biores Technol 181:105–113CrossRefGoogle Scholar
  49. 49.
    Saloheimo M, Paloheimo M, Hakola S, Pere J, Swanson B, Nyyssönen E, Bhatia A, Ward M, Penttilä M (2002) Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials. Eur J Biochem 269(17):435–442CrossRefGoogle Scholar
  50. 50.
    Sampedro J, Cosgrove DJ (2005) The expansin superfamily. Genome Biol 6(12):1–11CrossRefGoogle Scholar
  51. 51.
    Arantes V, Saddler JN (2010) JN: access to cellulose limits the efficiency of enzymatic hydrolysis: the role of amorphogenesis. Biotechnol Biofuels 3(1):275–277CrossRefGoogle Scholar
  52. 52.
    Qing Q, Wyman CE (2011) Supplementation with xylanase and β-xylosidase to reduce xylo-oligomer and xylan inhibition of enzymatic hydrolysis of cellulose and pretreated corn stover. Biotechnol Biofuels 4(1):1CrossRefGoogle Scholar
  53. 53.
    Zhang C, Zhuang X, Wang ZJ, Matt F, John FS, Zhu JY (2013) Xylanase supplementation on enzymatic saccharification of dilute acid pretreated poplars at different severities. Cellulose 20(4):1937–1946CrossRefGoogle Scholar
  54. 54.
    Cannella D, Chia-wen CH, Felby C, Jørgensen H (2012) Production and effect of aldonic acids during enzymatic hydrolysis of lignocellulose at high dry matter content. Biotechnol Biofuels 5(1):1CrossRefGoogle Scholar
  55. 55.
    Scott BR, Huang HZ, Frickman J, Halvorsen R, Johansen KS (2015) Catalase improves saccharification of lignocellulose by reducing lytic polysaccharide monooxygenase-associated enzyme inactivation. Biotech Lett 38(3):425–434CrossRefGoogle Scholar
  56. 56.
    Hu J, Chandra R, Arantes V, Gourlay K, van Dyk JS, Saddler JN (2015) The addition of accessory enzymes enhances the hydrolytic performance of cellulase enzymes at high solid loadings. Biores Technol 186:149–153CrossRefGoogle Scholar
  57. 57.
    Meng X, Ragauskas AJ (2014) Recent advances in understanding the role of cellulose accessibility in enzymatic hydrolysis of lignocellulosic substrates. Curr Opin Biotechnol 27:150–158CrossRefPubMedGoogle Scholar
  58. 58.
    Mesa L, Gonzã¡Lez E, Cara C, Castro E, Mussatto SI (2015) An approach to cellulase recovery from enzymatic hydrolysis of pretreated sugarcane bagasse with high lignin content. Biocatalysis 33(5–6):287–297CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Jian Du
    • 1
  • Wenxia Song
    • 2
  • Xiu Zhang
    • 1
  • Jian Zhao
    • 1
  • Guodong Liu
    • 1
  • Yinbo Qu
    • 1
    Email author
  1. 1.State Key Laboratory of Microbial TechnologyShandong UniversityJinanChina
  2. 2.School of Life ScienceQufu Normal UniversityQufuChina

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