3 Biotech

, 7:317 | Cite as

Enzyme kinetics of cellulose hydrolysis of Miscanthus and oat hulls

  • Ekaterina I. Makarova
  • Vera V. Budaeva
  • Aleksey A. Kukhlenko
  • Sergey E. Orlov
Original Article


Experiments were done to model enzymatic hydrolysis of Miscanthus and oat hulls treated with dilute solutions of nitric acid and sodium hydroxide in direct and reverse sequences. The enzymatic hydrolysis kinetics of the substrates was studied at an initial solid loading from 30 to 120 g/L. The effects of feedstock type and its pretreatment method on the initial hydrolysis rate and reducing sugar yield were evaluated. The fitting results by the developed models showed good agreement with the experimental data. These models designed for developing the production technology of concentrated glucose solutions can also be applied for glucose fermentation into ethanol. The initial solid loading of 60–90 g/L provides the reducing sugar concentration of 40–80 g/L necessary for ethanol synthesis. The kinetic model can also be applied to investigate enzymatic hydrolysis of other substrates (feedstock type, pretreatment method) under the similar conditions used herein, with adjusted empirical coefficient values.


Miscanthus Oat hulls Cellulose Enzymatic hydrolysis Kinetics Mathematical model 



Concentration of enzyme–substrate complex (g/L)


Equilibrium concentration of reducing sugars (g/L)


Concentration of reducing sugars (g/L)


Concentration of substrate (g/L)


Formation constant of enzyme–substrate complex (h−1)


Breakdown constant of enzyme–substrate complex [g/(L h)]


Formation constant of reducing sugars [g/(L h)]


Michaelis constant (g/L)


Disassociation constant of enzyme–substrate complex (g/L)


Nitric-acid method


Combined method


Reducing sugars


Miscanthus cellulose


Oat hull cellulose



This work was supported by the Federal Agency for Scientific Organizations of the Russian Federation [Grant No. 0385-2016-0001].

Compliance with ethical standards

Conflict of interest

The authors declare that there is no conflicts of interest regarding the publication of this article.


  1. Alvira P, Tomás-Pejó E, Ballesteros M, Negro MJ (2010) Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour Technol 101:4851–4861. doi: 10.1016/j.biortech.2009.11.093 CrossRefGoogle Scholar
  2. Alvira P, Moreno AD, Ibarra D, Sáez F, Ballesteros M (2013) Improving the fermentation performance of Saccharomyces cerevisiae by laccase during ethanol production from steam-exploded wheat straw at high-substrate loadings. Biotechnol Prog 29:74–82. doi: 10.1002/btpr.1666 CrossRefGoogle Scholar
  3. Bansal P, Hall M, Realff MJ, Lee JH, Bommarius AS (2009) Modeling cellulase kinetics on lignocellulosic substrates. Biotechnol Adv 27:833–848. doi: 10.1016/j.biotechadv.2009.06.005 CrossRefGoogle Scholar
  4. Briggs GE, Haldane JB (1925) A note on the kinetics of enzyme action. Biochem J 19:338–339CrossRefGoogle Scholar
  5. Budaeva VV, Makarova EI, Gismatulina YuA (2016) Integrated flowsheet for conversion of non-woody biomass into polyfunctional materials. Key Eng Mater 670:202–206. doi: 10.4028/ CrossRefGoogle Scholar
  6. Chaud LCS, Silva DDV, Mattos RT, Felipe MGA (2012) Evaluation of oat hull hemicellulosic hydrolysate fermentability employing Pichia stipites. Braz Arch Biol Technol 55:771–777. doi: 10.1590/S1516-89132012000500017 CrossRefGoogle Scholar
  7. Denisova MN, Makarova EI, Pavlov IN, Budaeva VV, Sakovich GV (2016) Enzymatic hydrolysis of hydrotropic pulps at different substrate loadings. Appl Biochem Biotechnol 178:1196–1206. doi: 10.1007/s12010-015-1938-y CrossRefGoogle Scholar
  8. Fan S, Chen S, Tang X, Xiao Z, Deng Q, Yao P, Sun Z, Zhang Y, Chen C (2015) Kinetic model of continuous ethanol fermentation in closed-circulating process with pervaporation membrane bioreactor by Saccharomyces cerevisiae. Bioresour Technol 177:169–175. doi: 10.1016/j.biortech.2014.11.076 CrossRefGoogle Scholar
  9. Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59:257–268. doi: 10.1351/pac198759020257 Google Scholar
  10. Gismatulina YuA, Budaeva VV, Sakovich GV (2015) Nitric acid preparation of cellulose from Miscanthus as a nitrocellulose precursor. Russ Chem Bull 64:2949–2953. doi: 10.1007/s11172-015-1252-4 CrossRefGoogle Scholar
  11. Gonzalez G, Caminal G, Mas C, Lopez-Santin J (1989) A kinetic model for pretreated wheat straw saccharification by cellulase. J Chem Technol Biotechnol 44:275–288. doi: 10.1002/jctb.280440404 CrossRefGoogle Scholar
  12. Gupta R, Kumar S, Gomes J, Kuhad RC (2012) Kinetic study of batch and fed-batch enzymatic saccharification of pretreated substrate and subsequent fermentation to ethanol. Biotechnol Biofuels 5:16. doi: 10.1186/1754-6834-5-16 CrossRefGoogle Scholar
  13. Hastie T, Tibshirani R, Friedman J (2009) The elements of statistical learning. Springer-Verlag, BerlinCrossRefGoogle Scholar
  14. Himmel ME, Ding SY, Johnson DK, Adney WS, Nimlos MR, Brady JW, Foust TD (2007) Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science 315:804–807. doi: 10.1126/science.1137016 CrossRefGoogle Scholar
  15. Ioelovich M (2015) Study of kinetics of enzymatic hydrolysis of cellulose materials. ChemXpress 8:231–239. doi: 10.13140/RG.2.1.3185.6806 Google Scholar
  16. Ioelovich M, Morag E (2012) Study of enzymatic hydrolysis of pretreated biomass at increased solids loading. Bioresources 7:4672–4682. doi: 10.15376/biores.7.4.4672-4682 CrossRefGoogle Scholar
  17. Jones MB, Walsh M (2001) Miscanthus for energy and fibre. Earthscan, LondonGoogle Scholar
  18. Kadam KL, Rydholm EC, McMillan JD (2004) Development and validation of a kinetic model for enzymatic saccharification of lignocellulosic biomass. Biotechnol Prog 20:698–705. doi: 10.1021/bp034316x CrossRefGoogle Scholar
  19. Marcos M, Garcia-Cubero MT, Gonzalez-Benito G, Coca M, Bolado S, Lucas S (2013) Optimization of the enzymatic hydrolysis conditions of steam-exploded wheat straw for maximum glucose and xylose recovery. J Chem Technol Biotechnol 88:237–246. doi: 10.1002/jctb.3820 CrossRefGoogle Scholar
  20. Miller GL (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31:426–428. doi: 10.1021/ac60147a030 CrossRefGoogle Scholar
  21. Obolenskaya AV, Yelnitskaya ZP, Leonovich AA (1991) Laboratornye Raboty po Khimii Drevesiny i Tsellyulozy [Laboratory works on wood and cellulose chemistry: textbook for higher educational institutions]. Ecology Publisher (in Russian), Moscow.
  22. Oh YH, Eom IY, Joo JC (2015) Recent advances in development of biomass pretreatment technologies used in biorefinery for the production of bio-based fuels, chemicals and polymers. Korean J Chem Eng 32:1945–1959. doi: 10.1007/s11814-015-0191-y CrossRefGoogle Scholar
  23. Olsen SN, Lumby E, McFarland K, Borch K, Westh P (2011) Kinetics of enzymatic high-solid hydrolysis of lignocellulosic biomass studied by calorimetry. Appl Biochem Biotechnol 163:626–635. doi: 10.1007/s12010-010-9068-z CrossRefGoogle Scholar
  24. Park CY, Ryu YW, Kim C (2001) Kinetics and rate of enzymatic hydrolysis of cellulose in supercritical carbon dioxide. Korean J Chem Eng 18:475–478. doi: 10.1007/BF02698293 CrossRefGoogle Scholar
  25. Pavlov IN, Denisova MN, Makarova EI, Budaeva VV, Sakovich GV (2015) Versatile thermobaric setup and production of hydrotropic cellulose therein. Cellul Chem Technol 49:847–852Google Scholar
  26. Peri S, Karra S, Lee YY, Karim MN (2007) Modeling intrinsic kinetics of enzymatic cellulose hydrolysis. Biotechnol Prog 23:626–637. doi: 10.1021/bp060322s CrossRefGoogle Scholar
  27. Prathyusha N, Kamesh R, Rani KY, Sumana C, Sridhar S, Prakasham RS, Yashwanth VV, Sheelu G, Kumar MP (2016) Modelling of pretreatment and saccharification with different feedstocks and kinetic modeling of sorghum saccharification. Bioresour Technol 221:550–559. doi: 10.1016/j.biortech.2016.09.007 CrossRefGoogle Scholar
  28. Rocha MSRS, Pratto B, de Sousa R Jr, Almeida RMRG, da Cruz AJ (2017) A kinetic model for hydrothermal pretreatment of sugarcane straw. Bioresour Technol 228:176–185. doi: 10.1016/j.biortech.2016.12.087 CrossRefGoogle Scholar
  29. Shill K, Miller K, Clark DS, Blanch HW (2012) A model for optimizing the enzymatic hydrolysis of ionic liquid-pretreated lignocellulose. Bioresour Technol 126:290–297. doi: 10.1016/j.biortech.2012.08.062 CrossRefGoogle Scholar
  30. Shumny VК, Veprev SG, Nechiporenko NN, Goryachkovskaya TN, Slynko NM, Kolchanov NA, Peltek SE (2010) A new form of Miscanthus (Chinese silver grass, Miscanthus sinensis–Andersson) as a promising source of cellulosic biomass. Adv Biosci Biotechnol 1:167–170. doi: 10.4236/abb.2010.13023 CrossRefGoogle Scholar
  31. Si S, Chen Y, Fan C, Hu H, Li Y, Huang J, Liao H, Hao B, Li Q, Peng L, Tu Y (2015) Lignin extraction distinctively enhances biomass enzymatic saccharification in hemicelluloses-rich Miscanthus species under various alkali and acid pretreatments. Bioresour Technol 183:248–254. doi: 10.1016/j.biortech.2015.02.031 CrossRefGoogle Scholar
  32. Sousa R Jr, Carvalho ML, Giordano RLC, Giordano RC (2011) Recent trends in the modeling of cellulose hydrolysis. Braz J Chem Eng 28:545–564. doi: 10.1590/S0104-66322011000400001 CrossRefGoogle Scholar
  33. Sun S, Sun S, Cao X, Sun RC (2016) The role of pretreatment in improving the enzymatic hydrolysis of lignocellulosic materials: a review. Bioresour Technol 199:49–58. doi: 10.1016/j.biortech.2015.08.061 CrossRefGoogle Scholar
  34. TAPPI method T222 om-83 (1999) Acid-insoluble lignin in wood and pulp. In: Test methods 1998–1999. TAPPI PressGoogle Scholar
  35. Thygesen A, Oddershede J, Lilholt H, Thomsen AB, Stahl K (2005) On the determination of crystallinity and cellulose content in plant fibres. Cellulose 12:563–576. doi: 10.1007/s10570-005-9001-8 CrossRefGoogle Scholar
  36. Yadav MP, Hicks KB, Johnston DB, Hotchkiss AT, Chau HK, Hanah K (2016) Production of bio-based fiber gums from the waste streams resulting from the commercial processing of corn bran and oat hulls. Food Hydrocoll 53:125–133. doi: 10.1016/j.foodhyd.2015.02.017 CrossRefGoogle Scholar
  37. Ye Z, Berson ER (2011) Kinetic modeling of cellulose hydrolysis with first order inactivation of adsorbed cellulose. Bioresour Technol 102:11194–11199. doi: 10.1016/j.biortech.2011.09.044 CrossRefGoogle Scholar
  38. Yu G, Afzal W, Yang F, Padmanabhan S, Liu Zh, Xie H, Shafy MA, Bella AT, Prausnitz JM (2014) Pretreatment of Miscanthus× giganteus using aqueous ammonia with hydrogen peroxide to increase enzymatic hydrolysis to sugars. J Chem Technol Biotechnol 89:698–706. doi: 10.1002/jctb.4172 CrossRefGoogle Scholar
  39. Zhang Y, Xu B, Zhou W (2014) On a novel mechanistic model for simultaneous enzymatic hydrolysis of cellulose and hemicellulose considering morphology. Biotechnol Bioeng 111:1767–1781. doi: 10.1002/bit.25244 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Laboratory of Bioconversion, Laboratory of Chemical Engineering Processes and ApparatusesInstitute for Problems of Chemical and Energetic Technologies, Siberian Branch of the Russian Academy of Sciences (IPCET SB RAS)BiyskRussia

Personalised recommendations