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Optimization of sulfuric acid hydrolysis conditions for preparation of nanocrystalline cellulose from enzymatically pretreated fibers


Preparation of nanocrystalline cellulose (NCC) by 62 and 65 % wt. sulfuric acid hydrolysis of cellulase-pretreated fibers was optimized to obtain the highest yield by applying two statistical plans. At optimal conditions (10 U/g odp cellulase, 25 min hydrolysis, 47 °C, 62 wt.% H2SO4), high yields (≥80 %) were obtained, including an increase of ~9 points due to the enzyme. Optimal conditions produced nanosized particles of around ~200 nm with reduced surface charge and sulfur content. The optimization allowed reduction of hydrolysis time by 44 % and increase of yield by more than 10 points compared with results in previous work. The effects of cellulase pretreatment were noticeable even under aggressive hydrolysis conditions, emphasizing its possibilities. Zeta potential and polydispersity index indicated that all studied conditions led to good-quality final products, with values around −50 mV and 0.2, respectively. Transmission electron microscopy (TEM) analysis confirmed the presence of NCC. Fourier-transform infrared (FTIR) spectroscopic analysis provided evidence that cellulase treatment increased the crystallinity of both cellulose fibers and NCC, as well as fiber accessibility, supporting the other analyses of NCC.

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Cellulase-treated fibers


NCC obtained from cellulase-pretreated fibers




Fourier-transform infrared


Control fibers


NCC obtained from control fibers


Nanocrystalline cellulose


Oven-dried pulp


Lateral order index


Total crystallinity index


Enzymatic activity unit


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Authors are grateful to Ministerio de Economía y Competitividad (Spain) for support of this work under the BIOSURFACEL (CTQ2012-34109, funding also from the Fondo Europeo de Desarrollo Regional, FEDER) and BIOPAPμFLUID (CTQ2013-48995-C2-1-R) projects and an FPI grant (BES-2011-046674). Special thanks are also due to the consolidated research group AGAUR 2014 SGR 534 with Universitat de Barcelona (UB). We are also grateful to Celsur and Fungal Bioproducts for supplying cotton linters and enzyme, respectively.

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Correspondence to M. Blanca Roncero.

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Beltramino, F., Roncero, M.B., Torres, A.L. et al. Optimization of sulfuric acid hydrolysis conditions for preparation of nanocrystalline cellulose from enzymatically pretreated fibers. Cellulose 23, 1777–1789 (2016).

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  • Nanocrystalline cellulose
  • Cellulose nanocrystals
  • Optimization
  • Cellulase
  • Enzymatic treatment
  • Yield increase