Transition of cellulose supramolecular structure during concentrated acid treatment and its implication for cellulose nanocrystal yield

Journal Pre-proof Transition of cellulose supramolecular structure during concentrated acid treatment and its implication for cellulose nanocrystal yield Lida Xing, Chuanshuang Hu, Weiwei Zhang, Litao Guan, Jin Gu

PII:

S0144-8617(19)31207-X

DOI:

https://doi.org/10.1016/j.carbpol.2019.115539

Reference:

CARP 115539

To appear in:

Carbohydrate Polymers

Received Date:

29 July 2019

Revised Date:

25 October 2019

Accepted Date:

26 October 2019

Please cite this article as: Xing L, Hu C, Zhang W, Guan L, Gu J, Transition of cellulose supramolecular structure during concentrated acid treatment and its implication for cellulose nanocrystal yield, Carbohydrate Polymers (2019), doi: https://doi.org/10.1016/j.carbpol.2019.115539

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Transition of cellulose supramolecular structure during concentrated acid treatment and its implication for cellulose nanocrystal yield Lida Xing, Chuanshuang Hu*, Weiwei Zhang, Litao Guan, Jin Gu* College of Materials and Energy, South China Agricultural University, Guangzhou, 510642, PR China *Corresponding Author Email: [email protected], [email protected]; Tel.: +86-2085282568; Fax: +86-

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2085281885.

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Graphical abstract

Highlights

Cellulose II nanocrystals were derived from H2SO4 treated cellulose I at restricted

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

conditions.

Cellulose crystalline structure transition resulted in a rapid change of nanocrystal

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

The regenerated cellulose II nanocrystals showed a twisted strip structure.

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

yield.



A tentative model of cellulose I and II nanocrystals formation was proposed.

Abstract Cellulose nanocrystals with cellulose I and II allomorphs (CNC-I and CNC-II) were 1

prepared from eucalyptus cellulose I substrate by controlling the sulfuric acid hydrolysis conditions, including acid concentration (56-64 wt%), reaction temperature (45 or 60 °C) and time (10-120 min). The crystalline structures were verified by XRD and 13C-NMR. CNC-II only appeared at very restricted reaction conditions. The rapid cellulose supramolecular structure transition under sulfuric acid concentration of around 60 wt% resulted in an abrupt change in CNC yield. A maximal CNC yield of 66.7 % was obtained at acid concentration of 58 wt% and reaction temperature of 60 °C. CNC-I exhibited spindle-shape, while CNC-II showed a twisted strip structure.

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The state of order in cellulose during the acid hydrolysis process has been studied using a coagulation method. A tentative model of CNC-I and CNC-II formation was then proposed. This work provided significant knowledge for the production of CNCs with high yield and controllable allomorph.

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Keywords: Cellulose nanocrystal; Sulfuric acid hydrolysis; Cellulose supramolecular

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structure; X-ray diffraction; Yield

1. Introduction

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Cellulose nanocrystals (CNCs) are a type of renewable nanomaterials commonly resulting from acid hydrolysis of native cellulose fibers. CNCs usually present short

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rod shape with a length of several hundred nanometers and a width of less than 100 nm. They have drawn a great interest in applying to food, additives, medical materials, template reagents, adsorbents, and energy storage materials, etc (Chu et al., 2015; Gu

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& Catchmark, 2013a, 2013b; Hu, Gu, Jiang, & Hsieh, 2016; Jiao et al., 2018; Moon, Martini, Nairn, Simonsen, & Youngblood, 2011; You et al., 2016), owing to their

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unique and excellent properties including high Young modulus, specific surface area, transparency and low thermal expansion coefficient, and biocompatibility (Huang, Chang, Lin, & Dufresne, 2014). Sulfuric acid, which could introduce negatively charged sulfate ester groups to CNCs and thus give rise to a stable suspension, is the most widely used acid to produce CNCs among all acid types (Moon et al., 2011). Previous research has 2

showed that sulfuric acid concentration, hydrolysis time and temperature have significant influences on the yield and properties of CNCs (Chen et al., 2015). Over the past few decades, the standard condition has been approximately 64 wt% sulfuric acid for producing CNCs with a yield usually less than 30 % (Araki, Wada, Kuga, & Okano, 1998; Bondeson, Mathew, & Oksman, 2006). However, recent studies indicated that 64 wt% was not the optimal acid concentration for CNC production (Chen et al., 2015). An abrupt change in CNC yield at acid concentrations between 56 and 58 wt% was observed using bleached eucalyptus wood pulp as cellulose source

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(Wang, Zhao, & Zhu, 2014; Wang et al., 2012). And over 70 % yield was achieved when acid concentration was controlled between 58-62 wt% (Chen et al., 2015). Although the morphology and yield of the CNCs produced in different acid conditions

have been well studied (Bo et al., 2016; Chen et al., 2015; Dong, Bortner, & Roman,

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2016; Wang et al., 2014), the supramolecular structure transition of cellulose during

transition was not well understood.

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this process have not been carefully analyzed and the mechanism behind this sharp

Cellulose contains highly ordered crystalline regions and disordered regions.

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Depending on molecular orientation and the hydrogen-bond network, different cellulose allomorphs (cellulose I–IV) have been identified (Moon et al., 2011; Trache,

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Hussin, Haafiz, & Thakur, 2017). Generally, native cellulose exhibits cellulose I allomorph with parallel chain orientation, while regenerated or mercerized cellulose is cellulose II with anti-parallel fashion. The allomorph of the cellulose has a significant

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impact on its optical, mechanical and thermal properties (Moon et al., 2011; Rinaldi & Schuth, 2009). During the acid treatment, it is widely accepted that the less ordered

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regions of cellulose are more susceptible to hydrolyze while the crystalline regions are relatively resistant to acid penetration. Thus, the resulting highly crystalline CNCs are expected to retain the same crystal allomorph as the original cellulose (Gong, Li, Xu, Xian, & Mo, 2017; Han, Zhou, Wu, Liu, & Wu, 2013). However, production of CNC-II from cellulose I sources by direct acid hydrolysis has been reported in very few studies (Ioelovich, 2012; Naduparambath et al., 2018; Neto et al., 2016; Sebe, 3

Ham-Pichavant, Ibarboure, Koffi, & Tingaut, 2012). In our previous study (Xing, Gu, Zhang, Tu, & Hu, 2018), when cellulose I sources were hydrolyzed using 64 wt% sulfuric acid at 45 °C, a partial transformation of cellulose allomorph (cellulose I cellulose II) was observed within 15 min. The cellulose II content in CNCs decreased with reaction time and only pure cellulose I nanocrystals were obtained after acid hydrolysis for more than 30 min. Concentrated sulfuric acid may mercerize or dissolve surface cellulose chains and result in cellulose allomorph transformation. While our previous study indicated that hydrolysis time had a significant impact on

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the CNC allomorph, the effect of acid concentration and reaction temperature on the transition of cellulose I to cellulose II have not been systematically studied in all those previous works. The detailed mechanism of the phenomenon is still not well known. CNC-II is different from CNC-I in morphology, self-assembling structure, and

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performances of their derived composites (Gong, Mo, & Li, 2018; Han et al., 2013; Neto et al., 2016) and shows potential applications in optical device and biomedicine

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(Neto et al., 2016).

In this work, by carefully controlling the sulfuric acid hydrolysis conditions,

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including acid concentration (56-64 wt%), reaction temperature (45 or 60 °C) and time (10-120 min), we systematically investigated the formation mechanism of

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cellulose I and II particles from eucalyptus cellulose I substrates. The crystalline structure and morphology of CNC I and CNC II were explored by X - ray diffraction (XRD), solid-state cross-polarization magic angle spinning (CP/MAS) nuclear

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magnetic resonance (13C-NMR), Fourier-transform infrared spectroscopy (FTIR), and transmission electron microscopy (TEM). A relationship between cellulose

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supramolecular structure transition and CNC yields has been established for the first time.

2. Materials and methods 2.1 Materials Cellulose I substrates used in this study were isolated from eucalyptus wood 4

(Eucalyptus urophylla × E. grandis, Jiangmen Mujiangweihua Inc.). All chemical reagents were provided by Guangzhou Chemical Reagents CO., including Toluene (99.5 wt%), ethanol (CH3CH2OH, 99.7 wt%), sodium chlorite (NaClO2, 80 wt%), acetic acid glacial (CH3COOH, 99.5 wt%), potassium hydroxide (KOH, 85 wt%) and concentrated sulfuric acid (H2SO4, 95-98 wt%). The deionized water used was purified by AIKE Advanced-II-OS water purification system (Chengdu Kangning Science and Technology Development Company). 2.2 Purification of cellulose from eucalyptus wood

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Eucalyptus wood was milled to pass through an 80-mesh screen. Cellulose was produced through a three-step chemical treatment process, namely toluene/ethanol extraction, acidified NaClO2 oxidation of lignin, and KOH dissolution of hemicelluloses, as in our previous study (Xing et al., 2018). The freeze-dried

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(SJIA-10NBA, Ningbo, China) cellulose product was pulverized to pass through an 80-mesh screen and stored in a sealed bag at ambient condition before use. The

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moisture content of the cellulose was measured to be 3.4 wt%. 2.3 Preparation of cellulose nanocrystals (CNCs)

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Preheated sulfuric acid (56-64 wt%) was rapidly added to eucalyptus cellulose with a cellulose to acid ratio of 1/10 (g/mL). Then the mixture was immediately heated to

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45 or 60 °C, and reacted for 10 to 120 min under magnetic stirring. Acid hydrolysis was stopped by diluting the mixture with 10-fold ice water, and the excess sulfuric acid was removed by centrifugation at 5000 rmp for 15 min at 5 °C (TGL-20000Cr,

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An Ting, Shanghai). The precipitate was transferred into dialysis membranes with a molecular weight cut off of 3500 and dialyzed against deionized water until constant

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pH reached. After the dialysis process, the suspension was sonicated (40 Hz, 80 watts, KH2200E, Kunshan Heli ultrasonic instruments Co., Ltd, China.) in an ice bath for 10 min and centrifuged again at 5000 rpm for 15 min to separate fine CNC supernatant from partially hydrolyzed, precipitable cellulose solid residue (CSR) (Chen et al., 2015; Xing et al., 2018). The CNC suspension was concentration adjusted to 0.1 wt% and stored at 4 °C until use. The CSRs were freeze-dried and stored in sealed bags 5

under room temperature for further characterization. In the following sections, all the CNC and acid concentrations mentioned are based on weight percentage in unit volume of solutions. We use (A in %, t in min, T in °C) to abbreviate acid hydrolysis conditions. 2.4 Transition of cellulose supramolecular structure during concentrated acid hydrolysis Eucalyptus cellulose was completely homogenized in preheated 64 wt% sulfuric acid (38 °C) by using a glass rod. Then the reaction temperature was raised to 45 °C.

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The reaction stream was sampled at 2, 10 and 30 min using a pipette to obtain time-dependent information of the cellulose supramolecular structure during acid hydrolysis. Each sample was evenly divided into three parts and poured into 20-fold ice water, 10 wt% NaOH solution and ethanol, respectively. The samples transferred

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into water and 10 wt% NaOH solution were washed with plenty of water, while the

sample with ethanol was still washed by ethanol. After that, the samples were quickly

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frozen by liquid nitrogen and freeze-dried. The freeze-dried samples were recorded as post water-treated cellulose (WC), sodium hydroxide-treated cellulose (SHC) and

2.5 Analytical methods

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2.5.1 CNC yields

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ethanol-treated cellulose (EtC).

The yields of CNC and CSR were calculated according to equation (1) and (2):

(1)

𝑐𝑉 𝑚

× 100

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𝑌𝐶𝑁𝐶 (%) =

𝑚𝐶𝑆𝑅 𝑚

× 100

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𝑌𝐶𝑆𝑅 (%) = (2)

where YCNC and YCSR are CNC and CSR yields, respectively; m, mCSR, c and V are the mass of initial cellulose, the mass of CSR, the concentration and volume of the CNC suspension, respectively. 2.5.2 CNC Morphology 6

The morphology of CNCs was observed by transmission electron microscope (TEM). CNC suspensions (0.005 %) of 10 μL were deposited onto glow-discharged carbon-coated TEM grids (300-mesh copper, formvar-carbon, Ted Pella Inc) and a filter paper was used to absorb the excess liquid after 2 min. Uranyl acetate solution of 2 wt% was applied to stain the specimens for 2 min, and the excess stain solution was removed by blotting with a filter paper. After that, the samples were allowed to dry at room temperature and observed using a transmission electron microscope (JEOL, JEM-2100F, Japan) operated at 100 kV accelerating voltage. The size

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distribution of the CNCs was determined by over 100 representative CNCs using ImageJ and Origin Pro software. 2.5.3 Crystalline structure analysis

XRD powder diffractometer (D8 Advance, Bruker) with Cu Kα radiation at an

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operating voltage of 40 kV and a filament current of 40 mA was used to study the crystal structure of eucalyptus wood flour, cellulose, CNCs and CSRs, as well as WC,

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SHC and EtC. The samples were grounded to powder and placed on the XRD sample plate followed by a slight press with a glass slide. The samples were scanned from 5

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to 50° (2θ) at a rate of 2° /min. XRD spectra were analyzed by means of MDI Jade6.0, and each peak profile was fit to the Pearson VII model as described in our previous

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study (Xing et al., 2018). The crystallinity index (CrI), the contents of cellulose I (CI) and cellulose II (CII) were calculated as reported previously (Xing et al., 2018). The dimension (Dhkl) of the crystallites perpendicular to the hkl diffracting planes was

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estimated from the peak broadening in the XRD profiles after deconvolution, using Scherrer's equation: 𝐾𝜆

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𝐷ℎ𝑘𝑙 = 𝛽

(3)

1/2 cos 𝜃

where K is a correction factor usually taken as 0.9, λ is the X-ray wavelength, θ is the diffraction angle and β1/2 is the peak width at half maximum intensity. The crystalline structure of CNCs was also study by solid-state

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C-NMR. The

measurements were performed on a Bruker AVANCE 400 spectrometer using a combination of cross-polarization, high power proton decoupling and magic angle 7

spinning (CP/MAS).

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C-NMR spectra were acquired at 298 K, with a 4 mm probe

operating at 100.13 MHz. MAS rotation was 12 kHz with CP contact time of 2 ms and repetition time of 1 s. 2.5.4 FTIR analysis FTIR spectra of eucalyptus cellulose and CNCs were obtained using a FTIR spectrometer (PerkinElmer, USA). For sample preparation, 1 mg of freeze-dried powder was grinded with 100 mg KBr and pressed into a circular pellet. The spectra were recorded with a resolution of 4 cm−1 over a range of 4500 to 450 cm−1 and

3. Results and discussion 3.1 Sulfuric acid hydrolysis of cellulose I substrates

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averaged from 60 scans in transmittance mode.

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Cellulose fibers were efficiently separated from eucalyptus wood flours following

the previously established three-step process described earlier (Gu et al., 2017; Xing

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et al., 2018). Infra-red and XRD results confirmed the removal of hemicelluloses and lignin (Figure S1 and S2). The cellulose fibers exhibited Iβ crystalline allomorph as

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natural higher-plant cellulose.

Cellulose fibers were subjected to sulfuric hydrolysis treatments with acid

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concentration and hydrolysis time varying in ranges between 56-64 % and 10-120 min at 45 or 60 °C. The appearance of the reaction mixtures during the hydrolysis process has been recorded in Table 1. When the reaction temperature was 45 °C, and with an

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acid concentration no more than 58 %, all suspensions were ivory white or light yellow during acid hydrolysis. However, with an acid concentration above or equal to

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60 %, the suspension became translucent or even transparent in the first 10-15 min, indicating swelling or gradual dissolution of cellulose. When the reaction temperature was 60 °C, with long reaction time and high acid concentration (≥62 %), the mixture exhibited a brownish color due to the over hydrolysis of cellulose. Translucent or transparent mixtures were observed with extended reaction time of 30-60 min at high acid concentration (≥60 %) perhaps associated with smaller cellulose particles in the 8

suspensions.

Table 1 Appearance of the eucalyptus cellulose - sulfuric acid reaction mixtures in different conditions. t/min

Appearance of mixtures during hydrolysis

°C

A/%

10 - 15 min

15 - 30 min

30 - 60 min

56

Ivory white

Ivory white

Light yellow

58

Ivory white

Ivory white

Light yellow

60

Translucent

Light yellow and translucent

Light yellow

62

Translucent

Light yellow and transparent

NE

64

Transparent

Light yellow and transparent

NE

56

Ivory white

Ivory white

Light yellow

58

Ivory white

Ivory white

60

Ivory white

62

Light yellow

Brown

64

Light yellow

Brown

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Light yellow

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60

Light yellow

Light yellow and transparent Brown and transparent Brown and transparent

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45

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T/

3.2 CNC yields

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NE: No experiment was carried out.

The yields of CNCs and corresponding CSRs were measured by equation (1) and

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(2), and the results were showed in Fig. 1. Temperature, acid concentration and reaction time all significantly affected CNC yields. Hydrolysis occurred slowly at low

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temperature. At 45 °C hydrolysis temperature, generally lower CNC yields were obtained. When the acid concentration was 56 or 58 %, a large amount of CSRs (55-82 %) were observed even with extended reaction time up to 120 min, indicating that cellulose was insufficiently depolymerized under these reaction conditions. The CNC yields (2-20 %) were extremely low under these reaction conditions. When the acid concentration was increased to 60 %, cellulose was effectively hydrolyzed resulting in higher CNC yield of 41.3 % within 60 min. However, if the acid 9

concentration was higher than 62 %, CNC yield (4-26 %) would again lower than those produced with 60 % acid, and there was no CSR after 30 min due to the rapid hydrolysis of cellulose. The yields of the CNCs increased in the first 20 min and then decreased with the reaction time up to 30 min. At 60 °C hydrolysis temperature, CNC yield rapidly increased with increasing hydrolysis time (within 60 min) using 56 or 58 % sulfuric acid. The big cellulose fibers were broken to yield small CNCs. These results could be attributed to the increase in hydrolysis rate in high reaction temperature, and perhaps also due to the

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enlargement of hydrolysis rate difference between disordered regions and crystal regions. The maximal CNC yield (66.7 %) was observed using 58 % acid and a

reaction time of 60 min. Interestingly, similar CNC yield (approximately 53 %) was achieved in various hydrolysis times using 60 % acid, which meant the disordered

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regions in cellulose almost complete depolymerized while crystal regions were

difficult to be penetrated in this hydrolysis condition. CNCs obtained using 62 and 64 %

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acid were greatly less than those derived using 58-60 % acid at all hydrolysis times. Similar to 60 % acid hydrolysis, change of CNC yields along with time was

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unapparent. The big cellulose fibers were quickly hydrolyzed into small molecules within 10 min, and the resulting small CNCs seemed to be relatively more resistant to

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acid hydrolysis.

Fig. 1 Effect of sulfuric acid hydrolysis conditions on the yield of CNCs and the corresponding CSRs. The reactions were carried out at either 45 or 60 °C. The 10

numbers above the bars indicate acid reaction time.

These results are consistent with a previous study using bleached eucalyptus kraft pulp as the starting material (Chen et al., 2015). In their study, CNC yield reached maximum (~70 %) when the sulfuric acid concentration was 58-62 %. They also demonstrate that acid concentration is the key parameter to control CNC productions. In a view of cellulose interaction with concentrated sulfuric acid, CNC yield is expected to be maximized if sulfuric acid selectively reacts with disordered regions

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and releases the crystalline regions. Early investigations with the purpose to develop methods for dissolution and hydrolysis of cellulose materials showed that at low

temperatures, the sulfuric acid with concentrations greater than 62-63 % caused swelling and dissolving of cellulose that accompanied by hydrolysis (Huang, Wang,

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Zhang, & Chen, 2016). Increased acid treatment temperature promoted both hydrolysis and dissolving of cellulose (Huang et al., 2016). Interestingly, the acid

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concentration (>62-63 %) requires for dissolution of cellulose was just a little above the acid concentration (58-62 %) optimal for maximum CNC yield. Thus one could

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quickly make an assumption that at increased concentration (>~62 %), sulfuric acid is capable to break the intermolecular hydrogen bonds and penetrate into crystalline

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domains of cellulose resulting in low CNC yield. 3.3 Investigation of cellulose crystallites structure by XRD The crystal structure of CNCs and CSRs was analyzed by XRD as shown in Fig.

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2 and Fig. S3. Depended on the hydrolysis conditions, typical cellulose I, cellulose II or mixture of cellulose I and II profiles were obtained. At 45 °C hydrolysis

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temperature, CNCs obtained at low sulfuric acid concentration (56 and 58 %) always exhibited typical cellulose I XRD patterns, with the characteristic diffraction peaks at 2θ angles of about 15, 16.5 and 22.5°corresponding to the cellulose I (11̅0), (110) and (200) crystal planes, respectively. However, with increased acid concentration but short reaction time, CNC (A60, t10, T45) exhibited typical cellulose II diffraction pattern, as identified by cellulose II diffraction peaks at 2θ=12, 20 and 22° relating to 11

the (11̅0), (110) and (200) lattice planes. With long hydrolysis time, CNC (A60, t20, T45) reemerged cellulose I diffraction patterns. Similarly, CNC (A62, t10, T45) showed cellulose II diffraction profile, while CNC (A62, t15, T45) and (A62, t20, T45) were cellulose I and II hybrids. Cellulose II components decreased with increasing hydrolysis time. When hydrolysis time reached up to 30 min, CNC (A62, t30, T45) was pure cellulose I. Similar trend also observed for CNCs prepared using 64 % sulfuric acid. Namely, cellulose II nanocrystals were prepared during the first 20 min, then CNC (A64, t30, T45) exhibited mixed cellulose I and II diffraction pattern (Xing

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et al., 2018). To understand the distribution of cellulose I and II components in the entire system, XRD profiles of the CSRs were also obtained (Fig. S3). Generally,

CSRs exhibited similar XRD patterns to their respectively CNC supernatant, but

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always contained relatively higher cellulose I content.

Fig. 2 XRD profiles of CNCs prepared at 45 °C (a) and 60 °C (b) for various acid concentrations and hydrolysis times. The XRD profiles of CNC (A64, t15, T45), (A64, t20, T45) and (A64, t30, T45) were taken from (Xing et al., 2018).

Interestingly, all CNC and CSR samples, derived at 60 °C under all acid 12

concentration (56-64 %) and hydrolysis time (10-60 min), still showed cellulose I XRD patterns which were the same as original cellulose I substrate. According to our previous study (Xing et al., 2018), a peak deconvolution method was used to estimate the contents of cellulose I (CI) and cellulose II (CII) components in the samples derived at 45 °C under 60, 62 and 64 % acid concentration, and the results are shown in Fig. 3. The content of CII in CNCs and CSRs decreased with reaction time. When the acid concentration was 60 %, CNC (A60, t10, T45) obtained with 10 min hydrolysis was pure CII, while the CII content in the

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corresponding CSR (A60, t10, T45) was only 13.0 %. Extending hydrolysis time to 15 min resulted in CNC (A60, t15, T45) with only 18.6 % CII, while the corresponding CSR (A60, t15, T45) was pure CI. Further increasing the hydrolysis time up to 20 or 30 min, both CNCs and CSRs became pure CI. When the acid

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concentration was 62 %, similar trend was observed. With the increase of hydrolysis

time, the content of CII in CNCs decreased. The contents of CII in CNCs prepared at

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10, 15, 20 and 30 min hydrolysis time were 100, 88.0, 34.4 and 0 %, respectively. And the content of CII in corresponding CSRs also showed a decreasing trend of 63.7,

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18.2 and 18.6 % (no CSR was obtained at 30 min). When the concentration of sulfuric acid increased to 64 %, the CNCs and CSRs obtained in 10, 15 or 20 min were

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completely CII (no CSR was obtained after 20 min). When the hydrolysis time

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reached 30 min, CI reappeared in CNC (A64, t30, T45), and its content was 97.3 %.

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Fig. 3 CI and CII percentages of CNCs and corresponding CSRs derived from sulfuric

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acid hydrolysis (60, 62 and 64 %) at 45 °C for various hydrolysis times.

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At low reaction temperature (45 °C) and higher acid concentration (≥60 %), acid molecules were able to reach cellulose crystalline zones, disrupt the hydrogen bonds, swell or dissolve crystalline cellulose. The dissolved cellulose recrystallized to

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generate cellulose II particles. Some cellulose II perhaps formed on the surface of small CNC-I that acted as seed, which is known as shish-kebab formation. The newly

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regenerated cellulose II would be subjected to degrade over reaction time (Xing et al., 2018). With increasing acid concentration, the solubility of cellulose increased.

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Similar results were reported by Ioelovich (Ioelovich, 2012). Cellulose could become completely swollen or dissolved in 64 % sulfuric acid as indicated by the transparent appearance of the mixture (Table 1) and the pure CII composition of CNC/CSR (A64, t10, T45) and (A64, t15, T45). At low reaction temperature (45 °C) and low acid concentration (≤58 %), sulfuric acid hardly reacted with crystalline regions. Transition of cellulose supramolecular structure during concentrated acid treatment has 14

significant impact on CNC yields. While cellulose was in a non-swollen state (A≤58 %), the intermolecular hydrogen bonds between crystalline cellulose chains precluded the entrance of acid and resulted in slow depolymerization. Dissolution of cellulose accelerated scissoring of polymer (A≥60 %). Thus an increase of CNC yield was observed using 60 % acid. However, acid penetration to crystalline regions also led to degradation of cellulose to small oligosaccharides and glucose, again giving rise to low CNC yield at higher acid concentration (>60 %). At high reaction temperature of 60 °C, cellulose II component was not observed

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in any CNC or CSR samples. Perhaps at elevated temperature, splitting cellulose chains quickly degraded to small oligosaccharides and no crystalline CNC-II could be

regenerated. CNC (A58, t60, T60) achieved a maximum yield of 66.7 % when the

disordered regions at an accelerated pace.

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sulfuric acid was barely blocked from the crystalline regions, but hydrolyzed the

To our knowledge, a very few studies also reported production of cellulose II

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nanocrystals by sulfuric acid hydrolysis of cellulose I. In Haafiz et al (Haafiz, Hassan, Zakaria, & Inuwa, 2014), CNCs with a mixture of CI and CII allomorphs were

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produced by sulfuric acid hydrolysis of oil palm microcrystalline cellulose I (64 % sulfuric acid, 40 °C, 60 min). In Neto et al (Neto et al., 2016), cellulose II

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nanocrystals were also prepared by sulfuric acid hydrolysis of eucalyptus cellulose I (64 % sulfuric acid, 40 °C, 20 min). In Naduparambath et al (Naduparambath et al., 2018), CNCs with co-existence of CI and CII were produced by sulfuric acid

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hydrolysis of sago seed shell cellulose I (64 % sulfuric acid, 45 °C, 45 min). In addition, Hu and Hashaikeh (Hu, Hashaikeh, & Berry, 2014) showed that at controlled

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short-time (25 min) hydrolysis of northern bleached softwood cellulose with 64 % sulfuric acid (45 °C), the insoluble part of cellulose exhibited CI crystalline structure, while the cellulose regenerated from acidic solutions always had CII crystalline allomorph. Although none of these studies systematically analyzed the reaction conditions for production of cellulose II nanocrystals, they all used low reaction temperature (40 or 45 °C) and high acid concentration (64 %). Crystallinity and 15

crystal size of the original cellulose materials may have an impact on the exact allomorph transition point. Moreover, variations of actual sulfuric acid concentration would also affect comparison of different studies. For example, commercially available sulfuric acid always has a concentration of 95-98 %. Moisture content of the cellulose materials may also vary. Besides transition of cellulose supramolecular structure, cellulose crystallinity also changed with different hydrolysis conditions (Table 2). Cellulose I substrates purified from eucalyptus wood had a CrI of 70.8 % (Xing et al., 2018). In general, at

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low hydrolysis temperature (45 °C), the CrI of CNCs or CSRs was usually higher than initial cellulose within 60 min when acid concentration was no more than 58 %. Degradation of cellulose had occurred at disordered regions with high accessibility.

Interestingly, while acid concentration was increased to 60-64 %, the CrI of the CNCs

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decreased in the first 10 min (CrI: 66.4-69.8 %) compared to initial cellulose, and then

increased with hydrolysis time up to 30 min (CrI: 74.8-76.9 %). These results agreed

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with our previous study, indicating cellulose could partially or completely dissolved in concentrated sulfuric acid. In other word, the hydrogen bonding network in crystalline

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regions was disrupted by hydrated sulfate ion resulting in more disordered regions. The newly formed disordered regions would later be hydrolyzed, leaving the

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crystalline kernels (Xing et al., 2018).

At high hydrolysis temperature (60 °C), almost all the CNCs and CSRs derived showed a higher CrI compared with the initial cellulose perhaps due to the hydrolysis

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of disordered regions. In addition, the CrI of the CNCs increased with the increase of acid concentration from 56 to 62 % and then slightly decreased with acid

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concentration up to 64 % at the same hydrolysis time. At acid concentration of 58 %, CNC yield reached to the maximum value of 66.7 %, while CrI was only 71.4 %, showing a minimal change with hydrolysis time. As a higher CrI (84.3 %) was obtained, CNC yields were relatively low at acid concentration of 60 or 62 %. In that case, the disordered and paracrystalline regions may all be hydrolyzed and the crystalline regions left. At 64 % acid concentration, the CrI and the yield of CNCs 16

were both low. Degradation of cellulose crystalline regions might happen (Chen et al., 2015).

Table 2 Crystallinity index (CrI) of CNCs and CSRs under different reaction conditions. t/min

CrI (CNC)/%

°C

A/%

10

15

20

30

60

10

15

20

30

60

56

NE

NS

NE

NS

67.1

NE

79.7

NE

80.4

80.7

58

NE

NS

NE

71.5

77.7

NE

82.1

NE

83.5

76.0

60

66.4

65.2

74.1

76.9

76.2

68.4

66.8

74.4

75.3

68.2

62

69.8

72.2

75.5

74.8

NS

75.9

72.4

69.3

NS

NS

64

69.3

66.5

64.2

75.5

NS

76.9

70.9

70.2

NS

NS

56

NE

73.5

NE

73.4

70.4

NE

80.9

NE

74.8

76.7

58

NE

73.6

NE

72.2

71.4

NE

75.0

NE

70.5

70.3

60

77.8

82.4

NE

78.8

77.1

76.5

75.2

NE

72.1

73.3

62

78.9

82.5

87.4

84.3

NS

74.2

NS

NS

NS

NS

64

75.2

72.2

77.6

76.7

NS

NS

NS

NS

NS

NS

-p

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60

re

45

CrI (CSR)/%

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T/

NE: No experiment was carried out. NS: No sample was derived under the hydrolysis

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condition. The CrI of CNC/CSR (A64, t15, T45), CNC/CSR (A64, t20, T45), CNC (A64, t30, T45) were taken from (Xing et al., 2018).

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The crystalline size (Dhkl) of ( 11̅0 ), (110), or (200) lattice planes of representative CNC-I (A58, t30, T45), (A64, t10, T60) and CNC-II (A64, t10, T45)

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was estimated by XRD (Table 3). CNC-I (A58, t30, T45) and (A64, t10, T60) had similar Dhkl of 4.21 and 4.47 nm at (11̅0), 4.29 and 4.23 at (110), 4.24 and 4.78 at (200) crystallographic planes, respectively. The values of crystalline size corresponding to (200) lattice planes are consistent with microcrystalline cellulose reported by Wada (Wada, Ike, & Tokuyasu, 2010). However, the crystalline sizes of CNC-II (A64, t10, T45) were 5.49 nm (11̅0), 7.68 nm (110) and 6.60 nm (200), 17

respectively, much larger than those of CNC-I. The same results were reported by Sèbe., et al. (Sebe et al., 2012) and Neto et al. (Neto et al., 2016). These values have to be considered as a lower bound since instrumental broadening and possible imperfections of the crystalline lattice are neglected by the method (Sebe et al., 2012).

Table 3 Crystallite size (Dhkl) of the CNC (A58, t30, T45), (A64, t10, T60) and (A64, t10, T45) from the deconvolution of XRD spectra. Dhkl (nm) 11̅0

allomorph

110

I

4.21

4.29

(A64, t10, T60)

I

4.47

4.43

(A64, t10, T45)

II

5.49

7.68

4.24 4.78 6.60

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(A58, t30, T45)

200

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Cellulose Samples

network by 13C-NMR and FTIR CP/MAS

13

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3.4 Investigation of cellulose crystalline, chemical structure and hydrogen bonding

C-NMR was used to further confirm the crystalline structure of

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representative CNC-I (A58, t30, T45), (A64, t10, T60) and CNC-II (A64, t10, T45) (Fig. 4a). Different resonances observed in

13

C-NMR spectra corresponded to 13

C-NMR spectra of CNC

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different conformations of anhydrous glucose units. The

(A58, t30, T45), (A64, t10, T60) and (A64, t10, T45) corresponded to the crystalline structure of cellulose I and II respectively, which were consistent with the XRD

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results. The signal between 60 and 70 ppm is attributed to C6. The next peak cluster between 70 and 80 ppm is associated to C2, C3 and C5. The signals in the range of 80

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to 95 ppm and 100 to 110 ppm regions come from C4 and C1, respectively. The NMR spectra of CNC-I and CNC-II show significant differences. The

13

C-NMR spectra of

CNC-I (A58, t30, T45) and CNC-I (A64, t10, T60) show the chemical shift of the crystal lattice of typical cellulose I crystal structure, with a singlet of C1 signal peak in the 100-110 ppm region. CNC-II (A64, t10, T45) shows an extra small shoulder peak at 107 ppm. In addition, C6 peak of CNC-II (A64, t10, T45) shows a chemical shift to 18

near field compared to that of CNC-I. The small peak at around 96 ppm is related to the C1 of the terminal glucose unit (the reduction end of cellulose C1). When the polymerization degree (DP) of cellulose is low enough, it could be detected. CNC-II seemed to have shorter cellulose chains than that of CNC-I. These results are consistent with a previous study (Hu et al., 2014), which showed the DP of the recrystallized acid-soluble cellulose was relatively low. The changes of chemical structure and hydrogen bonding network during the sulfuric acid hydrolysis were studied by FTIR (Fig. 4b). Cellulose characteristic peaks

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were observed in all spectra at around 3350, 2900 and 1063 cm−1 corresponding to stretching vibration of O-H, C-H and C-O bonds distortion in glucoside, respectively

(Jiang & Hsieh, 2015). The spectra of EC, CNC-I (A58, t30, T45) and CNC-I (A64, t10, T60) were similar. These results confirmed that the cellulose supramolecular

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structure retained the same at those hydrolysis conditions. Compared to CNC-I, the IR spectrum of CNC-II (A64, t10, T45) had two additional weak peaks at 3499 and 3445

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cm-1 relating to the intramolecular hydrogen bonds of cellulose II (Han et al., 2013). The stretching vibration peak of CH in methyl and methylene group shifted from

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2902 to 2892 cm-1 (Oh et al., 2005). The symmetric bending vibration peak of CH2 at 1428 cm-1 shifted to a lower wave number at 1420 cm-1, which indicated the

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formation of new inter- and intramolecular hydrogen bonds and change of CH2OH conformation from tg to gt (Han et al., 2013). The stretching vibration peak of C-O at C6 shifted from 1034 to 1024 cm-1 (Marta, Smith, Gidley, & Wilson, 2002). The band

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at 897 cm-1 assigned as asymmetric stretching vibration peak of C-O-C shifted to 895 cm-1. These results indicated the transformation of cellulose allomorph from cellulose

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I to cellulose II (Colom & Carrillo, 2002). In addition, the band at 1110 cm-1 contributed to planar stretching vibration of ring skeleton (Nelson & O'Connor, 1964), 1060 cm-1 corresponding to CO stretching vibration at C3 and C-C stretching (Marta et al., 2002), as well as C–O–H out-of-plane bending at 710 cm-1 for cellulose Iβ (Gumuskaya, Usta, & Kirci, 2003), decreased or even disappeared, which also indicated cellulose I converted into cellulose II (Han et al., 2013).These results are 19

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consistent with the XRD and 13C-NMR studies.

Fig. 4 (a) 13C- NMR of CNC-I and CNC-II. (b) FTIR spectra of a. EC, b. CNC-I (A58,

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t30, T45), c. CNC-I (A64, t10, T60), d. CNC-II (A64, t10, T45).

3.5 CNC micromorphology

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The morphology of typical CNC-I and CNC-II was studied by TEM imaging (Fig. 5). The shape and size of the three CNC samples were significantly different. Both

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CNC-I (A58, t30, T45) and (A64, t10, T60) exhibited spindle-shape that commonly appeared as the nanocrystals produced by conventional acid hydrolysis of native lignocellulose substrates (Gu et al., 2017; Lu & Hsieh, 2012), while CNC II (A64, t10,

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T45) exhibited a twisted strip structure (Neto et al., 2016). The size distribution of the CNCs is showed in Fig. S4. The length and width of CNC-I (A58, t30, T45) ranged

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from 35-350 nm and 4-30 nm, averaged 146.7±61.8 nm and 10.8±4.7 nm, respectively. While CNC-I (A64, t10, T60) was 40-300 nm in length and 4-20 nm in width,

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averaged 122.1±45.5 nm long and 9.3±3.5 nm wide. Additionally, A few tiny nanoparticles (about 4 nm wide) existed in both samples. CNC-II (A64, t10, T45) was 40-350 nm in length and 4-16 nm in width, averaged 129.5±58.5 nm long and 9.9±2.5 nm wide. Compared to the CNC-I (A58, t30, T45), CNC-I (A64, t10, T60) and CNC-II (A64, t10, T45) exhibited thinner and more uniform structure owing to the effective hydrolysis of disordered and paracrystalline regions in concentrated acid. The relatively smaller size of CNC-II (A64, t10, T45) is also consistent with its lower 20

molecular weight as indicated by NMR. The aspect ratios of three samples were calculated to be 13.6 (A58, t30, T45), 13.1 (A64, t10, T60) and 13.0 (A64, t10, T45), respectively.

CNC-I (A64, t10, T60) and c. CNC-II (A64, t10, T45)

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Fig. 5 TEM images of cellulose I and II nanocrystals: a, CNC-I (A58, t30, T45), b.

3.6 Transition of cellulose supramolecular structure during concentrated acid

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treatment

To further understand the supramolecular transition of cellulose during the

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hydrolysis process (64 % sulfuric acid, 45 °C), an indirect coagulation approach was applied to study the state of order in cellulose. After reacting with acid for various

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times (2, 10 and 30 min), the partly or completely dissolved cellulose was coagulated in different cellulose non-solvents, including ice water, 10 % sodium hydroxide solution and ethanol, respectively. The resulting cellulose products are named as WC,

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SHC and EtC, respectively. The EtC suspension always exhibited an ivory white appearance and gave rise to the highest sample mass than those of WC and SHC at all

ur

three reaction times. Acid hydrolysis of the cellulose for 2 min also yielded ivory white WC-2 and SHC-2 suspensions. However, with longer reaction time, WC-10 and

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WC-30 suspensions displayed a translucent appearance, while SHC-10 and SHC-30 were transparent and no solid samples could be produced in these conditions. XRD was used to analyze the crystal structure of all EtC, WC and SHC (Fig. 6).

EtC-2 exhibited typical amorphous cellulose XRD pattern as identified by the only broad peak at 19.7°, indicating that EtC-2 had uniformly disordered structure (Hu, Sheng, Yan, & Ke, 2018). Both WC-2 and SHC-2 were regenerated cellulose with a 21

small peak and two adjacent towering peaks at 11.5, 19.9 and 21.6°, respectively, representing (11̅0), (110) and (200) cellulose II crystal planes. EtC-10, WC-10 and EtC-30 showed typical cellulose II XRD diffraction patterns. Specially, EtC-10 and EtC-30 exhibited a less ordered structure compared to WC-10. Whereas WC-30 was almost cellulose I with diminutive peaks at 2θ = 12.0 ° and 20°, showing the

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re

-p

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coexistence of a small amount of cellulose II.

Fig. 6 XRD profiles of EtC, WC and SHC produced with 64 % sulfuric acid at 45 °C

et al., 2018).

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for 2, 10 and 30 min. The XRD profile of eucalyptus cellulose was taken from (Xing

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During the acid hydrolysis process, cellulose was dissolved in 64 % sulfuric acid at 45 °C. Then, the dissolved glucan chains may rapidly depolymerize into low

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molecular weight products. The solubility of glucans in a solvent decreased in the order of sodium hydroxide solution, water and ethanol. Thus, ethanol coagulation bath might have the potential to preserve the state of cellulose in sulfuric acid. With very short acid reaction time, it seems likely that cellulose became a uniformly disordered state as indicated by the XRD profile of EtC-2. With extended acid reaction time, the surface disordered cellulose molecules were depolymerized to low molecular weight 22

products. When the reaction was stopped by adding cellulose non-solvent, these low molecular weight products remained a disordered state in EtC and thus gave rise to the highest solid mass. Dissolved long-chain cellulose molecules may recrystallize to form cellulose II. However, when NaOH solution was used as coagulation bath, the formation of hydrogen bonds between the dissolved glucan chains was restricted, and only long glucan chains could recrystallize in the following water washing process to be regenerated cellulose. Hence, when the hydrolysis time was extended to 10 or 30 min, the cellulose products dissolved in NaOH bath, resulting in no solid samples.

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When water was used as coagulation bath, oligosaccharides were dissolved, and the remaining long glucan chains formed regenerated cellulose with low crystallinity. Interestingly, when the hydrolysis time was prolonged to 30 min, the recrystallized

WC-30 was almost completely cellulose I. There are at least two possible

-p

explanations for this phenomenon. First, in the early stage of hydrolysis process, only

the surface of the crystalline regions in cellulose was dissolved. With the deepening of

re

hydrolysis, those newly formed disordered regions were hydrolyzed and cellulose I crystalline cores left (Xing et al., 2018). The portion of the cellulose I crystalline

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cores might be too small to be detected by the XRD profiles of WC-2 and WC-10 (Fig. 6). The second explanation may be more plausible. Cellulose was uniformly dissolved

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in 64 % sulfuric acid at 45 °C. However, complicated acid-cellulose intermediates may form during the hydrolysis process. Cellulose would recrystallize to different crystalline structures in various reaction times and coagulation baths.

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Based on the discussion above, a tentative schematic model showing the formation of CNC-I, CNC-II and CNC-I/II in various acid hydrolysis conditions is

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given in Fig. 7. When high temperature (60 °C) or low temperature (45 °C) and low sulfuric acid concentration (56-58 %) was used, highly crystalline CNCs could be produced by the faster degradation of disordered regions than crystalline regions (Teixeira et al., 2011). It was an acid-catalyzed heterogeneous hydrolysis process (Rinaldi & Schuth, 2009). The hydronium ions releasing from acid molecules penetrated the disordered regions of cellulose and protonated the oxygen atom on the 23

β-1, 4 glycosidic bonds between two adjacent anhydroglucose units (Rinaldi & Schuth, 2009; Stephens, Whitmore, Morris, & Bier, 2008; Trache et al., 2017). This is also the generally accepted principle of CNC production. The hydrolysis rate of disordered regions dramatically increased when reaction temperature rose from 45 to 60 °C. With increase of acid concentration (≥60 %), crystalline regions would also be attacked, resulting in low CNC yield. Thus the highest CNC yield would achieve at

na

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-p

ro of

58 % acid hydrolysis at 60 °C.

Fig. 7 Schematic model of the CNC-I, CNC-II and CNC-I/II formation in various acid

ur

hydrolysis conditions.

When low temperature (45 °C) and moderate sulfuric acid concentration (60

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-62 %) was applied, cellulose may become partially dissolved. Alternatively, cellulose could be swollen by concentrated inorganic acid which was similar to the mercerization effect occurring in process of strong aqueous alkali treatment for cellulose (Neto et al., 2016; Sebe et al., 2012). Hydrated sulfate ion would reach the surface cellulose chains, resulting in a swollen shell as proved by the indirect coagulation method (Fig. S5). The less ordered shells were degraded by hydrolysis of 24

β-1, 4 glycosidic bonds. Furthermore, the repetitive swelling and dissolving process also happened in the median ordered paracrystalline zones. Cellulose II nanocrystal would be regenerated on the cellulose surface when the reaction was stopped by addition of ice water. Besides, the regeneration of cellulose II nanocrystals might also occur simultaneously with chain splitting in the hydrolysis process (Sebe et al., 2012). Cellulose I crystalline cores would not be affected in this procedure. With the deepening of hydrolysis, the newly formed swollen or disordered regions depolymerized but cellulose I cores retained, contributing to the final production of

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cellulose I nanocrystal. Hence, the allomorph of the CNCs would depend on the reaction time (Xing et al., 2018).

When low temperature (45 °C) and high sulfuric acid concentration (64 %) was

used, cellulose may become completely soluble in the first few minutes. Concentrated

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sulfuric aqueous solution was able to simultaneously interrupt the hydrogen bond

networks in both crystalline and disordered regions. Hydrogen ions reacted with the

re

oxygen atom of cellulose hydroxyl groups and bisulfate and sulfate anions reacted with hydrogen atom of cellulose hydroxyl groups. Homogeneous acid hydrolysis

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would happen, resulting in sharp degradation of cellulose and low CNC yield. However, as mentioned above, CNCs with different allomorphs would be derived in

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various reaction times and coagulation baths (water, 10 % NaOH solution and ethanol). It was highly possible that the crystalline cellulose dissolved in concentrated sulfuric acid still remained some degree of order. Namely, the arrangement of

ur

cellulose chains was not completely random, but might be similar to a highly swollen state. With short reaction time, the cellulose intermediate would recrystallize to be

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cellulose II which was thermodynamically more stable. With the deepening of hydrolysis, sulfuric acid might react with hydroxyl groups of the cellulose intermediate for its loose structure and formed cellulose-SO3H. The cellulose-SO3H intermediates might recrystallize to become cellulose I in aqueous solution.

4. Conclusions 25

By controlling the acid hydrolysis conditions, cellulose I nanocrystals (CNC-I) could be produced from cellulose I substrate at high temperature (60 °C) or low acid concentration (56-58 %), while cellulose II nanocrystal (CNC-II) could be derived from cellulose I substrate at a restricted range of conditions using concentrated acid (≥60 %) hydrolysis at 45 °C for short reaction time (10-20 min). Due to the transition of cellulose supramolecular structure, a transition point of CNC yield appeared at acid concentration of 58-60 %. The maximal yield gained with 58 % acid concentration at 60 °C instead of 45 °C. CNC-I exhibited spindle-shape, while the regenerated CNC-II

ro of

showed a twisted strip structure. The state of order in cellulose during the acid hydrolysis process has been studied using a coagulation method. The results indicate cellulose was uniformly dissolved with 64 % sulfuric acid at 45 °C, but some degree

of order in cellulose may still remain. A tentative model of CNC-I and CNC-II

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formation was then proposed. CNCs produced from lignocellulosic biomass with high yield, high crystallinity and controllable allomorph may have potentially diverse

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Acknowledgments

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applications.

The authors are very grateful that this research was financially supported by the

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Guangzhou Science and Technology Bureau (Project No. 201904010308) and Guangdong Government Science and Technology (Projects No. 2017B020238003 and 2014B050505019). We also acknowledge Yinglin Luo at SCAU for her assistance

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with the drawing of the 3D schematic model.

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