Insight into the Evolution of the Cellulose Microstructure Through the Enzyme Pretreatment Method

In this work, the changes of properties and microstructure of cellulose (bleached hardwood kraft pulp (BHKP)) subjected to different enzyme pretreatment times (0–10 h) were explored for further brillation. The various properties of the pretreated cellulose gradually decrease with the elapse of time relative to the pristine material, such as yield, water retention value, aspect ratio and degree of polymerization, etc. Enzyme pretreatment can promote the peeling of brils and loosen the amorphous areas of cellulose identied by Scanning Electron Microscope (SEM) and X-ray diffraction (XRD). A thorough investigation of the relation between pretreatment and evolution of inter-/intra-molecular H-bonds in cellulose was conducted including content and cleave sequence of H-bonds by Fourier transform infrared spectroscopy (FTIR), second derivative analysis and generalized two-dimensional correlation spectroscopy (2DCOS). The intermolecular H-bonds with the most signicant decrease in content was cleaved rst relative to the intramolecular H-bonds. These discoveries provide theoretical support to more effective pretreatment method for commercial production of brils from cellulosic bers.


Introduction
In recent years much more attention has been paid to nanoscaled bio-based material for various application. Numerous renewable and biodegradable bio-based ber composite materials have been developed to obtain the next generation of sustainable and green materials in this application eld (Jonoobi et al. 2015;Zhu et al. 2016). Cellulose is the most abundant renewable natural biopolymer and regaining importance as a renewable chemical resource to replace petroleum-based materials. In addition to biodegradability and renewability, the production of cellulose nano brils (CNFs) have added promising properties such as high mechanical properties, high speci c surface area and high transparency, which are widely used in the elds of food, cosmetic, pharmaceutical, exible displays and papermaking (Zhu et al. 2016). However, one of the bottlenecks in commercial production of CNF is the high energy consumption in the mechanical re ning process of CNF production.
Cellulose is a linear homopolysaccharide composed of β-1, 4-linked D-glucopyranose units with a high degree of polymerization (DP). The three hydroxyl groups of the monomer and their ability to form hydrogen bonds play an important role in leading the crystalline packing, which provide cellulose with a stable crystal structure and high crystallinity (Dufresne and Alain 2017; Zhu et al. 2016). Therefore, energy consumption is the main drawback for mechanical approaches to diminish cellulosic bers into nano brils (Daud et al. 2015). And CNF de brillation requires intensive mechanical treatment and less energy utilization will result in less cellulose brils and less nano ber production (Nechyporchuk et al. 2016). To overcome this shortcoming, researchers had basically proposed three different strategies for pretreatment of cellulose bers before mechanical treatment: (1) limit the hydrogen bonding in the system, and (2) add repulsive charges, and (3) reduce the DP or amorphous connection between individual laments (Lavoine et al. 2012). It is worth noting that the proper pretreatment of cellulose bers can promote the accessibility of hydroxyl groups, increase the inner surface, change the crystallinity and cleave the cellulose hydrogen bonds, improve the reactivity of the bers and effectively reduce the energy consumption during the brillation process, such as alkaline-acid, enzymatic hydrolysis, TEMPO-mediated oxidation and carboxymethylation (Asad et al. 2018; Chinga-Carrasco 2011; Ding et al. 2018;Nie et al. 2018;Saeed et al. 2018). Previous studies had shown that pretreatment (such as enzymes, chemicals) could help reduce the energy consumption of cellulose bers consumption to an amount of 1000 kWh/t from 20,000 to 30,000 kWh/t (Siró and Plackett 2010).
However, chemical pretreatment will result in a signi cant reduction in the mechanical strength and thermal degradation points of cellulose nano bers (Fukuzumi et al. 2009). Compared with the high capital cost and di cult drug recycling of chemical pretreatment, enzymatic pretreatment is considered a promising process for industrial applications due to its high selectivity, low chemical loading and environmentally friendly process (Bian et  The objective of this work was to study the microstructure and properties of BHKP cellulose bers pretreated with commercial endoglucanases. This type of pretreatment method was chosen because of its potential to commercialize nanocellulose. A thorough investigation of the effect of pretreatment time on the microstructure and properties of cellulose bers was conducted, including water retention value, aspect ratio, degree of polymerization, morphology, crystal structure and H-bonds pattern. This is critical for the future development of more economical pretreatment technologies and commercial promotion of nanocellulose.

Material
The cellulose source was never-dried bleached hardwood kraft pulp (BHKP) from Shandong Sun Paper Company. Commercial endoglucanase (OEM-9) was obtained from Doing-higher (Guangxi, China). The optimum pH and temperature for the OEM-9 were 5.5 and 40°C, respectively. Enzyme activity was 8.62 IU/ml measured by the dinitrosalicylic acid (DNS) method with D-glucose as the standard. (Sengupta et al. 2000) The cupriethylenediamine hydroxide solution was provided by Tianjin Zhentai Chemical Co., Ltd.
(Tianjin, China). All chemical reagents were purchased and used without further puri cation.

Enzymatic pretreatment
The procedure was carried out according to the previously published enzymatic pretreatment (Henriksson et al. 2007a). Brie y, 2wt% of BHKP was enzymatically hydrolyzed in citric acid-sodium citrate buffer (pH 5.5) for different times in an incubation shaker. The enzyme dosage and temperature were 10mg/g cellulose and 40°C respectively. After the hydrolysis, the slurry was centrifuged at 3000 rpm for 10 minutes to separate the solid phase and the liquid phase. The yield of cellulose was calculated by following formula: Y = m 1 /m 2 *100%. Where Y is the yield of cellulose (%), m 1 and m 2 are the weight of cellulose before and after pretreatment, respectively.

Average molecular weight and crystal structure
The viscosity of cellulose before and after pretreatment measured by ASTM method (ASTM D1795-94, 2001) was used to calculate the degree of polymerization (DP) according to the following formula:

Fourier transform infrared spectroscopy (FT-IR)
FTIR characterization was performed with a Vertex70 Hyperion FTIR spectrometer in absorbance mode using the KBr pellet technique. Spectra were acquired for a total of 32 scans in the range of 500-4000 cm − 1 with a resolution of 4 cm − 1 . The second derivative spectra (3700 − 3000 cm − 1 ) were calculated by the Savitzky-Golay method after the spectra were subjected to smoothing. Peak t software (v4.12) combined with Gaussian distribution function is used to t the spectrum in the range of 3700 − 3000 cm − 1 to analyze the changes in H-bonds. All generalized two-dimensional correlation spectral (2DCOS) analyses were performed in 2D-shige software (Buchanan and Wei 2018).

Physical and chemical properties of pretreated cellulose
Enzyme pretreatment have been used to loosen the cellulose bers to reduce energy consumption of isolation CNFs from cellulose prior to mechanical re ning. It was necessary to explore the mechanism of enzyme pretreatment on the physical and chemical properties and microstructure of cellulose, which may provide theoretical support for the commercial production of CNFs. Figure 1 exhibited the effect of pretreatment time on ber physical and chemical properties at a constant enzyme dosage of 10 mg/g, a physical temperature of 40°C and a pH of 5.5. It can be seen that the yield of cellulose gradually decreases and the water retention value gradually increases with the extension of the pretreatment time (Fig. 1a). The decrease in yield was due to the hydrolysis of cellulose by OEM-9 into soluble sugars or oligosaccharides (Kumar et al. 2016). And the more exposed hydroxyl groups can absorb more bound water to increase the WRV (Nie et al. 2018). The length and width of cellulose decreased by 78% and 21%, respectively, relative to the raw material when the pretreatment time was 10h (Fig. 1b). This tendency to decrease in the longitudinal direction resulted in a signi cant decrease in the aspect ratio of cellulose by 72%. It can be predicted that the pretreatment of OEM-9 will negatively affect the length of brils that disintegrate from the pretreated ber. In addition, the degree of polymerization and ne components had been reduced with the prolonging of pretreatment time. It proved that OEM-9 mainly cleaved β-1, 4glycosidic bonds of cellulose chains in the pretreatment stage and hydrolyzed into oligosaccharides

Microscopic and crystalline structure of pretreated cellulose
The surface topography of the bers with different pretreatment times were analyzed by SEM in Fig. 2. As the pretreatment time increases, the size of the pretreated ber gradually became smaller and the surface became wrinkled and rougher. This was consistent with the data measured by the Fiber Quality Analyzer (FQA) (Fig. 1b). In addition, signi cant brillation and breakage could be observed on the surface of pretreated bers, which indicated that OEM-9 could facilitate the peeling of brils.
X-ray diffraction analysis of cellulose ber was performed to study the effect of cellulose micro-crystal structure during the enzyme pretreatment process, as shown in Fig. 3. It could be seen that all the samples showed the cellulose-Iβ crystal structure which had a preferred orientation along the ber axis typical of plant bers (Fig. 3a). All XRD patterns showed obvious diffraction peaks at 15.2°, 16.5°, 22.5°a nd 34.5° respectively assigned to (1-10), (110), (200) and (004) re ections. It can be seen that the crystallinity index and crystallite size increase slightly with the extension of the pretreatment time (Fig. 3b). The crystallinity index and crystallite size of cellulose ber increased by 1.7% and 5.7%, respectively, when the pretreatment time was 10h. It can be predicted that OEM-9 pretreatment only loose the amorphous area of cellulose without causing hydrolysis. This can be explained by the random cleavage of β-1,4-glycosidic bonds in the cellulose chains and cellulose bers, resulting in the distortion of the crystallite size in the cellulose bers (Kumar et al. 2016). Figure 4 showed pretreatment-time-dependent FTIR spectra of cellulose ber with different enzyme pretreatment time. The broad region 3700-3000cm − 1 was assigned to -OH vibrations. The peak at 2900cm − 1 was related to the aliphatic saturated CH 2 and CH 2 OH stretching vibration of the cellulose. The absorption peak in the 1590cm − 1 band was due to bound water and carboxylate. The characteristic peaks from 1400 to 1300cm − 1 were attributed to the acetyl and uronic acid ester groups of cellulose. The absorption peak at 1030 cm − 1 was assigned as the CO stretch at the C 3 position. The absorption peak at 670 − 550 cm − 1 was related to CH deformation and OH out-of-plane bending. The hydrogen bond network structure inside cellulose is mainly composed of intramolecular and intermolecular hydrogen bonds (3700-3000cm − 1 ). It can be see that the absorbance of the 3000-3700cm − 1 region generated by the stretching vibration of the intermolecular and intramolecular H-bonds increased with the elapse of pretreatment time. According to previous reports, the band assignments for O-H stretching region in cellulose can be observed through the second derivative spectra (Watanabe et al. 2006). The second derivative spectra (3700-3000cm − 1 ) of cellulose with different enzyme pretreatment time as shown in Fig. 4 (b). There are three distinct peaks that can be identi ed in this region. These three peaks are assigned to the OH stretching modes of cellulose.

H-bonds pattern of pretreated cellulose
To explore the effect of pretreatment on the H-bonds of cellulose bers, FTIR in the range of 3700-  Table 1 and Fig. 5 (g). The content of O6H6···O3' (intermolecular H-bonds) decreased from 69% (raw cellulose) to 53% (Time 10 ) with the extension of the pretreatment time. And the peak position of O6H6···O3' shift to a higher wavelength as the pretreatment time elapses. The signi cantly larger wavenumber shift is probably due to the disruption of the H-bonds between the OH groups of the cellulose molecular chain.(Watanabe et al. 2006) The content and peak position of intramolecular H-bonds (O3H3···O5 and O2H2···O6) did not change signi cantly with the extension of the pretreatment time. This ambiguous shift indicated that the OEM-9 did not directly affect the intramolecular H-bonds and the interaction was very complicated (Laine and E. 1982). This result indicated that OEM-9 interact more easily with the O6H6···O3' rather than O3H3···O5 and O2H2···O6. This may be bene cial to improve the aspect ratio and tensile strength of the brils separated from the pretreated ber. To further study the sequence of OEM-9 acting on the H-bonds inside the ber during the pretreatment process, 2DCOS was used to analyze this region (3000-3700cm − 1 ). 2DCOS can directly extend the spectral signal to two dimensions to improve the spectral resolution and reveal the sequence of changes between groups (Noda 2016a; Noda 2016b). Figure 6 showed two-dimensional synchronous correlation spectrum generated from the pretreatment-time-dependent FTIR spectra variations in the time ranges 0-10h. Figure 6 (c) depicted the corresponding auto correlation spectra extracted from the synchronized 3D and 2D correlation spectra shown in Fig. 6 (a, b). It can be seen that a strong auto peak of Φ (3400, 3400) > 0 and two shoulders of Φ (3300, 3300) > 0 and Φ (3560, 3560) > 0, which means that the three bands intrachain H-bonds in cellulose occurred during the pretreatment process. No obvious crosspeaks are observed, indicating that there is no intermolecular interaction between the three H-bond models. In other words, the process of OEM-9 cleave these three H-bonds is independent in the pretreatment process. Figure 7 showed two-dimensional asynchronous correlation spectrum generated from the pretreatmenttime-dependent FTIR spectra variations in the time ranges 0-10h. The slice spectra extracted from the asynchronous 3D and 2D correlation spectra (Fig. 7a, b) at 3497cm-1 was depicted in Fig. 7(c). It can be seen that the slice spectra at 3497 cm − 1 had asynchronicity with the peaks appearing at 3350 and 3600

Conclusions
In conclusion, this study investigated the dynamic evolution of properties and microstructure of enzyme pretreated cellulose. We concluded that properties and microstructure of cellulose could be controlled dramatically by the pretreatment time, such as yield, water retention value, aspect ratio, degree of polymerization, crystallinity and H-bonds. The intermolecular H-bonds with the most signi cant decrease in content (16%) was cleaved rst relative to the intramolecular H-bonds. This unique study is of great value for the commercial production of brils through enzymatic hydrolysis.   Pretreatment-time-dependent FTIR spectra and second derivative spectra (3700-3000cm-1) of cellulose with different enzyme pretreatment time.

Supplementary Files
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