Isolation of Cellulose Nanobers (CNFs) based on Ball-milling Technique Combined with Supercritical Carbon dioxide/ethanol Pretreatment

Here, a new pretreatment method has been developed to produce CNFs from micro-brillated cellulose (MFC) by supercritical CO 2 pretreatment followed with ball-milling (SCB). MFC was obtained from cotton stalk by chemical purication.Experimental factors were controlled to enhance the properties of SCB-CNF, meanwhile a comparative study was conducted with the method of TEMPO oxidation and microuid homogenization (TMH). Compared to TMH-CNF, the SCB-CNF has such advantages as Energy saving, high eciency and environmental protection, indicating a wide application in heat-resistant materials, load materials and other elds. The solid yields of P-MFC after supercritical CO 2 pretreatment gradually decreased together with the temperature and the reaction time. Scanning electron microscope (SEM) images of the SCB-CNF and TMH-CNF show that the morphology of the SCB-CNF was basically acicular but that of the TMH-CNF was mainly soft brous. The SCB-CNF is smaller in width and shorter in length, and its size is between CNC and CNF. Thermal gravimetric results suggest that the thermal stability of the SCB-CNF was substantially higher than those of the TMH-CNF. XRD results indicate that the crystallinity showed an initial increasing trend and then declined with increasing temperature and reaction time, and the crystallinity value of SCB-CNF was larger than that of CNFs. The smaller SCB-CNF became rougher and had a larger surface area. High crystallinity make good thermal stability, short and coarse ber, easier to disperse than CNF, less energy consumption for dispersion, better than 3D mesh. It can be widely used in polymer composites, reinforcing agents, membrane materials and other elds.


Introduction
Cellulose nano bers (CNFs) from plant biomass are of considerable interest primarily thanks to their high surface area, low density, tunable surface functions, biocompatibility and biodegradability . [1,2] With these advantages, CNFs have become popular globally in recent years with a great potential in the development of new green nanocomposites [3][4] . CNFs have applications in the engineering eld that include and composite materials [5] and biology (drug delivery) [6] .
CNFs can be disintegrated from various lignocellulosic sources by separate or in combinated mechanical treatment [7,8] including re ning [9] , homogenizing [10] , micro-uidization [9][10][11][12] , grinding, cryocrushing [13][14][15] and high intensity ultra-sonication [16][17][18] . Comparing the CNFs produced by homogenization, micro grinding and micro uidization methods, the CNF produced by homogenization achieved the highest speci c surface area. [10] While, CNFs produced by micro grinding and micro uidization showed superior physical, mechanical and optical properties coupled with reduced energy consumption in comparison with homogenization. Unfortunately, all the above mechanical procedures are energy-intensive which lead to a steep decline in both yields and bril lengths. Thus, recent studies have shifted the focus to nding high-e ciency, environmentally friendly and low-cost methods to isolate CNF.
Pretreatment before mechanical methods has brought some positive results to the production of CNF [19] . Such as TEMPO oxidation pretreatment. The cellulose nano bers with uniform width, large aspect ratio, high crystallinity and complete nano dispersion can be prepared by TEMPO catalytic oxidation of cellulose under water medium and normal temperature and pressure. The treatment conditions of TEMPO oxidation system are mild, the raw materials are cheap and relatively environmental friendly. However, there are still some problems in the basic science and practical application of TEMPO pretreatment. For example, when tempo oxidized cellulose is homogenized, the concentration is small and the e ciency is low, which is not conducive to industrial promotion; at present, the construction of high concentration and low viscosity technology of tempo aqueous dispersion solution still needs to be explored by researchers [20] .
In recent years, supercritical solvents have been a proposed reaction system with the capability to enhance the effect of the pretreatment process [21] . Supercritical uids exhibit gas-like diffusivity and liquid-like dissolving capability, which could reduce the heat and mass transfer resistance during the reaction. For example, two-state CO 2 (gaseous and liquid) in the supercritical CO 2 system has higher dissolution capability of polar and non-polar substances, and the liquid CO 2 is acidic and has an autocatalytic effect on water removal from the amorphous region of cellulose. Subcritical water also promotes the hydrolysis of the amorphous and semi-crystalline regions of cellulose. This process shows great potential for cellulose nanocrystal production because it uses only water as the hydrolyzing agent, although it demands additional energy consuming on the use of a high pressurized reactor, the total process is less energy consumption because it does not require high-intensitive mechanical treatment and numerous washing steps. Although, more studies are needed to explore other reaction conditions in order to explain the mechanism of hydrolysis with subcritical water, many successful examples were carried out to improve CNF properties. A combination of pretreatment and re ning processes has led to the successful fabrication of uniform CNF with a high degree of brillation [22] . Similarly, Wang and Cheng [17] compared the application of high-intensity ultra-sonicationand combination with high-pressure homogenization and found that the combination of the two methods increased the effectiveness of brillation and produced uniform nano bers.
Here, a new pretreatment process has been explored for the preparation of CNF using supercritical carbon dioxide followed by ball milling(SCB). Firstly, the cotton stalk ber was chemically puri ed to obtain micro-brillated cellulose. Then, Supercritical carbon dioxide/ethanol pretreatment experiments were carried out on cotton stalk micro-brillated cellulose (MFC) using a laboratory autoclave. and the yields of pretreated MFC at different temperatures and timeswere examined.
The effect of pretreated conditions on the yields and the mechanism was discussed preliminarily, meanwhile a comparative study was conducted with the method of micro uid homogenization (TMH). The samples were characterized by using X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier transform infrared spectrometry (FTIR) and thermogravimetric analysis (TGA).

Preparation of MFC by chemical puri cation
In order to facilitate the isolation of NCFs from cotton stalk ber or parenchymal cells, chemical purification, which aims to remove all the non-cellulose polymers from cell walls. A certain amount of cotton stalk material was dewaxed in Soxhlet extractor with 2:1 (v/v) benzyl / ethanol mixture for 6 hours, and then placed in acidi ed sodium chlorite solution at 75 ℃ for 1 hour to remove lignin from the sample. The lignin removal process was repeated ve times until the whole cellulose ber was obtained and then washed with deionized water. Hemicellulose, residual starch and pectin in the whole cellulose ber were further removed by 2 wt% KOH at 90 ℃ for 2 hours. Finally, the solution was neutralized with 1 wt% hydrochloric acid, then washed with deionized water and dried.

Methods of preparation of CNFs
Particle size was further reduced by introducing an additional mechanical process using the suspension which was fully reacted with supercritical CO 2 in the ball mill for six hours. There were static and rotating grindstones in the ball-milling equipment and the pulp slurry material passed through these small stones together with the suspension. The goal of brillation in the grinder was to ensure the breaking down of the hydrogen bonds and cell wall core structure by shear forces and to reduce the pulp to nano scale bers [23] . Supercritical CO 2 pretreatment was therefore combined with ball-milling. Mechanical brillations by both ball-milling and supercritical CO 2 seemed to randomly dismantle the crystalline and amorphous regions of the cellulose. This fracture of cellulose crystals is thought to be a factor contributing to the dissociation of micro-brils and their bundles [24] . (see Fig.1

-A)
The MFC was pretreated by TEMPO oxidation, and then diluted with tap water in a ber unwinder. The suspension was circulated through a high-pressure homogenizer (tm-lmzo, micro uidics, Westwood, MA) for 20 cycles at 25000 psi to produce TMH-CNFs. (see Fig.1-B) 3.5 Characterization

Yield
Where, G1 represents the sample after drying and G0 the former MFC (2.5 g). The yield of P-MFC was also used as a response to the experimental design.

Scanning electron microscopy (SEM)
Scanning electron microscopy (SEM) was used for the analysis of the surface morphology of the nanobrillated cellulose. Images were captured at a magni cation of 10000× by means of a benchtop scanning electron microscope (Ultra55, Carl Zeiss AG, Oberkochen, Germany). The test was operated in high vacuum mode at an acceleration voltage of 15 kV. The samples were secured on a metal stub with carbon and coated with a thin layer of gold using an HR sputter coater (Agar Scienti c, Stansted, UK) prior to examination in order to render the products electro-conductive.

XRD
Dried neat CNW lms were separately mounted to a special holder through which a beam of x-rays passed through centrally. Scattering radiation was identi ed in the 2θ range from 2° to 40° at a scanning rate of 4° min -1 . The crystallinity index was evaluated from the XRD patterns using equation (2).

Fourier transform infrared spectrometry (FT-IR)
The chemical bonds of MFC and P-MFC were analyzed by FT-IR (Nicolet 5700, Thermo-Fisher Scienti c, Waltham, MA). The measurement was done using the KBr method for functionality changes in the range 400-4000 cm -1 (scans = 32).

Thermal gravimetric analysis
The thermal gravimetric analyzer used in this study was a Pyris Diamond DSC ( As shown in Fig. 2 (b), when the pretreatment time is set at 90 min, the solid-phase yield of p-MFC reaches the maximum at 135 ℃. Both increasing temperature and lowering temperature will reduce the yield of p-MFC.
When the reaction time was 30 min and the pretreatment temperature was 135 ℃, the solid yields of MFC without supercritical CO 2 pretreatment and with supercritical CO 2 pretreatment were 90.5% and 96.1%, respectively. This indicates that the supercritical CO 2 process may have a de nite enhancement effect on the amorphous zone hydrolysis reactions of MFC.
According to the above conclusions, the hydrolysis reaction in amorphous region of MFC increases with the increase of pretreatment temperature and time.
According to Lin & Dufresne [25] , the solid yield of p-MFC by supercritical carbon dioxide / ethanol pretreatment process is higher than that of MFC after sulfuric acid pretreatment. It is obvious that the amorphous region of p-MFC is hydrolyzed to a large extent. The higher the yield, the more hydrolysis in the amorphous zone, which proves that su cient temperature and time can ensure the hydrolysis.
However, with the increase of time and temperature, too severe conditions lead to hydrolysis in the crystallization zone.

SEM analysis
SEM micrographs of MFC pretreated under different conditions are shown in Fig. 3. The surface of MFC was smooth and the single bers were tightly bonded together. The morphology of P-MFC obtained from MFC pretreated at 135 ºC for 30 min showed that the bunch ber was divided into a single ber but the length and diameter of P-MFC were basically maintained (see Fig. 3b). As the pretreatment time increased to 150 min (see Fig 3c), the length and diameter of P-MFC clearly decreased in contrast to P-MFC which did not show a similar trend as the pretreatment temperature increased to 200 °C (see Fig.  3d). The trend in P-MFC morphology suggests that the pretreatment time has a greater effect on the MFC size than the pretreatment temperature. The explanation is that the hydrolysis rate of the reaction at 135°C was similar to that at 200 °C. A longer reaction time was required to reduce the MFC size by hydrolysis. The supercritical carbon dioxide/ethanol pretreatment can therefore decrease the length and diameter value of MFC under pretreatment conditions of 135 °C for 90 min.

XRD analysis
The XRD patterns of the original MFC material and the P-MFC obtained from MFC pretreatment at different reaction times and temperatures are shown in Figure 4. The diffraction patterns shown by the P-MFC and MFC were similar as shown in Fig. 4, indicating that the hydrolysis by supercritical CO 2 did not alter the crystalline structure of the MFC. All samples showed a sharp peak at 2θ = 23° on the left of the graphs, suggesting that the bers contained a substantial region of native cellulose I, as shown by the characteristic peak at 22.6°, which corresponds to the lattice plane of cellulose I [26] . This indicates that the hydrolysis by supercritical CO 2 did not change the crystalline structure of the MFC, and this agrees with the results obtained in previous studies [22,27] .
The crystallinity index in Fig. 4 shows that the XRD crystallinity for P-MFC pretreated with supercritical CO 2 increased to 73.98% compared with MFC. The above hypothesis, that the enzyme picked partially digested amorphous regions, is supported by the increase in crystallinity. The crystallinity index of P-MFC initially increased and then decreased as the reaction time increased and this indicates that the amorphous region of MFC hydrolyzed rst, and with increasing time the crystalline area was also broken. A reaction time of 90 min may therefore be considered the optimum reaction time in this experiment due to the higher crystallinity index of P-MFC.
Reaction temperature also exerted a substantial effect on the crystallinity of P-MFC. Fig. 4 shows the XRD crystallinity of P-MFC pretreated with supercritical CO 2 at different reaction temperatures. The trend of crystallinity was an initial increase followed by a decline. The amorphous region of the microcrystalline cellulose hydrolyzed rst and with increasing time the crystalline area also hydrolyzed. With supercritical CO 2 pretreatment the crystallinity index of MFC was 78.67% at 135 °C, and this may be regarded as the optimum pretreatment temperature for MFC.

SEM analysis
To determine the size of the SCB-CNF, a dilute suspension was examined using TEM and the images are displayed in Fig. 7. The length of SCB-CNF was about 200-400 nm and the ber diameter was about 20 nm, while the length of TMH -CNF was about 1-2 μm and the ber diameter was 20 nm. They bonded together and presented a three-dimensional nano-structured network comprising of randomly arranged nano brils. The networks by hydrogen bonds was enabled by the many hydroxyl groups in SCB-CNF. Compared to the MFC materials the surface of the SCB-CNF became rougher, and a trace of erosion by the supercritical CO 2 can be clearly seen. The length and the diameter were almost the same indicated that prolonging the reaction time beyond 90 min did not facilitate the characterization of the SCB-CNF. Meanwhile, the morphology of SCB-CNF is basically acicular, while that of TMH-CNF is mainly soft brous. Therefore, SCB-CNF with supercritical CO 2 pretreatment have already had the amorphous region removed. The pretreatment has an important role in reducing particle size and increasing speci c surface area, thereby enhancing thermal stability. The results indicate that this is a potent and environmentally friendly route for the preparation of SCB-CNF.

XRD and FTIR
Comparative XRD in Figs. 4 and 8 show crystalline peaks at 2θ = 14-16°, indicating an increase in the crystallinity of the SCB-CNF and the crystallinity value of SCB-CNF was larger than that of CNFs. It may be concluded that during acid hydrolysis, cellulose possessing a loose structure favors changes in the size of the crystallites. Tang et al. [34] suggested the explanation that changes in crystallite size during acid hydrolysis were a result of both the degradation of smaller crystallites and the growth of the defective crystallites.

Thermal gravity
The thermal stability of MFC, P-MFC (135 °C, 1.5 Mpa CO 2 , 150 min), and SCB-CNF (135 °C, 1.5 Mpa CO 2 , 150 min, ball-milled for 6 h) was determined by thermal gravimetric analysis. Particles exhibited a loss in weight due to increasing temperature and the rate of particle degradation is shown in Fig. 9. An initial weight loss was observed in the MFC (about 350~400 °C) and the SCB-CNF (135 °C, 1.5 Mpa CO 2 , 150 min) and is the result of the evaporation of water.
With further increasing temperature (≤ 800 °CC) the TG curves of the MFC and P-MFC (135 °C, 1.5 Mpa CO 2 , 150 min) change in a similar fashion. The rate of decomposition and weight loss of the SCB-CNF (135 °C, 1.5 Mpa CO 2 , 150 min, ball-milled for 6 h) and the initial degradation temperature of MFC was about 250 °C, lower than the MFC degradation temperature. These results may be explained by saying decreased polymerization and increased speci c surface, and consequently the loss of active groups. However, the thermal stability of the SCB-CNF produced by supercritical water hydrolysis was much higher than that of nano-brillated cellulose produced by sulfuric acid hydrolysis owing to the absence of sulfate groups in the SCB-CNF produced by hydrolysis with supercritical CO 2 [25] . According to an earlier study [29] , the weight loss of SCB-CNF produced by supercritical carbon dioxide is much less than that of nano-brillated cellulose produced by sulfuric acid.
The TG curves did not show large changes (≤ 800 °C) with increasing temperature, indicating higher thermal stability. Thus, the thermal stability of the SCB-CNF is clearly higher than those of the TMH-CNF and the raw material. The increased thermal stability of cellulose brils (compared to MFC) might be attributed to strong hydrogen bonding between the hydroxyl groups of the cellulose brils according to the supercritical carbon dioxide [30] . The higher thermal stability of the SCB-CNF can be attributed to their exibility and hence a greater possibility of entanglement of the nano brils. A comparable increase in thermal stability due to the tangling effect of exible micro-brils has been reported by Das et al. [31] . The melting peaks of the crystalline SCB-CNF were observed in the temperature range 230-250 °C. However, this was not observed in the case of any MFC because the removal of the amorphous portion by chemical treatments in SCB-CNF increased the crystallinity of the SCB-CNF [32] .
In most reactions the temperature was the important factor, but the P-MFC yield was predominantly in uenced by the timing. According to Fig. 9, carbon dioxide indicates a low signi cance of the factor. The thermal stability of cellulose whiskers is important, considering their potential application as a reinforcing ller in polymer composites, especially in thermoplastics, as processing temperatures often exceed 200 °C. [32] .

Conclusions
Here, a novel pretreatment method using supercritical CO 2 was found to be an effective and environmentally friendly reaction medium for cellulose without using an organic solvent. SEM shows that the morphology of SCB-CNF was basically acicular, while that of CNFs was mainly soft brous. Thermal gravimetry suggests that the thermal stability of the SCB-CNF was substantially higher than those of the CNFs and the raw material. XRD analysis also indicates that SCB-CNF have higher crystallinity than the MFC or CNFs. The supercritical CO 2 process may have a de nite enhancement effect on the amorphous zone hydrolysis reactions of MFC, and hydrolyzed to a greater extent than pretreatment with sulfuric acid, decrease the length and diameter value of MFC under pretreatment conditions of 135 °C for 90 min. The length of SCB-CNF is about 200nm, which is much smaller than that of TMH-CNFpretreated with sulfuric acid.

Declarations
Data Accessibility Data relating to this work are provided in the electronic supplementary material.