Nanocellulose and Cellulose Making with Bio-Enzymes from Different Particle Sizes of Neosinocalamus Affinis

Abstract

Cellulose is one of the most abundant, widely distributed and abundant polysaccharides on earth, and is the most valuable natural renewable resource for human beings. In this study, three different particle sizes (250, 178, and 150 μm) of Neosinocalamus affinis cellulose were extracted from Neosinocalamus affinis powder using bio-enzyme digestion and prepared into nanocellulose (CNMs). The cellulose contents of 250, 178, and 150 μm particle sizes were 53.44%, 63.38%, and 74.08%, respectively; the crystallinity was 54.21%, 56.03% and 63.58%, respectively. The thermal stability of cellulose increased gradually with smaller particle sizes. The yields of CNMs for 250, 178, and 150 μm particle sizes were 14.27%, 15.44%, and 16.38%, respectively. The results showed that the Neosinocalamus affinis powder was successfully removed from lignin, hemicellulose, and impurities (pectin, resin, etc.) by the treatment of bio-enzyme A (ligninase:hemicellulose:pectinase = 1:1:1) combined with NH₃·H₂O and H₂O₂/CH₃COOH. Extraction of cellulose from Neosinocalamus affinis using bio-enzyme A, the smaller the particle size of Neosinocalamus affinis powder, the more cellulose content extracted, the higher the crystallinity, the better the thermal stability, and the higher the purity. Subsequently, nanocellulose (CNMs) were prepared by using bio-enzyme B (cellulase:pectinase = 1:1). The CNMs prepared by bio-enzyme B showed a network structure and fibrous bundle shape. Therefore, the ones prepared in this study belong to cellulose nanofibrils (CNFs). This study provides a reference in the extraction of cellulose from bamboo using bio-enzymes and the preparation of nanocellulose. To a certain extent, the utilization of bamboo as a biomass material was improved.

1. Introduction

Bamboo is a woody perennial plant of the Gramineae family. It is a potential sustainable biomass energy feedstock. Bamboo includes 75 genera and 1250 species, most of which have a short growth period, reaching maturity in about 5 years [1]. There are about 1000 species of bamboo in Asia. China alone has 44 genera and about 300 species, covering about 33,000 km, accounting for 2%–3% of the total forest area of the country. Neosinocalamus affinis is one of the most planted and cultivated bamboo species in southwest China. It has long internodes, a high cellulose content (close to that of wood), and a large fiber length. The growth cycle is much shorter than that of wood. Scientific management of bamboo forests can be used forever without damaging the ecological environment. This is a characteristic that no other gramine has.

Cellulose is the main component of the cell wall of higher plants. It is the most abundant and important organic polymer, naturally renewable. It is a very high-demand alternative energy source worldwide, with the molecular formula (C₆H₁₀O₅)_n. It is a polysaccharide, consisting of hundreds to thousands of glucose unit chains, and is found in abundance on the earth, with varying amounts of cellulose in different plants. Research shows that cellulose is a promising renewable and sustainable chemical raw material [2,3]. It is widely used in paper, pharmaceuticals, packaging, textiles, automobiles, environmental management, and other fields [4,5].

With globalization and sustainable development, there is an increasing demand for biodegradable nanomaterials for nanotechnology. Pathak et al. prepared polycrystalline silver indium selenide films on Si (100) substrates by vacuum evaporation at high temperatures using chemometric powders. AIS nanorods were successfully synthesized by infrared radiation with incident 200 MeV Ag⁺ ions at a flux of 5 × 10 ions/cm² on Si (100) substrates [6]. Sagadevan et al. completed the controlled synthesis of TiO₂/SiO₂/CdS-nanocomposites by the hydrothermal-assisted method. Optical properties were obtained by UV-Vis absorption spectroscopy. The optical band gap of the nanocomposites was found to be 3.31 eV [7]. Pathak et al. prepared organic semiconductor films on glass substrates and annealed them at 55 °C. The engineered band gaps of the synthesized films were 2.18, 2.35, 2.36, 2.52 and 2.65 eV. This indicates that it can be used as a new lightweight and environmentally friendly carbon-based material for photovoltaic devices such as solar cells [8]. Hosseini synthesized MgAl₂O₄/NiTiO₃ nanocomposites for the removal of MeO dyes from water by the sol-gel method. The prepared nanocomposites showed excellent photodegradation of MeO under UV light. The photodegradation efficiency of MeO was 84% [9].

Biomass is considered an ideal alternative to fossil energy due to its low price, wide range of sources, and renewable and sustainable characteristics [10]. Therefore, research and development on the use of biomass energy have become a priority in many countries around the world. The global demand for new sustainable materials has grown rapidly in recent years, as evidenced by the UN 2030 Agenda [11]. A raw material with the potential for many new materials is wood and cellulose pulp fibers derived from wood. By separating the nanostructures that make up the fibers, it is possible to obtain materials with highly interesting properties—such as nanocellulose. As the most promising biomass nanomaterials. Nanocellulose has many advantages: biodegradable, low composite energy consumption, good processable type, good heat resistance, strong tensile properties, high specific surface area, high adsorption, non-cytotoxic and biocompatible. It is widely used in biomedical, energy storage, biosorption, food packaging, electronic devices and other fields [12,13]. There are many kinds of nanocellulose preparation methods, such as acid hydrolysis, physical-mechanical method, enzymatic method and solvent method. Seta et al. used a ball mill to pretreat the bamboo fibers and added maleic acid to hydrolyze the bamboo fibers to obtain CNC. A control sample without maleic acid was also prepared. The results showed that the CNC yields were 10.55%–24.50%. It was much higher than the control 2.80% [14]. Mahmud et al. prepared different forms and multiple morphologies of CNC by acid hydrolysis of medical cotton in sulfuric, hydrochloric and phosphoric acids [15]. OKsman et al. added DMF to 0.5 wt% lithium chloride solution as a swelling agent for cellulose, placed microcrystalline cellulose into the solution, stirred at 70 °C for 12 h then ultrasonically separated. The final nanocellulose with a width of about 10 nm was obtained [16]. Chen et al. used cellulase to hydrolyze cotton pulp fibers to prepare ribbon-shaped CNC. The results showed that when the concentration of cellulase was low, ribbon-shaped CNCs with a length of about 250–900 nm were prepared. When the concentration of cellulase increased at 300 μ/mL, the prepared CNCs were all granular [17]. Zhang et al. used the steam blasting method to pretreat poplar wood and then prepared nanocellulose by bio-enzymatic-assisted ultrasonication. Its width ranged from 20 to 50 nm and had a high aspect ratio and network entanglement structure [18]. Hayashi et al. enzymatically digested the microcrystalline cellulose of the algae of setae in a solution of cellulase at 48 °C and pH 4.8. Nanocellulose with an average length of 350 nm was obtained after 2–3 days [19]. Beltramino et al. investigated the effect of different cellulase doses and reaction times on NCC. The results showed that the yield of NCC exceeded 80% under optimal enzymatic conditions (20 U, 2 h). Moreover, the different intensities of enzyme treatment resulted in a decrease in fiber length and viscosity [20]. Currently, there are few studies related to the effect of particle size on the extraction of cellulose and the preparation of nanocellulose. However, in other fields, the particle size is equally important for the performance of the samples. Huang et al. prepared four different particle sizes of beet pulp powder by ultra-fine grinding and ordinary crushing methods. The effect of beet pulp particle size on the extraction and properties of pectin was investigated. The results showed that as the particle size of beet pulp decreased, the extraction rate and content of pectin gradually increased. Moreover, the viscosity showed a negative correlation with particle size [21]. Meng et al. investigated the effect of different particle sizes of nano-SiO₂ on the properties and microstructure of cement paste. The results showed that 50 nm nano-SiO₂ provided higher compressive strength than 15 nm nano-SiO₂. The 15 nm nano-SiO₂ provided a denser microstructure than the 50 nm nano-SiO₂ [22]. Cheng et al. prepared nano, micron and micron/nano ZnO/LDPE by melt blending using LDPE as matrix polymer and ZnO particles with diameters of 30 nm and 1 μ as inorganic fillers. The experimental results showed a tendency to reduce the AC breakdown field strength of all composites. However, the reduction in micron ZnO/LDPE is lower than that in nano ZnO/LDPE [23].

There are many reports on the preparation of nanocellulose by bio-enzymatic methods. However, there are fewer reports on the extraction of cellulose from bamboo using bio-enzymatic methods. Moreover, the range of cellulosic raw materials selected for the preparation of nanocellulose is relatively narrow, mainly including wood, pulp fibers and bacterial cellulose [24]. In comparison with other traditional methods, biological enzymes have specificity, mild enzymatic process conditions, and enzyme reagents are renewable resources. The bio-enzymatic method greatly reduces energy consumption, reduces the use of chemicals, and avoids problems such as pollution of the environment during the experimental process. It is of great significance to ecologically sustainable development. However, the reaction conditions (amount of enzyme, reaction time and reaction temperature, etc.) for the extraction of cellulose by bio-enzymatic methods and the preparation of nanocellulose are more demanding. The preparation efficiency is relatively slow. The presence of these factors hinders the industrialization of cellulose extraction by bio-enzymatic methods and the preparation of nanocellulose.

In this study, three different particle sizes (250, 178, and 150 μm) of Neosinocalamus affinis powder were selected, following the single variable principle. Cellulose was extracted by using bio-enzyme A (ligninase:hemicellulose:pectinase = 1:1:1) in combination with NH₃·H₂O and H₂O₂/CH₃COOH, and characterized and analyzed using Fourier Transform infrared spectroscopy (FTIR), X-ray Diffraction (XRD), Thermogravimetric analysis (TG) and Carbon nuclear magnetic resonance spectra (¹³C-NMR). The nanocellulose was prepared by bio-enzyme B (cellulase:pectinase = 1:1), and characterized and analyzed for morphology and structural dimensions. The results of this study will help to improve the utilization of bamboo materials, expand the sources of cellulose as well as nanocellulose raw materials and explore greener methods of cellulose extraction and preparation of nanocellulose.

2. Materials and Methods

2.1. Materials and Reagents

Material: Neosinocalamus affinis (Kunming, China). Neosinocalamus affinis was crushed through a high-speed multifunctional crusher, passed through 60 mesh sieve (250 μm particle size), 80 mesh sieve (178 μm particle size) and 100 mesh sieve (150 μm particle size). 105 ± 2 °C oven dried for 24 h and stored for spare.

Chemical reagents: Ammonia solution (NH₃·H₂O), Hydrogen peroxide (H₂O₂) and Glacial acetic acid (CH₃COOH) were purchased from Yunnan Shuoyang Biological Company, Ltd, Shuoyang, China. Pectinase (CAS: 9032-75-1), hemicellulose (CAS: 9025-56-3), ligninase (CAS: 80498-15-3) and cellulase (CAS: 9012-54-8) were purchased from Aladdin Biotechnology Co., Ltd. (Shanghai, China). The pectinase activity was 100,000 U/g, hemicellulase activity was 30,000 U/g, ligninase activity was 500 U/g and cellulase activity was 10,000 U/g. Analytical purity-grade reagents were used in all experiments.

2.2. Experiment Equipment

Constant temperature magnetic stirrer (C-MAG HS 7, Hangzhou Aipu Instruments Company Limited, Hangzhou, China). High-speed Multi-functional Crusher (XY-100, Yongkang Songqing Hardware Factory, Yongkang, China). Electric Heat Blast Dryer (DHG-9003, Shanghai Yiheng Scientific Instruments Company Limited, Shanghai, China). Electronic balance (BSM220, Shanghai Zhuojing Electronic Technology Company Limited, Shanghai, China). Thermostatic water bath (B220, Shanghai Yarong Biochemical Instrument Factory, Shanghai, China).

2.3. Cellulose Extraction

The method of extracting cellulose is shown in Figure 1. Take the above particle size of 250, 178, and 125 μm of Neosinocalamus affinis powder (10 g) first add the appropriate amount of NH₃·H₂O pretreatment 25 °C soaked for 24 h, using distilled water rinse filter to neutral, 105 ± 2 °C constant temperature drying. The purpose of this is to soften the Neosinocalamus affinis pellets and to treat the sample with alkali to remove some of the hemicellulose and lignin. Then, an appropriate amount of H₂O₂/CH₃COOH (1:1) was added to the pretreated cichlid pellets for bleaching and partial removal of lignin in a constant temperature water bath at 70 °C for 10 h. After acid treatment, the samples were filtered and washed with distilled water until the filtrate was neutral and dried at 105 °C ± 2 °C. Finally, 10% ligninase solution, 5% hemicellulase solution and 5% pectinase solution were configured and calibrated. Add an appropriate amount of 10% ligninase solution, 5% hemicellulase solution, and 5% pectinase solution (1:1:1) to the sample after ammonia pretreatment and acid treatment and put it into magnetic stirring. The samples were treated at a constant temperature of 50 °C and stirring speed of 800–1200 r/min for 240 min to remove residual lignin, hemicellulose and impurities (e.g., resins, pectin, etc.). Afterward, the filtered samples were rinsed with plenty of distilled water until clean and dried at a constant temperature of 105 ± 2 °C.

2.4. Preparation of Nanocellulose (CNMs)

Nanocellulose was prepared using a bio-enzyme method [24] with slight modifications, as shown in Figure 2. Take the above cellulose (0.5 g) extracted from different particle sizes into a beaker and add 20 g of zirconia grinding beads (0.1 mm diameter). Configure and fix the volume of 5% cellulase solution and 5% pectinase solution, and add 15 mL each of cellulase solution and pectinase solution to the beaker. Then place it in a 100 °C water bath for 20 min and sonicate for 30 min. The purpose of this is to inactivate bio-enzymes. Afterward, the precipitate was collected and diluted with distilled water by centrifugation at 8000 r/min for 10 min to wash away the biological enzyme impurities and repeated five times. Finally, stir (800–1200 r/min) at room temperature for 30 min, sonicate for 30 min and repeat several times until the suspension is formed. The suspension was freeze-dried into a powder to obtain Neosinocalamus affinis nanocellulose (CNMs).

2.5. Material Characterization

The chemical composition was determined and the extracted cellulose was characterized using Fourier transform infrared spectroscopy (FTIR), X-ray diffractometry (XRD), thermogravimetric analysis (TGA), and ¹³C-NMR. Morphological characterization of nanocellulose (CNMs) was performed using field emission scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

2.5.1. Determination of Chemical Composition

Lignin content was determined according to the Chinese standard GB/T2677. 8-94 (Determination of acid-insoluble lignin content of paper raw materials). Holo-cellulose content was determined according to the Chinese standard GB/T2677. 10-1995 (Determination of Holo-cellulose content of paper raw materials). α-cellulose content was determined according to the Chinese standard GB/T744-1989 (Determination of α-cellulose of pulp). The hemicellulose content was calculated according to Equation (1), and the experiment was repeated three times, and the results were averaged.

Hemicellulose content (%) = Holo-cellulose content (%) − α-cellulose content (%)

(1)

2.5.2. FTIR

The samples were dried with KBr, mixed at a mass ratio of 1:100 and then ground and pressed. The samples were examined by a Varian 1000 Fourier transform infrared spectrometer (FTIR, Varian, Palo Alto, CA, USA) with a scan range of 400–4000 cm⁻¹ at a resolution of 4 cm⁻¹ and the spectra were collected for an average of 32 scans. The main absorption peaks were determined using ORIGIN (2018) software.

2.5.3. XRD

XRD was used to study the crystallinity of the samples. The samples were scanned using a LabX6000 X-ray diffractometer from Shimadzu, Kyoto, Japan, at 40 kV and 30 mA current and in the diffraction angle range of 10°–60°. Equation (2) [25] was used to calculate the crystallinity index (CrI) of the samples:

$$CrI (\%)=\frac{I_{200}-I_{Am}}{I_{200}}\times 100\%$$

(2)

CrI is the crystallinity index. I₂₀₀ is the intensity of the diffraction peak near 2θ = 22° on the 200-crystal plane, which is the diffraction intensity of the crystalline region. I_Am is the intensity of the lattice diffraction peak near 2θ = 15.7°, which is the diffraction intensity of the amorphous region.

2.5.4. TGA

The samples were analyzed by TGA using a Pyris Diamond thermogravimetric analyzer from Pekin-Elmer, Waltham, MA, USA. The thermogravimetric (TG) curves and derivative thermogravimetric (DTG) curves were recorded from 30 to 600 °C at a constant heating rate of 10 °C/min under nitrogen gas.

2.5.5. ¹³C-NMR

A small amount of cellulose was dissolved in deuterated dimethyl sulfoxide, and ¹³C-NMR spectra of the samples were recorded using Bruker (Billerica, MA, USA) MSI400 spectroscopy at 25 °C and 62.9 MHz with a MAS rate of 3 kHz. Each spectrum was obtained by accumulating 5000 scans. The delay time was 60 s with a proton 90 (pulse width of 9 mm and contact time of 2 ms for cross-polarization).

2.5.6. Calculation of CNMs Yield and Viscosity Determination

The CNMs suspension was prepared by stirring, sonication and centrifugation by homogeneous dispersion of CNMs in water. The CNMs yield was determined by weight analysis and Yield (%) was calculated according to Equation (3) [26]:

$$Y (\%)=\frac{(m_1-m_2)V_1}{m_3{V}_2}$$

(3)

In Equation (3), Y-yield of CNMs, %; m₁-total mass of the sample and sealed bag after freeze-drying, g; m₂-mass of the sealed bag, g; m₃-mass of Neosinocalamus affinis cellulose, g; V₁-total volume of CNMs suspension, mL; V₂-volume of CNMs suspension used for freeze-drying, mL.

The viscosity of CNMs suspension was measured at 25 °C using an SNB-2 digital viscometer (Shanghai Heng ping Instrument Factory, Shanghai, China). The measurement was performed according to Chinese standard GB/T 14074-2017 (Test method for adhesives and their resins for the wood industry). The experiment was repeated three times, and the results were averaged.

2.5.7. TEM

The microstructure of nanocellulose was studied by transmission electron microscopy (TEM, JEM 2100, Japan Electronics Co., Ltd., Tokyo, Japan) at an accelerating voltage of 200 kV. A drop of nanocellulose suspension (0.1 wt%) was placed on a copper grid and dried at room temperature. CNMs powder was prepared from the CNMs suspension and homogeneously mixed with KBr.

2.5.8. SEM

The samples were placed on a copper grid, sprayed with gold by an ion sputterer, and then analyzed, observed and photographed by field emission scanning electron microscopy for sample morphology images. The samples were characterized using an SEM instrument (JEOL JSM-7100 F, Tokyo, Japan) at a voltage of 15 kV.

3. Analysis and Discussion

3.1. Chemical Composition

Table 1. Content of cellulose extracted from different particle sizes of Neosinocalamus affinis powder.

Chemical Compound	250 μm	178 μm	150 μm
Celluloses (%)	53.44	63.38	74.08
Hemicellulose (%)	14.72	10.62	7.59
Lignin (%)	7.60	4.35	3.55

shows the cellulose content extracted from three different particle sizes of Neosinocalamus affinis powder. Table 1 shows that the cellulose content of 250, 178 and 150 μm of Neosinocalamus affinis was 53.44%, 63.38% and 74.08%, respectively. The results showed that the highest cellulose content was extracted from 150 μm of Neosinocalamus affinis powder. This is because the smaller particle size has a larger surface area and thus a larger surface area is exposed. This allows smaller particle sizes to react more readily with bio-enzymes than larger particle sizes, resulting in the easier removal of lignin and hemicellulose [27].

3.2. FTIR Spectra of Cellulose

FTIR spectroscopy can visually determine the structural changes of the sample material. The FTIR spectra of cellulose extracted from three different particle sizes of Neosinocalamus affinis powder are shown in Figure 3. The attribution of some peaks of FTIR absorption is summarized in

Table 2. Characteristic peaks of some groups in the infrared spectrum.

Wave Number (cm−1)	Attribution of Characteristic Absorption Peaks	References
3370	O–H stretching vibration (hydrogen bonding)	[28]
2290	CH2 asymmetric stretching vibration	[29]
1640	O–H bending vibration (absorption of H2O), conjugated to C=C	[30]
1500	C=C stretching vibration	[31]
1162	C (1)–O–C (4) symmetric stretching vibration (sugar ring linkage bond characteristic peak)	[34]
1114	C (1)–O–C (5) intra-face pyranose ring asymmetric stretching vibration	[32]
1087	C (3)–OH stretching vibration	[35]
1030	C (6)–OH stretching vibration	[35]
890	C (1)–H asymmetric stretching vibration outside the face β-D glucosidic bond characteristic	[33]

. The infrared spectra showed that almost all absorption peaks of the Neosinocalamus affinis powder were present in the spectrum of the extracted cellulose. There are wider and stronger absorption bands near 3370 cm⁻¹ in the spectra of cellulose extracted from different particle sizes in Figure 3, which is the stretching vibration peak of O–H in cellulose. The C–H induced stretching vibration peak near 2900 cm⁻¹ [28,29]. Near 1640 cm⁻¹ is the stretching vibration peak caused by –OH, which is the signal absorption peak of water molecules in cellulose [30]. The absorption peak near 1500 cm⁻¹ is the stretching vibration peak caused by C=C in lignin [31]. The absorption peaks of curves b, c, and d here gradually weaken and disappear. The absorption peak near 1114 cm⁻¹ is a stretching vibration peak caused by C–O–C in pyranose. The peak near 890 cm⁻¹ is the absorption peak of the β-D glucosidic bond in cellulose, which is a characteristic peak of cellulose [32,33]. The cellulose characteristic peaks in curves b, c, and d were increased due to the removal of lignin and hemicellulose compared with curve a. The cellulose content of Neosinocalamus affinis was increased due to the removal of lignin and hemicellulose. The intensity of the cellulose characteristic peaks was relatively enhanced. FTIR spectroscopy showed that bio-enzyme A combined with NH₃·H₂O and H₂O₂/CH₃COOH could effectively remove lignin and hemicellulose from Neosinocalamus affinis. It disintegrates the strong surface structure of Neosinocalamus affinis and releases cellulose. It is favorable for the resource utilization of Neosinocalamus affinis cellulose.

3.3. XRD Spectral Characteristics of Cellulose

Cellulose consists mainly of crystalline and amorphous regions formed by intra- or intermolecular (hydrogen bonding, van der Waals forces) interactions [36]. The crystallinity indices of cellulose extracted from three different particle sizes of Neosinocalamus affinis powder were analyzed by XRD. The crystallinity of cellulose affects its mechanical properties. Moreover, high crystallinity is more effective in enhancing the properties of cellulose in composites [37]. The XRD diffraction pattern is shown in Figure 4. Three distinct crystalline peaks are observed in the cellulose diffraction pattern, all samples have diffraction peaks at 2θ = 15.7°, 2θ = 22.2°, 2θ = 34.7°. The diffraction peaks out of 22.2° and 34.7° are attributed to (200) and (004) reflections. It is shown that all samples have a typical cellulose type I crystal structure [38,39]. Although a series of chemical treatments did not destroy the crystalline structure of the cellulose, its crystallinity was altered. In Figure 4, it can be seen that curve c has a steeper diffraction peak for I₂₀₀ compared with curves a and b. After the calculation of Equation (2) and

Table 3. The crystallinity of cellulose extracted from different particle sizes of Neosinocalamus affinis powder.

Particle Size (μm)	CrI (%)
250	54.21%
178	56.03%
150	63.58%

, it is known that the crystallinity of 250 μm is 54.21%, 178 μm crystallinity is 56.03%, and 150 μm crystallinity is 63.58%. XRD further demonstrated that the combination of bio-enzyme A with NH₃·H₂O and H₂O₂/CH₃COOH successfully removed the most difficult to remove lignin from the biomass, and also removed hemicellulose and impurities (such as gum, resin, etc.). Thus, the crystallinity of cellulose is improved. The XRD results showed that the reaction contact area of the Neosinocalamus affinis powder became larger as the particle size became smaller. This has more degradation of cellulose non-crystalline region by using bio-enzymes, which makes cellulose crystallinity increase.

3.4. Thermal Characterization of Cellulose

Thermal analysis was performed on cellulose extracted from different particle sizes of Neosinocalamus affinis. Figure 5 shows the TG and DTG curves of cellulose extracted from different particle sizes. Near the first stage (0~110 °C), 250, 178, and 150 μm have about 6.28%, 7.50%, and 4.66% thermal weight loss, respectively. This is due to the evaporation of water from the sample [40]. In the second stage, between approximately 200~400 °C, the glycosidic bonds of cellulose begin to break and a large amount of cellulose begins to be cleaved [41]. The heat loss rate of 250, 178 and 150 μm at this stage is about 70%, 75% and 68%, respectively. The DTG curve shows that the weight loss rate of 150 and 178 μm is less than that of 250 μm, so the heat resistance of 250 μm is worse. The third stage starts from about 400 °C to the completion of pyrolysis [42]. The residues decomposed slowly, and the residues were mainly non-decomposable ash, with residual amounts of 13.09%, 11.15%, and 14.03%, respectively. The above pyrolysis-related parameters can be combined to assign a composite index D to characterize the ease of pyrolysis of cellulose. The smaller the composite index D, the better its thermal stability [43].

$$D=\frac{{(\raisebox{1ex}{$dw$}\!\left/ \!\raisebox{-1ex}{$dt$}\right.)}_{max}{(\raisebox{1ex}{$dw$}\!\left/ \!\raisebox{-1ex}{$dt$}\right.)}_{mean}{V}_{\infty }}{T_s{T}_{max}\Delta T_{\raisebox{1ex}{$1$}\!\left/ \!\raisebox{-1ex}{$2$}\right.}}$$

(4)

In addition, the following main parameters of the reaction pyrolysis characteristics commonly used are known from the TG/DTG curves and related calculations: pyrolysis initial temperature T_s; maximum pyrolysis weight loss rate (dw/dt)_max; peak temperature T_max corresponding to (dw/dt)_max; average pyrolysis weight loss rate (dw/dt)_mean; maximum pyrolysis weight loss rate V_∞; temperature interval ΔT_1/2 corresponding to (dw/dt)/(dw/dt)_max = 1/2 of the temperature interval ΔT_1/2. The D values are calculated by Equation (4) (

Table 4. Pyrolytic properties of cellulose.

Particle Size (μm)	(dw/dt)max (%/°C)	(dw/dt)mean (%/°C)	V∞ (%)	TS (°C)	Tmax (°C)	ΔT(1⁄2) (°C)	D
250	−17.30	−1.53	86.913	309.36	339.60	37.61	5.82 × 10−6
178	−19.99	−1.57	88.850	312.58	340.30	47.18	5.56 × 10−6
150	−17.31	−1.51	85.965	316.87	337.40	43.19	4.87 × 10−6

) and the above data show that the D values of 250, 178, and 150 μm cellulose are 5.82 × 10⁻⁶, 5.56 × 10⁻⁶, and 4.87 × 10⁻⁶, respectively. The TG/DTG curves further demonstrated the effective removal of hemicellulose and lignin by the treatment with bio-enzyme A combined with NH₃·H₂O and H₂O₂/CH₃COOH. Cellulose content was increased. In other words, the thermal stability of cellulose is improved due to the removal of lignin and hemicellulose, which have poor thermal stability. The results show that as the particle size becomes smaller, the overall index D value also becomes smaller. It indicates that the purity and thermal stability of cellulose are higher and better.

3.5. ¹³C-NMR Characterization of Cellulose

The cellulose was further analyzed in combination with ¹³C-NMR because of its more complex structure. Based on the above series of characterization, the best Neosinocalamus affinis cellulose (150 μm particle size) was selected and characterized by ¹³C-NMR. Figure 6 shows the ¹³C-NMR spectra of 150 μm Neosinocalamus affinis cellulose. The peak at 63.11 ppm is caused by the C₆ glucopyranose repeat unit in cellulose. Peaks around 72.54, 73.91 and 75.35 ppm are caused by C₂, C₃ and C₅. The peak at 84.4 ppm is caused by C₄ in the non-crystalline region of cellulose. No significant characteristic peak appears at 84.44 ppm in Figure 6. It indicates that the 150 μm Neosinocalamus affinis cellulose was preferentially degraded in the non-crystalline region after the treatment. The resulting increase in crystallinity of the cellulose is in general agreement with the XRD characterization results. The absorption peak at 101.66 ppm belongs to C₁ of glucose in cellulose [44].

3.6. CNMs Yield and Viscosity Analysis

To study in-depth the preparation of nanocellulose using bio-enzymes, the extracted cellulose was prepared into nanocellulose by bio-enzyme B. Figure 7 shows the preparation of CNMs from cellulose extracted from different particle sizes by bio-enzyme B. The yield of CNMs was calculated by Equation (3). The figure shows that the yield of CNMs was 14.27% for 250 μm, 15.44% for 178 μm and 16.38% for 150 μm, respectively. It was clearly shown that the yield of CNMs gradually increased as the particle size of Neosinocalamus affinis cellulose decreased and the contact area of the reaction became larger. Owing to the small particle size, the reaction contact area is large. By the synergistic action of pectinase and cellulase, the endonuclease in cellulase effectively promotes the breakage of molecular chains in the amorphous region of cellulose [45].

Figure 8 shows the CNMs suspensions prepared from three different particle sizes of Neosinocalamus affinis cellulose. The CNMs suspensions were in translucent colloidal form with concentrations of about 0.097 wt%, 0.102 wt%, and 0.119 wt%. The concentration of the nanocellulose suspension affects its dispersion state, from stable suspension to gel state as the concentration increases [46]. The CNMs suspensions prepared in this experiment were relatively low in concentration and presented a relatively stable suspension state.

From

Table 5. The viscosity of CNMs suspensions with different particle sizes.

Particle Size (μm)	Concentration (wt%)	Viscosity (mPa·s)
250	0.097	18.79
178	0.102	20.21
150	0.119	22.64

, it can be learned that the viscosities of the prepared CNMs suspensions were 18.79, 20.21, and 22.64 mPa·s. The viscosity of CNMs suspension was found to increase with the increase in concentration. Moreover, the viscosity was negatively correlated with the particle size. This may be due to the relatively high yield and concentration of CNMs with a particle size of 150 μm, which makes the viscosity of the suspension relatively large as well. Then the CNMs suspension was left at room temperature for a certain period. It was found that there was no significant change in the suspension of CNMs with 150 μm particle size (e.g., precipitation, stratification, etc.), and the suspension was still relatively turbid. The results showed that the suspension of CNMs with 150 μm particle size had better stability and the highest ratio of nano/microfibrils in the suspension [47,48].

Due to the relatively high concentration, yield and viscosity of the CNMs prepared at 150 μm particle size, the CNMs suspension was more stable and had the highest ratio of nano/microfibrils. Therefore, the CNMs prepared at 150 μm particle size were further analyzed (TEM, SEM) for their morphological characteristics and dimensions.

3.7. TEM Analysis

Figure 9 shows the transmission electron microscopy images of CNMs prepared from 150 μm particle size of Neosinocalamus affinis powder using bio-enzyme B. Figure 9 shows that the CNMs prepared in this study were in the form of fibrous bundles and cross-twisted with each other. It is due to the amorphous region in the internal structure of cellulose, which causes CNMs to exhibit aggregation [49]. Figure 9 shows that the CNMs prepared in this study are cellulose nanofilaments (CNFs).

3.8. SEM Analysis

Figure 10 shows the electron micrographs of CNFs prepared from Neosinocalamus affinis powder with a particle size of 150 μm. CNFs in Figure 10 show irregular fibrous bundles. It is because the hemicellulose outside the microfiber inhibits the cleavage and cleavage of the microfiber by cellulase [50]. The average diameter of the CNFs measured and calculated by Image J software was 74 nm. The microstructure of the prepared CNFs can be seen in Figure 10 as a network structure. The nanofibers entangle the fibers in the network structure. It is caused by the interaction between the hydroxyl groups [51]. This network formation capability is an important feature of nanocellulose. This structure greatly increases the regional interface for bonding with the composite and plays a toughening role in the composite [52]. The analysis showed that the prepared CNFs were in the nanoscale range. They can be added to the composites as a toughening component to improve the properties of the composites.

4. Conclusions

In this study, cellulose was extracted and nanocellulose was prepared from three different particle sizes (250, 178, and 150 μm) of Neosinocalamus affinis using a bio-enzyme method. The results showed that the highest cellulose content (74.08%), the highest crystallinity (63.58%), the highest purity and the best thermal stability were extracted from the Neosinocalamus affinis powder with a particle size of 150 μm. The highest yield (16.38%) of nanocellulose was prepared from Neosinocalamus affinis powder with a particle size of 150 μm. The concentration (0.119 wt%) and viscosity (22.64 mPa·s) of the nanocellulose suspension were relatively the highest. The nanocellulose exhibits a network structure. This network structure can play a toughening role. As a toughening component, it can be compounded with other materials to improve the toughness of the composite material.

Studies have shown that the smaller the particle size of Neosinocalamus affinis, the easier it is to react with bio-enzymes. The higher the content and purity of the extracted cellulose, the better the thermal stability and the higher the crystallinity. The smaller the particle size, the higher the yield of the prepared nanocellulose. The concentration and viscosity of the nanocellulose suspension are also relatively higher. The results illustrate the feasibility of cellulose extraction and preparation of nanocellulose from bamboo using the bio-enzyme method. The source of raw materials for cellulose extraction and preparation of nanocellulose using the bio-enzyme method was expanded to improve the utilization of bamboo as a biomass material.