Physicochemical Characterization of Acid-Treated Nanocrystal Cellulose and Amorphous Cellulose from Bamboo Sawdust

ABSTRACT The major by-product of bambooworking (i.e. making items from bamboo) is bamboo sawdust, which is a waste product from bambooworking operations and can cause human health hazards. Specifically, this experimental research investigates the effects of acid treatment methods on the physicochemical characteristics of nanocellulose fibers from bamboo sawdust. The experimental acid treatment methods are sulfuric acid hydrolysis for bamboo fiber nanocrystal cellulose (BBF-NCC) and sulfuric acid dissolution for bamboo fiber amorphous cellulose (BBF-AC). The physicochemical properties of nanocellulose from bamboo sawdust include the crystalline structure, morphology, and colloidal stability. The experimental results indicate that BBF-NCC possesses a higher crystallinity index (89.5%), in comparison with 35.4% for BBF-AC. The BBF-NCC is of rod shape with 10 nm in diameter and 50–100 nm in length, while BBF-AC coalesces into clusters of varying sizes. In addition, BBF-NCC possesses higher colloidal stability than BBF-AC, as indicated by a zeta potential of −30.93 mV for BBF-NCC, compared with −19.73 mV for BBF-AC. Essentially, this research is the first to experimentally convert bamboo sawdust into cellulose nanofibers as an eco-friendly and economically viable solution to the waste problem from the bamboomaking industry. Moreover, BBF-NCC and BBF-AC could potentially be adopted as a reinforcing agent in biocomposites or as a carrying agent in multiple applications.


Introduction
Bamboos are a diverse group of mostly evergreen perennial flowering plants making up the subfamily Bambusoideae of the grass family Poaceae.There are roughly 1,200 bamboo species, with approximately 40 species of bamboo growing in Thailand (Sungkaew and Teerawatananon 2017).Large tracts of natural bamboo forest are found in tropical Asian countries, including China, India, Myanmar and Thailand.
Bamboo is classified as a grass because of the monofilament stems and blade-like leaves.Bamboo trees can grow 1.5 inches per hour or 35 inches per day.The plant requires no fertilizer and no replanting is required as it can regenerate from its own root system (Hu et al. 2019).Besides, cellulose accounts for 40% of the total bamboo mass, followed by hemicellulose (31%), lignin (21%), protein (2%), extractives (4%), starch (1%), and ash (1%) (Kurei et al. 2021;Rabemanolontsoa and Saka 2013).
Bamboos are used in numerous products ranging from construction materials, bridges, furniture, handicraft, paper pulp, basketry to utensils (Sun et al. 2011).The major by-product of bambooworking is bamboo sawdust, which is a waste product from bambooworking operations such as sawing, sanding, milling, and routing; and is unfit for human consumption (Mohite et al. 2022).Besides, bamboo sawdust can cause eye and skin irritation.Once inhaled, the sawdust can cause respiratory irritation, nasal dryness, coughing, sneezing, wheezing, headache, and sinusitis (Kasangana et al. 2017).
Previous research studies experimentally extracted cellulose fibers from bamboo stems (Lin, Huang, and Yu 2021).However, there exist no research studies that attempt to extract cellulose from bamboo sawdust as an environmentally-friendly solution to converting bamboo sawdust into cellulose nanofibers.
Cellulose is a green alternative to fossil-fuel-based polymers (Chen et al. 2018).Cellulose is highly crystalline materials formed by the aggregation of long thread-like bundles of molecules stabilized laterally by hydrogen bonds between hydroxyl groups and oxygen adjacent molecules (Zhang et al. 2012).The advantages of cellulose include biodegradability, renewability, sustainability, and biocompatibility.The crystalline structure of cellulose could be transformed by acid treatment and temperature manipulation.Specifically, sulfuric acid hydrolysis under high temperatures generates nanocrystalline cellulose, while amorphous cellulose is obtained by dissolving cellulose in sulfuric acid at a low temperature.
Previous research studies experimentally extracted nanocrystal cellulose from different raw materials under variable conditions, including pineapple with 64% H 2 SO 4 at 45°C (Santos et al. 2013), white coir with 30% H 2 SO 4 at 60°C (Nascimento et al. 2014), banana with 65% H 2 SO 4 at 50°C (Mueller, Weder, and Foster 2014), and sisal fibers with 60% H 2 SO 4 at 55°C (Mariano, Cercená, and Soldi 2016).Meanwhile, Pantamanatsopa, Ariyawiriyanan, and Ekgasit (2022) experimentally extracted nanocrystal cellulose from water hyacinth under the optimal condition of 50% H 2 SO 4 at 60°C and 30 min.In this current research, the bamboo nanocrystal cellulose was hydrolyzed under the aforementioned optimal condition.Jorfi and Foster (2014) examined the use of NCC in a wide range of medical applications, including drug delivery, tissue repair and healing, and surface modification of medical implants.Dong et al. (2014) studied NCC bond with folic acid for targeted delivery of chemotherapeutic agents to increase receptor-positive cancer cells.
Amorphous cellulose is a type of chemically treated cellulose.Existing research on the extraction of amorphous cellulose from the natural fiber is less, including amorphized cellulose production by ball milling (Maier et al. 2005), In a study by Glas et al. (2015), amorphous cellulose was successfully extracted from cotton and filter paper (Avicel PH-101) using an ionic liquid.Furthermore, Burger et al. (2020) conducted experiments involving the functionalization of amorphous cellulose with zinc chloride (ZnCl 2 ) for a flame-retardant application.Their findings indicated that amorphous cellulose is highly compatible as a substrate for ZnCl 2 .Ioelovich (2013) experimentally extracted amorphous cellulose nanoparticle (ANP) and apply to cure skin function; and was found ANP can immobilize various therapeutically-active substances (TAS) containing basic functional groups.The ANP-TAS compatible use as remedies aimed for effective care and cure of the skin.Ioelovich (2021) experimentally extracted amorphous cellulose from cotton using the sulfuric acid dissolution method; and found that the optimal acid dissolution condition was 65-70%v/v sulfuric acid at 5-20°C, achieving the maximum amorphous cellulose yield of 65-70%.However, no research studies exist on amorphized cellulose from natural fiber, mainly bamboo sawdust.As a result, this current research attempted to experimentally extract amorphous cellulose fiber from bamboo sawdust using sulfuric acid dissolution.
Specifically, this research comparatively investigates the effects of different acid treatment methods on the physicochemical properties of nanocellulose fibers from bamboo sawdust.The experimental acid treatment methods are sulfuric acid hydrolysis for bamboo fiber nanocrystal cellulose and sulfuric acid dissolution for bamboo fiber amorphous cellulose.The physicochemical characteristics of the bamboo nanocellulose include the crystalline structure, morphology, and colloidal stability.Essentially, this research is the first to experimentally convert bamboo sawdust into cellulose nanofibers.The waste-to-nanofibers conversion is an eco-friendly and economically viable solution to the waste problem from the bamboomaking industry.

Preparation of bamboo cellulose fiber, nanocrystal cellulose and amorphous cellulose
Figure 1 shows the purification and extraction process of bamboo saw dust (raw bamboo fiber or BBF-R) for bamboo nanocrystal cellulose (BBF-NCC) and bamboo amorphous cellulose (BBF-AC).To obtain BBF-R, bamboo sawdust was sun-dried for 5 days to reduce the moisture content and filtered by a No. 18 sieve (1.0 mm nominal sieve opening with a wire diameter of 0.56 mm) for uniform raw fiber size.The sun-dried BBF-R was then boiled in 2 M NaOH solution at 90°C for 60 min and filtered by a No. 18 sieve and washed with distilled (DI) water to remove NaOH, so-called the alkaline-treated BBF (BBF-A).The BBF-A was subsequently bleached using 0.05 M aqueous NaOH buffer and 10% v/v aqueous H 2 O 2 at 90°C for 120 min with constant mechanical stirring (first bleaching).The bleached bamboo fibers (BBF-B) were filtered by a No. 18 sieve and washed with DI water.To further remove the impurities, the bleaching was repeated six times before oven-dried at 60°C for 4 h.
To prepare BBF-NCC, the oven dried BBF-B (after sixth bleaching) was hydrolyzed by diluted H 2 SO 4 solution (50% v/v) whereby 1 g of BBF-B was treated with H 2 SO 4 solution (1 g/10 ml) and constant magnetic stirring at 800 rpm for 30 min at 60°C (Pantamanatsopa, Ariyawiriyanan, and Ekgasit 2022).To stop the reaction, 150 ml of DI water was added to the mixture and centrifuged at 3,500 rpm for 15 min at 20°C (Frontier, PerkinElmer).The procedure was repeated until the aqueous BBF-NCC suspension was formed, as indicated by the flow birefringence.In this study, the flow birefringence of BFF-NCC was determined by using cross-polarized light setup.The aqueous BBF-NCC suspension was neutralized by dialysis process for 3 days using the dialysis membrane size 36.The process ended when the pH was 6. (Note: The initial pH of the DI water was 6.).
To prepare BBF-AC, the oven-dried BBF-B was dissolved using diluted H 2 SO 4 solution (60% v/v), whereby 1 g of BBF-B was treated with 40 ml of H 2 SO 4 solution by adding 5 ml of the solution every 5 min with constant magnetic stirring at 800 rpm at 0°C (placing in the ice bath).The procedure ended once the mixture became clear gel.To remove H 2 SO 4 , 1500 ml of DI water was added to the clear mixture and stirred using an overhead stirrer at 1500 rpm before centrifugation at 3500 rpm and 20°C for 3 min.The aqueous amorphous suspension was neutralized by adding 500 ml DI water and centrifuging at 3500 rpm for 5 min at 20°C.The procedure was repeated until the pH was 6. (Note: The initial pH of the DI water was 6.)

Color and microstructure of the bamboo fibers
The color of BBF-R, BBF-A, and BBF-B were visually inspected.The microstructure of the bamboo fibers were characterized by scanning electron microscopy (SEM; JEOL, JSM-5410LV) operated at an accelerating voltage of 5 kV.The dried samples of BBF-R, BBF-A and BBF-B were coated with gold layer prior to SEM analysis.

Chemical composition of the bamboo fibers
The chemical composition of BBF-R, BBF-A, and BBF-B were analyzed by Fourier transform infrared spectroscopy (FTIR; Frontier, Perkin Elmer).In the FTIR analysis, samples of bamboo fibers were first oven-dried at 50°C for 1 h and then individually scanned (16 times for each fiber sample) using the FTIR spectrometer in attenuated total reflectance mode at 4000-650 cm −1 wavelength and the spectra recorded in absorption mode.
The chemical composition of BBF-R and BBF-B were verified by nuclear magnetic resonance spectroscopy (NMR; JEOL, JNM-ECZR 500 MHz).In the NMR analysis, samples of BBF-R and BBF-B were oven-dried at 50°C for 1 h and analyzed by 13 C solid-state cross-polarization/magic angle spinning (CP/MAS) NMR at 10 kHz.The spectra of CP/MAS 13 C NMR were refined with the Delta 5.3.1 software.

Crystalline structure
The crystallinity index of BBF-B, BBF-NCC, and BBF-AC were determined using X-ray diffraction (XRD).In the XRD analysis, samples of the bamboo fibers were oven-dried at 50°C for 1 h and measured in the 2θ angular domain from 5° to 40°, given λ = 0.154 nm.The cellulose crystallinity index (I Cr ) was calculated based on the XRD spectra using equation (1).I Cr (%) =((I 002 -I am )/I 002 ) x 100% (1) where I 002 is the maximum intensity of the 002 crystalline peak between 20-23° and I am is the amorphous intensity at 18°.The crystalline structure of the bamboo fibers were analyzed by NMR with 13 C NMR.In the NMR analysis, samples of BBF-NCC and BBF-AC slurry (2 wt%) were oven-dried at 50°C for 8 h and analyzed by 13 C solid-state cross-polarization/magic angle spinning (CP/MAS) NMR at 10 kHz.The spectra of CP/MAS 13 C NMR were refined with the Delta 5.3.1 software.

Yields of bamboo nanocrystal cellulose and bamboo amorphous cellulose
The yields of BBF-NCC and BBF-AC were calculated by equation ( 2), where W 1 is the dried weight of BBF-NCC or BBF-AC and W 0 is the weight of bamboo fiber after alkaline treatment and bleaching before acid treatment. (2)

Morphology of bamboo nanocrystal cellulose and bamboo amorphous cellulose
The morphology of BBF-NCC and BBF-AC were characterized by transmission electron microscopy (TEM) (Talos, Thermo Scientific).TEM was utilized to determine the dimensions and physical appearance of BBF-NCC and BBF-AC.In the TEM analysis, the BBF-NCC and BBF-AC aqueous suspensions were diluted with DI water for BBF-NCC and BBF-AC suspensions with 0.2 wt%.The diluted suspensions were deposited on the surface of a carbon grid with a thin carbon film and left to dry at room temperature (25°C).The TEM analysis was carried out with an accelerating voltage of 80 kV.

Zeta potential of bamboo nanocrystal cellulose and bamboo amorphous cellulose
The zeta potential of BBF-NCC and BBF-AC aqueous suspensions (0.01 wt%) were determined using a particle analyzer (Delsa Nano C, Beckmam Coulter).To mitigate the effect of ionic strength, the aqueous suspensions were diluted in an aqueous standard solution with pH 7.

Microstructure of BBF-R, BBF-A and BBF-B
Figures 2a-d present the images of BBF-R, BBF-A, and BBF-B (after first and sixth bleaching), respectively.In Figure 2a, the color of BBF-R was brown and changed to light brown after alkalization, as shown in Figure 2b.Figures 2c, d show the BBF-B after the first and sixth bleaching, respectively.The color of BBF-B became whiter with increase in the number of bleaching.The change in fiber color was attributable to the removal of non-cellulosic materials (i.e., lignin and hemicellulose) and other impurities (i.e., pectin and wax) after alkalization and bleaching.Figures 3a-c show the SEM microstructure of BBF-R, BBF-A, and BBF-B (after sixth bleaching).In Figure 3a, BBF-R were bundled together, and the fibers were thick and rough.After the alkalinization, the BBF-A became unbundled, thinner, and smoother (Figure 3b).In Figure 3c, the bleaching further thinned out the bamboo fibers, resulting in the microfibers.The smoother and thinner fibers were attributable to the removal of non-cellulosic components and other impurities, The findings are consistent with Salih, Zulkifli, and Azhari (2020); Liu et al. (2019);Obi Reddy et al. (2012).

TEM morphology of BBF-NCC and BBF-AC
The TEM images of BBF-NCC are depicted in Figures 4a, b and those of BBF-AC are shown in Figures 4c-e.The BBF-NCC was of rod shape as the acid hydrolysis destroyed the amorphous domains of cellulosic microfibrils while retaining the straight crystalline domains.Figure 5a-c show the dimension, length, and aspect ratio of BBF-NCC, respectively.The diameters of BBF-NCC were between 4-20 nm (Figure 5a), the lengths were between 50-150 nm (Figure 5b), and the aspect ratios were between 5-10 (Figure 5c).The findings are consistent with Orellana, Wichhart, and Kitchens (2018).
Figure 4c-e depicts the TEM images of BBF-AC in which the cellulose fibers coalesced into clusters of varying sizes.The sulfuric acid completely dissolved the crystalline domain in the BBF-B prior to the regeneration of new crystalline domain of random particle size (Ioelovich 2021).

The yields of BBF-NCC and BBF-AC
Figure 6a shows the aqueous suspension of BBF-NCC under normal light, while Figure 6b depicts the aqueous suspension of BBF-NCC under cross-polarized light setup.In Figure 6a, the BBF-NCC aqueous solution was of clear yellow.In Figure 6b, the flow birefringence showed the cellulose nanocrystals in the BBF-NCC aqueous suspension.The NCC yield was 69%.Meanwhile, Figure 6c shows the aqueous suspension of BBF-AC under normal light.The BBF-AC aqueous solution was of milky white color.The AC yield was 65%.
In Figure 7d, the FTIR pattern of BBF-AC closely resembles that of BBF-B, suggesting that acid dissolution caused no damage to the cellulose chain.In Figure 7e, the FTIR of BBF-NCC exhibited a strong band peak at 3337 cm −1 , due to the stretching vibration of the O-H groups.The presence of OH-groups could be attributed to sulfuric acid-induced disruption of the amorphous domains.

NMR chemical composition and crystalline structure analysis
Figures 8a, b show the NMR spectra of BBF-R and BBF-B, respectively.In the figures, the peaks at 54 and 148 ppm were characteristic of lignin and that at 20 ppm was characteristic of hemicellulose.However, all the peaks associated with lignin and hemicellulose disappeared after alkalization and bleaching, suggesting that alkalization and bleaching effectively removed lignin and hemicellulose, consistent with Lin, Huang, and Yu (2021).
Figures 8c, d show the NMR spectra of BBF-NCC and BBF-AC.In the figures, the peak at 60 ppm was characteristic of C6, and those between 70 and 80 ppm were characteristic of C2, C3 and C5.The  peaks between 80 and 93 ppm; and between 102 and 108 ppm were characteristic of C4 and C1, respectively, the findings are consistent with Abiaziem et al. (2019).Specifically, the peaks between 86 and 92 ppm belonged to the crystalline domain, while those between 80 and 86 ppm to the amorphous domain.
In Figure 8c, the sharp crystalline peak of BBF-NCC at 86.5 ppm indicated that the acid hydrolysis effectively removed the amorphous regions.On the other hand, in Figure 8d, the broad crystalline peak of BBF-AC at 86.5 ppm demonstrated that the acid effectively dissolved the crystalline regions, consistent with Lin, Huang, and Yu (2021).

XRD crystalline structure of cellulose
Figures 9a-c show the XRD spectra of BBF-B, BB-NCC, and BBF-AC.The intensity of the dominant crystalline peak (I 002 ) of BBF-NCC (Figure 9b; 2θ ≈ 22°) was noticeably higher than BBF-B (Figure 9a; 2θ ≈ 22°).The result could be attributed to the effective removal of amorphous domains by acid hydrolysis.The maximum intensity of the dominant crystalline peak (I 002 ) at 22° indicated that BBF-B and BBF-NCC belonged to cellulose type I.In Figure 9c, I 002 of BBF-AC shifted to the lower 2θ (≈20°) as     compared to BBF-B, could be attributed to the effective removal of the amorphous regions by the acid hydrolysis.The lowest crystallinity index of BBF-AC was attributable to the dissolution of the crystalline domain and subsequent regeneration of the crystalline structure.

The zeta potential of BBF-NCC and BBF-AC
Table 2 tabulates the zeta potential of BBF-B, BBF-NCC, and BBF-AC.The zeta potential of BBF-NCC was −30.93 mV, followed by BBF-AC of −19.73 mV and BBF-B of −9.53 mV.According to Joseph and Singhvi (2019), a zeta potential below −30 mV or above 30 mV indicates high colloidal stability.In this research, the improved negative zeta potential was attributable to the insertion of H 2 SO 4 -induced charged sulfate ester groups onto the BBF-NCC and BBF-AC surface.

Conclusion
This experimental research investigated the effects of different acid treatment methods on the physicochemical characteristics of BBF nanocellulose from bamboo sawdust.The experimental acid treatment methods were sulfuric acid hydrolysis for BBF-NCC and sulfuric acid dissolution for BBF-AC.The physicochemical characteristics of BBF nanocellulose included the crystalline structure, morphology, and colloidal stability.The results showed that BBF-NCC possessed higher crystallinity index (89.5%),compared with 35.4% for BBF-AC.The significantly higher crystallinity index of BBF-NCC was attributable to the efficient removal of the amorphous regions by acid hydrolysis.Meanwhile, the low crystallinity index of BBF-AC was attributable to the destruction of crystalline regions by acid dissolution.The TEM results showed that BBF-NCC was of rod shape with 10 nm in diameter and 50-100 nm in length, while BBF-AC coalesced into clusters of varying sizes.Besides, the BBF-NCC exhibited higher colloidal stability than BBF-AC, as evidenced by a zeta potential of −30.93 mV for BBF-NCC and −19.73 mV for BBF-AC.Specifically, the acid treatment improved the zeta potentials of BBF-NCC and BBF-AC.Essentially, the BBF-NCC and BBF-AC could potentially be adopted as a reinforcing agent in biocomposites or as a carrying agent in numerous applications.Of particular relevance is that this experimental research is the first to convert bamboo sawdust (i.e., a waste product from bamboo working operations) into cellulose nanofibers as an eco-friendly and economically viable solution to the waste problem from the bambooworking industry.

Highlight of this paper
(1) This paper compares the effects of different acid treatment methods on the physicochemical characteristics of bamboo fiber (BBF) nanocellulose from bamboo sawdust.(2) In this paper, we are the first to report an experimentally convert bamboo sawdust into cellulose nanofibers (crystalline nanocellulose, BBF-CNC and amorphous cellulose, BBF-AC) as an eco-friendly and economically viable solution to the waste problem from the bamboo-making industry.
(3) The bamboo waste from industry is sawdust often found in many bamboo-making factories or communities which is regarded as waste, and most of this waste is burned or landfilled.If we can turn it into a value-added of BBF-CNC and BBF-AC we can help the reduction of waste problem.

Figure 1 .
Figure 1.The flowchart of the purification and extraction process for bamboo nanocrystal cellulose (BBF-NCC) and bamboo amorphous cellulose (BBF-AC).
the acid dissolved the crystalline domain in the BBF-B.The new peak (I 002 ) indicated the transformation from cellulose type I to type II, consistent withIoelovich (2021).The crystallinity index of BBF-B and BBF-NCC were calculated based on the dominant peak (I 002 ) at 22°, while that of BBF-AC based on I 002 at 20°.The crystallinity index of BBF-B, BBF-NCC, and BBF-AC were 76.2%, 89.5%, and 35.4%, respectively.The higher crystallinity index of BBF-NCC,

Table 1 .
Assignments of characteristic FTIR absorption bands of the bamboo fibers.