Enhanced Cellulose Isolation from Sugarcane Bagasse through Sequential Alkali and Oxidative Treatment

doi:10.21203/rs.3.rs-7360666/v1

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Enhanced Cellulose Isolation from Sugarcane Bagasse through Sequential Alkali and Oxidative Treatment

https://doi.org/10.21203/rs.3.rs-7360666/v1

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As the world seeks sustainable alternatives to fossil-based materials, agricultural residues like sugarcane bagasse are gaining prominence as valuable bioresources. Sugarcane bagasse, the fibrous waste left after juice extraction, is an agricultural residue that is particularly rich in cellulose, a biopolymer with immense potential for green material innovation. The present work utilised sugarcane bagasse, to obtain cellulose by optimising the Sodium hydroxide (NaOH) and Sodium hypochlorite (NaOCl) concentrations. Characterisation techniques such as Fourier transform infrared spectroscopy (FTIR), Field emission scanning electron microscope (FE-SEM) and X-ray Diffraction (XRD) were employed for detailed analysis. This article also encloses the effect of NaOH and NaOCl concentration on the cellulose yield and the various possible applications of the obtained cellulose. To evaluate statistical significance, one-way ANOVA was performed, complemented by Tukey’s multiple comparison test. Compositional analysis showed that the cellulose content of raw sugarcane bagasse was 42.6±1.5%. However, after alkali and bleaching treatment, the cellulose content was in the range 42.6±0.6% - 56.5±0.5%. FTIR analysis confirmed the successful cellulose extraction from sugarcane bagasse, as evidenced by the disappearance of lignin and hemicellulose associated peaks and characteristic cellulose absorption bands. XRD analysis revealed an increase in the crystallinity index from 29.8% in SCB to 53.7% in extracted cellulose. Morphological analysis employing FE-SEM highlighted significant surface differences in SCB and extracted cellulose. Statistical analysis unfolded that amendment with NaOH and NaOCl enhanced the cellulose yield significantly (p≤0.05) compared to raw sugarcane bagasse. The study highlights the immense potential of agricultural waste as a renewable and cost-effective source of cellulose. By leveraging these residues, industries can reduce dependence on conventional raw materials while promoting sustainable and environmentally responsible practices.

Agricultural residue

Cellulose

Sugarcane Bagasse

Alkaline treatment

Bleaching

The rapid generation of agricultural waste has become a growing environmental concern. It often leads to open-field burning as a method of disposal and this practice significantly contributes to greenhouse gas emissions and air pollution, exacerbating environmental and health issues (Ben et al. 2024; Debnath et al. 2021). This agricultural waste is an eco-friendly renewable resource and a natural carbon source; it is especially well-suited to be utilised as a precursor to create novel carbon-based materials (Sole et al. 2021). Despite its potential, less than 5% of LCB is currently utilized for productive purposes, with the rest remaining underexploited or discarded (Ashok et al. 2022). Effective utilization of agro-waste plays a vital role in reducing environmental harm while promoting the development of sustainable materials. In particular, the valorization of lignocellulosic biomass (LCB) offers a promising solution by transforming abundant agricultural residues into value-added products. This approach not only addresses the issue of waste management but also helps mitigate environmental pollution and decrease dependence on fossil fuel-based resources (Gangil & Wakudkar, 2013).

By harnessing LCB for the production of eco-friendly materials, significant progress can be made toward achieving environmental sustainability and advancing a circular bioeconomy. Lignocellulosic biomass (LCB), composed mainly of cellulose, hemicellulose, and lignin, can be sourced from sugarcane bagasse, wood chips, sawdust, rice husks, cotton linens, etc., highlighting its vast availability in nature (Khui et al. 2021). Among the components of LCB, cellulose stands out due to its biodegradability, renewability, and its ability to be engineered into micro- and nanoscale particles that exhibit novel physicochemical properties (Shao et al. 2020). The growing interest in cellulose is due to its potential applications in biodegradable plastics, textile industry, biomedical devices, drug delivery, and landscape mulch films (Raymond et al. 2024). Harnessing cellulose from agricultural waste such as sugarcane bagasse offers a dual benefit—efficient waste management and the development of eco-friendly materials for a greener future.

Waste products from agriculture, such as can be used as cheap, sustainable, and renewable raw materials to make cellulose and its derivatives for the Vast amounts of sugarcane bagasse waste are produced annually by processing sugarcane to make juice and alcohol (Saad et al. 2022). The accumulation of massive quantities of sugarcane bagasse poses a complex waste problem because, up until now, it scarcely found any commercial utilisation (Shaikh et al. 2009). Managing agricultural biomass has been regarded as an essential tactic in managing and using natural resources and maintaining environmental quality (Raymond et al. 2024). Nowadays, the sugar industry uses the majority of sugarcane bagasse as boiler fuel, feedstock, and manure. Nevertheless, the residual bagasse threatens the ecosystem (Mubarak et al. 2024). SCB primarily consists of cellulose, which is manufactured in enormous quantities worldwide (Mahmud and Ananya, 2021). According to its chemical makeup, Cellulose is a natural linear polymer composed of anhydroglucose units connected through β-glycosidic bonds between the first and fourth carbon atoms (Kadla et al. 2000). Cellulose structure is shown in Fig. 1. It is arranged into fibrils encased in a lignin and hemicellulose matrix. Although cellulose II chains are anti-parallel, cellulose I has a parallel chain orientation. The efficient use of sugarcane bagasse (SCB) as raw material for cellulose extraction has become more prevalent in recent years (Sun et al. 2004). Cellulose is used extensively as a diluent, thickener, cellulose ether, bioplastics, lubricant, regenerated textile fibres, film binder, and coating in the production of tablets and capsules (Wan et al. 2018; Aziz et al.2022).

Various pretreatment techniques, generally divided into four primary categories: physical, chemical, physicochemical, and biological, can be used to separate cellulose (Sun et al. 2016). Among the different pretreatment techniques, chemical procedures use chemicals to dissolve hemicelluloses and break the bond between cellulose and lignin. This strategy has several benefits, such as a simple extraction procedure, high extraction efficiency, superior thermal stability, advantageous crystallinity, simplicity in regulating reaction conditions, and cost-effectiveness (Lou et al. 2022). Bagasse's primary benefit is that it is a waste material that yields a very cost-effective, fully or partially biodegradable product after a few pretreatments, a crucial consideration in today's market. Furthermore, with the correct procedure, the extracted fibre can exhibit reasonably acceptable mechanical properties (Loh et al. 2013). Numerous investigations have explored the extraction of cellulose from agro residue through chemical treatments. For instance, Mubarak et al. 2024 utilised 200 ml (10–30%) of NaOCL to extract cellulose from 5g Sugarcane bagasse. However, this concentration of bleaching agent seems so high for a meager portion of bagasse. In another study, cellulose extraction from SCB employed 2–18% NaOH and 38% H₂O₂ (Rasheed et al. 2024). Moubarik et al. (2013) used a two-stage treatment including hot water and NaOH(15%) for cellulose extraction from SCB, yielding 42% of cellulose. No bleaching treatment was given here, and the NaOH(15%) concentration was high. Also, the obtained yield was not much higher than that of raw sugarcane bagasse. Thus, it is discernible that most of the researchers used a high concentration of NaOH and bleaching agents.

The present study attempts to find the optimum conditions for cellulose extraction. Also, it focuses on the effect of different sodium hypochlorite and sodium hydroxide concentrations on the yield and properties of extracted cellulose. The extracted cellulose was characterised with the help of FTIR, XRD and SEM. Through a straightforward recycling method that relies on removing the non-cellulosic component from SCB, the current work seeks to increase the economic value of sugarcane bagasse.

Materials and chemical substances

Raw sugarcane bagasse (SCB) was gathered from juice merchants in the Indian state of Haryana's Rohtak area. Sodium hypochlorite from Loba Chemie was used as a bleaching agent. CDH provided sodium hydroxide and hydrogen peroxide. All chemical compounds used in this experimental study were of analytical grade.

Raw Material Preparation

To guarantee total cleanliness, the substrate was cleaned adequately with running tap water to remove dirt and then sun-dried. Following this, the substrates were ground and sieved with a sieve of size 0.2 mm (Yadav et al. 2024). The sieved SCB powder was stored in a ziplock container at room temperature.

Determination of the Chemical composition of Sugarcane bagasse

The composition of SCB affects the bioconversion processes. A significant proportion of sugarcane bagasse consists of cellulose, cemicellulose and lignin. Apart from these compositional ingredients, it includes other elements like extractives, moisture, nitrogen, organic carbon, ash, and trace elements (Alokika et al. 2021). The SCB in this study was analysed for extractive, lignin, holocellulose, cellulose, and hemicellulose content. The process diagram for the compositional analysis of Sugarcane bagasse is shown in Fig. 2.

Extractives

In the Soxhlet extractor setup, 150 ml of acetone and 2.5 g of SCB were taken. During a 4-hour run period, the residence periods for the boiling and rising stages were meticulously set to 70 and 25 minutes, respectively, on the heating mantle. The extracted sample was allowed to air dry for a few minutes at room temperature. The removed material's weight remained constant in a convection oven set at 105°C. The extractive content can be calculated using Eq. (1) (TAPPI T 204 cm-17).

$$\:\text{E}\text{x}\text{t}\text{r}\text{a}\text{c}\text{t}\text{i}\text{v}\text{e}\text{s}\left(\text{%}\right)=\frac{W1}{W}x100$$

Where W = Initial weight of SCB and W1 = Final weight obtained after treatment.

Lignin

The Tappi T222 om-88 (Tappi test methods) determines the amount of lignin in biomass samples. 1 g of extractive-free biomass and a 72% H₂SO₄ solution were heated for two hours at room temperature. After diluting the sample with water to bring the sulphuric acid concentration down to 3 per cent, it is boiled for an additional four hours. The lignin is then filtered after being given time to settle. The residue is rinsed with hot water until a neutral pH is achieved. The extracted lignin content can be determined using Eq. (2) (Rizwan et al. 2021).

$$\:Lignin\left(\%\right)=\frac{W3}{W2}x100$$

Where, W2 = Initial sample mass and W3 = Obtained sample mass.

Holocellulose

With a few modifications, the conventional procedure outlined by Wise et al. (1946) was used to determine the holocellulose content. 10 g of SCB was bleached using hydrogen peroxide buffered with sodium hydroxide solution. The mixture was heated at 70°C for 4 hours. Once the mixture had cooled, the residue was filtered out, cleaned with water, dried, and weighed. Hollocellulose, a blend of cellulose and hemicellulose found in residues, can be quantified using Eq. (3) (Rizwan et al.2021).

$$\:\text{H}\text{o}\text{l}\text{o}\text{c}\text{e}\text{l}\text{l}\text{u}\text{l}\text{o}\text{s}\text{e}\left(\text{%}\right)=\frac{\text{W}5}{\text{W}4}\text{x}100$$

Where W4 = Weight of the original sample, and W5 = Weight of the obtained holocellulose.

Cellulose

For cellulose content determination, a 1 g sample of holocellulose and 25 ml of 17.5% NaOH were combined in a flask and heated at 95°C for one hour. 25 millilitres of distilled water (DW) was added, and the residue was filtered. After that, 25 millilitres of a 10% acetic acid solution was added, filtered once more, and repeatedly cleaned with distilled water. The amount of cellulose residue was determined after the sample was dried for 24 hours at 40°C. Silva et al. (2011). Cellulose content percentage is determined using Eq. (4) (Song et al. 2019).

$$\:\text{C}\text{e}\text{l}\text{l}\text{u}\text{l}\text{o}\text{s}\text{e}\left(\text{%}\right)=\frac{\text{W}7}{\text{W}6}\text{x}100$$

Where = Initial amount of holocellulose and W7 = Final amount of extracted cellulose.

Hemicellulose

The difference between the holocellulose and cellulose concentrations was calculated to determine the hemicellulose fraction (Eq. 5) (Balasubramani et al. 2024). The percentage that remains after deducting cellulose from holocellulose is hemicellulose. This formula is frequently used to analyse the composition of plant fibres in wood science, biomass research, and the paper industry.

Hemicellulose = Holocellulose − Cellulose (5)

Isolation of Cellulose

In this study, cellulose extraction was conducted with the help of combined and modified methods of Ungprasoot et al. (2021), Melesse et al. (2022) and Khiewsawai et al. (2023). The process of cellulose extraction from sugarcane bagasse is shown in Fig. 3, and it mainly involves three steps: dewaxing using ethanol, alkaline treatment using NaOH and bleaching treatment using NaOCl. The cellulose yield(%) is greatly affected by the amount of NaOH and NaOCl used. Thus, the two are used in varying concentrations, and six samples were prepared and labeled accordingly as given in Table 1.

Table 1
Chemical Processing Conditions for Cellulose Extraction from Sugarcane bagasse.
Sample	NaOH (%)	NaOCl (%)
SCBC1	2	1.5
SCBC2	2	3
SCBC3	5	1.5
SCBC4	5	3
SCBC5	8	1.5
SCBC6	8	3

Dewaxing

Dewaxing was done using a solution of ethanol and deionised water in 1:1 v/v for around four hours, then boiling the mixture for 1.5 hours. This procedure was repeated twice to remove dust, dirt, sugar extracts, and water-soluble contaminants. To remove any remaining sugar and surfactants, the mixture was rinsed with hot water first, followed by cold water. After that, it was oven-dried for a whole day.

Alkaline Treatment

Three varying concentrations 2%, 5%, and 8% of sodium hydroxide (NaOH) solution(200 ml ) were applied to a 10 g sample to perform the alkaline treatment. The samples were heated for two hours, stirring occasionally, to a temperature of 75°C. This process made the desired changes possible, guaranteeing that the sample and the alkaline solution interacted well. They were then extensively cleaned with distilled water to neutralise the solid residue that had been produced.

Bleaching

Two hundred millilitres of NaOCl at two differing concentrations 1.5% and 3% was used to bleach the residue left over after the alkaline treatment. For 30 minutes, residue obtained after alkali treatment was heated to 80°C while being constantly stirred and then to attain a neutral pH it was cleaned with distilled water.

Then for 16 hours, the isolated cellulose samples were oven-dried at 50°C. After oven drying, it was ground into a fine powder and kept in ziplock containers to retain moisture.

Yield

Cellulose yield indicates the proportion of cellulose successfully extracted, reflecting cellulose recovery efficiency. The yield was calculated as a percentage relative to the dry weight of the SCB after treatments. Accurate measurements of the initial and final weights of SCB and SCBC were used to determine the yield using Eq. (6) (Mubarak et al., 2024).

$$\:\text{Y}\text{i}\text{e}\text{l}\text{d}\left(\text{%}\right)=\frac{\text{W}9}{\text{W}8}\text{x}100$$

W8 = Initial weight of SCB, and W9 = Final weight of extracted cellulose.

Characterization

FTIR Analysis

Fourier transform infrared (FTIR) Spectroscopy was used to examine the functional groups. Bruker's Invenio ® FTIR spectrometer was employed. It had a 2 cm^− 1 resolution and was operated in gearbox mode. FTIR spectra were obtained over a range of wavenumbers from 4000 to 400 cm^− 1.

XRD

A multifunctional, adaptable X-ray diffractometer system of model Smartlab 3kW/Rigaku was used to conduct the X-ray diffraction analysis. With a phase of 0.04 and a scanning time of five minutes, the analysis was carried out in the 2β range value scanned from 10 to 50º. Segals method [(Eq. (7)] was used to calculate crystallinity index (Segal et al. 1959).

CrI (%) = [(I002 - Iam) / I002] × 100 (7)

Where, I002 = Intensity of the crystalline peak and Iam = Intensity of the amorphous background

FE-SEM

The micrographs were obtained using a Carl Zeiss field emission scanning electron microscope. The aluminium stubs were covered with a layer of gold after the dried, finely powdered SCB and SCBC3 samples were put on them. Better conductivity is guaranteed by this gold coating, which also improves the quality of the images acquired during the ensuing image processing procedure.

Statistical analysis

The experimental work was performed in triplicates. The results are given as the mean value and its corresponding standard deviation. The mean of altered treatments, Dunnett test for control, and analysis of variance (ANOVA) were assessed using Tukey’s test at the 0.5% statistical level in the IBM SPSS Statistics 25.0. Statistically significant differences among treatments are indicated by different letters in the figures, based on the Tukey multiple comparison test (p ≤ 0.05). Graphs were generated using Origin software.

Chemical Composition of Raw Sugarcane Bagasse

The raw SCB employed in this investigation contains 7.4 ± 0.9% extractives, 16.3 ± 0.5% lignin, 23.6 ± 2.08% hemicellulose, and 42.6 ± 1.5% cellulose. The chemical composition of SCB is shown in Fig. 4. The sugarcane bagasse (SCB) used in the aforementioned study has a cellulose, hemicellulose, and lignin concentration that falls within the ranges described in prior investigations. The cellulose content was close to that of Yadav et al. 2024, where the cellulose content was 43.2%. Nonetheless, they reported 16.6% and 26.5% of lignin and hemicellulose respectively.

The extractive and cellulose content of the present study were higher than that of the Rasheed et al. 2024, where 6% extractives and 41% cellulose were reported. The cellulose concentration was lower than the studies of Mohammed et al. 2023 and Ungprasoot et al. 2024 where sugarcane bagasse consists of 46% and 47% cellulose, respectively. In comparison to other agroresidues, the cellulose content was higher than wheat husk (36–39%) and lower than rice straw (48.5%) (Bledzki et al. 2011; Ungprasoot et al. 202). The lignin content of 23% (Lalucea et al. 2019), 21% (Rana et al. 2021) and 38% (Mubarak et al. 2024) were also found to be higher than in the current study, where lignin content is 16.3 ± 0.5%. The hemicellulose content was also higher in the few studies, ranging from 26.5%-35.2% (Laluce et al. 2019; Ungprasoot et al. 2021; Yadav et al. 2024).

Table 2 shows the comparative analysis of the chemical composition of SCB in the present study, as well as the chemical composition of SCB and different agricultural residues reported in the literature. Overall, the compositional data of sugarcane bagasse (SCB) closely aligns with values reported in the literature, highlighting its potential as a promising feedstock for various bio-based applications. Its well-balanced proportions of cellulose, hemicellulose, and lignin, combined with a moderate level of extractives, make SCB highly suitable for uses such as biofuel production, paper manufacturing, and the development of composite materials.

Table 2
Chemical composition of SCB and different agricultural residues.
Agro-residue	Cellulose (%)	Hemicellulose (%)	Lignin (%)	Extractive (%)	Reference
SCB	42.6 ± 1.5%	23.6 ± 2.08%	16.3 ± 0.5%	7.4 ± 0.9%	Present Study
SCB	46	28.7	22.6	2.04	Silva et al. (2011)
SCB	40	35	23	-	Laluce et al. (2019)
SCB	45.6	29.4	21.2	-	Rana et al. (2021)
SCB	41	18	16	6	Rasheed et al. (2024)
SCB	43.2	26.5	16.6	-	Yadav et al. (2024)
SCB	47	20.8	28	-	Ungprasoot et al. (2021)
SCB	46	16	38	-	Mubarak et al. (2024)
Wheat Husk	36–39%	18–21%	16%	-	Bledzki et al., (2010)
Wheat Bran	24	32	46	-	Stevenson et al. (2012)
Wheat Straw	33–45	19–32	8–16	-	Wang et al. (2012)
Calotropis procera	64.1 ± 1.6	19.5 ± 1.2	9.7 ± 1.2	-	Song et al. (2019)
Rice straw	48.5	35.2	5	-	Ungprasoot et al. (2021)
Chickpea husk	52.3	31.9	13.9	1.8	Lamo et al. (2024)

Impacts of alkali and bleaching treatment on the Cellulose yield.

This study examined the impact of varying sodium hydroxide and sodium hypochlorite concentrations on sugarcane bagasse cellulose production. It is generally recognised that hemicellulose is dissolved and removed from the biomass due to pretreatment with NaOH causing delignification, by significantly rupturing the cross-ester linkage between complex hemicellulose and lignin (Jung et al. 2020). According to Hashim et al. (2017), the lignin and hemicellulose dissolve in alkali solutions, forming a black liquid when the hydrogen bonds between the lignocellulosic components are broken, increasing the yield of cellulose. After alkali treatment, hemicellulose and lignin were still present in the extracted residue, and bleaching was required to eliminate them further (Geng and Han, 2023). Oxidising chemicals, such as sodium hypochlorite bleaching, were used to remove leftover lignin and other impurities affecting cellulose appearance and quality (Kapdi et al. 2024). The hypochlorite ion in NaOCl is a potent oxidant that can dissolve the ether bond in the lignin structure and enhance the pulp's white brightness (Sayakulu and Soloi, 2021). The brightness and purity of the cellulose were further enhanced by bleaching. Alkali pretreatment caused SCB's cellulose content to rise while hemicellulose and lignin levels were reduced. The pictorial representation of the various extracted cellulose samples can be seen in Fig. 5.

It was discovered that the yields for SCBC1 and SCBC2 were 46.1 ± 0.7% and 43.3 ± 0.6%, respectively. These are significantly less than the yield of 57.6% obtained in the study by Yadav et al. 2024. Hemicellulose can be partially broken down by 2% concentrations of NaOH, which results in some hemicellulose being removed from the sugarcane bagasse. Nonetheless, the pretreated biomass might still include a sizable amount of hemicellulose (Yadav et al. 2024). The use of NaOCl for bleaching could be the cause of the decreased concentration. It is evident from the study of Raymond et al. 2024 that a high concentration of bleaching agent reduced the yield of cellulose. In their study, using 10–20% NaOCl resulted in a lower yield, i.e., 32.2–40.2%. In another study 6% NaOCl alone was used for cellulose extraction, and the yield was 48% (Laluce et al. 2019). Thus, it is clear that the NaOCl concentration also affects the cellulose yield.

Sample SCBC 3 produced yield of 56.5 ± 0.2%. The Kapdi et al. (2024) study also demonstrated similar outcomes, with a 58% cellulose production from rice straw. The yield for SCBC 4 was 53.1 ± 0.4%. The cellulose yield was reduced significantly at high concentrations of NaOH (8%). SCBC5 and SCBC6 were 43.6 ± 0.8% and 42.6 ± 0.6% (Lowest Yield) respectively. The results are in harmony with the results of (Rasheed et al. 2024), where the yield using 10% NaOH and NaOCl was 44.2%. The yield percentage of extracted cellulose samples is shown in Fig. 6. It was discovered that raising the sodium hydroxide concentration reduced the yield because some of the cellulose chains would break down during the treatment procedure. The cellulose molecules were freely distributed in the solvent (NaOH) because of their stable open structure. A high concentration of NaOH will break up a few crystalline regions in cellulose and cause it to dissolve more readily in the solution treatment, lowering the amount of cellulose fibre produced (Azmin et al. 2020). A certain quantity of cellulose may dissolve in high concentrations of NaOH, lowering the percentage of cellulose (Martin-Bertelsen et al. 2020). This investigation's findings align with those of Kathirselvam et al. (2019), Melesse et al. (2022), Rahayu et al. (2022), and Saad et al. (2022), who also showed that a higher concentration of NaOH resulted in decreased cellulose yield. NaOCl's bleaching qualities enhanced the colour of the extracted samples and aided in the dissolution of any remaining lignin and hemicellulose. SCBC1 was light brown, possibly due to the low concentration of NaOCl (1.5%). SCBC2, SCBC3 and SCBC5 were found to be pale creamish yellow, whereas SCBC4 and SCBC6 were much brighter and white in appearance. Overall, Bleaching makes the finished cellulose product more aesthetically attractive and appropriate (Kapdi et al. 2024). It can be concluded that much higher and lower concentrations result in low yield and purity, respectively. Thus, the optimum concentration for cellulose extraction were 5% NaOH and 1.5% NaOCl, resulting in highest yield 56.5 ± 0.2% (SCBC3). The obtained cellulose yield reflects an effective isolation procedure and aligns well with previously reported values, further supporting the potential of sugarcane bagasse in value-added bioproduct development.

FTIR

Sugarcane bagasse (SCB) and extracted cellulose can be efficiently characterised using Fourier Transform Infrared (FTIR) spectroscopy. It aids in elucidating modifications to functional groups and molecular structures brought about by the pre-treatment procedure.The spectra of SCB and all extracted samples were broadly similar, with a few significant differences that indicate structural and compositional changes. Table 3 unfolds the Functional groupspresent in the FTIR spectra of Sugarcane bagasse and Sugarcane bagasse cellulose. The broad peak at 3326 cm⁻¹ and 3338–3339 cm⁻¹ in raw SCB and all the extracted cellulose samples, respectively, confirms the presence of hydroxyl (-OH) groups, indicating strong hydrogen bonding, a characteristic of cellulose (Nandiyanto et al. 2019). As the purification process advanced, the bell-shaped absorption band between 2980 and 3690 cm⁻¹ became somewhat narrower (Wang et al. 2018). Similar peaks at approximately 3434.5 cm⁻¹ and 3426.1 cm⁻¹were found for the bagasse and the extracted cellulose, respectively (Lu et al. 2012). Figure 7 presents the FTIR spectra of Raw SCB and extracted cellulose samples. The peak at 2890–2902 cm⁻¹in extracted cellulose samples and SCB were associated with C–H stretching from polysaccharides, validating the sample's organic composition by matching the C–H stretching vibrations of the methyl and methylene groups in polysaccharides (Saad et al. 2022; Mubarak et al. 2024).

Table 3
Functional groups in the FTIR spectra of Sugarcane bagasse and Sugarcane bagasse cellulose.
Wavenumber (cm^− 1)		Functional group assignment
Sugarcane bagasse	Sugarcane bagasse cellulose	Functional group assignment
3326	3338–3339	O-H Stretching vibrations
2890	2890–2902	C-H Stretching
1723	-	Carbonyl (C = O) stretching
1630	1612–1630	OH (Water Absorbed)
1511	-	Aromatic C = C Stretching
1292	-	Aromatic C-O Stretching
1152 − 1015	1123–1167	C-O-C Stretching
895	982–1018	C-O Stretching Vibration (Cellulose)
-	816–841	Ring Deformation
653	622 − 594	C-H Bending

The H–O–H bending of absorbed water is responsible for the peak at 1612–1630 cm⁻¹, indicating that the SCB and extracted cellulose contain some moisture. The moisture content of cellulose is marked by this area in other studies by Wang et al. (2020), Saad et al. (2022), Freitas et al. (2022) and Mubarak et al. (2024) with peaks in the range 1629 cm⁻¹to 1645 cm⁻¹. Further Peaks in the 1123–1167 cm⁻¹and 1018–982 cm⁻¹ ranges are present in extracted cellulose and in the range 1152 cm⁻¹-1015 cm⁻¹ and at 895 cm⁻¹ in SCB confirming C–O–C and C–O stretching respectively, which are traits of cellulose's glycosidic bonds which are crucial cellulose structural elements. The polysaccharide structure is confirmed by the literature, which shows that absorption bands in the 1162 cm⁻¹-899 cm⁻¹ range are linked to C–O–C and C–O stretching vibrations (Sayed and Khalaf, 2023; Yadav et al. 2024). SCB peaked at 653 cm⁻¹, corresponding to C–H bending. All the extracted cellulose samples exhibited Peaks at 816–841 cm⁻¹ and 622–594 cm⁻¹, and are associated with ring deformation and C–H bending, common in cellulose structures. These results align with the literature (Mubarak et al. 2024; Freitas et al. 2024).

Raw SCB exhibited peaks at 1723 cm⁻¹, 1511 cm⁻¹ and 1292 cm⁻¹, which correspond to C = O stretching that represents aldehyde, ketone, or carboxylic acids in hemicellulose, aromatic C = C stretching of lignin and C-O Stretching vibrations of lignin and hemicellulose, respectively. These peaks were either absent or were reduced in the extracted cellulose samples, which indicates the removal of hemicellulose and lignin, suggesting that cellulose has been successfully separated from sugarcane bagasse. Previous studies demonstrated similar findings (Viera et al. 2007; Zhao et al. 2018; Kapdi et al. 2024).Identifying the extracted sample as cellulose is strengthened by the close alignment of FTIR data with known cellulose spectrum features. While the water-related peak indicates some bound moisture, hydroxyl groups, glycosidic connections, and C–O stretching validate the cellulose structure. The FTIR peaks show that lignin and hemicellulose have been significantly removed. Typically, The current samples peak at 1612–1630 cm⁻¹, not the aromatic C = C stretching of lignin but rather H–O–H bending (absorbed water). The presence of cellulose is confirmed by the prominent peaks in your spectrum for O–H stretching (3338 cm⁻¹), C–O–C glycosidic linkages (1123–1167 cm⁻¹), and C–O stretching (1018 − 982 cm⁻¹). Rather than lignin or hemicellulose, the peaks at 816–841 cm¹ and 622–594 cm¹ are characteristic of cellulose. The presence and successful isolation of cellulose are confirmed by the preserved C–O–C and C–H peaks. Overall, the work was in harmony with (Sun et al. 2004; Viera et al. 2007; Perumal et al. 2018; Laluce et al. 2019; Katakojwala and Mohan 2020; Rasheed et al. 2024; Freitas et al. 2024). FTIR data indicates that the sugarcane bagasse has successfully undergone hemicellulose removal and delignification, leaving behind purified cellulose.

XRD

X-ray diffraction (XRD) is a crucial method for figuring out a material's crystallographic structure. This method includes subjecting a material to X-ray radiation and then determining the angles and intensities of X-ray scattering that result (Mubarak et al. 2024). Cellulose, lignin, and hemicellulose make up most of SCB; cellulose is crystalline, whereas lignin and hemicellulose are amorphous (Kundu et al. 2023). The present study examined structural changes and assessed the impact of alkali (NaOH) and bleaching (NaOCl) on the crystallinity of the resultant cellulose using XRD analysis of raw sugarcane bagasse (SCB) and SCBC3, which yielded the maximum amount of cellulose. The XRD spectra of SCB and SCBC3 are shown in Fig. 8.

There were discernible peaks at 2θ values of 15.16° and 21.96° in the XRD analysis of SCB, indicating the existence of the cellulose I. SCBC3 displays prominent peaks at 2°, measuring 15.84° and 22.68°, significantly stronger than in the bagasse. The existence of these peaks is proof that the treatments affected the extracted cellulose. These peak positions imply that the treated fibres' interlunar distance had grown (Liu et al. 2019). The crystallinity index was 29.8% and 53.2% for SCB and SCBC3, respectively. Due to its higher amorphous proportion of lignin and hemicellulose, the SCB has low relative crystallinity (Guilherme et al., 2013). The alkali (NaOH) and bleaching (NaOCl) treatment are the causes of the enhanced crystallinity index seen in extracted cellulose (Melesse et al. 2022). Alteration in structure and crystallinity of cellulose may be caused as a result of disruption of intra- and inter-chain H-bonding of cellulosic fibrils (Sharma et al. 2023 and Kundu et al. 2023). However, mild reaction conditions did not change the crystalline structure of the cellulose, and the extracted cellulose constituted mainly cellulose I in the structural form (Saad et al. 2022).

The finding of current study is consistent with earlier studies that found that extracting cellulose from sugarcane bagasse increased the crystallinity index. In a study by Yadav et al. (2024), XRD patterns continuously show peaks at roughly 2θ angles of 16.3 and 22.5°. Compared to the untreated SCB biomass, the SCB that had received sodium hydroxide pretreatment displayed a significantly raised peak. The CrI value of the raw SCB was 38.8%. Extracted celulose produced CrI levels of 53.64%. Saad et al. (2022) discovered that the bagasse fibre exhibits two distinct peaks around the 2θ value of 15.33º and 22.09° and the extracted cellulose showed prominent peaks at 2θ of 16.35º and 22.47º that were noticeably stronger. Also the CrI raised from 31.76–51.13%. The presence of the cellulose I structure is suggested by the peaks that can be seen in the bagasse fibre XRD analysis at 2θ values of 15.22° and 21.94°. Similarly, the extracted cellulose displays distinct peaks at 2°, measuring 15.02° and 22.19°, which are significantly more potent than those in the bagasse (Mubarak et al. 2024). The results of this study are also in agreement with several previous studies that reported an increase in this index value after biomass pretreatment (Galiwangoa et al. 2019;Kininge and Gogate 2022; Melesse et al. 2022; Bangar et al. 2023; Sharma et al. 2023; Sayed & Khalaf 2023; Lamo et al. 2024; Freitas et al.2024; Rasheed et al. 2024). This discovery highlights how alkali treatment affects the cellulose fibres' structural characteristics, which advances our knowledge of their properties and uses.

FE-SEM

The morphological differences between SCB and the extracted cellulose sample SCBC3 treated were analysed using Field emission scanning electron microscopy (FE-SEM), as presented in Fig. 9. The FE-SEM image of raw sugarcane bagasse reveals a compact, smooth, and intact surface with minimal visible porosity. This dense outer layer is characteristic of the naturally occurring lignocellulosic matrix, composed predominantly of cellulose, hemicellulose, lignin, waxes, and other extractives (Kumar et al. 2014; Khalid et al. 2021). In contrast, the treated sugarcane bagasse cellulose shows a highly porous, fibrillated structure, indicative of effective removal of non-cellulosic components, primarily lignin and hemicellulose (Feleke et al. 2023; Ding et al. 2025). Similar changes were seen in the morphological structure of SCB, followed by alkali pretreatment by Yadav et al. (2024). The untreated sample retains intact plant cell wall architecture, with minimal evidence of fibril exposure, suggesting low surface accessibility and reactivity. Conversely, the treated cellulose sample exhibits disrupted cellular networks and partially exposed cellulose microfibrils, suggesting significant structural breakdown and loosening of the plant matrix due to chemical pretreatment (Cao et al. 2006). A lack of visible porosity in the raw sample indicates the hydrophobic nature and inaccessibility of cellulose chains in their native state. In contrast, the treated sample presents a network of interconnected pores, cracks, and voids, beneficial for applications requiring water absorption or bonding (Velmurugan et al. 2024). The increased porosity directly correlates with improved functional properties, such as water uptake, surface modification, and biopolymer reinforcement (Razali et al. 2022).

Overall FE-SEM analysis confirms that pretreatment of sugarcane bagasse leads to profound morphological changes, especially in surface porosity (Rasheed et al. 2024), fibrillation (Phiri et al. 2024) and accessibility(Ding et al. 2024), facilitating enhanced surface area (Verma and Goh 2021), which is crucial for downstream applications. The results of the current study are also in harmony with the previous studies for SCB treated with acidified NaOCl and alkali treatment by Kumar et al. (2014), with acid and alkaline pretreatment and combined hydrothermal and alkaline pretreatment by Guilherme et al. (2015), with bleaching agent by Mubarak et al. (2024), with alkaline treatment and hydrogen peroxide bleaching by Rasheed et al. (2024). Additionally, these findings also show consistency with the results of FTIR and XRD, and validate the efficiency of the cellulose extraction process.

Statistical analysis

Results of statistical analysis revealed that the yield of cellulose was significantly enhanced (p ≤ 0.5) in SCBC1, SCBC2, SCBC3, SCBC4, and SCBC5 after the amendment with NaOH and NaOCl compared to raw sugarcane bagasse. Meanwhile, SCBC6 did not exhibit a significant change in yield. It was also observed that no significant (p ≤ 0.5) difference were observed between SCBC2 and SCBC5.

Application

An intriguing polymeric substance that is widely available on Earth is cellulose and agricultural leftovers are good source of cellulose. It is an auspicious raw material for replacing non-renewable feedstocks because of its widespread availability. Researchers have focused on natural cellulose because of its advantages over synthetic cellulose, which include its availability, affordability, perusability, biocompatibility, and minimal toxicity. It can also undergo chemical modification to enhance its chemical and/or physical characteristics. Commonly appealing uses of cellulose include printing, electronics, packaging, and healthcare materials (Aziz et al. 2022; Magalhaes et al. 2023). Various applications of cellulose are presented in Fig. 10.

The literature contains a wealth of research on modifying and using cellulosic materials. The efficient incorporation of SCB cellulose into a hydrogel composite using the gamma irradiation approach was noteworthy. This composite can be utilized as a platform for the adsorption of mercury ions (Khiewsawai et al. 2024). Cellulose was converted to carboxymethylcellulose (CMC) in a different study (Ungprasoot et al. 2021). Cellulose extracted from SCB was used to prepare microfibrillated cellulose (MFC). This improved the properties of microfibrillated cellulose, allowing it to be easily shaped into pliable sheets. The increased flexibility provides a straightforward method for creating eco-friendly, sustainable and biodegradable products (Wibowo et al. 2024). The conversion of cellulose into hydroxyethyl cellulose has numerous critical industrial applications (Halim, 2014).

Using sugarcane bagasse, Alirach et al. (2023) created and characterised bio-sheets for possible application as bio-sheet packaging material. The biosheets had 2.39 kg cm^− 2, 25.3 kgf and 11 kgf bursting strength, strip strength and stiffness, respectively. The results demonstrated that the bio-sheets under optimal conditions possessed sufficient physical qualities. These characteristics imply that the bio-sheets can also be improved to create bio-bags. Using ecologically friendly materials, specifically tapioca starch and sugarcane bagasse fibre (SBF), Asrofi et al. (2020) attempted to develop composite bioplastic. SBF is added to the tapioca matrix to reinforce the structural integrity of composite bioplastics. The findings demonstrate that adding ultrasonication improves the tensile strength of the composite bioplastic samples. The sample that underwent 15 minutes of ultrasonication at 2.5 MPa had the highest tensile strength. Ali et al. (2022) created a PVA/starch nanocomposite film reinforced with sugarcane bagasse cellulose nanofiber (CNF) as an alternative to the current biodegradable plastic packaging options.

Using ultrasonication and alkaline and mild acid treatment, cellulose nanofibers (CNF) were separated from sugarcane bagasse (SCB). It enhanced antimicrobial, thermal, and mechanical qualities. Using an N-dimethylacetamide/lithium chloride solvent, sugarcane bagasse nanofibers were converted into an all-cellulose nanocomposite (ACNC) film. The nanofiber sheet, ACNC and fibre sheet produced with a 10-minute dissolution time had respective tensile strengths of 8 MPa, 101 MPa, and 140 MPa. As the dissolution period increased, the ACNC film's water vapour permeability (WVP) rose proportionately. Because of its promising qualities, ACNC may be used in cellulose-based food packaging (Ghadheri et al. 2014). In 2020, Azmin et al. used sugarcane bagasse and cocoa pod husk to create biodegradable plastic films. Future research should concentrate on the environmentally friendly conversion of cellulose and its functional derivatives with increased efficiency and high selectivity.

Sugarcane bagasse exhibited great potential for cellulose extraction. The process conditions were optimised to maximise the cellulose yield. The process conditions with 5% NaOH and 1.5% NaOCl were the optimum, with the highest cellulose yield of 56.5 ± 0.2%. FTIR analysis revealed the removal of lignin and hemicellulose from sugarcane bagasse after treatment, as the peaks at 1723 cm⁻¹, 1511 cm⁻¹ and 1292 cm⁻¹ responsible for these components were missing in the extracted cellulose samples. However, the peaks relating to C–O–C and O–H confirmed the presence of cellulose. The XRD results showed an enhancement in cellulose crystallinity following alkali pretreatment. FE-SEM imaging effectively highlighted the structural transformations caused by the treatment, and an increase in surface porosity and accessibility was observed. Thus, it can be concluded that cellulose was successfully isolated from SCB. Statistical analysis also revealed a significant difference (p ≤ 0.05) in the yield of cellulose after applying the varying concentrations of NaOH and NaOCl as compared to raw SCB. The cellulose generated can find application in biofilm preparation after adding suitable binders. While the current study did not examine the effects of time and temperature, future investigations should explore how changes in these pre-treatment conditions influence the cellulose extraction to achieve a deeper understanding. Further evaluation of the cost-efficiency of these processes will provide critical insights for their adoption in sustainable industrial practices.

Conflict of Interest

The authors declare that they have no competing interests.

Funding

No funding was received for the preparation of this manuscript.

Author Contribution

This study was a collaborative effort, with contributions from all authors. Shikha Kumari conceptualized and designed the study, performed the experiments, and prepared the initial draft of the manuscript. Dinesh Arora conducted the statistical analyses and gave valuable sugestions for the improvement of manuscript. Dr. Manjeet Kaur and Dr. Geeta Dhania thoroughly reviewed the manuscript, offered valuable insights, and finalized the paper for submission. All authors read and approved the final version of the manuscript.

Acknowledgement

The author gratefully acknowledges the support and encouragement provided by the Department of Environmental Science, Maharshi Dayanand University, Rohtak, Haryana, India throughout the course of this research. The facilities, academic environment, and valuable guidance offered by the department played a significant role in the successful completion of this study.

Data Availability Statement

This study did not generate or analyze any datasets.

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Enhanced Cellulose Isolation from Sugarcane Bagasse through Sequential Alkali and Oxidative Treatment

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Abstract

Figures

Introduction

Materials and Methodology

Materials and chemical substances

Raw Material Preparation

Determination of the Chemical composition of Sugarcane bagasse

Extractives

Lignin

Holocellulose

Cellulose

Hemicellulose

Isolation of Cellulose

Dewaxing

Alkaline Treatment

Bleaching

Yield

Characterization

FTIR Analysis

XRD

FE-SEM

Statistical analysis

Results and Discussion

Chemical Composition of Raw Sugarcane Bagasse

FTIR

XRD

FE-SEM

Statistical analysis

Application

Conclusion

Declarations

Conflict of Interest

Funding

Author Contribution

Acknowledgement

Data Availability Statement

References

Supplementary Files

Status:

Version 1