Comparative study of lignin biocellulose and bacterial cellulose

doi:10.21203/rs.3.rs-4717370/v1

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Comparative study of lignin biocellulose and bacterial cellulose

https://doi.org/10.21203/rs.3.rs-4717370/v1

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The abundant oil palm empty fruit bunches possess attractive properties of lignin and biocellulose that present significant potential for conversion into a range of products. Nonetheless, this requires an appropriate pretreatment strategy that achieves a favorable equilibrium between effective lignin removal and mild cellulose extraction, to fully exploit these biopolymer constituents. This study demonstrated the effective utilization of OPEFB lignin and β-cellulose in developing an innovative biofilm product, underscoring the efficient utilization of lignin biocellulose. An encouraging technique involving bacterial cellulose, which is increasingly being explored for new applications in the food and medical fields, is evaluated for its suitability in OPEFB pretreatment. Nevertheless, it was observed that the current enzymatic approach is not ideal for overcoming the lignin protective barrier during OPEFB pretreatment. Areas for enhancement in this method are pinpointed, particularly in the production of ligninolytic enzymes that can rival emerging alkaline pretreatment methods.

OPEFB

lignin biocellulose

cellulose

bacterial cellulose

The abundant lignin content in oil palm empty fruit bunches (OPEFB) offers a significant opportunity for utilizing lignin biocellulose in various applications, such as antioxidants and ultraviolet functions (Mastrolitti et al. 2021). Numerous researchers have been exploring methods to extract OPEFB compound to obtain a high purity product, but they have faced limited success. The primary challenge lies in the complex structure of lignin monomer combined with holocellulose in an insoluble linked network within the OPEFB polymer, making direct extraction difficult (Shanmugarajah et al. 2019).

According to (Mastrolitti et al. 2021), a successful OPEFB pretreatment method should prioritize maximum lignin removal while minimizing the impact on holocellulose. This study has analyzed a suitable approach for extracting lignin biocellulose and improving its properties for various product uses. An optimal pretreatment condition has been developed using a modified alkali-based chemical process, which achieves a balance between effectively extracting lignin and preserving cellulose.

This leads to improved lignin purity and recovery, while maintaining cellulose crystallinity suitable for downstream product conversion. Additionally, the study demonstrates the promising feasibility of using lignin biocellulose from OPEFB in lignin CMC biofilm and includes a comparative analysis with bacterial cellulose extraction to develop a viable pretreatment technique.

In today's industrial applications, lignocellulosic biomass sourced from agricultural residues, woody crops, forestry residues, and wastepaper produces cellulose and lignin that are commonly utilized in a variety of industrial applications (Potoˇcnik et al. 2023). The current production of lignin relies on the conversion of a restricted supply of plant-based raw materials such as wood pulp and cotton (Mastrolitti et al. 2021).

Additionally, as suggested by (Perera et al. 2023) cellulosic d-glucose monomer derived from lignocellulosic biomass is utilized as an ingredient in polymers, composites, membranes, and cellulose fiber products. Although the ease of processing lignin allows for its use in different applications such as resin and adhesive, the limited availability of these raw materials and concerns about sustainability and deforestation have prompted the exploration of lignocellulosic biomass as a second-generation feedstock (Hidayat et al. 2022).

Specifically, OPEFB primarily consists of lignin biocellulose, with approximately 51.88% holocellulose (cellulose and hemicelluloses), 31.68% lignin, and 6.69% ash (Riaz et al. 2018). A study by (Law et al. 2020) suggested the relative quantities of the various OPEFB cell wall components contribute to it physiochemical properties in crystallinity, tensile strength, modulus, and moisture presence. The cell wall of OPEFB is commonly composed of cellulose, which contains polar functional groups, making it hydrophilic. Lignin, however, is mainly located in the interfibrous area, which contributes to the structural integrity of plants (Ajayi et al. 2023).

Accordingly, the different source of lignin provides physical and chemical behavior, and the high recalcitrance of OPEFB present difficulty for extracting it closer to the native lignin properties (Chia et al. 2023). Additionally, the presence of silica on the OPEFB surface as suggested by (Katahira et al. 2018) provide a physical barrier to hydrolysis that add to the delignification challenge.

This resulted into the extremely restricted utilization of OPEFB for in mere applications such as a fuel for boilers or soil conditioner (Lai et al. 2021). Therefore, to discover valuable applications for lignin biocellulose, it is imperative to dissolve the OPEFB into separate cellulosic and lignin monomers at high purity levels. This will enable the synthesis of targeted products with high value.

Consequently, this investigation examined the extraction method for obtaining these monomers from OPEFB, aiming to achieve a controlled composition and high purity of these molecules. A comparative assessment is conducted to evaluate various delignification methods including physical, chemical pretreatment, physio-chemical, and thermo-chemical techniques to determine their suitability for OPEFB pretreatment process.

Among these approaches, alkali-based chemical pretreatment is selected as the preferred method due to several reasons. Table 1 describes the various drawbacks associated with the other OPEFB pretreatment methods, which include excessive energy consumption, potential for corrosion, production of hazardous substances, variation in product composition, and a slower rate of hydrolysis.

Alkali-based chemical pretreatment method efficiently breaks down the chemical bonds that bind lignin monomer and other compounds within the cell structure (Chia et al. 2023). It has been discovered by (Haqiqi et al. 2021) that this technique enhances the swelling of cellulose, resulting in a higher inner surface area and a decrease in both the degree of polymerization and crystallinity. Hence, this approach holds the capability to attain a beneficial balance between efficient removal of lignin and mild extraction of cellulose, thereby maximizing the utilization of OPEFB components.

Additionally, the combination of alkaline treatment and ultrasonic sonication has the potential to enhance the extracted yield of lignin, while preserving its structure (Song and Othman 2022). However, further optimization is required to determine the optimal ultrasonic time, thermal stability, and the impact of structural properties on holocellulose.

Nevertheless, if conditions are not applied correctly, the majority of OPEFB compounds may undergo significant disintegration, resulting in the formation of intricate monomers that pose a challenge for extracting lignin from the broth mixture (Mastrolitti et al. 2021). Additionally, (Ajayi et al. 2023) suggests that this process could potentially give rise to unwanted by-products like biochar.

Therefore, to enhance the efficiency of OPEFB delignification, additional optimization is needed on key parameters such as the type of alkaline chemical used, the quantity of alkalinity present, the duration of pretreatment, and the operating temperature (Mohammad et al. 2020).

Table 1

Comparative performance of OPEFB pretreatment method (Ajayi et al. 2023; Mohammad et al. 2020; Song and Othman 2022)
Method	Major Features	Advantages	Disadvantages
Physical	The OPEFB particle size and crystallinity can be reduced through mechanical grinding. This process involves chipping, grinding, and milling.	The surface area of the biomass can be enlarged, leading to a decrease in the degree of polymerization.	The energy requirement is high, and it is not economically feasible.
Alkali-based chemical	Sodium, calcium, and ammonium hydroxide are typical solvents used in various applications. When exposed to NaOH, cellulose swells, leading to an increase in its inner surface area and a reduction in the degree of polymerization and crystallinity.	Capable of enhancing the digestibility of cellulose and the breakdown of lignin in OPEFB.	The pretreatment conditions are milder with this approach, but it needs to be optimized to minimize prolonged reaction times extended reaction durations.
Ultrasound assisted alkali-based chemical	Comparable conditions to alkali- chemical pretreatment coupled with the use of ultrasonic sonication.	There is a possibility of achieving a greater cleaving effect on the lignin ether bonds. Slight increase with carbon content when compared to alkaline lignin.	A longer duration of ultrasonic irradiation is necessary subject to further optimization. This technique can be applicable for delignification once holocellulose has been removed.
Acid-based chemical	The hemicellulose can be dissolved using this approach, which enhances the accessibility of the cellulose compound.	The use of dilute acids can achieve reasonably high hydrolysis rates, but they produce toxic decomposition products.	This technique is suitable for application with either strong or weak acid but results in the production of inhibitory substances and corrosive environments.
Physico-chemical pretreatment	Pretreatment approach with steam pressurization followed by rapid depressurization can be used. This method can also involve the partial hydrolysis and solubilization of hemicellulose, allowing for the extraction of lignin from biomass.	The use of steam pressure in this process takes a moderate amount of time for processing, and it can redistribute or extract the lignin to a specific extent.	Generates harmful substances and incomplete breakdown of hemicellulose occurs.
Thermo-chemical	The thermochemical procedure involves transforming biomass into a syngas intermediate,, at elevated temperature and pressure. The subsequent chemical conversion of the syngas takes place in a downstream process.	Capable of producing syngas mixture containing controlled ratio of hydrogen and carbon monoxide.	The need for elevated temperatures and pressures in processes such as gasification or pyrolysis leads to high operating costs. Instead of focusing on a specific chemical product, it generates a broader variety of fuel products.
Biological pretreatment	Biological pretreatment utilizes a range of microorganisms to facilitate mild synthetic conditions and eco-friendly environments. Among the microorganisms commonly used are white and soft rot fungi, actinomycetes, and bacteria for breaking down lignin.	The potential of this method lies in its ability to facilitate an extraction process that uses minimal energy, low chemical usage, operates under mild environmental conditions, and involves low investment costs. At the initial phase of proof-of-concept development, there is limited understanding of its capability for sorting or extracting cellulose.	There is not much information about whether this approach is economically viable. Compared to other methods, the main drawbacks are the slower hydrolysis rate and the need for a faster and more efficient delignification process from a new microorganism.

The extraction of lignin biocellulose is conducted out through a modified alkali based chemical process, which is designed to achieve a balance between effectively extracting lignin and balance the effective extraction of lignin with the mild extraction of cellulose. A combined method was used to sequentially extract OPEFB lignin and cellulose using optimized parameters, resulting in enhanced lignin purity and recovery, while preserving cellulose crystallinity.

An optimum pretreatment conditions are generated that comprises the use of 4 wt.% NaOH and the customized operation conditions determined from this study, that maximizes lignin purity, lignin recovery and holocellulose from OPEFB. A higher lignin purity of 88.2% was produced against 68.9–71.0% purified lignin as reported by [Ajayi et al. 2023; Mohammad et al. 2020]. In addition, a high lignin recovery of 83.5–98.7% is achieved from the untreated OPEFB that demonstrated this method suitability for delignification. This is in comparison to the lower OPEFB lignin recovery of 65.0-68.9% by using other chemical pretreatment conditions [Ajayi et al. 2023; Parit et al. 2018].

It is also shown that this method provides reasonably high holocellulose recovery of 59.0-63.2% whilst minimize alteration to the crystalline cellulose structure. The high content of extracted cellulose may also be a result of the dissolution of additional components in the sodium hydroxide solution. In comparison, the use of solvent for dissolving cellulosic into sugar monomers such as N-methylmorpholine N-oxide (NMMO) increase the pretreatment cost, lowering the recovery yield and limiting the direct application of OPEFB cellulose (Haqiqi et al. 2021).

Table 2

Comparative chemical composition (%) of pretreated OPEFB
Pretreated OPEFB	Holocellulose recovery (%)	Lignin purity (%)	Lignin recovery (%)
This study	59.0-63.2	88.2	83.5–98.7
References	-	68.9–71.0 [1, 13]	65.0-68.9 [1, 16]

Specifically, the alkali based chemical method performs the cleavage of ester bonds that connect lignin and hemicellulose, resulting in an augmentation of OPEFB surface area, as proposed by (Pasma et al, 2018). This study also found that this process causes most of the soluble lignin to migrate into a weak sodium hydroxide solution, leaving cellulose and hemicellulose with crystalline structure intact as solid byproducts.

Dissolving lignin or lignin salt in an alkaline solution leads to greater lignin removal compared to dissolving it in acid pretreatment (Mohammad et al. 2020). As a result, it is demonstrated in this study an optimized process that provide balance between the lignin recovery rate, achievable lignin purity and carbon content. Accordingly, this study presents an optimized process that demonstrates a balance between the recovery rate of lignin, the purity of lignin, and the carbon content.

The holocellulose extracted from OPEFB was found to exhibit similarities with complex cellulosic carbohydrates, featuring polymer chains linking unbranched β-1,4-polyacetal cellobiose, as described in our previous study (Othman et al. 2024). A combined pretreatment system was developed to acidify the OPEFB fiber with the use of a sodium chloride solution. By optimizing the fiber to solvent ratio parametrically using a chemically stable solvent under controlled conditions, non-degradable β-cellulose was effectively generated.

The effective use of lignin biocellulose was demonstrated by utilizing OPEFB lignin and β-cellulose to produce a new CMC biofilm embedded with lignin in the previous study (Song and Othman 2022). Nevertheless, there have been no reports in the literature about the conversion of raw OPEFB into CMC or the alteration of OPEFB cellulose. One notable exception is the research conducted by (Ajayi et al. 2023), where efforts were made to extract, analyze, and modify OPEFB cellulose in a hydrophobic manner.

As suggested by (Girard et al. 2024), the key CMC product properties on water solubility and chemical reactivity are primarily dependent on the degree of substitution (DS), and to lesser degree are the molecular weight and carboxymethyl substitution position in the polymer chain. In this study, DS of OPEFB CMC is calculated at 1.23, higher than CMC from agricultural cellulose sources such as water hyacinth (0.24–0.73), sago waste (0.33–0.82), sugar beet pulp (0.11–0.67) and durian husk (0.67), but lower than corn husk (2.41) and wheat straw (2.50), as detailed in Table 3.

Our previous study (Song and Othman 2022) has thoroughly examined the properties of lignin-CMC characterization, demonstrating its similar performance to commercially available CMC. This has created possibilities for converting lignin biocellulose into modified and bio-based CMC, presenting a novel and innovative product for a range of practical uses.

Table 3

Degree of substitution (DS) for the produced CMC relative to common sources (Song and Othman 2022).
CMC Types	DS	Remarks
Lignin CMC from OEPFB	1.23	OPEFB
CMC from water hyacinth	0.24–0.73	Agricultural cellulosic source
CMC from sugar beet pulp	0.11–0.67	Agricultural cellulosic source
CMC from sago waste	0.33–0.82	Sago waste
CMC from durian husk	0.67	Durian waste
CMC from corn husk	2.41	Corn waste
CMC from wheat straw	2.50	Grain waste

Based on (Hidayat et al. 2022], bacterial cellulose shares a comparable structure of $\:{\left({\varvec{C}}_{6}{\varvec{H}}_{10}\right)}_{\varvec{n}}$ with lignin biocellulose, yet they vary in terms of their physical and chemical characteristics. According to (Perera et al. 2023), the extraction of bacterial cellulose has the potential to be a cost-effective and environmentally friendly solution, as it does not produce toxic by-products or waste in contrast to chemical processes.

Lignin degradation enzymes such as lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase (Lac) are commonly used for lignocellulosic biomass (Parit et al. 2018). It has been suggested by (Suota et al. 2021) that the LiP enzyme has the potential to be effective in delignification, however, it is necessary to optimize the fermentation medium and operational conditions to address the current slow enzymatic activity. On the other hand, bacterial cellulose displays diverse characteristics such as crystallinity, structure, water absorption capacity, and mechanical strength (Perera et al. 2023).

Furthermore, the scarcity of research on the identification of OPEFB cellulose-degrading bacteria can be attributed to the substantial lignin content present in OPEFB. These lignin levels hinder the enzymes necessary for cellulose hydrolysis during the extraction process, as indicated by (Ajayi et al. 2023). However, an acid hydrolysis pretreatment is often the preferred method for OPEFB to eliminate lignin and improve the holocellulose surface area for more effective enzymatic reactions.

According to (Mohammad et al. 2020) an appropriately implemented pre-treatment strategy can reduce the population of indigenous microorganisms, particularly in the solid-state fermentation procedure. However, this leads to an increase in extraction expenses because of the additional requirement for neutralizing waste acids. As a result, several research studies (Mondylaksita et al. 2022; Parit et al. 2018; Rashid et al. 2021) have investigated microorganisms that are able to tolerate more intense pre-treatment conditions, such as elevated temperatures and a method based on ligninolytic enzymes.

(Rashid et al. 2021) further proposed that the direct fermentation of biocellulose from different biomass sources could potentially result in unique properties, making it a promising biochemical for innovative applications in the food and biomedical industries. This shifts the focus away from its use as an extraction medium to a lesser extent.

5.1 OPEFB quality improvement

The varying compositional properties of OPEFB necessitate the implementation of a control system to maintain consistency in the physiochemical properties of the extracted compound. As stated by (Sadeghifar, and Ragauskas 2018), factors such as fresh fruit bunch (FFB), geographical location, climate, soil composition, fertilization methods, harvesting, and storage conditions influence the composition and moisture levels of OPEFB.

The raw material is sourced from a palm mill in Klang Valley, where it undergoes a 24-hour crushing and sieving process to eliminate moisture content. A control mechanism is then enforced to ensure uniformity in the received OPEFB batches, focusing on aspects like hydrothermal pretreatment for moisture reduction, shredded fiber size, coarse impurities, and density.

The fresh OPEFB, with approximately 70% moisture, was subjected to a drying process at 105°C to decrease its moisture content by 8–10% by weight. Further measures were implemented to store the fresh OPEFB at temperatures ranging from 36–45°C to prevent decomposition and mold formation. Subsequently, the characterization of the fresh OPEFB was carried out to evaluate its physical properties such as ash, moisture, and metal impurities.

This analysis was conducted in accordance with the guidelines of ASTM E1755-01 (2020) for determining ash content in biomass, which also involved the examination of OPEFB's inorganic and mineral content. The organic composition and metal content of OPEFB were determined through proximate analysis using a CHNS elemental analyzer (Thermo Scientific, FlashSmart) and inductively coupled plasma optical emission spectrometry (Perkin Elmer Optima, DV5300). Furthermore, the microstructural analysis of OPEFB was performed using a field emission scanning electron microscope (Hitachi U8000, UHR FE-SEM cryoemission).

5.2 Lignin biocellulose extraction

The OPEFB that had been pretreated hydrothermally was subsequently subjected to an alkaline pretreatment using a diluted solution of sodium hydroxide, as shown in Fig. 2 following the guidelines outlined in references (Haqiqi et al. 2021; Katahira et al. 2018). The resulting OPEFB residue was filtered, neutralized, and dried at a moderate temperature until a constant mass was achieved. This process yielded crude cellulose, which was further characterized through bleaching to holocellulose, comprising α-cellulose and hemicellulose.

The characterization tests conducted considered the robust polymer structure of cellulose, known for its chemical stability and insolubility in both polar and nonpolar solvents. Subsequently, the synthesis of lignin-CMC from lignin biocellulose involved the esterification of α-cellulose hydroxyl groups with chloroacetic acid in the presence of an alkaline solution. This process was based by research conducted by (Girard et al. 2024; 26] to explore the essential processing variables and the production and analysis of lignin-infused CMC.

The experimental trials were conducted to experimentally verify the optimum OPEFB pretreatment conditions, followed by further testing to ensure the reproducibility of the results. As a result, a procedural model was developed to improve the permeability and wetting characteristics of OPEFB, optimizing the pretreatment process to reduce lignin levels without compromising the cellulose properties.

The pre-treated OPEFB underwent neutralization with diluted sulfuric acid to separate lignin from the solution for further refinement. OPEFB lignin exhibits similar behavior to lignocellulosic biomass as suggested by (Chia et al. 2023), particularly in terms of how its dissociation depends on pH levels. The purified lignin is then subjected to centrifugation and lyophilization before undergoing characterization tests.

The remaining solid residue of OPEFB was filtered, neutralized, and dried at a moderate temperature to create a dry raw cellulose mass. This mass was then bleached to produce cellulosic monomers, with alpha cellulose subsequently extracted for physiochemical analysis.

5.3 Purification and characterization

The lignocellulose extraction and purification parameters were determined with Design Expert v12 to determine the optimal acid concentration for maximizing compound recovery. This required finding a balance between compound recovery rate, attainable purity, and carbon content in the pretreatment process to improve monomer solubility in the solvent solution. The purity of the extracted monomer was assessed using Ultraviolet–visible spectroscopy (Hach DR6000 spectrophotometer) in accordance with the method previously described by (Haqiqi et al. 2021).

The absorptivity was determined using the Beer-Lambert law [3]:

$$\:\epsilon\:=\frac{A}{\left(c\times\:l\right)}$$

The absorptivity ε is represented in L $\:{\left[g.cm\right]}^{-1}$, while A stands for the absorbance (a.u.), c represents the concentration of the sample $\:{\left(g.L\right)}^{-1}$, and l is the pathlength of the measured cell (cm). Following this, the molecular weight of the monomer is established through gel permeation chromatography (Viscotek Gpcmax, Ve2001). The physical properties, including the mean molecular weight (Mw), mean molecular weight Z (Mz), mean molecular weight number (Mn), and polydispersity, are then obtained from the resulting chromatogram.

The degree of substitution of Lignin-CMC is subsequently determined based on previous studies by [4, 25]:

$$\:DS=\frac{162\:x\:\frac{0.3\:x\:V}{m}}{1-80\:x\:\frac{0.3\:x\:V}{m}}$$

The molecular weight of the anhydrous glucose unit is 162, with a net increment of 80 in the anhydrous glucose unit for every substituted carboxymethyl group. 𝑉 represents the acid volume, while 𝑚 stands for the mass of CMC. Further properties were analyzed, including the thermal durability of pure CMC and lignin CMC film using thermogravimetric analysis (Mettler Toledo Thermal Analysis). The analysis was carried out in an inert nitrogen environment to avoid sample combustion, ranging from room temperature to 900°C with a heating rate of 10 $\:\frac{℃}{min}$ (Siahkamari et al. 2022).

6.1 OPEFB quality improvement

It was determined that the OPEFB achieved uniform moisture content at this stage and was subsequently sampled for compositional analysis, as detailed in Table 3. The moisture content of OPEFB raw materials can be controlled to less than 8–10% by weight, with coarse dirt kept to a minimum of less than 3% by weight, and fiber size ranging from 1-3cm, enabling precise control of pre-treatment parameters during processing, and enhancing optimization.

By loading OPEFB under more stable conditions during pretreatment, the impact of mold decomposition on pretreatment performance can be avoided. Additionally, the reduction of dry fiber size allows for a higher fiber content, leading to improved biomass loading efficiency and a smaller pretreatment reactor.

Table 3

OPEFB raw material quality control parameters
Variables	Controlled Specifications	Remarks
Moisture content	8–10 wt.%	Storage temperature of 36–45 ℃ for 3 days can inhibit degradation and mold formation.
Coarse dirt (impurities and ash)	Maximum of 3 wt.%	Able to be removed by water washing
Press shredder	1–3 cm	Press shredder
Density	0.7–1.55 $\:\frac{g}{{cm}^{3}}$	Controlled density range allow for improved pretreatment control

SEM images of OPEFB samples at x400 and x1000 magnifications can be observed below. The images reveal the presence of round-shaped spiky silica extrudates on the fiber surface, covering a significant portion of the surface area. Previous studies (Haqiqi et al. 2021; Katahira et al. 2018) have indicated that these silica deposits serve as crucial physical obstacles for enzymatic hydrolysis, hindering access to the holocellulose surface. Consequently, an excessive delignification may result in harm to the OPEFB surface, such as destroying the cellulose structure and reduce its size, as demonstrated by previous research (Chia et al. 2023; Katahira et al. 2018).

6.2 Lignin biocellulose extraction

The results of the laboratory test for OPEFB compositional analysis, specifically ligno biocellulose, can be found in Table 4. These results not only reveal the composition characteristics but also offer valuable insights into the potential applications of lignin cellulose in rigid polymer structures and lignin CMC biofilms. Most of the lignin found in OPEFB was dissolved in the extracted aqueous solution, leaving behind a small amount of insoluble lignin.

This facilitates the purification procedure, leading to 62.1% alpha cellulose and 88.2% lignin by weight. A refined technique was achieved, allowing for the utilization of extracted cellulosic monomer from hemicellulose to produce high purity compound. The percentage composition of extracted lignin and cellulose is determined as follows:

$$\:\%\:lignin\:yield=\frac{Mass\:of\:extracted\:lignin}{Mass\:of\:OPEFB\:loading}$$

$$\:\%\:cellulose\:\:yield=\frac{Mass\:of\:extracted\:cellulose}{Mass\:of\:OPEFB\:loading}$$

Table 4

Compositional properties of the OPEFB and extracted lignin biocellulose
Composition	Untreated OPEFB [21]	Pretreated OPEFB
Alpha Cellulose (wt.%)	28.5	62.1
Hemicellulose (wt.%)	23.3	21.2
Extracted Lignin (wt.%)	31.7	88.2
Ash (wt.%)	6.7	1.4
Nitrogen (wt.%)	-	1.2

The optimized extraction method minimizes the effects on cellulose properties, while maximizing lignin yield. Consequently, a cellulose structure comparable to that of native OPEFB cellulose can be achieved, as demonstrated by the comparable Fourier-transform infrared spectroscopy (FTIR) spectra of both samples in the prior research (Katahira et al. 2018) on chemical delignification.

Furthermore, utilizing Design Expert for parametric optimization led to a sulfuric acid concentration of 69 wt.% to attain a minimum residual lignin content of 11.8 wt.%. The modeled reaction dependency parameters included retention time, pretreatment temperature, and OPEFB loading. This leads to a manageable purity range for the produced alpha cellulose and lignin, with ± 3.5 wt.% and ± 1.3 wt.% respectively.

6.3 Purification and characterization

This study revealed that the most efficient ligno biocellulose recovery was achieved through a two-step alkali based chemical method and acidic based precipitation. By acidifying the uniform solution of pretreated OPEFB following the procedure outlined in [5], complete precipitation of lignin from OPEFB was achieved. The two-step acid precipitation procedure conducted in this study facilitated the production of exceptionally pure lignin, as the lignin particles were able to increase in size at pH 6 before undergoing additional precipitation at a lower pH, where impurity levels were at their peak.

The extracted lignin purity from Ultraviolet–visible spectroscopy was determined by the absorbance spectrum measured at 280 nm, falling within the 0.3–0.8 a.u range (with a dilution factor of around 1:2500). Consequently, a workflow model was established to enhance the accessibility and wettability of OPEFB, optimizing the pretreatment process to decrease lignin content without compromising cellulose properties.

The analysis of the molecular weight through gel permeation chromatography on the isolated lignin specimen revealed Mn, Mw, and Mz values of 2,558 Da, 2,908 Da, and 3,483 Da, correspondingly, along with a suitable polydispersity index of 1, as detailed in our earlier study (Song and Othman 2022). Consequently, the alkali-treated cellulose, following the described method, resulted in the production of α-cellulose, which was identified as a viable source material for the creation of lignin-CMC.

Furthermore, the thermogravimetric analysis of this film demonstrated favorable thermal stability when subjected to incremental temperature increases from ambient temperature to 900°C. In general, uniform patterns in thermal properties were noted for all lignin-CMC films in our prior research (Song and Othman 2022) in line with the results by (Girard et al. 2024, Swingler et al. 2021).

6.4 Bacterial cellulose extraction and properties

This study found that an effective extraction method for bacterial cellulose from OPEFB has not been developed. This is primarily attributed to the significant obstacles faced during pretreatment, particularly in addressing the inherent resistance of EFB. One major challenge involves eliminating silica bodies present on the surface of EFB, which hinder the hydrolysis process.

One potential method involves utilizing T. reesei bacterial strains that have shown moderate effectiveness in extracting OPEFB cellulose and lignin, as suggested by (Swingler et al. 2021). Most microorganisms are observed to achieve maximum enzyme production between 24 to 72 hours. Nevertheless, this process exhibits a slower reaction rate when compared to alkali-based chemical methods and presents challenges in enzyme removal [19].

The use of lignin degradation enzymes in most of the research is restricted to the degradation of post-extracted OPEFB lignin into potential derivatives. (Parit et al. 2018) conducted a study on the organosolv approach for extracting OPEFB lignin and cellulose, resulting in 15.1 ± 1.3 wt.% and 63.3 ± 1.8 wt.% yields, respectively. A similar method was employed by (Mohammad et al. 2020) to enzymatically hydrolyze OPEFB lignin into glucan-rich, lignin-rich, and hemicellulose.

Lignin degradation enzymes face significant obstacles, such as the efficient extraction of lignin in OPEFB polymer network by microbial action and the elimination of silica residuals on the OPEFB surface. Moreover, the high macromolecular nature of lignin necessitates its separation into low molecular weight monomers before it can be utilized in specific products, requiring high-purity fractions for high-value applications. Similarly, alpha cellulose also requires attention to microbial activity, albeit to a lesser extent.

Hence, potential areas for future research in enhancing the production of ligninolytic enzymes lie in optimizing fermentation media, precise pH regulation, and synthesis parameters. There is potential for pretreatment strategies that merge chemical techniques with bacterial cellulose and compatible enzymes capable of tolerating high temperatures ranging from 45–80°C, as well as a broad pH spectrum.

The reduction in size of OPEFB dry fiber has been demonstrated to increase fiber content, resulting in enhanced biomass loading efficiency and a smaller pretreatment reactor. This streamlines the purification process, yielding 62.1% alpha cellulose and 88.2% lignin by weight. The refined extraction technique minimizes impacts on cellulose properties while maximizing lignin output.

As a result, a cellulose structure akin to that of native OPEFB cellulose can be attained. The holocellulose obtained from OPEFB displays resemblances to intricate cellulosic carbohydrates. When combined with the extracted lignin, a proven application in lignin CMC biofilm has exhibited promising feasibility.

This study found that an effective extraction method for bacterial cellulose from OPEFB has not been developed. This is mainly due to the major difficulties encountered during the pretreatment process. The latest progress in this area shows a slower rate of reaction in comparison to chemical methods based on alkali, and it poses difficulties in removing enzymes.

Competing interests

The authors have no relevant fnancial or non-fnancial interests to disclose.

Ethics approval

Not applicable.

Consent for publication

Not applicable.

Funding

This study did not receive any financial support.

Author Contribution

All authors participated in the study conception and design. Jofry Othman carried out material preparation, data collection, and analysis with the supervision of Norliza Abd Rahman, Jarinah Mohd Ali, and Siti Kartom Kamarudin. The final manuscript was reviewed and approved by all authors.

Acknowledgement

The authors would like to thank Universiti Kebangsaan Malaysia (UKM) for the overall support towards completion of this study.

Data availability

The authors declare that all data generated or analyzed during this study are included in this published article.

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Comparative study of lignin biocellulose and bacterial cellulose

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Abstract

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1. Introduction

2. Comparative performance on OPEFB pretreatment method

3. Lignin biocellulose extraction and properties

4. Bacterial cellulose extraction and properties

5. Material and Methods

5.1 OPEFB quality improvement

5.2 Lignin biocellulose extraction

5.3 Purification and characterization

6. Result and Discussion

6.1 OPEFB quality improvement

6.2 Lignin biocellulose extraction

6.3 Purification and characterization

6.4 Bacterial cellulose extraction and properties

7. Conclusions

Declarations

Competing interests

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Funding

Author Contribution

Acknowledgement

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References

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