# Ultrasound, pulsed electric fields, and high-voltage electrical discharges assisted extraction of cellulose and lignin from walnut shells

Yassine El Khayat Driaa, Hafida Maarir, Mehdi Mennani, Nabil Grimi, Amine Moubarik, Nadia Boussetta

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Yassine El Khayat Driaa, Hafida Maarir, Mehdi Mennani, Nabil Grimi, Amine Moubarik, et al.. Ultrasound, pulsed electric fields, and high-voltage electrical discharges assisted extraction of cellulose and lignin from walnut shells. International Journal of Biological Macromolecules, 2025, 292, pp.139319. 10.1016/j.ijbiomac.2024.139319. hal-05127197

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# Ultrasound, pulsed electric fields, and high-voltage electrical discharges assisted extraction of cellulose and lignin from walnut shells

Yassine EL KHAYAT DRIAA<sup>a,b,*</sup>, Hafida MAARIR<sup>b</sup>, Mehdi MENNANI<sup>c</sup>, Nabil GRIMI<sup>a</sup>, Amine MOUBARIK<sup>b</sup>, Nadia BOUSSETTA<sup>a,*</sup>

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

**Keywords:**

Ultrasounds

Pulsed electric fields

High-voltage electrical discharges

Alkali treatment

Cellulose

Lignin

Walnut shells

## ABSTRACT

Extracting The extraction of cellulose and lignin from biomass is essential for the development of sustainable bio-based materials. This study examines the effects of physical pretreatment techniques—ultrasound (US), pulsed electric fields (PEF), and high-voltage electrical discharges (HVED)—on the efficiency of alkali treatment for cellulose and lignin extraction from walnut shells. The primary objective was to enhance extraction yields and improve extract quality while evaluating the effectiveness of these methods. The results revealed that the combined treatments achieved lignin and cellulose yields of up to 17.03 % and 33.40 %, respectively, with US and HVED showing significantly higher extraction efficiencies compared to other techniques. Characterization of the extracted materials using FTIR, XRD, 2D HSQC NMR, TGA/DTG, and DSC confirmed the presence of key functional groups, crystalline structures, and detailed molecular features, while also providing insights into thermal stability. These findings underscore the potential of walnut shells as a sustainable source for bio-based materials and demonstrate the influence of physical pretreatments on the structural and thermal properties of extracted cellulose and lignin. This study paves the way for further research into industrial applications, such as wood adhesives. Future work should focus on optimizing extraction processes, scaling up operations, and investigating the effects of biomass variability on extraction outcomes.

## 1. Introduction

Interest in biomass utilization has grown significantly over recent decades, attracting attention from both academic and industrial sectors [1]. Lignocellulosic biomass, composed of cellulose, hemicellulose, and lignin, represents a key renewable resource [2]. Cellulose, the most abundant biopolymer on Earth, is extracted from biomass at an annual rate exceeding 10¹² tons [2]. Its applications continue to expand globally, including in regions with limited wood resources [3]. Lignin, the second most abundant natural polymer, also plays a vital role in the development of sustainable biomaterials. Technical lignin production, primarily derived from pulp and paper mills as well as cellulosic bio-refineries, ranges from 50 to 70 billion tons annually [4].

Cellulose and lignin can be extracted from a wide range of natural sources, including cane bagasse, giant reed, tomatoes, spent coffee

grounds, cactus, and olive stone [5,6]. However, significant quantities of walnut shells are discarded annually, leading to environmental concerns and underutilization of a valuable resource. These shells, which constitute approximately 50 % of the total fruit weight, are typically disposed of through landfilling, used as animal feed, litter, or as fuel for boilers. Repurposing walnut shells to produce value-added products offers a promising opportunity to diversify the agroindustry and create new markets for agricultural waste. Walnut shells are rich in cellulose and lignin, with reported compositions ranging from 24.2 % to 41.2 % for cellulose and 33.25 % to 53.9 % for lignin [7,8]. However, their dense and compact structure poses a significant challenge during extraction, as it limits access to the internal compounds compared to other lignocellulosic biomasses.

Conventional methods for extracting cellulose and lignin from lignocellulosic biomass primarily rely on chemical and physical

<footnote><sup>a</sup> Integrated Transformation and Renewable Matter TIMR (UTC/ESCOM), University of Technology of Compiegne- Alliance Sorbonne University, Centre of Research of Royallieu, Rue du docteur Schweitzer, CS 60319, 60203 Compiegne, France</footnote>

<footnote><sup>b</sup> Chemical Processes and Applied Materials Laboratory, Polydisciplinary Faculty, Sultan Moulay Slimane University, SBP: 592 Beni Mellal, Morocco</footnote>

<footnote><sup>c</sup> Materials Science, Energy and Nanoengineering (MSN) Department, Mohammed VI Polytechnic University, Lot 660-Hay Moulay Rachid, 43150 Ben Guerir, Morocco</footnote>

<footnote>* Corresponding authors at: Integrated Transformation and Renewable Matter TIMR (UTC/ESCOM), University of Technology of Compiegne- Alliance Sorbonne University, Centre of Research of Royallieu, Rue du docteur Schweitzer, CS 60319, 60203 Compiegne, France.
E-mail addresses: yassine.el-khayat-driaa@utc.fr (Y. EL KHAYAT DRIAA), nadia.boussetta@utc.fr (N. BOUSSETTA).</footnote>
processes, such as alkaline treatment, acid hydrolysis, and steam explosion. Alkaline treatments, such as sodium hydroxide extraction, are effective in removing lignin while preserving cellulose integrity; however, improper control can result in cellulose degradation [9]. Acid hydrolysis efficiently breaks down hemicellulose and lignin but may also reduce cellulose yield due to polymer degradation. Despite their effectiveness, these methods often degrade cellulose quality, as the harsh chemicals can disrupt the polymer structure, leading to lower yields of high-purity cellulose [9]. Additionally, the high energy demands of these processes—requiring elevated temperatures and pressures—contribute to significant environmental and economic costs, including a larger carbon footprint. The economic feasibility of large-scale cellulose extraction is further limited by the expenses associated with extraction and subsequent purification steps needed to produce usable cellulose [10]. These challenges underscore the urgent need for sustainable, energy-efficient extraction methods that minimize environmental impact while maximizing yield and cellulose quality.

To enhance extraction efficiency and cost-effectiveness, physical pretreatment methods such as ultrasound (US), pulsed electric fields (PEF), and high-voltage electrical discharges (HVED) have emerged as promising alternatives. Ultrasound, characterized by high-frequency sound waves (>20 kHz), induces cavitation phenomena when applied to the plant matrix [11,12]. This phenomenon involves the rapid formation and implosion of microbubbles, generating local microturbulence and exerting mechanical, thermal, and chemical effects on the biomass [13,14]. Pulsed electric fields (PEF) employ brief, high-intensity electrical pulses that induce transient electro-permeabilization of cell membranes, enhancing their permeability and facilitating the release of intracellular bioactive compounds [15–17]. In contrast, HVED applies high electrical voltages between electrodes submerged in the extraction medium, creating plasma channels. These channels produce strong shock waves, cavitation bubbles, UV radiation, and ozone, leading to significant physicochemical modifications in the extraction medium [13,15,18]. M. Brahim et al. [19] demonstrated the efficacy of US and HVED in enhancing delignification of rape straw. Ultrasound improved hemicellulose extractability, whereas HVED caused partial cellulose degradation. The study also highlighted the synergies and trade-offs associated with these treatments: US led to hemicellulose loss, while HVED impacted cellulose integrity. Both techniques, however, improved lignin recovery, yielding materials with enhanced thermal stability and physicochemical properties. Specifically, ultrasound promoted lignin polymerization, while HVED disrupted ferulate bridges. Despite their potential, combining physical pretreatments with chemical methods presents challenges. These include optimizing energy consumption, fine-tuning treatment parameters to prevent cellulose degradation, and ensuring the overall cost-effectiveness of the process.

This study aims to investigate the physicochemical extraction of cellulose and lignin from walnut shells using ultrasound (US), pulsed electric fields (PEF), and high-voltage electrical discharges (HVED) as pretreatments prior to alkaline treatment. The objectives include evaluating extraction efficiency, characterizing the extracted cellulose and lignin, and exploring their potential applications. A comprehensive analysis of the results is conducted using Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD), Heteronuclear Single Quantum Coherence Nuclear Magnetic Resonance (2D HSQC NMR), Thermogravimetric Analysis (TGA/DTG), Differential Scanning Calorimetry (DSC), and Scanning Electron Microscopy (SEM).

## 2. Materials and methods

### 2.1. Materials and chemicals

The walnut shells (WS) were obtained from local cooperatives located in Ouarzazate, within the Drâa-Tafilalet region in southern Morocco. These agricultural by-products underwent meticulous

preparation procedures, involving triple washing, sun drying, crushing into assorted sizes, and subsequent storage before utilization. The chemicals used in this research were supplied by Merck (Sigma-Aldrich, France).

### 2.2. Raw material composition

The moisture content was assessed using the Infrared Moisture Analyzer MA160 from Sartorius, Germany. Elemental analysis of the material was conducted using Energy-dispersive X-ray Spectroscopy (EDX) equipment provided by Bruker Nano GmbH in Berlin, Germany. Extractives and klason lignin contents were determined following the laboratory analytical procedure (LAP) outlined by the National Renewable Energy Laboratory (NREL) [20]. For the extraction procedure, approximately 3–4 g of powder (dry mass) were placed in a cellulose cartridge positioned over a 500 mL flask containing 300 mL of dichloromethane. The extraction process was conducted over 8 h. Subsequently, a rotary evaporator was employed to remove the solvent at 40 °C and recover the extractive. The flask containing the extractive and the treated powder was then subjected to 105 °C for 24 h in an oven and weighed after cooling. The treated powder underwent hydrolysis with 72 % sulfuric acid for 1 h, followed by autoclaving after dilution to 4 wt % sulfuric acid by water addition. After autoclaving, the samples were filtered, and the dried residue was weighed to determine the Klason lignin content. All analyses were conducted in triplicate, and standard deviations were calculated.

### 2.3. Alkali cellulose and lignin extraction

Control conditions for Walnut shells were established through alkaline treatment, following methods described previously [6,21]. Initially, 5 g of raw material was ground and sieved to obtain three distinct particle sizes (1–0.75 mm, 0.75–0.5 mm, and < 0.5 mm). These samples were then separately washed with hot water at 70 °C for 2 h to eliminate partially water-soluble materials and impurities. Subsequently, the washed samples underwent alkaline treatment at temperatures of 50, 70, 90, and 98 °C for 30, 60, 90, and 120 min, respectively. Aqueous sodium hydroxide solution (NaOH) at different concentrations (5%, 10%, 15%, and 20% w/v) was employed to achieve optimal conditions. The ratio of solid to liquid was maintained at 1:10 (w:v). After alkali extraction, the suspension was filtered to separate the solid (pulp) and the liquid (black liquor) parts. The resulting pulp underwent bleaching at 80 °C for 2 h using a bleaching solution containing acetate buffer (27 g NaOH and 75 mL glacial acetic acid, diluted to 1 L of distilled water) and (1.7 wt% NaClO₂ in water). The solid to liquid ratio for bleaching was 1:20 (w:v). To ensure further removal of amorphous components, the bleaching process was repeated twice. The cellulose obtained post-bleaching was washed with distilled water and oven-dried, resulting in Alkali treatment cellulose (Control-cellulose). Sulfuric acid was introduced into the obtained black liquor until reaching a pH of 2–3 to precipitate acidified lignin. Subsequently, the separated lignin underwent thorough washing until a neutral pH was reached. Finally, the obtained wet lignin was oven-dried, resulting in Alkali treatment lignin (Control-lignin). Three replicates were conducted for each material and concentration during the treatment, and standard deviations were calculated accordingly.

The yield of cellulose and lignin was determined in accordance with Eq. (1):

$$ \text{Yield (\%)} = \frac{W_f}{W_i} \times 100 \qquad (1) $$

Where $W_i$ is the initial total dry weight of the raw materials (g) and $W_f$ is the final dry weight of isolated cellulose or lignin.
## 2.4. Physical pretreatment

The physical pretreatment conditions for ultrasound (US), pulsed electric fields (PEF), and high-voltage electrical discharges (HVED) were established following previously described methods [19,22] (Fig. 1). In this study, a specific energy of 900 kJ/kg was selected based on preliminary results. For each treatment, 25 g of raw materials were introduced into the treatment chamber. Water, preheated at 70 °C, was added with a solid-to-liquid ratio of 1:10 (w:v).

For ultrasound treatment, a titanium probe with dimensions of 100 mm in length and 14 mm in diameter was used. This probe was connected to a generator (Hieldscher GmbH, Stuttgart, Germany). For the current study, the power and frequency of the generator were 400 W and 12 kHz, respectively.

The specific energy input (E, kJ/kg) for US treatment was determined using the following Eq. (2):

$$E = \frac{P_{us} \times t_{us}}{m} \qquad (2)$$

Where E represents the specific energy input in kJ/kg, PUS is the generator power (400 W), tUS is the treatment time, and m is the

suspension mass.

The equipment used for Pulsed Electric Fields (PEF) includes a pulsed high-voltage power supply (Basis, Saint-Quentin), along with a 1 L processing chamber. This chamber is composed of two parallel disk electrodes (diameter of 11 cm). The generated pulses exhibit a duration of 10 µs and a frequency of 1 Hz. Capable of producing exponential decay pulses, the generator achieves a maximum voltage of 40 kV and a maximum current of 10 kA.

The specific energy input (E, kJ/kg) for PEF treatment was determined using the following Eq. (3):

$$E = \frac{\sum_{i=1}^{n} W_{pef}}{m} \qquad (3)$$

In this context, WPEF represents the pulse energy per pulse (kJ/pulse), n is the pulse number and m denotes the suspension mass.

The determination of WPEF is based on the Eq. (4):

$$W_{pef} = \int_{0}^{t} UIdt \qquad (4)$$

Where U represents the voltage (V) and I represent the current (A).

The processing energy applied was 150 kJ/kg. The time of

<figure><img src="image_4.png" /><figcaption>Fig. 1. Material flow diagram for cellulose and lignin extraction from walnut shells.</figcaption></figure>
application of the PEF, $t_{pef}$, (s) was calculated as the product of the mean pulse width, $t_i$, (s) and the number of pulses $n_{pef}$ as the following Eq. (5):

$$t_{pef} = n_{pef} \times t_i \qquad (5)$$

The overall duration of solid-liquid interaction throughout the PEF treatment is given by Eq. (6):

$$t_{tot} = \frac{n_{pef}}{f} \qquad (6)$$

Where f represents the pulse frequency.

For the High Voltage Electrical Discharges (HVED) experiments, the generator is the same as that used for PEF. The only difference is the electrode geometry. The chamber is equipped with needle-plate geometry electrodes. The stainless-steel needle electrode had a diameter of 10 mm, while the grounded disk electrode possessed a diameter of 35 mm. The distance between the electrodes was set at 0.5 cm. A positive discharge voltage of 40 kV was applied to the needle electrode as the peak discharge voltage (U).

The specific energy input (E, kJ/kg) for HVED treatment, was determined using the following Eq. (7):

$$E = \frac{\sum_{i=1}^{n} W_{hved}}{m} \qquad (7)$$

In this context, $W_{hved}$ represents the pulse energy per discharge (kJ/ discharge), and m denotes the suspension mass.

The determination of $W_{hved}$ is based on the Eq. (8):

$$W_{hved} = \int_{0}^{t} UIdt \qquad (8)$$

Where U represents the voltage (V) and I represents the current (A).

The processing energy applied was 150 kJ/kg. The time of application of the HVED, $t_{hved}$, (s) was calculated as the product of the mean discharge width, $t_i$, (s) and the number of pulses $n_{hved}$ as the following Eq. (9):

$$t_{hved} = n_{hved} \times t_i \qquad (9)$$

The overall duration of solid-liquid interaction throughout the HVED treatment is given by Eq. (10):

$$t_{tot} = \frac{n_{hved}}{f} \qquad (10)$$

Where f represents the pulse frequency.

After each physical pretreatment, the alkali treatment was performed as previously described.

## 2.5. Characterizations

### 2.5.1. Structure and crystallinity analysis

In this study, analysis utilizing the Nicolet Summit LITE FTIR Spectrometer was conducted to identify the characteristic bands of each sample. Samples were ground and blended with dried KBr at a sample/ KBr ratio of 1/100, with the objective of preparing pastilles. The analysis encompassed a frequency range from 500 cm⁻¹ to 4000 cm⁻¹, employing a resolution of 0.6 cm⁻¹ and accumulating 16 scans.

The crystalline structure of all cellulosic samples was determined using an X-ray diffractometer (BRUKER Diffractometer, D8 ADVANCE). Each material was converted into powder form and then placed on the sample holder to ensure uniform X-ray exposure. Subsequently, the samples underwent scanning with Cu radiation (λ = 1.5406 Å) within the 2θ range of 5–40 °C, while maintaining the voltage and current at 40 kV and 40 mA, respectively. In order to examine the crystallinity of all cellulosic materials, the crystallinity index (CrI) was calculated using Segal's eq. (11) [23]:

$$CrI = \frac{I_{200} - I_{amorph}}{I_{amorph}} \qquad (11)$$

Here, $I_{200}$ represents the maximum intensity of the crystalline peak at around 2θ = 22.8°, and $I_{amorph}$ is the intensity from the amorphous phase at approximately 2θ = 18.6°.

2D HSQC NMR spectra were obtained for all lignin samples using a Bruker Ascend 400 NMR spectrometer. Initially, 50 mg of lignin was dissolved in 1 mL of DMSO-d₆. Spectral acquisition was carried out using standard Bruker pulse sequences optimized for heteronuclear correlation. Following acquisition, the spectra were processed using TopSpin 4.3.0 software, facilitating peak assignment and analysis. The resulting spectra offered valuable insights into the structure elucidation and interaction dynamics of the studied lignin samples. The H/G/S ratios were calculated as described in [24].

### 2.5.2. Thermal stability analysis

Thermal gravimetric analysis (TGA/DTG) and differential scanning calorimetry (DSC) were performed using the Setaram 131EVO instrument. Each sample, weighing 25 mg, underwent a heating process from 30 °C to 600 °C at a rate of 5 °C/min under a nitrogen atmosphere (20 mL/min). TGA/DTG provided information on the weight loss of the sample as a function of temperature, while DSC measured the heat flow associated with phase transitions and chemical reactions occurring within the sample.

### 2.5.3. Morphology analysis

The effect of physical pretreatment and chemical treatment on the morphology of cellulose and lignin was examined using a scanning electron microscope (SEM, FEI, QUANTA FEG 250) operating at 20 kV. To prepare the specimens, a thin layer of conductive carbon was applied using an ion-sputtering device.

## 2.6. Statistical analyses

Two-way analysis of variance (ANOVA) was conducted using OriginLab software (version 2019b 9.65, Massachusetts, USA) with a significance threshold of 5 %. For each analysis, a Tukey test was employed as a comparative method to evaluate the significance of the observed differences in the results.

# 3. Results and discussions

## 3.1. Chemical composition of raw materials

Walnut shells underwent characterization to determine moisture, extractives content, lignin content, and elemental composition, as summarized in Table 1. Walnut shells showed a moisture content of 9.17 ± 0.10 wt%, extractives content of 1.3 ± 0.21 wt%, and klason lignin content of 34.92 ± 0.66 wt%, in agreement with the findings of Morales et al. [8] and lower than those reported by Vita et al. [25]. Walnut shells contained approximately (55.57 % C, 42.46 % O, 1.29 % Ca, 0.22 % K, 0.22 % S). These results align well with previous studies conducted by Jovičić et al. [26] and Malayil et al. [27].

## 3.2. Alkali treatment parameters effects

Fig. 2 and Table 2 illustrate the lignin and cellulose yields from walnut shells subjected to different extraction conditions. The results indicate that the optimal extraction conditions was obtained with particle size under 0.5 mm at 90 °C for 60 min and 10 % of NaOH yielded 17.03 % lignin and 33.40 % cellulose. Lignin yield is comparable to Morales et al. [28], who reported 19 % lignin and 33 % cellulose using ethanol/water (70:30 v/v) at 200 °C for 90 min. However, Nishide et al. [29] achieved a significantly higher lignin yield of 34–35 % using
<table><caption>**Table 1**

Chemical composition of raw material.</caption><thead><tr><th rowspan="2">Biomass</th><th>Moisture</th><th>Extractives</th><th>Klason Lignin</th><th>C</th><th>O</th><th>Ca</th><th>K</th><th>S</th></tr><tr><th>%</th><th>%</th><th>%</th><th></th><th></th><th></th><th></th><th></th></tr></thead><tbody><tr><td>WS</td><td>9.17 ± 0.10</td><td>1.3 ± 0.21</td><td>34.92 ± 0.66</td><td>55.57</td><td>42.46</td><td>1.29</td><td>0.22</td><td>0.22</td></tr></tbody></table>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>**Fig. 2.** Effect of particle size (a), Temperature (b), Time of extraction (c), and Soda proportions (d) on cellulose and lignin extracted from walnut shells.</figcaption></figure>

<table><caption>**Table 2**

Comparison of cellulose and lignin extraction from walnut shells with different operating conditions.</caption><thead><tr><th rowspan="2">Biomass</th><th rowspan="2">Conditions</th><th>Lignin Yield</th><th>Cellulose Yield</th><th rowspan="2">References</th></tr><tr><th>%</th><th>%</th></tr></thead><tbody><tr><td rowspan="5">Walnut Shells</td><td>PS &lt;0.5 mm</td><td>17.03 ± 4.03</td><td>33.40 ± 0.84</td><td>This study</td></tr><tr><td>90 °C 60 min</td><td></td><td></td><td></td></tr><tr><td>10 % NaOH</td><td></td><td></td><td></td></tr><tr><td>200 °C 90 min</td><td>19</td><td>33</td><td>[28]</td></tr><tr><td>ethanol/water (70:30 v/v)</td><td></td><td></td><td></td></tr><tr><td></td><td>70 °C 24 h</td><td>34–35</td><td>–</td><td>[29]</td></tr><tr><td></td><td>EtOH or MeOH</td><td></td><td></td><td></td></tr></tbody></table>

ethanol or methanol at 70 °C for 24 h, although cellulose yields were not reported in their study.

### 3.2.1. Effect of sample particle size

Fig. 2a depicts the effect of particle size on the yields of lignin and cellulose. As particle size decreases from 1 mm–0.75 mm to <0.5 mm, a noticeable increase in lignin yield is observed. This can be attributed to the increased specific surface area of smaller particles, which enhances solvent and reagent penetration, thereby improving lignin extraction efficiency [30]. In contrast, cellulose yield remains relatively constant across the different particle sizes, suggesting that particle size reduction does not significantly influence cellulose extraction. The hard, dense structure of walnut shells, predominantly composed of lignin, provides substantial mechanical strength, making it difficult to access internal compounds during extraction. Thus, optimizing particle size is crucial for enhancing extraction efficiency. Reducing particle size increases the surface area available for chemical reactions and solvent interaction, which facilitates the breakdown of cell wall components [31].
### 3.2.2. Effect of extraction temperature

Fig. 2b shows the effect of extraction temperatures on the yields of lignin and cellulose. As the temperature increases, the lignin yield shows a notable upward trend, especially between 50 °C and 90 °C, reflecting the enhanced solubilization of lignin at higher temperatures. The lignin yield reaches a peak at 90 °C and remains steady with further temperature increases, indicating that this is the optimal temperature for lignin extraction. Conversely, the cellulose yield demonstrates a peak at around 70 °C but decreases at higher temperatures, suggesting potential thermal degradation of cellulose [32]. Thus, maintaining an optimal temperature is essential to balance efficient lignin extraction and cellulose preservation.

### 3.2.3. Effect of extraction duration

Fig. 2c presents the effect of extraction duration on lignin yield. There is a significant increase in lignin yield as extraction time extends from 30 min to 60 min, indicating that longer extraction times allow for more thorough solubilization and breakdown of lignin structures. However, beyond 60 min, the lignin yield reaches a plateau, suggesting that further extending extraction time does not significantly enhance yield. This plateau indicates that an extraction time of around 60 min is optimal for lignin extraction, balancing efficiency and practicality without unnecessarily prolonging the process. Prolonged extraction times can lead to cellulose degradation, potentially compromising fiber quality [33]. Furthermore, lignin's structure and solubility can be altered over extended extraction periods, influencing the overall extraction process [34].

### 3.2.4. Effect of hydroxide sodium concentration

Fig. 2d illustrates the effect of NaOH concentration on the extraction yields of lignin and cellulose. As the NaOH concentration increases from 5 % to 10 %, a significant rise in lignin yield is observed, demonstrating the alkali's effectiveness in breaking down lignin. However, beyond 10 %, the lignin yield plateaus, suggesting that higher NaOH concentrations do not further enhance lignin extraction. In contrast, cellulose yield declines with increasing NaOH concentration, particularly above 15 %, indicating potential cellulose degradation at elevated alkali levels. These findings highlight the importance of optimizing NaOH concentration to maximize lignin extraction while minimizing the detrimental effects on cellulose yield. During the extraction process, NaOH dissolves lignin and hemicellulose, forming soluble complexes [34], while the relatively lower reactivity of cellulose to NaOH allows it to remain intact and preserved.

## 3.3. Effect of physical pretreatment on cellulose and lignin

### 3.3.1. Effects on lignin and cellulose yield

Fig. 3 presents the extraction yields of cellulose and lignin from walnut shells using four distinct methods: Control, US, PEF, and HVED. The data reveals notable differences in extraction efficiency among these methods. The control extraction yielded 33.40 % cellulose and 17.03 % lignin, serving as a baseline. US treatment significantly enhanced cellulose yield to 43.46 %, demonstrating the method's efficacy in breaking down cellulose fibers through cavitation, which increases surface area for solvent interaction. This is attributed to the ability of high-intensity ultrasound to facilitate the breakdown of cellulose fibers, ultimately leading to a reduction in cellulose length and an increase in surface area [35]. The thermal energy associated with electrical discharges in HVED could facilitate the breakdown of lignin-carbohydrate bonds, aiding in lignin release from the biomass structure [36]. The lignin yield with US was slightly lower at 12 %, indicating selective efficiency. Pulsed electric fields (PEF) did not show a significant effect on lignin yield, which was 14.05 %, and the cellulose yield was 33.15 %. This suggests that PEF may not be particularly effective in lignin disruption as initially thought. The thermal energy associated with electrical discharges in HVED could facilitate the breakdown of lignin-carbohydrate bonds, aiding in lignin

<figure><img src="image_9.png" /><figcaption>Fig. 3. Effect of physical pretreatment on cellulose and lignin extracted from walnut shells.</figcaption></figure>

release from the biomass structure [36]. High-voltage electric discharge (HVED) also improved cellulose yield to 36.17 % and lignin yield to 17 %, attributed to the thermal energy and micro-shock waves facilitating the breakdown of lignocellulosic bonds. Comparatively, Nishide et al. [29] achieved a higher lignin yield (34–35 %) using ethanol or methanol at 70 °C for 24 h, while Morales et al. (2022) [28] reported similar cellulose yields (33 %) using ethanol/water at 200 °C for 90 min but slightly higher lignin yields (19 %). These findings highlight the relative efficiency of each method, with US and HVED showing significant promise for enhancing cellulose extraction, and PEF demonstrating superior lignin extraction capabilities.

### 3.3.2. Structure and crystallinity analysis

In Fig. 4, the FTIR spectra of raw walnut shells (WS), along with extracted cellulose and lignin by Control, US, PEF or HVED extraction are presented. Numerous distinct peaks indicative of the lignocellulosic composition can be identified. In the raw shells, a broad band around 3300–3400 cm⁻¹ represents OH bonding, attributed to the abundant hydroxyl group biopolymers (lignin, cellulose, hemicellulose, etc.) [37,38]. Bands at 2800–2900 cm⁻¹ are attributed to C—H stretching vibrations of methyl, methylene, and methoxyl [5,6]. Differences are observed between raw WS spectra and cellulose samples due to the absence of characteristic bands associated with hemicellulose and lignin compounds. Various treatment methods such as Control, US, PEF, and HVED are employed to enhance the extraction efficiency and purity of cellulose and lignin. For cellulose extraction, alkali treatment (Control) is recognized as a highly efficient technique, demonstrated by alterations in Control-cellulose spectra, including the vanishing of peaks at 1730 cm⁻¹ associated with hemicellulose molecules and the emergence of peaks at 897 cm⁻¹ related to β-glycosidic linkages of cellulose [8,28]. Similarly, US-Cellulose spectra display modifications with the disappearance of bands at 1730 cm⁻¹ and the presence of the 897 cm⁻¹ band. PEF-Cellulose spectra indicate effective removal of hemicellulose and lignin, shown by the disappearance of the 1730 cm⁻¹ peak and the appearance of the 897 cm⁻¹ peak. HVED-cellulose spectra exhibit similar changes with the disappearance of hemicellulose-associated peaks and the presence of cellulose-specific bands at 897 cm⁻¹. For lignin extraction, notable differences are observed between raw WS spectra and lignin samples. Alkali treatment (Control) emerges as one of the most successful methods for efficient lignin extraction. The spectra of Control-lignin indicate the disappearance of some bands at 1730 cm⁻¹ attributed to hemicelluloses and the appearance of a band at 1630 cm⁻¹ [39,40]. US-lignin spectra demonstrate similar changes with the disappearance of hemicellulose peaks and the emergence of characteristic lignin peaks. PEF-lignin spectra reveal the elimination of hemicellulose-
<figure><img src="image_10.png" /><figcaption>**Fig. 4.** FTIR spectra of (a) cellulose and (b) lignin extracted under Control, and assisted by US, PEF, and HVED conditions.</figcaption></figure>

related peaks and the presence of lignin-specific bands. HVED-lignin spectra also display the disappearance of hemicellulose peaks and the appearance of lignin-specific bands, highlighting the effectiveness of HVED in lignin extraction.

The crystal structure and crystallinity index of all cellulose samples extracted were investigated through X-ray diffraction (XRD) analysis, with the corresponding profiles shown in Fig. 5 and crystallinity indexes listed in Table 3. Notably, all samples demonstrate three primary peaks at 2θ = 16.4, 22.3, and 34.6, which can be attributed to the crystallographic planes of 110, 200, and 004, respectively. These findings align with the conventional crystal structure of cellulose I, as elucidated in existing literature [41,42], suggesting an absence of discernible alterations in the polymorphism of cellulose I following varied treatments. This confirms the classification of walnut shells as lignocellulosic materials. In Fig. 5, significant differences in crystallinity rates are observed depending on the extraction methods used. The raw material (RAW-WS) exhibits a crystallinity rate of 36.37 %, while the crystallinity rates of the extracted cellulose samples are 69.49 % for Control-cellulose, 59.68 % for US-Cellulose, 59.25 % for PEF-Cellulose, and 72.22 % for HVED-Cellulose. Although the differences between methods are not highly

<figure><img src="image_11.png" /><figcaption>**Fig. 5.** XRD Analyses for Walnut Shells and cellulose samples extracted under Control, and assisted by US, PEF, and HVED conditions.</figcaption></figure>

<caption>**Table 3**</caption>

Crystallinity Index, Thermal properties, and monolignol proportions of Raw Material, cellulose and lignin samples extracted under Control, and assisted by US, PEF, and HVED conditions.

<table><thead><tr><th rowspan="2">Samples</th><th>XRD</th><th colspan="2">TGA/DTG</th><th>2D HSQC NMR</th></tr><tr><th>CrI</th><th>T<sub>onset</sub></th><th>T<sub>max</sub></th><th>S/G/H</th></tr><tr><th></th><th>%</th><th>°C</th><th></th><th>%</th></tr></thead><tbody><tr><td>Raw-WS</td><td>36.37</td><td>265.43</td><td>361.71</td><td>N/A</td></tr><tr><td>Control-cellulose</td><td>69.49</td><td>288.78</td><td>332.33</td><td>N/A</td></tr><tr><td>US-cellulose</td><td>59.68</td><td>284.97</td><td>330.33</td><td>N/A</td></tr><tr><td>PEF-cellulose</td><td>59.25</td><td>281.30</td><td>328.62</td><td>N/A</td></tr><tr><td>HVED-cellulose</td><td>72.22</td><td>290.87</td><td>335.44</td><td>N/A</td></tr><tr><td>Control-lignin</td><td>N/A</td><td>207.88</td><td>235.44</td><td>75.5/6.5/12</td></tr><tr><td>US-lignin</td><td>N/A</td><td>242.49</td><td>258.75</td><td>41/12/47</td></tr><tr><td>PEF-lignin</td><td>NA</td><td>245.38</td><td>267.27</td><td>51/39/10</td></tr><tr><td>HVED-lignin</td><td>N/A</td><td>248.52</td><td>268.27</td><td>58.5/33/8.5</td></tr></tbody></table>

significant, they suggest that each physical pretreatment method has a unique effect on the degree of crystallinity of the extracted cellulose [43,44]. These results underscore the potential of utilizing alternative physical techniques to improve the effectiveness of cellulose extraction procedures and highlight the significance of choosing suitable pretreatment strategies according to particular goals for extracting highly crystalline cellulose. Significant differences in crystallinity rates are observed between extraction methods for the raw material (RAW-WS) and subsequent cellulose samples, indicating the efficiency of extraction methods in producing highly crystalline cellulose from walnut shells, with a remarkable increase in crystallinity rate compared to the raw material. Particularly, the high-voltage electrical discharge (HVED) pretreatment produces the highest crystallinity rate, closely followed by alkali treatment (Control). HVED could enhance the crystallinity through its unique combination of shock waves and localized heating, generating intense, short-lived electrical pulses that create cavitation bubbles and micro-shock waves in the medium. These forces promote the selective breakdown of the less-ordered, amorphous regions, such as hemicellulose and lignin, which are more susceptible to thermal and mechanical effects, leading to their removal. In contrast, the crystalline regions of cellulose, which are more compact and ordered, are resistant to these disruptions and remain intact. This selective removal of amorphous material results in a higher crystallinity index in HVED-treated cellulose. The FTIR results highlight the removal of hemicellulose and
lignin and the presence of characteristic cellulose peaks across different treatment methods (Control, US, PEF, HVED), indicating successful isolation of cellulose. Similarly, XRD results demonstrate significant increases in the crystallinity index of the extracted cellulose compared to raw walnut shells, with HVED and Control treatments showing the highest crystallinity. Furthermore, in sustainable applications, high crystallinity index cellulose enhances the mechanical properties and durability of wood adhesives, though its impact on enzymatic digestibility and long-term stability must be thoroughly assessed [45]. The consistency between FTIR and XRD findings underscores the efficacy of these extraction methods in enhancing the purity and crystallinity of cellulose, confirming that both techniques can effectively characterize the structural properties of the extracted cellulose. Furthermore, the crystallinity index (CI) of cellulose greatly influences adhesive performance. For instance, the CI affects the balance between strength and flexibility in the adhesive matrix, where an optimized crystallinity can enhance bond strength without compromising adaptability.

The 2D HSQC NMR analysis (Fig. 6) provides detailed insights into

the structural characteristics and monomeric arrangements of lignin extracted from walnut shells, with a particular focus on the primary monomers. The spectra offer a clear depiction of the composition and connectivity of syringyl (S), guaiacyl (G), and *p*-hydroxyphenyl (H) units, which are the fundamental building blocks of lignin (Fig. 6b). These monomers play a critical role in determining lignin's reactivity and properties [24,46], with a higher S/G ratio typically indicating improved processability [29]. Supporting these findings, Table 3 presents a compositional analysis of the various lignin samples obtained using 2D HSQC NMR spectroscopy. This analysis reveals the percentage composition of the syringyl (S), guaiacyl (G), and *p*-hydroxyphenyl (H) units. Control-lignin is characterized by a high proportion of syringyl units (75.5 %), suggesting that its structure is predominantly composed of these units and their linkages. In contrast, US-lignin shows a more balanced distribution of primary monomers, with syringyl (41 %), guaiacyl (12 %), and *p*-hydroxyphenyl (47 %) units. PEF-lignin displays a notable proportion of guaiacyl units (39 %) in addition to syringyl units (51 %). HVED-lignin has the highest proportion of syringyl units

<figure><img src="image_12.png" /><figcaption>**Fig. 6.** (a) Structural features and conformations for aromatic region for lignin (2D HSQC NMR) and (b) Primary lignin monomers [24] extracted samples under control, and assisted by US, PEF, and HVED conditions.</figcaption></figure>
(58.5 %) and a significant amount of guaiacyl units (33 %). The S/G ratio for HVED-lignin is 1.77, which is higher than the S/G ratio of 1.31 reported for lignin extracted by OrganoSolv assisted by HVED in Marwa Brahim's study [19]. This suggests that HVED-lignin exhibits enhanced thermal stability and rigidity, which may make it more suitable for applications in wood adhesives compared to lignin with a lower S/G ratio [47]. These compositional insights provide valuable quantitative data on the varying lignin types and their respective linkage preferences, which are essential for assessing their potential applications in bio-refinery and material sciences [48,49].

### 3.3.3. Thermal stability analysis

The comprehensive thermal analysis of cellulose and lignin extracted from walnut shells using alkali treatment (Control), ultrasound (US), pulsed electric fields (PEF), and high-voltage electrical discharges (HVED) demonstrates significant improvements in thermal stability compared to the raw material, as evidenced by TGA/DTG and DSC analyses (Fig. 7). The TGA/DTG results reveal enhancements in the initial decomposition temperature ($T_{onset}$) and maximum decomposition

temperature ($T_{max}$) for cellulose, reflecting improved thermal stability. Among the methods, HVED-cellulose exhibits the highest thermal stability ($T_{onset}$: 290.87 °C, $T_{max}$: 335.44 °C), followed by AT-cellulose ($T_{onset}$: 288.78 °C, $T_{max}$: 332.33 °C), US-cellulose ($T_{onset}$: 284.97 °C, $T_{max}$: 330.33 °C), and PEF-cellulose ($T_{onset}$: 281.30 °C, $T_{max}$: 328.62 °C). For lignin, HVED-lignin demonstrates superior thermal properties ($T_{onset}$: 248.52 °C, $T_{max}$: 268.27 °C), while PEF-lignin shows similar trends ($T_{onset}$: 245.38 °C, $T_{max}$: 267.27 °C). These results highlight HVED and PEF as particularly effective techniques for enhancing the thermal stability of both cellulose and lignin [19,50]. DSC analysis further elucidates the thermal behavior of the extracted materials by measuring heat flow during phase transitions and decomposition reactions. For cellulose, endothermic peaks (Fig. 7e) correspond to the energy required to break glycosidic bonds between glucose units during thermal decomposition. These peaks reflect the energy absorption necessary for the transition of cellulose from a solid state to a more reactive state. Conversely, lignin samples exhibit exothermic peaks (Fig. 7f), associated with the pyrolysis of aromatic structures, where energy is released during the breakdown of complex aromatic molecules into smaller

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>

<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /><figcaption>**Fig. 7.** TGA (a-b), DTG (c-d), and DSC (e-f) thermogram of cellulose and lignin extracted samples under Control, and assisted by US, PEF, and HVED conditions, respectively.</figcaption></figure>
fragments. This contrast in thermal behavior—endothermic for cellulose and exothermic for lignin—emphasizes the distinct chemical and structural properties of the two biopolymers [51,52]. The thermal stability trends observed in TGA/DTG and DSC analyses align with structural changes identified through FTIR and XRD. FTIR spectra revealed the removal of non-cellulosic components and the exposure of cellulose and lignin functional groups, as evidenced by the disappearance or shifting of key peaks, particularly for HVED and AT treatments. This structural purification correlates with the increased Tonset and Tmax values, indicating a more thermally stable cellulose product. Similarly, XRD analysis demonstrated increased crystallinity in extracted cellulose, with HVED-cellulose achieving the highest crystallinity index (CrI) of 72.22 %. The elevated crystallinity, indicative of the crystalline cellulose I structure, explains the enhanced thermal resistance observed in TGA/DTG results, as highly crystalline materials typically exhibit superior thermal stability. The improved thermal stability of cellulose and lignin is particularly advantageous for adhesive applications, where resilience to high temperatures during processing and usage is essential. Lignin's high glass transition temperature (Tg) contributes to the adhesive's overall heat resistance and structural stability, further validating its suitability for high-performance, bio-based adhesives [53].

### 3.3.4. Morphology analysis

The SEM analysis, presented in Fig. 8, reveals notable differences in the morphology of raw walnut shells (RAW-WS) and the extracted cellulose and lignin obtained through various methods (Control, US, PEF, HVED). The SEM images of RAW-WS show an irregular surface structure with visible traces of residues from the grinding process. In contrast, SEM images of cellulose extracted using different methods exhibit characteristic morphologies indicative of crystalline arrangements. Control-cellulose displays a compacted structure with elongated, regular features, suggesting a high degree of crystallinity [3]. US-cellulose shows a more fibrous and less compact morphology compared to Control-cellulose, while still retaining some structural regularity. Both PEF-cellulose and HVED-cellulose exhibit similar fibrous structures,

with noticeable compaction and densification, reflecting the effective removal of non-cellulosic components. For lignin, SEM analysis highlights distinct morphological differences across extraction methods. Control-lignin reveals a rough, fragmented structure with evident aggregation. US-lignin presents a more granular and less compact morphology compared to Control-lignin. In contrast, PEF-lignin shows a tangled, irregular structure indicative of particle agglomeration. HVED-lignin exhibits a similar trend, with significant aggregation and an irregular surface morphology. These observations underscore the influence of extraction methods on the structural and morphological characteristics of both cellulose and lignin. The relative uniformity and crystalline morphology of cellulose across all methods suggest effective removal of lignin and hemicellulose, resulting in a more ordered, compact structure [54,55]. Conversely, the consistent tendency of lignin to aggregate and form irregular structures, regardless of the extraction method, highlights the inherent complexity and variability in lignin's structural organization.

## 4. Conclusions

The comprehensive analysis of cellulose and lignin extraction from walnut shells using various methods—Control, ultrasound (US), pulsed electric fields (PEF), and high-voltage electrical discharges (HVED)—provides significant insights into optimizing biomass processing for sustainable applications. Characterization techniques, including Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD), Heteronuclear Single Quantum Coherence Nuclear Magnetic Resonance (2D HSQC NMR), Thermogravimetric Analysis (TGA/DTG), and Differential Scanning Calorimetry (DSC), revealed detailed structural and thermal properties of the extracted materials. FTIR analysis confirmed the presence of characteristic functional groups in cellulose and lignin, while XRD analysis highlighted the crystalline nature of cellulose and the amorphous characteristics of lignin. 2D HSQC NMR provided a detailed map of lignin's complex structure, identifying key linkages and monomeric units. TGA/DTG and DSC analyses demonstrated the

<figure><img src="image_19.png" /><figcaption>Fig. 8. SEM graphs of cellulose and lignin samples collected from Walnut Shells.</figcaption></figure>
thermal stability and degradation patterns of the extracted polymers, which are crucial for their suitability in high-temperature applications. The results showed that ultrasound treatment significantly enhanced cellulose yield by breaking down cellulose fibers through cavitation, thereby increasing the surface area for solvent interaction. HVED further improved extraction efficiency by disrupting lignocellulosic bonds through shock waves and localized thermal energy, which facilitated more effective extraction. Notably, HVED also enhanced the crystallinity of the extracted cellulose by selectively removing amorphous components, resulting in a more ordered and crystalline structure. Additionally, HVED-treated lignin exhibited improved thermal stability, as evidenced by higher onset and maximum decomposition temperatures. This enhanced thermal stability suggests that HVED-treated lignin could perform well in high-temperature applications. From an economic and environmental perspective, HVED offers a sustainable alternative to conventional chemical extraction methods. It requires less energy, minimizes the use of harsh chemicals, and reduces the overall carbon footprint, aligning well with sustainable processing goals. By potentially lowering operational costs and waste generation, these methods contribute to more sustainable biomass utilization. Moreover, the study highlights the promising applications of the extracted materials, particularly in biobased adhesive formulations. The crystallinity index of cellulose influences the balance between adhesive strength and flexibility, while the thermal stability of both cellulose and lignin enhances heat resistance and structural integrity. Integrating these biopolymers into adhesive products not only reduces reliance on petroleum-based materials but also promotes the utilization of agricultural residues, fostering a circular economy. Future research will focus on a deeper investigation of the techno-economic feasibility and environmental impact of these approaches. These efforts will further validate the potential of cellulose and lignin-derived from walnut shells for sustainable industrial applications, particularly in the production of high-performance wood adhesives.

Future perspectives include scaling up these extraction methods to pilot-scale operations and extending their application to other biomass sources. Further research should aim to refine the extraction processes to enhance yields while minimizing energy consumption, ensuring both efficiency and sustainability. Additionally, evaluating the performance of bio-based adhesives in diverse wood applications and optimizing their formulations to suit various environmental conditions will be critical for achieving commercial viability. These advancements will play a pivotal role in fostering the development of greener and more sustainable materials, aligning with global efforts to reduce environmental impact and promote the use of renewable resources.

### CRediT authorship contribution statement

**Yassine EL KHAYAT DRIAA:** Writing – original draft, Investigation, Formal analysis, Data curation, Conceptualization. **Hafida MAARIR:** Writing – original draft, Formal analysis. **Mehdi MENNANI:** Writing – original draft, Formal analysis. **Nabil GRIMI:** Visualization, Validation, Supervision. **Amine MOUBARIK:** Visualization, Validation, Supervision. **Nadia BOUSSETTA:** Visualization, Validation, Supervision.

### Declaration of competing interest

The authors declare no competing interests.

### Acknowledgments

We would like to express our gratitude to **Région Hauts-de-France** for their financing of this work. Their support has been invaluable in enabling us to conduct this research and advance our understanding of the field. We would like to express our gratitude to **Adama KONATE** for his valuable contributions through his analytical work. His insights have greatly enriched this research and have been instrumental in advancing

our findings.

### Data availability

Data will be made available on request.

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