Efficiency Determination of Water Lily (Eichhornia crassipes) Fiber Delignification by Electrohydrolysis Using Different Electrolytes

Abstract

Nowadays, biomass use has increased due to it being the most abundant raw material on the planet, and treating it is a difficult task, as a result of the number of existing methods and the applications’ diversification. This research work shows the results obtained using different delignification methods (physical and chemical) on water lily ((Eichhornia crassipes) fiber lignocellulosic biomass including a seldom exploited method, known as “electrohydrolysis” in order to determinate the removal efficiency of lignin and hemicellulose. The characterization of the physicochemical and morphological properties of the water lily (Eichhornia crassipes) fiber before and after the pretreatments were applied were by means of Fourier Transform Infrared (FT-IR), X-ray diffraction (XRD) and optical microscopy (OM). The results of FT-IR show a significant decrease in the bands associated with lignin and hemicellulose. By XRD, it was determined that the crystallinity of the cellulose increased by 60% for the treated samples with respect to the reference, and an increase in the surface roughness of the samples was observed by OM. In conclusion, it was determined that electrochemistry delignification is an efficient, environmentally friendly methodology to remove the soluble sugars, opening the possibility to use the water lily (Eichhornia crassipes) fiber to produce a green concrete.

1. Introduction

In recent years, the demand for the use of lignocellulosic biomass has been increasing because it is the most abundant raw material on the planet. Its chemical composition consists mainly of three biopolymers, namely cellulose, hemicellulose and lignin, which make it attractive for different applications in the energy sector (biogas, bioethanol, biobutanol) [1,2]. and production of biomaterials or green materials (green concrete, paper industry, biopolymers, bioremediators, among others) [3,4,5,6,7,8]. There is a great variety of lignocellulosic material from natural plant fibers such as cotton, sunflower, soybean and palm from the seed; flax, bamboo, hemp, jute and sugar cane from the stalk; sisal, cassava and banana from the leaf; and coconut and pineapple from the fruit, to list examples. This wide variety of lignocellulosic material makes it difficult to find a general delignification treatment design for raw materials [9]. There are different delignification pretreatments: physical, chemical (acid and alkaline), organic, biological, hybrid (a combination of two or more pretreatments) and new ones such as electrohydrolysis [1,2,10,11,12].

In this sense, recent research has addressed the use of hybrid treatments (e.g., combined physical–chemical pretreatment, alkaline–oxidant pretreatment, among others) which have stood out for having better efficiencies in the removal of soluble sugars [13,14,15,16]. The choice of delignification treatment depends mostly on the application in the use of lignocellulosic biomass and its chemical components, hence the importance of carefully choosing the pretreatment and the conditions, and observing the physicochemical and morphological changes before and after treatment in the biomass, and the efficiency as parameters before including them in any application [17].

The electrohydrolysis method has been little exploited despite having great advantages over other methods, highlighting that it is an environmentally friendly method. Electrohydrolysis is based on passing direct current through an electrolyte by means of electrodes to solubilize the sugars of the biomass, breaking the molecular bonds that exist in its structure and thus obtaining a solid fraction rich in cellulose and a liquid fraction of hemicellulose and lignin [1]. Electrohydrolysis has not been extensively studied and recent studies suggest the use of very high voltages (≥30 V) and long exposure times (≥80 min), generating greater energy expenditure and treatment time [1,10,12,18]. However, the method is considered promising not only as a delignification treatment but also as a catalyst in the hydrolysis stage, allowing the use of different electrolytes, depending on the application. On the other hand, it is necessary to optimize the method and deepen the analysis of the effect of variables such as time and voltage to determine its efficiency in the removal of soluble sugars from lignocellulosic materials to be considered a green alternative, looking for a reduction in the applied voltage, shorter exposure time and a reduced concentration of the electrolyte so that it can be adapted to any delignification process regardless of the lignocellulosic biomass.

Therefore, the objective of this research work is to determine the effect of the electrohydrolysis pretreatment conditions (electrolyte concentration, voltage and time) on the physicochemical and morphological changes that occur in the water lily (WL) (Eichhornia crassipes) before and after the pretreatment is applied and to compare it with existing hydrolysis pretreatments such as chemical hydrolysis (acid and alkaline) and thermal treatments. It is a novel fiber with abundant cellulosic content, belonging to the Pontederiaceae family, a floating macrophyte capable of colonizing around 10,000 specimens in a single day, and is considered one of the 100 most harmful invasive exotic species in the world. Therefore, it is necessary to extract it, treat it and give it an application to take advantage of its properties, such as its chemical composition and mechanical properties, and, at the same time, reduce the problems that its presence could cause in aquatic systems if its proliferation is not controlled.

2. Materials and Methods

2.1. Biomass and Reagents

The biomass was obtained from the water lily (Eichhornia crassipes), which was extracted from La Vega Escondida lagoon, located in front of the Casa de la Naturaleza in Tampico, Tamaulipas. The water lily (Eichhornia crassipes) in the study area is available in large areas as a result of the dredging of the La Vega Escondida lagoon year after year.

For its processing, a pretreatment was carried out, which consisted of de-stalking, separating leaf, root and stem. It was then washed with tap water to remove contaminants. Subsequently, the fiber was dehydrated, applying a heat treatment in an oven at 105 °C for 5 h to achieve a constant mass [3]. Once the fiber was dried, it was crushed and then passed through a series of sieves until 10 mm long fibers were obtained, corresponding to what was retained in the 3/8 insieve. Finally, a delignification pretreatment was carried out by different methods to determine which was the most efficient. The treatments used were the following: thermal, chemical, electrohydrolysis and hybrid (chemical assisted with voltage). Different media were used, such as tap water, distilled water, hydrogen peroxide and sodium percarbonate, respectively, depending on the delignification pretreatment of the fiber.

2.1.1. Heat Treatment

After obtaining the fiber, the thermal treatment was carried out, which consisted of a boiling process, where the WL remained immersed for four hours in boiling distilled water, after which the fiber was washed to eliminate impurities and traces of soluble sugars (lignin and hemicellulose). To eliminate moisture, they were dried in an oven at 105 °C for 6 h and prepared for characterization [5]. The heat treatment procedure was repeated 4 times on the same sample.

2.1.2. Chemical Treatment (Acid and Alkaline)

The chemical pretreatment of delignification for the elimination of soluble sugars consists of immersing the natural fiber in a solution composed of H₂O₂ or Na₂CO₃-1.5H₂O₂. The concentration varied between 2.5% and 5%, with a contact time of 30 and 60 min at room temperature. A biomass to reagent ratio of 1:20 w/v was used for all experiments. After pretreatment, they were washed with distilled water three to four times to remove reagent residues as well as already solubilized sugars until a neutral pH was obtained, and finally, they were dried in an oven at 105 °C for 6 h for subsequent characterization.

2.2. Electrohydrolysis

For the electrohydrolysis pretreatment, an electrochemical cell was used: a conventional cell with two stainless steel electrodes (cathode and anode) connected to a direct current (DC) power source. The electrodes were manufactured from stainless steel due to its ease of forming, availability and maintenance, as well as its efficiency in distributing the applied polarization.

A voltage of 1, 1.5 and 2 V was applied for times of 15 and 30 min. Different electrolytes were used, starting with distilled water, and the WL fiber was added until it was submerged in the distilled water. A multimeter was used to measure the applied current and voltage. Figure 1 shows a picture of the electrohydrolysis pretreatment setup.

2.3. Hybrid Electrohydrolysis

The hybrid electrohydrolysis pretreatment is also called chemical pretreatment assisted by potential. This consists of using the chemical treatment described above under equal conditions, but a potential or polarization was additionally applied. The electrolytes used were H₂O₂, Na₂CO₃-1.5H₂O₂ (varying the concentration across 2.5% and 5%), distilled water and tap water, and potentials of 1, 1.5 and 2 V were applied for times of 15 and 30 min at room temperature. A biomass to reagent ratio of 1:20 w/v was used for all experiments. This treatment generated a large experimental matrix, coupling the electrohydrolysis process with the chemical treatment.

3. Characterization

3.1. Fourier Transform Infrared Spectroscopy (FT-IR)

WL samples were analyzed by FT-IR before and after the delignification process to determine the effect on the functional groups and chemical components of the fiber studied. All samples were studied in powder form. A Perkin Elmer Spectrum One infrared spectrophotometer was used for the analysis, using an attenuated total reflection (ATR) diamond/ZnSe accessory. The samples were evaluated in the 4000 cm⁻¹ to 650 cm⁻¹ wavenumber range with a noise signal resolution of 4 cm⁻¹ and with a sweep of 32 scans for each sample.

3.2. X-Ray Diffraction (XRD)

To evaluate the percentage of crystallinity, interplanar distance and average crystal size of the samples, the XRD technique was used to determine the structural composition and mechanical properties of the samples studied. The samples were evaluated as powders. The analysis was performed in X-ray diffractometer equipment (Bruker brand, model D8 Advance) using a Bragg–Brentano configuration (θ-2θ), a voltage of 40 kV, a current of 40 mA and Cu Kα radiation (λ = 1.5406 Å). Samples were measured in the 2θ range from 10 to 100°.

3.3. Optical Microscopy (OM)

The optical microscopy technique was used to evaluate the WL fibers before and after delignification treatments to identify the main physical changes such as thickness and color of the samples, changes associated with the removal of soluble sugars resulting from delignification. The morphological characterization was performed using an Olympus optical microscope, model BX51, with an Olympus UIS (Universal Infinity System) optical correction system.

4. Results and Discussions

4.1. FT-IR

After the aquatic lily was weeded, parts of the fibers extracted directly from the lagoon, without any treatment or cleaning, and another sample from when the fiber was clean, were analyzed by FT-IR to determine the chemical composition characteristic of this fiber. The samples were labeled according to the part of the plant to which they correspond, i.e., leaf (L), root (R) or stem (S), as the initial, and with a second letter added, namely S (without washing) and L (with washing).

Figure 2 shows a broad and intense band around 3400–3300 cm⁻¹ attributed to the stretching vibration of the O-H bond and the presence of α-cellulose [19,20]. The bands at 2927 and 2850 cm⁻¹ indicate the stretching of C-H bonds associated with the presence of cellulose, hemicellulose and/or lignin. The band at 1730 cm⁻¹ is assigned mainly to the acetyl and uronic ester groups of hemicellulose or to the ester bond of the carboxylic group of the ferulic and p-coumaric acids of lignin and/or hemicelluloses [19,20]. The absorption band present at 1602 cm⁻¹ corresponds to the C=O stretching vibration of the aromatic ring attributed to lignin, and the absorption band at 1467 cm⁻¹ is associated with the CH₂ (C-H) and CH₃ (C-H) bending vibrations of the aliphatic groups of lignin. Finally, the absorption band at 830 cm⁻¹ corresponds to the aromatic rings of lignin, while the prominent band at 1033 cm⁻¹ corresponds to the stretching of the C-O bond, assigned to the C-O deformation in secondary alcohol and aliphatic ether, mainly from cellulose. Additionally, it can be observed that each part of the clean fiber presented the same absorption bands, determining that the chemical composition of the WL plant does not vary significantly, regardless of the zone to which it corresponds. Therefore, in this research, we worked with all the components of the plant (leaf, stem and root) as received, and identified it as water lily (Eichhornia crassipes) fiber (WLF).

4.2. Heat Treatment

Once the reference spectrum (WLF) was determined, it was decided to evaluate the delignification process according to the different methods used, starting with thermal treatment.

Figure 3 shows the spectra obtained from the WLF without treatment and with the application of delignification heat treatment. The comparison of the different absorption bands is made according to the absorption bands of the WLF identified in Figure 2. A total of five repetitions of this thermal treatment were carried out to eliminate the highest percentage of non-soluble sugars. When performing the first heat treatment (TT-1), a small decrease in the absorption bands of 2927, 2850 and 1730 cm⁻¹ characteristic of lignin and hemicellulose was observed. This behavior is mainly due to the fact that hemicellulose is the first structural compound to be thermally affected, even at low temperatures [21]. In the second and third thermal treatment (TT-2 and TT-3, respectively) a decrease in the absorption bands of 2927, 2850 and 1730 cm⁻¹ attributed to hemicellulose and lignin was observed; however, it is considered that the WLF sample still had residual non-cellulosic compounds. Additionally for the fourth and fifth treatment (TT-4 and TT-5), it can be observed that the absorption bands of 2927, 2850 and 1730 cm⁻¹ attributed to hemicellulose and lignin decrease considerably, as well as a significant decrease in the absorption bands at 1730 and 1240 cm⁻¹ attributed to the C=O stretching of the carbonyl group of hemicellulose and the absorption band at 1467 cm⁻¹ corresponding to the vibration of the aromatic ring and bending of the CH₂ (C-H) and CH₃ (C-H) of the aliphatic groups, respectively. This modification is mainly due to the fact that the degradation of WLF starts by deacetylation and, as a consequence, the released acetic acid acts as a catalyst for depolymerization, increasing its decomposition; therefore, the elimination of the soluble sugars present and/or delignification of WLF is accelerated. However, these effects were observed after repeating the heat treatment four or five times.

4.3. Chemical Treatment (Acid and Alkaline)

The FT-IR results obtained after the delignification treatment by acid hydrolysis are shown in Figure 4, where one can observe the decrease in the absorption bands of 2927 and 2850 cm⁻¹ attributed to the asymmetric and symmetric C-H stretching of the saturated aliphatic compounds corresponding to the aliphatic remains in lignin and hemicellulose, as well as the methyl and methylene groups of lignin, in addition to a significant decrease in the 1730 cm⁻¹ absorption band corresponding to the C=O stretching, assigned mainly to the ester groups of hemicellulose and to the C-O stretching of the acetyl groups of lignin. Similarly, the 1240 cm⁻¹ absorption band associated with lignin decreases significantly. Additionally, the absorption band associated with C-H stretching at 2927 and 2850 cm⁻¹ is considerably attenuated compared with the 5% concentration of hydrogen peroxide. This behavior may be due to the fact that by increasing the concentration and reaction time of hydrogen peroxide with the substrate, there will be greater enrichment of cellulose, as shown in the outstanding absorption band at 1033 cm⁻¹, but less lignin removal due to the production of the hydroperoxide radicals (HO₂), superoxides (O₂) and, to a lesser extent, hydroxyls (OH) [19,20,21]. The reaction mechanism of hydrogen peroxide when in contact with water is as follows [20,21]:

H₂O₂ + H₂O ↔ HOO⁻ + H₃O⁺

On the other hand, its by-products are oxygen and water, which are not harmful:

H₂O₂ ↔ H₂O + O₂

On the other hand, the results obtained after alkaline hydrolysis (Na₂CO₃-1.5H₂O₂) can be seen in Figure 4. Likewise, the concentration was varied across 2.5 and 5% for an immersion time of one and a half hours or 90 min. Sodium percarbonate (Na₂H₃CO₆) is an adduct of hydrogen peroxide and sodium carbonate, is soluble in water, colorless, hygroscopic and crystalline. Once dissolved in water, it dissociates into sodium Na⁺ cations, carbonate $C{O}_3^{2-}$ and a mixture of hydrogen peroxide that eventually dissociates into water and oxygen [22,23], as shown below:

$$2{\mathrm{Na}}_2{\mathrm{CO}}_3·3{\mathrm{H}}_2{\mathrm{O}}_2 \to 3{\mathrm{H}}_2{\mathrm{O}}_2+4{\mathrm{Na}}^{+}+2C{O}_3^{2-}$$

2H₂O₂ → 2H₂O + O₂

Dissociation by-products are found in the effluents and are not considered toxic to the ecosystem.

Figure 5 shows a considerable attenuation in the intensity of the absorption bands at 2927, 2850, 1730 and 1240 cm⁻¹ associated with the functional groups of lignin and hemicellulose. However, at a 5% concentration, a greater decrease in the bands is shown than at the 2.5% concentration of percarbonate. This behavior is in contrast to the behavior in acid hydrolysis and is mainly due to the fact that in the dissociation of the Na₂H₃CO₆ molecules, which separate into sodium (Na⁺) and carbonate cations ($C{O}_3^{2-}$), which raise the pH of the medium, reaching values higher than 11.5 pH and favoring the breakdown of the hemicellulose and lignin molecules and/or the removal of soluble sugars, enriching the presence of cellulose as a consequence of delignification. However, it is important to mention that although the pH was not recorded before the delignification process, it is recommended to monitor it, in order to determine the pH effect on the reduction of soluble sugars such as lignin and hemicellulose. On the other hand, the rest of the molecules dissociate into a mixture of hydrogen peroxide, which is the oxidant that degrades lignin and hemicellulose [23,24].

4.4. Electrohydrolysis

The electrohydrolysis treatment started by using distilled water as an electrolyte, applying a potential of 1, 1.5 or 2 V for times of 15 and 30 min. Figure 6a,b present the results obtained by FT-IR corresponding to the samples evaluated by varying the potential and the polarization time of 15 and 30 min. In all cases, the absorption bands of 2927 and 2850 cm⁻¹ attributed to the OH and C-H stretching of hemicellulose or lignin decreased, as well as the almost total decrease in the absorption band of 1730 and 1240 cm⁻¹, mainly assigned to the ester groups of hemicellulose and to the C-O stretching of the acetyl groups of lignin, respectively; these absorption bands are associated with the efficient removal of non-cellulosic compounds [25].

Figure 6a,b show that by increasing the potential, a greater attenuation is obtained in the absorption bands of 2927, 2850, 1730 and 1240 cm⁻¹ associated with the non-cellulosic components, obtaining the best results by applying 2 V.

Subsequently, the same methodology described above was applied under the same conditions, only the electrolyte was modified, now using tap water (potable). The results obtained in Figure 7a,b show that soluble sugars (lignin and hemicellulose) are removed efficiently, even more than in distilled water. The degradation of these soluble sugars is mainly due to the reduction reaction of the H₂O molecule (2H₂O + 2 e⁻ → H₂ + 2OH⁻), releasing hydroxyl radicals, which oxidize lignin and dissociate hemicellulose from WLF. Additionally, it can be observed that increasing the applied voltage accelerates the water reduction reaction, favoring the delignification of WLF [23].

4.5. Hybrid Electrohydrolysis

On the basis of the previous results, it was proposed to use alkaline and acid chemical treatment, applying a voltage, using Na₂H₃CO₆ and H₂O₂ as the electrolyte and varying the potential, concentration, time and electrolyte. In such a way, a hybrid treatment was applied by adjusting the electrohydrolysis previously studied and the chemical treatment also already discussed, resulting in a treatment called hybrid electrohydrolysis.

The resulting experimental matrix is shown in

Table 1. Hybrid electrohydrolysis treatment.

Medium	Polarization (V)	Concentration (%)	Time (min)
Na2H3CO6	1, 1.5, 2	2.5–5	15–30
H2O2	1, 1.5, 2	2.5–5	15–30

.

Hydrogen peroxide has a high redox potential of 1.77 V vs. a normal hydrogen electrode (ENH), which gives it unique chemical characteristics as an oxidizing agent, due to its interaction in aqueous solutions. The proposed mechanism considers the following reactions [10,11,18]:

H₂O₂ + 2H⁺ + 2e⁻ ↔ 2H₂O  E⁰ = 1.77 V vs. ENH

O₂ + H₂O + 2e⁻ ↔ H₂O₂   E⁰ = 0.68 V vs. ENH

HO₂⁻ + H₂O + 2e⁻ ↔ 3OH⁻  E⁰ = 0.867 V vs. ENH

This promotes the removal of soluble sugars, as shown in Figure 8a,b.

It is considered that increasing the H₂O₂ concentration and potential results in a greater attenuation of the absorption bands of 2927 and 2850 cm⁻¹ attributed to the O-H and C-H stretching of hemicellulose and lignin, as well as the almost total attenuation of the absorption bands of 1730 and 1240 cm⁻¹ mainly assigned to hemicellulose ester groups and the C-O stretching of the acetyl groups of lignin, respectively, which are absorption bands associated with the efficient removal of non-cellulosic compounds [23]. If we compare the results obtained from all the samples evaluated, the most efficient removal effect of the soluble sugars can be observed in the samples treated with 5% H₂O₂, applying 2 V. The removal of these soluble sugars is mainly due to the reduction reaction of H₂O₂, releasing hydroxyl radicals, which oxidize lignin and dissociate hemicellulose from WLF. However, all the samples evaluated showed a greater or lesser degree of soluble sugar removal.

On the other hand, the hybrid electrohydrolysis treatment used Na₂H₃CO₆ (sodium percarbonate) as the electrolyte under the same conditions as the pretreatment described above, varying the concentration, potential and time, considering the electrochemical reaction of the percarbonate as follows [21,22,26].

$$2{\mathrm{Na}}_2{\mathrm{CO}}_3·3{\mathrm{H}}_2{\mathrm{O}}_2 \to 3{\mathrm{H}}_2{\mathrm{O}}_2+4{\mathrm{Na}}^{+}+2C{O}_3^{2-}\hspace{1em}\hspace{1em}\hspace{1em}{\mathrm{E}}^0=1.7 \mathrm{V}$$

2H₂O₂ → 2H₂O + O₂

The results obtained are shown in Figure 9a,b, where it can be observed that, in the same way as seen for H₂O₂, a greater removal of soluble sugars occurred with a concentration of 5% Na₂H₃CO₆ and 2 V. The removal of soluble sugars is mainly due to the dissociation reaction of Na₂H₃CO₆ separating into sodium (Na⁺) and carbonate ($C{O}_3^{2-}$) cations, which increase the pH of the medium to values above pH = 11.5, favoring the breaking of hemicellulose and lignin bonds. However, all the samples evaluated showed soluble sugar removal.

Therefore, it was determined that the hybrid delignification treatment catalyzes the oxidation reactions of lignin and hemicellulose, promoting a higher and more efficient removal of non-cellulosic components with respect to the other methods.

Additionally, the best FT-IR results obtained from each of the delignification treatments studied were compared (Figure 10). Finally, they were analyzed by XRD and OM to confirm the physicochemical and structural changes observed by the FT-IR technique.

It can be observed that regardless of the delignification treatment used, a considerable attenuation of the characteristic absorption bands of the functional groups associated with lignin and hemicellulose is observed. However, considering the results obtained with the delignification treatment by electrohydrolysis using tap water as the electrolyte, it can be observed (pale green color) in Figure 10 that this delignification treatment is as efficient as the one obtained using distilled water, H₂O₂ or Na₂H₃CO₆. In addition, this treatment does not use any chemical reagents, nor does it represent an excessive expenditure of energy, so it is considered environmentally friendly and economical. Therefore, the delignification treatment to be selected is at the discretion of the research objective.

Finally, it was determined that electrohydrolysis could be an effective method for the delignification pretreatment of any lignocellulosic biomass for the removal of soluble sugars such as lignin and hemicellulose in less time, without excessive energy costs, as well as being environmentally friendly and economical.

4.6. Calculation of Delignification Efficiency

It is necessary to determine the percentage of delignification of the treated natural fibers to evaluate the efficiency of the process used, seeking to obtain the maximum removal of soluble sugars (lignin and hemicellulose) and, consequently, the enrichment of cellulose. WLF is made up of lignin (9.65–12.80%), hemicellulose (7.2–20%) and cellulose (59.86–65.40%) [27,28,29]. However, the efficiency was only determined for the treatments that showed the best response in the XRD and FT-IR characterizations.

There are different methods to quantify the percentage delignification of lignocellulosic materials; among them are the Klason method [30], UV spectrophotometry [31], the Weende method [32], the Kappa method [33] and Fourier Transform Infrared Spectroscopy (FT-IR) [34,35,36]. FT-IR was chosen because the vibrations of the molecules are directly related to the absorption bands in FT-IR spectroscopy. When a molecule absorbs infrared energy, its atomic bonds vibrate in different modes, such as stretching, bending, twisting and deformation. These vibrations are specific to each type of chemical bond and functional group present in the molecule. The intensity and position of these bands provide information about the presence and number of functional groups in the sample. However, it is necessary to complement a quantitative analysis, such as estimation of the area under the curve of the specific absorption bands of lignin and hemicellulose, from which the percentage removal of soluble sugars can be estimated by processing the data obtained mathematically.

The procedure begins with the normalization of the data obtained to make the spectra, as well as the drawing of a baseline. Subsequently, the absorption bands of interest are chosen; in this case the representative bands of lignin, hemicellulose and cellulose (2920, 2850, 1730 and 1033 cm⁻¹, respectively) were selected. Once identified, the area under the curve of each band in an interval [a,b] is calculated using the following equation [37]:

$${{\displaystyle \int }}_a^{b}f\left(x\right)dx$$

Figure 10 shows qualitatively the decrease in the bands at 2920, 2850 and 1730 cm⁻¹, which contributed to the partial removal of soluble sugars. After calculating the area under the curve, the percentage decrease of each band is quantified; likewise, the decrease in the area under the curve may also reflect changes in the chemical composition of the sample as the lignin is removed. The relative proportion of other components such as cellulose may increase, which can be observed in the FT-IR spectra (Figure 10) and in the calculation of the area under the curve in the band at 1033 cm⁻¹ (

Table 2. Calculated percentage of delignification (considering the reference bands as 0%) and comparative delignification results from the literature.

Sample	Removal Rate (%)	Removal Rate (%)	Increase Rate (%)	Average Delignification Rate (%)	Std. Dev.
5% H2O2, 0 V, 1.5 h	70.60	77.61	14.62	72.94	±0.0037
5% H2O2, 2 V, 15 min	78.01	92.14	23.44	82.72	±0.0167
TW, 2 V, 30 min	79.73	91.89	22.45	83.78	±0.0102
Corn stover (microwave irradiation) [38]	74.89	75.01	-	74.95	-
Wheat straw (hydrothermal) [39]	39.01	53.40	-	46.21	-
Wheat straw (distilled water) [10]	54.80	22.40	-	38.60	-

), where the enrichment in cellulose is clearly reflected.

Table 2 shows the results obtained from the calculation of the area under the curve of the bands (2920, 2850, 1730 and 1033 cm⁻¹) corresponding to the different delignification methods that presented better qualitative results. A significant increase in the removal of soluble sugars was observed in each of the calculated bands, which indicates that the delignification methods were efficient. Additionally, the standard deviation of the bands associated with hemicellulose and lignin was calculated from the average of the bands associated with lignin and hemicellulose. However, the results with the highest percentage of soluble sugar removal were obtained using the hybrid delignification treatments (acid electrohydrolysis at 5%H₂O₂ and 2 V for 15 min) and electrohydrolysis (TW, 2 V, 30 min). On the other hand, a lower efficiency has been reported for other delignification treatments [10,38,39]. In addition, the use of temperature, mechanical stress and harmful reagents has consequences, including high energy expenditure, prolonged times and harmful waste to the environment, opposite to the proposed treatments.

4.7. XRD

By means of XRD, the phases present in the WLF after the delignification treatments studied were determined, but only for the samples that presented the best FT-IR results. The phases present corresponding to Cellulose I, Cellulose Iβ and Cellulose II were identified, respectively, according to the intensities in the 2θ of the characteristic diffractograms of cellulose, using the crystallographic charts PDF 00-056-1719, 00-056-1718 and 00-056-1719.

In the obtained diffractograms (Figure 11), it can be observed that the cellulose obtained from WLF is of the semi-crystalline type [3,19,20]. The main intensities in the 2θ diffractogram of the WL reference are as follows: 15.15° with the diffraction plane (101) corresponds to Cellulose I, 20.75° (002), 24.36° (−201) and 29.26° (102) to Cellulose Ia and 35.95° (−301) corresponds to Cellulose Iβ. In the lignocellulose samples after the treatments, a reorganization of the cellulose crystalline structure was observed, identifying the characteristic patterns of Cellulose I and II. Cellulose Type II, the most ordered region, can be observed more tangibly, associated with a change in the intensity and amplitude of the peak of 2θ 20.75° to a narrower and more intense 2θ 21.95° (020).

All the diffractograms were intensified compared with the reference, which shows a reorganization of the crystalline structure, indicating an increase in its crystallinity that was almost complete from Cellulose I to II (mercerization) [30]. The characteristic pattern of the reference has broader and less intense peaks, indicating that it has less ordered or amorphous regions. After the delignification treatments, the percentage of crystallinity was calculated using the formula obtained from Romero et al. (2024) [3]. The results are shown in

Table 3. Structural parameters obtained via XRD.

Sample	Crystallinity Degree Xc	2θ (°)	Interplanar Distances d(Å)	Crystallite Size D(Å)
WLF	30.4 ± 2.2	22.05	8.149	255.789
5% H2O2, 0 V, 1.5 h	90.2 ± 1.1	21.59	4.111	22.446
5% H2O2, 2 V, 15 min	93.4 ± 1.2	21.538	4.041	25.488
TW, 2 V, 30 min	95 ± 1	21.59	4.028	25.589

TW, tap water; WLF, water lily fiber; V, voltage.

, where an increase in crystallinity is observed in all the samples analyzed, obtaining the best results using the delignification method using electrohydrolysis.

Additionally, according to X-ray diffraction, the structural parameters of the treated samples were determined according to the 2θ corresponding to each sample. A significant difference can be observed in the percentage of crystallinity, d(Å) interplanar distances and D(Å) crystallite sizes with respect to the untreated sample (reference), which are associated with the removal of soluble sugars such as lignin and hemicellulose, as well as the reorganization of the cellulose crystalline structure. This is reflected in a decrease in the interplanar distance, as well as the crystalline size and an increase in the degree of crystallinity. This behavior is associated with the efficiency of the delignification treatment via electrochemistry (electrohydrolysis) for the removal of soluble sugars, reaching a higher efficiency using tap water (TW) as the electrolyte, as shown in Table 3, obtaining a correlation in the delignification efficiency results determined by FT-IR and the crystallinity degree determined by XRD.

4.8. Optical Microscopy (OM)

In the micrograph observed in Figure 12a, corresponding to the WLF without delignification treatment, the fiber with dark pink color and irregular shapes in the cross-section and impurities can be seen. In contrast with the micrographs corresponding to the WLF samples after delignification treatment, a whitening occurs, characteristic of natural fibers previously delignified due to the solubilization of residual sugars such as hemicellulose and lignin in the fiber. In addition, no impurities were observed. The white color is proportional to the decrease in soluble sugars, i.e., the greater the bleaching, the greater the elimination of non-cellulosic components. Finally, no damage or weakening of the fibers obtained after delignification was observed (Figure 12b–d).

On the other hand, the cross-section of the delignified samples becomes smooth and a decrease in roughness can be observed. In this regard, the more aggressive the delignification treatments, the lower the roughness (

Table 4. Roughness calculation of reference WLF; 5% H₂O₂, 0 V and 1.5 h; 5% H₂O₂, 2 V and 15 min; and TW, 2 V and 30 min.

Sample	Ra			Average	Rq			Average
Reference	0.0719	0.0755	0.0668	0.0714	0.0952	0.0953	0.0864	0.0923
5% H2O2, 0 V, 1.5 h	0.0368	0.0340	0.0324	0.0344	0.0466	0.0439	0.0434	0.0446
5% H2O2, 2 V, 15 min	0.0377	0.0379	0.0397	0.0384	0.0580	0.0534	0.0556	0.0557
TW, 2 V, 30 min	0.0328	0.0391	0.0236	0.0318	0.0433	0.0498	0.0318	0.0416

Ra, roughness average; Rq, root mean square roughness.

).

5. Conclusions

In this research work, the efficiency of different delignification pretreatments of water lily (Eichhornia crassipes) fiber (WLF) was studied by FT-IR, with the main objective of identifying an environmentally friendly delignification method that does not generate toxic by-products or excessive energy costs. The results are as follows.

The FT-IR results showed that any of the delignification pretreatments studied (under the experimental conditions proposed in this work) present a high removal efficiency of residual sugars, which is mainly reflected in a decrease in the absorption bands at 2927, 2850 and 1730 cm⁻¹ associated with hemicellulose and lignin, highlighting that the highest delignification efficiency obtained was 83.78% for the hybrid acid electrohydrolysis and 82.72% for the electrohydrolysis method. The results correlate with the XRD results showing the increase in the percentage of crystallinity. This could have a direct positive effect on the mechanical properties of concrete and decrease the rate of fiber degradation if delignified fiber is added.

Additionally, by means of optical microscopy, it was determined that the WLF shows a whitening after delignification treatment, characteristic of natural fibers previously delignified, due to the removal of residual sugars such as hemicellulose and lignin.

Finally, of all the results obtained, the most efficient delignification pretreatment in the removal of soluble sugars such as lignin and hemicellulose were the sample electrohydrolysis pretreatment using tap water applying 2 V of potential for 30 min. And this delignification pretreatment is environmentally friendly, without generating or emitting toxic by-products, in addition to reducing energy expenditure, opening a path of opportunities to delignify any type of lignocellulosic natural fiber, and its versatility makes it easy to extrapolate in mass. The recovered cellulose can have applications as biofuels or biomaterials or in concrete.