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Article

Etching and Precursor Effects on Plasma-Modified Waste Polyethylene Terephthalate (PET) to Laccase Immobilization Applied in Catechol Biodegradation for Water Treatment

by
Reyna Orsua-Gaona
1,
Rosa Idalia Narro-Céspedes
1,*,
Anna Ilina
1,
Luis Fernando Mora-Cortés
2,*,
Yadira Karina Reyes-Acosta
1,
Gustavo Soria-Arguello
2,
Cynthia Luevano-Martínez
1,
Miriam Paulina Luévanos-Escareño
3 and
Claudia Gabriela Cuellar-Gaona
2
1
Facultad de Ciencias Químicas, Universidad Autónoma de Coahuila (UAdeC), Saltillo 25280, Coahuila, Mexico
2
Centro de Investigación en Química Aplicada (CIQA), Saltillo 25294, Coahuila, Mexico
3
Facultad de Ciencias Biológicas, Universidad Autónoma de Coahuila (UAdeC) Campus Torreón, Torreón 27275, Coahuila, Mexico
*
Authors to whom correspondence should be addressed.
Coatings 2025, 15(4), 421; https://doi.org/10.3390/coatings15040421
Submission received: 26 February 2025 / Revised: 19 March 2025 / Accepted: 20 March 2025 / Published: 2 April 2025
(This article belongs to the Special Issue Advanced Technology in Environmental Remediation and Resource Utilization, 2nd Edition)
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Versions Notes

Abstract

:
Polyethylene terephthalate (PET) is a polyester used in the beverage bottling industry that generates a serious pollution problem. Films were obtained to reuse discarded PET bottles, and their surface was modified to determine their effectiveness in supporting the immobilization of the laccase enzyme applied to a catechol biodegradation assay. Radiofrequency (RF) plasma was used to modify the PET surface using different precursors: (a) with the use of air as precursor, the effect of the etching caused by the air on the greater or lesser immobilization was observed; (b) with the use of ethylenediamine, a mixture of N2/H2, or aniline as precursors, it was observed which of these three precursors presented the greater or lesser number of amino groups deposited on the PET surface. After plasma modification, the films were cross-linked with glutaraldehyde to immobilize the laccase enzyme. Finally, the catechol test was performed. It was found that the best etching time using air as a precursor was 90 min, and the precursor that caused a higher insertion of amino groups on the surface was ethylenediamine, which reached a density of amino groups of 3.98 ± 0.10 g·mm−2. The highest percentage of laccase immobilization achieved on the surface of ethylenediamine-modified PET was 97.30%. In the catechol assay, the highest retention was 86.11%. This research reveals how the effect of plasma increases the surface area on a PET surface and, in conjunction with ethylenediamine as the best precursor of the three precursors evaluated, can immobilize a greater amount of enzyme and oxidize more catechol. There is no scientific evidence from previous studies that used air plasma technology to erode and then used three different precursors to modify a surface to immobilize the laccase enzyme and remove a water contaminant.

Graphical Abstract

1. Introduction

A serious and difficult-to-solve environmental pollution problem worldwide is the waste from PET bottles of water and soft drinks. The melting of these bottles contributes to climate change as they release toxic compounds when melted again [1]. Furthermore, these single-use bottles are easily disposed of in the environment, causing drainage problems in cities and pollution in rivers and seas. More than 722 thousand tons of PET bottles are produced annually in Mexico alone. This figure makes Mexico the country with the highest consumption of bottled water in the world [2] and is alarming because these bottles take between fifty and one hundred years to degrade [2,3]. Due to the great negative impact of this PET waste, associations such as Ecology and Business Commitment (ECOCE) have emerged to propose recycling and reuse alternatives since, according to statistics, in 2022, even though Mexico is the third country that recycles the most in the world only behind Germany and China, it recycled only 63% of PET [3]. This work supports this type of organization by proposing a new possibility of reusing this material.
Water pollution from industrial waste is a danger to society. Waste from various industries contains toxic compounds harmful to human health, flora, and fauna. One of these compounds is phenol, and its highly carcinogenic derivatives [4] are toxic due to the formation of free radicals that interact with cells. Phenolic derivatives are stable and bio-accumulative; they remain in the environment for long periods [5]. These phenolic derivatives are produced in processing polymers, leather, paper, textiles, pesticides, and dyes in oil refining and coking industries and the pharmaceutical industries. It is estimated that 73% of them are released into water [6]. Existing treatments for the degradation of phenols have been classified as physical, chemical, and biological. The physical therapies of coagulation, activated carbon adsorption, and membrane bioreactors require additional sludge treatment, carbon activation, and high effluent volumes, respectively. Chemical treatments such as electrochemical oxidation, photocatalytic degradation, and UV/H2O2 usually require sophisticated equipment and considerable amounts of ozone at high temperatures and pressures [7].
Within biological treatments, enzymes are presented as a promising alternative because they can biodegrade phenolic derivatives in an environmentally friendly and rapid manner [8]. A widely studied enzyme for structurally breaking down phenol and its derivatives is laccase [9,10,11,12,13,14,15]. Laccase enzyme (EC 1.10.3.2, p-diphenol: dioxygen oxidoreductase) is an extracellular glycoprotein belonging to the peroxidase family; it is a high molecular weight, copper-containing glycoprotein with the ability to reduce molecular oxygen to water and simultaneously oxidize organic and inorganic substrates [12]. One of the phenol derivatives with which the laccase enzyme has been reported is catechol [16,17,18,19]. Catechol is a compound that has become a model for evaluating the degradation of phenolic derivatives because it is a simple molecule compared to other phenol derivatives, such as 2-nitrophenol, 4-methylphenol, 4-aminophenol, butylated hydroxytoluene, 4-nonylphenol, and bisphenol A, the most well-known for its high toxicity [20].
In addition to the laccase enzyme, other oxidoreductase enzymes have used catechol to measure their efficiency on contaminants in phenolic removals, such as the nano en-zyme DNA–copper hybrid nanoflowers whose catalytic activity is laccase and has been reported in the reaction mechanism with 4-amino antipyrine through colorimetric detection of catechol [21].
Although it is a good alternative for the biodegradation of phenols, a problem of the laccase enzyme is that it has difficulty recovering from the reaction medium in which it acts [22]. Its immobilization could solve this problem. There are several methods to carry out enzyme immobilization, such as adsorption, entrapment, encapsulation, covalent bonding, and cross-linking [23]. Generally, these methods fall into two categories, physical and chemical, depending on the bond formed between the enzyme and the support. Physical methods involve Van der Waals force bonds, hydrogen bonds, ionic interactions, bonding affinity, encapsulation, and/or entrapment of the enzyme within the support with micelles or pores by adsorption, whereas, in chemical methods, strong bonds are formed, such as ionic, covalent, and cross-linking [24,25].
According to Daronch et al. [26], ideal support for enzyme immobilization has to be of low production cost and non-polluting, not react chemically with the medium with which it will be immersed, have thermal and mechanical resistance, avoid adsorption of the enzyme that is not necessary, withstand the pH and temperature variations that the enzyme needs to act more efficiently. It must increase the interactions between the active sites of the enzyme and the substrate molecules to finally store a maximum amount of enzyme. Therefore, considering these characteristics, PET can be viewed as a good alternative for reuse in this research and to act as a support in the immobilization of enzymes since, due to its excellent mechanical properties, even after being discarded [27], it meets all the aspects listed, which generates a competitive advantage over other supports. Once a single PET bottle is discarded, it can be reused, so there is no production cost [28], and it can also be considered for other new technological applications [29,30].
Although the waste PET retains most of the properties mentioned above [31,32], its surface must be previously conditioned so that the PET bottle can work as an immobilization support. Since it does not react chemically on its own, it is necessary to add chemical agents for reactions such as hydrolysis, glycolysis, and/or aminolysis, among others, to take place [33]. For the enzyme to adhere to the polymer, binding sites must be created by adding functional groups. Regarding the insertion of amine functional groups, the conventional treatment is known as chemical aminolysis, and its effectiveness has been investigated by various authors, such as Bech et al. [34] and Noel et al. [35], who reported densities higher than 10-NH2/nm2. However, this type of treatment is aggressive with the material and requires a high consumption of reagents and solvents during and after carrying out the reaction [36]. Due to these disadvantages, alternative treatments such as plasma have been proposed since it has proven effective in grafting this functional group, among others [36,37,38,39,40]. Furthermore, it does not use solvents. Therefore, it does not generate waste, does not interfere with the material’s chemical structure, and consumes little energy during the process, all representing a low environmental impact during its use [41,42]. Plasma can be a suitable tool to do this, as it can modify the surfaces of polymers without affecting the bulk material properties [43,44,45,46]. In addition, plasma is an environmentally friendly technology since it complies with the following principles of green chemistry: it does not generate chemical waste; it does not use solvents during plasma generation; there is energy efficiency because the procedure is carried out at room temperature and prevents contamination by not forming dangerous substances [47]. These characteristics give plasma a great advantage over conventional chemical treatment, in which flammable rea-gents and solvents are worked with at a temperature above 25 °C.
Plasma technology has been employed to modify polymer surfaces for different applications. Vesel et al. [48] modified polystyrene (PS) using oxygen plasma for surface functionalization with OH groups and showed that controlling the dose of functional groups is possible. Elammari et al. [45] reported the technical details of atmospheric pres-sure plasma modification, among other plasma sources, for various polymers. Burmeister et al. [49] modified polyethylene (PE) by atmospheric plasma-induced vinyl ben-zyl-sulfobetaine polymerization and evaluated its antifouling properties. Chytrosz-Wrobel et al. [50] applied cold oxygen plasma to generate specific functional groups, hydrophilicity, and nano topography in different polymers with different degrees of crystallinity: amorphous aromatic polyether-based polyurethane (PU), crystalline-amorphous poly (chloro para xylylene) (perylene C) and crystalline high-density polyethylene (HDPE). Nakulan et al. [51] improved the wettability property of polystyrene (PS) and polyethylene glycol (PEG) films with DC plasma glow discharge treatments of air, argon, oxygen, or nitrogen sources. Prasad et al. [52] modified polypropylene (PP) and polyethylene terephthalate (PET) films using dielectric barrier discharge (DBD) plasma at reduced pressure; the precursor gas used was air. They improved the wettability and roughness.
Additionally, this study empathizes with environmental care in two aspects: one, the reuse of discarded PET bottles for the immobilization of the laccase enzyme, and two, the biodegradation of phenols in aqueous media. The objective of the present study was the use of waste PET films as a possible economic and ecological support, where surface modification was performed with radiofrequency (RF) plasma in two steps: first: air etching, and the second using three different precursors, followed by the immobilization of the laccase enzyme and finally, its evaluation in the biodegradation of catechol for the possible elimination of phenols and their derivatives in wastewater treatment.

2. Materials and Methods

2.1. Surface Modification

PET films were obtained by cutting 3 cm long by 1 cm wide from the waste PET bottles. Afterward, they were washed in 2.5 mL of ethanol ((99.5% purity) Jalmek brand) for 15 min and placed in a desiccator for 24 h at room temperature. Finally, the films were stored in airtight bags to avoid contact with dust or other contaminants. A plasma reactor with an Advanced Energy model RFX600A (Ciudad de México, CDMX, México) radiofrequency (RF) generator was used. The surface modification of the films with RF plasma was performed in two stages: (1) in the first stage, the objective was to evaluate the effect of a larger surface area on the PET film through etching. To generate plasma, air was used as a precursor under the power of 50 W, pressure of 0.6 ± 0.2 mbar, and time of 0 (E0) and 6 h (E6). The marked difference in the times was to observe the etching effect; (2) In the second stage, three different precursors were used: ethylenediamine ((USP grade) Golden Bell Reactivos brand), an N2/H2 mixture ((95% N2-5% H2) AOC México, S.A de C.V brand), and aniline ((analytical grade- 98% purity) CTR-Scientific brand). Each was used independently in the plasma reactor to evaluate which one could graft more amino groups onto the PET surface. The parameters were a power of 50 W, pressure of 0.6 ± 0.2 mbar, and time of 90 min. Once the PET films were modified in the two stages, each of them with initial dimensions of 3 cm long by 1 cm wide was cut in half, leaving them with final dimensions of 1.5 cm long by 1 cm wide. This experiment was carried out in triplicate on each sample. A visual representation of the stages of the plasma modification of the films and the process of enzyme immobilization is shown in Figure 1.

2.2. Determination of Amino Group Density on PET Films

To quantify the amino groups deposited on the PET film surface, the films were stained with orange II (NII) dye (Golden Bell Reactivos brand) using the methodology reported by Noel et al. [35]. First, each film was immersed in a 100-ppm solution of NII dye at 40 °C for 30 min. Next, each film was washed thrice with a 1 M acidic HCl (Jalmek brand) solution adjusted to pH 3 and dried at room temperature. Subsequently, the films were immersed in a 1 M alkaline solution of NaOH (Jalmek brand) adjusted to pH 12 and left in agitation for 30 min. In the end, the films were removed. The absorbance of the obtained solutions was measured at 486 nm in a Thermo-Spectronic Genesys 20 UV-Vis spectrophotometer (MA, USA). The study was performed in triplicate.

2.3. Contact Angle (CA) Evaluation

The contact angle was measured on each plasma-modified PET film to evaluate changes in wetting and the degree of adhesion between the amino functional group deposited on the waste PET surface. The procedure involves depositing a 3 µL drop of water on the film with a micro syringe. A Kruss Drop Shape Analyzer goniometer (Nuevo Leòn, NL, Mexico) was used for the measurement. The study was performed in triplicate.

2.4. Fourier Transform Infrared Spectroscopy (ATR-FTIR)

Plasma-modified and unmodified PET films were analyzed by ATR-FTIR, with Nicolet iS5 equipment from ThermoFisher Scientific (MA, USA), with germanium plate (iD7-ATR-Ge), and the spectra were obtained by OMNIC software, with a total of 100 scans and a resolution of 0.8 cm−1 for each sample. Data analysis was performed using OriginPro 9.0 software.

2.5. Scanning Electron Microscopy (SEM)

The morphology of PET waste films before and after the two stages of plasma surface modification was evaluated using a JEOL model JCM 6000 field emission scanning electron microscope (MA, USA). The samples were placed in a stainless-steel sample holder attached to a copper tape, and then a gold/palladium (Au/Pd) coating was performed; this study was carried out with a secondary electron detector (SEI), using a voltage of 30 kV.

2.6. Immobilization of Laccase Enzyme

The laccase enzyme used was fungal Trametes versicolor (0.5 U mg−1) (Sigma brand). Plasma-modified PET films were individually immersed in 1.5 mL of glutaraldehyde ((II grade) Sigma-Aldrich brand). They were left to incubate at 25 °C for 3 h. Subsequently, they were washed with distilled water. After activating the films with glutaraldehyde, the immobilization reaction of the laccase enzyme by covalent bonding was carried out using the methodology Bradford reported by Sanchez et al. [53]. Laccase was prepared in 12 mL acetate buffer with a final concentration of 7 mg·mL−1. Each glutaraldehyde-activated film was subjected to 1.5 mL of the enzyme solution at 4 °C. The films were incubated for 12 h under orbital shaking at 100 rpm at 4 °C. At the end of the reaction, the films were removed from the solution and washed individually with distilled water three times and finally once with Tris-HCL buffer. The study was performed in triplicate.

2.7. Immobilized Protein

The Bradford colorimetric method [54] was used to quantify the immobilized enzyme. This method is based on calculating the residual protein. To 0.1 mL of each solution, 2 mL of Bradford reagent containing Coomassie Blue G-250 (Sigma-Aldrich brand) (was added. After 5 min, the absorbance was measured at 595 nm. Before quantification, a calibration curve was prepared with the bovine serum albumin (BSA) standard (LICON brand), which measures protein in a concentration range from 0 to 50 µg·mL−1. The study was performed in triplicate. The desorption amounts were calculated using the difference in concentrations.

2.8. Enzymatic Activity Assay

The activity of the enzyme immobilized on PET films was determined by the catechol oxidation assay following the methodology reported by Wang et al. [55]. The catechol solution was prepared at 0.1 M in 100 mM acetate buffer (Sigma-Aldrich brand) at pH 5. In addition, CuSO4 (Sigma-Aldrich brand) solution at a concentration of 2 mM was prepared in the same buffer. 250 µL of catechol was mixed with the 20 µL of CuSO4. 1.5 mL of the mixture was taken, and the films with the immobilized enzyme were immersed individually. They were left in incubation at 25 °C for 20 min. Then, each of the films was removed from the reaction medium. The absorbance was measured at 450 nm. The study was performed in triplicate.

3. Results and Discussion

3.1. Surface Etching of PET Films with Air Plasma: Morphological Analysis

Figure 2 shows the photographs and scanning electron micrographs (SEM) of the unmodified films at 0 h (E0) and of the air plasma-treated films at 6 h (E6). In Figure 2a, it can be observed that the unmodified waste PET film has a shiny and smooth appearance, and in the micrograph (Figure 2a*) a soft and defect-free surface of the same sample is observed, confirming that this is a typical characteristic of waste PET from water bottles that has not been surface modified. On the other hand, in contrast, it can be observed in Figure 2b that the PET film exposed to air plasma for 6 h (E6) presents a decrease in brightness and a slightly opaque white appearance with striations on the surface. Confirming this study, the micrograph obtained by SEM in Figure 2b* clearly shows corrugated areas and indentations in the film modified with air plasma, as indicated by the yellow arrows in this figure.
These photographs and micrographs show that cold plasma technology can produce the etching phenomenon, where electrons can travel at high speeds and cause the breaking of C-C bonds. Due to their magnification, the micrographs in Figure 2b* clearly confirm what was observed in the plasma-modified sample. Unlike the unmodified sample, it can be seen that this sample underwent a degradation process by etching caused by prolonged exposure to plasma [56]. Figure 3 shows a diagram of the etching process. In this process, the constant breaking of C-C bonds due to the bombardment of electrons colliding at high speeds on the PET surface is generated in the air plasma. The breaking of these C-C bonds causes the formation of free radicals and low molecular weight fragments that can be volatilized by the low-pressure system of the plasma equipment, generating imperfections on the material’s surface [57]. In general, this repetitive process leads to etching on the surfaces of PET and other materials.

3.2. ATR-FTIR Verification of Functional Groups in Air Plasma-Treated PET Films

Figure 4 shows the ATR-FTIR spectra of the PET waste film without modification (E0) and the film modified with air plasma for 6 h (E6). The bands corresponding to the different functional groups that chemically make up PET were identified in both cases. In the case of the vibrations of the terephthalate group, the stretching of the carbonyl of the ester that is attached to an aromatic ring is observed at 1718 cm−1, the stretching vibrations of the C=C aromatic double bond 1603 cm−1, and the stretching = C-H of aromatic carbon with sp2 hybridization at 3048 cm−1 in addition to the stretching of the C-O bond at 1261 cm−1 and the signal at 721 cm−1 is due to the deformation vibration of =C-H corresponding to benzene rings. Only methylenes (-C-H) were observed in the part of the hydrocarbon structure, whose asymmetric stretching band is 2945 cm−1, and the symmetric stretching band is 2850 cm−1. These signals were compared with those reported by Ramírez-Hernández et al. [58].
When comparing the spectrum of PET films modified with air plasma for 6 h with respect to unmodified PET (0 h), the spectrum of modified PET film shows an additional O-H band at 3405 cm−1 and an increase in the C=O band. Therefore, both bands are attributed to the air plasma modification that increases the sample’s surface hydrophilicity due to the rise in oxygen functional groups; this is corroborated in Section 3.5. These signals were compared with those reported by Savoji et al. [59]. During the etching process, polymers, as well as other materials, undergo a reduction in thickness, an increase in surface area, a decrease in the mechanical properties of the elements, and fluctuations in the topology that change the functional characteristics of the surface [57]. Activation also occurs simultaneously, creating chemical species on the surfaces according to the precursor gas. This is possible due to the breaking of chemical bonds that, in turn, initially form free radicals and that later combine with other chemical species from the plasma precursor to create new chemical bonds on the material [60].

3.3. Surface Activation of PET Films with Ethylenediamine Plasma, Aniline, and N2/H2 Mixture

Figure 5 shows the images of the PET waste films pretreated with air plasma and subsequently subjected to plasma treatment with three different precursors to deposit an amine group on the etching surface. These images are compared with the image of the not-modified PET film. The not-modified PET film (Figure 5a) has the characteristic appearance of PET, colorless and shiny. On the contrary, in the etching film modified with ethylenediamine (Figure 5b), an opaque and veined appearance is observed due to the previous etching and the treatment with ethylenediamine, which causes the surface deposition of the amino group. The film treated with the N2/H2 mixture is presented in Figure 5c. It shows a similar appearance to the one modified with ethylenediamine since both lost their initial shine and color and became slightly opaque. However, the N2/H2 film did not present streaks, but a non-smooth surface was observed. These differences can be explained by the fact that when gases such as nitrogen and hydrogen are used, the surface of the substrate is activated [61,62]. Figure 5d shows the aniline-modified PET film where a complete change in appearance from colorless to yellowish was observed. The color change is attributed to aniline being prone to oxidation. This happens because the NH2 group of aniline has a strong electron-donating effect (+R effect), and this causes the electron density on the benzene ring to increase since aniline is easily oxidized when exposed to air [63].

3.4. Qualitative and Quantitative Evaluation of PET Films’ Primary Amino Groups (Activation)

The quantification values of the -NH2 groups of the three precursors are listed in Table 1, as well as the results of the densities of the same films without etching. It is observed that films activated directly with ethylenediamine, N2/H2, or aniline plasma show lower densities than films pretreated with air plasma for 6 h (E6) and subsequently modified with ethylenediamine, N2/H2, or aniline, which is also corroborated by the low intensity of the color acquired by the film after quantification. All air plasma pre-modified films exhibit surface etching as observed in Section 3.3 above, increasing the surface area of the air plasma pre-modified films compared to non-air plasma pre-modified films, resulting in higher densities, and where an increase in the color intensity acquired by the film after quantification is observed, with the films that were etching and subsequently modified with ethylenediamine being the ones that presented the highest color intensity and were the ones that showed the highest densities of grafted amino groups with a maximum value of 3.98 ± 0.12 mg·mm−2. Mora Cortés et al. [64] carried out the surface insertion of amino groups from ethylene diamine in PET films with RF plasma at 200 W for 15 min and obtained a density of 4.10 ± 0.15 mg·mm−2. Both values are very close even though the maximum working power in this work was 50 W; therefore, more time was needed to perform the activation, in contrast to the 200 W power of the cited authors. The results obtained by this author and those of this research lead to the premise that by manipulating the reactor conditions, the procedure carried out can be made even more efficient. In addition, the results presented using together the two procedures: (a) modification with air plasma and (b) amination with three different precursors, are results that have not been published and even less so for the immobilization of an enzyme, which can eliminate phenols and their derivatives in contaminated water.
The chemical reaction of aminolysis involves substituting the acyl group R-CO, in which R can be a carbon, alkyl, or aryl substituent attached directly to the carbonyl group (-C=O). Removal of the hydroxyl group (-OH) from -RCOOH produces an acyl whose substitution is carried out on the carbonyl group of PET. This carbonyl carbon in PET acts as an electrophile. It is attacked by the nucleophilic amino group, resulting in the cleavage of an ester bond (-O=C-O) from the PET main chain and an amide covalent bond (-O=C-N) created between the amino groups. Part of this chemical reaction is presented in the scheme proposed by Noel et al. [35], which is shown in Figure 6. This section demonstrates that (a) the surface area is increased by eroding the waste PET film with air plasma, and (b) using any of the three precursors, a higher density of the amine grafted on the PET is obtained when previously modified with air plasma.
Compared to the three precursors, ethylenediamine presented a higher density of amino groups of 3.98 ± 0.10 mg·mm−2. This can be attributed to ethylenediamine being a small molecule with two amino groups (-NH2) attached to a hydrocarbon chain of (-CH2-CH2-). Compared to aniline, aniline is a molecule with only one amino group attached to an aromatic ring and is larger and bulkier. A higher amount of energy is required to separate the amine from the ring, which makes it difficult to release this functional group. As for the N2/H2 mixture, the PET films etching and subsequently modified with the N2/H2 mixture showed a higher amino group density of 1.79 ± 0.02 mg·mm−2, which was higher than the value obtained for aniline but lower than that for ethylenediamine. This behavior is because, with this gas mixture, amines are formed from different reactions in the plasma. These random reactions can generate primary, secondary, and tertiary amines [65]. The N2/H2 mixture molecules are more versatile because they are smaller than the larger aniline molecules. However, producing different types of amines, not just primary amines, turned out to be less effective than ethylenediamine but more effective than aniline. The etching PET films subsequently modified with aniline obtained a maximum density of 1.53 ± 0.1 mg·mm−2. The chemical interaction between PET and aniline can occur through the former’s C=O bonds and the latter’s amino group, respectively [66].

3.5. Contact Angle Hydrophilicity Test of PET Films

The contact angle allows us to determine changes in wetting, polarization, and degree of adhesion between a functional group deposited on a substrate. It also indicates whether a liquid can superficially wet a solid; that is, it gives information about the hydrophobicity of the solid. If the value of the angle formed between the liquid and the solid surface is less than 90°, the solid surface is hydrophilic, while for values greater than 90° it is a hydrophobic surface. Table 2 lists the average values of the contact angle of non-etching-non-activated E0 waste PET films (image a), E6 etching film after 6 h (image b); non-etching film activated with each of the three precursors: ethylenediamine E0-EtDA90 (image c), nitrogen/hydrogen mixture E0-N2/H290 (image e), and aniline E0-An90 (image g); film etching after 6 h and subsequently activated with each of the three precursors: ethylenediamine E6-EtDA90 (image d), nitrogen/hydrogen mixture E6-N2/H290 (image f) and aniline E6-An90 (image h). Regarding the etching and activated films, the lowest angle was that of the E6-EtDA90 film, with a value of 51.10°. This film produced a lower contact angle and a higher density of amino groups due to the joint impact of the air plasma and ethylenediamine plasma with respect to E0-EtDA90, which was not modified with air plasma. This is due to the joint effect of etching and amination that causes this behavior. However, when comparing the contact angle of E6-EtDA90 with respect to E6, an increase in the contact angle was observed with the incorporation of amino groups. This is explained by considering the study carried out by Casimiro et al. [38], who mention that the plasma can incorporate other polar species, such as oxygen, which promotes the obtaining of hydrophilic surfaces in greater proportion than the contact angle obtained by the incorporation of nitrogenous species. This means the amino groups are not as polar as the oxygen groups. It was observed that the modification carried out with the nitrogenous precursors in all cases caused an increase in the contact angle and a decrease in hydrophilicity with respect to E6, which was only modified with air plasma. The E6-An90 and E6-N2/H2 films presented contact angle values of 75.30° ± 0.10 and 60.60° ± 0.12, respectively, higher than that obtained by ethylenediamine of 51.10° ± 0.10, because the precursor structure also influences the results. In the case of films without etching and amination, higher contact angles and lower densities of amino groups were observed than the values calculated for the previously etched and aminated films. In general, it is observed that the increase in the hydrophilicity of the PET films (E0) is proportional to the increase in the exposure time in the plasma (E6) since a greater incorporation of functional groups occurs [58]. The decrease in the contact angle, according to the results observed in the three amination precursors, shows an inversely proportional relationship with the number of quantified amino groups; this means that the greater the number of amino groups on the surface of the substrates, the lower the contact angle and the greater the hydrophilicity. The same table also presents the average contact angle of the modified films not etching with precursors, where lower hydrophilicity is always observed compared to their counterparts modified with air plasma [67] and is attributed to the lower surface area available for the insertion of the amino groups.

3.6. Verification of the Chemical Environment in Activated PET Films Using ATR-FTIR

Figure 7 shows the ATR-FTIR spectra of the waste PET film without modification and the PET modified with ethylenediamine (E6-EtDA90). The bands corresponding to the different functional groups that chemically make up PET were identified in both cases. In the case of the vibrations of the terephthalate group, the stretching of the carbonyl of the ester that is attached to an aromatic ring is observed at 1715 cm−1, the stretching vibrations of the C=C aromatic double bond 1603 cm−1, and the stretching C-O bond at 1247 cm−1. These signals were compared with those reported by Ramírez-Hernández et al. [58]. The band that demonstrates the insertion of the amino groups on the surface of the E6-EtDA90 films is the band at 1545 cm−1 due to the symmetric deformation vibration of the -N-H bond [68]. There is an N-H stretching band, which should have been visible near 3433 cm−1; the O-H band likely overlapped this band due to the previous modification performed by the air plasma, so it could not be observed. However, in a previous study carried out by our work group, an XPS analysis was performed for sample E0 and the same sample E0 but aminated with ethylenediamine. With this technique, it was verified that the modification with plasma using ethylenediamine as a precursor effectively grafts amino groups on the PET surface, so there is already a precedent of the chemical behavior of these samples reported in reference [67].
In the ATR-FTIR spectra of the etching films 6 h and subsequently activated with the N2/H2 mixture (E6-N2/H290) and the etching film 6 h and activated with aniline (E6-An90), only the bands belonging to the PET matrix were found. This characterization technique did not see the signals corresponding to the N2/H2 gas mixture and aniline. This result is due to a lower insertion of amino groups with both precursors on the PET surface than the insertion of amino groups grafted with the ethylenediamine precursor. However, although this technique could not observe these bands, in Section 3.5, the effective insertion of amino groups was confirmed but with lower intensity in these last two precursors used.

3.7. SEM Analysis of Activated PET Films

Figure 8 shows the micrographs taken of the unmodified waste PET film (Figure 8a), the ethylene-diamine-activated etched PET film (Figure 8b), the N2/H2-activated etched PET film (Figure 8c), and the aniline-activated etched PET film (Figure 8d). Figure 8a shows a smooth and homogeneous surface compared to the other micrographs since it has no modification. In the E6-EtDA90 film (Figure 8b), larger and more pronounced protuberances are observed than in Figure 8c,d. This can be attributed to the higher number of chemical reactions occurring on the PET surface because ethylenediamine is a low molecular weight bifunctional molecule that has two amino groups in its structure, which can interact more quickly with the PET surface, forming a greater amount of imperfections compared to molecules with a single functional group, see indicated with yellow arrows. These results are consistent with those obtained in quantifying the amino groups, where the E6-EtDA90 film presented the highest density of amino groups (Table 1) and the lowest hydrophilicity (Table 2). In the case of E6-N2/H290 and E6-An90, protuberances were also observed, but they were much smaller and more distributed over the surface, not comparable with those of ethylenediamine. In E6-N2/H290, slightly larger and more abundant protuberances were observed, almost completely covering the PET surface compared to E6-An90. This phenomenon is due to the lower vapor pressure of this gas mixture, which is higher than that of aniline due to its larger molecular size [67,69]. This means that the gas mixture, being of low molecular weight, requires little vacuum pressure to transform from a gaseous state to a plasma, as the process is performed in a single step, and more molecules of this gas can pass into the plasma and react with the surface to generate imperfections. However, in the case of aniline, which has a higher molecular weight and is also in a liquid state, it must first pass from a liquid to a gaseous state and then transform into plasma. In this case, two steps are required to carry out the transformation to the plasma state. Higher reactor power or vapor pressure is needed to transform larger aniline molecules into plasma. In this study, the same vapor pressure and power were used in all experiments, resulting in fewer aniline molecules transforming to the plasma state, which caused fewer chemical reactions on the PET surface, thus presenting fewer bumps. The lower amount of chemical reactions on the PET surface subsequently also caused a lower amount of amino groups on the PET surface, Figure 1. This insertion of amino groups is because aniline is nucleophilic and participates in the formation of amide bonds with the polymer [63].

3.8. Quantification of the Immobilized Protein

The results of the immobilized protein on the films are shown in Table 3. All etching films, subsequently modified with any of the precursors, had better percentages of immobilized protein than the non-etching ones due to the increase in the surface area that promoted greater insertion of amine groups and a higher percentage of immobilized protein. The E6-EtDA90-G-EzL film presented the highest percentage of immobilized protein, with 97.30% and a desorption of 3.21 mg·mL−1. This indicates that the film with the highest amine density had the highest immobilized protein. Glutaraldehyde activating promotes stable binding by forming covalent bonds between the laccase enzyme and the amino groups generated by the plasma on the surface of the PET, fixing it to its surface with a stable union [70]. The results presented in Table 3 show that the use of glutaraldehyde promotes the fixation of the enzyme on the PET surface; without the use of glutaraldehyde, 0% of the immobilized enzyme was obtained. The decrease in the percentages of immobilized protein in E6-N2/H290-G-EzL of 94.30%, and E6-An90-G-EzL, with 93.10%, with respect to E6-EtDA90-G-EzL film modified with air and ethylenediamine, is related to the lower number of amino groups grafted on the PET surface observed in Figure 8 and Table 1. To E6-N2/H290-G-EzL and E6-An90-G-EzL desorption of 4.11 mg·mL−1 and 4.48 mg·mL−1, respectively, was observed. Despite having a considerable difference in -NH2 density, protein immobilization values close to each other were observed in the three films. This can be explained by steric problems between the enzyme and its binding to available amines. Rodrigues et al. [71] proposed that most of the enzyme-support bonds require “multipoint” binding, i.e., functional groups (as in this case, amines) in which the “multimeric” enzyme is anchored in all its subunits (molecules) with the support, taking into account the volume of these subunits, since, a single molecule can bind to only one point and/or to several points at a time, preventing other molecules from occupying such points. Therefore, similar amounts of immobilized enzyme can be obtained by having more points on one support or fewer points on another support.

3.9. Quantification of Enzyme Activity

The molar extinction coefficient [72] was considered (ε450 = 2211 M−1 cm−1) to calculate the initial enzymatic activity. This activity expresses the unit of activity (U) per milliliter (mL), where one unit of activity is defined as the necessary amount of enzyme to oxidize one µmol of catechol per minute under the described conditions. Table 4 shows the unit of enzymatic activity for each of the films that obtained the best percentages of enzyme immobilization, of which the E6-EtDA90-G-EzL film presented the highest initial enzyme activity of 0.36 U mL−1 (86.11%) with 97.30% immobilized protein. Different supports for laccase immobilization and catechol oxidation have been reported in the literature. Angelova et al. [16] immobilized laccase using a “fed-batch” process and obtained 79.20% catechol oxidation; Karami et al. [17] used gold-coated magnetic nanoparticles as a support, with which they reported 98% catechol oxidation; Liu et al. [18] used carbon nanoparticles doped with fluorine and nitrogen as immobilization support, with which they obtained 99.50% catechol oxidation and Li et al. [19] employed organometallic and zeolite scaffolds coated with bacterial cellulose and multi-walled carboxylated carbon nanotubes that reported 95% catechol oxidation. Although these results show high percentages of catechol oxidation with immobilized laccase, it is relevant to mention that the supports used in these works were obtained through processes that require a high investment in production and/or surface modification, such as nanoparticles and metals, organic structures and their coatings are based on expensive materials such as gold and carbon nanotubes. Some of these materials are difficult to recover after use, while PET can be recovered. On the other hand, the proposed technology in this research could significantly reduce costs and energy levels used to obtain products to eliminate contaminants in water with added value [73].

4. Conclusions

The results support that modified films obtained from waste PET bottles can immobilize the laccase enzyme and be applied in a catechol biodegradation test under the conditions used in this work.
It is concluded that the previous plasma etching on the PET surface increased the surface area of the PET, which led to a greater number of active sites, so a greater number of amino groups could be grafted onto the PET.
This research found that using etching surfaces also increases the percentage of immobilized enzymes due to the increase in the surface area available for the enzyme to be immobilized.
The results show that of the three precursors used, ethylenediamine obtained the highest number of amino groups on the surface, the highest amount of immobilized enzyme, and the highest percentage of catechol oxidation, followed by the N2/H2 mixture and aniline.
Other studies reported in the literature with other methods show that laccase oxidation activity efficiencies of 79.20%, 95%, 98%, and 99.5%, while the value obtained for this technique was found to be 86.11%. This proposal comes close to good efficiency with the advantage that it can be improved by changing the reactor conditions and using materials with more porous surfaces. In general, it reveals that plasma has the potential to be used as an alternative tool because it is environmentally benign and versatile, and its technology is relatively inexpensive.
On the other hand, the objective of using PET to obtain materials of great technological utility at low cost that can contribute to reducing the environmental impact of PET production was met.
These results are expected to encourage other researchers to use plasma technology as an alternative to improve water treatment processes and make them more economical. However, as future work, it is desirable to make this process more efficient by modifying the reactor conditions and using a porous material with a large surface area.

Author Contributions

R.O.-G.: investigation, experimentation, and writing; R.I.N.-C.: resources, investigation, supervising, writing, review, and validation of the final version of the manuscript; A.I.: supervising, review, and investigation; L.F.M.-C.: investigation, writing, and review; Y.K.R.-A.: supervising, review, investigation; G.S.-A.: characterization of experimentation; C.L.-M.: characterization of experimentation; M.P.L.-E.: is involved in methodology and investigation, review, and C.G.C.-G.: conceptualization, review, and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Fondo para la Investigación Científica y Tecnológica (FONCYT), with project code COAH-2021-C15-C111.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Acknowledgments

Thanks to the “Maestrìa en Ciencia y Tecnologìa Quìmica” program of the Facultad de Ciencias Quìmicas (Universidad Autònoma de Coahuila) and also thank you to the “Fondo para la Investigación Científica y Tecnológica” (FONCYT) for providing funding for the approved project COAH-2021-C15-C111. Thanks to the National Council of Humanities, Sciences and Technologies (CONAHCYT) for providing the doctoral scholarship number 1000741.

Conflicts of Interest

The authors declare no conflicts of interest.

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Coatings 15 00421 g001
Figure 1. Visual representation of plasma modification; stages (1) air plasma generation to etching process and N2/H2 mixture plasma generation; stages (2) ethylenediamine and aniline plasma generation.
Figure 1. Visual representation of plasma modification; stages (1) air plasma generation to etching process and N2/H2 mixture plasma generation; stages (2) ethylenediamine and aniline plasma generation.
Coatings 15 00421 g001
Coatings 15 00421 g002aCoatings 15 00421 g002b
Figure 2. Photographs of PET film: (a) not etching (E0); (b) etching with air plasma for 6 h (E6); Micrographs of PET film: (a*) not etching (E0); (b*) etching with air plasma for 6 h (E6).
Figure 2. Photographs of PET film: (a) not etching (E0); (b) etching with air plasma for 6 h (E6); Micrographs of PET film: (a*) not etching (E0); (b*) etching with air plasma for 6 h (E6).
Coatings 15 00421 g002aCoatings 15 00421 g002b
Coatings 15 00421 g003
Figure 3. Etching Process.
Figure 3. Etching Process.
Coatings 15 00421 g003
Coatings 15 00421 g004
Figure 4. ATR-FTIR spectra PET films: without air plasma (E0); etching with air plasma for 6 h (E6).
Figure 4. ATR-FTIR spectra PET films: without air plasma (E0); etching with air plasma for 6 h (E6).
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Coatings 15 00421 g005
Figure 5. PET films: (a) not etching (E0). Etching PET films with air plasma by 6 h (E6) and subsequently activated with: (b) ethylenediamine (E6-EtDA90); (c) N2/H2 mixture (E6-N2/H290); (d) aniline (E6-An90).
Figure 5. PET films: (a) not etching (E0). Etching PET films with air plasma by 6 h (E6) and subsequently activated with: (b) ethylenediamine (E6-EtDA90); (c) N2/H2 mixture (E6-N2/H290); (d) aniline (E6-An90).
Coatings 15 00421 g005
Coatings 15 00421 g006
Figure 6. Reaction scheme between PET and ethylenediamine. Adapted from reference [41]. Own elaboration.
Figure 6. Reaction scheme between PET and ethylenediamine. Adapted from reference [41]. Own elaboration.
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Coatings 15 00421 g007
Figure 7. ATR-FTIR spectra PET films: etching 6 h (E6); PET films etching 6 h and activated with ethylenediamine plasma 90 min (E6-EtDA90).
Figure 7. ATR-FTIR spectra PET films: etching 6 h (E6); PET films etching 6 h and activated with ethylenediamine plasma 90 min (E6-EtDA90).
Coatings 15 00421 g007
Coatings 15 00421 g008
Figure 8. SEM micrographs of PET films: (a) not etching (E0). Etching PET films with air plasma for 6 h (E6) and subsequently activated with: (b) ethylenediamine (E6-EtDA90); (c) N2/H2 mixture (E6-N2/H290); (d) aniline (E6-An90).
Figure 8. SEM micrographs of PET films: (a) not etching (E0). Etching PET films with air plasma for 6 h (E6) and subsequently activated with: (b) ethylenediamine (E6-EtDA90); (c) N2/H2 mixture (E6-N2/H290); (d) aniline (E6-An90).
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Table 1. Quantification of amino groups by the orange II dye method.
Table 1. Quantification of amino groups by the orange II dye method.
RF Plasma-Activated Films
EtDA90N2/H290An90
Density-NH2 (mg·mm−2)Density-NH2 (mg·mm−2)Density-NH2 (mg·mm−2)
No etchingEtchingNo etchingEtchingNo etchingEtching
1.02 ± 0.033.98 ± 0.100.86 ± 0.041.79 ± 0.020.33 ± 0.011.53 ± 0.10
Coatings 15 00421 i009Coatings 15 00421 i010Coatings 15 00421 i011Coatings 15 00421 i012Coatings 15 00421 i013Coatings 15 00421 i014
Table 2. The density of amino groups correlated with the measured contact angles.
Table 2. The density of amino groups correlated with the measured contact angles.
FilmDensity-NH2Contact Angle AverageContact Angle Illustration
E0-79.70° ± 0.11Coatings 15 00421 i001
image a
E6-50.20° ± 0.10Coatings 15 00421 i002
image b
E0-EtDA901.02 ± 0.0966.40° ± 0.10Coatings 15 00421 i003
image c
E6-EtDA903.98 ± 0.1051.10° ± 0.10Coatings 15 00421 i004
image d
E0-N2/H20.86 ± 0.0874.20° ± 0.11Coatings 15 00421 i005
image e
E6-N2/H21.79 ± 0.1060.60° ± 0.12Coatings 15 00421 i006
image f
E0-An900.33 ± 0.0173.90° ± 0.12Coatings 15 00421 i007
image g
E6-An901.53 ± 0.0875.30° ± 0.10Coatings 15 00421 i008
image h
Table 3. Quantification of the immobilized protein using the Bradford method.
Table 3. Quantification of the immobilized protein using the Bradford method.
FilmsImmobilized Protein
EtDA90-G-EzL22.46% ± 0.12%
N2/H290-G-EzL19.73% ± 0.10%
An90-G-EzL18.55% ± 0.11%
E6-EtDA90-EzL0.00%
E6-N2/H290-EzL0.00%
E6-An90-EzL0.00%
E6-EtDA90-G-EzL97.30% ± 0.10%
E6-N2/H290-G-EzL94.30% ± 0.12%
E6-An90-G-EzL93.10% ± 0.10%
Table 4. Enzymatic activity units using catechol oxidation test.
Table 4. Enzymatic activity units using catechol oxidation test.
FilmsImmobilized ProteinU (µmol·min−1) mL−1U %
E6-EtDA90-G-EzL97.30% ± 0.10%0.36 ± 0.0886.11% ± 0.09%
E6-N2/H290-G-EzL94.30% ± 0.12%0.27 ± 0.0777.03% ± 0.08%
E6-An90-G-EzL93.10% ± 0.10%0.21 ± 0.0771.35% ± 0.09%
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MDPI and ACS Style

Orsua-Gaona, R.; Narro-Céspedes, R.I.; Ilina, A.; Mora-Cortés, L.F.; Reyes-Acosta, Y.K.; Soria-Arguello, G.; Luevano-Martínez, C.; Luévanos-Escareño, M.P.; Cuellar-Gaona, C.G. Etching and Precursor Effects on Plasma-Modified Waste Polyethylene Terephthalate (PET) to Laccase Immobilization Applied in Catechol Biodegradation for Water Treatment. Coatings 2025, 15, 421. https://doi.org/10.3390/coatings15040421

AMA Style

Orsua-Gaona R, Narro-Céspedes RI, Ilina A, Mora-Cortés LF, Reyes-Acosta YK, Soria-Arguello G, Luevano-Martínez C, Luévanos-Escareño MP, Cuellar-Gaona CG. Etching and Precursor Effects on Plasma-Modified Waste Polyethylene Terephthalate (PET) to Laccase Immobilization Applied in Catechol Biodegradation for Water Treatment. Coatings. 2025; 15(4):421. https://doi.org/10.3390/coatings15040421

Chicago/Turabian Style

Orsua-Gaona, Reyna, Rosa Idalia Narro-Céspedes, Anna Ilina, Luis Fernando Mora-Cortés, Yadira Karina Reyes-Acosta, Gustavo Soria-Arguello, Cynthia Luevano-Martínez, Miriam Paulina Luévanos-Escareño, and Claudia Gabriela Cuellar-Gaona. 2025. "Etching and Precursor Effects on Plasma-Modified Waste Polyethylene Terephthalate (PET) to Laccase Immobilization Applied in Catechol Biodegradation for Water Treatment" Coatings 15, no. 4: 421. https://doi.org/10.3390/coatings15040421

APA Style

Orsua-Gaona, R., Narro-Céspedes, R. I., Ilina, A., Mora-Cortés, L. F., Reyes-Acosta, Y. K., Soria-Arguello, G., Luevano-Martínez, C., Luévanos-Escareño, M. P., & Cuellar-Gaona, C. G. (2025). Etching and Precursor Effects on Plasma-Modified Waste Polyethylene Terephthalate (PET) to Laccase Immobilization Applied in Catechol Biodegradation for Water Treatment. Coatings, 15(4), 421. https://doi.org/10.3390/coatings15040421

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