*Article* **Enzymatic Functionalization of Wood as an Antifouling Strategy against the Marine Bacterium** *Cobetia marina*

**Daniel Filgueira 1,2, Cristian Bolaño <sup>1</sup> , Susana Gouveia <sup>1</sup> and Diego Moldes 1,3,\***


**Abstract:** The protection of wood in marine environments is a major challenge due to the high sensitivity of wood to both water and marine microorganisms. Besides, the environmental regulations are pushing the industry to develop novel effective and environmentally friendly treatments to protect wood in marine environments. The present study focused on the development of a new green methodology based on the laccase-assisted grafting of lauryl gallate (LG) onto wood to improve its marine antifouling properties. Initially, the enzymatic treatment conditions (laccase dose, time of reaction, LG concentration) and the effect of the wood specie (beech, pine, and eucalyptus) were assessed by water contact angle (WCA) measurements. The surface properties of the enzymatically modified wood veneers were assessed by X-ray photoelectron spectroscopy (XPS), Fourier transforminfrared spectroscopy (FTIR). Antifouling properties of the functionalized wood veneers against marine bacterium *Cobetia marina* were studied by scanning electron microscopy (SEM) and protein measurements. XPS and FTIR analysis suggested the stable grafting of LG onto the surface of wood veneers after laccase-assisted treatment. WCA measurements showed that the hydrophobicity of the wood veneers significantly increased after the enzymatic treatment. Protein measurements and SEM pictures showed that enzymatically-hydrophobized wood veneers modified the pattern of bacterial attachment and remarkably reduced the bacterium colonization. Thus, the results observed in the present study confirmed the potential efficiency of laccase-assisted treatments to improve the marine antifouling properties of wood.

**Keywords:** laccase; lauryl gallate; wood; *Cobetia marina*; antifouling

## **1. Introduction**

Wood is a renewable, cheap, and biodegradable bioresource with interesting mechanical properties which make it a competitive material in construction applications. In marine environments, wood is used as a raw material for the construction of waterfront structures, e.g., groynes, jetties, dolphins [1] and classic boats. Besides, wood is used in marine platforms for the aquaculture of mollusks and crustaceous species. However, wood is highly hydrophilic and very sensitive to the attack of biological organisms [2]. These characteristics are a major challenge in marine environments, due to the regular wet conditions and the great number of living organisms in seawater. Under these conditions, all the immersed surfaces are rapidly colonized by different microorganisms leading to the formation of a biofilm, but also larger organisms could colonize the substrata after the biofilm formation. This phenomenon is commonly known as marine biofouling [3].

The biofilm formation starts with the adsorption of free organic material onto the surface of the substrata [3]. Then, bacteria and other microorganisms (e.g., diatoms, protozoa) are the first living organisms in colonizing the substrata [4]. Bacteria have the

**Citation:** Filgueira, D.; Bolaño, C.; Gouveia, S.; Moldes, D. Enzymatic Functionalization of Wood as an Antifouling Strategy against the Marine Bacterium *Cobetia marina*. *Polymers* **2021**, *13*, 3795. https://doi.org/10.3390/ polym13213795

Academic Editors: L'uboš Krišt'ák, Roman Réh and Ivan Kubovský

Received: 5 October 2021 Accepted: 29 October 2021 Published: 2 November 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

capacity to secrete extracellular polymeric substances (EPS) which are mainly composed of carbohydrates and proteins [5], forming a 3D structure of bacterial aggregates which facilitates their adhesion onto the substrata [6]. Such biofilm may change the physicochemical properties of the substrata, influencing the future adhesion of larger fouling organisms (e.g., barnacles, mollusks) [7]. A wide variety of paints and coatings have been used to limit the marine biofouling. Most of them involved the use of heavy metals (e.g., copper, zinc) or organic compounds that are toxic for fouling organisms, but also for non-target organisms such as algae, mollusks, crustaceans and fishes and they could be introduced in the food chain [8]. One of the most extended antifouling chemicals used in paints (tributyltin) was banned years ago and other hazardous treatments should be avoided to protect the marine ecosystems. Environmental factors and physicochemical properties of surfaces are the two main parameters which may favor or hinder the biofilm formation [9]. Since environmental factors are not controllable, the modification of the physicochemical properties of the substrata may disturb the pattern of bacterial colonization, minimizing the subsequent adhesion of marine fouling organisms. Based on such a mechanism, different non-biocide antifouling treatments have been proposed in the last decade [7,10]; most of them aim to interfere with the adhesion of marine microorganisms adjusting the surface free energy of the substrata between a specific range [11], which is usually controlled with hydrophobic compounds e.g., fluorinated polymers or silicones [12].

Another important issue regarding the protection of wood in marine environments is the leaching of the antifouling coatings and paints, since this release results in water pollution but also in the requirement of a new antifouling treatment. Covalent grafting of antifouling compounds onto surfaces could significantly reduce or avoid their leaching, providing stable protection against fouling organisms. Regarding wood as a material, a sustainable alternative to conventional chemical and thermal treatments is the use of laccase, which enable the stable grafting of several chemical compounds. Such lignolytic enzyme can oxidize phenolic and amine groups of a target compound [13,14], leading to the formation of radicals that may link to the aromatic structure of woody lignin by radical polymerization. By this mechanism, laccase could be used for functionalization of lignocellulosic materials or their components, mainly lignin. It is worth noting that laccase-assisted reactions are performed in mild conditions, minimizing the energy requirements. Thus, the physicochemical properties of wood can be tailored by means of an environmentally-friendly pathway. Enzymatic hydrophobization of wood veneers was successfully performed with different chemical compounds such as alkylamines [15] and fluorophenols [16]. Alkyl gallates are laccase-specific substrates with an aliphatic chain at the *para*-position that could provide stable hydrophobic properties if grafted onto wood. In fact, these compounds have been enzymatically grafted on different lignocellulosic substrates for such purpose [17,18], but also for providing antibacterial properties [19] since the aliphatic tail also presents this characteristic.

Therefore, the reaction conditions namely lauryl gallate (LG) concentration, laccase dose and time of reaction for the enzymatic hydrophobization of wood veneers were assessed in the present study. Water contact angle (WCA) measurements were carried out for the assessment of the wettability of the enzymatically-hydrophobized wood veneers. In addition, the surface of the hydrophobized wood samples was characterized by X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). Finally, the potential antifouling properties of the enzymatically-hydrophobized wood veneers were studied against the marine bacterium *Cobetia marina* by means of protein measurements and scanning electron microscopy (SEM), in order to assess the capability of hydrophobic functionalization of wood as a new antifouling strategy for such material.

#### **2. Materials and Methods**

#### *2.1. Materials*

Beech (*Fagus sylvatica*), eucalyptus (*Eucalyptus globulus*) and pine (*Pinus pinaster*) wood veneers were supplied by Foresa (Caldas de Reis, Spain). Beech and eucalyptus veneers were vaporized before supplying. These wood species were selected because of their importance regarding production and commercial interest in Galicia (NW of Spain).

Commercial laccase from *Myceliophthora thermophila* (NS51003) was provided by Novozymes (Bagsværd, Denmark). The activity of the enzyme was calculated by the 2,20 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) oxidation assay. One unit of activity is defined as the amount of enzyme that oxidizes 1 µmol of ABTS per minute at 25 ◦C and pH 7 in a 0.1 M phosphate buffer.

*Cobetia marina* (CECT4278) bacterium was purchased from the Colección Española de Cultivos Tipo—CECT (Valencia, Spain).

Bradford reactive was provided by Bio-Rad (Hercules, CA, USA).

All the other chemical reagents were acquired from Sigma-Aldrich (St. Louis, MO, USA) at reagent grade and used without any kind of purification.

#### *2.2. Enzymatic Hydrophobization of Wood Veneers*

Wood veneers from beech, pine or eucalyptus were cut in square plugs (50 × 50 mm), washed with distilled water at 50 ◦C for 30 min and oven-dried at 50 ◦C for 4 h. Each wood sample was then immersed in 58 mL of a phosphate buffer (pH 7, 0.1 M): acetone solution (60:40, *v*/*v*) at 50 ◦C. An experimental design was performed for assessing the treatment conditions which could provide the highest hydrophobic properties to wood veneers. Three levels of each of the selected parameters were chosen: laccase dose (3.36, 5.00 and 6.72 U/cm<sup>2</sup> of wood), LG concentration (2.5, 5 and 10 mM) and treatment time (1, 2 and 4 h). All the possible combinations of such parameters were assayed (Table S1). The enzymatically treated samples were then dried at a room temperature (18–20 ◦C) for 12 h and washed with an aqueous solution of acetone (50:50, *v*/*v*) at 50 ◦C for 1 h. Finally, the samples were washed with distilled water three times and oven-dried at 50 ◦C for 4 h. Control treatments without adding laccase and/or LG were performed in parallel. All the experiments were performed in triplicate.

#### *2.3. Water Contact Angle Measurements*

The hydrophobization of wood veneers was assessed by water contact angle (WCA) measurements. A goniometer MobileDrop GH11 (Krüss GmbH, Hamburg, Germany) was used to measure the contact angle of one drop of distilled water on the surface of wood veneers. DSA2 software (Krüss GmbH) was utilized to analyze the drop shape. WCA measurements were performed after water drop deposition with intervals of 30 s for 5 min. A minimum of two points per each side of the wood veneers were measured.

#### *2.4. X-ray Photoelectron Spectroscopy (XPS)*

The surface of wood veneers was analyzed by X-ray photoelectron spectroscopy (XPS). The analyses were performed in a Thermo Scientific K-Alpha device using a monochromized Al-K-α-X-ray source (1486.6 eV). As the samples were not conductive, an electron flood gun was used to minimize surface changing. A constant analyzed energy mode (CAE) with 100 eV pass energy for survey spectra was used to perform the measurements. Neutralization of the surface charge was implemented with a low-energy flood gun (electrons in the range 0–14 eV) and a low-energy Ar ions gun. Binding energy was adjusted using C1 (BE 284.6 eV). The elemental composition of the surface of wood samples was defined plotting on the standard Scofield photoemission cross-sections. The O/C ratio was calculated by means of the survey spectra collected (from 0 to 1350 eV) which showed the components C1s and O1s.

#### *2.5. Fourier Transform Infrared Spectroscopy (FTIR)*

The surface of wood pieces was also studied with a 4100 Fourier transform infrared spectrometer (Jasco), equipped with an attenuated total reflectance (ATR) device. A total of 32 scans were picked between 600 and 4000 cm−<sup>1</sup> with a resolution of 4 cm−<sup>1</sup> . The spectra were scale normalized and analyzed with OMNIC Suite software v 7.3 (Thermo Scientific, Waltham, MA, USA).

#### *2.6. Stock Bacteria and Culture Conditions*

Marine broth (5 g of bacteriological peptone, 3 g of yeast extract, 750 mL filtered marine water, 250 mL distilled water and pH adjusted to 7.4) was used as culture media in all assays.

*Cobetia marina* CECT4278 was provided lyophilized. It was resuspended in marine broth, which was then used for the inoculation of two flasks with 50 mL of fresh marine broth. Such culture media was incubated in an orbital shaker at 100 rpm and 27 ◦C for 24 h. Optical density (OD) was measured with Unicam Helios Beta spectrophotometer (Thermo Fisher Scientific) at 600 nm. When the value of OD was 1.0, 1.5 mL of bacterial culture medium was used to inoculate fresh culture media (3% of inoculum, *v*/*v*), in duplicate. After 24 h of incubation in an orbital shaker at 100 rpm and 27 ◦C, the culture medium was centrifuged (10,000 rpm) and stored as stock culture in aliquots of 3 mL with glycerol (30%) at −20 ◦C.

Working cultures were prepared in the same way, using stock cultures as inoculum but discarding the supernatant, suspending the pellet with 1.5 mL of culture media; then 750 µL of this suspension was used for inoculating 50 mL of culture media (3% of inoculum, *v*/*v*).

#### *2.7. Biofilm Assay*

Pine wood samples were hydrophobized according to the results obtained from the study of the treatment conditions. After the enzymatic treatment, the veneers were cut in square plugs (25 × 25 mm), dried at 50 ◦C for 24 h. Finally, the pine veneers were autoclaved at 121 ◦C for 20 min.

For the biofilm assay, each hydrophobized pine veneer was submerged in working cultures (50 mL of marine broth inoculated with *C. marina* 3% (*v*/*v*)). The flask was incubated in an agitator with orbital shaker at 100 rpm and 27 ◦C for 5 days. The colonized pine veneers were gently washed with distilled water to remove unattached bacteria from their surface. Control tests using untreated pine veneers and, also control test without *C. marina* were performed. Six replicas of biofilm colonization were carried out for each experiment.

#### *2.8. Bacterial Surface Hydrophobicity*

Surface hydrophobicity of *C. marina* was measured by means of the microbial adhesion to hydrocarbons (MATH) method. The test was performed according to Warne Zoueki et al. [20], who adapted the method previously proposed by Rosenberg et al. [21]. Firstly, 50 mL of marine broth were inoculated (3% (*v*/*v*) *C. marina*) and incubated for 24 h. Such culture was then centrifuged (8000 rpm) at 4 ◦C for 20 min into a Falcon tube. The supernatant was discarded, and the recovered biomass was suspended with KCl solution (150 mM, pH 7). Centrifugation and supernatant removal steps were performed twice. Then, the biomass was suspended with KCl solution (150 mM, pH 7) and diluted with the same solution in order to obtain an absorbance of 1.0 at 600 nm (ABS<sup>i</sup> ). After that, in our experiment, 5 mL of the diluted bacterial solution were mixed with 300 µL of dodecane in an acid washed Pyrex glass tube. Subsequently, the mixture was stirred for 10 min at 27 ◦C and left for 15 min to separate the aqueous phase from the organic phase. The aqueous phase was recovered, and its absorbance was measured at 600 nm (ABS<sup>f</sup> ). The partition coefficient (P) was calculated as follows:

$$\mathbf{P} = 1 - \text{(ABS}\_{\mathbf{f}} / \text{ABS}\_{\mathbf{i}}) \tag{1}$$

#### *2.9. Protein Measurement*

Each colonized pine veneer was immersed in 15 mL of distilled water in a 50 mL centrifuge tube. The tube content was then sonicated with a HD 2070 Sonoplus sonicator (Bandelin GmbH, Berlin, Germany) for 10 min (power 65%, cycle 70%) divided in two equal intervals of 5 min, with a pause of 2 min. After sonication, the wood samples were removed, and the aqueous solution was centrifuged at 7500 rpm for 40 min. Finally, the protein amount of the supernatant was measured using the Bradford's method, with slight modifications: 800 µL of the supernatant were mixed with 200 µL of Bradford's concentrated reagent and stirred with a vortex device; the mixture was left for 20 min and its absorbance was measured at 595 nm. A standard curve (5–25 mg protein/L) for protein quantification was performed using bovine serum albumin. Protein measurements of bacteria biomass on the wood veneers were performed after one, three and five days of incubation of *C. marina*.

#### *2.10. SEM Analysis*

Colonized pine veneers were introduced into plastic tubes with 2% of glutaraldehyde in cacodylate buffer (0.1 M, pH 7.4) and left at 4 ◦C for 2 h. Part of the solution was discarded avoiding the contact of the samples with atmospheric air and sodium phosphate buffer was added and the samples left for 30 min. Such a step was performed twice. Dehydration was performed with graded ethanol series, graded ethanol:amyl acetate substitution series and CO<sup>2</sup> critical point drying (73 atm, 31.3 ◦C). Then, the samples were coated with a layer of gold (thickness: 10–20 nm) (K550X Sputter Coater, Emitech, Ashford, UK). A Philips XL30 (SEMTech Solutions, Billerica, MA, USA) scanning electron microscope (SEM) was used to perform the SEM analysis. The applied acceleration voltage was 12kW and the magnifications were 100×, 200×, 500×, 1000×, 1500× and 2500×.

#### **3. Results and Discussion**

#### *3.1. Enzymatic Hydrophobization*

Nowadays, acetylation is the only processes available at industrial scale for the manufacturing of hydrophobic wood. However, acetylation is a high-cost process with a relatively high environmental impact. Hence, the wood industry needs to develop new sustainable routes for the manufacturing of hydrophobic wood. In this sense, laccase-assisted hydrophobization is a promising pathway to achieve an efficient hydrophobization of wood with a low environmental impact but also a competitive production cost.

Several parameters of laccase-mediated hydrophobization of beech veneers, namely laccase dose, LG concentration ([LG]) and time of reaction, were assessed to optimize the enzymatic hydrophobization process on beech veneers. The hydrophobicity of the treated beech veneers is showed as a function of the water contact angle (WCA).

It was expected that long reaction time combined with high [LG] improved the hydrophobicity of the beech veneers. Nevertheless, long reaction times (higher than 3 h) did not improve the surface hydrophobicity of the wood veneers. In fact, the time of reaction necessary to achieve the highest WCA on the surface of beech veneers was between 2–3 h for the different [LG] and laccase doses studied (Figure 1). It is worth to mention that besides the grafting of LG onto the surface of beech veneers, self-polymerization of LG monomers could be expected leading to the formation of oligomers [22]. In fact, several authors have already reported the laccase-catalyzed polymerization of phenolic compounds such as epigallocatechin [23], lignin model compounds [24], or condensed tannins [25]. Hence, stable LG adsorption without correct orientation and/or grafting/adsorption of LG oligomers without proper orientation after the grafting of LG monomers could explain the lower hydrophobicity obtained after 3 h of treatment. These results allow us to set 2 h as optimum time of treatment to achieve the highest enzymatic hydrophobization of beech veneers. The [LG] that yielded the highest WCA was in the range of 5–6 mM. Moreover, [LG] higher than 6 mM reduced the hydrophobicity of the wood samples. It is likely that a [LG] higher than 5 mM favored the grafting of LG oligomers which apparently did not provide high a hydrophobization of the wood veneers. Therefore, the optimum [LG] was set at 5 mM.

mum [LG] was set at 5 mM.

of beech veneers. The [LG] that yielded the highest WCA was in the range of 5–6 mM. Moreover, [LG] higher than 6 mM reduced the hydrophobicity of the wood samples. It is likely that a [LG] higher than 5 mM favored the grafting of LG oligomers which apparently did not provide high a hydrophobization of the wood veneers. Therefore, the opti-

**Figure 1.** Water contact angle values after one second of drop deposition of the hydrophobized beech veneers as a function of time of reaction and lauryl gallate concentration. Laccase dose: 3.36 U/cm2 wood (**A**); 5.00 U/cm2 wood (**B**); 6.72 U/cm2 wood (**C**). **Figure 1.** Water contact angle values after one second of drop deposition of the hydrophobized beech veneers as a function of time of reaction and lauryl gallate concentration. Laccase dose: 3.36 U/cm<sup>2</sup> wood (**A**); 5.00 U/cm<sup>2</sup> wood (**B**); 6.72 U/cm<sup>2</sup> wood (**C**).

Regarding the laccase dose, the results observed suggest that the higher the laccase dose the higher the WCA of the enzymatically treated beech veneers. The oxidation of the phenolic moieties of lignin present in the beech veneers enables the stable grafting of LG. Hence, the higher the laccase dose the higher the lignin oxidation and, therefore, a higher grafting of LG could be achieved. A medium laccase dose (5 U/cm2 of wood) was set as the optimum value considering the small differences in the WCA in the tested range. Thus, the experimental design permitted to identify the conditions to improve the hydrophobicity of treated beech veneers while minimizing the requirements of energy and both chemical and enzymatic reagents. Regarding the laccase dose, the results observed suggest that the higher the laccase dose the higher the WCA of the enzymatically treated beech veneers. The oxidation of the phenolic moieties of lignin present in the beech veneers enables the stable grafting of LG. Hence, the higher the laccase dose the higher the lignin oxidation and, therefore, a higher grafting of LG could be achieved. A medium laccase dose (5 U/cm<sup>2</sup> of wood) was set as the optimum value considering the small differences in the WCA in the tested range. Thus, the experimental design permitted to identify the conditions to improve the hydrophobicity of treated beech veneers while minimizing the requirements of energy and both chemical and enzymatic reagents.

The WCA of the beech veneers treated with laccase and LG was compared with those samples treated only with laccase or LG. As noted in Figure 2, veneers treated with both laccase and LG showed a stable WCA and always higher than 125°, after 5 min of water drop deposition. In addition, samples treated only with laccase showed a remarkable hydrophobicity, but much lower than those samples treated with both laccase and LG. Probably, laccase oxidized the hydroxyl groups of lignin's phenolic structures [26], leading to the formation of oxidized functionalities which, apparently, increased the hydrophobicity of lignin. Samples treated with LG alone showed a WCA similar to the observed on the untreated beech veneers. Thus, the activity of laccase was necessary to achieve a stable grafting of LG onto wood veneers surface. The WCA of the beech veneers treated with laccase and LG was compared with those samples treated only with laccase or LG. As noted in Figure 2, veneers treated with both laccase and LG showed a stable WCA and always higher than 125◦ , after 5 min of water drop deposition. In addition, samples treated only with laccase showed a remarkable hydrophobicity, but much lower than those samples treated with both laccase and LG. Probably, laccase oxidized the hydroxyl groups of lignin's phenolic structures [26], leading to the formation of oxidized functionalities which, apparently, increased the hydrophobicity of lignin. Samples treated with LG alone showed a WCA similar to the observed on the untreated beech veneers. Thus, the activity of laccase was necessary to achieve a stable grafting of LG onto wood veneers surface.

Regarding the different wood species tested (beech, pine, and eucalyptus), enzymatically treated beech veneers showed a WCA slightly higher than pine and a much higher hydrophobicity than eucalyptus veneers (Figure 3). Nonetheless, the highest change in hydrophobicity was achieved in the enzymatically hydrophobized pine veneers, since their WCA, after 5 min of drop deposition, was about 120◦ whereas untreated pine veneers had already absorbed the water droplet. Enzymatically hydrophobized beech veneers showed a WCA around 70% higher than untreated samples and such gap was much lower (20%) for eucalyptus wood veneers. It is worth mentioning that LG was expected to be enzymatically

grafted on lignin moieties and both eucalyptus and beech are hardwood species with a similar lignin content (≈25%). Lignin biopolymer is composed of phenylpropane units (C6–C3) which differ one each other in their methoxy substitutions on the aromatic ring, e.g., guaiacyl (G), syringyl (S) and *p*-hydroxyphenyl (H). The S/G ratio in eucalyptus wood is proximately to 6.25 which is much higher than the S/G ratio in beech wood, 0.71 [27,28]. In addition, the lignin of pine has a clear predomination of G units [29,30]. Thus, these results obtained with the WCA measurements suggest that the laccase-assisted grafting of LG was more efficient in wood species in which there is a relative low amount of S units. G units present higher tendency to addition reaction in comparison with S units, due to the lower content of methoxy groups in *ortho*-position. Otherwise, the radicals produced by laccase in S units should be more stable than those produced in G units, since S units have two electro-donating groups (methoxy) in *ortho*-position instead one methoxy group which possess G units [31]. This extra methoxy group of S units seems to have a remarkable effect in the laccase-mediated polymerization of technical lignins [32]. However, our results suggest that the free *ortho*-position in G units apparently favored the laccase-mediated grafting of LG. *Polymers* **2021**, *13*, x FOR PEER REVIEW 7 of 15

**Figure 2.** Water contact angle of beech wood veneers. Untreated (UW); treated with lauryl gallate (LG); laccase (L); and laccase and lauryl gallate (L+LG). **Figure 2.** Water contact angle of beech wood veneers. Untreated (UW); treated with lauryl gallate (LG); laccase (L); and laccase and lauryl gallate (L + LG). *Polymers* **2021**, *13*, x FOR PEER REVIEW 8 of 15

radicals produced by laccase in S units should be more stable than those produced in G units, since S units have two electro-donating groups (methoxy) in *ortho*-position instead one methoxy group which possess G units [31]. This extra methoxy group of S units seems **Figure 3.** Water contact angle of wood veneers. Enzymatically hydrophobized beech wood (EHBW); pine wood (EHPW); and eucalyptus wood (EHEW). Untreated beech wood (UBW); pine wood (UPW); and eucalyptus wood (UEW). **Figure 3.** Water contact angle of wood veneers. Enzymatically hydrophobized beech wood (EHBW); pine wood (EHPW); and eucalyptus wood (EHEW). Untreated beech wood (UBW); pine wood (UPW); and eucalyptus wood (UEW).

to have a remarkable effect in the laccase-mediated polymerization of technical lignins [32]. However, our results suggest that the free *ortho*-position in G units apparently fa-

[39,40]. Nevertheless, XPS analysis of some lignocellulosic species, e.g., softwood must be carefully conducted due to the migration of lipophilic extractives to the surface of the material which could be induced by the vacuum conditions necessary to perform the analysis [41]. XPS analysis of beech wood samples provided the elemental composition e.g., carbon (C1s), oxygen (O1s) and nitrogen (N1s), but also the functional groups which are present in the surface of the wood veneers. Thus, the results obtained were a powerful tool to study the changes induced by the laccase-assisted grafting of LG onto beech veneers.

The elemental composition results confirmed that beech samples treated enzymatically with LG showed a clear increase of carbon atoms (4.8%) and a decrease of oxygen (5.9%) which were related to the presence of the aliphatic tail of LG onto the veneers surface (Table 1). In addition, wood samples treated with laccase showed a higher nitrogen content than untreated wood samples which suggests that laccase remained partially adsorbed on the veneers surface even after the washing process. Importantly, adsorption of

**Table 1.** Elemental quantification (%) and functional groups relative percent (%) of beech veneers. Untreated beech wood veneers (UW); beech veneers treated with lauryl gallate (LG); treated with

UW 65.29 30.85 1.29 47.25 38.98 11.30 2.47 16.82 83.19 0.48 1.21 LG 66.60 29.06 1.72 53.79 30.63 11.05 4.54 17.30 82.71 0.44 1.76 L 65.39 26.75 6.42 38.70 42.46 16.60 2.25 30.40 69.61 0.41 0.91 L+LG 70.09 24.95 3.24 53.68 31.99 11.77 2.56 26.68 73.32 0.36 1.68

Four different components were obtained after deconvolution of C1s spectra. C1 was related to C-C or C-H bonds; C2 comprised a carbon bonded to single non-carbonyl

**C1s O1s N1s C1% C2% C3% C4% O1% O2% Ratio** 

**tio** 

**C1/C2** 

**Sample Elements C1s Components O1s Components O/C Ra-**

X-ray photoelectron spectroscopy (XPS) analysis was performed to obtain a better knowledge of the surface chemistry of the enzymatically hydrophobized beech veneers. Such technique has been previously used to study the surface chemistry of several wood

laccase seems to be reduced when LG is present in the reaction [33].

laccase (L); treated with laccase and lauryl gallate (L+LG).

vored the laccase-mediated grafting of LG.

*3.2. XPS Study* 

#### *3.2. XPS Study*

X-ray photoelectron spectroscopy (XPS) analysis was performed to obtain a better knowledge of the surface chemistry of the enzymatically hydrophobized beech veneers. Such technique has been previously used to study the surface chemistry of several wood species [33,34], pulp and paper [35,36], cellulose nanocrystals [37,38] and biocomposites [39,40]. Nevertheless, XPS analysis of some lignocellulosic species, e.g., softwood must be carefully conducted due to the migration of lipophilic extractives to the surface of the material which could be induced by the vacuum conditions necessary to perform the analysis [41]. XPS analysis of beech wood samples provided the elemental composition e.g., carbon (C1s), oxygen (O1s) and nitrogen (N1s), but also the functional groups which are present in the surface of the wood veneers. Thus, the results obtained were a powerful tool to study the changes induced by the laccase-assisted grafting of LG onto beech veneers.

The elemental composition results confirmed that beech samples treated enzymatically with LG showed a clear increase of carbon atoms (4.8%) and a decrease of oxygen (5.9%) which were related to the presence of the aliphatic tail of LG onto the veneers surface (Table 1). In addition, wood samples treated with laccase showed a higher nitrogen content than untreated wood samples which suggests that laccase remained partially adsorbed on the veneers surface even after the washing process. Importantly, adsorption of laccase seems to be reduced when LG is present in the reaction [33].

**Table 1.** Elemental quantification (%) and functional groups relative percent (%) of beech veneers. Untreated beech wood veneers (UW); beech veneers treated with lauryl gallate (LG); treated with laccase (L); treated with laccase and lauryl gallate (L + LG).


Four different components were obtained after deconvolution of C1s spectra. C1 was related to C-C or C-H bonds; C2 comprised a carbon bonded to single non-carbonyl oxygen atom (C-O); C3 corresponded to a carbonyl group (C=O) or a carbon bonded to two non-carboxyl oxygen atoms (O-C-O); C4 was assigned to a carbon bonded to a carbonyl group and non-carbonyl oxygen (O-C=O). Beech veneers treated with laccase showed a remarkable increase of C3 component which was related with the oxidation of hydroxyl groups in the phenolic moieties of lignin to form carbonyl groups. The presence of hydrophobic compounds on wood surface could be related with both a low O/C ratio and a high C1/C2 ratio [42]. Samples treated only with laccase showed an important reduction of C-C or C-H bonds and a higher proportion of both ether (C2) and carbonyl (C3) groups which could explain the increase in the hydrophobicity of the laccase-treated wood samples (Figure 2). Samples treated with LG without laccase addition showed a high C1/C2 ratio but also a relative high O/C ratio which means that LG could be adsorbed on the wood surface but not stably bonded. Nevertheless, the LG-treated wood samples did not improve their hydrophobicity (Figure 2) which suggests that the adsorption of LG was not meaningful, or the adsorbed LG was not properly oriented. On the contrary, beech veneers enzymatically treated with LG showed the lowest O/C ratio and a much higher C1/C2 ratio than untreated samples. Therefore, the increase of the C1/C2 ratio (39%) and the decrease of the O/C ratio (25%) suggest that LG was stably bonded onto the wood samples by means of the laccase-mediated treatment.

#### *3.3. FT-IR Analysis 3.3. FT-IR Analysis*

Several works have shown that FT-IR spectra could be a useful technique to assess the grafting of hydrophobic compounds onto the surface of biobased materials after their laccase-mediated hydrophobization [18,19,43]. Generally, the main differences between both hydrophobized and unmodified lignocellulosic materials are due to the vibration of the chemical groups which are present in the aliphatic chain of the grafted compound. Thus, FT-IR spectra analysis of the enzymatically hydrophobized beech veneers showed two small peaks at 2921 and 2854 cm−<sup>1</sup> which were related with the stretching of methyl (-CH3) and methylene (-CH2-) groups of the 12-carbon aliphatic chain of LG (Figure 4). However, such small peaks were not detected in the spectra of unmodified samples. Therefore, FT-IR spectra analysis evidenced that there was a stable link between LG and beech veneers surface after the laccase-assisted treatment. Several works have shown that FT-IR spectra could be a useful technique to assess the grafting of hydrophobic compounds onto the surface of biobased materials after their laccase-mediated hydrophobization [18,19,43]. Generally, the main differences between both hydrophobized and unmodified lignocellulosic materials are due to the vibration of the chemical groups which are present in the aliphatic chain of the grafted compound. Thus, FT-IR spectra analysis of the enzymatically hydrophobized beech veneers showed two small peaks at 2921 and 2854 cm−1 which were related with the stretching of methyl (- CH3) and methylene (-CH2-) groups of the 12-carbon aliphatic chain of LG (Figure 4). However, such small peaks were not detected in the spectra of unmodified samples. Therefore, FT-IR spectra analysis evidenced that there was a stable link between LG and beech veneers surface after the laccase-assisted treatment.

oxygen atom (C-O); C3 corresponded to a carbonyl group (C=O) or a carbon bonded to two non-carboxyl oxygen atoms (O-C-O); C4 was assigned to a carbon bonded to a carbonyl group and non-carbonyl oxygen (O-C=O). Beech veneers treated with laccase showed a remarkable increase of C3 component which was related with the oxidation of hydroxyl groups in the phenolic moieties of lignin to form carbonyl groups. The presence of hydrophobic compounds on wood surface could be related with both a low O/C ratio and a high C1/C2 ratio [42]. Samples treated only with laccase showed an important reduction of C-C or C-H bonds and a higher proportion of both ether (C2) and carbonyl (C3) groups which could explain the increase in the hydrophobicity of the laccase-treated wood samples (Figure 2). Samples treated with LG without laccase addition showed a high C1/C2 ratio but also a relative high O/C ratio which means that LG could be adsorbed on the wood surface but not stably bonded. Nevertheless, the LG-treated wood samples did not improve their hydrophobicity (Figure 2) which suggests that the adsorption of LG was not meaningful, or the adsorbed LG was not properly oriented. On the contrary, beech veneers enzymatically treated with LG showed the lowest O/C ratio and a much higher C1/C2 ratio than untreated samples. Therefore, the increase of the C1/C2 ratio (39%) and the decrease of the O/C ratio (25%) suggest that LG was stably bonded onto the

*Polymers* **2021**, *13*, x FOR PEER REVIEW 9 of 15

wood samples by means of the laccase-mediated treatment.

**Figure 4.** FT-IR spectra between 2000 and 4000 cm<sup>−</sup>1 of untreated beech wood veneers (U Wood) and beech wood treated with laccase and lauryl gallate (L+LG Wood). **Figure 4.** FT-IR spectra between 2000 and 4000 cm−<sup>1</sup> of untreated beech wood veneers (U Wood) and beech wood treated with laccase and lauryl gallate (L + LG Wood).

#### *3.4. Biofilm Assay 3.4. Biofilm Assay*

The biofilm assay was performed for the assessment of the potential antifouling properties provided by the laccase-mediated grafting of LG onto the surface of pine veneers surface. Pine wood was the specie used for the antifouling assay due to its high The biofilm assay was performed for the assessment of the potential antifoulingproperties provided by the laccase-mediated grafting of LG onto the surface of pine veneers surface. Pine wood was the specie used for the antifouling assay due to its high hydrophobicity (Figure 3) but also its economic importance in the Atlantic area of Europe. The conditions for the enzymatic grafting of LG were those found in the factorial design (5 mM LG, 5 U of laccase/cm<sup>2</sup> of wood and 2 h of treatment).

Regarding the antimicrobial properties of LG, [44] found that the antimicrobial activity of alkylphenols was related with the hydrophobicity of the *para*-substituent. In addition, it was proved that LG has antibacterial properties, specifically against Gram-positive bacteria due to inhibition of their membrane respiratory chain [45]. However, the bacterium (*C. marina*) in this study is a Gram-negative bacterium which suggest that the potential antifouling properties of the enzymatically-hydrophobized pine veneers were mostly related with their hydrophobicity. It was showed that the antifouling properties of a substrate are directly linked to its surface energy [46]. It was observed that the lowest surface retention of fouling organisms was achieved when the surface energy of the substrata was between 20–30 mN/m. Since surface energy is inversely proportional to WCA, hydrophobic substrata present low surface energy. In fact, they showed that the atomic groups that performed best antifouling properties were hydrophobic domains such as methyl groups. Thus, it was expected that the long aliphatic chain of LG provided a hydrophobicity high enough to hinder the *C. marina* adhesion onto pine veneers.

At the same time, bacterial surface chemistry is also an important parameter since its hydrophilicity/hydrophobicity character will determine its chemical compatibility and the strength of its initial attachment with the hydrophobized pine veneers. Therefore, the surface chemistry of *C. marina* was studied to assess its hydrophilicity degree. Hence, *C. marina* was suspended in a solution containing both aqueous and organic phases and the migration of the bacteria to the organic phase was measured. According to Karunakaran and Biggs [47], a bacterial migration higher than 50% would mean that bacteria possess a hydrophobic surface. Nevertheless, the partition coefficient was 19.65 ± 2.86% which means that more than 80% of *C. marina* remained in the aqueous phase. Therefore, the surface chemistry of the *C. marina* strain (CECT4278) was mostly hydrophilic.

#### *3.5. Protein Measurement*

The potential antifouling properties of the enzymatically-hydrophobized pine veneers were studied by incubating the wood samples in a marine broth inoculated at 3% of *C. marina* (*v*/*v*) for 1, 3 and 5 days. The amount of bacteria onto the veneers surface was indirectly quantified by measuring the protein content on the surface of the wood samples. Such protein measurement of the colonized wood samples showed that the laccase-assisted grafting of LG reduced substantially the *C. marina* adhesion (Figure 5). After one day, the hydrophobized pine veneers showed a protein content of 44.83% lower than untreated veneers. Such trend was also observed after 3 and 5 days of incubation of the wood veneers in the marine broth inoculated with *C. marina*. Apparently, the hydrophobicity induced by the laccase-assisted grafting of LG onto the surface of pine veneers modified the pattern of *C. marina* adhesion, restricting the bacterial attachment and/or the secretion of EPS. *Polymers* **2021**, *13*, x FOR PEER REVIEW 11 of 15

**Untreated Pine Wood Hydrophobized Pine Wood**

**Figure 5.** Protein measurement of the hydrophobized pine veneers submerged for 5 days in a marine broth containing a *C. marina* culture. **Figure 5.** Protein measurement of the hydrophobized pine veneers submerged for 5 days in a marine broth containing a *C. marina* culture.

*3.6. SEM Analyses*  The surface of the colonized pine veneers was studied by SEM to confirm the results obtained through the protein measurements. It was expected to detect the EPS produced by *C. marina* after their attachment onto veneers surface [52]. Such EPS are a real problem since they may protect bacteria against biocides and antibiotics [53]. In addition, EPS have chelating properties which are used by bacteria to enhance the nutrients availability [54]. Therefore, by hindering the production of EPS it could be easier to attack the bacteria and reduce their biofouling activity. It is worth noting that LG was grafted onto the aromatic moieties of lignin, which means that the hydroxyl groups of cellulose and hemicelluloses were not significantly modified [22]. Therefore, the surface of the enzymatically treated pine veneers resembles to an amphiphilic surface, since both hydrophilic (hydroxyl groups from cellulose and hemicellulose) and hydrophobic (alkyl groups from LG) domains are present on the veneers surface. Such particular chemical composition could hinder the adhesion of a wide range of microorganisms [48,49], but also reduce the impact of the secreted proteins which has an important role in the microorganisms attachment [50,51]. The results proved the efficiency of the laccase-assisted functionalization to provide antifouling properties to wood.

#### cubation in the marine broth and, such EPS density was even higher after the fifth day of *3.6. SEM Analyses*

ronments.

test (Figure 6A,B). These pictures suggest that C. marina can adhere easily to the surface of untreated pine veneers. On the contrary, SEM pictures of the enzymatically hydrophobized pine veneers showed a much lower density of EPS compared with untreated veneers. Moreover, there were not significant differences in the apparent EPS density of the hydrophobized pine veneers between the first and the fifth day of incubation in the The surface of the colonized pine veneers was studied by SEM to confirm the results obtained through the protein measurements. It was expected to detect the EPS produced by *C. marina* after their attachment onto veneers surface [52]. Such EPS are a real problem since they may protect bacteria against biocides and antibiotics [53]. In addition, EPS have chelating properties which are used by bacteria to enhance the nutrients availability [54].

marine broth. These results agreed with the data obtained in the measurements of the amount of protein which was present on the veneers surface. There are two plausible

the untreated and the enzymatically hydrophobized pine veneers. One the one hand, the 12-carbons aliphatic tails of the LG that was enzymatically grafted onto the surface of pine veneers could disrupt notably the normal secretion of EPS. On the other hand, the hydrophobization of the pine veneers could delay or directly hinder the surface colonization of

It is worth noting that both untreated and hydrophobized pine veneers showed an important surface roughness which apparently affected to the bacterial adhesion. In fact, the higher density of EPS was observed on the angled edges of the veneers surface, which means that such angled zones likely favored the attachment of *C. marina* (Figure 7). Nonetheless, hydrophobized pine veneers showed a much lower density of EPS onto such angled edges than untreated veneers which, confirm the effectiveness of the enzymatic grafting of LG. These results confirm that the laccase-assisted grafting of LG modified the normal pattern of *C. marina* adhesion and/or the secretion of EPS. Thus, the marine antifouling properties of the pine veneers were remarkably improved by means of the laccaseassisted grafting of LG onto the pine veneers surface. These results could lead to the development of a new environmentally friendly treatment to protect wood in marine envi-

Untreated pine veneers showed a relatively high density of EPS after one day of in-

*C. marina* and, therefore the EPS could not be secreted yet.

Therefore, by hindering the production of EPS it could be easier to attack the bacteria and reduce their biofouling activity.

Untreated pine veneers showed a relatively high density of EPS after one day of in-cubation in the marine broth and, such EPS density was even higher after the fifth day of test (Figure 6A,B). These pictures suggest that C. marina can adhere easily to the surface of untreated pine veneers. On the contrary, SEM pictures of the enzymatically hydrophobized pine veneers showed a much lower density of EPS compared with untreated veneers. Moreover, there were not significant differences in the apparent EPS density of the hydrophobized pine veneers between the first and the fifth day of incubation in the marine broth. These results agreed with the data obtained in the measurements of the amount of protein which was present on the veneers surface. There are two plausible mechanisms that could explain the big differences observed between the SEM pictures of the untreated and the enzymatically hydrophobized pine veneers. One the one hand, the 12-carbons aliphatic tails of the LG that was enzymatically grafted onto the surface of pine veneers could disrupt notably the normal secretion of EPS. On the other hand, the hydrophobization of the pine veneers could delay or directly hinder the surface colonization of *C. marina* and, therefore the EPS could not be secreted yet. *Polymers* **2021**, *13*, x FOR PEER REVIEW 12 of 15

**Figure 6.** SEM pictures of pine wood veneers colonized by *C. marina*. Untreated pine wood veneers after one day of anti-biofilm assay (**A**); and after five days (**B**). Enzymatically hydrophobized pine wood veneers after one day of anti-biofilm assay (**C**); and after five days (**D**). All the pictures were acquired at 100× magnification. **Figure 6.** SEM pictures of pine wood veneers colonized by *C. marina*. Untreated pine wood veneers after one day of anti-biofilm assay (**A**); and after five days (**B**). Enzymatically hydrophobized pine wood veneers after one day of anti-biofilm assay (**C**); and after five days (**D**). All the pictures were acquired at 100× magnification.

**Figure 7.** SEM pictures of colonized pine wood veneers after five days of antibiofilm assay. Un-It is worth noting that both untreated and hydrophobized pine veneers showed an important surface roughness which apparently affected to the bacterial adhesion. In fact, the higher density of EPS was observed on the angled edges of the veneers surface, which means that such angled zones likely favored the attachment of *C. marina* (Figure 7). Nonetheless, hydrophobized pine veneers showed a much lower density of EPS onto such angled edges than untreated veneers which, confirm the effectiveness of the enzymatic grafting of LG. These results confirm that the laccase-assisted grafting of LG modified the normal pattern of *C. marina* adhesion and/or the secretion of EPS. Thus, the marine antifouling properties of the pine veneers were remarkably improved by means of the laccase-assisted grafting of LG onto the pine veneers surface. These results could lead to the development of a new environmentally friendly treatment to protect wood in marine environments.

treated pine wood veneers (**A**); and enzymatically hydrophobized pine wood veneers (**B**). The pic-

ties of wood has been proposed in the present study. Such new green strategy is based on the laccase-assisted grafting of LG onto wood veneers. Different wood species (beech, pine, and eucalyptus) were effectively hydrophobized through the enzymatic treatment. It was observed that the reaction conditions played an important role on the extent of hydrophobization, but the treated wood species were also a major factor. Based on these results, pine wood was selected to study the impact of the laccase-mediated hydrophobization on the marine antifouling properties of wood. SEM pictures and protein measurements confirmed that the hydrophobized wood veneers modified the colonization pattern of *Cobetia marina*, revealing that the proposed enzymatic methodology could act as a new marine antifouling treatment for lignocellulosic materials. Future studies should analyze the antifouling properties of the enzymatically treated wood against other marine microorganisms and the "in situ" response of the hydrophobized wood under marine environments. The combination of the proposed treatment with other conventional ones is also of

tures were acquired at 1000× magnification.

**4. Conclusions** 

interest for future research.

**Figure 6.** SEM pictures of pine wood veneers colonized by *C. marina*. Untreated pine wood veneers

wood veneers after one day of anti-biofilm assay (**C**); and after five days (**D**). All the pictures were

**Figure 7.** SEM pictures of colonized pine wood veneers after five days of antibiofilm assay. Untreated pine wood veneers (**A**); and enzymatically hydrophobized pine wood veneers (**B**). The pic-**Figure 7.** SEM pictures of colonized pine wood veneers after five days of antibiofilm assay. Untreated pine wood veneers (**A**); and enzymatically hydrophobized pine wood veneers (**B**). The pictures were acquired at 1000× magnification.

#### **4. Conclusions 4. Conclusions**

tures were acquired at 1000× magnification.

acquired at 100× magnification.

A new environmentally friendly strategy to improve the marine antifouling properties of wood has been proposed in the present study. Such new green strategy is based on the laccase-assisted grafting of LG onto wood veneers. Different wood species (beech, pine, and eucalyptus) were effectively hydrophobized through the enzymatic treatment. It was observed that the reaction conditions played an important role on the extent of hydrophobization, but the treated wood species were also a major factor. Based on these results, pine wood was selected to study the impact of the laccase-mediated hydrophobization on the marine antifouling properties of wood. SEM pictures and protein measurements confirmed that the hydrophobized wood veneers modified the colonization pattern of *Cobetia marina*, revealing that the proposed enzymatic methodology could act as a new marine antifouling treatment for lignocellulosic materials. Future studies should analyze the antifouling properties of the enzymatically treated wood against other marine microorganisms and the "in situ" response of the hydrophobized wood under marine environments. The combination of the proposed treatment with other conventional ones is also of interest for future research. A new environmentally friendly strategy to improve the marine antifouling properties of wood has been proposed in the present study. Such new green strategy is based on the laccase-assisted grafting of LG onto wood veneers. Different wood species (beech, pine, and eucalyptus) were effectively hydrophobized through the enzymatic treatment. It was observed that the reaction conditions played an important role on the extent of hydrophobization, but the treated wood species were also a major factor. Based on these results, pine wood was selected to study the impact of the laccase-mediated hydrophobization on the marine antifouling properties of wood. SEM pictures and protein measurements confirmed that the hydrophobized wood veneers modified the colonization pattern of *Cobetia marina*, revealing that the proposed enzymatic methodology could act as a new marine antifouling treatment for lignocellulosic materials. Future studies should analyze the antifouling properties of the enzymatically treated wood against other marine microorganisms and the "in situ" response of the hydrophobized wood under marine environments. The combination of the proposed treatment with other conventional ones is also of interest for future research.

> **Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/polym13213795/s1, Table S1. Treatment conditions for the enzymatic grafting of lauryl gallate (LG) on beech veneers tested in the experimental design.

> **Author Contributions:** D.F. conducted the experiments of wood modification and wrote the manuscript. C.B. designed and conducted the biofouling experiments and edited the manuscript. S.G. conducted the experiments of wood modification and edited the manuscript. D.M. supervised the work, designed the experiments, and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

> **Funding:** This research was funded by ERDF and Xunta de Galicia (Grant Numbers 09TMT012E, EM2014/041 and Eq3-GRC2017-I751).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The support of CACTI from the University of Vigo for XPS analysis and acquisition of microscopy images is appreciated. The authors want to thank to Foresa (Caldas de Reis, Spain) for providing wood samples and to Novozymes (Bagsværd, Denmark) for supplying the enzymes.

**Conflicts of Interest:** The authors declare no conflict of interest.

## **References**


**Igor Wachter \*, Tomáš Štefko, Peter Rantuch, Jozef Martinka and Alica Pastierová**

Department of Integrated Safety, Faculty of Materials Science and Technology in Trnava, Slovak University of Technology in Bratislava, Botanická 49, 917 24 Trnava, Slovakia; tomas.stefko@stuba.sk (T.Š.); peter.rantuch@stuba.sk (P.R.); jozef.martinka@stuba.sk (J.M.); alica.pastierova@stuba.sk (A.P.)

**\*** Correspondence: igor.wachter@stuba.sk; Tel.: +421-904-398-793

**Abstract:** Optically transparent wood is a type of composite material, combining wood as a renewable resource with the optical and mechanical properties of synthetic polymers. During this study, the effect of monochromatic UV-C (λ—250 nm) radiation on transparent wood was evaluated. Samples of basswood were treated using a lignin modification method, to preserve most of the lignin, and subsequently impregnated with refractive-index-matched types of acrylic polymers (methyl methacrylate, 2-hydroxyethyl methacrylate). Optical (transmittance, colour) and mechanical (shore D hardness) properties were measured to describe the degradation process over 35 days. The transmittance of the samples was significantly decreased during the first seven days (12% EMA, 15% MMA). The average lightness of both materials decreased by 10% (EMA) and 17% (MMA), and the colour shifted towards a red and yellow area of CIE *L\*a\*b\** space coordinates. The influence of UV-C radiation on the hardness of the samples was statistically insignificant (W+MMA 84.98 ± 2.05; W+EMA 84.89 ± 2.46), therefore the hardness mainly depends on the hardness of used acrylic polymer. The obtained results can be used to assess the effect of disinfection of transparent wood surfaces with UV-C radiation (e.g., due to inactivation of SARS-CoV-2 virus) on the change of its aesthetic and mechanical properties.

**Keywords:** transparent wood; UV-C radiation; optical properties; basswood; hardness; chromophores deactivation

#### **1. Introduction**

Wood, as a renewable and earth-abundant resource, is a well-established material in many applications due to its good physical and chemical properties, including high strength, low thermal conductivity, non-toxicity, and biodegradability [1,2]. The future of sustainable development depends on how humans will transfer their dependability from finite fossil-based materials to sustainable and renewable materials to combat the climate change. Recently, there is an increasing number of articles dealing with eco-friendly composites [3,4].

Wood composite materials are engineered and produced with tailored physical and mechanical properties appropriate for a wide variety of applications, both known and not discovered yet [5].

Transparent wood is a composite material consisting of a modified wood component (deactivated chromophores or delignification) and an in situ polymerized, transparent component. Transparent wood has received much attention, owing to its great potential for applications in light-transmitting buildings, which can partially replace artificial light with sunlight and therefore save energy [6,7]. Transparent wood can be used to produce building [8,9], solar cells [10] and magnetic materials [11]. Additional functionalization has been demonstrated, such as lasing [12], heat shielding [13], thermal energy storage [14], electro-

**Citation:** Wachter, I.; Štefko, T.; Rantuch, P.; Martinka, J.; Pastierová, A. Effect of UV Radiation on Optical Properties and Hardness of Transparent Wood. *Polymers* **2021**, *13*, 2067. https://doi.org/10.3390/ polym13132067

Academic Editor: Roman Réh

Received: 31 May 2021 Accepted: 21 June 2021 Published: 23 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

luminescent devices [15], and combined with conducting polymers in electromechanical devices [16].

To fabricate transparent wood, two steps are typically involved: completely removing the light-absorbing lignin from the cell walls of natural wood by a solution-based immersion method and infiltrating a refractive index matching polymer into the delignified wood matrix to minimize light absorption and scattering, respectively [9,17–20].

Alternatively, many studies have focused on physically and/or chemically modifying lignin structures to reduce lignin colour and impart new functionalities, including fractionation [21], grind [22], acetylated lignin [23,24], fragmented lignin [25,26] and metaldecorated lignin [27,28].

Although considered critical in previous publications, delignification processes are time consuming and not necessarily environmentally friendly because of the production of odorous components and chlorinated compounds. Moreover, the removed lignin significantly weakens the wood structure so it can be challenging to work with such a fragile material, and this also lowers the number of suitable wood species for transparent wood preparation; pine and spruce, for example, breaks into pieces after the delignification step. The lignin modification method is superior to the delignification process in the following four aspects [29]:


Also, it is important to note that wood consists of around 30 wt% of lignin which provides structural support and therefore the transparent wood fabricated by this process could be considered more environmentally friendly because less synthetic polymer is needed for its fabrication.

There is an increasing number of studies with various applications for the use of transparent wood where it needs to withstand outdoor weather conditions from which UV radiation may cause its degradation. Such applications include perovskite solar cells assembled directly on transparent wood substrates [30], anisotropic transparent paper with high efficiency as a light management coating layer for GaAs solar cell [10], smart photo-responsive windows with energy storage capabilities [31], radiative cooling structural materials [32], smart and energy-saving buildings applications [9,33], and structural elements in architectural construction. [34,35] In addition, due to the worldwide pandemic caused by COVID-19, the use of UV-C radiation for sanitation of surfaces and internal spaces has risen dramatically.

Based on the arguments described above, it is necessary to understand the influence of UV radiation on this type of material that has huge potential applications in the future. According to a review made by [36], there are questions which should be addressed in the future studies to allow industrialization of the technology, such as optical and mechanical stability and the desirability of increased cellulose content. The increased cellulose content was addressed by [29].

A study, conducted by [37], evaluated colour, chemical and optical (transmittance) changes of a transparent wood composite made from poplar wood and epoxy resin with a UV absorber when exposed to UV-A (340 nm) light. To the best of our knowledge, to this date, it is the only study dealing with this issue. Therefore, the aim of this study is to further examine the effect of UV radiation (UV-C, 250 nm) on optical (transmittance and colorimetry) and mechanical stability (hardness) of lignin retaining transparent wood obtained by lignin chromophores deactivation.

#### **2. Materials and Methods**

#### *2.1. Materials and Chemicals*

Radially cut basswood (*Tilia*) was purchased from JAF Holz Slovakia s. r. o. (density: 0.53–0.56 g cm−<sup>3</sup> ). Sodium silicate, sodium hydroxide, magnesium sulfate, DTPA and

H2O<sup>2</sup> (35%), propanol, acetone were purchased from CentralChem s.r.o. Deionised water was prepared directly in the laboratory. MMA and 2,20 -azobis(2-methylpropionitrile) were purchased from Sigma-Aldrich and 2-hydroxyethyl-methacylate with activator were purchased from Epoxy s.r.o.

#### *2.2. Lignin Modification*

The lignin modification procedure was originally proposed by [29]. Basswood samples with dimensions of 100 × 50 × 1.2 mm (±0.1 mm) were submerged into a lignin modifying solution at 70 ◦C until the wood became white. The solution was prepared by mixing chemicals in the following order: deionized water, sodium silicate (3.0 wt%), sodium hydroxide solution (3.0 wt%), magnesium sulphate (0.1 wt%), DTPA (0.1 wt%), and then H2O<sup>2</sup> (4.0 wt%). Gradually, H2O<sup>2</sup> (35% vol) was added to the solution until the samples became completely white. The samples were then washed with hot deionized water to remove traces of residual chemicals. Finally, the samples were dehydrated with propanol and acetone, subsequently, and stored until polymer infiltration.

## *2.3. Transparent Wood Preparation*

Before polymer infiltration, wood samples were dehydrated with ethanol and acetone sequentially. Each solvent-exchange step was repeated three times. MMA was pre-polymerized before infiltration to remove the dissolved oxygen. Pre-polymerization was carried out at 75 ◦C for 15 min with 0.3 wt% 2,20 -azobis(2-methylpropionitrile) as initiator and the solution was then cooled to room temperature. Subsequently, the bleached wood template was fully vacuum-infiltrated in a pre-polymerized PMMA solution. Finally, the infiltrated wood was sandwiched between two glass slides, packaged in aluminium foil, and the polymerization was performed in an oven at 75 ◦C for 4 h. Infiltration of 2 hydroxyethyl-methacylate was carried out without pre-polymerization. Activator (0.2 wt%) was mixed with 2-hydroxyethyl-methacrylate and it was allowed to dissolve for 1 h. After full vacuum-infiltration the samples were sandwiched between two glass slides, packaged in aluminium foil, and the polymerization was performed in an oven at 90 ◦C for 4 h. In total 20 pieces of samples were prepared (10 for each methacrylate). After fabrication of the samples, the resulting product can be considered to be around 40% renewable.

#### *2.4. Characterization*

According to the study by Li et al. [29], the transparent wood samples retained up to 80 wt% of lignin leading to a stronger wood template compared to the de-lignified alternative. In this study, the weight loss of the wood component due to the modification of lignin was 21.9 ± 0.9%. After polymer infiltration, a high-lignin content transparent wood with a transmittance of 83%, a haze of 75%, a thermal conductivity of 0.23 Wm K−<sup>1</sup> , and work-to fracture of 1.2 MJ m−<sup>3</sup> (a magnitude higher than glass) was obtained (MMA samples). Samples prepared for this study did not reach the values of previously mentioned research because of the use of different (more dense) wood. Figure 1 shows a boxplot of wood and acrylate polymer weight in the samples. The average proportion of wood component in the W+MMA and W+EMA samples was 27% and 29%, respectively.

The colorimetry of the samples was performed using by a Colorimeter NR200 Precision (Threenh Technology Co., Ltd.; Shenzhen, China) with the following characterizations: Measuring aperture Φ8 mm, Colour space CIE *L\*a\*b\** and Light Source D65. The colour change caused by UV radiation was monitored using the CIE *L\*a\*b\** colour space coordinates. In this way, the colour of the measured surface is expressed using three coordinates:


**Figure 1.** Boxplot of wood and acrylate polymer weight in the samples. **Figure 1.** Boxplot of wood and acrylate polymer weight in the samples.

The colorimetry of the samples was performed using by a Colorimeter NR200 Precision (Threenh Technology Co., Ltd.; Shenzhen, China) with the following characteriza-To describe the total shift in this colour space, the total colour difference is used, which can be expressed as follows:

$$dE\_t = \sqrt{\left(L\_t^\* - L\_0^\*\right)^2 + \left(a\_t^\* - a\_0^\*\right)^2 + \left(b\_t^\* - b\_0^\*\right)^2} \tag{1}$$

<sup>∗</sup> is the

coordinates. In this way, the colour of the measured surface is expressed using three coordinates: • *L\**—coordinate on the axis indicating lightness where *dE<sup>t</sup>* is the total colour difference at time *t*, *L* ∗ *t* is the value of *L\** at time *t*, *L* ∗ 0 is the value of *L\** before exposure to UV radiation, *a* ∗ *t* is the value of *a\** at time *t*, *a* ∗ 0 is the value *a\** before exposure to UV radiation, *b* ∗ *t* is the value of *b\** at time *t*, *b* ∗ 0 is the value of *b\** before exposure to UV radiation.

• *a\**—coordinate on the axis between red and green • *b\**—coordinate on the axis between yellow and blue The transmittance was measured using a modified photometer RMG2.1 (Heil Metalle GmbH, Mülheim, a. d. Ruhr, Germany). The measuring area was 20 mm × 20 mm.

To describe the total shift in this colour space, the total colour difference is used, which can be expressed as follows: For the measurement of the hardness Digital Shore D Hardness Tester—Sauter HD (Sauter GmbH, Balingen, Germany) was used.

݀ܧ௧ = ඥሺܮ௧ ܮ − <sup>∗</sup> <sup>∗</sup> ሻଶ + ሺܽ௧ <sup>∗</sup> − ܽ <sup>∗</sup> ሻଶ + ሺܾ௧ <sup>∗</sup> − ܾ ∗ሻଶ (1) where dEt is the total colour difference at time t, ܮ௧ <sup>∗</sup> is the value of L\* at time t, ܮ value of L\* before exposure to UV radiation, ܽ௧ <sup>∗</sup> is the value of a\* at time t, ܽ <sup>∗</sup> is the value a\* before exposure to UV radiation, ܾ௧ <sup>∗</sup> is the value of b\* at time t, ܾ <sup>∗</sup> is the value of b\* before exposure to UV radiation. The transmittance was measured using a modified photometer RMG2.1 (Heil Metalle GmbH, Mülheim, a. d. Ruhr, Germany). The measuring area was 20 mm × 20 mm. The UV ageing (an accelerated weathering test) has been carried out in a UV chamber. The samples were irradiated for 35 days. All measurements have been done after 7 days of UV exposition. The ageing has been done under a temperature of 50 ◦C. As a source of UV-C radiation, 4 germicidal fluorescent lamps Philips TUV 15 W (Piła, Poland) were used. The efficiency of the fluorescent lamp was 32%. UV-C radiation (wavelength 250 nm) reached a power output of 4.9 W and the volume of the chamber was 50 L. The samples were placed 100 mm from the UV lamps in every direction. The irradiance flux density was 16.07 W m−<sup>2</sup> and the inner surface of the chamber was made of stainless steel with a 50% reflectance factor.

For the measurement of the hardness Digital Shore D Hardness Tester—Sauter HD (Sauter GmbH, Balingen, Germany) was used. The UV ageing (an accelerated weathering test) has been carried out in a UV chamber. The samples were irradiated for 35 days. All measurements have been done after 7 days of UV exposition. The ageing has been done under a temperature of 50 °C. As a For FTIR analysis, Varian FT-IR Spectrometer 660 (Agilent Technologies, Inc., Santa Clara, CA, USA) samples were directly applied to a diamond crystal of ATR GladiATR (PIKE Technology Inc., Madison, WI, USA) and the resulting spectra were corrected for background air absorbance. The spectra were recorded using a Varian Resolutions Pro and samples were measured in the region 4000–400 cm−<sup>1</sup> ; each spectrum was measured 146 times, at resolution 4.

source of UV-C radiation, 4 germicidal fluorescent lamps Philips TUV 15 W (Piła, Poland) were used. The efficiency of the fluorescent lamp was 32%. UV-C radiation (wavelength 250 nm) reached a power output of 4.9 W and the volume of the chamber was 50 L. The All of the measurements were carried out using Stat Soft STATISTICA 10 (StatSoft s.r.o., Praha, Czechia) software. The impact of the exposure time of UV radiation on the total colour difference, transmittance and hardness were evaluated by the Duncan's test.

samples were placed 100 mm from the UV lamps in every direction. The irradiance flux density was 16.07 W m−2 and the inner surface of the chamber was made of stainless steel

(PIKE Technology Inc., Madison, WI, USA) and the resulting spectra were corrected for background air absorbance. The spectra were recorded using a Varian Resolutions Pro

For FTIR analysis, Varian FT-IR Spectrometer 660 (Agilent Technologies, Inc., Santa

with a 50% reflectance factor.

#### **3. Results 3. Results**  *3.1. Colorimetry*

#### *3.1. Colorimetry*

146 times, at resolution 4.

In terms of colour change due to UV radiation, the most significant changes occurred during the first 7 days. This fact is clearly visible in Figure 2. A significant change was observed in all three coordinates, which was subsequently reflected in the value of the overall colour difference. In terms of colour change due to UV radiation, the most significant changes occurred during the first 7 days. This fact is clearly visible in Figure 2. A significant change was observed in all three coordinates, which was subsequently reflected in the value of the overall colour difference.

and samples were measured in the region 4000–400 cm−1; each spectrum was measured

All of the measurements were carried out using Stat Soft STATISTICA 10 (StatSoft s.r.o., Praha, Czechia) software. The impact of the exposure time of UV radiation on the total colour difference, transmittance and hardness were evaluated by the Duncan's test.

*Polymers* **2021**, *13*, 2067 5 of 13

**Figure 2.** Time dependence of colour components in CIE L\*a\*b\* space and total colour difference: (**a**) Lightness; (**b**) the coordinate *a\**; (**c**) *b\** coordinate; (**d**) total colour difference \*, \*\*, \*\*\* denoted exposure times of W+EMA and \*\*\*\* denoted exposure times of W+MMA at which the total colour differences were statistically significant based on the results of Duncan's test (difference between 0 days of exposure and other exposure times is obvious without statistical test). **Figure 2.** Time dependence of colour components in CIE *L\*a\*b\** space and total colour difference: (**a**) Lightness; (**b**) the coordinate *a\**; (**c**) *b\** coordinate; (**d**) total colour difference \*, \*\*, \*\*\* denoted exposure times of W+EMA and \*\*\*\* denoted exposure times of W+MMA at which the total colour differences were statistically significant based on the results of Duncan's test (difference between 0 days of exposure and other exposure times is obvious without statistical test).

The average lightness of both measured materials was approximately 73.5. However, after the first 7 days of UV exposure, it decreased to values of around 66 for W+EMA samples and to an average of 64 for W+MMA samples, which represents a reduction of 10% and 17%, respectively. The average lightness of both measured materials was approximately 73.5. However, after the first 7 days of UV exposure, it decreased to values of around 66 for W+EMA samples and to an average of 64 for W+MMA samples, which represents a reduction of 10% and 17%, respectively.

The *a\** coordinate also changed most rapidly at the onset of UV exposure. In 7 days, its average value increased from 2 to almost 6 (W+EMA) and from 3.4 to 9 (W+MMA). A less pronounced increase subsequently continued until day 28 of the test. Subsequently, The *a\** coordinate also changed most rapidly at the onset of UV exposure. In 7 days, its average value increased from 2 to almost 6 (W+EMA) and from 3.4 to 9 (W+MMA). A less pronounced increase subsequently continued until day 28 of the test. Subsequently, there was a very slight decrease. As with *L\**, W+EMA samples proved to be less susceptible to changes due to UV radiation.

In the case of the *b\** values, it is possible to see a similar course as in the case of *a\**, but after the initial significant increase it changes only slightly over a period of more than 7 days. Although the *b\** of both materials is very similar in the samples before exposure

to UV radiation, the samples of W+MMA acquire higher values than W+EMA due to its influence. The results of the overall colour difference reflect the changes described in the individual coordinates. A significant colour change occurs mainly during the first 7 days, fol-

there was a very slight decrease. As with *L\**, W+EMA samples proved to be less suscepti-

In the case of the *b\** values, it is possible to see a similar course as in the case of *a\**, but after the initial significant increase it changes only slightly over a period of more than 7 days. Although the *b\** of both materials is very similar in the samples before exposure to UV radiation, the samples of W+MMA acquire higher values than W+EMA due to its influence.

*Polymers* **2021**, *13*, 2067 6 of 13

ble to changes due to UV radiation.

The results of the overall colour difference reflect the changes described in the individual coordinates. A significant colour change occurs mainly during the first 7 days, followed by only a slight increase. lowed by only a slight increase. As already mentioned, the graphs corresponding to the values of *a\** and *b*\* have a

As already mentioned, the graphs corresponding to the values of *a\** and *b*\* have a similar course. The following equations can be determined from the graphical representation of the measured values (Figure 3): similar course. The following equations can be determined from the graphical representation of the measured values (Figure 3): ܾௐାாெ ∗ = 9.96 + 26.24 × log ܽௐାாெ <sup>∗</sup> (2)

$$a\_{W+EMA}^{\*} = 9.96 + 26.24 \times \log a\_{W+EMA}^{\*}\tag{2}$$

$$b^\*\_{W+MMA} = 1.83 + 33.91 \times \log a^\*\_{W+MMA} \tag{3}$$

where *b* ∗ *<sup>W</sup>*+*EMA* is the coordinate *b\** for the sample W+EMA, *a* ∗ *<sup>W</sup>*+*EMA* is the coordinate *a\** for the sample W+EMA, *b* ∗ *<sup>W</sup>*+*MMA* is the coordinate *b\** for the sample W+MMA and *a* ∗ *<sup>W</sup>*+*MMA* is the coordinate *a\** for the sample W+MMA. The coefficients of determination in these cases are 0.9376 (W+EMA) and 0.8191 (W+MMA). where ܾௐାாெ for the sample W+EMA, ܾௐାெெ <sup>∗</sup> is the coordinate *b\** for the sample W+MMA and ܽௐାெெ <sup>∗</sup> is the coordinate a\* for the sample W+MMA. The coefficients of determination in these cases are 0.9376 (W+EMA) and 0.8191 (W+MMA).

**Figure 3. Figure 3.**  Interdependence of colour space coordinates Interdependence of colour space coordinates *a\** and *b\* a\** : ( and **a**) W+MMA; ( *b\**: (**a**) W+MMA; ( **b**) W+EMA. **b**) W+EMA.

The comparison of colour changes in transparent wood infiltrated by 2-hydroxyethylmethacrylate (a,b) and methyl methacrylate (c,d) before and after 840 h of UV-C irradiation is shown in Figure 4a,b, and in Figure 4c,d. The significant colour darkening (photo yellowing) was observed within the first few hours of exposure, which increases with further exposure. The comparison of colour changes in transparent wood infiltrated by 2-hydroxyethyl-methacrylate (a, b) and methyl methacrylate (c,d) before and after 840 h of UV-C irradiation is shown in Figure 4a,b, and in Figure 4c,d. The significant colour darkening (photo yellowing) was observed within the first few hours of exposure, which increases with further exposure.

**Figure 4.** Change of transmittance and colour: W+EMA (**a**) before UV-C irradiation; (**b**) after 35 days of UV-C irradiation; W+MMA (**c**) before UV-C irradiation; (**d**) after 35 days of UV-C irradiation (Source of logo: https://www.mdpi.com/journal/polymers, accessed on 28 May 2021). **Figure 4.** Change of transmittance and colour: W+EMA (**a**) before UV-C irradiation; (**b**) after 35 days of UV-C irradiation; W+MMA (**c**) before UV-C irradiation; (**d**) after 35 days of UV-C irradiation (Source of logo: https://www.mdpi.com/journal/polymers, accessed on 28 May 2021).

#### *3.2. Transmittance 3.2. Transmittance*

Depending on the acrylic polymer used, the transmittance values differ significantly, even for samples not exposed to UV-C radiation (Figure 5). While W+EMA transmits almost 69% of light, W+MMA is about 58%. After 7 days, these values decrease to 57% resp. 43% and consequently their change is almost negligible. Throughout the experiment, the W+EMA samples remained significantly more transparent, with the difference between them highlighted by the action of UV-C radiation, as is shown in Figure 5. Depending on the acrylic polymer used, the transmittance values differ significantly, even for samples not exposed to UV-C radiation (Figure 5). While W+EMA transmits almost 69% of light, W+MMA is about 58%. After 7 days, these values decrease to 57% resp. 43% and consequently their change is almost negligible. Throughout the experiment, the W+EMA samples remained significantly more transparent, with the difference between them highlighted by the action of UV-C radiation, as is shown in Figure 5.

#### *3.3. FTIR Analysis*

Changes to the chemical structure (bond scission/forming) of W+EMA and W+MMA sample after UV-C irradiation are displayed in Figure 6 as infrared spectrum. There are two characteristic bands attributed to the stretching C–O and CH3–O of methyl ester, peak at wavenumber 2916 cm−<sup>1</sup> and 2848 cm−<sup>1</sup> the –CH stretching aliphatic band of the ethylene segment. It is seen, a very strong peak is visible at 1720 cm−<sup>1</sup> due to carbonyl (–C=O) stretching vibration of the acrylate ester group, in both samples. Two peaks at 1435 and 1381 cm−<sup>1</sup> can be attributed to CH<sup>3</sup> symmetric and asymmetric deformation. At wavenumber 958 cm−<sup>1</sup> can be seen C–O–C stretching vibration and at 746 cm−<sup>1</sup> is band characteristic for C–H stretching [38–41]. However, after irradiation by UV-C, major changes were observed evidencing chemical changes in the polymer samples. The bands that undergo prominent changes are the functionalities of hydroxyl O–H, carbonyl C=O and ester (C–O–C) in region of wavenumber from 746 to 1435 cm−<sup>1</sup> . Other photo products, e.g., carbonyl groups or double bonds may be weakened from the surfaces, leading to reduced absorption [42].

**Figure 5.** Change of transmittance during the experiment \* denoted exposure time of W+EMA at which the total colour differences was statistically significant (in interval from 7 to 35 days) based

them highlighted by the action of UV-C radiation, as is shown in Figure 5.

The comparison of colour changes in transparent wood infiltrated by 2-hydroxyethyl-methacrylate (a, b) and methyl methacrylate (c,d) before and after 840 h of UV-C irradiation is shown in Figure 4a,b, and in Figure 4c,d. The significant colour darkening (photo yellowing) was observed within the first few hours of exposure, which increases

**Figure 4.** Change of transmittance and colour: W+EMA (**a**) before UV-C irradiation; (**b**) after 35 days of UV-C irradiation; W+MMA (**c**) before UV-C irradiation; (**d**) after 35 days of UV-C irradiation

Depending on the acrylic polymer used, the transmittance values differ significantly,

even for samples not exposed to UV-C radiation (Figure 5). While W+EMA transmits almost 69% of light, W+MMA is about 58%. After 7 days, these values decrease to 57% resp. 43% and consequently their change is almost negligible. Throughout the experiment, the W+EMA samples remained significantly more transparent, with the difference between

(Source of logo: https://www.mdpi.com/journal/polymers, accessed on 28 May 2021).

with further exposure.

*3.2. Transmittance* 

**Figure 5.** Change of transmittance during the experiment \* denoted exposure time of W+EMA at which the total colour differences was statistically significant (in interval from 7 to 35 days) based on results of Duncan's test (difference between 0 days of exposure and other exposure times is obvious without statistical test). **Figure 5.** Change of transmittance during the experiment \* denoted exposure time of W+EMA at which the total colour differences was statistically significant (in interval from 7 to 35 days) based on results of Duncan's test (difference between 0 days of exposure and other exposure times is obvious without statistical test). (C–O–C) in region of wavenumber from 746 to 1435 cm−1. Other photo products, e.g., carbonyl groups or double bonds may be weakened from the surfaces, leading to reduced absorption [42].

**Figure 6.** Changes to the chemical structure of W+EMA and W+MMA samples before and after the

**Figure 6.** *Cont*.

UV-C irradiation.

Changes to the chemical structure (bond scission/forming) of W+EMA and W+MMA sample after UV-C irradiation are displayed in Figure 6 as infrared spectrum. There are two characteristic bands attributed to the stretching C–O and CH3–O of methyl ester, peak at wavenumber 2916 cm−1 and 2848 cm−1 the –CH stretching aliphatic band of the ethylene segment. It is seen, a very strong peak is visible at 1720 cm−1 due to carbonyl (–C=O) stretching vibration of the acrylate ester group, in both samples. Two peaks at 1435 and 1381 cm−1 can be attributed to CH3 symmetric and asymmetric deformation. At wavenumber 958 cm−1 can be seen C–O–C stretching vibration and at 746 cm−1 is band characteristic for C–H stretching [38–41]. However, after irradiation by UV-C, major changes were observed evidencing chemical changes in the polymer samples. The bands that undergo prominent changes are the functionalities of hydroxyl O–H, carbonyl C=O and ester (C–O–C) in region of wavenumber from 746 to 1435 cm−1. Other photo products, e.g., carbonyl groups or double bonds may be weakened from the surfaces, leading to reduced

**Figure 6.** Changes to the chemical structure of W+EMA and W+MMA samples before and after the UV-C irradiation. **Figure 6.** Changes to the chemical structure of W+EMA and W+MMA samples before and after the UV-C irradiation. *3.4. Hardness* 

#### *3.4. Hardness* The hardness of both types of materials was at a very similar level. Its values were

**4. Discussion** 

strength.

*3.3. FTIR Analysis* 

absorption [42].

The hardness of both types of materials was at a very similar level. Its values were initially around 86. In contrast to the optical properties, the hardness of the measured samples is significantly less affected by the UV-C radiation to which the transparent wood samples were exposed. Figure 7 shows its course as a function of the time of UV-C treatment. The hardness of both types of samples was practically the same during the experiment and no differences are apparent between them. initially around 86. In contrast to the optical properties, the hardness of the measured samples is significantly less affected by the UV-C radiation to which the transparent wood samples were exposed. Figure 7 shows its course as a function of the time of UV-C treatment. The hardness of both types of samples was practically the same during the experiment and no differences are apparent between them.

**Figure 7.** Change of hardness during the experiment (the Duncan's test proved no statistically significant differences between hardness at all investigated times for both investigated samples). **Figure 7.** Change of hardness during the experiment (the Duncan's test proved no statistically significant differences between hardness at all investigated times for both investigated samples).

Schäfer [44]. The W+EMA samples had a hardness of 86 ± 2.3 and were therefore among

The hardness of PMMA in the Shore D scale can be found in a higher number of works than in the case of PEMA. According to Poomali, Suresha and Lee, its value is 90 ± 1 [45]. Seeger et al. state a value of 87.5 ± 0.4 [46] and Akinci, Sen and Sen 79 [47]. The data measured in this study for W+PMMA are 85.4 ± 1.9, which is in agreement with the reported values. Because of these similarities as well as the high proportion of acrylic polymer in the samples (approximately 35%), it can be stated that the hardness is much more significantly influenced by the properties of the resin as a component of delignified wood. It was also observed that the samples were more brittle during hardness testing. No cracks and fractures were observed directly after UV-C irradiation. This behaviour was confirmed by the study of [48] where ultraviolet radiation altered PMMA stiffness, resulting in changes in tensile properties, such as reduction in elongation at break and tensile

Light exposure is a major cause of wood degradation, leading to colour change and

UV degradation of poly(methyl methacrylate) and its vinyltriethoxysilane containing copolymers, was tested using a mercury lamp with a wavelength of 259 nm, situated 10 cm away from the samples and found out that UV irradiation causes changes in the me-

loss in mechanical properties [49–51]. Significant changes were observed after 4 h of irradiation. All *m/z* signals of lignin were either absent or their intensity was considerably reduced, suggesting that lignin underwent an extensive degradation. The irradiation pro-

moted a reduction in the transparency, due to the yellowing [47].

the data of the mentioned authors.

chanical properties of PMMA [52].

#### **4. Discussion**

Shore A hardness of PEMA is according to Hourston, Satgurunathan and Varma 78 [43]. A value higher than 95 can be deducted from the graph in the work of Hourston and Schäfer [44]. The W+EMA samples had a hardness of 86 ± 2.3 and were therefore among the data of the mentioned authors.

The hardness of PMMA in the Shore D scale can be found in a higher number of works than in the case of PEMA. According to Poomali, Suresha and Lee, its value is 90 ± 1 [45]. Seeger et al. state a value of 87.5 ± 0.4 [46] and Akinci, Sen and Sen 79 [47]. The data measured in this study for W+PMMA are 85.4 ± 1.9, which is in agreement with the reported values. Because of these similarities as well as the high proportion of acrylic polymer in the samples (approximately 35%), it can be stated that the hardness is much more significantly influenced by the properties of the resin as a component of delignified wood. It was also observed that the samples were more brittle during hardness testing. No cracks and fractures were observed directly after UV-C irradiation. This behaviour was confirmed by the study of [48] where ultraviolet radiation altered PMMA stiffness, resulting in changes in tensile properties, such as reduction in elongation at break and tensile strength.

Light exposure is a major cause of wood degradation, leading to colour change and loss in mechanical properties [49–51]. Significant changes were observed after 4 h of irradiation. All *m/z* signals of lignin were either absent or their intensity was considerably reduced, suggesting that lignin underwent an extensive degradation. The irradiation promoted a reduction in the transparency, due to the yellowing [47].

UV degradation of poly(methyl methacrylate) and its vinyltriethoxysilane containing copolymers, was tested using a mercury lamp with a wavelength of 259 nm, situated 10 cm away from the samples and found out that UV irradiation causes changes in the mechanical properties of PMMA [52].

Wochnowskia et al. [53] irradiated PMMA by UV-laser light with different wavelengths (193 nm, 248 nm and 308 nm) in order to investigate the photolytic degradation of the physico-chemical molecular structure and reported that, during the UV-irradiation (248 nm), there was the existence of methyl formate, a great amount of methanate, methanol and additionally the occurrence of methyl and other molecule fragments of the polymer side-chain even at a low irradiation dose. At this irradiation dose, side chain cleavage from the polymer main chain takes place yielding mechanical densification of the polymeric material due to Van-der-Waals forces with a subsequent increase in the refractive index.

From the above-mentioned arguments we can conclude that the change of favourable optical properties of transparent wood (transmittance and colour) was caused by the degradation of both components, the acrylic polymer as well as the wood itself.

#### **5. Conclusions**

Transparent wood, combining many advantageous properties, is an emerging new material for light-transmitting and environmentally friendly applications. There is an increasing number of research teams who introduce new methods of fabrication and new ways to use transparent wood. Therefore, it is crucial to know how this material behaves under various conditions.

Exposure to UV-C sources has a significant effect on the colour of transparent wood. It was mostly pronounced from the beginning of the test (during the first 7 days). Samples became darker with increasing exposure time and their colour shifts towards shades of red and yellow which can be possibly explained by the reactivation of chromophores. The values of the coordinates *a*\* and *b*\* show an interdependence that appears to be logarithmic. W+MMA samples are more prone to discolouration due to UV-C radiation than W+EMA samples.

The transmittance of light through the measured samples of transparent wood was significantly affected by the action of UV-C radiation. As in the case of colour changes, the UV-C effect was most pronounced at the beginning and had only a minimal effect in the

later stages. W+EMA had higher light transmission and its reduction due to UV-C was less pronounced than in the case of W+MMA.

The influence of UV-C on shore D hardness of W+EMA and W+MMA is significantly lower than in the case of optical properties. The differences between these materials are not statistically significant. The measured values show that the resulting hardness of transparent wood depends mainly on the hardness of the acrylic polymer used.

In a previously mentioned study, the impact of UV-B radiation on the optical and mechanical properties of transparent wood has been investigated. The UV-B radiation was used for ageing acceleration. Due to the SARS-CoV-2 virus pandemic, the UV-C radiation (for virus deactivation purpose) began to be used massively. However, there were no data concerning the impact of UV-C radiation on transparent wood key properties before this study. This is the first study revealing the impact of UV-C radiation on key optical and mechanical parameters of transparent wood. Obtained results also proved that UV-C radiation (at irradiance flux of 16 W·m−<sup>2</sup> during 35 days) has virtually no effect on the transparent wood (W+EMA and W+MMA) shore D hardness. Obtained results also proven that the impact of UV-C radiation on the optical characteristics of transparent wood (at stated irradiance flux) is significant only for the first 7 days (in the following days the impact was only negligible).

In future research, it is necessary to evaluate the effect of different wavelengths on the properties of transparent wood and also to describe the time period during which the highest degradation occurs.

**Author Contributions:** Conceptualization, I.W. and T.Š.; methodology, I.W.; software, P.R.; validation, I.W., T.Š. and P.R.; formal analysis, I.W., A.P. and T.Š.; investigation, I.W.; resources, I.W. and J.M.; data curation, I.W., T.Š., J.M. and P.R.; writing—original draft preparation, I.W.; writing—review and editing, I.W. and A.P.; visualization, I.W. and P.R; supervision, I.W.; project administration, I.W.; funding acquisition, I.W. and J.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by the Slovak Research and Development Agency under the contract No. APVV-16-0223. This work was also supported by the KEGA agency under the contracts No. 016STU-4/2021 and No. 001TU Z-4/2020. This work was also supported by the Institutional project—FiTraW of MTF STU No. 1617.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

## **References**


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