Raw material

Pinus elliottii Engelm. wood boards were obtained from a sawmill located in the city of Pelotas, Brazil. The boards were cut into wood small samples with dimensions of 2,5 x 2,5 x 0,9 cm for the fungal tests and with 2,5 x 2,5 x 10 cm for the color tests. The wood moisture content was set at around 12% before the impregnation of TiO₂ nanoparticles. The nanoparticles were synthesized from titanium oxysulfate (TiOSO₄) and sodium hydroxide (NaOH) precursors, all of them supplied by Sigma-Aldrich.

Production and characterization of TiO ₂ nanoparticles

The method used to produce the TiO₂ nanoparticles was the microwave-assisted hydrothermal. Two solutions containing 0,01 mol of TiOSO₄ (precursor solution) and 0,6 mol of NaOH (mineralizer solution) in distilled water were prepared with constant stirring. After the homogenization, the solutions were mixed, remaining under constant stirring until to reach 50°C and pH 14. This solution was transferred to a Teflon reactor and heated using microwave radiation for 20 minutes at 160°C with heating rate of 160°C/min.

At the end of the synthesis, the white powder formed in the reaction was washed by centrifugation at room temperature until neutral pH (~7). Then, it was dried at 60°C for 8 hours.

For the determination of the crystallization and the identification of different TiO₂ phases, a Bruker diffractogram (D8 Advance model) with CuK (1,5406 A) and scan rate of 0,02°/s in a range of 20° to 80° was used. The morphology of TiO₂ nanoparticles was investigated by Scanning Electron Microscopy (SEM, JSM - 6610LV), operating at high vacuum and 15 kV.

Wood impregnation with TiO ₂ nanoparticles

TiO₂ nanoparticles were dispersed in distilled water (7,5 mg/ml). This dispersion remained on a magnetic stirrer for homogenization. The wood modification was performed by vacuum-pressure and simple immersion methods. In the first method, the nanocomposites were produced in an autoclave under vacuum-pressure. The wood samples were subjected to an initial vacuum of 600-700 mmHg for 30 minutes. Then, TiO₂ dispersion was placed inside the autoclave, remaining at constant pressure of 8 bar for 1,5 hours. Subsequently, a final vacuum of 600-700 mmHg was performed for 15 minutes to remove waste material (not impregnated). In the second method, the samples were immersed for 7 days into the TiO₂ dispersion without vacuum and pressure. After both procedures, the wood samples were kept in a climatic chamber (20°C and 65% RH) to reach 12 % equilibrium moisture content.

The untreated and treated samples were characterized by SEM coupled with Energy Dispersive Spectrometry (EDS) to investigate the degree of TiO₂ impregnation. SEM images were taken at high vacuum and under lower power energy to prevent wood degradation by the ion beam. EDS analysis was performed to determine the chemical composition of the samples.

Wood impregnation with CCB

CCB (chromated copper borate) solution with 2,5% of active ingredients was used as positive treatment. The CCB solution was prepared by dissolving 63,5% sodium dichromate, 26% copper sulfate and 10,5% boric acid in distilled water (^{Galvão et al. 2004}). The conditions of treatment were the same used in TiO₂ impregnation by vacuum-pressure and simple immersion.

Color evaluation

Color changes of the wood samples were measured using a colorimeter Konica Minolta (model CR 400). The equipment was set for use a light source D25 and illumination angle of 10°, according to the Commission International de L'Eclairage (CIE-L*a*b*) standard. Two measurements in the tangential direction of each wood sample were performed to obtain the parameters L* (lightness), a* (red-green chromatic coordinate) and b* (blue-yellow chromatic coordinate). Chroma (C*), hue angle (h) and color difference (ΔE) were determined by Equations 1, 2 and 3, respectively.

()1

()2

()3

C* = chroma; h = hue angle; a* = red-green chromatic coordinate; b* = blue-yellow chromatic coordinate; ΔE = color difference; ΔL*, Δa* e Δb* = variation of lightness, red-green chromatic coordinate and blue-yellow chromatic coordinate of untreated and impregnated wood samples.

Decay resistance

The accelerated laboratory tests of natural decay resistance of untreated and impregnated wood were performed according to the ASTM D2017 standard (ASTM 2005). The decay resistance was analyzed using a colony of white-rot fungus Ganoderma applanatum (Persoon ex Wallroth) Patouillard, provided by the Setor de Biodegradação e Preservação da Madeira, Laboratório de Produtos Florestais (LPF) from the Serviço Florestal Brasileiro (SFB) located in the city of Brasília (Brazil). The mycelium was cultivated in malt-agar extract for 21 days under an incubation temperature of 25 ± 3 °C and 12 h in the presence of day light. The malt-agar extract was prepared by dissolving 20g of malt and 20g of agar in one liter of distilled water.

Glass containers of approximately 600mL were filled with 100g of commercial soil with capacity of 40% water retention and pH 6. The moisture of the soils was checked and adjusted to 130% of retention capacity. Six replicates for each treatment were used.

In each container, a support plate of Pinus elliottii sapwood (0,3 x 2,9 x 3,5 cm) were placed, serving as substrate for the initial establishment of the fungal colony. All containers containing moistened soil and the support plate were autoclaved at 120°C and 1,9 atm for 40 minutes. Then, a disc of 5 mm in diameter of fungal mycelium was aseptically transferred on each support plate, and the flasks were incubated for 30 days in a climatic chamber (20°C and 65% RH) without light.

The untreated and the impregnated wood samples were dried at 50°C to reach constant mass and they were sterilized in the autoclave at 120°C for 1 hour. Then, each sample was put in contact with the support plate in each bottle, where the fungus had already established itself for 30 days. The fungus remained in contact with wood for 16 weeks under the same incubation conditions mentioned above. The ASTM D2017 standard was adapted for exposure of the TiO₂-treated wood samples to 12h of light, as previously stated by ^{Foster et al. 2011}, Markowska et al. 2011 and ^{Harandi et al. 2016}. These authors affirmed that TiO₂ absorbs UV radiation and can form reactive oxygen species through the recombination electron/hole, which can degrade the fungi wall, inhibiting their respiration and, consequently, resulting in their death.

After 16 weeks, the mycelia were removed and all the wood samples were dried at 50°C to measure the mass loss by fungal attack. The decay resistance was determined according to the rating established by ASTM D2017 standard (Table 1). SEM images were captured from the cell wall of the wood samples after the exposure to the fungus.

Statistical analysis

The average values of the colorimetric parameters, and mass loss after the accelerated decay tests were evaluated by analysis of variance (ANOVA) at 95% of confidence level. When the null hypothesis was rejected, the mean values were compared with Tukey test at 5% probability of error.

Structural characterization of TiO ₂

The analysis of X-Ray Diffraction (XRD) showed the formation of anatase (PDF 21-1272) and rutile (PDF 21-1276) TiO₂ phases (Figure 1). The nanoparticles presented diffraction patterns of broad peaks and low crystallinity. This enlargement is common in particles with small size (^{Mendes et al. 2012}). The diffractogram shows the crystalline planes of TiO₂ nanoparticles and they confirmed that the presence of anatase phase was predominant. The microwave-assisted hydrothermal method and the presence of SO4^-2 ions resulting from the precursor TiOSO₄ can inhibit the formation of rutile phase, stabilizing the anatase (^{Sun et al. 2014}). An enlargement of the peaks can be observed, in which there is the presence of both phases due to the overlapping of the diffraction peaks of the individual phases. Therefore, besides the instrumental broadening and the size of the TiO₂ nanoparticles, the overlay contributes to this enlargement. This overlap occurred at 25,31° and 27,43° of anatase and rutile phases, respectively. A baseline was drawn to correct the background generated in the diffractogram due to amorphous fraction of TiO₂ phase. The intensity of the peak (200) did not correspond to a preferred orientation of the anatase phase due to this baseline.

Figure 1: XRD patterns of TiO₂ powders synthesized by microwave-assisted hydrothermal method. 

The TiO₂ powders formed a roughly spherical morphology with grains measuring around 50 nm. The enlargement of the diffraction peaks corroborated with the small size of the TiO₂ nanoparticles. Such nanoparticles formed agglomerates with approximately 500 nm (Figure 2), which are typically formed by van der Waals forces. The agglomeration of these nanoparticles came from induced dipoles in the synthesis method, which carry out short-range interactions and can be easily broken by the action of pressure during the impregnation.

Figure 2: SEM images of TiO₂ nanoparticles synthesized by the microwave-assisted hydrothermal method. 

Characterization of TiO ₂ impregnated wood

The appearance of the surface and of the microstructures of coniferous wood is well described in previous studies (^{Wimmer 2002}, ^{Siegloch and Marchiori 2015}). The surface is rough (Figure 3a) and the softwood is basically represented by tracheids with diameter bigger than 10 micrometers (Figure 3a_(ii))_, which characterizes its high porosity. These SEM images illustrated the efficiency of the impregnation of TiO₂ nanoparticles into the pinewood structure due to the size of the tracheids and the nanoparticles. The nanoparticles impregnated by vacuum-pressure were allocated preferably into the surface, forming a homogeneous coating (Figure 3b_(i)), and they are deposited into the tracheids (Figure 3b_(ii)). The interaction of these nanoparticles may be due to physical interactions or hydrogen bonds occurring between the OH present in the polymers of the cell wall and the hydrolyzed TiO₂ (^{Wang et al. 2014}, ^{Pori et al. 2016}). On ther other hand, the simple immersion method was not able to unlock the access to the inner part of the tracheids (Figure 3c_(ii)). Thus, only a superficial coating with low amount of TiO₂ was obtained, since no vacuum-pressure was applied, allowing the nanoparticles decantation. The absence of surfactants contributed to the decantation of the TiO₂ nanoparticles.

Figure 3: SEM images of untreated Pinus elliottii wood (a), wood impregnated with TiO₂ by vacuum-pressure (b) and wood impregnated with TiO₂ bysimple immersion (c). Where: (i) wood surface; (ii) wood tracheids. 

The chemical mapping by EDS (Figure 4) confirmed the effectiveness of the vacuum-pressure method. The application of vacuum and pressure during the wood impregnation with TiO₂ nanoparticles resulted in a higher intensity of the Ti peak compared to the simple immersion method, which the last did not present a significant peak.

Figure 4: Energy dispersive X-ray spectroscopy of Pinus elliottii wood impregnated with TiO₂ nanoparticles using vacuum-pressure (a) and simple immersion (b) methods. 

Color evaluation

TiO₂ impregnation did not change the natural color of Pinus elliottii wood. On the other hand, the wood impregnated with CCB became greenish, limiting their use for esthetic purposes, especially for decoration and furnitures. The TiO₂ nanoparticles did not change significantly the lightness (L*), red-green chromatic coordinate (a*) and hue angle (h) (Table 2). The parameter L* remained stable after the TiO₂ impregnation, while the impregnation with CCB resulted in wood darkening, reducing a* due to the oxidation of copper adsorbed and fixed onto the wood cell wall. The saturation (C*) decreased significantly in the wood treated with CCB, where as its remained constant for wood impregnated with TiO₂.

Table 2: Average values of lightness (L*), chromatic coordinates (a* and b*), chroma (C*) and hue angle (h*) of untreated and TiO₂-impregnated wood. 

*Average values followed by the same letter do not differ statistically from each other by the Tukey's test at 5% of probability of error. **Significant at 5% of probability of error by the F test of the analysis of variance.

Decay resistance

Table 3 shows the mass loss of untreated and TiO₂-impregnated wood. The untreated wood showed high deterioration in its structure. The mass loss reachs 23-32%, and the untreated wood was classified as moderately resistant. The wood impregnated with TiO₂ by vacuum-pressure did not exceed 13% of mass loss, which means a resistant wood. The use of vacuum and pressure for TiO₂ impregnation increased the effectiveness, as well as, the adhesion and the interaction of the nanoparticles with the wood cell wall. The small deterioration of wood impregnated with TiO₂ by vacuum-pressure is due to the physical barrier formed by the adhesion of TiO₂ into the cell wall, avoiding the wood deterioration through the digestive enzymes released by the fungus. The ability of the TiO₂ to assign a hydrophobic character to the surfaces (^{Sun et al. 2010}) should be considered. TiO₂ nanoparticles may have decreased the capacity of water absorption of wood, difficulting the fungal colonization, since these organisms need moisture for their development.

Table 3: Mean values for the apparent specific gravity at 12% of moisture content before and after the fungal attack, and mass loss of untreated and treated Pinus elliottii wood. 

*Average values followed by the same letter do not differ statistically from each other by the Tukey's test at 5% of probability of error. **Significant at 5% of probability of error by the F test of the analysis of variance.

The decay resistance of wood impregnated with TiO₂ by simple immersion was similar to the resistance of untreated wood. The variation of mass loss was from 5 to 32%, remaining in the same class of resistance of untreated wood. However, this variation may be due to the low impregnation efficiency during the immersion step. The absence of vacuum and pressure may difficult the penetration of the nanoparticles inside the wood cell wall, as previously observed in Figure 3. The simple immersion resulted just in a slight surface coating, which helps the easy proliferation of the fungus into the wood cell wall.

The wood samples treated with CCB presented the same degree of deterioration for both vacuum-pressure and simple immersion methods. The mass loss was lower than 6% and the wood was classified as highly resistant. The mass loss of wood impregnated with TiO₂ by vacuum-pressure method did not differ statistically from the wood treated with CCB, proving the efficiency of the TiO₂ nanoparticles to protect wood against white-rot fungus. Nevertheless, from an environmental point of view, the CCB is more toxic than the TiO₂ nanoparticles, which makes the last a good alternative for wood preservation with low environmental impact.

Figure 5a shows the mycelial growth in the untreated wood. The penetration of the hyphae inside the wood cell wall resulted in an increase of the wood roughness. This high deterioration causes greater disintegration of the microfibrils. The mycelial growth and the presence of hyphae in the TiO₂-impregnated wood (Figure 5b and 5c) was lower than in the untreated wood, since no significant changes in the roughness were observed in both cases. These results confirm the effectiveness of TiO₂ nanoparticles to increase the wood decay resistance.

Figure 5: SEM images of untreated wood (a), hyphae penetrating into the untreated wood (b), CCB-treated wood treated no roughness (c) and wood impregnated with TiO₂ by vacuum-pressure no roughness (d) after Ganoderma applanatum attack. 

The TiO₂ impregnation by the vacuum-pressure method was efficient to protect wood against the white-rot fungus under different environments, either in the presence or absence of light. In such cases that wood is deteriorated by fungi, this treatment with TiO₂ nanoparticles can be a good alternative to decrease/avoid fungal proliferation, providing to wood a self-cleaning mechanism. These observations are in accordance with the statements presented by ^{De Filpo et al. (2013}) and ^{Harandi et al. (2016}). Both studies identified significant increase of TiO₂ effectiveness in the presence of light.