Durability of tannin-citric acid modified Scots pine against weathering and fungal exposures

2.1 Tannin impregnation

Pine sapwood samples were pressure-impregnated with a tannin-based solutions using an impregnating plant (Scholz, Maschinenbau SCHOLZ GmbH & Co. KG, Germany) with a capacity of 0.074 m³. The process began with applying a vacuum (−91.19 kPa) to the impregnation chamber for 60 min, followed by filling the chamber with the tannin solution. A pressure of 700 kPa was then applied for 60 min, after which the liquid was drained. The untreated heartwood and sapwood samples served as reference controls. One modification condition was prepared to use sole tannin (code: T). The samples were treated with the impregnation solutions by placing them on a metal grid on their narrow side down with at least a 10 mm gap between them and left to rest at room temperature for 24 h. To prevent cracking, the samples were gradually dried at temperatures of 35 °C, 45 °C, 65 °C, 85 °C, and finally 100 °C for 18 h at each step. They were then cured at 140 °C for 10 h in a drying oven. After treatment, the wood darkened, especially at higher CA concentrations (Figure 1). All samples were conditioned at 20 °C and 65 % RH for four weeks prior to testing.

Figure 1:

Modified wood after curing. Tannin (T) impregnated sapwood (SW) samples with citric acid (CA) concentrations of 0 (T), 2 (TCA2), 10 (TCA10) and 20 (TCA20) wt% of total tannin solid.

2.2 Accelerated weathering test

The modified and unmodified samples with dimensions of 70 mm (width) × 15 mm (thickness) × 300 mm (length) were used for accelerating weathering test conducted using the QUV Accelerated Weathering Tester, QUV/spray (Q-Lab Co., Westlake, NJ, USA), following the standard SS-EN 927-6 (2006). A total of four samples were used per modification condition (n = 24). The QUV system, equipped with the AUTOCAL system, simulates real-world environmental conditions such as sunlight, rain, and dew to assess the weatherability, light stability, and corrosion resistance of products. The test lasted for 672 h, with each complete cycle lasting one week (168 h), consisting of 24 h of condensation at 45 °C, followed by 2.5 h of UV radiation at 60 °C (0.89 W/m²/nm, wavelength of 340 nm), and 0.5 h of water spray (6–7 L/min). The color of the samples was measured using a Chroma Meter CR-410 (Minolta Co. Ltd., Japan) before and after weathering. The CIELAB color system was used to describe color in three-dimensional L^*a^*b^* coordinates, where L^* represents lightness (0 for black, 100 for white), +a^* indicates the red direction, −a^* the green direction, +b^* the yellow direction, and −b^* the blue direction. The color change (∆E^*_ab) was calculated using the following equation:

where ∆L^∗, ∆a^∗, and ∆b^∗ are the color changes after accelerated weathering. The values were measured on the two points of the flat side for each set of samples. Reference marks were used to ensure that the measurements were taken at the same position for each sample. In addition, the number length of cracks on the surface of weathered wood samples was counted and measured under a stereo microscope at 1.5× magnification.

2.3 Contact angle

The contact angle between the wood surfaces and 10 μL distilled water droplets at room temperature was determined at 1, 30 and 60 s through the sessile drop method using A high-resolution drop shape analysis system (DSA 10-MK2, KRUESS, Germany) (model DSA25; KRÜSS GmbH). The charge-coupled device (CCD) camera captured the side view of water drop and the contact angle was measured in the recorded image by using drop shape analysis software, Scientific Drop Shape Analysis Software, DSA 1, Version 1.70 (KRUESS GmbH, Germany). Contact angle was measured on the flat surfaces for all conditioned (20 °C and 65 % RH) specimens (unweathered and weathered) per treatment or control, and at least three locations per specimen.

2.4 Mould resistance

The samples with dimensions of 70 mm (width) × 15 mm (thickness) × 150 mm (length) were prepared for the assessment of mould resistance, according to the method described previously (Ahmed et al. 2013a). A total of eight samples per group were tested (n = 48) in a Memmert HCP 246 humidity chamber (Memmert GmbH, Germany) at 30 °C and 95 % RH by suspending from the top of the chamber with the flat surface set vertical and parallel to the other sample surfaces with a 10 mm gap between in a randomly ordered fashion to minimize the positioning error. Three Scots pine sapwood (P. sylvestris L.) samples naturally infested mainly with Aspergillus, Rhizopus, and Penicillium genera were placed on the bottom of the climate chamber as the sources of mould inocula. After 4 weeks of incubation, the experiment was stopped because of abundant mould growth on some of the sample surfaces. Both flat surfaces of the samples were evaluated visually and graded on a scale of 0 (no infestation) to 6 (extremely heavy infestation).

2.5 Basidiomycete decay test

The resistance of the modified wood against brown rot fungus decay was evaluated in a malt agar incubation test according to SS-EN 113-1 (2020). The modified and unmodified samples of 50 mm (length) × 25 mm (width) × 15 mm (thickness) were incubated for 16 weeks. A total of four replications were used for each group. Samples were oven-dried at 103 ± 2 °C till constant mass and weighed to the nearest 0.001 g to determine oven-dry mass. After steam sterilization in an autoclave at 120 °C for 30 min, sets of two specimens of the same group were placed on fungal mycelium grown on 70 mL malt extract agar in 400 mL Kolle flasks. The brown rot fungus Coniophora puteana (Schumach.) P. Karst was used for the tests. Further, 6 replicates made from Scots pine sapwood were used as virulence controls. All specimens were incubated for 16 weeks at 22 °C and 70 % RH.

After incubation, specimens were cleansed from adhering fungal mycelium, weighed to the nearest to 0.001 g, oven-dried at 103 ± 2 °C, and weighed again to the nearest 0.001 g to determine mass loss through wood-destroying basidiomycetes as follows:

where ML _F is the mass loss by fungal decay (%), M_0,inc is the oven-dry mass after incubation (g), and M₀ is the oven-dry mass before incubation (g).

2.6 Microscopical analysis

The control sapwood, heartwood, and TCA-20 from basidiomycete decay test were selected for microscopic analysis. Small blocks measuring 5 mm (radial) × 5 mm (tangential) × 10 mm (longitudinal) were soaked in water overnight. Thin hand-cut sections approximately 15–20 µm thick were prepared using a Leica microtome blade. These sections were mounted on glass slides and a drop of lactophenol blue (Sigma-Aldrich Sweden AB) was added to stain fungi mycelium, followed by a subsequent wash with water. One drop of glycerin was then added before covering the sections with coverslips. The prepared slides were examined under a motorized Olympus BX63F light microscope (Olympus, Tokyo, Japan) equipped with a DP73 color CCD cooled camera (maximum resolution 17.28 megapixels) and OLYMPUS cellSens Dimension software, version 1.18 (Olympus, Tokyo, Japan).

2.7 Statistical analysis

For statistical evaluation, the IBM SPSS Statistics software package, Version 30.0.0.0 (IBM Corporation, New York, USA) was used to determine differences of color change, mould rating and mass loss due to decay among the treatments. One-way analysis of variance (ANOVA) at 0.05 significance level was applied to determine significant differences. Turkey’s-b multiple comparisons test was performed to separate the effects of the cross linker when significant differences were observed.

3.1 Weathering stability

The weathering stability of the unmodified and modified samples was evaluated by means of color changes and crack formation after 672 h of exposure to an accelerated weathering device. The color of the treated wood was darker with the higher concentration of CA used in the system (Table 2). This could be due to the increasing acidity of the system producing furanic compounds from the wood polymeric carbohydrates (Zhang et al. 2023). After the weathering test, the surface of the specimens became darker. After weathering, the most significant color changes were detected on the wood surfaces of untreated heartwood and sapwood. Heartwood showed a slightly higher color change than sapwood, but the difference was found to be statistically insignificant. A similar trend was also obtained after exposure of sapwood and heartwood to ultraviolet light by Laskowska (2018). Tannin-modified wood with different CA concentrations did not show any specific trend with significant differences but demonstrated significantly lower color changes (ΔE^*) compared to unmodified sapwood or heartwood. Tannin modification without the cross-linker (T) resulted in the least color change (ΔE^*) after weathering; however, the difference was not statistically significant compared to the other modified wood samples treated with varying levels of cross-linkers. Higher CA concentrations resulted in an increased Δa^* value, thus darkening the samples after modification. During weathering, lignin degrades first due to its high UV absorption capacity (Yalcin et al. 2017). Degradation products of lignin are conjugated ketones, aldehydes, and quinones, which make the weathered surface darker (Kanbayashi et al. 2018). Those degraded products are washed away from the surface during the condensation cycle and leave cellulose as the primary component, which exhibits greater color stability against UV light (Kránitz et al. 2016). Therefore, the color of untreated sapwood, heartwood and different TCA-treated wood faded, exhibiting a decrease in ΔL^* value after 672 h weathering (Table 2). The total color change (ΔE^*) is a crucial parameter for evaluating the color stability of weathered samples. After weathering, all color coordinates in the modified samples decreased, likely due to the tannin leaching during the condensation cycles. However, wood treated with or without CA, demonstrated better resistance to color change after the weathering process. Tannin-impregnated wood samples had more stability against discolorations agains artificial weathering and this experimental results are in line with Tondi et al. (2012).

Tannic acid stabilizes wood during weathering by absorbing UV light through its hydroxyl groups, converting it into heat, and scavenging free radicals to prevent degradation (Wang et al. 2023). However, this process leads to the hydrolysis of tannic acid into gallic acid and the formation of quinone structures, consuming tanninc acid over time and thus resulting the formation of chromophores causes a darker color (Peng et al. 2021).

After 672 h of weathering, longitudinal microcracks were observed on the exposed surface (Figure 2). The length and number of these microcracks decreased as the CA concentration in the formulation increased. The highest number and length of the crack were observed in the unmodified sapwood sample with respective average values of 22.5 and 19.3 mm and the lowest was detected in TCA20- treated wood with respective values of 11.6 and 13.0 mm. The phenomenon of crack formation is associated with the photodegradation of lignin, which acts as the binder of cellulose microfibrils in wood cell wall layers (Huang et al. 2013). Peng et al. (2021) demonstrated that the tannin acid-modified wood exhibited excellent photostability, which effectively reduced the frequency of crack formation. Authors explained that the leaching of degraded products, the exposure of new surfaces, and the appearance of more pits on the tracheids became noticeable. In addition, after 480 h of accelerated weathering, the tannin acid- treated wood showed a flatter surface compared to the control samples, possibly due to the migration of tannin acid particles that filled the holes and microcracks on the exposed surface. As exposure time increased, these particles accumulated and contributed to a coarser surface and significantly related with the weathering duration (Yalcin and Ceylan 2017). Long exposure to artificial and natural weathering thus could lead to higher number of cracks in tannin-hexamine-treated wood (Tondi et al. 2012).

Figure 2:

Surfaces of the sapwood (SW), heartwood (HW) and tannin (T)-modified samples after accelerated weathering for 672 h. Treatment represents citric acid (CA) concentrations of 0 (T), 2 (TCA2), 10 (TCA10) and 20 (TCA20) wt% of total tannin solid. Scale bar = 2 mm.

3.2 Contact angle

Figure 3 illustrates the contact angle of water droplets on different wood samples before and after weathering. Before weathering, the contact angles remain relatively high across all treated wood samples, with minor variations between the treatments. However, a significant decrease in contact angle was observed after weathering, particularly for untreated sapwood and heartwood samples, suggesting increased wettability. In contrast, tannin-modified samples, especially those treated with higher CA concentrations, retain higher contact angles after weathering, demonstrating improved water repellency. Even after 60 s of measurement, tannin-treated wood (10 and 20 wt% CA of total tannin solids) retained contact angle similar to or higher than that measured at 1 s for the control sapwood and heartwood. A higher CA concentration in tannin-treated wood resulted in improved hydrophobicity, and this result aligns with previous findings by Ahmed et al. (2025), which demonstrated better dimensional stability in tannin-treated wood with higher CA concentrations.

Figure 3:

Contact angle of water droplet with the surfaces of unweathred and accelerated weathering samples measured at 1, 30 and 60 s. Treatment represents tannin (T)-modified wood with citric acid (CA) concentrations of 0 (T), 2 (TCA2), 10 (TCA10) and 20 (TCA20) wt% of total tannin solid. The error bars in columns indicate the standard deviations.

Figure 3 clearly illustrates that the contact angle decreases after weathering. Weathering increases the wettability of wood by reducing the water-repellent effect of extractives and degrading its hydrophobic components, such as lignin (Kalnins and Feist 1993). As a result, the wood surface becomes richer in hydrophilic cellulose and hemicelluloses. Additionally, microcracks caused by weathering (Kishino and Nakano 2004) may further enhance wettability by allowing liquids to penetrate more easily into the surface. This effect is particularly evident in the control sapwood and heartwood samples, which display a more hydrophilic surface after artificial weathering. In contrast, TCA-modified wood exhibited improved hydrophobicity after weathering, especially in samples treated with a higher concentration of CA (20 wt% of total tannin solids). This could be attributed to the tannin treatment reducing the wettability of the wood surface while also enhancing resistance to leaching, thereby preventing water from easily penetrating the wood material (Tondi et al. 2013).

The acidic nature of the TCA system, particularly at higher concentrations of CA, is evident from the pH values presented in Table 1. It is well recognized that strongly acidic environments (pH 2–3), as observed in formulations containing 20 % CA, can promote the hydrolysis of polymeric carbohydrates in wood, potentially leading to a deterioration in mechanical strength. Furthermore, the addition of CA may contribute to the hydrolysis of carbohydrate fractions naturally present in the tannin extract, typically ranging from 6.8 to 9.0 % (Bianchi et al. 2015). This reaction could also partially explain the observed color darkening at higher CA concentrations. Recent developments in self-neutralizing CA-based systems offer a promising direction for future work. Notably, Zhou et al. (2025) and Xu et al. (2024) have demonstrated that self-neutralizing formulations in both starch- and tannin-based adhesives can maintain performance while reducing the risks associated with low pH. Incorporating such systems may provide a viable solution to address acidity-related limitations in TCA wood treatments and thus could be addressed to the continuation of this current research work.

3.3 Durability against mould

The mould growth rate was evaluated visually after 4 weeks of testing and graded from 0 to 6 (Figure 4). The results showed that the mould growth was apparently low in unmodified heartwood and followed by unmodified sapwood. The unmodified control samples showed statistically lower mould growth rates than the modified samples (ANOVA, α = 0.05). Although the mould growth was slightly decreased with increasing the crosslinker, the differences between the TCA-modified wood samples were statistically insignificant.

Figure 4:

Mould surfaces (top) and grades (bottom) of untreated sapwood (SW), heartwood (HW) and tannin (T)-modified samples. Treatment represents citric acid (CA) concentrations of 0 (T), 2 (TCA2), 10 (TCA10) and 20 (TCA20) wt% of total tannin solid. Mean values followed by different letter within each group indicate that there is a significant difference (P ≤ 0.05) as determined by ANOVA and Turkey’s-b multiple comparisons test. The error bars in columns indicate the standard deviations.

The mould resistance of pine and spruce heartwood was reported previously by Ryparová and Rácová (2024) and Lie et al. (2019), who discussed the low amount of nutrients as a major reason for limited mould growth in the heartwood. In contrast, sapwood and also tannin may contain low-molecular-weight sugars such as glucose, fructose, and sucrose depending on the extraction process (Khanbabaee and van Ree 2001; Sharma 2019; Terziev et al. 1993), which can provide nutrients for mould and determines the intensity of mould infestation (Ahmed et al. 2013b). The higher amounts of low-molecular-weight sugars in the outer sapwood, which gradually decrease towards the innermost sapwood and heartwood (Saranpää and Höll 1989), contribute to greater susceptibility to mold in sapwood. In contrast, TCA-modified wood exhibited even greater mold growth than both sapwood and heartwood, possibly due to the presence of low-molecular-weight sugars in tannin.

3.4 Durability against decay fungi

The decay resistance of wood samples was assessed after 16 weeks of incubation against the brown rot fungus C. puteana (Figure 5). The unmodified pine heartwood was used for comparison purposes and served as an additional control. The unmodified sapwood showed a mass loss mean value of 54.9 %, which is considerably higher than the 20 % threshold value that is commonly used for validation of the fungal activity according to the standard EN 113-1 (2020). Although the unmodified heartwood showed an average mass loss of about 35.1 %, which was significantly lower than the sapwood samples, it was still considerably high. This is particularly in comparison with tannin-modified samples with expressively low mass loss. The mass loss values of the unmodified samples, both sapwood and heartwood, found in this experiment are in agreement with previous findings (Metsä-Kortelainen and Viitanen 2009; Sehlstedt-Persson and Wamming 2010). A slightly higher resistance of heartwood to brown rot fungal decay than sapwood might be due to the presence of phenolic compounds like stilbenes pinosylvin and pinosylvin monomethyl ether, resin acid and other extractives (Lu et al. 2016; Venäläinena et al. 2003). The differences between unmodified and tannin-modified samples were statistically significant (α = 0.05). No significant mass changes were observed among the modified samples by the addition of cross-linking agent, ranging from 2.6 % in sole tannin-modified samples to 3.7 % in TCA-modified wood samples. In comparison to sorbitol-CA treatement, higher concentration (10 %, 20 %, 30 % and 50 %) leads to lower mass loss of modified Scots pine wood sample from 26 to 2 % (Kurkowiak et al. 2023). The enhanced durability of SorCA-treated wood is attributed to the cell wall bulking and the cross-linking sorbitol-CA polyesters with wood polymers (Kurkowiak et al. 2021). Higher concentration of CA in the tannin formulation increased the bulking and moisture realetd properties of modified pine as reported previously (Ahmed et al. 2025).

Figure 5:

Mass loss of untreated sapwood (SW), heartwood (HW) and tannin (T)-modified samples with the brown rot fungus Coniophora puteana. Treatment represents citric acid (CA) concentrations of 0 (T), 2 (TCA2), 10 (TCA10) and 20 (TCA20) wt% of total tannin solid. Mean values followed by different letter within each group indicate that there is a significant difference (P ≤ 0.05) as determined by ANOVA and Turkey’s-b multiple comparisons test. The error bars in the columns indicate the standard deviations.

The antifungal resistance of brown rot fungi (Fomitopsis palustris and Gloeophyllum trabeum) in Scots pine samples modified with mimosa (Acacia mollissima) and quebracho (Schinopsis lorentzii) bark extracts was observed by Tascioglu and cowrkers (2012). The high antifungal activity was attributed to the presence of a high concentration of condensed tannins in these extracts. Condensed tannins make a covalent bond with hemicellulose, which strengthens or plasticizes the cell walls and improves the strength, fire resistance and durability of wood (Bariska and Pizzi 1986; Thévenon et al. 2009). The decay resistance of tannins against wood-decaying organisms is attributed to their phenolic structure (Venäläinena et al. 2003), which undergoes minimal degradation in brown rot fungi (Zabel and Morrell 1992). Brown rot fungi primarily degrade carbohydrates while leaving behind modified lignin during the decay process. They utilize both enzymatic and non-enzymatic depolymerization mechanisms, including the Fenton reaction (H₂O₂/Fe³⁺) (Hosseinpourpia and Mai 2016a,b,c). Coniophora puteana is able to break down the structural carbohydrates mainly by hydrolytic enzymes, with cellulose degradation facilitated by endo-1,4-β-glucanase, exo-1,4-β-glucanase, and 1,4-β-glucosidase, while hemicellulose degradation involves endo-1,4-β-xylanase, 1,4-β-xylosidase, their mannan equivalents, and several acetyl esterases (Zabel and Morrell 1992). Tannins enhance decay resistance by forming soluble or insoluble complexes with fungal proteins (Hart and Hillis 1972; Laks et al. 1988). Dirol and Scalbert (1991) proposed that these complexes may inactivate fungal enzymes such as cellulases or reduce enzyme accessibility to wood cell-wall polysaccharides. In addition, the authors suggested another potential antifungal mechanism involving tannins complexing with essential metal ions, such as iron, which fungi require for metabolic processes. Tannin treatment may inhibit the Fenton reaction by interacting with ferric ions in the wood, thereby further disrupting fungal degradation pathways (Tomak and Gonultas 2018), although the crosslinked tannin complex may react differently. In addition, moisture exclusion through the reduction of cell wall voids lowers the maximum moisture capacity of the wood. This limits the space available for water molecules, thereby restricting the diffusion of fungal reductants and impeding the formation of hydroxyl radicals. This may consequently prevent the penetration of hydrolyzing enzymes into the wood cell walls (Thybring 2013).

After 16 weeks of incubation, distinct differences were observed between the unmodified and modified wood samples (Figure 6). An apparent shrinkages were observed in unmodified samples due to the formation of numerous cracks and clefts within the secondary wall. The mass loss in sapwood was approximately 1.5 times greater than that of heartwood. Latewood cells exhibited relatively higher resistance (Figure 6c), which appears to be linked to their greater degree of cell wall lignification. This lignification hinders the diffusion of cellulolytic enzymes into the cell wall, thereby increasing resistance to decomposition (Schwarze et al. 2003). However, C. puteana also reported for the degradation of all cell wall layers including lignin-rich middle lamellae for the production of laccase activity (Lee et al. 2004), and thus, the unmodified wood showed considerably higher weight loss than the tannin-modified samples. The high extractive content in heartwood (Khviyuzov et al. 2021) might also be a reason for restricting the wood decay (Martínez-Iñigo et al. 1999). Microscopic observations showed significantly thinner cell walls in the tracheids of both unmodified sapwood and heartwood compared to tannin-modified wood (Figure 6b and c). Although fungal hyphae were visible in the tracheid lumen of tannin-modified wood (Figure 6d), the cell walls remained almost intact, resulting in minimal mass loss.

Figure 6:

Decayed sapwood (SW), heartwood (HW) and tannin (T)-modified wood after 16 weeks of incubation period (top). Treatment represents citric acid (CA) concentrations of 0 (T), 2 (TCA2), 10 (TCA10) and 20 (TCA20) wt% of total tannin solid. Optical micrographs of transverse sections showing undecayed sapwood (a), earlywood of decayed unimpregnated SW (b), latewood of HW (c), and tannin-treated wood with a citric acid (CA) concentration of 20 wt% of the total tannin solids on earlywood cross (d), radial (e) and tangential (f) sections. Black arrowheads showing fungal mycelia inside the cell lumen. White arrowhead showing cured tannin inside the cell lumen of tracheids. Scale bar = 50 µm.