Bio-Durability and Engineering Characteristics of Heat-Treated Poplar Wood Biološka trajnost i tehnička svojstva toplinski modificirane topolovine

• The aim of this study was to evaluate the effect of brown rot fungus Coniophora puteana activity on physical and mechanical properties as well as biological resistance of heat-treated poplar wood. Two poplar wood species (Populus deltoids and Populus nigra) were heat-treated by thermo-wood (Thermo-D) method. Control and heat-treated specimens were exposed to brown rot fungus C. puteana for 16 weeks. Physical and mechanical characteristics of specimens including density, compression strength parallel to the grain and impact strength were evaluated before and after exposure to fungus. Mass loss of specimens caused by fungal activity (MLF) was also calculated. In addition, the effect of thermal modification on laccase production by C. puteana was assayed. The highest mass loss due to fungal deterioration was observed in control specimens, coinciding with the highest substrate-enzyme interactions and constant decrease in detectable laccase levels. According to the results, thermal modification can be used effectively to protect poplar wood against brown rot fungus attack.


INTRODUCTION 1. UVOD
Wood as a natural polymer has been widely used in structures for many years.However, some properties of this natural material such as moisture absorption, dimensional instability, biological degradation, weathering, etc., limit its use.So, numerous new methods of wood modification have been developed to remove these disadvantages and expand its functionality.Wood heat treatment is one of the modification methods, which is carried out by different processes.This kind of modification was first studied scientifically by Stamm and Hansen in the 1930s in Germany and in the 1940s by White in the United States.The most comprehensive research in this field has been carried out by VTT (International Thermo-Wood Association) in Finland, whose product is called thermo-wood.A temperature of 160-260 °C is usually used to produce thermowood (Militz, 2002).Thermal modifications have some positive and negative effects on wood.In some cases, thermal treatments lead to positive changes in chemical structure of wood, colour, dimensional stability, thermal insulation properties, biological resistance to biological degradation.However, these treatments also reduce some mechanical properties such as wood bending strength.
Heat treatment of wood in the temperature range of 140-260 ºC with a long holding time causes an irreversible reduction of moisture absorption (González-Peña et al., 2004; Obataya and Tomita, 2002); it leads to dimensional stability (Krause et al., 2004), less moisture movement (Militz and Tjeerdsma, 2001), and improved biological resistance (Welzbacher and Rapp, 2004;Farahani et al., 2001).However, one of the major drawbacks of these treatments is the reduction of wood mechanical properties (González-Peña and Hale, 2007).
Brown rot fungi have an enzyme system that produces hydrolytic and oxidative enzymes, which act on cell wall component degradation such as lignin, cellulose and hemicelluloses (Baldrian and Gabriel, 2003).
Evaluation of heat treatment effect on bio-durability of a wood species showed that it significantly increases wood durability against fungal action due to the reduction of hydroxyl groups (Mburu et al., 2007).Kamperidou (2019) evaluated the biological durability of thermo-chemically modified pine and spruce wood against white and brown rot fungi.The results showed that thermal modification presented lower mass loss caused by used fungi compared to the unmodified wood.In other words, heat treatment increases the biological resistance of wood.
According to Gao et al. (2016), the colour of wood became darker and there was an improvement in dimensional stability as well as reduction in modulus of elasticity of heat-treated poplar wood at temperatures of 140 to 200 °C for 1-3 hours.In addition, biodurability of heat-treated specimen was better against white rot fungus than brown rot fungus.
Furthermore, Corleto et al. (2020) investigated the effect of thermal modification on properties of padauk wood, indicating that heat treatment at high temperature caused significant loss in bending strength and bending stiffness of padauk wood.Moreover, the results of chemical analysis showed that cellulose and lignin proportion increased, while that of hemicellulose decreased substantially following thermal modification.Another study showed that durability and dimensional stability of European birch, European aspen, Norway spruce as well as Scots pine improved by thermal modification (Karlsson et al., 2011;Militz and Altgen, 2014).Kaygin et al. (2009) investigated the effect of mass loss of mechanical properties of heat-treated Paulownia at 160, 180 and 200 °C.The results showed that mechanical properties including compression strength, modulus of elasticity, bending strength and impact strength decreased with increasing of heat treatment temperature.In fact, it was determined that, due to thermal modification, the mass loss significantly affected mechanic properties of Paulownia wood.
Heat treatment can be used effectively against fungal attack for Scotch pine, oak and beech wood species (Ayata et al., 2017).At high temperature of wood thermal modification, the lignin content increases because of autocondensation and its higher thermal stability.There is a slight decrease in the extractives content.Moreover, the cellulose and its DP decrease, while cross-linking reactions occur in the cellulose (Čabalová et al., 2019).
Brown rot fungi have the ability to degrade cellulose by producing extracellular fungal cellulose degrading enzymes.The extracellular enzymes produced by these fungi are greatly affected by the type of substrate being decayed.Our study focuses on measuring oxidative enzyme laccases secreted by C. puteana in the presence of poplar woods (control and heat-treated specimens) as substrates.Therefore, this study aimed to find the effect of heat treatment on biological resistance of thermo-wood against degradation by brown rot fungus Coniophora puteana.Meanwhile, physical and mechanical properties of thermo-wood and control specimen after exposure to C. puteana were investigated.In addition, levels of laccase enzyme on both substrates (control and modified specimens) were assayed.

MATERIJALI I METODE
The lumbers of two poplar wood species (Populus deltoids and Populus nigra) were prepared and treated in Mazand wood company.After kiln loading, the temperature was rapidly raised to a level of around 100 ºC and then increased steadily to 130 ºC.Thereafter, the temperature inside the kiln was increased to around 212 °C and remained constant for 3 hours.The final stage was cooling and moisture conditioning by using water spray system.When the temperature reached 80 ºC, re-moisturizing took place to bring the wood moisture content to a level of around 6 %.In order to evaluate biological resistance as well as physical and mechanical properties of treated timbers, six specimens were prepared in accordance with EN 113-2:2020 standard.
In this study, brown rot fungus Coniophora puteana was used to evaluate biological resistance of control and modified specimens.Modified and control specimens were exposed to fungal mycelium in Kolleflasks.As a nutrient medium for fungal mycelium, Malta Extract Agar (Merck KGaA, Darmstadt, Germany) was used.After preparing nutrient medium and sterilization in autoclave (temperature 120 °C, time 20 minutes, pressure 1.5 kg/cm 2 ), small pieces of fungal mycelium were inoculated in Kolle flasks.Then the flasks with nutrient medium and mycelium were incubated in the climate chamber at a temperature of 22 °C and 65±5 % relative humidity for 2 to 3 weeks until the entire surface of the nutrient medium was overgrown by mycelium (as shown in Figure 1).Six modified and control specimens were placed in each flask and were incubated in the same climate chamber for another 16 weeks according to EN 113-2: (2020) standard (Figure 1).After 16 weeks, all Kolle flasks were taken out from the climate chamber.
Ligninolytic enzyme laccase was extracted at 25 ºC in two extraction cycles, 5 and 24 hours, with 50 mM sodium acetate buffer (pH=5.5)with 0.1 g/L Polysorbate 20 (Tween ® 20; from Sigma Aldrich: Steinheim, Germany).It should be noted that two extraction cycles of 5 and 24 hours were used to obtain the optimal time for maximum enzyme extraction.An amount of 1.5 gr chips prepared from decayed specimens were first soaked in 50 mL extraction buffer for 5 hours.The second extraction was performed with 25 mL extraction buffer for 24 hours.For this purpose, six repetitions were considered.Then, the supernatants collected from all extractions were filtered, centrifuged at 5.000 rpm for 15 minutes and used to test enzyme activity.
Laccase activity was assayed by measuring the oxidation of 2,6-DMP.An aliquot of enzyme solution was incubated in 1 ml of 5 mM of 2,6-Dimethoxyphenol (2,6-DMP) in 0.1 M sodium acetate buffer (pH= 3.6) at 30 ºC.Absorbance was monitored at 469 nm in a spectrophotometer.One unit of laccase activity was defined as the amount of the laccase that oxidized 1 μmol of 2,6-DMP per minute at 30 ºC (Field et al., 1993).
In order to evaluate the durability of wood specimens against brown rot fungus, the mass loss was calculated.After 16 weeks of incubation, the wood specimens were taken out of the Kolle flasks and carefully cleaned from the mycelium and then oven dried for 24 hours at the temperature of 103±2 °C until the constant mass was reached, and then weighed.The mass loss and the repellent effect of thermal modification on MLF of each specimen were calculated according to equation 1 and 2, respectively: M.L= ((D 1 -D 2 )/D 1 ) ×100 (1) Where: M.L -mass loss (%) D 1 -oven dry mass of specimen before exposure to fungi (g) D 2 -oven dry mass of specimen after exposure to fungi (g) R.E= ((M.L 1 -M.L 2 )/ M.L 1 ) ×100 (2) Where: R.E -repellent effect of thermal modification on MLF (%) M.L 1 -mass loss of control specimen caused by fungi (%) M.L 2 -mass loss of heat-treated specimen caused by fungi (%) In this research, only the properties that could be determined based on the specimens that can be placed in Kolle flasks, based on standard dimensions, were considered.The physical and mechanical properties of control and heat-treated specimens were tested according to ASTM D 143-09.Also, the standard ASTM D256 was used to measure impact strength, because the dimensions of the specimens in this standard were suitable to be placed in the Kolle flask.Moreover, the standard EN 113 was used to evaluate bio-durability.It should be noted that 6 replications were considered for each parameter.The obtained data from the tests were statistically analyzed based on One-Way ANOVA method.This method was used to determine any statistically significant differences between the means of the two groups (control and modified specimens) for each wood species separately.

REZULTATI I RASPRAVA
In this research, the physical and mechanical properties of two wood species (P.deltoids and P. ni-gra) were evaluated in two stages: the first after thermal modification and the second after fungal degradation to show the effect of heat treatment on fungal functionality as well as physical and mechanical properties.Table 1 summarizes the results of statistical analysis of One-Way ANOVA test for physical and mechanical properties of two poplar wood species, specifying significant levels.The numbers presented in Table 1 show whether the heat treatment had a significant effect on the investigated properties.

Procjena pojave lakaze
The results of laccase evaluation of specimens after 16 weeks of exposure to C. puteana showed that the laccase value in controls was lower than in heattreated specimens in both poplar wood species (Figure 2).In fact, the reduction of this enzyme in controls compared to thermo-wood indicates greater consumption and effectiveness of this enzyme in untreated specimens.As heat treatment reduces the amount of hemicellulose and holocellulose, hydroxyl groups, which are an active site for chemical reactions, are reduced.As a result, the activity and effectiveness of laccase enzyme produced by brown rot fungi in modified wood is reduced (Gaff et al., 2019;Mburu et al., 2007;Wentzel et al., 2019).Faraz et al. (2003) reported that, when Eucalyptus grandis wood chips were exposed to Ceriporiopsis subvermisporaon, the greatest mass loss was directly associated with a significant reduction of laccase enzyme, which supports the results of this study.

Gubitak mase zbog djelovanja gljiva (MLF)
The results revealed that heat treatment had a significant effect on the decay action of brown rot fungi, leading to mass loss (Table 1).The MLF in thermowood specimens were significantly lower than in control specimens.This indicates the inhibition of heat treatment on fungal activity (Figure 3).As indicated in Figure 3, the repellent effect of thermal modification on MLF obtained 72.28 and 82.69 % in P. deltoids and P. nigra, respectively.On the other hand, as shown in Figure 2, laccase activity in heat-treated specimens is greater than in controls, which is a sign of thermowood resistance against fungal degradation.Considering the substrate-enzyme interactions, it seems that in the control specimens, laccase enzyme had more interaction with the substrate and therefore the mass in these specimens was reduced to a greater extent compared to the treated specimens.According to the results of this study, there are other reports that show the deep adsorption of laccases in substrates suitable for their degradation (Tu et al., 2009).Moreover, improvement of bio-durability of thermo-wood against fungal degradation is related to the reduction of hydroxyl groups due to heat treatment (Mburu et al., 2007).

Gustoća
This physical property was evaluated as follows.

After thermal modification 3.3.1. Nakon toplinske modifikacije
The results showed that heat had a significant effect on the density 1), so that the thermo-wood process reduced the density of both wood species (Figure 4).The reduction of density due to heat treatment in P. deltoids and P. nigra was 8.81 and 5.17 %, respectively.The change of wood chemical structure at high temperature causes mass loss, which leads to density decrease.The reduction of density due to heat treatment is related to destruction and change of chemical structure of wood, which intensifies at high temperatures (Militz, 2002).

After fungal degradation 3.3.2. Nakon razgradnje gljivama
After density assessment of control and heat-treated specimens in section 3.3.1,these specimens were exposed to brown rot fungus C. puteana.The results showed that heat treatment had no significant effect on fungal functionality in reduction of density in both poplar wood species (Table 1).As shown in Figure 4, the density difference between the control and thermo-wood specimens is little in both poplar species.However, heat treatment has been able to reduce the activity of brown rot fungus in both wood species, so that the density reduction due to fungal action in thermo-wood specimen was less than in controls (Table 2).This indicates that heat treatment has a repellent effect on fungal activity.

Compression strength parallel to grain Čvrstoća na tlak paralelno s vlakancima
This mechanical property was evaluated as follows.

After thermal modification 3.4.1. Nakon toplinske modifikacije
The results showed that thermal modification had no significant effect on the compression strength parallel to the grain of P. nigra but it did in P. deltoids (Table 1).However, as indicated in Figure 5, heat treatment has improved this mechanical property in both wood species, so that the value of compression strength parallel to the grain in P. deltoids and P. nigra was 27.46 and 11.21 %, respectively (Figure 5).As a matter of fact, wood chemical structure changes at high temperature during thermal modification.Consequently, heat treatment causes cross-linking of lignin as well as increasing of lignin and cellulose values in wood structure, which leads to the improvement of compression

After fungal degradation 3.4.2. Nakon razgradnje gljivama
The results revealed that heat treatment had no significant effect on fungal efficiency in terms of compression strength parallel to the grain (Table 1).However, comparison of the results in Figure 5 shows that this kind of modification was able to reduce the activity of brown rot fungi in both wood species.Consequently, the reduction of compression strength parallel to the grain due to fungal activity thermo-wood specimen was less than that in the control, which indicates a repellent effect of heat treatment on fungal activity (Table 2).As shown in Figure 2, laccase activity in heattreated specimens was more than that in the control group, which indicates bio-durability of thermo-wood.Heat treatment of wood at high temperatures reduces hydroxyl groups, which leads to the reduction of enzymatic hydrolysis (Mburu et al., 2007).

Impact strength Čvrstoća na udarac
This mechanical property was evaluated as follows:

After thermal modification 3.5.1. Nakon toplinske modifikacije
The results revealed that heat treatment had a significant effect on impact strength of both wood species (Table 1).Thermal modification had negative effect on this mechanical property in both species, as shown in Figure 5.The reduction of impact strength was 8.24 and 31.91 % in P. deltoids and P. nigra, respectively (Figure 6).The reduction of impact strength of thermowood might be related to brittle structure of heat-treated specimens.In fact, the reduction of this mechanical property is directly related to chemical structure changes, while mass loss of specimens occurs due to heating (Kaygin et al., 2009).

After fungal degradation 3.5.2. Nakon razgradnje gljivama
The results showed that heat treatment had a significant effect on fungal action in terms of impact strength (Table 1).Although thermal modification can reduce the activity of the brown rot fungi, the reduction of impact strength in thermo-wood exposed to brown rot fungi was greater than in control group (Table 2).The effect of heat treatment on structural changes of wood as well as destruction of cellulose and holocellulose by brown rot fungi can be effective agents in the reduction of impact strength of thermo-wood specimens.

ZAKLJUČAK
The results of the present study revealed that the degradation of two poplar wood species (P.deltoids and P. nigra) by C. puteana could be affected by thermal modification.Although the activity of C. puteana and laccase production occurred in both control and heat-treated specimens, the lowest detectable laccase levels were observed in controls, coinciding with the highest mass loss.Considering the substrate-enzyme interactions, it seems that in the control specimens, laccase enzyme had more interaction with the substrate and therefore the mass in these specimens was reduced to a greater extent compared to the treated specimens.Therefore, the result of laccase activity assessment also showed that the laccase value in controls was less than

Table 2
Average reduction values of physical and mechanical properties due to fungal degradation Tablica 2. Prosječne redukcijske vrijednosti fizičkih i mehaničkih svojstava zbog razgradnje gljivama Effect of thermal modification on impact strength and its variation under fungal attack Slika 6. Utjecaj toplinske modifikacije na čvrstoću na udarac i promjene te čvrstoće zbog napada gljiva that in modified specimens.Overall, the results revealed that thermal modification improves bio-durability of both poplar wood species; however, it reduces some mechanical properties.On the other hand, this kind of modification prevents the reduction of mechanical properties due to biological degradation.terial from industrial scale processes.In: Proceedings of 35 th Annual Meeting, International Research Group on Wood Protection, IRG/WP 04-40279.25.Wentzel, M.; Fleckenstein, M.; Hofmann, T.; Militz, H., 2019: Relation of chemical and mechanical properties of Eucalyptus nitens wood thermally modified in open and closed systems.Wood Material Science & Engineering, 14 (3): 165-173.https://doi.org/10.1080/17480272.2018.1450783 26.***EN 113:1997 Wood preservatives.Test method for determining the protective effectiveness against wood destroying basidiomycetes.27.*** ASTM D 143-09:2014 Standard methods of testing small clear specimens of timber.28.*** ASTM D 256:2018 Standard test methods for determining the Izod pendulum impact strength of plastics.
Corresponding address:REZA HAJIHASSANI Wood and Forest Products Science Research Division, Research Institute of Forests and Rangelands, Agricultural Research Education and Extension Organization (AREEO), Tehran, IRAN, e-mail: Reza.hajihassani@gmail.com