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The Resistance of Modified Manii Wood with Boric acid and Chitosan/Glycerol and Heating Against Fungi and Termites

Manii wood (Maesopsiss eminii Engl.) is a fast grown species that has low durability (class IV) and requires preservation. Boric acid is generally used in wood preservation. However, its application needs to be combined with other treatments to prevent from leaching. This study aimed to evaluate the resistance of manii wood after double impregnation of boric acid and chitosan or glycerol and heat treatment. Heating temperatures were 70ºC and 140ºC. The durability test was carried out against white rot fungus (Schizophyllum commune), dry wood termites (Cryptotermes cynocephalus), subterranean termites (Coptotermes curvignathus) according to SNI 7207-2014 standard, and field test based on ASTM D 1758-08 standard. The retention of boric acid in manii wood was 15,2 ± 1,0 kg ma−3. The interaction of boric acid and chitosan/glycerol impregnations and heat treatment significantly affected the wood resistance against decay fungi and termites. Impregnation of boric acid and glycerol by heating at 140ºC increased the resistance of wood against dry wood and subterranean termites. Double impregnation of boric acid and chitosan followed by heating at 140ºC was the best treatment in this research that improved significantly the resistance of manii wood against white rot fungi, dry wood termites and subterranean termites.


Introduction
The need of wood is increasing along with population growth, especially high quality wood that is easy to process, and resistant to attacks by degrading organisms. Most of commercial timber (80-85%) on the market has low durability, especially fast grown woods from plantation and community forests. Low durability woods are was easily attacked by degrading organisms [1]. According to Jasni among 4000 of wood species in Indonesia about 15% are high durability wood (I and II) while the remaining 85% are undurable wood including class III-IV and V [2].
Manii wood (Maesopsis eminii Engl.) is one of the fast grown species and can be used as building material. Manii wood has almost white sapwood and a yellowish-brown gold heartwood, has a rather rough wood texture and crossed fibers [3]. Manii wood is widely available in the market, but its durability is low hence its service life is short [4]. The resistance of manii wood against Schizophyllum commune belongs to the durability class IV [5]. Therefore, preservation is very important to a longer service life. In addition, other alternatives to improve low durability is by wood modifications.
Wood modification is a promising method for improving the quality of fast grown species. The wood modification by furfuryl alcohol impregnation can improve the dimensionaal stability of wood [6]. Impregnation with boron and MMA improved the dimensional stability and durability of wood [7]. Wood modifications generally aims to reduce the various weaknesses of low-quality wood. The modifications used in this study were impregnation and heat-treatment. Impregnation method is a 2 technique of wood modification by using pressure that facilitates a solution penetration into the wood and produces better retention and penetration [8]. Chemicals penetrate into the vessel, lumen, or wood cell wall. The chemical impregnation fills the cell walls and forms bonds [7].
This study applied double impregnation with boric acid and chitosan or glycerol. Boric acid (H3BO3) is a widely used boron-based preservative [9]. Boric acid is odorless, does not change wood color, has fungicide and insecticide effect [10]. However, boron preservative is leachable by water [11]. Tomak et al. revealed that boron compounds were leached from the wood in 14 days of test so that the durability of wood was almost the same as the control [12].
Chitosan has the potential as an environmentally friendly wood preservative, abundant, cheap, antibacterial, and antifungi Zivonic et al. [13]. Some researchers have tested the antifungal properties of chitosan. Chitosan inhibited the growth of Penicilium digitatum [14]. Salman showed that 2% chitosan treatment inhibited the highest growth of Aspergillus foetidus [15]. According to Oldertroen et al. the existence of chitosan in wood can inhibit the growth of staining fungus, and stable in wood [16]. Glycerol can be used as preservatives, solvents, and plasticizers [17]. Glycerol can increase the calorific value of wood [18]. In addition, Dauvergne et al. said that the combination of boron and glycerol can reduce the growth of Poria placenta in pine wood [19].
Heat modification can increase the biological resistance of wood against fungal attacks, decrease hemicellulose, reduce hygroscopic properties, and increase dimensional stability [20]. Wood heattreatment temperatures are generally above 100°C, or between 150-230°C [21]. Heat-treatment improved wood quality with decreasing hygroscopic properties of wood, improved stability, and increased wood durability [22]. Heating is one of wood modification techniques that is environmentally friendly [23]. It can improve wood dimensional stability and resistance against wood degrading organisms [24,25].

Materials preparation
Manii wood (Maesopsis eminii Engl.) were cut from a tree that had height of 15 meters and a diameter of 50 cm in the arboretum of the Faculty of Forestry and Environment, IPB University. Manii log's were cut using circular table saw to be some board's with a thickness of 6 cm. The boards were dried in an experimental kiln at 50°C to gain moisture content of 14%. The sample size for the test of white-rot fungus (Schizophyllum commune), dry wood termites (Cryptotermes cynocephalus), subterranean termites (Coptotermes curvignathus), and field test were 5 cm × 2.5 cm × 1.5 cm, 2.5 cm × 2.5 cm × 0.5 cm, 2.5 cm × 2.5 cm × 5 cm and 2 cm × 2 cm × 37 cm, respectively. Each test used five replications. Boric acid, chitosan, and glycerol were used as impregnantion materials. Boric acid solutions were made at 5% concentration for impregnation I and chitosan 2% or glycerol 10% for impregnation II.

Impregnation process and heat treatment
The sample tests were dried in the oven at a temperature of 40°C until 9% moisture content (MC). The impregnation treatments were in two stages then followed by heat treatments at temperatures 70°C or 140°C for 4 hours, as described in Table 1. Impregnation of boric acid was conducted in a pressurized tank at 7 kg cm -2 for 4 hours. Subsequently, the samples were drained and weighted to calculate the retention of boric acid. After that, the impregnation of chitosan was carried out in a pressurized tank at 6 kg cm -2 for 2 hours Casado-sanz et al. while glycerol impregnation at 7 kg.cm -2 for 4 hours [26,19]. Heat treatments were conducted after impregnation II at temperatures of 70°C or 140°C for 4 hours in a heating oven. Before and after the impregnation II, the sample were oven-dried at 103±2°C to calculate the value of weight percent gain (WPG). -

White-rot fungal resistance testing
Sample tests were dried at 70ºC to 9% MC and weighed (W0). Then, the sample test were wetted using aquades for 1 minute, then plastic wrapped, and put in the microwave for sterilization in 1 minute. Furthermore, the sample test was inserted into a petri dish PDA culture with mycelium of white-rot fungus (Schizophyllum commune). The inoculation was perfomed aseptically in laminar air flow. Incubation was carried out for 12 weeks at temperature of ±25°C. The test sample was removed and cleaned from attached mycelium, then ovened at 103±2ºC to a constant weight and weighed (Wk1). Futhermore, of the initial dry weight (Wk0) and weight loss (WL) were calculated by the equation (1) and (2)

Dry Wood Termites Resistance Testing
Sample tests were oven dried at 70ºC to 9% MC and weighed (W0). Paralon tubes were wet sterilized using autoclaves at a temperature of ± 121°C for 15 minutes the tube end was glued on a wood surface. After that, 50 dry wood termites (Cryptotermes cynocephalus) were put in each paralon tube. Then the test was kept in a dark room for 12 weeks and observed every month. The samples were cleaned and dried in the oven at 103±2ºC and weighed (Wk1). Futhermore, the initial dry weight (Wk0) and weight loss (WL) of the samples were calculated by equation (1) and (2).

Subterranean termites resistance testing
Wood samples of 2.5 × 2.5 × 0.5 cm were oven dried at 70ºC to ±9% MC and weighed (W0). The test sample was put into a glass jar by standing at the base, and 200 grams of moist sand having 9% below water holding capacity were added. Furthermore, 200 workers of subterranean termites (Coptotermes curvignathus) were placed. The experimental unit was stored in a dark room. After 4 weeks, the test sample was removed from the glass jar and cleaned from the sand. The wood samples were dried in an oven at 103±2ºC and weighed (Wk1). Futhermore, the initial dry weight (Wk0) and weight loss (WL) of the samples were calculated by the equation (1) and (2).

In Ground Field Test of Biodeterioration
Wood samples of 2 cm × 2 cm × 37 cm were dried at 70°C, weighed (W0) and dimensionally measured. Sengon (Falcataria moluccana) wood was used as comparative samples having durrability class IV. Furthermore, the sample tests were buried randomly with a depth of 2/3 of the sample height in the soil at the Arboretum Faculty of Forestry and Environment IPB University with a distance between woods 30 cm × 60 cm. After three months of exposure period, the samples were cleaned and visually evaluated. The test samples were dried in an oven at 103±2ºC and weighed (Wk1). Initial dry weight (Wk0) and weight loss were calculated using equation (1). The percentage of cross-sectional damage determines the wood biodeterioration grading system due to subterranean termite attacks [27] in table 2. No attack, 1 to 2 small nibbles permited 9 Nibbles to 3% cross-section 8 Penetration 3 to 10% of cross-section 7 Penetration reaches 10 to 30% of cross-section 6 Penetration reaches 30 to 50% of cross-section 4 Penetration reaches 50 to 75% of cross-section 0 Failure

Data analysis
The effect impregnation of boric acid, chitosan or glycerol and heating treatments on the resistance of manii wood were statistically analyzed using a factorial completely randomized design (2 × 3 ×2). The experimental design included three variable factors of treatment with five replications. The factors were preservative (no preservative, boric acid), impregnant (chitosan, glycerol, or without glycerol chitosan), and heating temperatures (70°C and 140°C). Analysis of variance was followed by Duncan test when there was a significant effect at the 95% confidence interval. The data were processed using Microsoft Excel 2013 and IBM SPSS Statistic 26.0

Evaluation of impregnation process
Retention is a success parameter of a preservation process. Boron preservative retention is required at 8.2 kg m -3 for under-roof uses and 11.3 kg m -3 for off-roof uses [28]. The results showed that the retention of boric acid in manii wood was 15.2 ± 1.0 kg m -3 . The retention value of this study was lower than Mursyidah [24] that was 30 kg m -3 in manii wood at 10 kg cm -2 for 6 hours [25]. A high retention related to its molecular weight. According to Yaras the molecular weight and density of boric acid were 61.83 g mol -1 and 1.44, respectively [29]. Manii wood was easily treated boric acid preservative. According to Krisdianto et al. manii wood belongs to treatability class I [5]. Weight percent gain (WPG) is a percentage of additional weight of wood due to impregnation compared to the initial weight of wood. The results showed that WPG of glycerol was higher than that of chitosan in manii wood (figure 1).  The analysis of variance and Duncan test at the 95% confidence interval showed that the interaction of boric acid impregnation and heat treatment had a significant effect on WPG of chitosan/ glycerol in manii wood. Based on Duncan test, WPG of glycerol was significantsign higher than that of chitosan. In addition, the WPG of glycerol in wood previously treated with boric acid (BG70) was significant lower than that untreated one (NG70), while WPG of chitosan in wood contained boric acid (BC) was no different from that without boric acid (NC). Boron treated wood had significant lower WPG of glycerol/ chitosan then untreated wood. Heated wood at 140°C had lower WPG of glycerol and chitosan than heated wood at 70°C. The WPG of glycerol higher than chitosan, which could be caused by the lower viscosity of glycerol than chitosan. According to Pagliaro and Rossi chitosan has a melting point of 264°C and a boiling point of 118°C, while glycerol has a melting point of 20°C and a boiling point of 290°C [30].

The resistance against white-rot fungi
Wood resistance against white-rot fungi was analyzed based on weight loss value. Weight loss of wood indicated damage to the cell walls of both hemicellulose, cellulose, and lignin components due to the attack of white-rot fungi [31]. The results (figure 2) showed that boric acid impregnation as well as heating 140°C generally increased the resistance of manii wood against white-rot fungi. Based on SNI 7207: 2014, heated boric acid treatment at 140°C (BT140) and heated boric acid and chitosan treatment at 140°C (BC140) increased the durability class III of control (NT70) to a durablity class II in the test of decay resistance against white-rot fungi. The results (figure 2) showed that the weight loss of treated manii wood were lower than that of control wood (NT70) which was 3.96%. The lowest weight loss value was 140°C at 0.48%. The analysis of variance at the 95% confidence interval showed that the interaction of impregnation treatment of boric acid, chitosan/ glycerol, and heating exerted a very significant effect (p<0.01) on the weight loss value of manii wood in the resistance test against white-rot fungi. The Duncan test showed that heated boric acid at 70°C treatment (BT70) resulted in significant lower weight loss than control (NT70). This suggests that boric acid was able to withstand the attack of white-rot fungi. Temiz et al. showed that boric acid reduce weight loss (1-2%) on Pine sylvestris when exposed to decay Postia placenta [9]. Dywnda and Zainul stated that boric acid can be used as an antiseptic, insecticide, pH buffer, or neutron absorbent [32].
The Duncan test also showed that the weight loss of wood treated by boric acid, chitosan and heating at 140°C (BC140) was significant lower than control (NT70) and boric acid treatment (BT70). According to Gorgij et al. chitosan 2% has good performance in inhibiting the growth of fungi [33]. Salman also stated that treatment with a 2% chitosan concentration in PDA media showed the smallest diameter growth in the Aspergillus foetidus [15]. The mechanism of action of chitosan in inhibiting the growth of microbes in the presence of interactions between the positive charge on chitosan molecules and the negative charge on microbial cell membranes leads to the release of protein elements and other elements of microbial intracellular constituents [34]. According to Sedjati chitosan has a positively charged amino group that can bind negative charge from other compounds that are different from other neutrally charged polysaccharides [35].
Heating 140°C also increased the protection of boric acid against white-rot fungi as evidenced by lower weight loss in wood treated with boric acid and heating at 140°C (BT140) than wood treated with boric acid and heating 70°C (BT70). This is supported the research of Calonego et al. that heat treatment at 140°C on Eucalyptus grandis wood reduced weight loss by 34.32% in resistance test against Picnoporus sanguineus [36]. Weiland and Guyonnet revealed that the heat treatment on pine and beech woods caused the transformation of hemicellulose from hydrophilic and easily digestible into hydrophobic [37].

The resistance against dry wood termites
In this test, weight loss of wood indicates a termite attack in wood. The higher weight loss value, the less resistant wood of wood against dry wood termites [38]. The results (figure 3) showed that each treatment of boric acid, chitosan or glycerol, and heating in general increased wood resistance against dry wood termite attacks. This was indicated by a decrease in the weight loss value of tested wood. figure 3 showed the treated wood with boric acid and glycerol (BG) was more resistant against attacks  of dry wood termites with lower weightloss value than control (NT70). Based on SNI 7207-2014, the treatment of boric acid, chitosan and heating at 140°C (BC140) as well as the treatment of boric acid, glycerol and heating at 140°C (BG140) in the resistance test against dry wood termites. The analysis of variance at the 95% confidence interval proved that the interaction between boric acid, chitosan/ glycerol and heating temperature had a very significant effect (p<0.01) on the weight loss value of manii wood in the resistance test against dry wood termites. Furthermore, Duncan test showed that the weight loss value of wood treated with boric acid and heating at 140°C (BT140) was significant lower than that of control (NT70). This revealed that boric acid has insecticidal properties against dry wood termites. Boron compounds such as borax (Na2B4O7) and boric acid (H3BO3) are used as insecticidal preservatives [39]. The double impregnation of boric acid and chitosan or glycerol and heating at 140°C (BC140 or BG140) resulted in a significant lower weight loss than the control (NT70). Chitosan is a polysaccharides that can serve a barrier to the entry of protozoa. Batista et al. states that Eucalyptus grandis wood treated with 140°C heat has low resistance to Cryptotermes sp. attacks compared to controls weigth loss value of less than 1 % [40].

The resistance against subterranean termites in laboratory scale
The results (figure 4) showed that in general the impregnation of boric acid, chitosan, glycerol or heating 140 °C is able to reduce the weight loss of wood against the attack of subterranean termites. Based on SNI 7207:2014, wood impregnation with boric acid and chitosan or glycerol as well as heating of 140 °C (BG140 and BC140) resulted in an increase in the class of durable IV on control (NT70) to durable class II in resistance test to subterranen termites. The analysis of variance at 95% confidence level proved that the treatment interaction of boric acid treatment, chitosan/ glycerol and heating temperature had a significant effect (p<0.05) on the weight loss value in the manii wood resistance test against subterranean termites. Duncan test showed that the treatment of boric acid (BT70), boric acid and glycerol (BG70) or boric acid-chitosan (BC70) produced a significant lower value of control (NT70). According to Ahmed et al. the weight loss value of wood preserved with boric acid is lower than the control in testing against termite attack Coptotermes acinaciformis; boric acid also has effectiveness as a slow-working toxicity and low toxicity that it can disturb the colony of subterranean termites Odontotermes sp. [41,42]. According to Hayashi boric acid can stick stablely to the wood due to the high adhesion of chitosan on the wood so as to with against of subterranean termites [43],. The chitosan treatment (NC70) also increases the resistance of lower weight loss from control (NT70).
The heating treatment 140°C (NT140) also showed a significant lower weight loss value than control at 70°C (NT70). This is due to changes in the chemical composition of wood as heating temperatures increase from 140, 180, 200, and 220°C for 2, 4, and 6 hours [44]. In addition, the double impregnation of boric acid and chitosan acid by heating 140°C (BC140) resulted in significant lower weight loss than the control and treatment of boric acid (BT70) as well as the lowest value in the manii wood resistance test of subterranean termite attacks.

Field test
The field test based on ASTM D 1758-02 is one of the methods that describes the actual condition, strongly influenced by the environment, place and weather so that termites or fungus can attack the tested wood. The durability of manii wood after the field test was reviewed based on weight loss value and visual evaluation. Based on visual evaluation (figure 5) shows the treated of chitosan or glycerol with heating 140°C in general increased the resistance value of wood. The best resistance against subteranean termites was obtained in the treated wood with boric acid and chitosan impregnation followed by heating at 140°C (BC140). The analysis of variance at 95% confidence level proved that the interaction between boric acid treatment, chitosan/ glycerol and heating temperature had a significant effect (p<0.05) on the weight loss of manii wood in field test. Duncan test revealed that the treatment of boric acid (B140) and boric acid-chitosan (BC140) with a heating of 140°C resulted in a significant lower weight loss than the control sample (NT70). This because boric acid is fungicide and insecticide [45] and chitosan has bioactive properties. The heating treatment at 140°C in boric acid-chitosan treatment (BC140) also showed a significant lower value compared to boric acid-chitosan treatment with heating at 70°C (BC70) and boric acid treatment with heating 70 °C (BT70). Heat treatments causes significant degradation of amorp polysaccharides components so that a decrease in nutrient content can lead to increased wood resistance [46] The results of termite identification based on key determination by Nandika et al. showed that the types of termites that attack wood in field tests for three months ( figure 6) were Microtermes sp., Macrotermes sp., and Capritermes sp [47]. Arinana et al. found four species of subterranean termites that attack wood samples at the Arboretum Faculty of Forestry IPB, among them are Schedorhinotermes sp., Microtermes sp., Capritermes sp., and Macrotermes sp [48]. The types of subterranean termites from the family Termitidae which is also against termites commonly found in the tropics [47]. Capritermes sp. is not found in residential areas, while Microtermes sp. is the most dominant species of subterranean termites attacking wood. Natawiria states that Macrotermes sp. can live in hard, wet, and moist soils [48]. According to Sulistyawati et al. Macrotermes gilvus Hagen. was found in the Arboretum of the Faculty of Forestry and Environment of IPB University [50].

Conclusion
The interaction of boric acid, chitosan/ glycerol and heat treatments had a very significant effect (p<0.01) on the resistance of manii wood against white-rot fungi and dry wood termites, as well as had a significant effect (p<0.05) on the resistance against subterranean termite attacks in laboratory and field test. Glycerol or chitosan treatment resulted in a significant lower weight loss than controls in wood  10 resistance tests against white-rot fungi, dry wood termites, and subterranean termites. The heat treatment 140°C caused significant lower weight loss than heating at 70°C in each wood resistance test against white-rot fungi, dry wood termites, subterranean termites, and field test.
The recommended treatment for modifying manii wood is the double impregnation of boric acid and chitosan accompanied by a heating at 140ºC which is generally best at improving the resistance of manii wood from all the destroying organisms.