Biodegradation and Micro- Scale Treatability Pattern of Loblolly Pine Heartwood Bioincised by Bacillus Subtilis and Physisporinus Vitreus

One strategy for improving the treatability of refractory wood species is biological incising, and its efficiency depends on how the microorganisms modify the porous structure of the wood. Evaluation of the bioincised wood treatability on a micro-scale can thus help to better understand the treatability enhancing mechanisms. In the present study, the biodegradation pattern and micro-scale treatability of Loblolly pine (Pinus taeda L.) heartwood were determined after bioincising with the white-rot fungus Physisporinus vitreus (Pers.: Fr.) P. Karsten isolate 136 and bacterium Bacillus subtilis UTB22. Oven-dried specimens with dimensions of 50 mm × 25 mm × 15 mm (L × T × R) were incubated with the microorganisms at (23±2) °C and (65±5) % relative humidity for six weeks. The control and exposed wood blocks were then pressure treated by 1 % fluorescent dye (fluorescein)containing water to study the treatability pattern under a fluorescence microscope. The longitudinal and tangential air permeability and compression strength parallel to the grain of the specimens were also determined at the end of the incubation period. Scanning electron microscopic (SEM) studies showed that degradation by B. subtilis UTB22 was limited to the pit membranes, but the cell walls were also degraded to some extent by P. vitreus. The fungus caused a higher mass loss compared to the bacterium, whereas the permeability enhancing ability of the bacterium was more pronounced. The fluorescent dye tracer also showed that higher treatability with more uniformity was obtained by B. subtilis UTB22. The improvement in treatability by both microorganisms was mainly due to the degradation of the earlywood tracheids.

with macro-scale analyses. The current study gives some detailed information on the micro-scale treatability of bioincised wood which could be helpful in achieving an in-depth understanding of the treatability-enhancing mechanisms.

Wood sample preparation 2.1. Priprema uzoraka drva
Wood specimens with dimensions of 50 mm × 25 mm × 15 mm (L × T × R) were prepared from the heartwood of air-dried Loblolly pine (Pinus taeda L.) boards according to EN 113:2004 for fungal and bacterial incubation. Loblolly pine is native to the southeastern United States and widely grown on plantations as well. Wood samples from the trees planted in northern Iran were used for this study. The treatability class of Loblolly pine heartwood varies from III to IV, indicating that it is difficult or extremely difficult to treat (EN 350: 2016). Compression strength parallel to the grain of the specimens with dimensions of 60 mm × 20 mm × 20 mm (L × T × R) was also determined according to DIN 52185:1976 Most wood species are not sufficiently durable against wood-destroying organisms and have to be treated with chemical preservatives to ensure the desired service life or to improve other relevant properties like resistance to fire and UV stability. Preservatives Adequate penetration of the wood by the preservatives during the treatment process is necessary to achieve a successful protection against biological degradation. However, the penetration depth of active ingredients alone is not sufficient to judge the wood treatability; the uniformity of the penetration should be evaluated, e.g., by microscopic studies of the treated wood (Tarmian et al., 2020). Wood decay can occur from the non-uniform impregnation of the wood cells (Omidvar and Schneider, 2004). This type of penetration is sometimes called marble penetration.
Various strategies can be used to enhance the penetration of the preservatives into refractory wood species, among which mechanical incising is commonly used in industry for this purpose (Hansmann et  . For example, it has been known for a long time that some bacteria (e.g., Bacillus subtilis) are able to increase the permeability of some refractory coniferous wood species during ponding or water sprinkling by deteriorating the bordered pit membranes (Pánek and Reinprecht, 2008;Yildiz et al., 2012).
The white-rot fungus Physisporinus vitreus has been used as a bioincising fungus to enhance the permeability of some refractory softwood species, like for 20 minutes, and then impregnation was carried out at atmospheric pressure for 30 minutes. Transverse sections of about 10-15 μm thickness were cut from the treated specimens using a sledge microtome (GSL1) and studied by fluorescence microscopy (BEL FLUO-3, Italy). Under the UV light of the microscope (exciting spectrum area: 330-400nm), the treated area gave off a green color.

Measuring air permeability 2.4. Mjerenje propusnosti zraka
There is a strong correlation between wood treatability and air permeability (Siau, 1984). Thus, air permeability of the specimens was also measured. For this purpose, cylindrical samples 18 mm in diameter and 20 mm in length were cut along the longitudinal and tangential directions of the wood specimens with ten replicates for each treatment. The lateral surfaces of the specimens conditioned to equilibrium moisture content (EMC) of about 12 % were first coated by epoxy resin to avoid lateral air leakage. Superficial gas permeability) was then determined under steady-state conditions based on Darcy's law using a well-known falling-water displacement method suggested by Siau (1984) (see Figure 1 for details of the apparatus): (1) Where V d = p·r 2 ·Dz (m 3 ), P atm is the atmospheric pressure (mHg), L is the sample length (m), is the average height of water over surface of reservoir during period of measurement (m), A is the cross-sectional area of specimen (m 2 ), t is the time (s) required for the water drop through ∆h and C is the correction factor for gas expansion: (2) The wood samples were oven dried at (103±2) °C for 24 h, steam-sterilized at 121 °C and 124.1 kPa for 20 min, and weighed before fungal and bacterial exposure. Physisporinus vitreus (Pers.: Fr.) P. Karsten isolate 136 was obtained from the University of Hamburg (Bari et al., 2015). The woodblocks were exposed to fungal mycelia grown in Kolle flasks containing 4.8 % malt extract agar (Merck, Germany).
Bacillus subtilis UTB22 was obtained from the Department of Plant Protection at the University of Tehran. One ml of B. subtilis UTB22 was transferred to sterilized Luria Bertani aqua culture medium and put in a shaker incubator for 120 min -1 at 28 °C for 24 h. The bacterium was harvested by centrifugation for 6 minutes at 8000 min -1 . A suspension of bacterium and distilled water was supplied, and the optimum population of bacteria was determined using a spectrophotometer. Incubation by both microorganisms was carried out at a temperature of (23±2) ºC and (65±5) % relative humidity for 6 weeks. At the end of incubation, the samples were oven dried and weighed to calculate the mass loss. A Scanning Electron Microscope (SEM; Zeiss DSM 960 A, Germany) was used to observe the biodegradation pattern of the incubated specimens.

Micro-scale treatability test 2.3. Ispitivanje impregnacije na mikrorazini
The control and incubated wood specimens with a moisture content of 12 % were pressure treated with 1 % fluorescent dye (fluorescein)-containing water. Before the impregnation process, the end sections of the specimens were completely coated with epoxy resin to limit solution penetration through the transverse direction. An initial vacuum of 30 kPa was first applied   (Bucur, 1995).

Wood degradation 3.2. Razgradnja drva
Both microorganisms degraded the tracheid bordered pits and cross-field pits, which resulted in an opening of the aspirated pits (Figures 2 and 3). Pit degradation by P. vitreus was more severe than that by B. subtilis UTB22. The extent of the fungal degradation was not similar for all pits (Figures 3 a, b, and e). The degradation for some pits was limited to the tori (Figure 3b, white arrow), but the membranes of some were completely degraded (Figure 3b (2000) and Schwarze et al. (2006Schwarze et al. ( , 2008 is questionable, even at early stages of wood colonization. These contradictory results, however, may be due to different incubation conditions used by the authors, which implies that fungal activity is highly dependent upon the bioincising conditions. The degradation of pit membranes can be more selective by optimizing the incubation parameters (Lehringer, 2011). Degradation by B. subtilis UTB22 was confined to the pit tori (Figures 2c-d)

. A non-uniform
Where V r is total volume of apparatus above point 1 including volume of hoses (m 3 ), and ∆h is change in the water drop level during the test (m). Finally, the specific permeability coefficient (K) (m 3 m -1 ), which is only a function of the porous medium properties, was calculated by: (3) Where µ is the viscosity of air at 20 °C (µ=1.81·10 -5 Pa·s).

Statistical analysis 2.5. Statistička analiza
Statistical analysis was conducted using SPSS software (Version 20) by one-way analysis of variance (ANOVA). Duncan multiple range test was used to test the statistical significance at the α = 0.05 level.

Mass loss and compression strength parallel
to the grain 3.1. Gubitak mase i čvrstoća na tlak paralelno s vlakancima P. vitreus caused higher dry mass loss than B. subtilis UTB22. The average mass losses were less than 1 % with B. subtilis UTB22 and 8.4 % with P. vitreus (Table 1). Low mass loss after exposure to B. subtilis UTB22 might not be the sole consequence of the bacterial degradation; the leaching of extractives was also observed. Some previous works have reported smaller mass losses after colonization of the wood with the same isolate of P. vitreus for six weeks; for example  (Schwarze and Landmesser, 2000). Such a variation in the mass loss definitely implies that the fungal activity changes largely with the wood species, the wood block size, and incubation conditions. The compression strength parallel to the grain of the wood specimens was reduced by 16 % and 7 % after incubation with B. subtilis UTB22 and P. vitreus, respectively (Table 1). Other authors have also reported that a short period of wood incubation by P. vitreus caused a slight reduction in mechanical strength (  penetration of fungal hyphae in the porous structure of wood (Figures 3a and c) was observed in the current study. A heterogeneous fungal colonization in timbers due to differences in the heartwood percentage, moisture content, and availability of nutrients is one of the main drawbacks of such biological incising methods on an industrial scale (

Propusnost zraka i impregnacija na mikrorazini
Both P. vitreus and B. subtilis UTB22 were able to significantly enhance the permeability of Loblolly pine heartwood (Table 1), which was increased by around 56 % in the tangential direction and 124 % in the longitudinal direction after fungal exposure. The improvement was more remarkable when the specimens were exposed to B. subtilis UTB22 (485 % for tangential permeability and 676 % for longitudinal permeability). Several studies have also shown that different isolates of P. vitreus can significantly improve wood permeability, even after a short period of colonization (Schwarze and Landmesser, 2000;Schwarze et al., 2006;Lehringer et al., 2010;Fuhr et al., 2011;Emaminasab et al., 2015Emaminasab et al., , 2016; however, the changes in permeability vary with the wood species, the fungal isolate, and incubation conditions (Schwarze et al., 2006). The improvement in permeability is mostly attributable to the degradation of the pit membranes (Schwarze et al., 2006;Schwarze and Schubert, 2011;Fuhr et al., 2011). It is believed that the secretion of pectinase enzyme by B. subtilis plays an important role in improving wood permeability through the decomposition of pectin compounds and hemicellulose of the pit membrane (Yildiz et al., 2012). Possible reasons for the lower improvement in permeability by P. vitreus compared to B. subtilis UTB22 are the heterogeneous fungal activity, accumulation of fungal hyphae, and degradation products within the cell lumens ( Figure  3d), which may block the fluid flow paths (Emaminasab et al., 2015).
Micro-scale treatability studies under a fluorescence microscope showed that the treatability of the tracheid cells increased after incubation by both microorganisms ( Figure 4). The present results clearly demonstrate the higher bioincising efficiency of B. subtilis UTB22 compared to P. vitreus (Pers.: Fr.) P. Karsten isolate 136. While most tracheid lumina were filled with the impregnating solution after bacterial exposure (Figures 4c, e), many of the lumina remained empty in the wood specimens bioincised by P. vitreus (Figures  4b, d).
A non-uniform penetration of the solution into the specimens exposed to P. vitreus (Figure 4b) may be visually inspected the uptake of bluish dye Neolan Glaucin E-A by Norway spruce and silver fir (Abies alba) wood blocks incubated with two isolates of P. vitreus (EMPA 642 and EMPA 643), and found that the uptake within P. abies was less homogeneous. Lehringer et al. (2009a) also reported that the uptake and penetration depth of aqueous modification substances after vacuum impregnation of the bioincised wood were highly variable due to the heterogeneous colonization of the wood by P. vitreus.
After bioincising, earlywood showed better treatability than latewood ( Figure 5). Only a few latewood cells were treated even after the fungal or bacterial incubation, which indicates the weaker ability of the microorganisms to degrade latewood cells. Therefore, this treatability enhancing strategy may be less successful in improving the treatability of softwoods with high latewood percentage, like the Pinaceae family.

ZAKLJUČAK
The present work comparatively studied the degradation pattern, permeability, and micro-scale treatability of Loblolly pine heartwood after incubation with two bioincising microorganisms (white-rot fungus Physisporinus vitreus (Pers.: Fr.) P. Karsten isolate 136 and bacterium Bacillus subtilis UTB22). Both microorganisms improved treatability significantly, mainly because of degradation of the pit membranes. However, a higher treatability with more uniform impregnation of the wood cells was obtained with B. subtilis UTB22 despite a higher mass loss with P. vitreus. SEM observations showed that the lower treatability enhancing efficiency of the fungus was possibly due to the heterogeneous pit degradation and blockage of the tracheid lumens with the fungal hyphae. Earlywood was more treatable than latewood after bioincising because of the higher degradation of the former.
It can be concluded that degradation by B. subtilis UTB22 was more selective than that by P. vitreus (Pers.: Fr.) P. Karsten isolate 136. Degradation by the bacterium was mainly confined to the tori of the pits, whereas some pits were completely degraded by the fungus and the cell walls were also attacked. Based on the current results, a heterogeneous impregnation of the bioincised wood cells can also be an important challenge for scaling up the bio-incising of wood with P. vitreus to an industrial scale.