Mechanical, physical and compositional effects of Meruliporia incrassata on Southern Yellow Pine

2.1 Wood decay

The decay protocol implemented was adapted from EN 113 (European Committee for Standardization 1996). Test specimens were cut from clear wood portions of two-by-four dimensional lumber sourced from a building materials supply company. The nominal dimensions of each test specimen were 20 mm by 15 mm by 50 mm. A four-week conditioning period was utilized for wood specimens. Following cutting, specimens were conditioned at 25 °C and 60 % RH for three weeks, steam sanitized, and conditioned for one additional week at 25 °C and 60 % RH to ensure a moisture content of approximately 12 %.

Cultures of M. incrassata (Berk. & Curt.) Murr. (MAD-563; ATCC 11236) were prepared on potato-dextrose-yeast agar (PDY). Petri dishes of fungus were cultured for four-weeks at 25 °C and 60 % RH. The culture vessels utilized were 100-mm diameter plastic jars with screw-top lids. A 17 mm hole was cut in each lid and filled with sterile cotton to facilitate aseptic air exchange. Vessels were autoclaved, and approximately 50 mL of PDY agar mixed with approximately 1 g of steam sanitized SYP sawdust saturated with deionized water was poured into each vessel. Sawdust was added to the agar to stimulate production of fungal decay compounds during the culture period, prior to specimen inoculation. After setting, a 20 mm-by-20 mm slice of agar was cut from the culture vessel and replaced with a slice of agar covered by cultured fungus from the petri dishes. Culture vessels were then incubated in the conditioning chamber for four weeks to allow the fungus to fill the vessel. Any vessels showing signs of contamination were discarded.

After the four-week conditioning (wood specimens) and incubation (culture vessels) period, wood specimens were inoculated by placing specimens into the culture vessels. Three-millimeter-thick acrylic supports were utilized to keep the wood pieces from contacting the agar surface. Testing was performed on five randomly selected specimens prior to decay and at two-week intervals for up to 12 weeks (Figure 1). At each test interval the fungal material was scraped from the surface from each specimen using a razor blade. The dimensions in all directions, mass, and moisture content were recorded. Ultrasonic pulse velocity (UPV) testing was then performed on each test specimen to estimate each stiffness parameter. Samples were then dried in an oven to determine the moisture content. Finally, samples were harvested from each test specimen for compositional analysis.

Figure 1:

Wood specimens in a culture vessel during sampling.

2.2 UPV

A James Instruments Non-destructive Testing Equipment V-Meter Mk IV™ (Chicago, USA) ultrasonic pulse velocity system was utilized to estimate the stiffness of each test specimen across each dimension. Petroleum jelly was utilized as the lubricant. Testing was conducted by recording the time for an acoustic wave to propagate through each dimension of the wood. The pulse velocity was then calculated by dividing the time by the dimension of the specimen tested. Stiffness was estimated via a well-established correlation with material density (Bader et al. 2012a; Kohlhauser and Hellmich 2012). Following testing, specimens were cleaned of any residual jelly.

2.3 Thermogravimetric analysis (TGA)

TGA is a well-established methodology for measuring the hemicellulose, cellulose, and lignin weight fractions in organic materials, including wood (Anca-Couce et al. 2020; Gašparovič et al. 2010; Grønli et al. 2002; Saadatkhah et al. 2020; Skreiberg et al. 2011). TGA was performed using a Mettler Toledo TGA DSC 3+ (Columbus, USA) to evaluate the wood composition every two weeks throughout decay, similar to (Alfredsen et al. 2012). Test specimens were made by cutting an approximately 3 mm thick slice from the center of the wood block; the slice was then milled with a knife mill for approximately 20 s. Samples were sieved through a #20 and #40 mesh sieve, and samples were harvested from material passing the #40 mesh. Three 6 mg test samples from each test specimen were evaluated. A 5 °C/min heating rate was employed from ambient temperature to 450 °C. Nitrogen purge gas was utilized at an 80 mL/min flow rate. Following testing, the derivative thermogravimetric (DTG) curves were deconvoluted utilizing nonlinear regression analysis to determine the weight fractions of hemicellulose, cellulose, and lignin (Anca-Couce et al. 2020; Grønli et al. 2002). First the DTG and second derivative curves were calculated from the experimental data (Figure 2A). Next, the key temperatures were extracted from these two curves. Finally, peak deconvolution of three phases was conducted to fit the Arrhenius equation to each wood constituent utilizing least-squares regression implemented in MATLAB (Equation (1), Figure 2B), where A _r represents the pre-exponential factor of each phase; E _r is the activation energy of that phase, measured in kilo joules; R is the gas constant; T is the temperature in degrees Kelvin; a _r is the conversion of phase r, and n _r is the degree of the reaction. The mass fraction of each phase, c _r , was then solved for utilizing least-squares regression in order to minimize the error between the fitted model for each constituent (Equation (2)) and the experimental DTG curve of each sample. Initial values and bounds for the Arrhenius constants were taken from data presented in (Anca-Couce et al. 2012, 2020), and bounds for the mass fractions were estimated based on the best fit of the data (Table 1). The bounds for hemicellulose mass fraction were set to linearly increase throughout decay by roughly half of the total mass decomposition during decay. This was done to account for the effects of mass loss in the material due to fungal decomposition that cannot be captured within the DTG curves. Cellulose mass fraction was assumed to be one minus the sum of the hemicellulose and lignin mass fractions.

Figure 2:

TGA analysis curves: (A) conversion and derivatives and (B) peak deconvolution of DTG curve.

3.1 Change in physical properties

The initial value of moisture content in samples was 11.6 ± 1.6 % (mean value ± one standard deviation). Throughout decay, the moisture content of wood samples increased greatly, doubling by four-weeks, and decreased to 20 % below initial values by week ten (Figure 3). The changing moisture content is indicative of the level of fungal activity in the wood samples: as fungal activity increased, it drew water into the specimen. M. incrassata produces a characteristically high amount of metabolic water during degradation (Burdsall 1991; Verrall 1968). As fungal activity decreased, the specimens began to dry out. Moisture transport through wood during decay is correlated with fungal hyphal action when samples are held at a constant relative humidity (Money 2007). Hyphae require water as a media to diffuse the depolymerizing enzymes into the wood cell wall (Ringman et al. 2019). Once the fungus breaks into the cell wall and begins to break down the constituents, the bound water in the cell wall is released, as is water from fungal respiration, which may then be absorbed by surrounding “intact” wood material. The appearance of a dry, brown wood material is representative of why brown rot was mistakenly referred to as “dry rot” for many years (Dinwoodie 2000; Singh 1999).

Figure 3:

Changes in physical properties throughout decay period: (A) density and (B) moisture content.

Density at harvest had an average initial value of 626 kg/m³ while dry density had an average initial value of 624 kg/m³. Density at harvest generally linearly declined by 8–10 % throughout the 12 week decay period. Dry density decreased by 10–12 % during the study period. However, while the moisture content increased considerably during the first four weeks, the density of test specimens did not increase during the first four weeks. While mass was added to the sample through added moisture, there were minimal changes in volume. Consequently, the constant density of wood samples at harvest indicates that the rate of mass loss through decay was roughly opposite and equal that of moisture gain in the first four weeks. The decrease in dry density further verifies the loss of material due to fungal action.

3.2 Visual characterization

Changes in the wood structure were observed qualitatively throughout the testing period. Samples presented in Figure 4 represent characteristic images of both the long-grain and the end grain cross section of samples from each testing period. The long grain photos represent the face in contact with fungal cultures. For end grain photos, the bottom edge in the photos of each specimen represents the exposed face. Photos were taken after samples had been kiln dried following removal from the fungus and represent samples in an equivalent moisture state. Staining within the wood sample was apparent from fungal activity within the first two weeks. By four weeks, early stages of cracking and annual ring delamination began. By six weeks, severe annual ring degradation was apparent and significant section loss was noted on some samples. This trend continued into 12 weeks where earlywood rings were apparently completely consumed, and there was significant degradation throughout the depth of the sample. Additionally, specimens with wider annual rings appeared to have more significant radial cracking, particularly in earlywood. Specimens with narrower annual rings displayed more longitudinal cracking along the annual ring interface. Bouslimi et al. (2014) and Schwarze (2007) similarly noted extensive decay of earlywood over that of latewood in microstructural investigations of brown rot decay. Other data suggest that this is more characteristic of early stage decay and that latewood degradation supercedes that of earlywood during advanced decay (Belt et al. 2022).

Figure 4:

Qualitative wood visual characterization: (A) long grain images and (B) end-grain images.

3.3 Change in stiffness

Average initial values for stiffness were 17.3 ± 5.8 GPa in the longitudinal direction (L–L), and 1.38 ± 0.12 GPa across the height (H–H), and 1.41 ± 0.64 GPa across the width (W–W) of samples. Throughout the 12 weeks of decay, longitudinal stiffness decreased by approximately 31 %, and stiffness perpendicular to the grain decreased by approximately 20–25 % (Figure 5). It is significant to note that this stiffness decrease was observed as early as 2 weeks for longitudinal stiffness and 4 weeks for the perpendicular to the grain specimens. Others have also noted similar losses in longitudinal stiffness for brown rot degradation of softwoods within two to six weeks of decay (Wilcox 1978). Bouslimi et al. (2014) reported similar magnitudes of longitudinal stiffness degradation over three decay states in a study of decayed Eastern White Cedar trees. The increase in perpendicular to the grain stiffness at two-weeks can be accounted for both by the variability in the measured values as well as the increased moisture content. Stiffness estimates had large variability due to the high natural variation in the decay process as well as variations in how ultrasonic pulses traveled through decayed samples. The most variability was observed in the W–W direction, and average values calculated at 8- and 12-weeks decay are not here reported due to extreme variability in values. Samples performed essentially transversely isotropic perpendicular to the grain (W–W and H–H); this was expected as there was no preferential orientation for wood samples. Samples were cut with both radial and tangential grain patterns across the cross sections. Bader et al. (2012a) noted a slower decrease in degradation of all stiffness parameters than this study but similar final magnitudes in a study of Gloeophyllum trabeum effects on Scots Pine wood. This is to be expected because M. incrassata may have higher oxalate production – the primary driver of non-enzymatic decay – than other brown rot species (Jellison et al. 2004).

Figure 5:

Changes in stiffness throughout decay period.

3.4 Change in composition

The mass loss adjusted weight fraction of each constituent throughout decay as measured by DTG is shown in Figure 6. The void fraction shown represents the measured mass loss in wood specimens throughout the decay period; because TGA cannot capture mass loss, the measured experimental values were used to normalize the total mass fraction of chemical constituent proportions. A summary of the evaluated Arrhenius constants is found in Table 2. Decay of cellulose and hemicellulose components continued steadily throughout the decay period with hemicellulose content decreasing from an average of 35 % initially to 31 % and cellulose content decreasing from an average of 45–40 % over the decay period as the mass loss increased to approximately 10 %. Lignin content remained relatively stable throughout this period, likely due to rapid re-polymerization during non-enzymatic decay (Goodell et al. 2017). These findings are consistent with the decay mechanisms of brown rot fungus (Bader et al. 2012b; Goodell et al. 2017, 2008); brown rot fungi are known to preferentially attack hemicellulose and cellulose components, starting from the easily accessible hemicellulose and continuing inward in the wood cell walls to the crystalline cellulose core of the cellulose microfibrils.

Figure 6:

Changes in composition throughout decay period.