Illustration of the E ﬀ ects of Five Fungi on Acacia saligna Wood Organic Acids and Ultrastructure Alterations in Wood Cell Walls by HPLC and TEM Examinations

: In the present study, Acacia saligna (Labill.) H.L.Wendl. wood blocks with dimensions of 0.5 × 1 × 2 cm were inoculated with ﬁve molds ( Aspergillus niger , A. ﬂavus , Alternaria tenuissima , Fusarium culmorum, and Trichoderma harzianum ) and the changes in the organic acids (oxalic, citric, tartaric, succinic, glutaric, acetic, propionic, and butyric) of powdered wood were analyzed by HPLC. The e ﬀ ects of the ﬁve inoculated fungi on the alterations to the wood cell wall ultrastructures were examined by TEM. The wood became more acidic as it was inoculated with the studied fungi. From the HPLC analysis, the oxalic acid (293.34 µ g / g o.d.) in the A. saligna, A. tenuissima (167.33 µ g / g o.d.), and T. harzianum (245.01 µ g / g o.d.) wood decreased, but it increased in the A. ﬂavus (362.08 µ g / g o.d.), A. niger (1202.53 µ g / g o.d.), and F. culmorum (431.85 µ g / g o.d.) inoculated wood. Citric acid was observed in the wood inoculated with A. ﬂavus (110 µ g / g o.d) and A. niger (2499.63 µ g / g o.d). Tartaric (1150.98 µ g / g o.d), acetic (2.04 µ g / g o.d), and propionic (1.79 µ g / g o.d) acids were found in the wood inoculated with A. niger . Butyric acid was found in small amounts. A loss of wood substances appeared as the electron-lucent increased in the middle lamella and the layers of the secondary wall. Within the secondary cell wall regions, checks and splits were also noted, which resulted from the e ﬀ ects of the acids on the carbohydrates, according to the fungus type and the acids. In conclusion, increasing the amount of organic acids in the wood samples through inoculation with fungi results in more degradations in the wood, especially in the wood inoculated with A. niger .


Introduction
Wood, as a natural organic material, consists of hemicellulose, cellulose, lignin, and minor amounts of organic extractives, as well as ash as an inorganic mineral [1]. Wood is susceptible and readily degraded by bacteria, fungi, and termites [2][3][4]. In particular, wood is attacked by fungi when the conditions are suitable, such as a high wood moisture content (>20%), the presence of oxygen, and temperatures in the range of 25-40 • C [5]. Fungi colonize wood by degrading the components of the

High-Performance Liquid Chromatography (HPLC)
The organic acid compositions of the inoculated A. saligna wood with each fungus were separated by HPLC Knauer, Berlin, Germany, with a Rezex@ column. The flow rate and UV detector were set at 0.6 mL/min and 214 nm, respectively. The temperature of the column oven was kept constant at 65 • C, and the mobile phase was 0.005 M H 2 SO 4 . The data were analyzed using clarityChrom software. The environmental conditions for the separation techniques were set at 26.7 • C and RH 32.90%. Standard organic acids of analytical grade of oxalic, citric, tartaric, succinic, glutaric, acetic, propionic, and butyric acids were used for the HPLC analysis.

Transmission Electron Microscope (TEM)
Small sticks of some of the selected wood samples were removed with a scalpel. They were immediately placed in freshly prepared aqueous 1% w/v KMnO 4 and left for 1 h at room temperature. After continuous washing with distilled water to remove KMnO 4 , the samples were dehydrated using ethanol and then an ethanol-acetone series (10 steps). They were flat embedded in Spurr's epoxy resin. Resin polymerization proceeded at 70 • C for 24 h, then the samples were sectioned with an ultramicrotome (Reichert FC4). After sectioning, these samples were post-stained using 1% w/v KMnO 4 . Observation was done with a transmission electron microscope (Zeiss E-M10) at 60-80 kv. Table 1 presents the pH values of the powdered wood samples of A. saligna after inoculation with each fungus. The wood samples became more acidic after being inoculated with the studied fungi, ranging from 4.39 to 5.81 for inoculated the wood inoculated with A. niger and F. culmorum, respectively, compared with the control treatment (pH 6.06). Another study reported that a decrease in the pH of wood may be indicated at early stages of impregnated and unimpregnated Norway spruce (Picea abies (L). Karst) sapwood decayed by brown-rot fungi (T. versicolor and Schizophyllum commune) [44].

HPLC Analysis of Organic Acids
The HPLC chromatogram peaks suggest the presence of oxalic, citric, tartaric, succinic, glutaric, acetic, propionic, and butyric acids in the wood samples of A. saligna inoculated with A. niger, A. flavus, A. tenuissima, F. culmorum, and T. harzianum (shown in Figure 1). The changes in organic acids from the HPLC analysis of the A. saligna wood after inoculation with fungi compared with the control treatment (without fungi) are shown in Table 2. Oxalic acid was present in the wood samples of A. saligna (293.34 µg/g o.d.); decreased in the wood incubated with A. tenuissima and T. harzianum; and increased in wood incubated with A. flavus, A. niger, and F. culmorum. The wood samples inoculated with A. flavus and A. niger showed the highest amounts of citric acid.
Tartaric acid was found in high amounts (1150.98 µg/g o.d. sample), while acetic and propionic acids were observed in low amounts, only in the wood samples inoculated with A. niger. Succinic acid was found only after the wood was inoculated with A. niger and A. tenuissima. Glutaric acid was found in the wood samples that were inoculated with A. flavus, A. niger, F. culmorum, and T. harzianum, but not detected in the wood inoculated with A. tenuissima or in the control treatment.
Butyric acid was found in small amounts in the control treatment, but the values increased in the wood inoculated with A. flavus, A. niger, and T. harzianum, and decreased in the wood sample inoculated with A. tenuissima.
The wood sample inoculated with A. niger showed the presence of all of the organic acids, with high amounts of oxalic, citric, tartaric, succinic, and glutaric acids. Oxalic acid was found, in either high or low amounts, in all of the samples inoculated with fungi, suggesting that this acid is dominant in A. saligna wood. During the alkaline pulping of Acacia, a significant amount of oxalate was formed and released from the wood, where a higher hydroxide concentration led to a higher oxalate content in the black liquor during the early stages of Acacia wood cooking [45].
During fermentation, fungi produce not only oxalic acid, but also other organic acids, such as citric, gluconic, glucuronic, and malic, depending on the operating conditions [28,[46][47][48]. Oxalic acid is involved in wood-rotting fungi (brown-and white-rot fungi) mediated processes, including lignocellulose degradation and the breakdown of xenobiotic pollutants [18,49]. Oxaloacetase and glyoxylate oxidase are two oxalate enzymes produced from wood-rotting fungi [50]. Brown-rot fungi produce large amounts of oxalic acid [49]. The synthesis of oxalate is an energy efficient process, and the excreted oxalic acid, as a by-product of sugar oxidation, proves that oxaloacetic acid can be formed through the carboxylation of pyruvate in the cytoplasm [51].
After two weeks of exposure to A. vaillantii, a significant reduction in the pH was observed [44]. The reason for this drop may be from the oxalic acid produced by brown-rot fungi [52]. Oxalic acid has an important role in the first, pre-visual, non-enzymatic stages of decay [53,54]. At a pH higher than 6, A. niger effectively secretes oxalic acid, while if the fermentation is carried out under acidic conditions (pH 2 to 3), the main product is usually citric acid [46,47].
It was reported that A. niger is an established organism for the industrial production of citric and oxalic acids, as well as the enzymes gluconic acid and ethanol [55][56][57]. In low concentrations, A. niger strains are known to produce succinic acid [58], as well as a minor co-product production of citric acid [59]. Succinic acid was directly produced from minimally pretreated lignocellulosic biomass with employing a mixed lignocellulolytic and acidogenic A. niger and T. reesei co-culture [60].
At elevated carbohydrate concentrations, organic acids of malic, citric, succinic, and fumaric acids were identified mostly in liquid cultures, while oxalic acid was dominant among the acids produced by A. niger, and its production was decreased by P. citrinum at high carbohydrate concentrations [61]. Carbohydrates, especially fructose, are mainly involved in biosynthetic processes [62], which is suitable for the formation of organic acids in the Krebs cycle, such as fumaric, malic, and succinic acid synthesis. T. harzianum was reported to decompose cellulose and other carbohydrates to glucose within the parenchyma cells and the pits, while it lacks ligninolytic activity [63,64]. Another study showed that no cell wall degradation signs appeared in the wood samples inoculated with T. harzianum [8,65].

TEM Examination of the Inoculcated A. saligna Wood with Fungi
TEM studies show that erosion and losing parts of the secondary wall occurred. The middle lamella remained intact in the majority of samples. The formation of cavities in the cell walls was the dominant feature. The formation of cavities within the S 1 , S 2 , or S 3 layers and erosion depend on the fungus type [11,12].
As it can be seen from the TEM micrographs, the most affected samples were those infested with A. niger, where the effect of the fungus on the wood cells appeared in the presence of soft-rot cavities, especially within the secondary cell walls of the S 1 and S 3 layers (Figure 2b-e) compared to control (Figure 2a). Soft-rot decay type II (erosion) was obvious in the samples infested with A. flavus, starting from the S 2 layer and extending to the S 1 layer. An increase in the electron-lucent appearance of some regions in the middle lamella and secondary wall layers suggests a loss of wood substance (Figure 3a,c). In addition, checks and splits were frequently noted within the secondary cell wall regions (Figure 2b).
The wood samples infected with F. culmorum showed general laceration in the wood tissue because of sectioning, which indicates the weakness of the sample. In addition, soft-rot decay type II (erosion) starting from the S 2 layer and extending to the S 1 layer was apparent (Figure 4). A. tenuissima caused ultrastructure alterations in the wood samples similar to soft-rot fungi, regarding cavities (soft-rot decay type I) within the S 3 layer ( Figure 5). Moreover, T. harzianum caused two types of soft-rot decay in wood samples, namely: cavities within the S 3 and S 1 layers and erosion within the S 3 layer ( Figure 6). These results, regarding the patterns and mechanisms of degradation, are consistent with previous studies [11,12]. TEM studies show that erosion and losing parts of the secondary wall occurred. The middle lamella remained intact in the majority of samples. The formation of cavities in the cell walls was the dominant feature. The formation of cavities within the S1, S2, or S3 layers and erosion depend on the fungus type [11,12].
As it can be seen from the TEM micrographs, the most affected samples were those infested with A. niger, where the effect of the fungus on the wood cells appeared in the presence of soft-rot cavities, especially within the secondary cell walls of the S1 and S3 layers (Figure 2b-e) compared to control (Figure 2a). Soft-rot decay type II (erosion) was obvious in the samples infested with A. flavus, starting from the S2 layer and extending to the S1 layer. An increase in the electron-lucent appearance of some regions in the middle lamella and secondary wall layers suggests a loss of wood substance (Figure  3a,c). In addition, checks and splits were frequently noted within the secondary cell wall regions (Figure 2b).
The wood samples infected with F. culmorum showed general laceration in the wood tissue because of sectioning, which indicates the weakness of the sample. In addition, soft-rot decay type II (erosion) starting from the S2 layer and extending to the S1 layer was apparent (Figure 4). A. tenuissima caused ultrastructure alterations in the wood samples similar to soft-rot fungi, regarding cavities (soft-rot decay type I) within the S3 layer ( Figure 5). Moreover, T. harzianum caused two types of softrot decay in wood samples, namely: cavities within the S3 and S1 layers and erosion within the S3 layer ( Figure 6). These results, regarding the patterns and mechanisms of degradation, are consistent with previous studies [11,12]. , showing the effect of the fungus on the wood cells, which appears in the presence of soft-rot cavities, especially within the S1 and S3 layers. ML-middle lamella; S1, S2, S3secondary cell wall layers of wood. , showing the effect of the fungus on the wood cells, which appears in the presence of soft-rot cavities, especially within the S1 and S3 layers. ML-middle lamella; S1, S2, S3secondary cell wall layers of wood.         . TEM photos of wood samples infected with A. tenuissima, showing cavities within the S3 layer similar to soft-rot decay type I (a). Furthermore, checks and fissures (shown by arrows), are found within the S1 and S2 layers, with extensive erosion of the middle lamellae region (b). Figure 6. TEM photos (a,b) of wood samples infected with T. harzianum, showing cavities within the S3 and S1 layers, similar to soft-rot decay type I, and erosion within S3 (soft-rot decay type II). In addition, checks can be seen within the S2 layer.

Conclusions
In this study, A. niger, A. flavus, A. tenuissima, F. culmorum, and T. harzianum were bio-assayed for their effects on organic acids and ultrastructure alterations in wood in the cell walls of A. saligna. These measurements were done using HPLC and TEM techniques. An increase in the electron-lucent appearance of some regions in the middle lamella and secondary wall layers suggests a loss of wood substance; in addition, checks and splits were frequently noted within the secondary cell wall regions, indicating the effect of acids on the carbohydrates (cellulose and hemicellulose), according to the fungus type and the acids. Although the wood samples were inoculated with different fungi for the same period of time, the effect of these fungi on the wood acidity differs according to the fungus type. A. niger severely increased the samples' acidity, leading to the destruction of the carbohydrate-rich layers in the wood cells, resulting in more degradations of the cell wall ultrastructure. A. tenuissima and T. harzianum have almost the same effect on wood acidity and wood cells. What is more, the wood samples inoculated with F. culmorum and A. flavus have a lower pH Value compared with the other fungi, but their content of oxalic acid and the degradation in their structure was bigger than those inoculated with A. tenuissima and T. harzianum.

Conflicts of Interest:
The authors declare no conflict of interest. Figure 6. TEM photos (a,b) of wood samples infected with T. harzianum, showing cavities within the S 3 and S 1 layers, similar to soft-rot decay type I, and erosion within S 3 (soft-rot decay type II). In addition, checks can be seen within the S 2 layer.

Conclusions
In this study, A. niger, A. flavus, A. tenuissima, F. culmorum, and T. harzianum were bio-assayed for their effects on organic acids and ultrastructure alterations in wood in the cell walls of A. saligna. These measurements were done using HPLC and TEM techniques. An increase in the electron-lucent appearance of some regions in the middle lamella and secondary wall layers suggests a loss of wood substance; in addition, checks and splits were frequently noted within the secondary cell wall regions, indicating the effect of acids on the carbohydrates (cellulose and hemicellulose), according to the fungus type and the acids. Although the wood samples were inoculated with different fungi for the same period of time, the effect of these fungi on the wood acidity differs according to the fungus type. A. niger severely increased the samples' acidity, leading to the destruction of the carbohydrate-rich layers in the wood cells, resulting in more degradations of the cell wall ultrastructure. A. tenuissima and T. harzianum have almost the same effect on wood acidity and wood cells. What is more, the wood samples inoculated with F. culmorum and A. flavus have a lower pH Value compared with the other fungi, but their content of oxalic acid and the degradation in their structure was bigger than those inoculated with A. tenuissima and T. harzianum.