2024 Volume 66 Issue 1 Pages 67-71
In nature, only white-rot fungi can completely decompose wood biomass in the environmental carbon cycle. However, their degradation strategy is still unclear. Despite many studies on the lab-scale wood-decay process, investigations on the open-field process have been limited by their difficulty. Here, we aimed to elucidate the degradation strategy of white-rot fungi in nature by monitoring the chemical composition changes of logs exposed to the white-rot fungus, Grifola frondosa, over 4 y of cultivation using Fourier transform infrared spectroscopy. Grifola frondosa began to decompose lignin and deacetylate hemicellulose in the first 2 y and then degraded polysaccharides in the next 1 y. Finally, lignin decomposition recurred after the third year. Thus, our study revealed that G. frondosa decayed wood in a repetitive two-stage process: lignin and polysaccharide degradation. The switching cycle may promote efficient degradation.
Grifola frondosa is one of the most popular edible mushrooms worldwide. This mushroom has attracted attention as a functional food because it contains physiologically active metabolites with hypocholesterolemic and antioxidant effects (Illana-Esteban, 2008). In Japan, G. frondosa mushrooms are artificially cultivated using fungal bed or wood-log cultivation. Wood-log cultivation produces mushrooms with a significantly higher umami content and sweetness components (monosodium glutamate-like components, 5′-GMP, etc.) than does fungal bed cultivation, and it is therefore highly valued (Tabata et al., 2004). In addition, mushroom wood-log cultivation contributes to the effective use of wood resources. Degraded wood and spent mushroom waste used in mushroom cultivation that have been delignified can be converted into useful products, such as bioethanol (Kamei et al., 2012, 2014). Therefore, mushroom cultivation using wood can contribute to the production of functional foods and valuable resources, making it important to understand its mechanism.
Woody biomass is the largest biological resource that captures and stores carbon in the terrestrial parts of the earth and has therefore attracted attention as a renewable resource (Alonso et al., 2017; Mainka et al., 2015; Ragauskas et al., 2014). Biomass is primarily composed of polysaccharides (cellulose and hemicellulose) and the persistent aromatic compound, lignin. Among all microorganisms, only white-rot fungi and some ligninolytic bacteria can decompose lignin (Bugg et al., 2011; Pollegioni et al., 2015). Many edible mushrooms, including G. frondosa, are classified as white-rot fungi. Wood treated with the marine white-rot fungus Phlebia sp. is characterized by the preferential decomposition of lignin and an increase in the proportion of polysaccharides in the wood (Kamei et al., 2012). White-rot fungi with a decay pattern similar to that of Phlebia sp., such as G. frondosa, are called selective white-rot fungi (Schwarze et al., 2000). In addition, the deletion of genes encoding lignin-modifying enzymes in white-rot fungi results in the upregulation of genes encoding polysaccharide-degrading enzymes (Nakazawa et al., 2023). These findings suggest that white-rot fungi efficiently utilize wood in nature by separating the lignin and polysaccharide decomposition stages.
Therefore, in this study, we aimed to elucidate the wood utilization strategy of white-rot fungi in nature by determining the wood component ratios of G. frondosa-inoculated wood logs at different cultivation stages using Fourier transform infrared (FT-IR) spectroscopy.
The experimental design and sampling protocol are shown in Figure 1. Logs of Quercus serrata were used as test wood logs in this study. They had a diameter of 10-15 cm and length of 12-16 cm. In Jan of each year, the sawdust spawn of G. frondosa was added to sterilized wood logs, which were then placed in plastic bags and incubated for 5-6 mo at 20-25 °C. In Jul, the G. frondosa-inoculated wooden logs were buried in sand in an artificial Japanese cedar forest (Misato, Miyazaki). In Nov 2022, we collected the following five wood log samples for this study: control wood logs (buried in sand from Jul to Nov without inoculation), first-year wood logs (1 y of cultivation), second-year wood logs (2 y of cultivation), third-year wood logs (3 y of cultivation), and fourth-year wood logs (4 y of cultivation). The wood log samples were split at the boundary between the heartwood and sapwood, and approximately 20 g fragments were taken from the center of each section. Each fragment was freeze-dried and then ground in a blender. Thus, we used 20 heartwood and sapwood samples each (five cultivation stages × four replicates) (40 samples in total). Detailed information on the same samples has been described previously (Chen et al., 2023).
In this study, FT-IR spectroscopy was used to measure the relative amounts of the wood components. In a previous study (Pandey & Pitman, 2003), wood components in hardwood treated with white-rot fungi were analyzed by FT-IR spectroscopy, and this analysis referred to that study. Spectra were recorded on an IRTracer-100 spectrometer equipped with a DLATGS detector (Shimadzu Corp., Kyoto, Japan). Peak heights were measured using LabSolutions IR (Shimadzu Corp.). The FT-IR spectra of the uninoculated and cultivated Quercus logs are shown in Figure 2. In this study, we focused on peaks related to lignin (ⅰ: 1526 cm−1 for aromatic skeletal in lignin), polysaccharides (ⅱ: 1366 cm−1 for C-H deformation in cellulose and hemicellulose, ⅲ: 1150 cm−1 for C-O-C vibration in cellulose and hemicellulose, ⅳ: 895 cm−1 for C-H deformation in cellulose), and acetyl group of hemicellulose (ⅴ: 1755 cm−1 for unconjugated C=O in xylans) (Fig. 2). We calculated the ratios of lignin to polysaccharide: (ⅰ: 1526 cm−1)/(ⅱ: 1366 cm−1), (ⅰ: 1526 cm−1)/(ⅲ: 1150 cm−1), and (ⅰ: 1526 cm−1)/(ⅳ: 895 cm−1) and the ratios of acetyl group of hemicellulose to polysaccharide: (ⅴ: 1755 cm−1)/(ⅱ: 1366 cm−1), and (ⅴ: 1755 cm−1)/(ⅲ: 1150 cm−1) to investigate the wood degradation model of G. frondosa in wood log cultivation. The non-parametric Kruskal-Wallis test combined with Dunn's multiple comparison test were performed to analyze the significance of the results using the R package PMCMRplus (Pohlert, 2022).
The ratio of lignin to polysaccharide peak heights decreased significantly until the second year of cultivation in both heartwood and sapwood, except at 1526 cm−1/1150 cm−1 in sapwood (Fig. 3). The peak height ratio of lignin to polysaccharides increased significantly in both heartwood and sapwood from the second to third year of cultivation, except at 1526 cm−1/1366 cm−1 and 1526 cm−1/1150 cm−1 in sapwood (Fig. 3). Thereafter, from the third to fourth year of cultivation, the peak height ratio of lignin to polysaccharides decreased; this decrease was significant at 1526 cm−1/1366 cm−1 (Fig. 3A) and 1526 cm−1/895 cm−1 (Fig. 3E) in heartwood and at 1526 cm−1/1366 cm−1 (Fig. 3B) in sapwood. Interestingly, there was no significant difference in the peak height ratio of lignin to polysaccharides between the control and the third-year wood logs for any comparative condition (Fig. 3). In a previous study, we found that the abundance of cellulolytic bacteria was significantly increased in wood logs inoculated with G. frondosa compared to the control, suggesting that these bacteria may help decompose cellulose in the wood logs, especially from the second to third year of cultivation (Chen et al., 2023). We also discovered that G. frondosa was dominant in wood logs from the first to fourth year of cultivation through a quantitative PCR to determine the copy numbers of the 16S rRNA gene from bacteria and cytochrome C gene (cytC) from G. frondosa in wood logs (Chen et al., 2023). These results indicate that the wood logs were mainly decayed by G. frondosa, which progresses from the lignin decomposition stage spanning 2 y to the polysaccharide decomposition stage spanning 1 y, and that polysaccharides are decomposed until the lignin to polysaccharide ratio is equivalent to that of the control, after which the next cycle of lignin decomposition proceeds (i.e., after the third year of cultivation). Previous studies have suggested that white-rot fungi decompose wood by separating the lignin and polysaccharide decomposition stages. Phlebia sp. selectively degrades lignin under aerobic conditions; however, when nutrients are added and cultured under anaerobic liquid conditions, the metabolism switches to polysaccharide decomposition (Kamei et al., 2012). Additionally, the expression of putative cellulolytic and xylanolytic genes is upregulated in delignification-defective mutants of the edible white-rot fungus, Pleurotus ostreatus (Wu et al., 2020). Moreover, a brown-rot fungus, Postia placenta, expresses lignin modification-related genes and polysaccharide degradation-related genes alternately and independently, depending on the decay stage (Zhang et al., 2016). In addition, in P. placenta, lignin-modifying genes (oxidoreductase genes) are strictly suppressed in the presence of all soluble sugars, and polysaccharide degradation-related genes (cellulase and hemicellulase genes) are upregulated in the presence of soluble cellobiose and polymeric carbon sources (Zhang & Schilling, 2017). From the results of the present study and previous reports, we speculate that selective white-rot fungi such as G. frondosa decay wood by repeating two stages in which they preferentially decompose lignin followed by polysaccharides.
On the other hand, the ratio of the acetyl group of hemicellulose to the polysaccharide peak height decreased significantly, except for the peak height ratio of 1755 cm−1 to 1366 cm−1 in heartwood (Fig. 4A) until the second year of cultivation (Fig. 4). The peak height ratio of 1755 cm−1 to 1366 cm−1 in the heartwood significantly decreased during the third year of cultivation (Fig. 4A). Considering that G. frondosa decompose polysaccharides in wood log between the second and third year of cultivation, G. frondosa deacetylates polysaccharides as a pre-step to polysaccharide decomposition. It has been reported that 11-17% of hardwood holocellulose contains acetyl groups, and approximately 40% of acetyl glucuronoxylan contains acetyl groups (Bouveng, 1961; Hägglund et al., 1956). Moreover, acetylation of wood (Pinus sylvestris) improves resistance to decay caused by P. placenta (Ringman et al., 2014), and a decrease in the number of acetyl groups in acetylated pine leads to the progression of wood decay caused by the same fungus (Ringman et al., 2019). Brown-rot fungi selectively degrade polysaccharides from the wood components. Based on the results of the present study and that from previous reports, white-rot fungus, G. frondosa, appears to deacetylate hemicellulose before degrading polysaccharides.
In conclusion, this study showed that wood decay by the white-rot fungus G. frondosa involved repeated cycles of two decay stages: the first stage involved decomposition of lignin and deacetylation of hemicellulose, while the second stage involved decomposition of polysaccharides. It was speculated that the switching of the decomposition target in this manner led to the efficient decomposition of wood by white-rot fungi. The molecular mechanisms underlying the regulation of the two decay stages require further evaluation. Furthermore, we demonstrated that FT-IR spectroscopy is an effective modality for evaluating wood decay given its ability to facilitate rapid evaluation of the deacetylation of hemicellulose and the decomposition of the main wood components.
The authors declare that they have no conflicts of interest.
MT designed this study, analyzed the study data, and was a major contributor in writing the manuscript. CF and AD contributed to the discussion of the results. All the authors read and approved the final manuscript.
The authors would like to thank Mr. Takesi Imanisi, Prof. Yoshio Kijidani, Prof. Ichiro Kamei, Dr. Taku Tsuyama and Dr. Takeshi Nitta for their contributions to sample collection. We would like to thank Editage (www.editage.jp) for English language editing. This work was supported by JSPS KAKENHI Grant Numbers 23K19309 and 24K17942.
