Growth Characteristics of Polyporales Mushrooms for the Mycelial Mat Formation

MYCOBIOLOGY 2021, VOL. 49, NO. 3, 280–284 https://doi.org/10.1080/12298093.2021.1911401

RESEARCH NOTE Growth Characteristics of Polyporales Mushrooms for the Mycelial Mat Formation

Ã Ã Bin Bae , Minseek Kim , Sinil Kim and Hyeon-Su Ro Department of Bio and Medical Big Data (BK4 program) and Research Institute of Life Sciences, Gyeongsang National University, Jinju, Republic of Korea

ABSTRACT ARTICLE HISTORY Mushroom strains of Polyporales from the genera Coriolus, Trametes, Pycnoporus, Ganoderma, Received 26 January 2021 and Formitella were explored in terms of mycelial growth characteristics for the application Revised 28 March 2021 of mushroom mycelia as alternative sources of materials replacing fossil fuel-based materials. Accepted 29 March 2021 G. lucidum Among the 64 strains of Polyporales, LBS5496GL was selected as the best candi- KEYWORDS date because it showed fast mycelial growth with high mycelial strength in both the saw- Polyporales; Mycelial mat; dust-based solid medium and the potato dextrose liquid plate medium. Some of the Ganoderma; SEM analysis Polyporales in this study have shown good mycelial growth, however, they mostly formed mycelial mat of weak physical strength. The higher physical strength of mycelial mat by G. lucidum LBS5496GL was attributed to its thick hyphae with the diameter of 13 mmas revealed by scanning electron microscopic analysis whereas the hyphae of others exhibited less than 2 mm. Glycerol and skim milk supported the best mycelial growth of LBS5496GL as a carbon and a nitrogen source, respectively.

Fossil fuels have been used as an important source served as a key link between carbon dioxide in the of energy and material production in human life atmosphere and plant biomass as primary decom- since they appeared in human history as an import- posers [4]. Apart from their role in the ecosystem, ant energy source for the Industrial Revolution, agaricomycetes are grown as edible crops and are which began in the mid-eighteenth century. recognized as important agricultural products. However, with industrial development and popula- Recently, efforts have been underway to develop tion growth, the demand–supply of materials has new eco-friendly industrial materials, away from the exploded, and humans face two risks: resource limited use of fungi, including mushrooms, in trad- depletion and environmental destruction due to itional industries such as food, antibiotics, and resource abuse. Therefore, securing eco-friendly enzyme production. In particular, various mush- energy sources that minimize the use of fossil fuels, rooms have been studied in the development of bio- developing new recycling methods of materials, and composite materials using mushroom mycelia and developing restoration technologies for the agricultural byproducts [5–7]. Common agricultural destroyed environment are key tasks to open a sus- byproducts such as rice straw, wheat straw, and tainable future. sawdust are combined with mushroom mycelia and Fossil fuels are known to be originated primarily used in the production of various living and indus- from plant materials that flourished in the carbon- trial components such as insulation, interior materi- iferous period by creating underground sedimentary als, furniture, and decorative items. These mycelial layers without decomposition before the agaricomy- biocomposites, combined with 3D printing technol- cetes started to break down dead plants [1,2], sug- ogy, show the potential to develop into an import- gestively together with climate and crustal ant eco-friendly material for new industry [8]. In fluctuations [3]. The capability of agaricomycetes to addition, research on the production of fungal meat decompose dead plant is attributed to the destruc- and leather, which mimic animal meat and leather, tion of lignin by the activity of several ligninolytic respectively, have been conducted and partially com- enzymes [4]. This suggests that agaricomycetes have mercialized [9,10].

CONTACT Hyeon-Su Ro [email protected] Department of Bio and Medical Big Data (BK4 program) and Research Institute of Life Sciences, Gyeongsang National University, Jinju, Republic of Korea Ã These authors contributed equally to this work. Supplemental data for this article can be accessed here. ß 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of the Korean Society of Mycology. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. MYCOBIOLOGY 281

Figure 1. Growth characteristics of the selected strains of Polyporales in sawdust medium (A,B) and in PDB plate medium (C,D). The results are summarized in Supplementary Table S1.

The filamentous growth characteristics of fungi fraxinea, stocked in the Center for Mushroom enable the formation of mycelial networks. If fungal Molecular Genetics, GNU were screened in terms of network grows vertically and horizontally together mycelial growth. The strains were routinely grown with occasion hyphal fusion, it can create thick on potato dextrose agar medium (PDA; Oxoid, mycelial tissue which can show the characteristics of Hampshire, UK) at 25 C. The mushroom strains animal skins or meat if properly processed. In add- were firstly screened in a sterilized sawdust medium ition, the mycelial network penetrates into plant containing 50 g of oak tree sawdust and 100 mL of substrates, it strengthens the connection between water in a polypropylene container (120 mm x the substrates, enabling the formation of plant-based 80 mm, Phytohealth, SPL Life Sciences, Pocheon, biocomposite. The physicochemical properties of the Korea) by inoculating the mycelial culture broth fungal biocomposite depend on the unique physio- grown in potato dextrose broth (PDB; Oxoid) for a logical properties expressed by the genetic informa- week at 25 C. The inoculated medium was incu- tion of the fungus as well as physicochemical bated at 25 C for 2 weeks under 80% relative conditions for the production of the composite, humidity. The degree of mycelial growth was meas- such as substrate type, growth temperature, pH, and ured by a 5-point scale. As a result, five strains of F. carbon dioxide concentration. For example, the fraxinea, including LBS9639FF, LBS9630FF, mycelial network of Schizophillum commune has LBS9257FF, LBS2337FF, and LBS9541FF, and a sin- been shown to be affected by the expression of gle strain of C. versicolor, LBS2279CV, were found hydrophobin and carbon dioxide concentration [11]. to be the fastest growing strains (Figure 1(A) and For the development of industrial material using the Supplementary Table S1). On the other hand, fungal mycelial network, the growth rate of fungal LBS1894CB (C. brevis), LBS8925CV (C. versicolor), strain, density of cultured mycelium, and strength of LBS1819CV (C. versicolor), LBS9327CH (C. hirsu- individual hyphae are important. tus), LBS2291TG (T. gibbosa), and LBS9680TG (T. In this study, we explored mushroom strains gibbosa) grew poorly (Figure 1(B) and belonging to the order Polyporales which have been Supplementary Table S1). known to grow well and form dense mycelial net- Because the mycelial growth in the sawdust work. To this end, 64 strains of Polyporales, includ- medium took too long incubation time, we next ing Coriolus versicolor, Trametes, Pycnoporus investigated the mycelial growth in potato dextrose coccineus, Ganoderma lucidum, and Formitella liquid plate medium in a sterile rectangular culture 282 B. BAE ET AL.

Figure 2. Scanning electron microscopy analyses of mycelial mats from different polypore mushrooms in two different magni- fications (Â250 and Â900). (A,B) Coriolus brevis LBS1894CB. (C,D) Fomitella fraxinea LBS4388FF. (E,F) Ganoderma lucidum LBS5496GL. (G,H) F. fraxinea LBS2351FF. (I,J) C. versicolor LBSCVEYCV. (K,L) F. fraxinea LBS2349FF. The samples were prepared using the mycelial mats obtained from the PDB plate medium culture. Arrows indicate the hyphal diameters of the mycelium in the mycelial mat. The hyphal diameter of F. fraxinea was less than 1 mm, as indicated. plate (126.4 Â 126.4 Â 20 mm, Square dish, SPL Life mycelia. Among them, three strains, LBS1894CB, Sciences), containing 50 mL PDB plus 50 mL of 1 LBS5496GL, and LBS2283CV, produced a relatively week PDB grown inoculum. The mushroom strains uniform white mycelium. These results show that were incubated at the same conditions as the saw- the growth characteristics of mushroom strains and dust medium and the mycelial growth was recorded the nature of mycelial mats can vary significantly by a 5-point scale. As a result, the strains of C. ver- depending on mushroom strains, substrate, and sicolor (LBS3442CV, LBS9665CV, LBS5512CV, environmental conditions. LBS2283CV, and LBS1140CV), T. gibbosa Scanning electron microscopy (SEM) analysis was (LBS1162TG, LBS9303TG, and LBS9421TG), C. bre- conducted after drying the mycelium for 24 h at vis (LBS1672CB and LBS1894CB), and G. lucidum 60 C obtained through the above liquid (LS5496GL) showed better growth (Figure 1(C), culture. The dried mycelial mat was gold-coated Supplementary Table S1). Among them, using vacuum sputter coater (DSR; element Pi, LBS3442CV, LBS1672CB, LBS9665CV, and Beaverton, OR) and examined under a SEM micro- LBS5512CV were equally well-growing fungi in saw- scope (Jeol JSM-7610F; Joel, Tokyo, Japan). The sur- dust mediums, but LBS1894CB and LBS1162TG face analysis of the fungal mat of F. fraxinea rarely grew in sawdust medium (Figure 1(D), showed a thin layer of primary crumbly membranes, Supplementary Table S1). C. versicolor LBS9783CV with a thin layer of mycelium (within 1 mm diam- and LBS9281CV, which showed excellent growth in eter) at the bottom, with a low mycelial density sawdust medium, showed, in contrast, poor growth (Figure 2). C. brevis LBS1894CB, which produces a in liquid culture. Unlike sawdust medium, F. fraxi- uniform white mycelium, had very uniform mycelial nea LBS9639FF, LBS9630FF, LBS9257FF, tissue at around 2.6 mm in hyphal diameter, but the LBS2337FF, and LBS9541FF showed moderate strength of mycelium was weak enough to break by growth characteristics. Meanwhile, the color of hand (Figure 2(A,B)). In the case of LBSCVEYCV, mycelia formed in the liquid medium also differed which has a relatively good growth characteristic, significantly depending on the type of mushroom the strength of the mycelium was stronger than that strains. Most of the strains used in the experiment of the C. brevis, and the mycelial density was rela- were brown, with the LBS9524CV, LBS9787CV, tively higher ((Figure 2(I,J)). Finally, in the case of LBS9293CV, and LBS1819CV making yellow G. lucidum LBS5496GL, the strength of the MYCOBIOLOGY 283

Figure 3. Effects of carbon and nitrogen sources on the mycelial growth of Ganoderma lucidum LBS5496GL. (A) Effect of carbon sources. Two different concentrations (4 and 10 g/L) of different carbon sources were examined in minimal medium. (B) Effect of nitrogen sources. 5 g/L of each nitrogen source was examined in minimal medium. (C) Effect of the concentrations of skim milk. All the experiments were triplicated. The error bars indicate standard errors of each experimental sets. (D) Mycelial mat generated from scale-up experiment using the optimal conditions. mycelium was incomparably stronger than that of First, 5 mL of PDB-grown culture broth was inocu- other fungi. The mycelial mat was characterized by lated to the minimal medium composed of yeast the formation of a thick hyphae with a diameter of nitrogen base (6.7 g/L) and 4 g/L or 10 g/L of indi- 13 mm(Figure 2(E,F)) and by the coverage of the vidual carbon sources, including galactose, glucose, mat with a large amount of uncharacterized macro- glycerol, maltose, sorbitol, and sucrose. Nitrogen molecules. In fact, the mycelial mat of LBS5496GL sources (5 g/L), such as casein, peptone, skim milk, was strongest among all examined strains in this and soytone, were also examined under the same study. Mechanical strength of mycelial network is conditions. As a result, galactose, glucose, maltose, important factor in the industrial application [12]. and glycerol showed similar mycelial growth while Tacer-Caba et al. demonstrated the generation of skim milk was the best nitrogen source (Figure bio-composites with high compressive strength 3(A,B)). The mycelial growth was dependent on the when mushroom strains, such as Agaricus bisporus, concentration of skim milk, resulting in the max- Pleurotus ostreatus, and G. lucidum, were cultivated imum growth at the concentration of 60 g/L (Figure on rapeseed cake [7]. Ganoderma SP. grown on cot- 3(C)). Based on these results, we conduct a scale-up ton plant materials was also applied to produce bio- experiment for large-scale mycelial mat production. degradable packaging material [13]. First, G. lucidum LBS5496GL was cultured in Since G. lucidum is known as a medicinal mush- 500 mL PDB supplementary with 10 g/L glycerol and room, and thus is primarily interested in the fruiting 30 g/L skim milk. The culture was poured into a body cultivation, there are very few studies on polypropylene container (24 Â 18 Â 6 cm) which mycelial growth in solid culture [14] and liquid cul- contained layers of synthetic cotton in 2 cm and nat- ture for the production of bio-active compounds ural cotton in 0.5 cm. The container was incubated [15,16]. Because our sceening experiments con- for one week at 30 C with 85% relative humidity. firmed that G. lucidum LBS5496GL produced the The experiment resulted in a mycelial mat of best physical characteristic of mycelium, we investi- 24 Â 18 Â 0.6 cm (Figure 3(D)). gated the effects of carbon and nitrogen sources In an effort to replace fossil fuel-based materials, focusing on the growth of G. lucidum LBS5496GL. attempts to employ fungal mycelia in the fabrication 284 B. BAE ET AL. of eco-friendly composite materials have emerged [5] de Lima GG, Schoenherr ZCP, Magalh~aes WLE, recently. Particularly, basidiomycetes, such as et al. Enzymatic activities and analysis of a myce- Daedaleopsis confragosa, A. bisporus, P. ostreatus, lium-based composite formation using peach palm (Bactris gasipaes) residues on Lentinula edodes. and G. lucidum, have been sought for the industrial Bioresour Bioprocess. 2020;7(1):58. application in coatings, papers, membranes, packag- [6] Elsacker E, Vandelook S, Brancart J, et al. ing materials, composite materials, and leathers due Mechanical, physical and chemical characterisation to their physical strength and growth characteristics of mycelium-based composites with different types [17,18]. Accordingly in this study, screening of 64 of lignocellulosic substrates. PLoS One. 2019;14(7): e0213954. strains of Polyporales in terms of growth rate and [7] Tacer-Caba Z, Varis JJ, Lankinen P, et al. the capability to form mycelial mat was performed, Comparison of novel fungal mycelia strains and resulting in the finding of G. lucidum LBS5496GL as sustainable growth substrates to produce humid- a potential candidate for further industrial applica- ity-resistant biocomposites. Mater Des. 2020;192: tion. Subsequent medium optimization and scale-up 108728. [8] Bhardwaj A, Vasselli J, Lucht M, et al. 3D printing experiments allowed the formation of big size myce- of biomass-fungi composite material: a preliminary lial mat. These results are expected to serve as the study. Manuf Lett. 2020;24:96–99. basis for new industrial applications of fungi, [9] Cho SY, Ryu GH. Effects of mushroom compos- including mushrooms, in the future. ition on the quality characteristics of extruded meat analog. Kor J Food Sci Technol. 2020;52(4): 357–362. Disclosure statement [10] Ahirwar R, Jayathilakan K, Jalarama RK, et al. Development of mushroom and wheat gluten The authors report no conflicts of interest. based meat analogue by using response surface methodology. Int J Adv Res. 2015;3(1):923–930. [11] Appels FVW, Dijksterhuis J, Lukasiewicz CE, et al. Funding Hydrophobin gene deletion and environmental This work was supported by a grant from the New growth conditions impact mechanical properties of Breeding Technologies Development Program [Project mycelium by affecting the density of the material. No. PJ01516502], Rural Development Administration, Sci Rep. 2018;8(1):4703. Republic of Korea. [12] Hartmann F, Baumgartner M, Kaltenbrunner M. Becoming sustainable, the new frontier in soft robotics. Adv Mater. 2020;2020:2004413. [13] Holt G, Mcintyre G, Flagg D, et al. Fungal myce- ORCID lium and cotton plant materials in the manufac- ture of biodegradable molded packaging material: Hyeon-Su Ro http://orcid.org/0000-0003-1128-8401 evaluation study of select blends of cotton byproducts. J Biobased Mat Bioenergy. 2012;6(4):431–439. [14] Jung IC, Kim SH, Kwon YI, et al. Cultural condi- References tion for the mycelial growth of Ganoderma lucidum on cereals. Kor J Mycol. 1996;24(1):81–88. [1] Eastwood DC. Evolution of fungal wood decay. In: [15] Hu G, Zhai M, Niu R, et al. Optimization of cul- Schultz TP, Goodell D, Nicholas DD, editors. ture condition for ganoderic acid production in Deterioration and protection of sustainable bio- Ganoderma lucidum liquid static culture and mass. Washington (DC): American Chemical design of a suitable bioreactor. Molecules. 2018; Society (ACS Symposium Series; vol. 1158); 2014. 23(10):2563. – p. 5 93. [16] Meng L, Bai X, Zhang S, et al. Enhanced ganoderic [2] Hawksworth D. Coal measure formation and lig- acids accumulation and transcriptional responses nin-degrading fungi. IMA Fungus. 2012;3(2): of biosynthetic genes in Ganoderma lucidum fruit- – 55 58. ing bodies by elicitation supplementation. IJMS. [3] Nelsen MP, DiMichele WA, Peters SE, et al. 2019;20(11):2830. Delayed fungal evolution did not cause the [17] Jones M, Mautner A, Luenco S, et al. Engineered Paleozoic peak in coal production. Proc Natl Acad mycelium composite construction materials from Sci USA. 2016;113(9):2442–2447. fungal biorefineries: a critical review. Mater Des. [4] Floudas D, Binder M, Riley R, et al. The paleozoic 2020;187:108397. origin of enzymatic lignin decomposition recon- [18] Jones M, Gandia A, John S, et al. Leather-like structed from 31 fungal genomes. Science. 2012; material biofabrication using fungi. Nat Sustain. 336(6089):1715–1719. 2021;4(1):9–16.