RESEARCH NOTE

Growth Characteristics of Polyporales Mushrooms for the MycelialMat Formation

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

ABSTRACTMushroom strains of Polyporales from the genera Coriolus, Trametes, Pycnoporus, Ganoderma,and Formitella were explored in terms of mycelial growth characteristics for the applicationof mushroom mycelia as alternative sources of materials replacing fossil fuel-based materials.Among the 64 strains of Polyporales, G. lucidum LBS5496GL was selected as the best candi-date because it showed fast mycelial growth with high mycelial strength in both the saw-dust-based solid medium and the potato dextrose liquid plate medium. Some of thePolyporales in this study have shown good mycelial growth, however, they mostly formedmycelial 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 13mm asrevealed by scanning electron microscopic analysis whereas the hyphae of others exhibitedless than 2mm. Glycerol and skim milk supported the best mycelial growth of LBS5496GL asa carbon and a nitrogen source, respectively.

ARTICLE HISTORYReceived 26 January 2021Revised 28 March 2021Accepted 29 March 2021

KEYWORDSPolyporales; Mycelial mat;Ganoderma; SEM analysis

Fossil fuels have been used as an important sourceof energy and material production in human lifesince they appeared in human history as an import-ant energy source for the Industrial Revolution,which began in the mid-eighteenth century.However, with industrial development and popula-tion growth, the demand–supply of materials hasexploded, and humans face two risks: resourcedepletion and environmental destruction due toresource abuse. Therefore, securing eco-friendlyenergy sources that minimize the use of fossil fuels,developing new recycling methods of materials, anddeveloping restoration technologies for thedestroyed environment are key tasks to open a sus-tainable future.

Fossil fuels are known to be originated primarilyfrom plant materials that flourished in the carbon-iferous period by creating underground sedimentarylayers without decomposition before the agaricomy-cetes started to break down dead plants [1,2], sug-gestively together with climate and crustalfluctuations [3]. The capability of agaricomycetes todecompose dead plant is attributed to the destruc-tion of lignin by the activity of several ligninolyticenzymes [4]. This suggests that agaricomycetes have

served as a key link between carbon dioxide in theatmosphere and plant biomass as primary decom-posers [4]. Apart from their role in the ecosystem,agaricomycetes are grown as edible crops and arerecognized as important agricultural products.Recently, efforts have been underway to developnew eco-friendly industrial materials, away from thelimited use of fungi, including mushrooms, in trad-itional industries such as food, antibiotics, andenzyme production. In particular, various mush-rooms have been studied in the development of bio-composite materials using mushroom mycelia andagricultural byproducts [5–7]. Common agriculturalbyproducts such as rice straw, wheat straw, andsawdust are combined with mushroom mycelia andused in the production of various living and indus-trial components such as insulation, interior materi-als, furniture, and decorative items. These mycelialbiocomposites, combined with 3D printing technol-ogy, show the potential to develop into an import-ant eco-friendly material for new industry [8]. Inaddition, research on the production of fungal meatand leather, which mimic animal meat and leather,respectively, have been conducted and partially com-mercialized [9,10].

CONTACT Hyeon-Su Ro rohyeon@gnu.ac.kr Department of Bio and Medical Big Data (BK4 program) and Research Institute of Life Sciences,Gyeongsang National University, Jinju, Republic of Korea�These authors contributed equally to this work.

Supplemental data for this article can be accessed here.

� 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.

MYCOBIOLOGY2021, VOL. 49, NO. 3, 280–284https://doi.org/10.1080/12298093.2021.1911401

The filamentous growth characteristics of fungienable the formation of mycelial networks. If fungalnetwork grows vertically and horizontally togetherwith occasion hyphal fusion, it can create thickmycelial tissue which can show the characteristics ofanimal skins or meat if properly processed. In add-ition, the mycelial network penetrates into plantsubstrates, it strengthens the connection betweenthe substrates, enabling the formation of plant-basedbiocomposite. The physicochemical properties of thefungal biocomposite depend on the unique physio-logical properties expressed by the genetic informa-tion of the fungus as well as physicochemicalconditions for the production of the composite,such as substrate type, growth temperature, pH, andcarbon dioxide concentration. For example, themycelial network of Schizophillum commune hasbeen shown to be affected by the expression ofhydrophobin and carbon dioxide concentration [11].For the development of industrial material using thefungal mycelial network, the growth rate of fungalstrain, density of cultured mycelium, and strength ofindividual hyphae are important.

In this study, we explored mushroom strainsbelonging to the order Polyporales which have beenknown to grow well and form dense mycelial net-work. To this end, 64 strains of Polyporales, includ-ing Coriolus versicolor, Trametes, Pycnoporuscoccineus, Ganoderma lucidum, and Formitella

fraxinea, stocked in the Center for MushroomMolecular Genetics, GNU were screened in terms ofmycelial growth. The strains were routinely grownon potato dextrose agar medium (PDA; Oxoid,Hampshire, UK) at 25 �C. The mushroom strainswere firstly screened in a sterilized sawdust mediumcontaining 50 g of oak tree sawdust and 100mL ofwater in a polypropylene container (120mm x80mm, Phytohealth, SPL Life Sciences, Pocheon,Korea) by inoculating the mycelial culture brothgrown in potato dextrose broth (PDB; Oxoid) for aweek at 25 �C. The inoculated medium was incu-bated at 25 �C for 2 weeks under 80% relativehumidity. The degree of mycelial growth was meas-ured by a 5-point scale. As a result, five strains of F.fraxinea, including LBS9639FF, LBS9630FF,LBS9257FF, LBS2337FF, and LBS9541FF, and a sin-gle strain of C. versicolor, LBS2279CV, were foundto be the fastest growing strains (Figure 1(A) andSupplementary Table S1). On the other hand,LBS1894CB (C. brevis), LBS8925CV (C. versicolor),LBS1819CV (C. versicolor), LBS9327CH (C. hirsu-tus), LBS2291TG (T. gibbosa), and LBS9680TG (T.gibbosa) grew poorly (Figure 1(B) andSupplementary Table S1).

Because the mycelial growth in the sawdustmedium took too long incubation time, we nextinvestigated the mycelial growth in potato dextroseliquid plate medium in a sterile rectangular culture

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.

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plate (126.4� 126.4� 20mm, Square dish, SPL LifeSciences), containing 50mL PDB plus 50mL of 1week PDB grown inoculum. The mushroom strainswere incubated at the same conditions as the saw-dust medium and the mycelial growth was recordedby a 5-point scale. As a result, the strains of C. ver-sicolor (LBS3442CV, LBS9665CV, LBS5512CV,LBS2283CV, and LBS1140CV), T. gibbosa(LBS1162TG, LBS9303TG, and LBS9421TG), C. bre-vis (LBS1672CB and LBS1894CB), and G. lucidum(LS5496GL) showed better growth (Figure 1(C),Supplementary Table S1). Among them,LBS3442CV, LBS1672CB, LBS9665CV, andLBS5512CV were equally well-growing fungi in saw-dust mediums, but LBS1894CB and LBS1162TGrarely grew in sawdust medium (Figure 1(D),Supplementary Table S1). C. versicolor LBS9783CVand LBS9281CV, which showed excellent growth insawdust medium, showed, in contrast, poor growthin liquid culture. Unlike sawdust medium, F. fraxi-nea LBS9639FF, LBS9630FF, LBS9257FF,LBS2337FF, and LBS9541FF showed moderategrowth characteristics. Meanwhile, the color ofmycelia formed in the liquid medium also differedsignificantly depending on the type of mushroomstrains. Most of the strains used in the experimentwere brown, with the LBS9524CV, LBS9787CV,LBS9293CV, and LBS1819CV making yellow

mycelia. Among them, three strains, LBS1894CB,LBS5496GL, and LBS2283CV, produced a relativelyuniform white mycelium. These results show thatthe growth characteristics of mushroom strains andthe nature of mycelial mats can vary significantlydepending on mushroom strains, substrate, andenvironmental conditions.

Scanning electron microscopy (SEM) analysis wasconducted after drying the mycelium for 24 h at60 �C obtained through the above liquidculture. The dried mycelial mat was gold-coatedusing vacuum sputter coater (DSR; element Pi,Beaverton, OR) and examined under a SEM micro-scope (Jeol JSM-7610F; Joel, Tokyo, Japan). The sur-face analysis of the fungal mat of F. fraxineashowed a thin layer of primary crumbly membranes,with a thin layer of mycelium (within 1 mm diam-eter) at the bottom, with a low mycelial density(Figure 2). C. brevis LBS1894CB, which produces auniform white mycelium, had very uniform mycelialtissue at around 2.6 mm in hyphal diameter, but thestrength of mycelium was weak enough to break byhand (Figure 2(A,B)). In the case of LBSCVEYCV,which has a relatively good growth characteristic,the strength of the mycelium was stronger than thatof the C. brevis, and the mycelial density was rela-tively higher ((Figure 2(I,J)). Finally, in the case ofG. lucidum LBS5496GL, the strength of the

Figure 2. Scanning electron microscopy analyses of mycelial mats from different polypore mushrooms in two different magni-fications (�250 and �900). (A,B) Coriolus brevis LBS1894CB. (C,D) Fomitella fraxinea LBS4388FF. (E,F) Ganoderma lucidumLBS5496GL. (G,H) F. fraxinea LBS2351FF. (I,J) C. versicolor LBSCVEYCV. (K,L) F. fraxinea LBS2349FF. The samples were preparedusing the mycelial mats obtained from the PDB plate medium culture. Arrows indicate the hyphal diameters of the myceliumin the mycelial mat. The hyphal diameter of F. fraxinea was less than 1mm, as indicated.

282 B. BAE ET AL.

mycelium was incomparably stronger than that ofother fungi. The mycelial mat was characterized bythe formation of a thick hyphae with a diameter of13 mm (Figure 2(E,F)) and by the coverage of themat with a large amount of uncharacterized macro-molecules. In fact, the mycelial mat of LBS5496GLwas strongest among all examined strains in thisstudy. Mechanical strength of mycelial network isimportant factor in the industrial application [12].Tacer-Caba et al. demonstrated the generation ofbio-composites with high compressive strengthwhen mushroom strains, such as Agaricus bisporus,Pleurotus ostreatus, and G. lucidum, were cultivatedon rapeseed cake [7]. Ganoderma SP. grown on cot-ton plant materials was also applied to produce bio-degradable packaging material [13].

Since G. lucidum is known as a medicinal mush-room, and thus is primarily interested in the fruitingbody cultivation, there are very few studies onmycelial growth in solid culture [14] and liquid cul-ture for the production of bio-active compounds[15,16]. Because our sceening experiments con-firmed that G. lucidum LBS5496GL produced thebest physical characteristic of mycelium, we investi-gated the effects of carbon and nitrogen sourcesfocusing on the growth of G. lucidum LBS5496GL.

First, 5mL of PDB-grown culture broth was inocu-lated to the minimal medium composed of yeastnitrogen base (6.7 g/L) and 4 g/L or 10 g/L of indi-vidual carbon sources, including galactose, glucose,glycerol, maltose, sorbitol, and sucrose. Nitrogensources (5 g/L), such as casein, peptone, skim milk,and soytone, were also examined under the sameconditions. As a result, galactose, glucose, maltose,and glycerol showed similar mycelial growth whileskim milk was the best nitrogen source (Figure3(A,B)). The mycelial growth was dependent on theconcentration of skim milk, resulting in the max-imum growth at the concentration of 60 g/L (Figure3(C)). Based on these results, we conduct a scale-upexperiment for large-scale mycelial mat production.First, G. lucidum LBS5496GL was cultured in500mL PDB supplementary with 10 g/L glycerol and30 g/L skim milk. The culture was poured into apolypropylene container (24� 18� 6 cm) whichcontained layers of synthetic cotton in 2 cm and nat-ural cotton in 0.5 cm. The container was incubatedfor one week at 30 �C with 85% relative humidity.The experiment resulted in a mycelial mat of24� 18� 0.6 cm (Figure 3(D)).

In an effort to replace fossil fuel-based materials,attempts to employ fungal mycelia in the fabrication

Figure 3. Effects of carbon and nitrogen sources on the mycelial growth of Ganoderma lucidum LBS5496GL. (A) Effect of car-bon 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 ofskim milk. All the experiments were triplicated. The error bars indicate standard errors of each experimental sets. (D) Mycelialmat generated from scale-up experiment using the optimal conditions.

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of eco-friendly composite materials have emergedrecently. Particularly, basidiomycetes, such asDaedaleopsis confragosa, A. bisporus, P. ostreatus,and G. lucidum, have been sought for the industrialapplication in coatings, papers, membranes, packag-ing materials, composite materials, and leathers dueto their physical strength and growth characteristics[17,18]. Accordingly in this study, screening of 64strains of Polyporales in terms of growth rate andthe capability to form mycelial mat was performed,resulting in the finding of G. lucidum LBS5496GL asa potential candidate for further industrial applica-tion. Subsequent medium optimization and scale-upexperiments allowed the formation of big size myce-lial mat. These results are expected to serve as thebasis for new industrial applications of fungi,including mushrooms, in the future.

Disclosure statement

The authors report no conflicts of interest.

Funding

This work was supported by a grant from the NewBreeding Technologies Development Program [ProjectNo. PJ01516502], Rural Development Administration,Republic of Korea.

ORCID

Hyeon-Su Ro http://orcid.org/0000-0003-1128-8401

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