Growth Characteristics and Ergosterol Content of Grifola frondosa in Various Solid-state Substrates

Growth characteristics of medicinal mushroom Grifola frondosa mycelia were studied in solid-state cultivation on various solid-state substrates in different setups. The mycelial growth rate was determined in racing tubes by the measurement of ergosterol content. The fastest growth rate of 3.76 mm d–1 and the highest biomass amount of 54 mg g–1 were achieved. The results were scaled-up in a horizontal stirred bioreactor, where higher amounts of biomass (53.2 mg g–1 in 42 days) were achieved. These results represent an available platform for large-scale production of medicinal fungi biomass in bioreactors.


Introduction
Grifola frondosa (Dicks: Fr) S.F.Gray is a white rot basidiomycete, belonging to Basidiomycotina, Homobasidiomycetes, Aphyllophoromycetideae, Polyporales, Family Meripilaceae 1 .The mushroom (Figure 1) is known under several English names (Dancing mushroom, Cloud mushroom, Hen-of-the-woods, the Dancing Butterfly mushroom, Sheep's head), in Japan as Maitake, and in China as Hui Shu Hua.Fresh and dried G. frondosa fruit bodies, which are commonly available in food markets of Asia, especially of Japan, China and Korea, have also become available in other countries, such as the USA, Canada, and EU.Numerous nutraceutical preparations based on G. frondosa powdered fruit bodies, dried extracts or purified polysaccharides are emerging in different forms -as capsules, tablets, as well as additives to food formulations.
Grifola frondosa mycelium and its polysaccharides can be produced in liquid media 2 as well as solid-state cultivation on sunflower seed huls 3 and different grain types 3,4 , while fruiting bodies are usually cultivated on broadleaf sawdust-based substrates with addition of different supplements, such as wheat bran, crushed cord seeds 5 , olive oil press cakes 6 and others.Commercially, polypropylene bags or bottles are used as substrate containers from which fruiting bodies emerge.
Grifola frondosa fruit bodies often occur as a heavy mass at the base of stumps and on the roots of dead or dying deciduous hardwoods, such as Quercus, Ulmus, Acer, Nyssa, Larix and Fagus; sporadically also on Castanea, Prunus, Pseudotsuga and Pinus.The optimal growing conditions exist within a limited range of temperature, moisture, humidity, and other environmental factors.Parts of northeastern Japan are especially hospitable for this mushroom, although it can also be found in the northern temperate forests of Asia, Europe, and eastern North America 9 .
Beside the references cited above, other broadleaf sawdust types could have a potential use for G. frondosa biomass and fruiting bodies cultivation.In this article, G. frondosa mycelium growth traits on selected substrates composed of beech, Norwegian spruce, European larch and common grape vine sawdust were conducted, and fungal biomass were determined.This work represents the investigation of presently unexploited raw materials for G. frondosa biomass and fruiting bodies production.Substrate compositions with the best characteristics for G. frondosa growth were evaluated, selected, and applied in a pilot solid-state horizontal stirred bioreactor.

Microorganism and seed culture preparation
Grifola frondosa (Dicks: Fr) S.F.Gray strain GF3, original strain from Slovenian forests used in this study, was isolated and is deposited at the Fungal bank of the Biotechnical Faculty, Department of Wood Science and Technology, University of Ljubljana, Slovenia.The culture was maintained at 28 °C on potato dextrose agar -PDA (Difco, USA), and transferred every two weeks onto freshly prepared media.Liquid inoculum for PDA plate inoculation was prepared by mixing a two-week-old PDA plate overgrown with G. frondosa mycelium in 200 mL sterilized deionized water in a S700 blender (Waring products, Calhoun, GA, USA), three times, for 20 seconds.

Mycelium growth in vitro
Twenty-one petri dishes (H =13 mm, r = 43 mm) containing 35 mL of sterilized PDA were inoculated in the center of the PDA plate with 10 µL of liquid inoculum.Incubation was performed at 28 °C.Mycelium growth was followed with digital images analysis using the Motic Images Plus software (Motic China Group LTD) for 21 days, and expressed as radius of the circular growth zone.Once the mycelium overgrew the petri plate, it was carefully collected from the media surface, dried, weighed, and analysed for ergo sterol content.

Mycelium growth in racing tubes
Growth rate measurements, biomass production, and ergosterol content were studied in solid-state bioprocessing conditions using "racing tubes".The method for growth rate measurements in the samples was developed.Glass tubes 175×25 mm, (Figure 2) were filled with 28 g of substrate (substrate density of 0.35 g mL -1 ), closed on both sides with a plastic cap, and sterilized for 45 minutes at T = 121 °C; p = 1.2 • 10 5 Pa.After cooling, the substrates were inoculated on one side of the tube with a 16-mm disc from a two-week-old PDA culture, and wrapped in parafilm to prevent moisture loss.The inoculated substrate in the racing tubes was incubated at 28 °C and mycelium growth was monitored periodically using a stereo microscope joined with a precision measuring tool.The length of the linear growth zone of mycelia in the racing tubes in different time-points represents the growth rate.All experiments were performed in at least triplicate.

Mycelium growth in glass jar bioreactors
Cylindrical glass jars of 700 mL volume were used as containers for fungal biomass cultivation.They were filled with 400 mL of substrate, and closed with a cotton filter lid for air exchange.The substrates were sterilized for 45 min at T = 121 °C; p = 1.2 • 10 5 Pa, inoculated with 5 mL of liquid inoculum, thoroughly mixed and incubated at 28 °C for 7 days.Samples were obtained every three days, and the ergosterol and moisture content determined.All experiments were performed in triplicate.Electron micrographs of G. frondosa mycelium on solid-state substrates were performed with a Field-emission Scanning Electron Microscope at an accelerating voltage of 1 kV (Carl Zeiss Supra 35 VP).

Mycelium growth in horizontal stirred tank bioreactor
The best substrate formula (and perhaps time of solid-state cultivation) from the previous stepthe glass jar bioreactors, were scaled up in an ad hoc 15-L horizontal stirred tank bioreactor.Details of the construction of this device are shown in Figure 3.The reactor was filled to two-thirds depth with substrate (3500 g of beech sawdust substrate), sterilised in situ for 120 minutes (T = 121 °C; p = 1.2 • 10 5 Pa), and cooled to the inoculation temperature.Substrate was inoculated with 500 mL of seed culture, and incubated at 28 °C.Periodical mixing (80 rpm) by horizontal blades attached to a central shaft, for two minutes/day, and aeration over a hollow shaft of the impeller (5 L h -1 air flow) were applied.The same principles of process optimization as inoculation, sterilization, mixing, aeration, temperature, and humidity were applied as in our previous works 6,10 .

Ergosterol and biomass determination
Ergosterol was determined applying a modified method described by Gregori (2014) 11 .In brief, 10 mg of dry fungal biomass obtained from three petri dish PDA plates were extracted for 90 minutes with 1 mL of a solution of 10 % (w/v) polyvinylpyrrolidone (Sigma, Germany) in 96 % ethanol, at 4 °C on a rotary shaker at 200 rpm.The resulting extract was centrifuged (10000 rpm, 15 min, T = 4 °C), and filtered through a 0.45-µm pore size filter.The fil-trate (100 µL) was injected into a HPLC system (Varian, USA) with 1:1 (v/v) methanol:acetonitrile (Sigma, Germany) mobile phase at 1 mL min -1 flow rate on a Synergi MAX-RP C12 column (Phenomenex, USA).Ergosterol was identified using standard retention time and specific peak between 260 and 300 nm obtained by calibration curve preparation using purified ergosterol standard (Sigma, Germany).For ergosterol determination in solid-state cultivation substrates, a 2-g sample was extracted with 20 mL of the same extraction mixture and subjected to the same procedure 12 .

Moisture content determination
The moisture content of substrate samples (grams) was determined with a HR83 Moisture Analyzer (Mettler-Toledo, Switzerland).

Statistics
All of the cultivation experiments were performed at least in three runs.The results were analyzed for statistical significance by one-way analysis of variance (ANOVA) test using the Statistical Package of the Social Science (SPSS) version 11.0 (SPSS Inc., Chicago, IL, USA).Statistical significance of the test effects was evaluated at p < 0.05.Data were expressed as mean ± SD.

Mycelium growth in vitro and ergosterol content determination
Ergosterol is a specific component of fungal cell membranes 12 .With ergosterol being in correlation to fungal biomass quantity, its determination F i g .3 -Scheme of horizontal stirred tank bioreactor -according to Berovic and Ostroveršnik (1997)  can serve as an indicator for fungal growth rate.Figure 4 shows the mycelium growth until day 21 when it reached the borders of the petri plate in all experimental units.
Regarding the ergosterol content in dry biomass, it was found that it was constant throughout the 21 days of the vegetative mycelium growth.The average content of ergosterol in the dried mycelium from the surface of PDA was 5.80 mg g -1 ± 0.04 mg g -1 .This value was used as reference to calculate the biomass content in the solid-state fermented substrates.

Mycelium growth in racing tubes
The growth rate of G. frondosa on different solid-state bioprocessed fermented substrates using the racing tubes test is shown in Figure 5.The highest growth rate occurred on beech sawdust-based substrate (3.76 mm d -1 ), followed by larch-(3.61mm d -1 ), spruce-(2.95mm d -1 ), and common vine (1.76 mm d -1 ) based substrates.Larch-and sprucebased substrates presented a long lag phase that lasted about 12 days, while beech and common vine substrate had no noticeable lag phase.

Mycelium growth in glass jars
The mycelium growth rate of G. frondosa in solid-state bioprocessed substrates in glass jar bioreactors is shown in Figure 6.The highest dry biomass yield of 54 mg g -1 solid-state bioprocessed substrate was obtained at day 47 on the beech sawdust-based substrate.Next, were vine (49 mg g -1 ), spruce (40 mg g -1 ) and larch sawdust (30 mg g -1 ) based substrate.Comparing the results from the race tubes and glass jar bioreactors, it was concluded that substrates composed of beech sawdust were more favorable for mycelial growth compared to other substrates.This is well in accordance with the fact that G. frondosa in natural habitats occurs mostly on beech wood 8 .The results for growth rates on substrates composed of spruce sawdust are comparable in terms of relative growth rate and biomass accumulation.The growth rate on spruce sawdust substrate was 78 % higher, while in this case the accumulated biomass was only 74 % of that obtained on beech sawdust substrate.
The growth rate on larch sawdust-based substrate was 96 % of the growth on beech substrate, while the amount of biomass was only 56 % of the previous one.This was also visible when comparing the density of mycelia in racing tubes and glass jars.The mycelium was markedly less dense.Interestingly, the data for vine sawdust substrate was more favorable in terms of biomass accumulation, which achieved 90 % of biomass accumulation on beech sawdust substrate, but showed lower growth rate (56 % of the growth rate of beech sawdust substrate).Mycelium growth in the horizontal stirred tank bioreactor After the lag phase, mycelium grew concentrically on the surface of the solid particles and coaxially from the perforated shaft of impeller towards the walls of the bioreactor (Figure 3).During growth, the polysaccharides were secreted at the tips of young hyphae (Figure 7a).These molecules were used as sticky materials to produce the anchorage points for the movement of young hyphae, as well as transport medium for secretion of lignocellulolytic enzymes and a protective agent (Figure 7b).
From our results, it was found that growth in the horizontal stirred tank reactor was closely parallel with the growth in the glass jar fermenter achieving the most comparable amount of biomass (53.2 mg g -1 ) in only 42 days.The quicker growth rate can be attributed to aeration, as the glass jar fermenter only had passive ventilation.The total biomass produced in the solid-state fermenter after 42 days of incubation was 65 g dry weight.

Conclusions
Various substrates based on lignocellulosic sawdust particles from beech, Norwegian spruce, European larch and grapevine (sizes from 0.125 mm -0.5 mm) were tested.The average ergosterol content in fungal biomass in measured samples was 5.80 mg g -1 ± 0.04 mg g -1 .The growth rates and biomass accumulation were different on each of the prepared solid substrates.As expected, the best results were obtained on beech sawdust substrate (3.76 mm d -1 and 54 mg g -1 ).The remarkable differences were indicated on substrates composed of larch and vine sawdust substrates.The former was better in terms of growth rate and the latter in biomass production.
The results from the cultivation process in the horizontal stirred bioreactor, in terms of biomass per amount of solid substrate, compared at the same amount of biomass, indicated a faster cultivation process in aerated bioreactor.The growth area increased linearly (at 10.55 ± 0.02 SD mm d -1 ), while the biomass increased exponentially.Final dry biomass achieved at 21 days of incubation was 88.3 ± 0. 028 SD mg.

F i g . 1 -
Fruiting body of G. frondosa in artificial cultivation on beech sawdust supplemented with wheat branF i g . 2 -Racing tubes showing solid-state cultivation on beech sawdust substrate.Mycelial growth is visible with the naked eye.

F i g . 4 -
Mycelium growth in vitro.The growth radius and total dry biomass of G. frondosa mycelium on PDA medium are presented.

F i g . 5
-G. frondosa mycelium growth in racing tubes.The growth in millimeters (■) and the biomass dry weight (♦) are presented for different substrates formulations.

F i g . 6 -
Mycelium growth in glass jar fermenters.Results are expressed as mg of dry biomass per g of solid-state fermented substrate.Main components of substrates are beech sawdust (▲), larch sawdust (■), spruce sawdust (♦) and vine sawdust (▬).

F i g . 7 -
Mycelium from horizontal stirred tank bioreactor.(a) Fungal polysaccharide on the tip of young hyphae.(b) Sticky anchorages of hyphae tips on the cellulose matrix.(c) Mycelium growth on spruce sawdust (d) Vine sawdust-based substrate in the horizontal stirred bioreactor.