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Mycelium growth kinetics and optimal temperature conditions for the

cultivation of edible mushroom species on lignocellulosic substrates

Article in Folia Microbiologica · February 2001

DOI: 10.1007/BF02818539 · Source: PubMed

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Folia Microbiol. 46 (3), 231-234 (2001) http://www.biomed.cas.cz/mbu/folia/

Mycelium Growth Kinetics and Optimal Temperature

Conditions for the Cultivation of Edible Mushroom Species
on Lignocellulosic Substrates
3 b 3 b
G. Z E R V A K I S , A. P H I L I P P O U S S I S , S. I 0 A N N I D 0 U , P. D l A M A N T O P O U L O U

"Institute of Kalamata, National Agricultural Research Foundation, 241 00 Kalamata, Greece

h
Institute of Agricultural Machinery and Constructions, National Agricultural Research Foundation, 135 61 Athens, Greece
e-mail zervakis@kal.forthnet.gr

Received 23 November 2000

Revised version 26 March 2001

A B S T R A C T . The influence of environmental parameters on
mycelial linear growth of Pleurotus ostreatus, P. eryngii,
P. pulmonarius, Agrocybe aegerita, Lentinula edodes, Volva-
riella volvacea and Auricularia auricula-judae was determined
in two different nutrient media in a wide range of temperature,
forming the basis for the assessment of their temperature opti-
ma. V. volvacea grew faster at 35 °C, P. eryngii at 25 °C,
P. ostreatus and P. pulmonarius at 30 °C, A. aegerita at 25 or
30 °C and A. auricula-judae at 20 or 25 °C depending on the
nutrient medium used and L. edodes lit 20 or 30 °C depending
on the strain examined. The mycelium extension rates were
evaluated on seven mushroom cultivation substrates: wheat
straw, cotton gin-trash, peanut shells, poplar sawdust, oak
sawdust, corn cobs and olive press-cake. The mycelium exten-
sion rates (linear growth and colonization rates) were deter-
mined by the 'race-tube' technique, and were found to be the
highest on cotton gin-trash, peanut shells and poplar sawdust
for Pleurotus spp. and A. aegerita. Wheat straw, peanut shells
and particularly cotton gin-trash supported fast growth of
V. volvacea, whereas wheat straw was the most suitable sub-
strate for L. edodes and A. auricula-judae. Supplemented oak
sawdust and olive press-cake were poor substrates for most spe-
cies examined, while almost all strains performed adequately on
corn cobs.

The large quantities of agro-industrial wastes that are produced world-wide often cause environ-
mental problems associated with their safe disposal. This, together with the ever-growing need of producing
cheap nutritious food, led to the fast development of the mushroom cultivation industry. Most species of
edible mushroom fungi possess the appropriate enzymic mechanisms to successfully bioconvert ligno-
cellulosic wastes into value-added products such as food, fodder or plant fertilizers and soil conditioners
(Mayson and Verachtert 1991; Nerud and MiSurcova 1996; Philippoussis et al. 2000; Poppe 2000; Tripothi
and Yadar 1992). Current market diversification calls for the introduction of new mushroom products
through the cultivation of exotic species, which provides an array of novel choices for the consumer and pro-
motes social development in rural areas (Philippoussis and Zervakis 2000; Zervakis and Venturella 2000).
However, growing mushrooms requires an in-depth understanding of the fungus physiology and
nutrition preferences. An essential part of this process is the phase of substrate colonization with the fungal
biomass after inoculation and prior to the initiation of primordia formation. During this particular stage, the
effect of environmental parameters plays an essential role in mycelium growth, and contributes significantly
to the success of the entire cultivation procedure. In addition, the duration of the substrate colonization phase
is of direct economic importance, since the media that are non-thoroughly impregnated with the hyphae are
sensitive to fungal and bacterial infections resulting in reduced yields. Likewise, the valorization of agro-
industrial wastes for mushroom cultivation demands extensive experimentation with various species and
growing conditions.
In this line of study, 34 edible mushroom strains were subjected to peer screening as regards their
mycelium growth rates on solidified media (Ioannidou et al. 2000), and the temperature optima of growth
were established for seven qualified strains belonging to the species Pleurotus ostreatus, P. eryngii, P. pul-
monarius, Agrocybe aegerita, Lentinula edodes, Volvariella volvacea and Auricularia auricula-judae.
Furthermore, seven agro-industrial wastes (wheat straw, cotton gin-trash, peanut shells, poplar sawdust, oak
sawdust, corn cobs and olive press-cake) were examined as regards their suitability as cultivation substrates
of the previously mentioned mushroom species. The efficacy of these species to colonize the substrate at the
determined optimal environmental conditions for mycelium growth was compared.
232 G. ZERVAKIS el al. Vol. 46

MATERIALS AND METHODS

Organisms. Seven wild dikaryotic strains (each one representing one species) were studied as to
their mycelium growth rates on different nutrient media: (/') Pleurotus eryngii ( D . C . : F R . ) QUEL. (LGAM
Ρ101, isolated in Greece), (/'/) Pleurotus ostreatus (JACQ.:FR.) K.UMM. (LGAM P 6 9 , isolated in Greece),
(Hi) Pleurotus pulmonarius ( F R . ) QUEL. (LGAM P26, isolated in Greece), (rv) Agrocybe aegerita (BRING.)
SING. (SIEF 0834, isolated in China), (v) Auricularia auricula-judae (BULL.) WETTST. (NAGREF IK 11,
isolated in Greece), (vi) Lentinula edodes (BERK.) PEGLER (SIEF 0231, isolated in China), and (vii) Vol-
variella volvacea (BULL.:FR.) SING. (SIEF 1318, isolated in China). All biological material was maintained
in the fungal culture collections of the authors' Institutes on agar slants or submerged into distilled water
at 4 °C.
Nutrient media and cultivation substrates. Potato dextrose agar (PDA; Difco) and cellulose medium
(CM; Eggins and Pugh 1962) were used for the assessment of linear growth rates and temperature optima.
Mycelium extension rates during the substrate colonization phase were also established for seven
agro-industrial wastes: wheat straw, cotton gin-trash, peanut shells, poplar sawdust, oak sawdust, corn cobs
and olive press-cake. Preparation of the substrates included grinding them into small particles (size < 20 mm),
adjustment of their moisture content (65-70 % ) , addition of wheat bran for regulating the C/N ratio to 2 0 ^ 0
(10-30 %, WIW, depending on the substrate used) and mixing with calcium carbonate (2 % dry mass) for pH
adjustment.
Measurements of linear growth rates. The temperature optima for mycelium linear growth were
assessed on both PDA and CM over the range of 15^15 °C with a 5 °C graduation. Experiments were con­
ducted in Petri dishes (Zervakis and Balis 1996), and measurements were taken every day for all species
examined with the exception of V. volvacea (measurements taken every 4 h).
Mycelium extension rates on the selected agro-industrial wastes were determined by a method simi­
lar to that used by Straatsma et al. (1989): Glass 'race-tubes' (150 mm height, 20 mm diameter) were
uniformly filled with 100 mL of each substrate and were sterilized by autoclaving (103.5 kPa, 121 °C, 1 h).
After cooling, inoculation was performed by mycelium plugs (6 mm diameter) taken from the periphery of
4-d-old colonies grown on PDA and transferred onto the top of the substrate. Cultures were incubated at
optimal growth temperatures (as previously determined). Growth rates were determined by measuring the
mycelial growth front distal from the inoculum and by averaging the growth measurements at four equi­
distant points around the circumference of each tube. Mycelial progression was calculated as mm/d, and
measurements were taken every 1 d.
Data analysis. Mycelium growth rates (kT) were calculated by fitting the linear growth function
y - kr χ + c (where y is the distance, and χ is the time) and were expressed in mm/d.
All experiments were conducted in five replicates and statistical analysis was performed using Tukey's
test at a significance level of ρ = 0.05.

RESULTS AND DISCUSSION

In preliminary experiments several edible mushroom species were screened in respect of their
growth rates on laboratory nutrient media (data not presented). Temperature optima of mycelium growth
were determined in strains representing seven widely cultivated species of high commercial interest, due to
their ability to efficiently decompose a large array of lignocellulosic wastes and to their market demand (i.e.
P. ostreatus, P. eryngii, P. pulmonarius, A. aegerita, L. edodes, V. volvacea and A. auricula-judae).
The growth rate of Pleurotus spp. and V. volvacea was satisfactory at 25 °C for P. eryngii, at 25 and
30 °C for P. ostreatus and P. pulmonarius, and at 35 °C for V. volvacea. It decreased sharply at higher
temperatures (45 °C in V. volvacea, 35 °C in all other cases; Table I). In previous studies, the optimum tem­
perature for the growth of V. volvacea was established at 30-37 °C (Chandra and Purkayastha 1977; Chang-
Ho and Yee 1977; Chang and Steinkraus 1982). The behavior of L. edodes was strain-dependent: strain
SIEF 0231 presented an optimum at 30 °C while other strains examined in preliminary experiments had
growth optima at 20 °C. Furthermore, the L. edodes strains failed to grow on CM irrespective of the tem­
perature used. Our results on L. edodes confirmed data of Han et al. (1981) on the strain-dependent behavior
and optimum growth temperature of 20-28 °C. A. aegerita and A. auricula-judae strains demonstrated dif­
ferent optimal temperature (25 or 30 °C and 20 or 25 °C, respectively) depending on the nutrient medium
used, and this is in agreement with the results of previous screening tests (Ioannidou et al. 2000).
2001 CULTIVATION OF EDIBLE MUSHROOM SPECIES ON LIGNOCELLULOSIC SUBSTRATES 233

3
Table I. Linear growth rates (mm/d) of selected mushroom species at different air temperatures (15—40 °C) on potato dextrose agar
(PDA) and cellulose medium (CM)

15 20 25 30 35 40
Species
PDA CM PDA CM PDA CM PDA CM PDA CM PDA CM

P. ostreatus 1.9 2.4 2.4 2.1 3.0 2.9 4.4 4.2 0 0 _

P. pulmonarius 2.0 1.9 3.3 2.9 5.6 4.5 6.6 4.7 0 0 -
P. eryngii 2.7 2.6 3.7 3.0 4.2 4.5 3.3 3.1 0 0 -
A. aegerita 1.4 1.8 2.5 3.0 3.9 5.2 4.7 4.8 0 0 -
L. edodes 2.3 0 4.1 0 3.7 0 4.4 0 0 0 -
A. auricula-judae 2.9 3.5 5.3 4.6 6.2 2.4 4.0 3.1 0 0 -
V. volvacea 0 0 5.0 7.0 7.1 9.9 12.8 18.8 25.0 32.2 7.8 5.2

a
N o growth was observed at 45 °C.

The 'race-tube' experiments provided an estimate of the potential of wheat straw, cotton gin-trash,
peanut shells, poplar sawdust, oak sawdust, corn cobs and olive press-cake to serve as alternative cultivation
substrates; these data form a basis for their valorization.
For all Pleurotus spp., colonization rates were the highest on cotton gin-trash, peanut shells, poplar
sawdust and corn cobs; these wastes were better than the conventional wheat straw substrate. Cotton gin-
trash, wheat straw and peanut shells supported fast growth for V. volvacea (Table II). Peanut shells proved to
be the best substrate for the growth of A. aegerita followed by poplar sawdust, corn cobs and cotton gin-
trash. A. auricula-judae grew better on wheat straw than on peanut shells, poplar sawdust and corn cobs.
Similar pattern was found with L. edodes which grew best on wheat straw and peanut shells.

3
Table II. Linear growth rates (mm/d) of selected mushroom species grown on seven agro-industrial wastes

Poplar Olive press Cotton

Species Wheat straw Peanut shells Oak sawdust Corn cobs
sawdust cake gin-trash

P. eryngii 3.8 blab 4.6 c/bc 5.0 c/bc 3.3 a/a 4.6 clc _ 4.5 bc/a
P. ostreatus 6.2 bid 8.6 did 7.5 cd/e 6.0 bib 7.1 c/e 3.4 a/- 7.4 cd/be
P. pulmonarius 4.4 bled 6.6 e/c 5.7 cd/bed 4.1 a/a 5.3 bc/d 3.9 a/- 6.7 dab
A. aegerita 2.6 a/a 4.0 bib 3.7 ab/o 3.2 ab/a 3.4ab/o - 3.4 ab/a
V. volvacea 12.5 c/e 12.2 c/e 5.9 aid 0 8.8 blf - 12.5 clc
A. auricula-judae 5.1 eld 3.9 bda 3.3 ab/a 0 2.8 a/a - -
L. edodes 4.3 d/b 4.1 elb 3.4 bib 3.5 b/a 2.8 a/a - 2.7 a/a

Values accompanied by identical letters across the same row and/or the same column (in italics) do not differ significantly.

In general, wheat straw proved to be suitable for fast colonization by L. edodes and V. volvacea;
peanut shells were a satisfactory substrate for most strains examined; poplar sawdust and corn cobs were
better with Pleurotus spp. and A. aegerita; while cotton gin-trash was very good for most species with the
exception of L. edodes. On the other hand, oak sawdust and olive press cake were poor substrates for most
strains tested.
The present work and our previous study (Philippoussis et al. 2001), show that in general, earliness
(/'. e. the time period between inoculation of the substrate and fructification) is favored in substrates ensuring
high linear extension rates. Hence, cotton gin-trash produced the best earliness values followed by wheat
straw and peanut shells for all Pleurotus spp. This correlates well with the results of the 'race-tube' experi-
ments. In contrast, poplar sawdust produced high extension rates for Pleurotus spp., but was a poor substrate
for carpophore induction. Earliness and mycelial growth correlated well for cotton gin-trash and wheat straw
with V. volvacea, for wheat straw and poplar sawdust with L. edodes, and for peanut shells with A. aegerita.
Poplar sawdust failed to support the production of basidiomata.
If mushroom yield and biological efficiency (Philippoussis et al. 2001) are correlated with the my-
celium extension rates during the substrate colonization phase, then cotton gin-trash (and to a lesser extent
wheat straw) gives consistent results for Pleurotus spp. and V. volvacea, and wheat straw for L. edodes only.
For A. aegerita, no correlation was detected between hyphal growth and yield on the substrates tested.
A similar lack of correlation was found with Agaricus subfloccosus or A. bisporus cultivated in composted
 234 G. ZERVAKIS et al. Vol. 46

substrates, where the thermophilic microflora seems to exert a significant influence on mushroom mycelial
growth (Noble et al. 1995; Smith et al. 1995). Also, hyphal extension rate was not directly associated with
mushroom biomass increase in the case of pasteurized Agaricus compost samples (Wiegant et al. 1992;
Smith et al. 1995). For white-rot fungi growing in sterilized media, the correlation between basidioma yield
and mycelial growth rate seems to depend mostly on the nature of the cultivation substrate.
In ecological terms, fast mycelial extension is often interpreted as indication of hyphal progression
on a nutritionally poor or unfavorable medium. A slower and denser growth can be attributed to favorable
conditions and exploitation of the nutrient resources of the medium by the fungus. The substrates used in this
study maintained a relatively constant nutrient status and the linear growth rates recorded indicate their suit­
ability as mushroom cultivation substrates. Especially at temperatures optimal for mycelial growth, high
colonization rates not only make competitor mould establishment less likely, but they could also contribute
to an improvement of the mushroom crop.

This work was financially supported by the Greek General Secretariat of Research and Technology (EPETII-SYN133
project) and the National Agricultural Research Foundation (DIMITRA95 project). Thanks are extended to Dr. F. Nerud (Institute of
Microbiology. Academy of Sciences of the Czech Republic) for critically reviewing the manuscript.

REFERENCES

CHANDRA Α., PURKAYASTHA R.P.: Physiological studies on Indian edible mushrooms. Trans.Brit.Mycol.Soc. 69, 63-70 (1977).
C H A N G S C . , STEINKRAUS K . H : Lignocellulolytic enzymes produced by Volvariella volvacea, the edible straw mushroom. Appl.
Environ. Microbiol. 43, 4 4 ( M 4 6 (1982).
C H A N G - H o Υ . , Y E E N T . : Comparative study of the physiology of Volvariella volvacea and Coprmus cinereus. Trans.Brit.Mycol.Soc.
68, 167-172 (1977).
EGGINS H . O . W . , P U G H G . J . F . : Isolation of cellulose-decomposing fungi from the soil. Nature 193, 9 4 - 9 5 (1962).
HAN Y . H . , U E N G W . T . , C H E N L.C., C H E N G S.: Physiology and ecology of Lentinus edodes ( B E R K . ) SING. Mush.Sci. 11, 623-658
(1981).
IOANNIDOU S., ZERVAKIS G . , PHILIPPOUSSIS Α., D I A M A N T O P O U L O U P . : Influence of the air temperature and the composition of nutrient
media on the linear growth rates of selected edible mushroom species, in L. Van Griensven (Ed.): Proc. 15th Internal. Congr.
Science and Cultivation of Edible Fungi. Balkema, Rotterdam (Netherlands) 2000.
MAYSON E., VERACHTERT H . : Growth of higher fungi on wheat straw and their impact on the digestibility of the substrate. Appl.
Microbiol.Biotechnol. 36, 421-424 (1991).
N E R U D F., MISURCOVA Z . : Distribution of ligninolytic enzymes in selected white-rot fungi. Folia Microbiol. 41, 264-266 (1996).
N O B L E R., GROGAN H . , ELLIOTT T . J . : Variation in morphology, growth and fructification of isolates in the Agaricus subfloccosus com­
plex. Mycol.Res. 99, 1453-1461 (1995).
PHILIPPOUSSIS Α., D I A M A N T O P O U L O U P . , ZERVAKIS G . , IOANNIDOU S.: Potential for the cultivation of exotic mushroom species
by exploitation of Mediterranean agricultural wastes, pp. 523-530 in L. Van Griensven (Ed.): Proc. 15th Internal. Congr.
Science and Cultivation of Edible Fungi. Balkema, Rotterdam (Netherlands) 2000.
PHILIPPOUSSIS Α., ZERVAKIS G . , DIAMANTOPOULOU P . : Bioconversion of agricultural lignocellulosic wastes through the cultivation of
the edible mushrooms Agrocybe aegerita, Volvariella volvacea and Pleurotus spp. World J.Microbiol.Biotechnol., in press
(2001).
PHILIPPOUSSIS Α., ZERVAKIS G . : Cultivation of edible mushrooms in Greece: presentation of the current status and analysis of future
trends, pp. 843-848 in L. Van Griensven (Ed.): Proc. 15th Internal. Congr. Science and Cultivation of Edible Fungi. Bal­
kema. Rotterdam (Netherlands) 2000.
Poppe J.: Use of agricultural waste materials in the cultivation of mushrooms, pp. 3-23 in L. Van Griensven (Ed.): Proc. 15th Internal
Congr. Science and Cultivation of Edible Fungi. Balkema, Rotterdam (Netherlands) 2000.
SMITH J . F . , W O O D D.A., THURSTON C . F . : Growth measurement of Agaricus mycelium in composted substrates as an indicator of
compost selectivity and mushroom productivity, pp. 293-301 in T. Elliott (Ed.): Proc. Nth Internat. Congr. Science and
Cultivation of Edible Fungi. Balkema, Rottterdam (Netherlands) 1995.
STRAATSMA G., G E R R I T S J . P . G . , G E R R I T S T . M . , H U U B J.M. O P D E N C A M P , VAN GRIENSVEN L.: Growth kinetics of Agaricus bisporus
mycelium on solid substrate (mushroom compost). J.Gen.Microbiol. 137, 1471-1477 (1991).
TRIPOTHI J.P., Y A D A R J . S . : Optimization of solid substrate fermentation of wheat straw into animal feed by Pleurotus ostreatus
- a pilot effort. Anim.FeedSci.Technol. 37, 59-72 (1992).
W I E G A N T W . M . , W E R Y J., BUITENHUIS E.T., D E B O N T J.A.M.: Growth-promoting effect of thermophilic fungi on the mycelium of the
edible mushroom Agaricus bisporus. Appl.Environ.Microbiol. 58, 2654-2659 (1992).
ZERVAKIS G., BALIS C : A pluralistic approach on the study of Pleurotus species, with emphasis on compatibility and physiology of
the European morphotaxa. Mycol.Res. 100, 717-731 (1996).
ZERVAKIS G., VENTURELLA G.: Mushroom breeding and cultivation favors ex situ conservation of Mediterranean Pleurotus taxa. Proc.
Internat. Conf. Science and Technology for Managing Plant Genetic Diversity in the 21st Century. IPGRI, Kuala Lumpur
(Malaysia), in press (2000).

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