Significance of thermophilic fungi in mushroom compost preparation : effect on growth and yield of Agaricus bisporus (Lange) Sing

Journal of Agricultural Technology
Significance of thermophilic fungi in mushroom compost
preparation: effect on growth and yield of Agaricus bisporus
(Lange) Sing.



R.K. Salar1* and K.R. Aneja2

1Department of Biotechnology, Chaudhary Devi Lal University, Sirsa – 125 055, India.
2Department of Microbiology, Kurukshetra University, Kurukshetra – 136 119, India.

Salar, R.K. and Aneja, K.R. (2007) Significance of thermophilic fungi in mushroom compost
preparation: effect on growth and yield of Agaricus bisporus (Lange) Sing. Journal of
Agricultural Technology 3(2): 241-253.

Eighteen species of thermophilic and thermotolerant fungi were isolated from mushroom
compost. Growth of Agaricus bisporus, was studied on sterile compost pre-colonized with four
thermophilic fungi viz., Chaetomium thermophile, Malbranchea sulfurea, Thermomyces
lanuginosus and Torula thermophila. All the four fungi were inoculated singly and in different
combinations on sterilized compost to evaluate their potential to promote growth and yield of
A. bisporus. A mixed inoculum of Malbranchea sulfurea and Torula thermophila was found to
be the best amongst the various treatments that promoted the growth of A. bisporus to the
plateau of 7.7 mm day-1 and the yield of the mushroom was almost twice compared to the
pasteurized control. The effect of T. lanuginosus when inoculated singly or in combination
with other thermophilic fungus/fungi in compost was insignificant resulting in lower growth
rates. Mycelial extension rate, pH of the compost before spawning, yield of mushrooms and
biological efficiency for various treatments were studied. The study reveals that thermophilic
fungi provide for compost selectivity and protection against negative effects of compost
bacteria on mycelial growth of A. bisporus. This finding is of relevance for the commercial
production of high-yielding mushroom compost for A. bisporus.

Key Words: biological efficiency, crop yield, growth rate, mixed culture

Introduction

The white button mushroom (Agaricus bisporus) (Lange) Sing. is
cultivated on a substrate consisting of a composted mixture of straw bedded
horse manure, wheat straw, chicken manure and gypsum. Compost is prepared
in a sequence of processes. Conventionally two phases of composting are
distinguished (Sinden and Hauser, 1950; Fermor et al., 1985). After mixing
and moistening the ingredients, the mixture is left for a short period and

*Corresponding author: Raj Kumar Salar, E-mail: rajsalar@rediffmail.com

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subjected to phase I composting process in the open air. During phase-I NH3
and unpleasant smelling compounds are emitted into the environment. It is
followed by phase-II: an indoor, temperature-controlled process. Subsequently,
conditioning of the compost is carried out at approximately 45°C. This can
either be performed in limited quantities in mushroom houses or in bulk in
'tunnels. In mushroom houses self-heating of the compost is controlled by
ventilating air around layers of up to 30 cm thick contained in trays or
polyethylene bags. Whereas in tunnels fresh air is forced through the body of
the compost, allowing an accurate control, and layers up to 250 cm can be
processed (Derks, 1973; Gerrits 1988a). Several other investigators suggested
that combined phase-I and phase-II indoor composting is feasible (Laborde et
al., 1987; Perrin and Gaze, 1987; Gerrits, 1987; Straatsma et al., 1989, 1991,
1994a & b; Wiegant, 1992).
Thermophilic fungi are believed to contribute significantly to the quality
of compost (Seal and Eggins, 1976; Eicker, 1977; Ross and Harris, 1983;
Gerrits, 1988b). The effect of these fungi on the growth of mushroom mycelia
and mushroom yield have been described at three distinct levels (Wiegant,
1992). First, they decrease the concentration of ammonia in the compost,
which otherwise would counteract the growth of the mushroom mycelium.
Second, they immobilize nutrients in a form that apparently is available to the
mushroom mycelia. And third, they may have a growth promoting effect on the
mushroom mycelia, as has been demonstrated for Scytalidium thermophilum
and for several other thermophilic fungi (Wiegant et al., 1992). The
effectiveness of S. thermophilum in compost preparation for A. bisporus has
been shown by Straatsma et al. (1994a) which obtained a two folds increase in
the yield of mushrooms on inoculated compost when compared to the
pasteurized control.
In order to produce compost with constant high quality that does not emit
ammonia and odour into the environment, the artificial inoculation and
controlled preparation of compost is highly desirable. This study focuses on the
significance of thermophilic fungi in reducing the time for compost preparation
and their effect on growth rate and crop yield of A. bisporus by single and dual
culture inoculation.

Materials and methods

Substrates

The formulation used for compost preparation for the cultivation of white
button mushroom consisted of wheat straw (chopped 8-20 cm long), 300 kg;
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wheat bran, 15 kg; chicken manure, 125 kg; urea, 5.5 kg; gypsum, 20 kg and
BHC (10%), 125 g. Ingredients of the compost were obtained from the local
market. Traditional scheme of mixing and moistening the ingredients for
compost preparation were applied. Here the term 'young compost' is used for
any substrate prior to phase-II. The growth of thermophilic fungi was tested on
young compost that was obtained from a local mushroom farmer at
Kurukshetra.

Isolation of thermophilic fungi

One kg of compost was randomly sampled in 100 g portions, mixed
thoroughly and was used for isolation. Thermophilic fungi were isolated from
different composting phases by using serial dilution method on YpSs agar
(Yeast extract, 4.0 g; K2HPO4, 1.0 g; MgSO4.7H2O, 0.5 g; Soluble starch, 15 g;
Agar, 20.0 g; Distilled water, 750 ml; Tap water, 250 ml) plates supplemented
with streptomycin and rose bengal @ 50 mg/L each. In this method, 10 g of
compost sample was taken in a 250 ml Erlenmeyer flask containing 90 ml of
sterile water and shaken on a rotary shaker for 1 hour. Various dilutions (10-2,
10-3, and 10-4) were used for the isolation of thermophilic fungi. Plates were
incubated at 45°C in the dark and were screened daily for up to 5 days.
Representative isolates were purified and maintained on YpSs agar slants at
4°C.

Thermophilic fungi tested

Four species of thermophilic fungi namely, Chaetomium thermophile La
Touch, Malbranchea sulfurea (Miehe) Sigler and Carmichael, Thermomyces
lanuginosus Tsiklinskya and Torula thermophila Cooney and Emerson were
isolated and selected for growth stimulation of A. bisporus. The criterion for
selection was their high affinity for cellulose breakdown (Rosenberg, 1978).
Though T. lanuginosus is a non-cellulolytic fungus yet it was selected as it
enhances cellulose breakdown by ce11ulolytic fungi in combination (Deacon,
1985). All the four fungi were tested singly and in different combinations on
sterilized compost for their potential to promote A. bisporus growth. A control
without inoculation was simultaneously run. Culture tubes (160 x 25 mm),
each holding a glass tube of 10 mm diameter, were fil1ed with young compost.
The glass tube was removed to leave a ventilation channel in the compost (Fig.
1A). The young compost contained in culture tubes was pasteurized for 4 h at
70°C and after stabilizing at room temperature, the compost was inoculated in
replicates of three with an 8 mm agar disc of the isolate to be tested singly and

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in various combinations (Table 2). The inoculated tubes were placed in
polyethylene bags and incubated at 45°C in humidified containers. Growth was
recorded daily for the successful1 colonization of the isolate/s. This compost
treated with various thermophilic fungi was later used for the inoculation of
A. bisporus mycelia in test tubes.

Culture of mushroom mycelia in compost in test tubes and Petri dishes

Ten spawn grains covered with A. bisporus were put at the bottom of
each sterilized culture tube (160 x 25 mm) as an inoculum. Thirty gram
substrate (compost treated as above with various thermophilic fungi) was
added in the test tubes with sterilized forceps. The culture tubes were closed
with sterile cotton plugs and incubated upright at 24+1°C in the dark. All the
treatments had three replicates. From first day onwards the position of the
mycelial front was recorded with a marker at various intervals. Growth rate
was expressed as Kr (mm d-1).
Radial growth rates of A. bisporus were also studied in Petri-dishes.
Sterile compost and sterile compost fully grown with T. thermophila and M.
sulfurea and a combination of both these fungi (having passed a selection for
better growth stimulation) was taken in 90 mm Petri dishes at 30 g per dish.
The dishes were inoculated with A. bisporus spawn in the center. The lids of
the inoculated Petri dishes were fixed with adhesive tape and marked with radii
that crossed the inoculum. The dishes were placed in humidified container to
prevent desiccation and incubated at 24+1°C in the dark. The position of the
mycelial front was marked on third day of incubation and growth was recorded
at 2 days interval. Growth rate (GR) was calculated in mm d-1.

Cropping of Agaricus bisporus

Cropping trials for A. bisporus were done in polyethylene bags (18 x 12
inches). Young compost (5 kg) was filled in polyethylene bags and pasteurized
at 70°C for 8 hr. in an incubator. After pasteurization, the compost bags were
stabilized at room temperature and were inoculated with T. thermophila and
M. sulfurea singly and in dual cultures in replicates of five. The bags were
incubated at 45°C for 6 days in humidified incubators. Five bags processed
similarly but not inoculated with the test organisms served as controls. Side
vents in the incubators helped in air circulation. After conditioning of the
compost at 45°C, the bags were stabilized at room temperature and inoculated
with A. bisporus spawn @ 0.5%. After spawning, the compost was pressed
hard to make it compact, covered with newspaper sheets sprayed with 2%
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formalin and incubated for 15 days at 24+1°C. Mushroom was cultivated and
yield was taken as an average of five replications.

Statistical analysis

Within experiments, each treatment performed three to five replicates.
Calculations were done with data from independent experiments. Means of n
experiments are given. Coefficient of variations (CV) was calculated by
analysis of variance (ANOVA) as outlined in Gomez and Gomez (1984).
Significant differences were calculated by pair comparison using Duncan
Multiple Range Test (DMRT) at P = 0.05.

Results and discussion

Survey

Eighteen species of thermophilic and thermotolerant fungi were isolated
from the mushroom compost (Table 1) and these represent most of the known
thermophilic taxa. Fast growing species were very common, and after 2 days of
incubation counting was possible only if the number of colonies per plate was
as small as 15-20. Therefore, higher dilutions (10-3) were used for the isolation
of fungi in pure form. During phase-I isolation, it was found that most of the
time; the plates were overcrowded with the ubiquitous Aspergillus fumigatus
and Rhizomucor spp. The isolation of a few isolates producing only sterile
mycelium proved problematic. Macroscopically, young cultures of Torula
thermophila and Chaetomium thermophile resembled closely and were fast
growing. Rhizomucor pusillus, R. miehei and Absidia corymbifera were also
very similar in gross morphology and were fast growing. Seven thermophilic/
thermotolerant fungi were isolated from phase-I compost, rest of the fungi
were isolated from phase-II compost (Table 1). The zygomycetous fungi
(Table 1) are very common in wheat straw compost (Fergus, 1964; Chang and
Hudson, 1967; Chahal et al., 1976; Straatsma et al., 1994b). During phase-I
total count was quite low as revealed by low CFU g-1 (Table 1). Fungi isolated
from Phase-II compost were Chaetomium thermophile, Emericella nidulans,
Thermoascus aurantiacus, Myriococcum albomyces, Humicola insolens,
Malbranchea sulfurea, Torula thermophila, Stilbella thermophila and
Thermomyces lanuginosus. Two basidiomycetous species were also visually
observed in phase-II compost. A species producing only sterile mycelium was
also isolated from phase-II. Most species almost disappeared after phase-II
composting. Fungi recovered from end phase-II compost were almost

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exclusively T. thermophila, H. insolens and C. thermophile. The population
density of T thermophila was the highest (15849 CFU g-l) as compared to other
two fungi (Table 1).
The existence of phase-I fungi was limited to the short period before the
temperature reached its maximum. With the exception of T. lanuginosus they
did not reappeared in the phase II compost. The first phase consisted of
primary colonizers or primary sugar fungi. In the second phase, the fungi
recovered were almost all cellulolytic, suggesting a role in decomposition of
organic matter (Rosenberg, 1978; Srivastava et al., 1981) The ratio of
thermophilic to mesophilic fungi rose during composting, resulting in a much
higher proportion of thermophiles (Chang and Hudson, 1967). The general
point that can be made from the survey is that the thermophilic fungi can
tolerate the high peak heating phase and their spores persist and remain viable
in the compost for a long time. This is suggested by their recolonization of
compost after it had cooled down.
Our survey of thermophilic fungi in composts provided us with valuable
isolates of T. thermophila, C. thermophile, M. sulfurea and T. lanuginosus. The
first three fungi are known to adapt and colonize the compost frequently
(Straatsma et al., 1994 a,b) and T. lanuginosus is known to promote
decomposition rate when grown in combination with cellulolytic fungi
(Deacon, 1985).

Growth of thermophilic fungi

Four fungi viz. Chaetomium thermophile, Malbranchea sulfurea,
Thermomyces lanuginosus and Torula thermophila singly and in various
combinations were tested on pasteurized compost, and most, in particular T.
thermophila grew well (Fig. 1A). Of the other species tested, C. thermophile
and M. sulfurea were successfu1 colonizers of compost. Torula thermophila
grew best in combination with M. suIfurea and C. thermophile and rapidly
colonized the pasteurized compost in test tubes (Fig. 1A). The common
compost species T. lanuginosus grew poorly when inoculated singly and/or in
combination, indicating low competitive abilities. The inoculation of
thermophilic fungi showed that compost colonization by selected isolates was
successful and that microbial manipulation of phase-II composting is possible.





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Table 1. Thermophilous fungi isolated from the mushroom compost phase I
and II.

Fungus Log10 CFU g-1 Reference(s)a
Zygomycetes

Absidia corymbifera* 2.9
2,5,14
Rhizomucor miehei* 3.1
6,14
Rhizomucor pusillus*
3.4 2-7,10,11,13,14
Ascomycetes

Chaetomium thermophile 3.7 2-7,10,13
Emericella nidulans
3.0 2,4,14
Talaromyces emersonii*
2.9 14
Talaromyces thermophilus*
2.9 5-7,14
Thermoascus aurantiacus
3.6 3,14
Myriococcum albomyces
3.5 8,13,14
Basidiomycetes

Basidiomycetes 1


Basidiomycetes 2



Hyphomycetes

Aspergillus fumigatus* 3.9
2-7,10-14
Humicola insolens
3.9

Malbranchea sulfurea
3.1
5,6,14
Torula thermophila 4.2
2-7,10-14
Stilbella thermophila
2.8 4,7,11,13,14
Thermomyces lanuginosus*
3.7 2-7,10,11,13,14
Unidentified taxon
2.8
1. Anonymous (1992), 2. Basuki (1981), 3. Bilai (1984), 4. Renard and Cailleux (1973), 5.
Chang and Hudson (1967), 6. Eicker (1977), 7. Fergus (1964), 8. Fergus (1971), 9. Fergus and
Sinden (1969), 10. Fermor et al. (1979), 11. Hayes (1969), 12. Olivier and Guillaumes (1976),
13. Seal and Eggins (1976), 14 Straatsma et al. (1994b)
* Isolated from phase-I compost only, rest of the fungi were isolated from phase II compost

Growth promotion of Agaricus bisporus

The linear growth rate of mycelium of A. bisporus on sterilized compost
in tubes (Fig. 1B) was 6.1 mm per day. After an initial lag phase of one day,
the growth rates were constant (data not shown). The effects of T. lanuginosus
when inoculated singly or in combination with other thermophilic fungus/fungi
on compost were not significant, resulting in lower growth rates as compared
to control (Table 2). This could be due to the competition between organisms
for producing hydrolytic enzymes. Therefore, it is difficult to establish the
initial growth rates of thermophilic fungi. The other three species viz. C.
thermophile, M. sulfurea and T. thermophila tested, promoted growth of A.
bisporus as the rates of 6.3, 7.1 and 6.6 mm per day respectively when
inoculated singly. A mixed inoculum consisting of M sulfurea and T.

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thermophila was the best and promoted growth of A. bisporus mycelium to the
plateau of 7.7 mm per day (Table 2) and was significantly higher from all other
treatments. This finding indicates some specificity of the growth promoting
factor(s).



Fig. 1. A. Young compost colonized by four thermophilic fungi, 1. Chaetomium thermophile,
2. Torula thermophila, 3. Thermomyces lanuginosus, and 4. Malbranchea sulfurea; B.
Colonization of Agaricus bisporus mycelium on compost treated with various thermophilic
fungi as in A; C-F: Mycelial growth of Agaricus bisporus 8 days after inoculation on (C)
sterile compost, (D) compost treated with M. sulfurea, (E) compost treated with T. thermophila
and (F) compost treated with M. sulfurea + T. thermophila.
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Table 2. Growth rates of Agaricus bisporus on sterilized compost inoculated
with different thermophilic fungi singly and in combinations.

Mycelial extension rate
Radial growth rate (GR)
Species
(Kr) in tubes (mm/day)a
in Petri dishes (mm/day)
Control 6.1e
4.9+0.5
Chaetomium thermophile 6.3d
ND
Malbranchea sulfurea 7.1b
6.1+0.9
Thermomyces lanuginosus
5.4g ND
Torula thermophila
6.6c 5.3+0.8
C. thermophile + M. sulfurea
7.1b ND
C. thermophile + T. lanuginosus
6.0e ND
C. thermophile + T. thermophila
6.6c ND
M. sulfurea + T. lanuginosus
5.8f ND
M. sulfurea + T. thermophila
7.7a ND
T. lanuginosus + T. thermophila
6.0e ND
C. thermophile + M. sulfurea +
6.5c 9.0+1.0
T. lanuginosus + T. thermophila


CV = 1.5%, ND = not determined, + S.D.
aTreatments receiving the same letter are not significantly different (DMRT; P<0.05)

Growing Agaricus bisporus when grown in Petri dishes on compost
inoculated with M. sulfurea,(Fig.1D) T. thermophila (Fig. 1E) and M. sulfurea
+ T. thermophila (Fig.1 F) showed radial growth rates of (GR) 6.1, 5.3 and 9
mm/day, respectively (Fig.1) The GR on control was low (4.9 mm per day).
These two species of thermophilic fungi appeared to be the most promising and
were used for more controlled preparation of the substrate for A. bisporus
cultivation. The growth of A. bisporus on control dishes was densed as
compared to growth on compost treated with either M. sulfurea or T.
thermophila. Fluffy growth occurred when compost treated with both the fungi
that were used as substrate for A. bisporus growth (Fig. 1 D, E).
The effect of thermophilic fungi on growth rate of mushroom mycelia in
sterilized compost is quite remarkable. Radial growth rate of mushroom
mycelia on any laboratory medium never exceeds 3 mm per day (Last et al.,
1974). Based on our experimental data, we were convinced that thermophilic
fungi in particular T. thermophila and M. sulfurea provides a trigger for
enhanced growth of A. bisporus acting by an unknown mechanism. This may
be taken as an indication that the results of this study could be extrapolated to
what actually happens during the production of mushroom compost.
Unfortunately a growth-promoting effect of Scytalidium thermophlium (syn. =
Torula thermophila) on A. bisporus is not found on agar media (Renard and
Cailleux, 1973). Actinomycetes and other bacteria might play a role in
successful colonization of S. thermophilum during composting (Straatsma et

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al., 1989). Till (1962) showed that good yield of mushrooms can be obtained
on a non-composted sterile mixture containing mainly straw and organic
nitrogen. The high hyphal extension rates of A. bisporus on compost in the
presence of thermophilic fungi may have an ecological significance: it may be
able to grow as fast as possible, thereby colonizing as much substrate as
possible. Once the substrate has been occupied, the mushroom mycelium
seems to be able to prevent the occupation by other microorganisms, either by
consuming them (Fermor and Wood, 1981; Fermor and Grant, 1985) or by
excretion of carbon monoxide (Stoller, 1978), which effectively inhibits
growth of most competing organisms but inhibits the growth of the mushroom
mycelium itself only partly (Derikx et al., 1990). Carbon dioxide
concentrations in the range of 0.3 to 1.0% generate a higher extension rate of
mushroom mycelium (Wiegant et al., 1992).
The lower growth rates observed in Petri dishes than in culture tubes
remain unexplained. This has also been reported for other fungi (Dickson,
1935; Trinci, 1973). The probable reason of higher growth rate in culture tubes
may be the ventilation caused by the ventilation channel in tubes.

Table 3. Compost inoculation with thermophilic fungi and cropping of
Agaricus bisporus.

pH of compost
Yield of mushrooms
Biological
Treatment
Before spawning
g/ 5 Kg of compost)a
efficiency (%)
Control 6.9
1020
20.4
M. sulfurea
6.5
1910
38.2
T. thermophila 5.3
1355
27.1
M. sulfurea + T. thermophila
6.0 1990
39.8
CV = 2.18%
aAverage of five replications, the differences among treatments
are significant (DMRT; P<0.05)

Cropping of Agaricus bisporus

After inoculation with A. bisporus spawn at the rate of 0.5% (w/w) and
incubation for 15 days, the inoculated composts were fully colonized by A.
bisporus mycelium. The pH of inoculated compost before spawning was 6.5
and that of the control was 6.9, indicating weaker colonization (Gerrits,
1988b). Mushroom yields from compost inoculated with M. sulfurea + T.
thermophila were high (1990 g/5 kg of compost), almost twice that from
pasteurized control. The yield from control compost was clearly lower (1020
g/5 kg of compost) than inoculated compost and was significantly lower as
shown by pair comparison using DMRT (Table 3). Low yields of A. bisporus
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