Temperature and pH conditions for mycelial growth of Agaricus brasiliensis on axenic cultivation

Temperature and pH conditions for mycelial growth of Agaricus

brasiliensis on axenic cultivation

Condições de temperatura e pH para o crescimento micelial de

Agaricus brasiliensis em cultivo axênico

Nelson Barros Colauto1*; Patrícia Midori Aizono2; Lis Ribeiro Magalhães de

Carvalho3; Luzia Doretto Paccola-Meirelles4; Giani Andrea Linde5

Abstract
Few studies have been done to determine Agaricus brasiliensis Wasser et al. (A. blazei; A. subrufescens)
basic mycelial growth characteristics on axenic cultivation. This study aimed to determine the optimal
temperature and initial pH for mycelial growth of A. brasiliensis on malt extract agar medium to develop
axenic cultivation techniques. Studied initial pH values for mycelial growth were adjusted to 3.0, 4.0, 5.0,
5.5, with HCl, 6.0, 7.0, 8.0, with NaOH, and again 7.0 and 8.0, with CaCO3. Studied temperatures for
mycelial growth were 22 ºC, 25 ºC, 28 ºC, 31 ºC and 34 ºC. It was concluded that A. brasiliensis can grow
in axenic cultivation at temperature range from 22 oC to 34 ºC, with optimal temperature range from 28 oC
to 31 ºC and optimal temperature value of 30.5 ºC ± 0.3 ºC. It also grows in initial pH range from 4.0 to 7.0,
adjusted with HCl or NaOH but not CaCO3, with optimal initial pH range from 5.5 to 6.0 and optimal initial
pH value of 5.56 ± 0.05. Mycelial growth is inhibited with pH of 3.0 or lower, 8.0 or higher, or when CaCO3
is used to adjust pH in the substratum to 7.0 or higher.
Key words: Growth conditions, growth inhibition, Agaricus brasiliensis, Agaricus blazei

Resumo
Poucos estudos foram desenvolvidos para determinar as condições básicas de crescimento micelial do
fungo Agaricus brasiliensis Wasser et al. (A. blazei, A. subrufescens). O objetivo deste trabalho foi
determinar a faixa ótima de temperatura e pH para o crescimento micelial, em agar-extrato-de-malte, de A.
brasiliensis, visando o desenvolvimento de técnicas de cultivo axênica. Os valores de pH estudados
foram 3,0, 4,0, 5,0 e 5,5, ajustados com HCl, 6,0, 7,0 e 8,0, ajustados com NaOH, e 7,0 e 8,0, ajustados com
CaCO3. As temperaturas de crescimento estudadas foram 22 ºC; 25 ºC; 28 ºC; 31 ºC e 34 oC. Concluiu-se
que A. brasiliensis cresce em uma faixa de temperatura ótima de 28 oC a 31 ºC, com valor ótimo de
temperatura de 30,5 ºC ± 0,3 ºC. A faixa de pH inicial ótimo no substrato é de 5,5 a 6,0 e o valor de pH inicial
ótimo é de 5,56 ± 0,05. O crescimento do micélio é inibido com pH de 3,0 ou inferior, 8,0 ou superior, ou
quando CaCO3 é utilizado para ajuste do pH para 7,0 ou superior.
Palavras-chave: Condições de crescimento, inibição do crescimento, Agaricus brasiliensis, Agaricus blazei

1
Prof. Dr. Titular da Universidade Paranaense – Laboratório de Biologia Molecular - [email protected].
2
Nutricionista – Universidade Paranaense.
3
Bióloga - Universidade Paranaense.
4
Prof.a Dr.a Associada da Universidade Estadual de Londrina – CCB/BIO.
5
Prof.a Dr.a Adjunta da Universidade Paranaense - Laboratório de Biologia Molecular.
* Autor para correspondência
Recebido para publicação 31/05/07 Aprovado em 05/02/08
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 Colauto, N. B. et al.

Introduction narrow pH range, affecting fungus nutrient

metabolism (MILES; CHANG, 1997). For A.
Agaricus brasiliensis Wasser et al. (Agaricus
brasiliensis, pH value of 4.5 was considered ideal
blazei Murrill ss. Heinemann) is a native fungus to
for mycelial growth in submerged cultivation
Brazil (WASSER et al., 2002), also denoted Agaricus
(KAWAGOE et al., 2004) and pH from 6.5 to 6.8
subrufescens (KERRIGAN, 2005), has been studied
was ideal for compost cultivation (IWADE;
because of its therapeutic (MIZUNO, 2002;
MIZUNO, 1997). For A. bisporus cultivation CaCO3
WATANABE et al., 2003; SOUZA-PACCOLA et
or CaSO4 or both are added on compost to keep pH
al., 2004; KIMURA at al, 2004; KIM et al., 2005;
at 7.5 in the end of Stage II (pasteurization and
SILVA et al., 2005; MANTOVANI et al., 2006) and
conditioning) and to control NH4+ dissociation into
sensory (STIJVE et al., 2002; ESCOUTO et al., 2005)
NH3 which is toxic to fungus (GERRITS, 1988;
properties. However, few studies describe specific
RINKER, 1993).
techniques and parameters for A. brasiliensis
cultivation, which is generally produced empirically or Because of dissonance for basic variables as
based on Agaricus bisporus cultivation in Brazil temperature and pH ranges and lack of specific
(BRAGA et al., 1998; EIRA; BRAGA, 2003). parameters for axenic cultivation of A. brasiliensis,
the aim of this study was to determine the optimal
Composting process is a very used technique for
temperature and pH for mycelial growth of A.
mushroom cultivation; however, axenic cultivation is
brasiliensis strains in order to develop techniques
an alternate technique due to better substrate use,
for axenic cultivation.
medium standardization and pest control, as
Sciarideae fly that may cause up to 50% production
loss (EIRA et al., 2005). In compost cultivation the
Material and Methods
mycelial growth in the substratum must be fast to
avoid the development of competing microorganisms Agaricus brasiliensis 97/11, 99/25, 99/26, 99/28
(BRAGA et al., 1998; EIRA; BRAGA, 2003) and in and 99/29 strains (COLAUTO et al., 2002), from
the axenic cultivation it has to be fast to reduce the the fungus collection of the Molecular Biology
spawn and substratum mycelial colonization period. Laboratory at UNIPAR were coded as L1, L2, L3,
In this stage, it is important to control basic variables L4 and L5, respectively. Each strain, maintained at
that are associated to mycelial growth as temperature 20 oC in malt extract agar (MEA) (24 g/L), was
and hydrogen ion concentration in solution expressed subcultured in Petri dishes with MEA (48 g/L) and
in terms of potential of hydrogen (pH). kept in the dark at 28 oC for 10 days. Cylinders with
4mm-diameter from the edge of the mycelial growth
Temperature affects enzymatic activity and
were used as inoculum. Special care was taken to keep
vitamin synthesis and may accelerate or inhibit fungus
the cylinder mycelium in direct contact with culture
growth (MILES; CHANG, 1997). The temperatures
medium. All experiments were quadruplicated.
reported for A. brasiliensis growth are dissonant
and vary from 18 o C to 30 ºC (OKUBO; For temperature study, culture medium was 48 g/
KURAMOTO; OHKUBO, 1991), 22 oC to 26 ºC L of MEA (pH 5.6) autoclaved at 121 °C for 15 min.
(IWADE; MIZUNO, 1997), 25 oC to 28 ºC (EIRA; After inoculated culture media were incubated in the
BRAGA, 2003) for compost cultivation and 30 °C dark at 22 oC, 25 oC, 28 oC, 31 oC and 34 oC. For pH
for submerged cultivation (KAWAGOE et al., 2004). study, culture medium was 48 g/L of MEA autoclaved
at 121 °C for 15 min; then pH was adjusted to 3.0,
Most fungi have vegetative growth at pH values
4.0, 5.0, 5.5, with HCl (1M), 6.0, 7.0, 8.0, with NaOH
from 6.5 to 6.8 and excrete extra cellular enzymes in
(1M) and another adjusted to 7.0 and 8.0, with CaCO3
the substrate; these enzymes present activity in a
(1M). All solutions were sterilized by filtration (0.22
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 Temperature and pH conditions for mycelial growth of Agaricus brasiliensis on axenic cultivation

µm pore size filter). After that, culture media were 30.2 ºC for L3, 30.4 ºC for L4 and 30.8 ºC for L5.
inoculated and incubated in the dark at 28 °C. Considering all strains the mycelial optimal
temperature value was 30.5 oC ± 0.3 oC. This
For each replication, the mycelial growth was
temperature value is compatible to the ones in tropical
verified by calculating the average of three different
countries, where this fungus comes from, and
measurements of the diameter (in mm) 21 days after
represents an economic advantage to mycelial growth
inoculation. Four replications were made to calculate
in tropical countries. However, higher temperatures
the mycelial growth diameter average for each
imply in more expenses to control moisture and CO2
treatment. Obtained data were evaluated using
levels for fungus cultivation. Quimio, Chang and
variance analysis and significant differences were
Royse (1990) reported 28 °C to 30 °C as optimal
determinated by Tukey’s test with a significance level
temperature for Agaricus bitorquis growth, a tropical
p < 0.05. The optimal values for mycelial growth on
basidiomycete, whereas for Agaricus bisporus, a
both temperatures and pHs for each strain were
fungus in which A. brasiliensis cultivation is based
determined by non-linear regression and after that
on, the recommended temperatures are from 22 oC
an average of all strains was calculated.
to 25 °C. The results found in this research are similar
to the ones that reported 30 oC as optimal temperature
Results and Discussion for the mycelial growth of A. blazei in submerged
cultivation (KAWAGOE et al., 2004). Other authors
Mycelial growth results of different A.
as Eira and Braga (2003) and Iwade and Mizuno
brasiliensis strains in function of temperature
(1997) did not studied temperatures higher than 28
showed optimal results between 28 ºC and 31 ºC o
C on compost cultivation. Thus, it was possible to
determinated by Tukey’s test (Figure). The optimal
verify a new range of temperature for A. brasiliensis
temperature for mycelial growth determined by non-
growth in spawn production, axenic cultivation and
linear regression was 30.5 ºC for L1, 30.7 ºC for L2,
possibly in compost cultivation.

L1
c
c L2
80
L3
Mycelial diameter (mm)...

L4
L5
60
b

40 a a

20

0
19 22 25 28 31 34 37
Temperature (ºC)

Figure. Mycelial growth of Agaricus brasiliensis strains (L1 to L5) at 21 days of cultivation on malt extract agar
(48 g/L) with initial pH value of 5.6 at different temperatures. Different letters represent significant differences (p < 0.05)
among temperatures.

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 Colauto, N. B. et al.

Mycelial growth in the substrate with initial pH initial pH of 5.0 but the optimal mycelial growth was
values of 4.0, adjusted with HCl, 7.0 and 8.0, adjusted between 5.5 and 6.0 by Tukey’s test in axenic
with NaOH or CaCO3, showed different degrees of cultivation (Table). This pH range value for mycelial
mycelial growth inhibition (Table). Unexpected the growth is compatible to the more acid Brazilian soil
substrate adjusted with CaCO 3, because of its (MALAVOLTA, 1987), in which pH values are lower
buffering effects, inhibited mycelial growth (Table) than 6.0, where A. brasiliensis was collected from.
causing possibly metabolic disorders. Thus, pH Thus, A. brasiliensis, in axenic cultivation, probably
control through the traditional addition of CaCO3 excrete extra cellular enzymes in the substrate with
(BRAGA et al., 1998) affects A. brasiliensis growth optimal activity in a different pH range than it was
negatively in axenic cultivation and it is likely to affect reported by Miles and Chang (1997) for most fungi
A. brasiliensis in other cultivation methods. This (pH from 6.5 to 6.8) or by Iwade and Mizuno (1997)
procedure of pH control with CaCO3 is usual as well (pH from 6.5 to 6.8) to A. blazei on compost
in spawn formulations in A. brasiliensis culture cultivation or by Kawagoe et al. (2004) (pH of 4.5)
(BRAGA et al., 1998) and could be improved with for A. blazei on submerged cultivation. By non-linear
the correct buffer and pH range in the substratum. regression the optimal pH for mycelial growth was
On the other hand, the pH adjusted with NaOH determined as 5.56 for L1, 5.55 for L2, 5.57 for L3,
allowed a natural adjustment of the substratum pH 5.59 for L4 and 5.54 for L5. The optimal initial pH
during mycelial growth besides there was a minor considering all strains was 5.56 ± 0.05. Gerrits (1988)
mycelial growth inhibition with pH of 4.0 (adjusted and Rinker (1993) reported that a pH of 7.5 was
with HCl) and 7.0 (adjusted with NaOH) but a major used in the substratum for Agaricus bisporus on
inhibition when pH was 3.0 (adjusted with HCl) or compost cultivation because it makes easier control
8.0 (adjusted with NaOH or CaCO 3 ), which the dissociation of NH4+ into NH3, that is toxic to
emphasizes the pH limits for A. brasiliensis growth basidiomycetes. It is as well a basic and usual
in axenic cultivation independently of the CaCO3 procedure on A. brasiliensis cultivation that should
buffering effect for pH of 7.0 or higher in the substrate be reviewed after the results showed in this research,
(Table). A better mycelial growth was verified with mainly when CaCO3 is added in the substratum.

Table. Mycelial growth average of Agaricus brasiliensis strains (L1 to L5) at 21 days of cultivation on malt extract agar
(48 g/L), kept at 28 oC with different initial pH values adjusted with HCl, NaOH or CaCO3.

pH L1 L2 L3 L4 L5 Average (mm) Tukey’s test

3.0* 4.2 3.7 3.5 3.1 3.6 3.6 a
4.0* 58.2
69.0 67.0 43.2 50.8 61.0 c
5.0* 70.6 68.4 54.9 52.8 68.6 63.1 cd
5.5* 78.2 70.1 59.0 76.4 75.8 71.9 d
6.0 ** 77.5 70.5 62.2 78.0 77.5 73.1 d
7.0 ** 69.8 42.0 50.0 66.2 69.8 59.6 c
8.0 ** 6.3 8.3 9.2 5.9 6.3 7.2 a
7.0*** 38.1 37.2 24.4 26.0 38.4 32.8 b
8.0*** 4.8 2.3 5.7 3.6 2.9 3.9 a

*Substratum adjusted with HCl. **Substratum adjusted with NaOH. ***Substratum adjusted with CaCO3. Different
letters represent significant differences (p < 0.05) by Tukey’s test.

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 Temperature and pH conditions for mycelial growth of Agaricus brasiliensis on axenic cultivation

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