Biotechnology Letters (2005) 27: 1537–1541  Springer 2005

DOI 10.1007/s10529-005-1779-9

Response surface analysis of solid state growth of Pleurotus ostreatus

mycelia utilizing whey permeate

Ghibom Bhak, Minkyung Song, Seungyong Lee & Seokhwan Hwang*

School of Environmental Science and Engineering, Pohang University of Science and Technology, San 31,
Hyoja-dong, 790-784, Nam gu, Pohang, Gyungbuk, Korea
*Author for correspondence (Fax: +82-54-279-8299; E-mail: shwang@postech.ac.kr)

Received 8 July 2005; Accepted 28 July 2005

Key words: mycelial cultivation, optimization, Pleurotus ostreatus, response surface analysis, whey
permeate

Abstract
A novel approach to utilizing whey permeate, the cultivation of mycelia of the edible mushroom Pleurotus
ostreatus, is introduced. Response surface analysis (RSA) was successfully applied to determine the com-
bination of substrate concentration, temperature and pH that would result in a maximal mycelial extension
rate under solid state cultivation. The conditions to maximize the mycelial extension rate were predicted to
be 44 g lactose l)1, pH 6.0 and 24.2
C. Subsequent verification of these levels agreed with model
predictions.

Introduction toma and antisarcoma activities (Wang et al.

Whey permeate is that portion of milk that re-
mains after most milk proteins have been
removed during cheese production followed by
protein recovery processes such as ultrafiltration
of the remaining whey. This potential pollutant
should be considered as a by-product that can be
used as a growth media for economically valu-
able products because whey permeate is rich in
carbohydrate (i.e., 4–5% lactose) and mineral
content, including approximately half of the
original nutrients of milk (Lee et al. 2003).
Mushrooms have long been valued as nutri-
tional foods and essential components of medi-
cines by many societies throughout the
world (Smith et al. 2002). Researches into the
mushroom Pleurotus ostreatus have revealed
the presence of various bioactive compounds
increasing macrophases and lymphocyte activities
(Kurashige et al. 1997), reducing cholesterol con-
tent (Bobek et al. 1998), and increasing antihepa-
2000). The perceived medical benefits along with
its appealing taste have made it one of the most
popular edible mushroom in the global market
(Kurashige et al. 1997).
The various bioactive compounds contained
in mushrooms can be extracted from the mycelia
of the species without waiting for a full fruiting
body to develop (Hatvani & Mécs 2001). Conse-
quently, mycelial cultivation has received great
interest as an efficient method for industrial pro-
duction of valuable metabolites and various
agro-industrial by-products have been tried as
economical growth substrates (Fang & Zhong
2002, Hatvani 2001, Yang & Liau 1998). The
purpose of this research was to use whey perme-
ate as a growth medium for mycelia of the mush-
room species P. ostreatus and to optimize
conditions with respect to the simultaneous
effects of temperature, pH and substrate concen-
tration where the maximum mycelial growth
occurred using whey permeate as a substrate.
1538
Materials and methods
Whey permeate and mycelial strain
Dried whey permeate powder was dissolved in
distilled water to obtain differing concentrations
of lactose (Table 1). Since the purpose of the re-
search was to provide information about the
treatment of raw cheese whey permeate with the
mushroom mycelia, no additional nutrients were
added. Commercial agar (Becton Dickinson and
Co, Sparks, Md., USA) was added to the differ-
ent concentrations in the ratio of 1.5% (w/v) and
the solutions were then mixed and autoclaved at
120
C for 20 min. Before pouring the solution
into petri dishes, the pH was adjusted by addi-
tion of 0.5 M HCl or 0.5 M NaOH as needed to
meet the experimental parameters in Table 1.
The solution was then poured into petri dishes
and allowed to solidify for solid state cultivation
of the mycelia.
Pleurotus ostreatus (KCTC 6735, Korean Col-
lection for Type Cultures) was transferred to pet-
ri dishes containing PDA media and incubated at
25
C for 4 days. Mycelial agar discs (5 mm)
were used as inocula for subsequent experiments.
Analysis of mycelial growth using response surface
methodology
Growth of mycelia was on PDA medium in petri
dishes. Growth was measured as the diameter of
the colony in four different places along lines
crossing at right angles and the average value
was used as the size of the colony.
The radial extension rate estimated was as-
sumed to be the mycelial growth rate under the
solid state culture at given conditions (Sang et al.
2001). These rates were then used to perform
response surface analysis (RSA) along with their
corresponding environmental conditions and
obtain the optimal conditions that would maxi-
mize the mycelial growth rate. The experiment
(Table 1) was based on a 3
2 orthogonal design
(pH, temperature and substrate concentration
at two levels each) with a center point being
replicated 5 times as previously described
(Adinarayana et al. 2003, Lee et al. 2003). This
type of design was used to minimize the number of
trials needed to obtain statistically relevant results.
Results and discussion
For this experiment, ranges of temperature and
pH for growth of P. ostreatus were based on liter-
ature values (Hatvani 2001). Because whey per-
meate has not previously been used for the
mycelial cultivation of P. ostreatus, we performed
preliminary experiments to estimate the maxi-
mum growth rate of P. ostreatus in various con-
centrations of whey permeate using a modified
bisectional method (Chapra & Canale 1998). The
radial extension rates of P. ostreatus at different
substrate concentrations were then fitted to an
equation suggested by Shi et al. (1999) (Figure 1).
The r-squared value for this modified equation
was 0.92 and the standard error of estimate was
0.09. Analysis of model parameters was signifi-
cant for all terms at 5% a level. Thus, it was con-
cluded that the model adequately described the
effect of whey permeate concentration on mycelial
growth of P. ostreatus. The substrate concentra-
tion that maximized the mycelial growth rate was
assumed to be 50 g lactose l)1, which was used as
a center point for RSA model building.
A total of 19 trials, including a center point,
were run to approximate the response surface for
the mycelial production of P. ostreatus. To find a
maximum response (i.e. highest radial extension
rate) in the RSA, increasingly complex equations
from linear to partial cubic were sequentially tes-
ted to model the data obtained from the trials in
Table 1. When the data were analyzed using the
various models, the p-value of regression was sig-
nificant at the 5% a level while lack of fit was
not significant at the same confidence level only
for the partial cubic model (Equation 1).
g ¼  104 þ 1:5x1 þ 27:9x2 þ 2:5x3
 4:6
101 x1 x2  1:2
102 x1 x3
 3:1
101 x2 x3  8:5
104 x21
ð1Þ
 1:9x22  2:8
102 x23 þ 2:9

104 x22 x23 þ 2:8
103 x1 x2 x3
þ 3:4
102 x1 x22
where
g: experimental value of the mycelial growth
rate (mm d)1)
xk: independent variable k (k = lactose
concentration, pH and temperature in order)
 1539

Table 1. Experimental design and observed radial extension

rate of Pleurotus ostreatus mycelia grown with reconstituted
dry whey permeate on solid medium.

Independent variables

Trials Lactose Media Incubation Radial

(g l)1) pH temperature extension
(
C) rate
(mm d)1)

Linear 1 40 5 20 0.7
design 2 60 5 20 0.5
3 40 7 20 0.7
4 60 7 20 0.4
5 40 5 30 0.8
6 60 5 30 0.9
Fig. 1. Observed and predicted mycelial growth rate of Pleu- 7 40 7 30 0.2
rotus ostreatus at different lactose concentrations: (d) ob- 8 60 7 30 1.0
served mycelial growth rate; (–) model predictions. An 9a 50 6 25 1.3±0.2
equation suggested by Shi et al. (1999) was used to fit the Quadratic 10 40 6 25 1.3
radial extension rates of P. ostreatus with different substrate
design 11 60 6 25 0.8
concentrations.
12 50 5 25 1.1
13 50 7 25 1.1
The regression coefficient and residual standard 14 50 6 20 0.8
deviation of the partial cubic model were 0.92 15 50 6 30 0.6
and 0.17, respectively. This indicated that the Validation 16 44.1 6 24.2 1.7±0.1
partial cubic model was an accurate representa-
tion of the data and that curvature existed within
the defined response surface. Therefore, this show a definite peak and constant contour lines,
equation was used to analyze the response sur- suggesting that the control variables involved are
face and determine a set of conditions that not interdependent. The third interaction (i.e.,
would maximize mycelial growth rate by setting lactose concentration
temperature), however,
the partial derivatives of the equation to 0 with was significant on the mycelial growth at the 5%
respect to the independent variables. The condi- a level. This indicates an interaction where the
tions determined as providing maximal mycelial values obtained for lactose concentration and
growth rate were 44.1 g lactose l)1 concentration, temperature will vary based on the values of one
pH 6.0 and 24.2
C. Maximum growth rate un- another. This relationship is shown in Figure 3
der these conditions was estimated to be and the interaction between the variables mani-
1.4±0.5 mm d)1. fests itself in the form of an elongated ellipse.
Statistical analysis of the variables involved in The rounded ridge, inside the design boundary,
the model show that incubation temperature was runs diagonally on the plot from lower right to
significant for mycelial growth rate at 1% a level. the upper left.
The three-way interaction of the variables was In order to verify the accuracy of the model
not significant at the 5% a level, suggesting that predictions, an additional five trials of the experi-
all three variables were not simultaneously inter- ment were run under the optimal growth condi-
dependent. Of the three possible two-way inter- tions predicted by the model (24.2
C, pH 6.0
actions among the variables, two of the and 44.1 g lactose l)1); afterwards, residual plots
interactions (lactose concentration
pH and pH for all experimental values were examined for

temperature) were not significant at the 5% a any weakness in using the model (Draper & John
level. Figure 2, for example, shows the relation- 1988). These trials resulted in an average growth
ship of the non-significant interactions between rate of 1.7 mm d l)1 with a standard deviation of
the pH and temperature. These response surfaces 0.1, which was similar to the model output. The
1540
residual plots for the model and the experimental
data set showed no patterns or trends (Figure 4).
A check of the constant variance assumption
(Draper & John 1988) could also be addressed
because a random plot of residuals meant homo-
geneous error variances across the observed val-
ues. Therefore, it can be concluded that the
equation was able to accurately model the
response surface of and predict optimal growth
Fig. 2. Two- and three-dimensional contour plots of the par-
tial cubic model for the mycelial growth of Pleurotus ostrea-
tus. An orthogonal design for the experiments was used to
develop the empirical model, providing a quantitative inter-
pretation of the relationships between the two variables (i.e.,
pH and temperature). The designed intervals were
5 £ pH £ 7 and 20 £ temperature (
C) £ 30, respectively.
conditions for P. ostreatus mycelia when grown
using whey permeate as a substrate.
Conclusions
The results of this experiment suggest that whey
permeate could provide a viable growth substrate
for the cultivation of P. ostreatus mycelia. The
Fig. 3. Two- and three-dimensional contour plots of the par-
tial cubic model for the mycelial growth of Pleurotus ostreatus
with respect to lactose concentration and temperature within
the orthogonal design boundaries. The designed intervals
were 40 £ lactose concentration (g l)1) £ 60 and 20 £ tem-
perature (
C) £ 30, respectively.

Fig. 4. Residual plots of the partial cubic model for mycelial
growth rate. Each residual was calculated using Equation (1).
RSA designed to optimize substrate concentra-
tion, pH and temperature during the cultivation
of P. ostreatus mycelia using whey permeate was
effective in providing statistically sound conclu-
sions. These conditions were determined to be
44.1 g lactose l)1, pH 6.0 and temp 24.2
C. The
optimal conditions determined by this experiment
are valid for mycelia of the edible mushroom P.
ostreatus when grown on whey permeate under
solid state culture.
Acknowledgements
This research was supported in part by the Kor-
ean Institute of Environmental Science and Tech-
nology (EcoTechnopia-21) and U.S. Dairy
Management Inc. (National Research Plan)
programs.
1541
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