jmb

J. Microbiol. Biotechnol. (2013), 23(12), 1757–1764

http://dx.doi.org/10.4014/jmb.1302.02057 Research Article

Media Optimization for Laccase Production by Trichoderma harzianum

ZF-2 Using Response Surface Methodology
Huiju Gao†, Xiang Chu†,Yanwen Wang*, Fei Zhou, Kai Zhao, Zhimei Mu, and Qingxin Liu
College of Forestry, Shandong Agricultural University, Shandong 271018, China

Received: February 25, 2013

Revised: August 16, 2013
Accepted: September 12, 2013
First published online
September 17, 2013
*Corresponding author
Phone: +86-538-824-1315;
Fax: +86-538-824-9164;
E-mail: ywang@sdau.edu.cn
†
These authors contributed
equally to this work.
pISSN 1017-7825, eISSN 1738-8872
Trichoderma harzianum ZF-2 producing laccase was isolated from decaying samples from
Shandong, China, and showed dye decolorization activities. The objective of this study was to
optimize its culture conditions using a statistical analysis of its laccase production. The
interactions between different fermentation parameters for laccase production were
characterized using a Plackett-Burman design and the response surface methodology. The
different media components were initially optimized using the conventional one-factor-at-a-
time method and an orthogonal test design, and a Plackett-Burman experiment was then
performed to evaluate the effects on laccase production. Wheat straw powder, soybean meal,
and CuSO4 were all found to have a significant influence on laccase production, and the
optimal concentrations of these three factors were then sequentially investigated using the
response surface methodology with a central composite design. The resulting optimal medium
components for laccase production were determined as follows: wheat straw powder 7.63 g/l,
soybean meal 23.07 g/l, (NH4)2SO4 1 g/l, CuSO4 0.51 g/l, Tween-20 1 g/l, MgSO4 1 g/l, and
KH2PO4 0.6 g/l. Using this optimized fermentation method, the yield of laccase was increased
59.68 times to 67.258 U/ml compared with the laccase production with an unoptimized
medium. This is the first report on the statistical optimization of laccase production by
Trichoderma harzianum ZF-2.

Copyright © 2013 by
Keywords: Trichoderma harzianum ZF-2, media optimization, laccase production, response
The Korean Society for Microbiology surface methodology
and Biotechnology

Introduction Fungi have been shown as the most promising sources

Laccases (benzenediol: oxygen oxidoreductase, EC 1.10.3.2)
are oxidoreductases. They belong to the phenol-oxidase
family and can catalyze the oxidation of both phenolic and
aromatic amines by using molecular oxygen as the electron
acceptor. The broad substrate spectrum of laccases has
resulted in a large number of biotechnological applications.
In particular, their ability to mineralize a wide range of
synthetic dyes makes this biocatalyst suitable for application
in several bioprocesses, including bioremediation, biobleaching,
biopulping, and the treatment of industrial wastewater [1,
10, 18, 35]. Laccases have also been used in enzyme
immunoassays as a marker enzyme [34], in the design of
various biosensors [12], and in energy transformation
systems [5].
for laccase production [3, 7, 8, 19, 28], especially basidiomycete
fungi, including Trametes versicolor [16], Phlebia radiata [15],
and Penicillium chrysogenum [26]. Whereas there have only
been a few reports on ascomycete and deuteromycete laccases,
there have been many studies of Trichoderma species, from
the moniliales subdivision of deuteromycotina, as potential
sources of cellulase enzymes for the commercial hydrolysis
of cellulosic materials and as active participants in the
biodegradation of cellulose in nature. Assavanig et al. [2]
described extracellular laccases in 14 different natural
isolates of Trichoderma sp. Moreover, extracellular laccases
have also been isolated from Trichoderma atroviride,
Trichoderma harzianum, and Trichoderma longibrachiatum [11,
13]. Some white-rot fungi increase laccase production
when mixing the culture with certain Trichoderma sp. [27,

December 2013 ⎪ Vol. 23 ⎪ No. 12

1758 Gao et al.
33]. Thus, it is of interest to determine whether Trichoderma
strains with laccase activity are capable of more extensive
natural substrate hydrolysis than strains without laccase
activity.
Laccases can be either constitutive or inducible enzymes,
plus their production occurs during secondary metabolism
and is subject to complex regulation by nutrients (C, N,
inducers, and copper) [6, 17, 20, 29]. These regulators affect
the transcription levels of laccase and other genes [23, 25,
30]. Many efforts have already been made to enhance laccase
production by optimizing the nutrient media compositions
in fungal fermentation [14, 21, 24, 31, 32].
In our previous studies, a Trichoderma sp. strain producing
laccase, named ZF-2, was isolated from decaying samples
from Shandong, China. The taxonomy of the fungus was
confirmed as Trichoderma harzianum based on morphological
and 5.8S rDNA/ITS analyses (Accession No. HM05119).
Brilliant Blue KN-R, Basic Violet, and Congo Red were all
decolorized by its laccase. Accordingly, to maximize its
laccase production, this study optimized the different media
components using the conventional one-factor-at-a-time
method, and then further optimized the critical parameters
using the response surface methodology (RSM).
Materials and Methods
Microorganism
Trichoderma harzianum ZF-2 was isolated from decaying samples
from Shandong, China and maintained on slants of Potato Dextrose
Agar (PDA ) at 4oC. Subculturing was performed every fifteenth
day to maintain its viability.
Culture Conditions and Enzyme Extraction
The basal medium contained 20 g glucose, 1 g (NH4)2SO4, 1 g
MgSO4, 0.6 g KH2PO4, and distilled water to make 1 liter. The ZF-2
strain was inoculated into the liquid culture medium and mixed
thoroughly by keeping the flasks on a rotary shaker at 150 rpm for
36 h at 28oC. The culture liquid was then centrifuged at 10,000 rpm
for 20 min at 4oC, and the supernatant collected and used for the
enzyme assay.
Laccase Activity Assay
The laccase activity was measured spectrophotometrically using
guaiacol as the substrate according to Gao et al. [9]. Briefly, an
aliquot of the enzyme solution was incubated in 4.0 ml of a 50 mM
sodium acetate buffer (pH 4.5) containing 1.0 mM guaiacol at 30oC.
The changes in absorbance due to the oxidation of guaiacol in the
reaction mixture were monitored at 465 nm (ε465 = 12,100 M-1 cm-1).
One unit of enzyme activity was defined as the amount of enzyme
that oxidized 1 µmol of guaiacol per minute under the above
conditions.
J. Microbiol. Biotechnol.
Medium Optimization Using One-Factor-at-a-Time
The production of laccase was optimized based on varying the
carbon sources, nitrogen sources, carbon/nitrogen ratios, surfactants,
and concentrations of copper as part of the culture conditions.
Eight carbon sources (glucose, maltose, lactose, sucrose, soluble
starch, wheat straw powder, corn flour, and bran), nine nitrogen
sources (organic nitrogen sources: urea, soybean meal, yeast
extract, beaf extract, and peptone; inorganic nitrogen sources:
ammonium chloride, sodium nitrate, ammonium sulfate, and
ammonium nitrate), five surfactants (Tween-80, Tween-20, resveratrol,
guaiacol, and gallic acid), and five gradient concentrations of
copper (0.01, 0.05, 0.1, 0.2, and 0.4 g/l) were used.
Plackett-Burman Experimental Design
Based on the results of a preliminary study on the production of
laccase by strain ZF-2, a Plackett-Burman (PB) experiment was
performed to identify the significant variables affecting the enzyme
production. A set of 12 experiments was conducted. Each variable
was set at two levels, high and low, denoted by (+1) and (-1),
respectively, where the low level was the optimal condition and
the high level was 1.5 times the low level. The response value
represented the laccase activity, and three replicates were performed
per treatment.
Central Composite Design and Response Surface Methodology
A central composite design was adopted to optimize the effect
of the major variables (wheat straw powder, soybean meal, and
CuSO4) on laccase production. The effect of each variable was
studied at five different levels, namely -α, 1, 0, +1, +α, and the set
of 20 experiments was performed in triplicate. The central coded
value for all the variables was set at zero. The data obtained from
the RSM on laccase production were subjected to analysis of
variance (ANOVA). The results of the RSM were used to fit a
second-order polynomial, Eq. (1), to represent the behavior of the
system:
Y = b0 + b1X1 + b2X2 + b3X3 + b1b2X12 + b2b2X22 + b3b3X32 + b1b2X1X2
+ b1b3X1X3 + b2b3X2X3 (1)
where Y is the response variable representing laccase activity, b0 is
the intercept, b1, b2, b3 are the linear coefficients, b1,1, b2,2, b3,3 are
the squared coefficients, b1,2, b1,3, b2,3 are the interaction coefficients,
and X1, X2, X3, X12, X22, X32, X1X2, X1X3, X2X3 are the levels of the
independent variables. The data analysis and generation of the
response surface graphs were conducted using the statistical
software Design Expert (Design-Expert 7.0).
Results
Effects of Carbon Source, Nitrogen Source, Copper, and
Surfactant on Laccase Production
The effects of seven carbon sources on laccase production
were investigated by replacing the glucose in the basal
 Media Optimization for Laccase Production by Trichoderma 1759

Table 1. Effects of different compounds on laccase production Table 2. Analysis of variance for orthogonal test.
by strain ZF-2. Element Mean square F-value Pr > F
Laccase Wheat straw powder 50.3473 221.68 <0.0001
Compounds
activity (U/ml)
Soybean meal 3.0145 13.27 0.0047
Carbon sources Glucose 1.127
(NH4)2SO4 0.8535 3.76 0.0788
Maltose 1.234
Lactose 0.464
Sucrose 0.482 laccase production, but peptone, beef extract, and yeast
Soluble starch 1.167
extract had an even better effect. Conversely, urea produced
a small decrease in laccase activity (Table 1). Although the
Wheat straw powder 5.242
inorganic nitrogen sources were not as effective as the
Corn flour 3.178
organic sources, (NH4)2SO4 was shown to be better than the
Bran 3.296
others for the production of laccase (Table 1).
Organic nitrogen sources Urea 5.985
To determine whether the copper concentration in the
Soybean meal 9.614 basal mineral medium would affect the laccase activity, the
Yeast extract 7.652 basal medium was supplemented with various concentrations
Beaf extract 8.499 of CuSO4. Whereas the lower and higher concentrations
Peptone 7.411 both partially inhibited laccase activity, the highest laccase
Inorganic nitrogen sources Ammonium chloride 4.849 activity of 27.883 U/ml was achieved with a CuSO4
Ammonium sulfate 5.9745 concentration of 0.2 g/l (Table 1).
Sodium nitrate 2.879 The effect of a surfactant on laccase production was
Ammonium nitrate 4.962 investigated by supplementing the basal medium with
CuSO4 concentration (g/l) 0.01 17.15
Tween, resveratrol, gallic acid, and guaiacol. The results
showed that only Tween-20 significantly increased the
0.05 20.73
laccase activity up to 34.452 U/ml. Meanwhile, Tween-80,
0.1 23.92
resveratrol, and guaiacol had no obvious effect on laccase
0.2 27.88
production, and gallic acid even had a negative effect
0.4 22.15
(Table 1).
Surfactant CK 27.17
Tween-80 28.15 Effect of Carbon/Nitrogen (C/N) Ratio on Laccase Production
Tween-20 34.45 An L16 (43) orthogonal test design was applied to optimize
Resveratrol 26.42 the levels of wheat straw powder, (NH4)2SO4, and soybean
Guaiacol 26.12 meal for laccase production. The results showed that the
Gallic acid 20.12 maximum laccase activity of 12.49 U/ml was achieved with
1% wheat straw powder, 0.8% soybean meal, and 0.4%
(NH4)2SO4. The analysis of variance also showed that the
medium. Among the carbon sources screened, the organic wheat straw powder and soybean meal had a more
carbon sources all improved the laccase activity (Table 1). significant effect on laccase production (P < 0.01), while the
However, corn flour and bran showed a lower improvement effect of (NH4)2SO4 was less significant (P > 0.05). Thus, the
than wheat straw powder, whereas maltose and soluble best C/N ratio was determined as 1% wheat straw powder,
starch produced the lowest laccase levels. When the glucose 0.8% soybean meal, and 0.1% (NH4)2SO4, which was used
in the basal medium was replaced with wheat straw for the PB testing (Table 2).
powder, the laccase activity increased to 5.242 U/ml. Thus,
wheat straw powder was used as the carbon source for the Identification of Significant Variables Using Plackett-
next fermentation. Burman Design
(NH4)2SO4 was the nitrogen source in the basal medium The first optimization step identified the significant factors
for laccase production, and it was replaced by various for laccase production using a 12-run Plackett-Burman
organic and inorganic nitrogen sources. Among the organic design. Variations ranging from 20.606 to 48.371 U/ml in the
nitrogen sources tested, soybean meal had a good effect on production of laccase were observed (Table 3), which

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1760 Gao et al.

Table 3. Experimental conditions and response values of Plackett-Burman test for laccase production by strain ZF-2.
Factors (coded levels)
Laccase
X1 X2
Run X3 X4 D1 D2 D3 activity
Wheat straw Soybean
CuSO4 Tween-20 Dummy Dummy Dummy (U/ml)
powder meal
1 −1 1 −1 −1 −1 1 1 41.571
2 1 −1 −1 −1 1 1 1 20.606
3 −1 −1 −1 −1 −1 −1 −1 34.463
4 1 −1 1 −1 −1 −1 1 26.143
5 1 1 −1 1 −1 −1 −1 22.011
6 1 1 −1 1 1 −1 1 24.757
7 −1 1 1 1 −1 1 1 47.658
8 1 1 1 −1 1 1 −1 32.562
9 −1 −1 −1 1 1 1 −1 33.664
10 −1 −1 1 1 1 −1 1 32.479
11 1 −1 1 1 −1 1 −1 23.994
12 −1 1 1 −1 1 −1 −1 48.371

Table 4. Analysis of variables for Plackett-Burman test.

Model term Degree of freedom Coefficient T-value Pr
Intercept 1 32.356 39.57 0.000a
X1 1 −7.344 −8.98 0.001 a
X2 1 3.798 4.64 0.010 a
X3 1 2.844 3.48 0.025 a
X4 1 −1.596 −1.95 0.123
a
Statistically significant at 95% of confidence level (Pr < 0.05).

indicated that optimization is important to attain higher
productivity. Statistical analyses of the responses were
performed and the results are presented in Table 4. A value
of Pr < 0.05 indicates that the model term is significant. As
a result, three factors were found to have a significant
effect on laccase production: wheat straw powder, soybean
meal, and CuSO4. Wheat straw powder showed the highest
significance and a negative effect, and thus its level was
reduced in the subsequent experiments. Meanwhile, soybean
meal and CuSO4 had a positive effect on laccase production,
where soybean meal had a stronger effect than CuSO4.
Therefore, the levels of soybean meal and CuSO4 were both
increased in the subsequent experiments. As Tween-20 had
no significant effect on laccase production, it was kept at a
low level. The observed responses were used to compute
the model coefficients using the least-square method. The
model (Eq. (2)) for laccase production was calculated as
Y = 32.4 - 7.34X1 + 3.80X2 + 2.84X3 - 1.60X4
where X1, Wheat straw powder; X2, Soybean meal; X3,
CuSO4; and X4, Tween-20. Since the statistical model was
able to explain 96.8% of the variability in the response, it
was concluded that the model fitted well with the measured
data.
Statistical Optimization Using Central Composite Design
Based on the PB tests, statistical optimization was used to
determine the optimal concentrations of wheat straw
powder, soybean meal, and CuSO4 for laccase production.
The effect of each variable on enzyme production was
characterized at five different levels, namely -1.68, 1, 0, +1,
+1.68. Thus, a set of three factors with five levels and a total
of 20 treatments were performed. The results of the response
surface experiments to determine the effects of wheat straw
power, soybean meal, and CuSO4 are presented in Table 5.
The determination coefficient (R2) value of 0.9645 indicates
that the statistical model was able to explain 96.45% of the
(2) variability in the response. In addition, the value of the

J. Microbiol. Biotechnol.
 Media Optimization for Laccase Production by Trichoderma 1761

Table 5. Experimental plan for central composite design F = 30.22 and P > F-value < 0.0001. The results indicated
performed for selected parameters. that the linear effects of wheat straw powder (X1) and the
Variables Laccase interaction effect of X1X3 were significant (P < 0.05). Moreover,
Run X1 Wheat X2 Soybean activity the linear effects of soybean meal (X2) and CuSO4 (X3), the
X3 CuSO4
straw powder meal (U/ml) quadratic effects of wheat straw powder (X12), soybean
1 −1 −1 −1 51.98 meal (X22), and CuSO4 (X32), and the interaction effect of
2 1 1 −1 59.92 X2X3 were more significant than the other factors (P < 0.01).
3 0 0 1.68 59.97 Thus, the response of laccase production (Y) by ZF-2 could
4 0 0 0 62.22
be expressed in terms of the following regression equation:
5 1 −1 1 59.36
Y = 64.35 + 1.04X1 + 2.78X2 + 1.93X3 + 0.36X1X2 + 1.16X1X3
6 0 0 −1.68 51.92
- 1.56X2X3 - 2.95X12 - 2.33X22 - 2.91X32 (3)
7 −1 1 1 57.53
8 0 0 0 63.98
where X1 is wheat straw powder, X2 is soybean meal, and
9 0 0 0 65.82 X3 is CuSO4.
10 −1.68 0 0 53.71 A 3D response surface was drawn based on the model
11 −1 1 −1 57.95 equation to investigate the interaction among the variables
12 1 −1 −1 48.92 and determine the optimum concentration of each factor
13 0 1.68 0 62.47 for maximum laccase production by ZF-2 (Figs. 1A-1C).
14 0 −1.68 0 52.66 The model predicted that the maximum laccase production
15 0 0 0 64.07 was up to 65.457 U/ml when the medium was wheat straw
16 1 1 1 60.53 powder 7.63 g/l, soybean meal 23.07 g/l, and CuSO4
17 1.68 0 0 57.96
0.51 g/l.
18 0 0 0 64.87
Validation of Model
19 0 0 0 65.23
To confirm the accuracy of the model, a validation
20 −1 −1 1 54.21
experiment was performed using three different solutions
(wheat straw powder, soybean meal, and CuSO4), as
adjusted determination coefficient (adjusted R2 = 0.9326) suggested by the software results (Table 7). The results
was also very high, indicating the high significance of the showed a good agreement between the predicted and
model. experimental values, thereby validating the model. The
The significance of each term in the model is presented in maximum laccase production observed was 67.258 U/ml
Table 6. The ANOVA for the selected quadratic model and the optimal medium was wheat straw powder 7.63 g/l,
showed that the model was significant with a Model soybean meal 23.07 g/l, (NH4)2SO4 1 g/l, CuSO4 0.51 g/l,

Table 6. ANOVA for response surface quadratic model.

Source Sum of squares (SS) Degree of freedom (df) Mean square (MS) F-value Pr > F
Model 474.86 9 52.76 30.22 <0.0001
X1 14.78 1 14.78 8.46 0.0156
X2 105.50 1 105.50 60.42 <0.0001
X3 51.03 1 51.03 29.22 0.0003
X1 X 2 1.04 1 1.04 0.59 0.4588
X1 X 3 10.67 1 10.67 6.11 0.0330
X2 X 3 19.47 1 19.47 11.15 0.0075
2
X1 125.06 1 125.06 71.62 <0.0001
X2 2 78.51 1 78.51 44.97 <0.0001
X3 2 121.77 1 121.77 69.74 <0.0001

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1762 Gao et al.

Tween-20 1 g/l, MgSO4 1 g/l, and KH2PO4 0.6 g/l. As a

result of optimizing the culture conditions, a 59.68-fold
increase in laccase activity was achieved compared with
that (1.127 U/ml) previously obtained under basal conditions.

Discussion

Traditional optimization methods change one independent

Fig. 1. Three-dimensional response surface plots of laccase
production by ZF-2 under conditions optimized using RSM.
(A) Effect of interaction of soybean meal and wheat straw; (B) effect of
interaction of copper sulfate and wheat straw; (C) effect of interaction
of copper sulfate and soybean meal.
variable, while keeping the other variables fixed at a
certain level. However, this single-dimensional search is
laborious, time-consuming, and incapable of reaching a
true optimum owing to the interactions among the variables.
The response surface methodology (RSM), first described
by Box and Wilson in 1951 [4], is an effective strategy for
seeking the optimum conditions for a multivariable system.
Whereas this statistical method for media optimization has
already been successfully utilized to improve laccase
production from white-rot fungi, there are no reports on
the optimization of laccase production from Trichoderma
harzianum. Therefore, this study applied RSM to optimize
the medium conditions for laccase production by Trichoderma
harzianum ZF-2. The obvious important factors (wheat
straw power, soybean meal, and CuSO4) that influenced
the production of laccase were obtained using a Plackett-
Burman experimental design, and the optimal concentrations
of these three factors were then sequentially investigated
using the response surface methodology with a central
composite design. The final optimal medium components
included wheat straw powder 7.63 g/l, soybean meal
23.07 g/l, (NH4)2SO4 1 g/l, CuSO4 0.51 g/l, Tween-20 1 g/l,
MgSO4 1 g/l, and KH2PO4 0.6 g/l. When using this medium,
the yield of laccase was increased 59.68 times (67.258 U/ml)
compared with the laccase production in an unoptimized
medium (1.127 U/ml).
Among the important factors affecting laccase production,
wheat straw, an inexpensive and easily available agro-
residue, was the most suitable carbon source for laccase
production and showed higher significant effect than the
other substrates. In agreement with this result, Patel et al.
[22] previously reported that wheat straw was the best
substrate for the production of laccase by the basidiomycete
fungal isolate Pleurotus ostreatus HP-1. Soybean meal is also

Table 7. Experimental verification of combined effect of optimized medium on laccase production.

Substrate Concentration of optimization (g/l) Predicted response (U/ml) Observed response (U/ml)
Wheat straw powder (X1) 7.63
Soybean meal(X2) 23.07 65.457 67.258
CuSO4 (X3) 0.51

J. Microbiol. Biotechnol.

an inexpensive organic nitrogen source and had a positive
effect on laccase production. Therefore, this study provides
useful information regarding the use of cheaper carbon and
nitrogen sources for maximizing the production of laccase.
Furthermore, CuSO4 supplementation at 0.051% was
effective in improving the laccase production by ZF-2. This
expressive effect is also supported by the fact that Cu2+ is a
laccase cofactor, where every four Cu2+ are associated with
one single polypeptide chain.
In conclusion, this research is the first systematic medium
optimization for laccase production by Trichoderma harzianum
ZF-2 and showed the effect of each component. Although
some scale-up experiments are yet to be performed, the
optimum medium will be helpful for further studies of
laccase production by Trichoderma harzianum ZF-2 using
large-scale batch fermentation. Further studies will also
explore the role of laccase in the delignification and
biodegradation of cellulose in nature by ZF-2.
Acknowledgments
This works were funded by the National Basic Research
Program of China (No. 2012CB114600), the National
Natural Science Foundation of China (No. 31200450), China
Agriculture Research System (No. CARS-22), Postdoctoral
Science Foundation of China (No. 2013M541944).
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