World J Microbiol Biotechnol (2009) 25:1929–1939
DOI 10.1007/s11274-009-0091-6
ORIGINAL PAPER
Optimization of amylase production by Aspergillus niger
in solid-state fermentation using sugarcane bagasse as solid
support material
Renato Pérez Rosés Æ Nelson Pérez Guerra
Received: 8 February 2009 / Accepted: 15 June 2009 / Published online: 28 June 2009
Ó Springer Science+Business Media B.V. 2009
Abstract Synthesis of amylase by Aspergillus niger
strain UO-01 under solid-state fermentation with sugarcane
bagasse was optimized by using response surface meth-
odology and empirical modelling. The process parameters
tested were particle size of sugarcane bagasse, incubation
temperature and pH, moisture level of solid support
material and the concentrations of inoculum, total sugars,
nitrogen and phosphorous. The optimum conditions for
high amylase production (457.82 EU/g of dry support)
were particle size of bagasse in the range of 6–8 mm,
incubation temperature and pH: 30.2°C and 6.0, moisture
content of bagasse: 75.3%, inoculum concentration:
1 9 107 spores/g of dry support and concentrations of
starch, yeast extract and KH2PO4: 70.5, 11.59 and 9.83 mg/
g of dry support, respectively. After optimization, enzyme
production was assayed at the optimized conditions. The
results obtained corroborate the effectiveness and reliabil-
ity of the empirical models obtained.
Keywords Amylase
Optimization

Solid-state fermentation
Sugarcane bagasse
Abbreviations
RSM Response surface methodology
SLF Submerged liquid fermentation
R. P. Rosés
Department of Pharmacy, Natural Science Faculty,
University of Oriente, Santiago de Cuba, Cuba
N. P. Guerra (&)
Nutrition and Bromatology Group, Department of Analytical
and Food Chemistry, Food Science and Technology Faculty,
Ourense Campus, University of Vigo, As Lagoas,
32004 Ourense, Spain
e-mail: nelsonpg@uvigo.es
SSF Solid-state fermentation
gds g of dry support
TAA Total amylase activity
EU Enzymatic units
T Temperature
IC Inoculum concentration
M Moisture content
TS Total sugars concentration
N Nitrogen concentration
P Phosphorous concentration
Introduction
The Aspergillus species produce a large variety of extra-
cellular enzymes of which amylases are of world-wide
interest in fermentation, food, pharmaceutical, textile and
paper industries (Bhargav et al. 2008; Uma Maheswar Rao
and Satyanarayana 2007). Production of amylases by
Aspergillus strains in both submerged liquid fermentation
(SLF) and solid-state fermentation (SSF) by using different
food wastes or agricultural residues has been thoroughly
studied (Ellaiah et al. 2002; Francis et al. 2002, 2003;
Murado et al. 1997; Salas et al. 2006). However, compar-
ative studies between SLF and SSF claim higher yields and
other advantages for products obtained by SSF, such as low
energy requirements, lower availability of water that
reduces the possibilities of contamination by bacteria and
yeast, small volumes of polluting effluents and low
downstream processing cost (Guerra et al. 2003; Raimbault
1998).
The production of amylases in SSF is affected by a variety
of physicochemical factors, including the type and nutrient
composition of the substrate, incubation temperature, pH,
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aeration, concentration and type of the carbon, phosphate
and nitrogen sources, concentration and age of the inoculum,
particle size and moisture level of the substrate (Balkan and
Ertan 2007; Ramachandran et al. 2004; Rodrı́guez and
Sanromán 2006). Therefore, after selecting a culture med-
ium for amylase production, the fermentation conditions
must be optimized to improve enzyme production at a low
production cost (Balkan and Ertan 2007; Spier et al. 2006).
In recent years, there has been an increasing interest
towards more efficient utilization of different agro-indus-
trial residues, including sugarcane bagasse, wheat bran,
wheat straw, rye straw and corncob leaf and oil cakes, for
amylases production (Balkan and Ertan 2007; Bhargav
et al. 2008; Ramachandran et al. 2004). Sugarcane bagasse
has been used as a solid inert support for the production of
value-added products including L-glutamic acid, ergot
alkaloids, penicillin, fruity aroma, lactic acid, pigments and
pectinases (Pandey et al. 2000). However, to our knowl-
edge, there is not information available on the use of
sugarcane bagasse as a support material for the production
of amylases by Aspergillus strains.
Considering the substantial availability of sugarcane
bagasse at very low prices by local sugar cane factories of
Santiago de Cuba, its use as a support material could offer
an attractive possibility for a low cost production of amy-
lases by Aspergillus niger strain UO-01 in SSF. Since the
amylase production by this strain was studied before in
SLF (Salas et al. 2006), the aim of the present study was to
optimize enzyme production by SSF conditions. Particle
size of sugarcane bagasse was firstly optimized in a single
parameter study. Subsequently, other important process
parameters were optimized in a sequential order by using
response surface methodology (RSM) and empirical mod-
elling. The other process variables optimized were incu-
bation pH and temperature (second experiment), moisture
level, inoculum and total sugars concentrations (third
experiment) and the concentrations of phosphorous and six
nitrogen sources (fourth experiment). Finally amylase
production was assayed at the optimized conditions to
verify the effectiveness and the accuracy of the empirical
models obtained.
Materials and methods
Microorganism
Aspergillus niger strain UO-01, was acquired from the
Biotechnology Center of the University of Oriente (Santi-
ago de Cuba, Cuba) and it was maintained on potato
dextrose agar slants at 4°C (Ellaiah et al. 2002; Salas et al.
2006).
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Inoculum preparation, culture media and fermentation
conditions
Inocula were prepared by transferring 2-ml of 60-h old
slant culture in 50 ml of medium (250 ml Erlenmeyer)
composed by (g l-1): glucose, 20; (NH4)2SO4, 6.6;
KH2PO4, 3.5; FeSO4
7H2O, 0.15; MgSO4
7H2O, 0.10;
MnCl2
2H2O, 0.45 and mycological peptone, 3.0; at pH
6.8. The culture was incubated at 30°C and 220 rev/min for
48 h (Salas et al. 2006).
Sugarcane bagasse obtained from a local sugar cane
factory of Santiago de Cuba was used as a solid support
material. It was treated with NaOH [0.12 g of NaOH/g of
dry support (gds)] and autoclaved at 121°C for 20 min
(Gutiérrez-Correa and Tengerdy 1997) to remove the core
and noncore lignin fractions (Doran et al. 1994). Then, the
samples of sugarcane bagasse were thoroughly washed
with tap water, subsequently with distilled water until
neutrality and dried at 80°C (Doran et al. 1994).
Fermentations were conducted in 250-ml Erlenmeyer
flasks containing 5 g of dry bagasse which was moistened
with the liquid media (salt solution and distilled water)
previously supplemented with soluble potato starch to get
desired level of sugar in the solid support material. The salt
solution was prepared to provide (%, g/g of dry bagasse),
NaNO3, 1; KH2PO4, 1; NaCl, 0.2; MgSO47H2O, 0.2
(Francis et al. 2003).
The contents of the flasks were mixed thoroughly and
then autoclaved at 121°C for 15 min. After cooling, the
sterile media were inoculated with 1.25 ml of inoculum
containing the appropriate cell suspension (spores/gds) and
thoroughly mixed. Then, the flasks were incubated at the
corresponding temperature in a chamber with temperature
and humidity control. Samples as whole flasks in triplicate
were withdrawn each 12 h.
Extraction of the enzyme
After fermentations, the whole sample of each flask was
extracted by the addition of distilled water containing 0.1%
Tween-80, to a total extract volume of 100 ml. Contents
were mixed thoroughly (150 rev/min, at room temperature
for 1 h) in a rotary shaker and filtered using a Whatman
filter paper No. 1. The culture filtrates were used as the
crude enzyme extracts (Francis et al. 2003).
Analytical methods
Total amylase activity (TAA) was determined at 40°C by
mixing appropriately the diluted crude enzyme extract with
0.15 M citrate-phosphate buffer (pH 5.0) and 4% soluble
starch (previously maintained at 40°C/15 min) and deter-
mining the reducing sugars (glucose equivalents) after
World J Microbiol Biotechnol (2009) 25:1929–1939 1931

10 min (Murado et al. 1993). One unit of amylase activity Table 1 Experimental domain and codification of the variables used
[enzymatic units (EU)/ml] was defined as the amount of in the three experimental designs
enzyme releasing 1 mg/ml of reducing sugars/min under Coded values Actual values
the assay conditions. Then, the amylase activity units were
T pH M IC TS N P
expressed as EU/gds.
Method for determination of total sugars in the super- -1.525 – – 58.5 4.2 9.5 – –
natants was described or referred to in a previous work -1.267 24.0 3.0 – – – 4.3 4.3
(Salas et al. 2006). All determinations were carried out in -1 25.5 3.5 63.0 5.0 20.0 12.0 12.0
triplicate. 0 31.0 5.5 71.5 6.5 40.0 41.0 41.0
1 36.5 7.5 80.0 8.0 65.0 70.0 70.0
Optimization of process parameters in SSF 1.267 38.0 8.0 – – – 77.7 77.7
1.525 – – 84.5 8.8 70.5 – –
The particle size (PS) was optimized by using the ‘‘one
T temperature (°C), M moisture content (%), IC inoculum concen-
variable at a time’’ method for amylase production. Thus, tration [log (number of spores/gds)], TS total sugars concentration
in a first experiment, the bagasse was milled to different (mg/gds), N nitrogen source concentration (mg/gds), P phosphorous
particle sizes (\1, 1–2, 2–4, 4–6, 6–8, 8–10, 10–12, 12–14, source concentration (mg/gds)
14–16 and 16–20 mm), which were used as support
materials to determine the appropriate particle size for inoculated with 1.25 ml of a suspension containing the
maximal enzyme production in SSF. The dried bagasse was appropriate amount of spores to obtain the corresponding
impregnated with 2.5 ml of the salt solution buffered at pH inoculum concentrations (Table 1). All the media were
6.0 with potassium hydrogen phthalate-HCl buffer (0.1 M) then incubated at 30°C for 84 h.
and 16.25 ml of distilled water previously supplemented In the third optimization step, the concentrations of
with soluble potato starch to get 26 mg of total sugars/gds. the nitrogen (N) and phosphorous (P) sources were opti-
After sterilization, the media were inoculated with 1.25 ml mized by including the appropriate nitrogen source
of inoculum to give an inoculum concentration of [NH4CH3COO, (NH4)2HPO4, NaNO2, NaNO3, yeast
1 9 107 spores/gds. The final moisture content after inoc- extract or (NH4)2SO4] and KH2PO4 in the salt solution to
ulation was calculated to be as 80%. The flasks were give the concentrations indicated in Table 1. This last
incubated at 30°C until the total sugars were completely experiments were carried out in the optimal conditions (PS,
consumed. T, pH, M, IC and TS) previously calculated. After sterili-
Then, a three-step optimization strategy using RSM and zation and inoculation, the media were incubated at 30°C
empirical modelling (Uma Maheswar Rao and Satyanara- for 84 h.
yana 2007) was employed to optimize amylase production. Results were analyzed by Experimental Design Module
In the first step, initial culture pH and temperature (T) were of the Statistica software package (Statistica 5.1 for Win-
optimized using the optimum particle size of the sugarcane dows computer program manual; StatSoft Inc. Tulsa, OK,
bagasse calculated in the previous experiment. In this USA). The Student’s t-test was employed to check the
assay, the liquid medium was buffered at different pHs statistical significance of the regression coefficients. The
with the appropriate buffer (0.1 M potassium hydrogen Fisher’s F-test for analysis of variance (ANOVA) was
phthalate-HCl buffer for pHs 3.0, 3.5 and 5.5 or 0.1 M performed on experimental data to evaluate the statistical
sodium phosphate buffer for pHs 7.5 and 8.0) and the flasks significance of the model.
were incubated for 84 h at the corresponding temperature
according to the experimental matrix defined by the design
used (Table 1). Results
In the second step, the levels of three factors: moisture
content (M), inoculum concentration (IC) and total sugars The first optimization experiment was performed to check
concentration (TS) were then optimized in the optimal the influence of different bagasse particle sizes on amylase
conditions (PS, T and pH) calculated in the two previous production by A. niger UO-01 in SSF. The results obtained
experiments. In this assay, sugarcane bagasse was are shown in Fig. 1. Maximum enzyme production
impregnated with 2.5 ml of the salt solution and an (109.12 EU/gds) was observed when the particle size of
appropriate volume of distilled water (both at pH 6.0) bagasse was in the range of 6–8 mm, decreasing for lower
containing different amounts of soluble potato starch to of higher particle sizes. In subsequent experiments, there-
give the different moisture contents and total sugars con- fore, a particle size between 6–8 mm was used for the
centrations defined in Table 1. The sterile media were production of amylase by A. oryzae FQB-01.

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A
120 120
< 1 mm 1 - 2 mm 2 - 4 mm 4 - 6 mm 6 - 8 mm
90 90

60 60
TAA and TS

TAA and TS
30 30

0 0
8 - 10 mm 10 - 12 mm 12 - 14 mm 14 - 16 mm 16 - 20 mm
90 90

60 60

30 30

0 0
0 30 60 90 0 30 60 90 0 30 60 90 0 30 60 90 0 30 60 90 120
Time (h)

B 120

90
TAA (EU/gds)

60

30

0
<1 mm 1-2 mm 2-4 mm 4-6 mm 6-8 mm 8-10 mm 10-12 mm 12-14 mm 14-16 mm 16-20 mm
Particle size

Fig. 1 a Profiles of TS (, in mg of TS/gds) and TAA (h, in EU/gds) production in the A. niger UO-01 cultures in SSF with different bagasse
particle sizes. b Maximum TAA levels obtained after 84 h of incubation for each bagasse particle size. Means of three analytical replications

After optimizing the particle size of bagasse, the following
experiments were focused on the determination of the opti-
mum levels of seven factors (T, pH, M, IC, TS, N and P) for
high amylase production by using second-order orthogonal
designs. The corresponding analysis of variance (ANOVA)
of each empirical model obtained along with the values of the
determination coefficient (R2) and the adjusted determination
coefficient (adj. R2) are presented in Tables 2, 3 and 4. In
each case, the lack-of-fit analysis gave non-significant
P-values ([0.050) and F-values lower than the correspond-
ing tabulated F-values, thus ensuring that the models
obtained were highly significant. In addition, the high value
of R2 (0.960 or higher) indicated that the fitted models could
explain at least 96% of the total variation in the responses.
These facts indicate that the quadratic models were appro-
priate to fit and describe satisfactorily the experimental data.

Table 2 Analysis of variance (ANOVA) of the model (1) obtained Table 3 Analysis of variance (ANOVA) of the model (2) obtained
for amylase production as a function of incubation temperature and for amylase production as a function of the moisture content, and
pH inoculum and total sugars concentrations
SS df MS F P SS df MS F P

Lack of fit 73.8 4 18.5 3.67* 0.118 Lack of fit 575.7 7 82.2 1.42* 0.361
Pure error 20.1 4 5.0 Pure error 289.1 5 57.8
Total SS 2,335.6 12 Total SS 35,973.3 19
R2 value 0.960 R2 value 0.976
Adj. R2 value 0.940 Adj. R2 value 0.962
SS sum of squares, df degrees of freedom, MS mean square SS sum of squares, df degrees of freedom, MS mean square
* Significant at F44 (a = 0.050) \ 6.39 * Significant at F57 (a = 0.050) \ 4.88

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Table 4 Analysis of variance (ANOVA) of the empirical models (3), (111.58 EU/gds) was obtained at a pH value of 6.0 and a
(4), (5), (6), (7) and (8) obtained for amylase production as a function temperature of 30.2°C. This result is in perfect agreement
of the concentrations of the nitrogen and phosphorous sources
with the TAA level (109.12 EU/gds) obtained in the
SS df MS F P previous experiment with the optimal particle size (6–8 mm),
which was performed at T = 30°C and pH 6.0.
Model (3)
On the other hand, the polynomial model obtained for
Lack of fit 960.8 4 240.2 4.31* 0.093
amylase production as a function of moisture content (M),
Pure error 222.7 4 55.7
inoculum concentration (IC) and total sugars concentration
Total SS 117,262.6 12
(TS) was:
R2 value 0.990
Adj. R2 value 0.985 TAA (EU=gds) ¼ 246:6 þ 25:1  M þ 6:0  IC þ 32:4
Model (4)  TS  10:4  M  IC þ 9:7  IC
Lack of fit 929.0 4 232.2 5.98* 0.056  TS  24:0  M 2  22:6  IC2
Pure error 155.3 4 38.8 ð2Þ
Total SS 89,383.8 12
The significant coefficients for the quadratic terms (M2
R2 value 0.988
and IC2) in model (2) implied the existence of optimum
Adj. R2 value 0.982
values for M and IC inside the experimental domain. Since
Model (5)
the coefficient for the quadratic term TS2 was found to be
Lack of fit 1,117.3 4 279.3 5.49* 0.064
non-significant and the linear term TS had a positive
Pure error 203.4 4 50.9
coefficient, the optimum value for this variable is located in
Total SS 100,757.1 12
the maximum level (70.5 mg/gds) assayed.
R2 value 0.987
The response surfaces curves generated according to
Adj. R2 value 0.980
model (2) show the relative effects of two variables at a
Model (6)
time on amylase production, when the coded value of the
Lack of fit 759.3 4 189.8 3.37* 0.133 third variable is maintained at fixed levels of ‘‘-1’’ or
Pure error 225.6 4 56.4 ‘‘1’’ (Fig. 3a, b, c). The results obtained showed that an
Total SS 91,286.6 12 initial moisture content of 75.3%, inoculum concentration
R2 value 0.989 of 1 9 107 spores/gds and starch concentration of
Adj. R2 value 0.984 70.5 mg/gds were the best conditions to produce the high
Model (7) amounts of enzyme (308.24 EU/gds) with A. niger UO-01
Lack of fit 542.0 4 135.5 2.71* 0.179 in SSF.
Pure error 199.9 4 50.0
Total SS 79,304.0 12
R2 value 0.991
Adj. R2 value 0.986
Model (8)
Lack of fit 1,569.2 4 392.3 5.16* 0.070 100
100
Pure error 304.1 4 76.0
TAA (EU/gds)

Total SS 103,604.1 12 75
75
R2 0.982
50
Adj. R2 0.973 50

SS sum of squares, df degrees of freedom, MS mean square 25

25
* Significant at F44 (a = 0.050) \ 6.39
0
0
After eliminating the non-significant terms, the final 1.3 1.3
response equation for amylase production (TAA) in response 0 0
to pH and temperature (T), in coded units, is given as follows: -1.3

TAA (EU=gds) ¼ 110:4  4:1  T þ 6:7  pH

T pH
ð1Þ
 13:5  T 2  12:9  pH2
Fig. 2 Response surface showing the effect of temperature (T) and
The three-dimensional response surface (Fig. 2) pH on TAA production by A. niger UO-01 in SSF. Variables T and
obtained from model (1) shows that the maximum TAA pH are in coded values

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Fig. 3 Response surfaces
showing the effect of moisture
content (M), inoculum (IC) and
total sugars (TS) concentration
on TAA production by A. niger
UO-01 in SSF. Variables M, IC
and TS are in coded values
From the comparison between the results obtained in
submerged (Salas et al. 2006) and SSF (Fig. 3), it can be
noted that amylase production ceased to increase from a
starch concentration of 30 g l-1 in the submerged cultures,
while in SSF this effect was not observed (Fig. 3b, c). In fact,
the empirical model (2) predicted a linear increase in amy-
lase concentration when the TS concentrations increased
from 9.5 to 70.5 mg/gds (Fig. 3b, c). The same trend was
observed for amylase production by A. oryzae CBS 125-59
by SSF on polyurethane foams (Murado et al. 1997).
On the other hand, the enzyme yield coefficients
expressed as amylase concentration on initial total sugars
(YTAA/iTS in EU/g of initial TS) obtained in the submerged
cultures with initial starch concentrations of 10, 20, 30
and 40 g l-1 (Salas et al. 2006) were calculated to be as
1,757, 2,067, 2,400 and 4,200 EU/g of initial TS,
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respectively. These values are lower than the YTAA/iTS
value obtained in the optimal conditions determined in
this experiment, which was calculated to be as 4,372 EU/
g of initial of TS.
The corresponding empirical equations obtained for
TAA production as a function of the concentrations of
NH4CH3COO (model 3), (NH4)2HPO4 (model 4), NaNO2
(model 5), NaNO3 (model 6), yeast extract (model 7) or
(NH4)2SO4 (model 8) and the phosphorous source
(KH2PO4) were:
TAA (EU=gds) ¼ 289:9  74:5  N  91:5  P  34:9
 N 2  42:6  P2 ð3Þ
TAA ðEU=gdsÞ ¼ 284:3  66:8  N  76:6  P  37:8
 N 2  35:3  P2 ð4Þ
World J Microbiol Biotechnol (2009) 25:1929–1939
TAA ðEU=gdsÞ ¼ 278:7  69:4  N  84:0  P  31:6
 N 2  41:0  P2 ð5Þ
TAA (EU=gds) ¼ 240:7  59:7  N  85:1  P  28:2
 N 2  40:0  P2 ð6Þ
TAA (EU=gds) ¼ 389:9  84:5  N  46:8  P  41:6
 N 2  21:8  P2 ð7Þ
TAA (EU=gds) ¼ 245:6  76:0  N  79:5  P  39:3
 N 2  35:8  P2 ð8Þ
The response surfaces obtained according to the above
six models showed that the enzyme production was highly
influenced not only by the phosphorus concentrations but
also by the type and concentrations of the nitrogen source
(Fig. 4). In general, a linear increase in TAA production
was observed when the concentrations of N and P
increased up to the optimal values, and thereafter TAA
production declined sharply. In addition, the optimal N and
P values as well as the maximum TAA levels obtained with
the use of the six nitrogen sources were clearly different
(Table 5). The highest amylase concentration (457.82 EU/
gds) was obtained with the use of yeast extract followed by
NH4CH3COO (378.88 EU/gds), NaNO2 (359.79 EU/gds),
(NH4)2HPO4 (355.47 EU/gds), (NH4)2SO4 (326.48 EU/
gds) and NaNO3 (317.50 EU/gds).
Verification of the predicted results in the optimal
conditions
Verification of the predicted results was accomplished by
using the optimized conditions in triplicate incubation
experiments for each nitrogen source (Fig. 5). Under the
calculated optimal culture conditions, the mean values
of TAA (in EU/gds) after 84 h of incubation were
380.55 ± 2.06 (in case of NH4CH3COO), 353.32 ± 2.64
(in case of (NH4)2HPO4), 358.12 ± 1.84 (in case of
NaNO2), 319.85 ± 2.41 (in case of NaNO3), 455.16 ±
3.89 (in case of yeast extract) and 325.04 ± 1.38 (in case
of (NH4)2SO4). These values were found to be very close to
the predicted values (Table 5) obtained from the empirical
models (3), (4), (5), (6), (7) and (8). Therefore, the models
developed can be considered to be accurate and reliable for
predicting amylase production by A. niger UO-01 in SSF
with sugarcane bagasse as a support material.
Discussion
Particle size, incubation temperature, pH, initial moisture
content of solid material, inoculum concentration, as well
as the concentrations of sugars, nitrogen and phosphorous
1935
sources are important factors that influence amylase pro-
duction by A. oryzae strains in SSF (Gigras et al. 2002;
Murado et al. 1997; Francis et al. 2003). However, the
effects of these variables on enzyme production depend on
the producer strain and the solid substrate used. For
example, optimal particle size, moisture level and inocu-
lum concentration for a-amylase production in SSF by a
Penicillium chrysogenum strain were [1 mm, 75 and 20%
for corncob leaf, [1 mm, 65 and 20% for wheat straw,
1 mm, 65 and 20% for wheat bran and [1 mm, 55 and
30% for rye straw (Balkan and Ertan 2007). However,
initial moisture content of 70%, inoculum concentration of
1 9 107 spores/g dry substrate and an incubation temper-
ature in the range of 25–30°C were necessary to achieve
the maximum a-amylase production by A. oryzae NRRL
6270 on spent brewing grains (Francis et al. 2002). In the
same way, the optimal moisture levels for glucoamylase
production by Aspergillus sp. A3 in wheat bran (Ellaiah
et al. 2002) and for a-amylase and glucoamylase produc-
tion by A. niger LPB 28 in cassava starch and sugarcane
bagasse (Spier et al. 2006) were 80 and 90%, respectively.
Taking into account these observations, amylase pro-
duction by A. oryzae FQB-01 under SSF using sugarcane
bagasse as a support material was optimized by determin-
ing the optimal values of eight important process variables
(PS, T, pH, IC, M, TS, N and P).
A particle size in the range of 6–8 mm gave the highest
amylase production in comparison with the enzyme levels
obtained with other particle sizes (\1, 1–2, 2–4, 4–6, 8–10,
10–12, 12–14, 14–16 and 16–20 mm). Thus, the optimum
particle size (6–8 mm) probably provides the most effec-
tive support material for attachment of the fungal strain
(Laxmi et al. 2008), or facilitates the mass transfer per-
formance (gas and nutrient diffusion) greatly, resulting in a
better respiration/aeration efficiency and an increased
availability of nutrients (Balkan and Ertan 2007; Mazutti
et al. 2007; Pandey et al. 1999). These facts favour both the
growth and enzyme production (Balkan and Ertan 2007;
Pandey et al. 1999).
Aspergillus niger UO-01 exhibited its best performance
for enzyme production in the mesophilic range (T =
30.2°C), as it was reported before for A. niger LPB 28, which
produce the highest amylase level at 30°C (Spier et al. 2006).
Temperature values higher than 30°C may lead to enzymatic
inactivation (Mazutti et al. 2006) or suppression of cell via-
bility (Francis et al. 2002). In contrast, low temperature val-
ues may reduce the metabolism of the microorganism
(Mazutti et al. 2006) and consequently, the enzyme synthesis.
In this study, it was also verified that A. niger UO-01 has a
preference to pH around 6.0 for amylase production, but its
production capacity decreased for pH levels higher and lower,
probably as a consequence of a reduction in the metabolic
activity of the amylase-producing strain (Ellaiah et al. 2002).
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Fig. 4 Response surfaces

showing the effect of the initial
concentrations of six nitrogen
sources (N) and KH2PO4 (P) on
TAA production by A. niger
UO-01 in SSF. Variables N and
P are in coded values

Table 5 Optimum N and P concentrations that provided the highest amylase (TAA) productions by A. niger UO-01 in the cultures performed
with six different nitrogen sources and KH2PO4, and estimated costs associated with the use of the optimum concentrations of each nitrogen
source and KH2PO4
Optimum values Estimated cost (€/ton bagasse)
N source [N] mg/gds [P] mg/gds [TAA] EU/gds

NH4CH3COO 10.03 9.84 378.88 785.20

(NH4)2HPO4 15.33 9.55 355.47 827.63
NaNO2 9.15 11.30 359.79 785.20
NaNO3 10.31 10.19 317.50 730.97
Yeast extract 11.59 9.83 457.82 1,208.49
(NH4)2SO4 12.98 8.79 326.48 662.23

The effect of initial moisture content on enzyme pro- solid particles (Ellaiah et al. 2002). Thus, higher moisture
duction in SSF has usually been related to the interference content could reduce the porosity of the solid substrate,
that this variable produces on the physical properties of the thus interfering with the oxygen transfer (Lonsane et al.

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World J Microbiol Biotechnol (2009) 25:1929–1939
Fig. 5 Profiles of TS (, in mg
of TS/gds) and TAA (h, in
EU/gds) production by A. niger
UO-01 in SSF at the optimized
(T, pH, M, IC, TS, N and P)
conditions. Means of three
analytical replications
1985). On the contrary, lower moisture levels can lead to a
reduction in the solubility of nutrients of the substrate, low
degree of swelling and high water tension (Zadrazil and
Brunnert 1981).
Inoculum concentration is other important factor that
influences the production of metabolites under SSF (Balkan
and Ertan 2007; Mazutti et al. 2007; Pandey 2003). An
inoculum concentration higher than the optimum value
may produce a high amount of biomass which rapidly
depletes the nutrients necessary for growth and product
synthesis (Baysal et al. 2003; Selvakumar and Pandey
1999). On the other hand, lower inoculum levels may give
insufficient biomass and allow the growth of undesirable
organisms in the production medium. This increases the
necessary time to grow to an optimum number to consume
the substrate and synthesise the desired product (Balkan
and Ertan 2007; Kashyap et al. 2002).
On the other hand, yeast extract was the best nitrogen
source for amylase production, probably due to its high
content in minerals, vitamins, coenzymes and nitrogen
components (Guerra and Pastrana 2002). Although the
optimal concentrations of nitrogen and phosphorous were
different for each nitrogen source used, at concentrations
higher than the optimal values both the nitrogen and
phosphorous sources caused inhibition of enzyme synthe-
sis. Since the liquid medium was initially buffered at pH
6.0 in the six experiments, this inhibition could be related
with the repression effect that the high concentrations of
1937
nitrogen (Abu and Ado 2004; Michelena and Castillo 1984)
and phosphorous (Michelena and Castillo 1984) produced
on amylase production. This repression effect has been
observed before for cellulose production by Aspergillus
terreus (Ali et al. 1991).
Some studies on the effect of nutrients (carbon, nitrogen
and phosphorous) on amylase production in both SSF and
SLF have been carried out using a fixed concentration of
both nutrients (Ellaiah et al. 2002; Francis et al. 2002;
Kunamneni et al. 2005). Although this approach allows the
selection of the best N and P sources, determination of their
optimal concentrations is not possible. Other studies based
on the optimization of N and P levels for amylase pro-
duction, used the medium optimization by the traditional
‘‘one variable at a time’’ technique (Kekos et al. 1987;
Kunamneni et al. 2005; Michelena and Castillo 1984).
However this method is time consuming and could lead to
an incomplete interpretation of the behaviour of the sys-
tem, resulting in a lack of predictive ability, mainly when
there are interactions between the independent variables. In
contrast, determination of the effects of N and P concen-
trations and their interaction on amylase production by
using central composite designs is an effective way to
obtain the best nitrogen and phosphate source as well as
their optimal concentrations. With this procedure, the cost
of amylase production in SSF could be reduced by
selecting the cheapest N and P source. With regard to this,
the optimum N and P values obtained in the present work
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1938
for high enzyme production by A. oryzae UO-01 were
different for each nitrogen source used (Table 5). This is an
important factor that must be considered for a large scale
production, because the amounts of nutrients added to the
culture media have a direct relation with the production
cost. Thus, in the present study, the estimated costs asso-
ciated with the use of the optimum amounts of each
nitrogen source and KH2PO4 were calculated by taking into
account the price of each nutrient. As it can be observed in
Table 5, the combined use of yeast extract and KH2PO4 for
amylase production gave the highest amylase levels but
also the highest cost. On the other hand, the use of
NH4CH3COO provided an optimum amylase concentration
1.2 times lower than yeast extract, but gave a cost 1.5 times
lower than that of yeast extract. These results suggest that
NH4CH3COO could be the most appropriate nitrogen
source for a low-cost amylase production by A. niger
UO-01 in solid-state cultivation with sugarcane bagasse.
Conclusions
The results obtained in this work showed that sugarcane
bagasse is a valuable support material for amylase pro-
duction and provided useful information and reference for
the optimization of medium composition for other SSF
processes with the same support material. Statistical opti-
mization method for fermentation process using response
surface methodology could overcome the limitations of
classic empirical methods and proved to be a powerful tool
for the optimization of amylase production by A. niger
UO-01 in SSF. On the other hand, validation experiments
showed that the predicted values by the empirical models
agreed with the experimental values well. These results
therefore proved the validity and corroborate the effec-
tiveness and reliability of the empirical models obtained.
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