Biochemical Engineering Journal 15 (2003) 107115

Use of response surface methodology for optimizing process parameters

for the production of -amylase by Aspergillus oryzae
Febe Francis
a,b
, Abdulhameed Sabu
a
, K. Madhavan Nampoothiri
a
,
Sumitra Ramachandran
a
, Sanjoy Ghosh
b
, George Szakacs
c
, Ashok Pandey
a,
a
Biotechnology Division, Regional Research Laboratory, CSIR, Trivandrum, Kerala 695019, India
b
Centre for Biotechnology, Anna University, Chennai 600025, India
c
Department of Agricultural Chemical Technology, Budapest University of Technology and Economics, 1111 Budapest, Gellerter 4, Hungary
Received 7 May 2002; accepted after revision 20 November 2002
Abstract
Optimization of three parameters (incubation temperature, initial substrate moisture and inoculum size) was attempted by using a
BoxBehnken design under the response surface methodology for the optimal production of -amylase by Aspergillus oryzae NRRL
6270 in solid-state fermentation (SSF). Spent brewing grains (SBG) was used as sole carbon source. The experimental data was tted
into a polynomial model for the yield of enzyme and an optimum level was arrived at which nutrient supplements were optimized. A
PlackettBurman design was employed to screen nineteen nutrient components for their inuence on -amylase production by the fungal
culture. Three components (soybean meal, calcium chloride and magnesium sulphate) were selected based on their positive inuence on
enzyme formation. A BoxBehnken design was employed to optimize their composition, which showed that an incubation temperature of
30

C, an initial moisture of 70% and an inoculum rate of 1 10

7
spores/g dry substrate were the best conditions to produce -amylase
with A. oryzae NRRL 6270 on SBG. Under optimized conditions of SSF, about 20% increase in enzyme yield was observed.
2002 Elsevier Science B.V. All rights reserved.
Keywords: -Amylase; Aspergillus oryzae; Spent brewing grains; Response surface methodology; Solid-state fermentation
1. Introduction
Large quantities of agricultural and agro-industrial
residues are generated round the year from diverse agricul-
tural and industrial practices. These residues represent one
of the most energy-rich resources on the planet [7]. They
are in fact, one of the best reservoirs of xed carbon in
nature. Utilization of such resources has been in the fron-
tier of industries practicing solid-state fermentation (SSF)
[9,1114,16]. Such resources are particularly attractive as
they provide an inexpensive industrial substrate; moreover
because, it offers elimination of large-scale accumulation of
biomass. Spent brewing grains (SBG) is one such residue
that has gained attention as a substrate for the production
of enzymes under SSF [18].
The amylase family of enzymes nds potential application
in a number of industrial processes especially in food, tex-
tiles and paper industries. -Amylases (endo-1,4--d-glucan

Corresponding author. Tel.: +91-471-251-52-79;

fax: +91-471-249-17-12.
E-mail address: pandey@csrrltrd.ren.nic.in (A. Pandey).
glucohydrolase EC 3.2.1.1) are extra-cellular enzymes
that randomly cleave the 1,4--d-glucosidic linkages be-
tween adjacent glucose units in the linear amylose chain.
-Amylase is secreted as a primary metabolite and its
production is reported to be growth associated [19,20].
The optimization of fermentation conditions, particu-
larly physical and chemical parameters are of primary
importance in the development of any fermentation process
owing to their impact on the economy and practicability of
the process. The diversity of combinatorial interactions of
medium components with the metabolism of the cells as
well as the large number of medium constituents necessary
for cell growth and production do not permit satisfac-
tory detailed modeling. The one-dimensional search with
successive variation in variables is still employed, even
though it is well accepted that it is practically impossible
for the one-dimensional search to achieve an appropriate
optimum in a nite number of experiments. Single vari-
able optimization methods are not only tedious, but also
can lead to misinterpretation of results, especially because
the interaction between different factors is overlooked
[24].
1369-703X/02/$ see front matter 2002 Elsevier Science B.V. All rights reserved.
PII: S1369- 703X( 02) 00192- 4
108 F. Francis et al. / Biochemical Engineering Journal 15 (2003) 107115
Statistical experimental designs have been in use for
several decades [2,17]. These experimental layouts can be
adopted at various phases of an optimization process, such
as for screening experiments or for nding the optimal con-
ditions for targeted results. Of late, the results analyzed by
a statistically planned experiment are better acknowledged
than those carried out by the traditional single variable
experiments. Some of the popular choices in statistical de-
sign includes the PlackettBurman design [22], the central
composite design [4,21,23], the BoxBehnken design [22]
and the GraecoLatin square design [5]. Response surface
methodology has by now been established as a convenient
method for developing optimum processes with precise
conditions and has also minimized the cost of produc-
tion of many a process with efcient screening of process
parameters [23].
1.1. Response surface methodology, the BoxBehnken
design
BoxBehnken designs [1] are response surface designs,
especially made to require only three levels, coded as 1,
0, and +1. They are formed by combining two-level facto-
rial designs with incomplete block designs. This procedure
creates designs with desirable statistical properties but, most
importantly, with only a fraction of the experiments required
for a three-level factorial. Because there are only three lev-
els, the quadratic model is appropriate. The coefcients of
the quadratic model may be calculated using standard re-
gression techniques. The present study employed this design
twice. At rst, it was used to study the interaction of signif-
icant physical parameters and later to nd the optimal com-
position of selected nutrient components screened using the
PlackettBurman design.
1.2. The PlackettBurman design
The PlackettBurman experimental design assumes that
there are no interactions between the different media con-
stituents, x
i
in the range of variables under consideration
[17]. A linear approach is considered to be sufcient for
screening.
Y =
0
+
i
x
i
(i = 1, . . . , k)
where Y is the estimated target function and
i
are the regres-
sion coefcients. The PlackettBurman experimental design
is a fractional factorial design and the main effect (the con-
trast coefcient) of such a design may be simply calculated
as the difference between the average of measurements made
at the high level (+1) of the factor and the average of mea-
surements at the low level (1).
The experience of safe commercial use of Aspergillus
oryzae for the production of -amylase is well established.
This reputation of A. oryzae is put to use in the develop-
ment of an appropriate bioprocess by utilizing SBG for the
production of -amylase under SSF in the present study.
Response surface methodology has been adopted here as a
tool to obtain best process conditions.
2. Materials and methods
2.1. Microorganism
A strain of A. oryzae NRRL 6270 was used for the
present study. It was maintained on potatodextroseagar
(Hi-Media, Bombay). Subculturing was carried out once in
every 3 weeks and culture was stored at 4

C.
2.2. Spent brewing grains (SBG)
SBG was used as the substrate. It was obtained from a
brewery in Budapest, Hungary.
2.3. Inoculum preparation
Spores of A. oryzae NRRL 6270 from 7 days old PDA
slants were dislodged into 10 ml sterile distilled water con-
taining 0.1% Tween-80. This spore suspension was used as
the master suspension, which was appropriately diluted for
the required density of spores. The number of viable spores
in the inoculum was determined by the pour plate count
technique.
2.4. Substrate preparation
Five grams of SBGwas weighed into a 250 ml Erlenmeyer
ask and to this a supplementing salt solution was added
to the desired moisture level. The composition of the salt
solution was as follows (in percentage (g/g) of dry substrate),
NH
4
NO
3
: 1; KH
2
PO
4
: 1; NaCl: 0.2; MgSO
4
7H
2
O: 0.2. The
contents were thoroughly mixed and autoclaved at 121

C
(15 psi) for 20 min.
2.5. Solid-state fermentation (SSF)
The sterilized solid substrate was inoculated with 1 ml
of inoculum. The contents were mixed thoroughly and in-
cubated at the appropriate temperature. Samples as whole
asks in duplicate, were withdrawn after 96 h of incubation.
Experiments were conducted according to the statistical de-
sign. Variations in the process parameters were maintained
according to the design.
2.6. Enzyme extraction
The crude enzyme from the fermented material was ex-
tracted by simple contact method. For this, the fermented
substrate was mixed thoroughly with distilled water contain-
ing 0.1% Tween-80, to a total extract volume amounts of
100 ml. Contents were mixed thoroughly by shaking for 1 h
at room temperature in a rotary shaker (Certomat, B. Braun
Biotech) at 150 rpm. At the end of the extraction, the suspen-
sion was centrifuged at 7000 rpm for 10 min (C-24 Cooling
F. Francis et al. / Biochemical Engineering Journal 15 (2003) 107115 109
Centrifuge, REMI) and the supernatant was collected and
used as the crude enzyme for further analysis.
2.7. -Amylase assay
-Amylase activity was determined as described by Okolo
et al. [8]. The reaction mixture consisted of 1.25 ml 1%(w/v)
soluble starch (Merck) solution, 0.25 ml, 0.1 M sodium ac-
etate buffer (pH 5.0), 0.25 ml of distilled water, and 0.25 ml
of properly diluted crude enzyme extract (10320). After
10 min of incubation at 50

C, the liberated reducing sugars

(glucose equivalents) were estimated by the dinitrosalicylic
acid method [6]. Appropriate blanks were used. One unit
(U) of -amylase is dened as the amount of enzyme releas-
ing 1 mol glucose equivalent per minute under the assay
conditions.
3. Results and discussion
3.1. Optimization of physical parameters
Temperature, initial moisture content of substrate and in-
oculum size were identied as most inuential among phys-
ical parameters for the production of -amylase on SBG.
A BoxBehnken design was employed to analyze the inter-
active effect of these parameters and to arrive at an opti-
mum. The base points for the design were selected from a
single-parameter study (data not shown). A summary of the
variables and their variation levels is given in Table 1.
SSF was carried out according to the design for 96 h.
The fermented samples were extracted and assayed for
-amylase activity and soluble protein contents. The results
were analyzed on a PC running under Windows OS, using
Design expert 6.0 (StatEase Inc., Minneapolis, USA) sta-
tistical software and the response surface generated using
STATISTICA (StatSoft Inc., Tulsa, USA).
The design and results of experiments carried out with the
BoxBehnken design are given in Table 2. The analysis of
variance (ANOVA) was employed (data not shown) for the
Table 1
Summary of variables for the BoxBehnken design for the optimization
of physical parameters
Factor Basic
level
Variation
interval
Value of
the
factor
Coded value
Temperature (

C) 30 5 25
30 0
35 +
Moisture content (%) 70 2 68
70 0
72 +
Log (number of spores) 6 1 5.5
6.5 0
7.5 +
Table 2
Experimental design (BoxBehnken) used to optimize the physical pa-
rameters for the production of -amylase on SBG by A. oryzae NRRL
6270
Medium
code
Temperature
(

C)
Moisture
content
(%)
Log (number
of spores)
-amylase activity
(U/g dry substrate)
A 25 68 6.5 5100
B 35 68 6.5 3801
C 25 72 6.5 4417
D 35 72 6.5 6523
E 25 70 5.5 4634
F 35 70 5.5 4651
G 25 70 7.5 5048
H 35 70 7.5 5335
I 30 68 5.5 2734
J 30 72 5.5 4100
K 30 68 7.5 5048
L 30 72 7.5 5990
M 30 70 6.5 6356
determination of Signicant parameters. ANOVA consists
of classifying and cross classifying statistical results and
testing whether the means of a specied classication differ
signicantly. This was carried by Fishers statistical test for
the analysis of variance. The F-value is the ratio of the mean
square due to regression to the mean square due to error
and indicates the inuence (signicance) of each controlled
factor on the tested model.
The model equation tted by regression analysis is given
by
Y =1, 048, 272 4883T 19T
2
+30, 346M
231M
2
+16, 521S 966S
2
+85TM
+14TS 53MS
where Y is the -amylase activity (U/g dry substrate), T the
temperature (

C), M the moisture content (% (w/w)) and

S the log
10
(spores/g dry substrate). The Model F-value of
11.44 implied that the model was signicant. There was
only a 0.2% chance that a Model F-value this large could
occur due to noise. Values of Probability > F less than
0.0500 indicated that model terms were signicant. In this
case the model terms M, S, M
2
, S
2
and TM were found
signicant. The model determination coefcient R
2
(0.9363)
suggested that the tted model could explain 93.63% of the
total variation. This implies a satisfactory representation of
the process by the model. The tted response surface for the
production of -amylase by the above model was generated
using STATISTICA and is given in Figs. 13.
Fig. 1 shows the effect of interaction of incubation tem-
perature and initial moisture content of substrate on the
production of -amylase. A decrease in production was ob-
served at temperatures outside the mesophilic range, which
was in good agreement to the fact that A. oryzae belonged to
mesophilic group. The contours were slightly inclined to the
horizontal showing that there was a signicant interaction
110 F. Francis et al. / Biochemical Engineering Journal 15 (2003) 107115
Fig. 1. Effect of incubation temperature and initial moisture on production of -amylase by A. oryzae on SBG.
between the two parameters. Asingle parameter study would
overlook this entity. An interaction of temperature and mois-
ture content was obvious as temperature was a factor that
inuenced humidity and water activity, which in turn was a
governing factor of the transport phenomena across cellular
membranes [16]. In SSF, during fermentation, there is a gen-
Fig. 2. Effect of incubation temperature and inoculum size on production of -amylase by A. oryzae on SBG.
eral increase in the temperature of the fermenting substrate
due to respiration [9,10]. However, these problems are gen-
erally encountered during the scale-up of SSF. In laboratory
studies using asks, no such difculty was noticed.
Fig. 2 depicts the interaction of moisture content and in-
oculum size. The contours were parallel to the two axes
F. Francis et al. / Biochemical Engineering Journal 15 (2003) 107115 111
Fig. 3. Effect of inoculum size and initial moisture on production of -amylase by A. oryzae on SBG.
suggesting that the two parameters were quite independent
of each-other. Fig. 3 shows a similar trend between inocu-
lum size and temperature. Age and size of the inoculum is
usually found to relate with time-course of incubation rather
than temperature or moisture content.
4. Optimization of nutrient supplements
A total of 19 components were analyzed for their effect
on -amylase production when supplemented to SBG us-
ing the PlackettBurman design. These included the four
compounds used in previous experiments (sectionsubstrate
preparation), as well as some organic and inorganic nitrogen
sources. A few metallic salts were also included. Table 3
shows the different levels of each of the constituents used
in the PlackettBurman design. Codes A to S were used
to designate each of the components. The comparison of
-amylase production in different media is given in Table 4
along with the design of the experiment. Codes 1 to 20
were used to designate the media supplementation to SBG in
each trial. The analysis showed that SBG supplemented with
medium 15 gave the maximum yield followed by medium
13 and medium 7. Corn steep liquor, soybean meal and
CaCl
2
were present in higher titres in these three media.
The analysis of the contrast coefcient (b) showed that soy-
bean meal, CaCl
2
and MgSO
4
7H
2
O had pronounced inu-
ence on the production of -amylase. The effect of Ca
2+
and Cl

ions on the stability of -amylase have been well

documented [3,15,22]. The organic nitrogen sources, such
as corn steep liquor and soybean meal have an edge over
Table 3
Nutrient supplements for screening using PlackettBurman design
Nutrient
code
Compound (+) Level (%)
(g/g dry substrate)
() Level (%)
(g/g dry substrate)
A NH
4
NO
3
0.2 0.1
B KH
2
PO
4
0.2 0.1
C NaCl 0.1 0.05
D MgSO
4
0.1 0.05
E Yeast extract 0.2 0.1
F Corn steep liquor 0.2 0.1
G Peptone 0.2 0.1
H Casein 0.2 0.1
I Soybean meal 0.2 0.1
J Urea 0.2 0.1
K NH
4
H
2
PO
4
0.2 0.1
L (NH
4
)
2
SO
4
0.2 0.1
M NH
4
Cl 0.2 0.1
N NaNO
3
0.2 0.1
O CaCl
2
0.1 0.05
P ZnCl
2
0.1 0.05
Q FeCl
3
0.1 0.05
R MnSO
4
0.1 0.05
S KCl 0.1 0.05
inorganic sources as they also have trace mineral and ions
that could enhance the production of the enzyme. The other
components were neglected and optimum combinations of
these three were further analyzed by a BoxBehnken design.
5. Optimization of nutrient supplements
Experiments were carried out in duplicates to arrive at
an optimum combination of the three nutrients using a
1
1
2
F
.
F
r
a
n
c
i
s
e
t
a
l
.
/
B
i
o
c
h
e
m
i
c
a
l
E
n
g
i
n
e
e
r
i
n
g
J
o
u
r
n
a
l
1
5
(
2
0
0
3
)
1
0
7

1
1
5
Table 4
Experimental design using PlackettBurman method for screening of nutrients
Medium
code
A B C D E F G H I J K L M N O P Q R S Activity
(U/g dry substrate) at 96 h
1 + + + + + + + + + + 6057
2 + + + + + + + + + + 7436
3 + + + + + + + + + + 4757
4 + + + + + + + + + + 1885
5 + + + + + + + + + + 4963
6 + + + + + + + + + + 3876
7 + + + + + + + + + + 8216
8 + + + + + + + + + + 4012
9 + + + + + + + + + + 5400
10 + + + + + + + + + + 3841
11 + + + + + + + + + + 4575
12 + + + + + + + + + + 5074
13 + + + + + + + + + + 8425
14 + + + + + + + + + + 6581
15 + + + + + + + + + + 8753
16 + + + + + + + + + + 5735
17 + + + + + + + + + + 2012
18 + + + + + + + + + + 2702
19 + + + + + + + + + + 1342
20 3806
b 307 203 511 1124 502 338 488 142 1972 555 129 1174 893 288 2206 210 596 1571 1132
F. Francis et al. / Biochemical Engineering Journal 15 (2003) 107115 113
Table 5
Composition (% (w/w)) of supplementing nutrients added to the substrate
Nutrient 0 +
Soybean meal 0 0.5 1
CaCl
2
0 0.1 0.2
MgSO
4
0 0.1 0.2
Table 6
BoxBehnken design for optimizing supplementation of SBG with nutri-
ents
Medium
code
Soybean
meal (%)
CaCl
2
(%)
MgSO
4
(%)
-Amylase activity
(U/g dry substrate)
A 0 0 0.1 62
B 1 0 0.1 4252
C 0 0.2 0.1 68
D 1 0.2 0.1 6410
E 0 0.1 0 75
F 1 0.1 0 4605
G 0 0.1 0.2 1348
H 1 0.1 0.2 5185
I 0.5 0 0 4719
J 0.5 0.2 0 3905
K 0.5 0 0.2 4691
L 0.5 0.2 0.2 3750
M 0.5 0.1 0.1 4285
BoxBehnken design. Table 5 gives the variation levels at
which these components were supplemented to SBG. Table 6
gives the design and results of experiments carried out by
the BoxBehnken design. The results obtained were sub-
Fig. 4. Effect of supplementation of SBG with soybean meal and CaCl
2
on production of -amylase by A. oryzae (with 0.1% supplement of MgSO
4
7H
2
O).
mitted to analysis of variance on Design expert 6.0 and the
regression model is given as
Y =452 +10, 095.25S 3311.25C +3272.5M
6100S
2
6200C
2
+4325M +10, 760SC
3465SM3175CM
where S is the concentration (%) of soybean meal, C that of
CaCl
2
and M MgSO
4
7H
2
O, respectively. A Model F-value
of 14.45 was observed and implied the model to be signi-
cant. There was only a 0.10% chance that a Model F-value
this large could occur due to noise. The analysis showed
that S and S
2
were signicant model terms. Hence, soy-
bean meal could be considered as to have the most effect
among the three nutrients studied. The value of the deter-
mination coefcient R
2
(0.9489) suggested that the tted
model could explain 94.89% of the total variation. The t-
ted response for the above regression model is plotted in
Figs. 46. Fig. 4 depicts the variation in enzyme production
with addition of soybean meal and CaCl
2
as supplements to
SBG. The yield showed a quadratic dependence on the con-
centration of soybean meal. However, increase in produc-
tion was only marginal with the addition of CaCl
2
. A slight
increase was observed at higher concentrations. A similar
observation can be made from Fig. 5 on the inuence of
soybean meal on enzyme production. However, with the in-
crease in MgSO
4
concentration, -amylase production was
not observed to vary much. Fig. 6 showed that the presence
of CaCl
2
had a better impact on -amylase production than
114 F. Francis et al. / Biochemical Engineering Journal 15 (2003) 107115
Fig. 5. Effect of supplementation of SBG with soybean meal and MgSO
4
7H
2
O on production of -amylase by A. oryzae (with 0.1% supplement of CaCl
2
).
MgSO
4
. The optimum composition of the components were
selected based on the signicant parameters; the analysis
showed only soybean meal to be signicant, however, CaCl
2
was also incorporated as its stabilizing effect on -amylase
Fig. 6. Effect of supplementation of SBG with CaCl
2
and MgSO
4
7H
2
O on production of -amylase by A. oryzae (with 0.5% supplement of soybean meal).
has been well-documented [3,22]. The optimum combina-
tion was found to be soybean meal: 1.0%, CaCl
2
: 0.2%
and MgSO
4
7H
2
O: 0.0% (w/w of dry substrate). The model
showed that MgSO
4
7H
2
O was not essential for -amylase
F. Francis et al. / Biochemical Engineering Journal 15 (2003) 107115 115
Table 7
Comparison between the original and optimized media
Nutrient
supplement
Composition (%)
(w/w of dry SBG)
Observed yield
(U/g dry substrate)
Original
medium
NH
4
NO
3
1.00 5464
KH
2
PO
4
1.00
NaCl 0.20
MgSO
4
7H
2
O 0.20
Optimized
medium
Soybean meal 1.00 6583
CaCl
2
0.20
production along with a nutrient source such as soybean
meal. Table 7 gives the comparison between the yield of
-amylase from original and optimized media under the ex-
perimental conditions. The increase in yield (6583 U/g dry
substrate) is noteworthy. Use of Spent brewing grains has
been beneciary, as the yield of -amylase has been high
in comparison to -amylase production reported under SSF
using other substrates, such as amaranthus grains [22].
6. Conclusion
Spent brewing grains (SBG) was found to be a good sub-
strate for the production of -amylase by lamentous fungi
under solid-state fermentation. Statistical analysis proved to
be a useful and powerful tool in developing optimum fer-
mentation conditions. The statistical analysis based on a
BoxBehnken design showed that an incubation tempera-
ture of 30

C, an initial moisture of 70% and an inoculum

of 1 10
7
spores/g dry substrate were the best conditions
to produce -amylase with A. oryzae NRRL 6270 on SBG.
Incorporation of most suitable conditions and supplements
to the SSF medium resulted about 20% increase in enzyme
yield.
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