Biochemical Engineering Journal 43 (2009) 149–156

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Biochemical Engineering Journal

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To study the influence of different components of fermentable substrates on

induction of extracellular ␣-amylase synthesis by Bacillus subtilis DM-03 in
solid-state fermentation and exploration of feasibility for inclusion of ␣-amylase
in laundry detergent formulations
Ashis K. Mukherjee a,∗ , Munindra Borah b , Sudhir K. Rai a
a
Department of Molecular Biology and Biotechnology, School of Science and Technology, Tezpur University, Tezpur 784 028, Assam, India
b
Department of Mathematical Sciences, School of Science and Technology, Tezpur University, Tezpur 784 028, Assam, India

a r t i c l e i n f o a b s t r a c t

Article history: Study on potential of different agro-industrial waste residues for supporting ␣-amylase production by a
Received 13 May 2008 thermophilic strain of Bacillus subtilis DM-03 in solid-state fermentation (SSF) was initiated and potato
Received in revised form peel was found to be the best substrate for ␣-amylase production. Linear multiple regression analysis
13 September 2008
showed that higher starch contents of the fermentable substrates enhanced the ␣-amylase synthesis
Accepted 20 September 2008
while the higher sugar content of the substrates imposed a negative effect presumably due to catabolic
repression of the enzyme synthesis. Furthermore, C/N ratio of the substrates also played a critical role
Keywords:
in induction of ␣-amylase synthesis. The result of batch fermentation showed 532 ± 5 U of ␣-amylase
Amylase
Bacillus subtilis
production per gram of dry substrate by B. subtilis DM-03 post-72 h of incubation under optimized SSF
Enzyme production condition. The crude ␣-amylase enzyme from B. subtilis DM-03 strain demonstrated excellent stability
Fermentation and compatibility with the laundry detergents that favors its inclusion in commercial laundry detergent
Linear regression formulations.
Solid-state fermentation © 2008 Elsevier B.V. All rights reserved.

1. Introduction
Amylases, which hydrolyze starch molecules to fine diverse
products such as dextrins, oligosaccharides and glucose molecules,
are among the most important hydrolytic enzymes having the
great industrial significance [1–4]. Alpha-amylases, hydrolyze ␣-
1,4 bonds of starch molecules, can be derived from several sources,
such as plants, animals and microorganisms however, it is rather the
enzymes from latter sources that generally meet industrial demand
[1,4]. Survey of literature shows that apart from starch hydroly-
sis, microbial ␣-amylases have found potential applications in the
fine chemicals, pharmaceutical, textile, food, bakery, and laundry
detergent industries [1–4].
Since the industrial use of alkaline amylases is expected tremen-
dously to grow in the coming decade therefore, during the recent
years, efforts have been directed to explore the means to reduce
the amylase production costs through improving the yield, and the
use of either cost-free or low-cost agricultural byproducts as sub-
∗ Corresponding author. Tel.: +91 3712 267007/8/9x5401;
mobile: +91 9957 184351; fax: +91 3712 267005/6.
E-mail address: akm@tezu.ernet.in (A.K. Mukherjee).
strate(s) for ␣-amylase production [5–8]. Over the past couple of
years, solid-state fermentation (SSF) involving growth of microbes
on moist solid substrate(s) in the absence of free-flowing water,
has gained a tremendous momentum owing to certain advantages
over the conventional submerged fermentation (SmF), like low
production cost, saving of water and energy, less waste effluent
problem and stability of the product due to less dilution in the
medium [9–12]. Besides, SSF technology has a tremendous scope
for the employment generation particularly in developing coun-
tries [13]. Furthermore, it has been reported that wild-type strains
of microorganisms (fungi or bacteria) have a tendency to do bet-
ter performance in SSF system compared to genetically altered
microorganisms, and thus reducing the energy and cost require-
ment even further [14].
In the recent past, many agro-industrial byproducts such as
wheat bran, rice bran, molasses bran, barley bran, maize meal, soy-
bean meal, potato peel, coconut oil cake, etc. have been screened
as low-cost solid substrates for microbial production of ␣-amylase
in SSF [5–8]. A perusal of literature shows that some of the solid
substrates possess higher potential as compared to the other solid
substrates for supporting/inducing the ␣-amylase (or even other
enzymes) production by microbes. For example, wheat bran and

1369-703X/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2008.09.011
150 A.K. Mukherjee et al. / Biochemical Engineering Journal 43 (2009) 149–156
potato peel are reported to be superior as compared to the other
tested solid substrates for optimum ␣-amylase production by many
microorganisms under the identical SSF condition [6,8,15]. How-
ever, the exact reason(s) for such a higher enzyme production by
microbes while growing on some specific substrates has never been
explored and till date no logical explanation has been provided for
the above observed effect.
It is well documented that the production of thermostable ␣-
amylases in solid culture is mostly limited to Bacillus and many
species of Bacillus such as B. subtilis, B. polymyxia, B. vulgarus, B.
megaterium, B. coagulans and B. licheniformis have been reported
to produce ␣-amylase [6,8,15–17]. Our previous study has also
shown that Bacillus subtilis DM-03 isolated from a traditional fer-
mented food secretes significant amount of extracellular alkaline
␣-amylase in SmF system using starch as a substrate [17] and
therefore, we selected this strain for ␣-amylase production study
by using various low-cost solid substrates. The first objective of
the present study was to explore which component(s) of the fer-
mentable solid substrates influences the ␣-amylase synthesis by B.
subtilis DM-03 strain in SSF. To achieve this goal, we have screened
a variety of agro-industrial waste residues as substrates for ␣-
amylase production by B. subtilis DM-03 in SSF and analyzed the
proximate composition of each substrate. The second objective was
optimization of fermentation conditions with respect to moisture
content of the best fermentable material, moist agents, and sup-
plementation of co-carbon and co-nitrogen sources for alkaline
␣-amylase production in SSF. Last, suitability of the B. subtilis DM-03
␣-amylase for application in laundry detergents was investigated.
2. Materials and methods
2.1. Microorganism
A B. subtilis strain DM-03 previously isolated from the fer-
mented food was shown to produce significant amount of alkaline
␣-amylase in the culture medium under thermophilic growth con-
dition [17]. The bacteria were sub-cultured on nutrient-agar plates
before use as inoculum for ␣-amylase production under SSF condi-
tion.
2.2. Preparation of substrates
Procedure for the preparation of different agro-industrial waste
materials, viz. mustered oil cake (MOC), wheat bran (WB), rice bran
(RB) Imperata cylindrica (IC) grass and banana leaves (BL) for use in
SSF has been described elsewhere [11]. Potato peel was prepared
by our previously elucidated procedure [12].
2.3. Solid-state fermentation and optimization of process
conditions
Initially 5.0 g of coarse solid substrates were taken in 500 ml
Erlenmeyer flasks and 2.5 ml of 100 mM K-phosphate buffer, pH 8.0
was added, mixed thoroughly and autoclaved at 121 ◦ C, 15 lbs pres-
sure for 15 min. The flasks were cooled to room temperature and
then inoculated with 2.0 ml of 24-h grown bacterial culture (OD at
600 nm 0.50 ± 0.01) under sterile conditions and incubated at 50 ◦ C
under static condition for various time periods (24, 36, 48, 72, 96
and 120 h). To investigate the influence of other culture parame-
ters on ␣-amylase production, effects of initial moisture content of
the substrate (25%, 50%, 100%, 150%, 200% and 300%, v/w), different
moistening liquids (0.1 M K-phosphate buffer, pH 8.0, 0.1N Tris–HCl,
pH 8.0, 0.001N NaOH, pH 11.0, distilled water with pH adjusted to
6.0–10.0 with 0.1 M HCl or 0.1N NaOH for decreasing or increasing
the pH of water), inoculum size (10, 20, 50, 100, 150 and 300%, v/w),
co-carbon sources (glucose, fructose, galactose, lactose, maltose,
and starch at 10.0%, w/w) and co-nitrogen sources (NH4 Cl, NH4 NO3 ,
(NH4 )2 SO4 , yeast extract, beef extract, casein and peptone at 1.0%,
w/w) were studied. For initial moisture content, solid substrate was
mixed with predetermined amount of moistening liquid and 100%
moisterization was achieved by adding 1.0 ml water to 1.0 g sub-
strate and vice versa [18]. Un-inoculated flasks served as negative
controls whereas the inoculated flasks without any co-carbon or
co-nitrogen source served as positive control. Alpha-amylase pro-
duction was expressed as mean and standard deviations based on
the results obtained with triplicate flasks.
2.4. Extraction of ˛-amylase from the fermented matter
For isolation of ␣-amylase produced in SSF, a known quantity of
fermented matter (PP) was mixed with distilled water (1:5, w/v)
by stirring on a magnetic stirrer for 30 min at room temperature
(∼25 ◦ C). The slurry was then squeezed through a cheese cloth fol-
lowed by centrifuging the whole content at 10,000 × g for 10 min
at 4 ◦ C to remove the insoluble matters. The clear supernatant
was used for the ␣-amylase assay and the amylase recovery was
expressed as total units (U) of crude ␣-amylase obtained per gram
of dry substrate (gds).
2.5. Batch fermentation under SSF
100 g of substrates mixture (90% PP and 10% starch, w/w)
was taken in rectangular trays of approximate dimension
286 mm × 120 mm × 80 mm mixed thoroughly with 100 ml of dis-
tilled water (pH was adjusted to 8.5 with 0.1N NaOH). The
moistened powder was spread evenly on the tray in layers, typi-
cally between 0.5 and 1.0 cm deep in tray with an open top. The
tray was then closed with aluminum foil followed by autoclav-
ing at 15 lbs pressure and at 121 ◦ C temperature for 15 min, cooled
to room temperature and inoculated with B. subtilis DM-03 strain
(optical density 0.5 ± 0.1 at 600 nm), mixed well and the tray was
re-covered with aluminum foil. The operation was carried out
aseptically under a laminar flow. The tray was incubated at 50 ◦ C
temperature for 72 h under static condition, the fermented matter
was transferred to a 500 ml glass beaker and to this, 250 ml of dis-
tilled water (pH adjusted to 8.5) was added. The enzyme extraction
and assay were carried out as described previously.
2.6. Alpha-amylase assay
Amylolytic activity was assayed by measuring the reducing sug-
ars released during the reaction, using starch as the substrate [19].
One unit of enzyme activity was defined as the liberation of reduc-
ing sugars equivalent to 1 ␮mol of d-glucose/min under the assay
condition.
2.7. Proximate analysis
Total nitrogen, crude protein, lipid and carbohydrate, fiber,
nitrogen-free extract (NFE) and ash content of substrates were
determined by the methods described by Kalita et al. [20], whereas
the carbon content was calculated as described by Mukherjee et al.
[11]. For determining the specific activity of the ␣-amylase, protein
content of the cell-free supernatant was estimated as described by
Lowry et al. [21]. For the isolation and estimation of starch con-
tent of the substrates, the procedure described by Sawhney and
Singh [22] was followed whereas the reducing sugar contents was
determined by using 3,5-dinitrosalicyclic acid reagent [19].
 A.K. Mukherjee et al. / Biochemical Engineering Journal 43 (2009) 149–156 151

2.8. Compatibility test with various commercial laundry

detergents

In order to confirm the potential of B. subtilis DM-03 ␣-amylase

as a laundry detergent additive, its compatibility and stability
towards some commercial laundry detergents available in local
market such as Surf excel® , Rin advanced® , and Wheel® (Hindus-
tan Lever Ltd., India), Tide® and Ariel® (Procter & Gamble, India),
Henko® (Henkel India Ltd.), and Nirma® (Nirma Ltd., India) were
examined. Aqueous solutions of detergents (7.0 g l−1 to stimulate
washing condition) were heated at 100 ◦ C for 90 min to denature
the indigenous enzyme activity (if any), and this was confirmed by
␣-amylase assay. Then detergent and the crude ␣-amylase enzyme
were mixed in a ratio of 1:1 (v/v), incubated at 45 ◦ C for 1 h, followed
by measuring the residual ␣-amylase activity by standard assay
procedure and compared with the control (enzyme diluted to 1:1
in tap water without detergent). The relative activity of ␣-amylase
in presence of detergent was expressed in percentage activity con-
Fig. 1. Screening of different waste residues such as MOC (), WB (), RB (), IC (×),
sidering the activity of control as 100%. PP ( ), BL (䊉), and TL () for the production of alkaline ␣-amylase by Bacillus subtilis
DM-03 strain at different time intervals. The pH of the moistening liquid used was
0.1 M K-phosphate buffer, pH 8.0. Values are mean ± S.D. of five experiments.
2.9. Statistical analysis

The influence of different components of fermentable substrates 3. Results and discussion

on induction of extracellular ␣-amylase synthesis by B. subtilis DM-
03 in SSF was evaluated by statistical analyses. The design variables
were starch, reducing sugar and protein contents as well as C/N ratio
of substrates while the response variable was enzyme (␣-amylase)
yield (U/gds). The experimental data were fitted according to the
different linear regression models (Eqs. (1)–(6)) including individ-
ual and cross-effect of each variable:
model 1, y = a + bx1 + cx2
model 2, y = a + bx1 + cx2 + dx1 x2
√ been reported by many authors for microbial ␣-amylase synthesis
model 3, y = a + bx1 + cx2 + d x1 x2
model 4, y = a + bx1 + cx2 + dx3
model 5, y = a + bx1 + cx2 + dx3 + ex4
model 6, y = a + bx1 + cx2 + dx4
In all these models, x1 –x4 are variables (starch, reducing sugar,
protein contents and C/N ratio, respectively), Y is the predicted
response (predicted enzyme yield), a is the intercept term, b, c,
d and e denotes the measures of the effects (regression coeffi-
cients) of variables. A computer programme was developed by us
in FORTRAN-77 for fitting the multiple linear regression models
using the method of least squares [23]. The regression coefficients
of the linear regression models have been estimated and the sig-
nificance of the coefficients is tested by using Student’s t-test [24].
The acceptability of each model or satisfactory model adequacy was
evaluated by 2 (Chi-square goodness of fit)-test, p-value analysis
and the coefficient of multiple determination R2 [23]. The effective
size for multiple regression (f2 ) value was determined by using Sim-
ple Interactive Statistical Analysis Software (http://statpages.org).
The ranking of the models was decided based on R2 and f2 values.
The model showing the highest values of R2 and f2 was ranked first
and the model showing next values of these statistical parameters
was ranked second and so on [23].
All the results are represented as mean ± S.D. of at least three
experiments. A probability level of p < 0.05 was considered statisti-
cally significant [23].
3.1. Screening of different waste materials for alkaline ˛-amylase
production
It has been well documented that Bacillus species served as
the most important sources of ␣-amylase and either SmF or SSF
system is employed for the production of this enzyme by many
species of Bacillus. However, many of the scientists vouch for the
(1) SSF process as potential tool for achieving economy in ␣-amylase
production and starch hydrolysis [6,7]. As shown in Fig. 1, produc-
(2) tion of ␣-amylase by B. subtilis DM-03 varied with the types of the
substrates and also dependent on incubation time, a fact that has
(3)
[6,7,8,16,25]. Present study showed that potato peel (PP) followed
(4) by wheat bran (WB) supported maximum ␣-amylase productions
post-72 h and 48 h of incubation, respectively documenting these
(5) substrates influenced the ␣-amylase synthesis machinery in bac-
teria (Fig. 1). Potato peel was found to be a superior substrate for
(6) the production of ␣-amylase in SSF by B. subtilis and B. licheniformis
[8] and biosurfactant by B. subtilis DM-04 strain in both SmF and
SSF conditions [12]. Minimum ␣-amylase production (3.1 U/gds)
was observed when waste TL was used as a fermentable sub-
strate/support material (Fig. 1). Due to the superior potential of
PP in inducing the ␣-amylase production by B. subtilis DM-03 in
SSF, this substrate was selected for further study (optimization of
process parameters).
3.2. Proximate composition analysis and multiple regression
analysis to determine the influence of components of substrates
on ˛-amylase production
It has been observed that the nature of the solid substrate is
one of the most important factor for enzyme production in SSF and
therefore, the chemical composition of fermentable substrate(s)
should be given a due consideration in SSF technology [17,26].
The proximate compositions of substrates used as fermentable
materials in the present study are displayed in Table 1. The carbon-
to-nitrogen ratio was found to be lowest for MOC followed by PP
whereas highest total carbohydrate as well as starch contents was
observed in PP. Banana leaves and MOC were characterized to pos-
sess highest sugar and protein contents, respectively. It is to be
152 A.K. Mukherjee et al. / Biochemical Engineering Journal 43 (2009) 149–156

Table 1
Proximate composition analysis of various agro-industrial wastes used as fermentable substrates for ␣-amylase production by Bacillus subtilis DM-03. Values are mean ± S.D.
of triplicate determinations.

Substrate C content (g%) N content (g%) C:N Total carbohydrate (g%) Starch (g%) Sugar (g%) Protein (g%) Sugar:starch

Mustured oil cake 52.9 ± 2.1 5.8 ± 2.5 9.1 ± 1.2 22.8 ± 1.1 6.1 ± 1.1 1.3 ± 0.3 36.3 ± 2.1 0.21 ± 0.1
Wheat bran 51.6 ± 1.9 2.1 ± 0.8 24.6 ± 1.7 34.0 ± 2.4 20.0 ± 1.6 0.6 ± 0.1 13.1 ± 1.1 0.03 ± 0.01
Rice bran 50.2 ± 2.2 2.3 ± 1.0 21.3 ± 2.3 36.0 ± 2.1 12.1 ± 1.4 1.8 ± 0.6 14.4 ± 1.2 0.2 ± 0.02
Imperata cylindrica 54.7 ± 3.1 1.2 ± 0.4 45.6 ± 1.5 15.7 ± 1.8 2.1 ± 0.5 1.0 ± 0.2 7.5 ± 1.0 0.5 ± 0.02
Potato peel 51.7 ± 1.8 2.6 ± 0.9 19.9 ± 1.2 62.2 ± 3.3 49.3 ± 2.3 2.6 ± 1.0 16.3 ± 2.1 0.04 ± 0.01
Banana leave 48.3 ± 2.2 1.2 ± 0.4 40.3 ± 1.0 51.4 ± 2.5 12.1 ± 1.6 27.5 ± 1.3 7.5 ± 1.8 2.3 ± 0.2
Used tea leaves 52.2 ± 1.1 1.4 ± 37.2 ± 2.1 1.0 ± 0.2 0 0 0 0

noted that components such as starch, sugar and protein could not
be detected in tea leaves (Table 1).
The observed and predicted values for ␣-amylase production
by B. subtilis DM-03 based on various linear regression models are
shown in Table 2a, while the Chi-square test, p-value, Student’s
t-value, the effective size for multiple regressions (f2 ) and coeffi-
cient of multiple determinations (R2 ) for each model are shown in
Table 2b. Since tea leaves were devoid of starch, sugar and protein
contents therefore, the marginal amount of ␣-amylase synthesized
by B. subtilis DM-03 while growing on tea leaves may be consid-
ered as constitutive enzyme. In order to calculate the yield (U/gds)
of ␣-amylase in presence of starch, sugar and protein contents of
the substrates (inducible ␣-amylase), the value of enzyme yield
(3.1 U/gds) by B. subtilis DM-03 while growing on tea leaves in SSF
was subtracted from the highest values of ␣-amylase production by
B. subtilis DM-03 on other fermentable substrates under identical
conditions (Table 2a). The statistical analysis (p-value) shows that
predicted values for ␣-amylase production by all the models are
in close agreement with the experimental (observed) values for ␣-
amylase production and there is no significant difference between
two sets of data within 95% confidence limit.
The sample coefficient of determination (R2 ) measures the
closeness of fit of the sample regression equation to the observed
value of y [24]. When R2 is large, then, the regression has accounted
for a large proportion of the total variability in the observed value
of y which favors the regression equation model. However, the
value of multiple coefficient of determination, R2 , should always be
between 0 and 1. The closer the value of the R2 to 1.0, the stronger is
the model and better it predicts the response [23,24]. The highest
value of R2 (0.9984) was determined for the model 3 followed by
model 5 (R2 = 0.9979) which implied that these two fitted models
could explain 99.84% and 99.79%, respectively of the total variation
(Table 2b). Identically, the highest value for f2 (the effective size for
multiple regression) which is a measure of the strength between
two variables, was calculated for model 3 (624.0) followed by model
5 (475.19) reinforcing the highest acceptability of these two models
compared to the other proposed models. The significance of each
model was further evaluated by Student’s t-test and p-values, and
the larger magnitude of the t-value and smallest p-value vouches
for the significance of the corresponding coefficient of each model.
The Chi-square distribution is another important theoretical
distribution that is used to compare the agreement between the
observed and expected (predicted) frequency distribution and
therefore, 2 is a measure of the extent to which in a given situation,
pairs of observed and expected frequencies agree [24]. When there
is a close agreement between observed and expected frequencies
2 is small, and when the agreement is poor 2 is large [24]. The
Chi-square goodness-of-fit test of the predicted models (Table 2b)
reinforces that model 3 having the least 2 value is the best model
followed by model 5 showing the second lowest value of 2 .
In the multiple regression model we assume that a linear rela-
tionship exists between some variable Y (dependent variable) and
n independent variables x1 , x2 , x, . . ., xn [24]. If in the population the
relationship between x and y is linear, ˇ, the slope of the line that
describes this relationship, will be either positive, negative, or zero.
Based on the result obtained with the multiple regression analysis,
starch and glucose contents of the substrates were found to be the
major variables in induction of ␣-amylase synthesis by B. subtilis
DM-03 in SSF condition. The best fitted overall second-order poly-
nomial equation (rank 1) (Table 2b) for ␣-amylase production can
be well represented by model 3 (see Eq. (3)) as
√
Y = 53.390 + 6.2595x1 − 9.6750x2 + 11.6092 x1 x2 . . . (7)
According to the best fitted model 3 (rank 1), a negative regres-
sion correlation observed between the enzyme yield and sugar
content of substrates explains the reason for declining of ␣-amylase
production with corresponding increase in the sugar content of
the substrates, whereas a positive regression correlation between
enzyme production and starch content of the substrates advocates
that starch acts as an inducer of ␣-amylase synthesis by B. subtilis
DM-03 strain. However, the regression coefficients for ␣-amylase
synthesis as calculated by various linear regression models show
that the geometrical mean of x1 and x2 interaction is positive imply-
ing interaction between starch and sugar has a positive impact
on ␣-amylase production (Table 3). Prediction of ␣-amylase syn-
thesis based on second-order polynomial regression equations did
not produce satisfactory results (observed and predicted values
were significantly different) and therefore, the models based on
second-order polynomial regression equations were not taken into
consideration for evaluating the impact of cross-effects of differ-

Table 2a
The observed and predicted values for ␣-amylase production in SSF based on various models. The observed ␣-amylase yieldsa are mean ± S.D. of triplicate determinations.

Maximum ␣-amylase yielda (U/gds) Predicted ␣-amylase yield (U/gds)

Model 1 Model 2 Model 3 Model 4 Model 5 Model 6

106.9 ± 3.2 [MOC] 107.9 108.0 111.7 110.1 107.2 113.5

217.9 ± 11.2 [WB] 226.5 226.3 213.0 226.0 230.4 226.0
174.9 ± 10.9 [RB] 156.6 156.6 165.9 156.3 166.9 158.0
67.9 ± 4.3 [IC] 75.5 75.6 73.7 74.1 65.7 69.7
464.9 ± 20.1 [PP] 465.3 465.4 468.3 465.4 461.7 464.7
74.9 ± 3.8 [BL] 75.5 75.6 74.8 75.5 75.5 75.5
a
Inducible ␣-amylase; within square bracket the fermentable substrate is indicated.
 A.K. Mukherjee et al. / Biochemical Engineering Journal 43 (2009) 149–156 153

Table 2b
Statistical analyses of predicted models for satisfactory model adequacy.

Parameters Values

Model 1 Model 2 Model 3 Model 4 Model 5 Model 6

2 3.2388 3.2590 1.2861 3.1224 1.1735 2.5454

p 0.5187a 0.3534a 0.7324a 0.3731a 0.5561a 0.4671a
Student’s t 0.7068 1.0954 0.3753 1.0443 0.7006 0.8306
f2 237.09 237.09 624.00 242.90 475.19 276.77
R2 0.9958 0.9958 0.9984 0.9959 0.9979 0.9964
Rank 5 5 1 4 2 3
a
The difference between observed and predicted values of ␣-amylase synthesis is not statistically significant.

ent components of substrates on ␣-amylase production (data not
shown).
The regression models presented in the present study are well
suited to explain the differential ␣-amylase production by same
bacterium while growing on different solid substrates of having
different chemical compositions. Among the tested solid wastes
PP and WB possessing the highest and second highest starch con-
tents, respectively (Table 1) supported the maximum and second
best ␣-amylase production. Similarly, although the RB and BL pos-
sess identical amount of starch (12.1 g%); however, the higher sugar
content of the BL (27.5 g%) as compared to RB (1.8 g%) repressed the
␣-amylase synthesis by B. subtilis DM-03 strain when grown on
banana leaves. Therefore, it might be reasonable to assume that
sugars such as glucose and fructose caused a catabolic repression
of ␣-amylase synthesis, which was just to prevent the wastage of
energy by synthesizing extracellular polysaccharide hydrolyzing
enzymes such as ␣-amylase when sugars required for the microbial
growth were readily available in the fermentable solid substrate
[17].
Interestingly the model 5, which was considered as the second
best fitted model describing the roles played by protein and C/N
ratio besides starch and sugar contents of the substrates in induc-
tion of ␣-amylase synthesis by bacteria, showed that the former two
variables (protein and C/N ratio of the substrates) produced nega-
tive regression coefficient with respect to x2 , x3 and x4 (Table 3). This
implies that both higher protein content as well as C/N ratio of the
solid fermentable substrates had a negative impact on ␣-amylase
production by B. subtilis DM-03 strain in SSF condition. This is accor-
dance to our previous report demonstrating influence of the C/N
ratio of fermenting material in the protease production by B. subtilis
DM-04 in SSF and it was observed that an optimum C/N ratio sup-
ported maximum protease yield [11]. Therefore, a higher C/N ratio
of the substrate is detrimental to the ␣-amylase synthesis by B. sub-
tilis DM-03 and present study suggests that for selecting a suitable
substrate for microbial ␣-amylase production in SSF, the C/N ratio
of the fermentable substrates should be given a due consideration.
Identically, a higher protein concentration exerted a negative effect
on ␣-amylase production by B. subtilis DM-03 strain in SSF condi-
tion, probably by shifting the C/N ratio of the substrate from the
ideal condition that favors the induction of ␣-amylase synthesis.
3.3. Effect of initial moisture content and moistening liquids on
alkaline ˛-amylase production
A major critical factor in the SSF system that influences the
enzyme yield is the initial moisture content of the substrate
[5,6,7,11], because growth of microbes and product (enzyme) for-
mation takes place at or near the surface of moist solid substrate [1].
Since the requirement of moisture content may vary from microbe
to microbe therefore, optimization of the moisture content that
controls the water activity (Wa) of the fermenting substrate is the
most crucial step for achieving maximum yield of the desirable
product.
In the present study, with an increase in the initial moisture
content of the substrate (PP) from 25% to 100% alkaline ␣-amylase
production by B. subtilis DM-03 was concomitantly enhanced from
319 to 484 U/gds and further increase in the moisture content of
substrate resulted in a steady decline in enzyme yield (Fig. 2).
Shukla and Kar [8] reported that optimum ␣-amylase yield was
observed at 80% and 70% (v/w) moisture contents of PP when B. sub-
tilis and B. licheniformis, respectively were grown on this substrate.
Similarly, 150% (v/w) moisture content of wheat bran induced the
maximum ␣-amylase produced by Bacillus sp. PS-7 [17]. There-
fore, it may be inferred that different species or (strain) of Bacillus
may have a different requirement of moisture level. It has been
suggested that lower moisture content causes a reduction of the
solubility of the nutrients of the solid support resulting in a lower
degree of swelling and higher water tension [16]. The moisture
contents beyond the optimum level decrease the porosity as well
as change the structure of the particles of the fermentable solid
substrate that subsequently promotes development of stickiness,
reduce gas volume and exchange and lower oxygen transfer which
in turn interfere with the microbial activity [6,11,16–18].
Depending upon the nature of producing microorganisms, the
type and pH of the moistening liquid also plays a crucial role in
enzyme synthesis. Among the different moistening liquids tested
for alkaline ␣-amylase production by B. subtilis DM-03, the distilled
water or tap water adjusted between pH 8.5 and 9.0 was found to
be most efficient moistening liquid for optimum ␣-amylase pro-
duction in SSF using PP as substrate (470 ± 20.1 U/gds) compared
to used in the present study as moistening agent for ␣-amylase

Table 3
Regression coefficients for ␣-amylase production as calculated by various linear regression models.

Models Regression coefficient of independent variables

√
Intercept (a) x1 x2 x1 x2 x1 x2 x3 x4

Model no. 1 61.063 8.3657 −3.1543 NA NA NA NA

Model no. 2 61.387 8.3356 −3.3086 0.0129 NA NA NA
Model no. 3 53.399 6.2595 −9.6750 NA 11.6092 NA NA
Model no. 4 59.0571 8.3692 −3.1139 NA NA 0.1140 NA
Model no. 5 127.1459 8.0039 −2.8385 NA NA −1.4206 −1.4828
Model no. 6 69.668 8.2980 −2.9757 NA NA NA −0.3169

NA: not applicable.

154 A.K. Mukherjee et al. / Biochemical Engineering Journal 43 (2009) 149–156
Fig. 2. Influence of initial moisture content of the substrate (PP ()) on alkaline
␣-amylase production by B. subtilis DM-03. Values are mean ± S.D. of triplicate
determinations.
production (371 ± 18.3 U/gds) by B. subtilis DM-03 under identical
condition. Similar reports are available on the use of distilled water
or tap water as moistening liquid in SSF processes [11,16,17]. It might
be reasonable to assume that high salt concentration of the buffers
may have a negative impact on ␣-amylase production by B. subtilis
DM-03. However, the distilled water is devoid of salts and the tap
water used in the present study contains negligible salts thus favors
the amylase synthesis.
3.4. Effect of inoculum size on ˛-amylase production
There is a great influence of inoculum’s concentration in enzyme
production and therefore, this parameter should be given a proper
consideration for optimum level of ␣-amylase production. As
shown in Fig. 3, with an increase in inoculum concentration from
10 to 100% (v/w) ␣-amylase production by B. subtilis DM-03 was
enhanced concomitantly and increase in the inoculum size beyond
100% resulted in a steady decline in ␣-amylase production by B. sub-
tilis DM-03 which might be due to exhaustion of nutrients in the
fermentation mash. In contrast, the lower inoculum size requires
longer time for the cells to multiply to sufficient number for the
maximum substrate utilization resulting in lower enzyme yield
[7,11].
Fig. 3. Influence of inoculum size on alkaline ␣-amylase production by B. subtilis
DM-03 strain using PP () as a substrate in SSF. Values are mean ± S.D. of triplicate
determinations.
Fig. 4. Effect of supplementation of co-carbon source (10%, w/w) to PP (90%, w/w)
on alkaline ␣-amylase production by B. subtilis DM-03 in SSF. Values are mean ± S.D.
of three determinations.
3.5. Effect of supplementation of co-carbon and co-nitrogen
sources on ˛-amylase production
The choice of the nitrogen and carbon sources has a major
influence on the yield of ␣-amylase enzyme. As shown in Fig. 4,
␣-amylase production by B. subtilis DM-03 strain was enhanced
significantly (p < 0.05) when starch (10%, w/w) was supplemented
as co-carbon source to the PP (90%, w/w) fermentation mash. This
finding is in accordance with the reports from many other labora-
tories where starch was found to be the best inducer of ␣-amylase
synthesis in many bacteria as well as fungi [7,27]. It was observed
that the ␣-amylase production in the present study was growth
associated and it could be possible that the carbon of the starch
represented the energetic source to stimulate the growth of the
microorganism [7]. However, supplementation of other co-carbon
sources (sugars) resulted in suppression of ␣-amylase produc-
tion by B. subtilis DM-03 strain which might be due to catabolic
repression of ␣-amylase synthesis [17]. In a sharp contrast to this
observation, many of the authors have reported either absence or a
minor catabolic repression of ␣-amylase synthesis by microbes in
SSF in presence of glucose [7,16]. Therefore, it might be reasonable
to assume that the effect of glucose on induction of ␣-amylase syn-
thesis is dependent on the microbial species. Addition of maltose to
PP also resulted in decrease in ␣-amylase production by B. subtilis
DM-03 (Fig. 4) which reinforces the previous reports on ␣-amylase
production by microbes in presence of maltose [7,28].
However, none of the tested co-nitrogen sources were found to
enhance the ␣-amylase production by B. subtilis DM-03 strain in SSF
using PP as a substrate (data not shown), although we have reported
that in SmF using 10% (w/v) starch as a substrate (in M9 medium),
supply of 0.1% (w/v) NH4 Cl as co-nitrogen source enhanced the ␣-
amylase production by the same strain [9]. Therefore, it appears
that nitrogen sources present in PP are sufficient and this substrate
does not require additional supplementation of any co-nitrogen
source for extra ␣-amylase production by B. subtilis DM-03. This
is in accordance with our previous study where we have shown
that carbon and nitrogen sources present in Imperata cylindrica and
PP can serve as complete basal and standardized medium and does
not require supplementation of any co-nitrogen/carbon source for
protease production by B. subtilis DM-04 [11].
3.6. Batch fermentation
The result of batch fermentation showed 532 ± 5 U of ␣-amylase
production per gds post-72 h of incubation at 50 ◦ C temperature
 A.K. Mukherjee et al. / Biochemical Engineering Journal 43 (2009) 149–156
Fig. 5. Stability and compatibility of crude ␣-amylase from B. subtilis DM-03 strain
against commercial laundry detergents at pre-incubation temperature 37 ◦ C ()
and 45 ◦ C (). A control was run without detergents and experimental values were
compared with that. Values are means ± S.D. of three determinations.
and under optimized SSF condition (90%, w/w PP, 10%, w/w starch,
initial moisture content 100% (v/w), pH of distilled water was
adjusted to 8.5).
3.7. Compatibility of ˛-amylase with commercial laundry
detergents
All the commercial detergents contain hydrolytic enzymes and
these enzyme-based detergents known as “green chemicals” find
a wide range of applications in laundry, dishwashing, textile and
other related industries [2,4]. The amylases are used in the deter-
gents to degrade the residues of starchy foods such as potatoes,
gravies, custard, chocolate, etc. to dextrins and other smaller
oligosaccharides. The suitability of any hydrolytic enzymes for
inclusion in detergent formulation is dependent on its stability
and compatibility with detergent components [11,29]. Except some
limited examples [29], there is a dearth of report on the stabil-
ity of ␣-amylase with commercial laundry detergents. As shown
in Fig. 5, ␣-amylase from B. subtilis DM-03 exhibited a significant
stability and compatibility with all the tested commercial laundry
detergents. The enzyme retained 63.0–114.0% and from 82.0% to
147.0% of its original activity in presence of detergents at 37 and
45 ◦ C, respectively. The ␣-amylase activity was maximum with the
laundry detergent Rin advance® at both the tested temperatures.
The stability of any enzyme in detergent formulations is mainly
dependent on different ingredients, such as surfactants particu-
larly the anionic surfactants, sequestrants, bleaching agents and
stabilizers used for the detergent formulations [30,31]. Therefore,
partial loss of ␣-amylase activity in some of the detergents may be
attributed to inhibitory effect(s) of component(s) of these deter-
gents. In contrast, some of the components of the detergent(s),
for example ethoxylated surfactants and sucrose may have a stim-
ulatory effect on B. subtilis DM-03 ␣-amylase [30–32] resulting
in observed increase in enzyme activity in presence of detergent
compared to control (enzyme activity without detergent). Such an
increase in the activity of other hydrolytic enzymes in presence of
detergent components has already been reported [31]. However,
further detailed studies are necessary to elucidate the mechanism
of inhibition/activation of B. subtilis DM-03 ␣-amylase by different
components of detergents.
In conclusion, the ␣-amylase enzyme from B. subtilis DM-03
strain demonstrated excellent stability and compatibility with
the commercial laundry detergents at two different temperatures
generally used for washing purpose that favors its inclusion in com-
mercial laundry detergent formulations.
155
Acknowledgements
Authors thank Mr. Soumen Mukherjee, Department of Busi-
ness Communication, Institute of Management Studies, Ghaziabad
for editing the manuscript. Mr. S.K. Rai was a recipient of Junior
Research Fellowship from the Department of Biotechnology, New
Delhi. This work was supported by financial assistance to AKM from
the Department of Biotechnology, Ministry of Science and Technol-
ogy, Govt. of India.
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