Biochemical Engineering Journal 37 (2007) 294–297

Production of bacterial ␣-amylase by B. amyloliquefaciens

under solid substrate fermentation
M. Saban Tanyildizi a,∗ , Dursun Özer a , Murat Elibol b
a Department of Chemical Engineering, Faculty of Engineering, Fırat University, 23119 Elazig, Turkey
b Department of Bioengineering, Ege University, 35100 Bornova, Izmir, Turkey

Received 3 February 2006; received in revised form 8 May 2007; accepted 18 May 2007

Abstract
Production of ␣-amylase by Bacillus amyloliquefaciens, under solid substrate fermentation (SSF) was investigated in shaken-culture. The
maximum ␣-amylase activity was obtained under the following optimized conditions: corn gluten meal (CGM) 30 g/l, yeast extract (YE) 10 g/l,
agitation rate 150 rpm and fermentation temperature 33 ◦ C. The results showed that ␣-amylase production in a medium with CGM was five times
higher than that in the medium contained starch and other components. The temperature of fermentation was found to be most crucial factor in
␣-amylase production.
© 2007 Elsevier B.V. All rights reserved.

Keywords: Solid substrate fermentation; Corn gluten meal; B. amyloliquefaciens; ␣-amylase

1. Introduction
␣-Amylases are extracellular enzymes that randomly cleave
the ␣-1,4 glucosidic bonding of linear amylose and branching
amylopectin. They are the most important group of enzymes
produced commercially. Bacterial ␣-amylases have several
applications in many food and textile processes [1–3].
␣-Amylase can be produced by different species of microor-
ganisms using both submerged fermentation (SMF) and solid
substrate fermentation (SSF). Most of the production has been
carried out using SMF; however, SSF systems appear promising
due to the natural potential and advantages they offer [4]. SSF
is generally characterized by the growth of microorganism on
and/or within particles of a solid substrate in the presence of
varying amounts of water. The solid substrate acts as a source of
carbon, nitrogen, minerals and growth factors, and has a capacity
to absorb water, necessary for microbial growth. As the microor-
ganisms in SSF are growing under conditions similar to their
natural habitats, they may be able to produce certain enzymes
and metabolites more efficiently than in submerged fermentation
[5–7].
∗ Corresponding author. Tel.: +90 424 237 0000/5529; fax: +904242415526.
E-mail address: mtanyildizi@firat.edu.tr (M.S. Tanyildizi).
SSF has many advantages over SMF, including superior pro-
ductivity, simple technique, low capital investment, low energy
requirement and less water output, better product recovery and
lack of foam build up and reported to be the most appropri-
ate process for developing countries. A further advantage of
SSF is that cheap and easily available substrates, such as agri-
culture and food industry by-products [8]. Crude or partially
purified enzymes produced by SSF have industrial applications
(e.g. pectinases used for fruit juice clarification, ␣-amylase for
saccharification of starch) [9].
Inexpensive agriculture and agro-industrial residues repre-
sented one of the most energy-rich sources on the planet can be
used as a substrate in SSF. These residues are in fact, one of the
best reservoirs of fixed carbon in nature [10]. In the SSF, the
solid substrate not only supplies the nutrients to the culture, but
also serves as an anchorage for the microbial cells [11].
The composition and concentration of media and fermen-
tation conditions greatly affect the growth and production of
extracellular enzymes from microorganisms. Cost and availabil-
ity are important considerations, and therefore the selection of an
appropriate solid substrate plays an important role in the devel-
opment of efficient SSF processes [12]. On preliminary cost
analysis, a net savings of about 60 and 50% on fermentation
medium cost and the expenditure on down-stream processing,
respectively, as compared to the presently employed SMF tech-
nique was evident [13].

1369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.bej.2007.05.009
 M.S. Tanyildizi et al. / Biochemical Engineering Journal 37 (2007) 294–297 295

Corn gluten meal (CGM), a by-product of corn wet milling,

generated large quantities from starch industrial practice is rel-
atively cheap substrate. Traditionally, CGM has been used for
animal feed, and, therefore, it is desirable to find new uses for
CGM [14]. It contains rich proteins (≥60%) and vitamin and
other minerals.
It is known that SSF is mainly confined to processes involv-
ing fungi and not suitable for bacterial cultures because of
higher water activity requirements. However, successful bac-
terial growth and production ␣-amylase by using the SSF
technique is known in many natural fermentations [11]. The
genus Bacillus is major source of industrial enzymes and B.
amyloliquefaciens one of the most widely used species for the
bulk production of ␣-amylase [15].
The main objective of this study was to investigate into
␣-amylase production by B. amyloliquefaciens under SSF con-
dition with CGM.
2. Materials and methods
2.1. Strain and medium
The B. amyloliquefaciens NRRL B-645 was obtained from
Agricultural Research Service Culture Collection in USA. The
strain was maintained on agar slopes at 4 ◦ C. A standard inocu-
lum medium containing (g/l) glucose 15, peptone 2.5, yeast
extract (YE) 2.0, NaCl 1.5, KH2 PO4 0.5, MgSO4 0.5 and CaCl2
0.1 was inoculated into 250 ml. Erlenmayer flask which was then
kept at 37 ◦ C and 150 rpm for 18 h. The initial pH of the medium
was adjusted to 7.0. 1% (v/v) inoculum was transferred into
250 ml Erlenmayer flasks containing 50 ml production medium.
The production medium contained CGM and tap water only.
However, in order to determine the effect of different concen-
trations of medium constituents and process conditions on the
production of ␣-amylase, enzyme production medium based on
that described by Anderson et al. was also used [16]. The pH
of medium was initially adjusted to 7 and allowed to follow its
natural course throughout the fermentation.
2.2. Enzyme analysis
Fig. 1. Time course ␣-amylase production by B. amyloliquefaciens on CGM
(Initial pH: 7.0, CGM concentration: 10 g/l, fermentation temperature: 37 ◦ C,
agitation rate: 150 rpm).
the B. amyloliquefaciens utilized the CGM effectively, produc-
ing ␣-amylase. The physico-chemical parameters and amount
of media components apparently influenced the production of
the enzyme. The results reported here are the averages of three
values. The production of ␣-amylase reached a peak of 663 IU at
24 h. The production of enzyme relatively decreased after 30 h
for this basal medium. In order to follow the profile of enzyme
production, the fermentation was run for a long time of period
(72 h).
In this study, two different sizes of CGM (50 and 100 mesh)
were used. Commercially produced CGM has very small parti-
cles, so it cannot be ground more. The results show that there
is no difference in the values of enzyme and time to reach max-
imum enzyme production in both experiments. Therefore, it
was decided that the natural size of CGM was sufficient for
␣-amylase production.
The Fig. 2 shows the effect of nitrogen source (peptone
and YE) in fermentation medium on ␣-amylase production.
In all concentrations, the higher enzyme activity was obtained
when YE was used as a nitrogen source. The figure also shows
that 10 g/L of YE is the optimum for maximum ␣-amylase
production. However, the activity decreased higher and lower
concentrations of YE. Bajpai and Santos also reported similar
results in their works [20,21].

The fermented broth was taken after 30 h and centrifuged at

7000 rpm for 15 min, and then supernatant was used for estima-
tion of enzyme activity. The activity was measured by decrease
in iodine color reaction showing dextrinization of starch. The
activity was measured against the control in which no enzyme
was added. The detail of the method is given elsewhere [17]. One
unit of enzyme activity is defined 0.0284 optical density reduc-
tion of blue color intensity of starch iodine solution at 37 ◦ C
[18].

3. Results and discussions

In SSF, the selection of a suitable substrate for a fermentation

process is a critical factor [19]. The change of ␣-amylase pro- Fig. 2. Effect of the YE and peptone concentration on ␣-amylase production.
duction with incubation time, in which medium contained CGM (Initial pH: 7.0, CGM concentration: 10 g/l, fermentation temperature: 37 ◦ C,
and tap water only, is shown in Fig. 1. The results showed that agitation rate: 150 rpm).
296 M.S. Tanyildizi et al. / Biochemical Engineering Journal 37 (2007) 294–297
Fig. 3. Effect of CGM on ␣-amylase production. (Initial pH: 7.0, YE concen-
tration: 10 g/l, fermentation temperature: 37 ◦ C, agitation rate: 150 rpm).
One of the most important parameters in fermentation sys-
tems is the level of substrate used. In this study, seven different
CGM concentrations varying from 5 to 40 g/l were used (Fig. 3).
Maximum ␣-amylase activity was achieved at 30 g/l CGM con-
centration. Enzyme activity increased with increasing substrate
concentration until 30 g/l; any further increase in substrate con-
centration however, resulted in a decrease in enzyme production.
It is well known that substrate concentration has a direct effect
on water content in fermentation medium, because water level in
the fermentation medium decreases with an increase in substrate
concentration. When the quantity of the water becomes insuffi-
cient and does not allow a good diffusion of solutes and gas, the
cell metabolism slows, or can stop, because of a lack of substrates
or through too high concentration of inhibitive metabolites in or
near the cell [22].
Likewise, the effects of MgSO4 and CaCl2 on ␣-amylase
production were investigated in a medium containing CGM, YE
and tap water. However, there was no significant effect in the
use of these salts. The enzyme activity values obtained with and
without these salts were 2687 and 2645 IU respectively, which
are almost the same values. Therefore, it was decided that these
salts had no effect on the enzyme production. Although there are
many reports indicating an enhancement of ␣-amylase produc-
tion by these salts [23–25], the salt requirement for production
Fig. 4. Effects of rotary velocity of the shake flask on ␣-amylase production. (Ini-
tial pH: 7.0, CGM concentration: 30 g/l, YE concentration: 10 g/l fermentation
temperature: 37 ◦ C).
Fig. 5. Effects of fermentation temperature on ␣-amylase production. (Ini-
tial pH: 7.0, CGM concentration: 30 g/l, YE concentration: 10 g/l, agitation
rate:150 rpm,  25 ◦ C,  30 ◦ C,  33 ◦ C, × 37 ◦ C, ♦ 40 ◦ C, + 45 ◦ C).
of this particular enzyme was apparently provided by the nature
of CGM and tap water. These are of great important in terms of
the cost of production of enzyme.
Enzymes are susceptible to mechanical force, which may
disturb the elaborate shape of complex molecule to such a degree
that denaturation occurs. ␣-amylase production was investigated
at three different speeds, i.e., 100, 150 and 250 rpm. The highest
␣-amylase activity was obtained at 150 rpm (Fig. 4).
Fermentation temperature is also very important for SSF
since growth and production of enzymes or metabolites are usu-
ally sensitive to temperature [26]. In this study, six different
incubation temperatures varying from 25 to 45 ◦ C were used.
The results are shown in Fig. 5. The highest ␣-amylase produc-
tion was recorded at 33 ◦ C. The enzyme production however
decreased at higher temperatures. In a previous study, ␣-amylase
production using complex medium containing starch, the opti-
mum incubation temperature was 37 ◦ C (not shown here). The
thermal characteristics of substrate and the low moisture content
in SSF are especially difficult conditions for heat transfer. Heat
removal is inadequate for dissipating metabolic heat due to the
poor thermal conductivity of most solid substrates and result in
unacceptable temperature gradients [27].
Microbial growth and metabolism inevitably lead to a change
in the hydrogen ion balance and hence, the pH of the culture
medium [12]. To study the effect of pH on enzyme production
the initial pH was varied from 5.0 to 8.0 each at 1.0 interval. After
the sterilization in the autoclave, all these media having different
initial pH, were showed the same pH. It was then thought that
CGM contained high proteins (about 60%) serve as a buffer.
Therefore, in the subsequent experiments, the initial pH of the
fermentation medium was adjusted to 7.0.
4. Conclusions
Commercial ␣-amylase production is usually produced by
submerged fermentation; however, SSF appear promising due
to the natural potential and advantages they offer. In this study,
CGM was found to be a suitable substrate for ␣-amylase pro-
duction. The study of the CGM as solid substrate for ␣-amylase
production by B. amyloliquefaciens is of interest because the
 M.S. Tanyildizi et al. / Biochemical Engineering Journal 37 (2007) 294–297
substrate was sufficient enough with only a supplement of YE
(10 g/l). The medium compounds, which are essential in sub-
merged fermentation for maximum ␣-amylase production are
not required in SSF. The knowledge about, obtained higher
enzyme activity in case of using SSF than SMF are given in
literature [6]. Activity of ␣-amylase, obtained in this study, is
nearly five times higher than our previous study [28] related
to using of SMF method. The CGM, which is a by-product of
starch industries, is not expensive and readily available. It was
also determined that one of the most important parameters in
␣-amylase production was the fermentation temperature.
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