J. Basic Microbiol.

42 (2002) 5, 320 – 326

(Biotechnology Division, Regional Research Laboratory, CSIR, Trivandrum – 695 019, India;
1
Department of Agricultural Chemical Technology, Technical University of Budapest, 1111 Budapest,
Geller ter 4, Hungary)

Synthesis of a-amylase by Aspergillus oryzae

in solid-state fermentation
FEBE FRANCIS, A. SABU, K. MADHAVAN NAMPOOTHIRI, GEORGE SZAKACS1 and
ASHOK PANDEY*

(Received 26 March 2002/Accepted 16 April 2002)

Spent Brewing Grains (SBG) was evaluated for its efficacy to be used as sole carbon source for the
synthesis of a-amylase in solid-state fermentation using a fungal strain of Aspergillus oryzae NRRL
6270. Enzyme production was superior when the culture grew on mesophilic temperatures and best
yields were at 25 °C. At 30 °C, yields were almost comparable. Maximum production of a-amylase
[6870 U/g dry substrate (gds)] was obtained when SSF was carried out at 30 °C for 96 h using SBG
medium, which had initial moisture of 70% and was inoculated using a spore suspension containing
1 × 107 spores/ml. Supplementation of SBG with external carbon sources such as mono-, di and
polysaccharides caused repression in enzyme synthesis by the fungal culture.

Solid State Fermentation (SSF) has, of late, emerged as an appropriate technology for the
management of agro-industrial residues and for their value addition (PANDEY 1992, PANDEY
et al. 2000a, 2001). The food and beverages industry is one that produces large quantities of
residues that pose serious problems of disposal even if they are valuable sources of biomass
and nutrients. A number of such substrates have been successfully put to use under SSF for
various end-products such as enzymes and secondary metabolites. Recently, however, many
agro-industrial residues are targets of microbial conversion, especially for value-addition
(PANDEY et al. 2000b, 2000c, 2000d).
Spent Brewing Grain (SBG) consists of the residual seed hull and fiber of barley after the
malting process of the brewing industry (CASIDA 1999). The spent grains separated from the
wort, without drying, find application as cattle feed; they are also dried to about 10% mois-
ture to provide an animal feed product, commercially known as dried brewer’s grains.
While majority of the dried grains are used up as cattle feed, the rest create serious problems
of disposal. A potential method to convert SBG in to a value added product such as a pro-
tein-rich animal feed supplement and production of enzyme by microbial fermentation are
being analyzed. The SSF technology may prove to be a relatively simple and economical
method towards this. The potential of SBG to be used as a substrate for the production of
many other enzymes including cellulase and xylanase under SSF has been analyzed (SIM
et al. 1990). WANG et al. (2001) have reported on the efficacy of SBG as a substrate for
mushroom production. They have reported on the increase in crude protein content as well
as the decrease in the ratio of lignin to cellulose of SBG by the growth of fungi. The ability
of spent grains to be used as a useful sorbent for lead and cadmium under batch condition
has also been reported (LOW et al. 2000).
The production of a-amylase by Aspergillus oryzae has been well studied and the use of
the same for commercial production of α-amylase has been established. As a Koji mold, it
has been used safely in the food industry for several hundred years (BENNETT 1985). TERUI

* Corresponding author: Prof. A. PANDEY; e-mail: pandey@csrrltrd.ren.nic.in

© 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0233-111X/02/0310-0320 $ 17.50+.50/0
Synthesis of a-amylase by Aspergillus oryzae 321

and MORIMOTO (1961) have discussed about higher production of Taka amylase A in solid
state fermentation than in submerged fermentation by A. oryzae.
The aim of the present work was to evaluate the feasibility of spent brewing grains as
solid substrate in SSF for the production of a-amylase by a fungal strain of Aspergillus
oryzae.

Materials and methods

Microorganism and its maintenance: A strain of Aspergillus oryzae NRRL 6270 was used for this
study. This was maintained on potato-dextrose-agar medium (Hi-Media, Bombay). Sub-culturing was
carried out once in every three weeks by growing the PDA slants at 30 °C for 7 days to obtain full
sporulation. The slants were stored at 4 °C.

Substrate preparation: Spent brewing grains (SBG), obtained from a brewery in Budapest was used
as substrate. Five grams (dry) of solid substrate was weighed into a 250-ml ERLENMEYER flask and to
this a supplementing salt solution in distilled waster was added to obtain the desired moisture level.
The composition of the salt solution was as follows (%, g/g of dry substrate): NH4NO3: 1; KH2PO4: 1;
NaCl: 0.2; MgSO47H2O: 0.2. The contents of the flasks were mixed thoroughly and autoclaved at
121 °C (15-psi) for 20 minutes.

Preparation of inoculum: The spores of A. oryzae were dislodged from a fully sporulated (7 days
old) PDA slant culture using 10-ml of sterile distilled water containing 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
technique.

Solid-state fermentation: The sterilized solid substrate was inoculated with one ml of the prepared
inoculum. The contents were mixed thoroughly and incubated at the appropriate temperature. Samples
as whole flasks in duplicate, were withdrawn after the required time for incubation.

Enzyme extraction: The crude enzyme from the fermented matter was extracted by simple contact
method. For this, the fermented substrate was mixed thoroughly with distilled water containing 0.1%
Tween-80, to a total extract volume amounts of 100-ml. Contents were mixed by shaking for one hour
at 30 °C on a rotary shaker (Certomat, B. BRAUN Biotech) at 150 rpm. At the end of the extraction, the
suspension was centrifuged at 7000 rpm for 10 minutes (C-24 cooling centrifuge, REMI) and the
supernatant was collected and used as the crude enzyme for further analysis.

a-Amylase assay: a-Amylase activity was determined as described by OKOLO et al. (1995). The
reaction mixture consisted of 1.25 ml 1% (w/v) soluble starch (MERCK, Mumbai) solution, 0.25 ml
0.1 M sodium acetate buffer (pH 5.0), 0.25 ml of distilled water, and 0.25 ml of properly diluted crude
enzyme extract (10 – 320×). After 10 minutes of incubation at 50 °C, the liberated reducing sugars
(glucose equivalents) were estimated by the dinitrosalicylic acid method (MILLER 1959). Appropriate
blanks were used. One unit (U) of a-amylase is defined as the amount of enzyme releasing one mmol
glucose equivalent per minute under the assay conditions.

Determination of moisture of the substrate: The moisture content of the substrate was analyzed by
a Mettler LP16 infra-red analyzer.

Results and discussion

A fungal strain of Aspergillus oryzae NRRL 6270 was used for α-amylase production. A
single parameter optimization approach was employed to optimize various process parame-
ters such as incubation time, incubation temperature, moisture content and inoculum size
that influenced the rate of enzyme production.
322 F. FRANCIS et al.

4000

3000
α -amylase activity (U/gds)

2000

1000

0
0 12 24 36 48 60 72 84 96 108 120 132 144
Incubation time (H)
Fig. 1
Effect of incubation period on a-amylase production by A. oryzae NRRL 6270

Optimization of incubation time

SSF was carried using SBG to optimize the time-course of incubation. Initial moisture
of the substrate was adjusted to 58 and incubation temperature was maintained at 30 °C.
Samples were withdrawn every 12 hour and extracted. Maximum enzyme production
(3515 U/gds) was observed at the end of 96 h. The enzyme production showed a growth-
relatedness (Fig. 1). Previous studies on a-amylase production by Aspergillus sp. (SUDO
et al. 1994) showed that it was growth associated and reached a maximum around 96 h.
After 96 h, the production seemed to decrease as the growth of the organism would have
reached a stage, from which it could no longer balance its steady growth with the availabil-
ity of nutrient resources. It entered to its stationary phase of growth, where it started the
production of secondary metabolites.
One of the advantages of using agro-industrial residues in SSF is the unique buffering
action of these materials (PANDEY et al. 2001). Such potentials of agro-industrial residues
rule out the need for initial adjustments of pH of the medium. In view of this, we analyzed
the initial and final (after different periods of SSF) pH of the substrates. SBG depicted good
buffering capacity as there were practically only marginal or no changes in the pH values
(data not shown). This observation advocated the potential advantage of using SBG for
enzyme production, as it did not require any effort to control pH of the medium.

Optimization of incubation temperature

SSF was carried out at different incubation temperatures ranging from 25–50 °C. The initial
moisture content was maintained at 58% and the samples were extracted after 96-hours of
fermentation. The organism exhibited its best performance for enzyme production in the
Synthesis of a-amylase by Aspergillus oryzae 323

4000

3500

3000
α - amylase activity

2500

2000

1500

1000

500
25 30 35 40 45 50
0
Incubation temperature ( C)
Fig. 2
Effect of incubation temperature on α-amylase production by A. oryzae NRRL 6270

mesophilic range (Fig. 2); it yielded a maximum production of 3600 U/gds at 25 °C.
At 30 °C incubation temperature, the yields were almost comparable (3515 U/gds). The
significance of temperature in the development of a biological process is such that it could
determine the effects of protein denaturation, enzymatic inhibition, promotion or suppres-
sion of the production of a particular metabolite, cell viability and death (PANDEY et al.
2001).
In SSF, during fermentation there is a general increase in the temperature of the ferment-
ing substrate due to respiration (PANDEY 1992, PANDEY and RADHAKRISHNAN 1992). Heat
build up in fact is a drawback in SSF system. There are some reports that describe efficient
application of forced aeration to control it (PANDEY et al. 2001). However, these problems
are generally encountered during the scale-up of SSF. In laboratory studies using flasks, no
such difficulty was noticed.
Since in these experiments, there was not much difference in the enzyme production at
25 or 30 °C, and since maintenance of 30 °C could be considered easier (in terms of energy
expenses), for further studies 30 °C was used as the incubation temperature.

Optimization of moisture content

The influence of initial moisture of the substrate was studied by carrying out SSF at varying
levels of moisture (50–75%, w/w). The samples were incubated at 30 °C and extracted after
96 hours of fermentation. The study revealed that the enzyme activities were lower when
substrate moisture was higher or lower than 70% (Fig. 3). The decrease in enzyme activity
with increase in substrate moisture may be attributed to the phenomenon of flooding of
inter-particle space of the substrate. This reduces effective transfer of air needed by
324 F. FRANCIS et al.

5500

5000
α - amylase activity (U/gds)

4500

4000

3500

50 55 60 65 70 75
Moisture content (%)
Fig. 3
Dependence of a-amylase production on moisture content of the substrate

the organism for respiration and in turn reduces the growth and proliferation of the fun-
gal mycelium (PANDEY et al. 1992). Fungi would prefer unbound moisture for its survi-
val in such quantities that it does not hamper with its metabolic pathways. A related
criterion with this is the water activity of the medium, which is considered as the funda-
mental parameter for mass transfer of water and solutes across the cell membrane. When
water is made available in a lower or higher quantity than that is optimally required, the
productivity of the process is significantly affected. The control of this parameter can be
used to control and modify the metabolic activity of the microorganism (PANDEY et al.
1994).

Optimization of inoculum size

A master spore suspension was made from a PDA slant and varying levels of inoculum size
was achieved by the method of serial dilution. SSF was carried out with samples that were
inoculated with one ml of these spore suspensions that had varying levels of viable spores
with initial moisture of 70% (w/w), at 30 °C for 96 hours. The organism yielded a maxi-
mum of 6870 U/gds at an inoculum size of 107 spores/gds (Fig. 4). The size of inoculum
determines biomass production on the solid medium (PANDEY et al. 2000a). An increase in
the number of spores in inoculum would ensure a rapid proliferation and biomass synthesis.
However, when the competition for nutrients becomes evident, there would be a decrease in
the metabolic activity of the organism. At the optimum inoculum size for enzyme producti-
on, there is a balance between proliferating biomass and availability of nutrients that sup-
ports production of the same.
Synthesis of a-amylase by Aspergillus oryzae 325

7000

6500
α - amylase activity (U/gds)

6000

5500

5000

103 104 105 106 107 108

Inoculum size (spores/gds)

Fig. 4
Variation in a-amylase production with inoculum size

Effect of supplementation of carbon sources

SBG was supplemented with various carbon sources separately at 1.0% (w/w, dry weight
basis) concentration to assess their impact on enzyme production. None of the tested carbon
sources were found to have any positive influence on a-amylase production (Fig. 5). In fact,

Fig. 5
Effect of supplementation of substrate with different carbon sources on the production of a-amylase
by A. oryzae NRRL 6270
326 F. FRANCIS et al.

all of them were repressive for fungal activity of enzyme synthesis and among the various
carbon sources studied, glucose had the most repressive effect. The repressive action
of mono- and di-saccharides were evident as they counteracted the very purpose for which
a-amylase was synthesized by the organism (KICHAKOVA 1991). An easy assimable energy
source (carbon source) would suppress the need for the cellular machinery to produce
enzymes that has the function of releasing energy molecules. It can be deduced that SBG in
itself was sufficient to promote the production of a-amylase.
From the results, it could be concluded that spent brewing grain was a suitable substrate
for the production of a-amylase by SSF. It was transformed into a value-added product by
fermentation using A. oryzae in 96 h. Under optimized conditions, the fungal strain of
A. oryzae NRRL 6270 produced 6870 U/g dry substrate a-amylase. These results could be
of good commercial implications.

References
BENNETT, J. W., 1985. Molds, manufacturing and molecular genetics. In: W. E. TIMBERLAKE, (ed.),
Molecular Genetics of Filamentous Fungi. Alan R. Liss, Inc. NY.
CASIDA. L. E., 1999. Industrial Microbiology. New Age Intl Publishers. Delhi, 274 – 299.
KICHAKOVA, N. A., 1991. The effect of the carbon sources and calcium ions on the activity of thermo-
stable alpha-amylase in Bacillus sp. 86. Mikrobiol. Zh., 53, 44 – 48.
LOW, K. S., LEE, C. K. and LIEW, S. C., 2000. Sorption of cadmium and lead from aqueous solutions
by spent grain. Process Biochem., 36, 59 – 64.
MILLER,G. L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal.
Chem., 31, 426 – 429.
OKOLO, B. N., EZEOGU, L. I. and MBA, C. N., 1995. Production of raw starch digesting amylase by
Aspergillus niger grown on native starch sources. J. Sci. Food Agric., 69, 109 – 115.
PANDEY, A., SOCCOL, C. R., RODRIGUEZ-LEON, J. and NIGAM, P., 2001. Solid state fermentation in
biotechnology. Asiatech Publishers Delhi, p. 221.
PANDEY, A., SOCCOL, C. R. and MITCHELL, D., 2000 a. New developments in solid-state fermentation.
I. Bioprocesses and applications. Process Biochem., 35, 1153 – 1169.
PANDEY, A., SOCCOL, C. R., NIGAM, P. and SOCCOL, V. T., 2000b. Biotechnological potential of agro-
industrial residues. I. Sugarcane bagasse. Bioresource Technol., 74, 69 – 80.
PANDEY, A., SOCCOL, C. R., NIGAM, P., SOCCOL, V. T., VANDENBERGHE, L. P. and MOHAN, R., 2000 c. Bio-
technological potential of agro-industrial residues. II. Cassva bagasse. Bioresource Technol., 74, 81 – 87.
PANDEY, A., SOCCOL, C. R., NIGAM, P., BRAND, D., MOHAN, R. and ROUSSOS, S., 2000 d. Biotechno-
logical potential of coffee pulp and coffee husk for bioprocesses. Biochemical Engineering Journal,
6, 153 – 162.
PANDEY, A., ASHAKUMARY, L., SELVAKUMAR, P. and VIJAYALAKSHMI, K. S., 1994. Influence of water
activity on growth and activity of Aspergillus niger for glucoamylase production in solid state fer-
mentation. World J. Microbiol. Biotechnol., 10, 485 – 4 86.
PANDEY, A., 1992. Recent developments in solid-state fermentation. Process Biochem., 27, 109 – 116.
PANDEY, A. and RADHAKRISHNAN, S., 1992. Packed bed column bioreactor for production of enzymes.
Enzyme Microb. Technol., 14, 486 – 488.
SIM, T. S. and OH, J. C. S., 1990. Spent brewery grains as substrate for the production of cellulase by
Trichoderma reesei qm9414. J. Ind. Microbiol., 5, 153 – 158.
SUDO, S., ISHIKAWA, T., SATO, K. and OBA, T., 1994. Comparison of acid stable α-amylase production by
Aspergillus kawachii in solid-state and submerged cultures. J. Fermentation. Bioengg., 77, 484 – 489.
TERUI, G. and MORIMOTO, T., 1961. Time course of metabolism in rice Koji III. Hakkokogaku, 39
p. 196 – 200.
WANG, D., SAKODA, A. and SUZUKI, M., 2001. Biological efficiency and nutritional value of Pleurotus
ostreatus cultivated on spent beer grain. Bioresource Technol., 78, 293 – 300.

Mailing address: Professor ASHOK PANDEY, Biotechnology Division, Regional Research Laboratory
(CSIR), Trivandrum – 695 019, India
Phone: +91-471-515 279; Fax: +91-471-491 712
E-mail: pandey@csrrltrd.ren.nic.in