Full text of DOI:10.1038/sj.jim.2900811

Journal of Industrial Microbiology & Biotechnology (2000) 24, 237–243
2000 Society for Industrial Microbiology 1367-5435/00 $15.00
www.nature.com/jim
Production of high activity thermostable phytase from
thermotolerant Aspergillus niger in solid state fermentation
TN Mandviwala and JM Khire
Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008, India
The thermotolerant fungus, Aspergillus niger NCIM 563, was used for production of extracellular phytase on agricul-
tural residues: wheat bran, mustard cake, cowpea meal, groundnut cake, coconut cake, cotton cake and black bean
flour in solid state fermentation (SSF). Maximum enzyme activity (108 U g−1 dry mouldy bran, DMB) was obtained
with cowpea meal. During the fermentation phytic acid was hydrolysed completely with a corresponding increase
in biomass and phytase activity within 7 days. Phosphate in the form of KH2PO4 (10 mg per 100 g of agriculture
residue) increased phytase activity. Among various surfactants added to SSF, Trition X-100 (0.5%) exhibited a 30%
increase in phytase activity. The optimum pH and temperature of the crude enzyme were 5.0 and 50°C respectively.
Phytase activity (86%) was retained in buffer of pH 3.5 for 24 h. The enzyme retained 75% of its activity on incubation
at 55°C for 1 h. In the presence of 1 mM K+ and Zn2+, 95% and 55% of the activity were retained. Scanning electron
microscopy showed a high density growth of fungal mycelia on wheat bran particles during SSF. Journal of Industrial
Microbiology & Biotechnology (2000) 24, 237–243.
Keywords: phytase; Aspergillus niger; phytic acid
Introduction
increase the bioavailability of phosphate, decreasing phos-
phorus pollution in areas of intensive animal agriculture.
The thermostability of this enzyme suggests potential
biotechnological application in the pulp and paper industry
as a novel biological agent to remove plant phytic acid.
The enzymatic degradation of phytic acid will not produce
mutagenic and highly toxic byproducts; thus exploitation of
enzymes in the industrial process would be environmentally
friendly and would assist in development of novel techno-
logies [23].
Although there are reports on phytases from micro-
organisms there are few reports on production of high
activity phytase by SSF of agricultural residues using fungi.
The present communication reports production of high
activity phytase by solid state fermentation using the therm-
otolerant fungus Aspergillus niger NCIM 563.
Phytic acid [myo-inositol(1,2,3,4,5,6) hexakis phosphate] is
the major storage form of phosphorus in seeds and pollen
[26,38]. Because of its strong chelating properties it is
regarded as an anti-nutritive factor. Moreover, it forms
insoluble complexes with nutritionally important metals
such as calcium, zinc, magnesium and iron, decreasing their
bioavailability [8,10]. Phytase (myo-inositol hexaphosphate
hydrolase) hydrolyzes phytic acid to myo-inositol and
phophoric acid. Two types of phytases are known: 3-phy-
tase (E.C. 3.1.3.8) and 6-phytase (E.C. 3.1.3.26), indicating
the predominant attack of the susceptible phosphoester
bond. Phytases are widely distributed in nature [6,45]. They
have been studied most intensively in seeds of plants, such
as wheat, barley, bean, corn, soybean, rice and cotton
[20,32]. Phytase activity in microorganisms has been found
most frequently in fungi [48], in particular Aspergilli
[18,19,42–47]. It occurs also in bacteria viz E. coli [11],
Bacillus subtilis [20–22,35], yeasts [4,31,40] and rumen
microorganims [37]. There are recent reports on expression
of the Aspergillus niger phytase gene (PhyA) in the yeasts
Pichia pastoris [13] and Saccharomyces cerevisiae [14] and
E. coli phytase gene in pig colon [39]. X-ray crystallo-
graphic analysis of phytase from Bacillus amyloliquefaci-
ens has also been reported [12]. Among Aspergilli there
are few reports of phytase production by SSF ie A. ficcum
[7,15,27] and A. carbonarius [1,2].
Materials and methods
Chemicals
Phytic acid sodium salt, was purchased from Sigma Chemi-
cal Company, St Louis, MO, USA. All other chemicals
used were of analytical grade and obtained from leading
manufacturers including BDH, Sigma and Glaxo. Various
agricultural residues like wheat bran, coconut cake, ground-
nut cake, soya residue and soya flour were purchased from
a local feedstuff outlet.
Phytases are of interest for biotechnological applications,
especially for the reduction of phytate in food and feedstuff.
Supplementation of animal feedstuff with phytases will
Microbial strain and its maintenance
Screening of fungal and bacterial cultures was carried out to
identify a strain capable of producing thermostable phytase.
Approximately 55–60 fungi and 15 thermophilic bacteria
were screened for phytase production using wheat bran as
a carbon source under SSF. Four fungal cultures were
found to be phytase producers. Out of these, thermotolerant
Aspergillus niger NCIM 563 was selected for further work
Correspondence: Dr JM Khire, Division of Biochemical Sciences,
National Chemical Laboratory, Pune 411008, India. E-mail: jmkhireȰ
yahoo.com
Received 7 June 1999; accepted 18 December 1999

Phytase from Aspergillus niger
TN Mandviwala and JM Khire
238
because it exhibited comparatively high phytase activity. It
was maintained on potato dextrose agar (PDA) slants. The
fungus was maintained by periodic transfer and stored at
4°C.
Phytic acid reduction
Phytic acid reduction from the wheat bran during SSF was
monitored by removing 2 g of solid state culture and
extracting the phytic acid by using 2.4% HCl, with continu-
ous shaking (200 rpm) for 1 h. After extraction, the suspen-
sion was centrifuged (6000 × g, 15 min), the supernatant
collected and phytic acid measured by the Haug and
Lantzch method [16].
Phytase production in solid state fermentation
Erlenmeyer flasks (250 ml) containing 10 g of wheat bran
(or any other agricultural residue) moistened with 10 ml
distilled water were autoclaved at 121°C for 40 min,
cooled, inoculated with 1 ml spore suspension containing
106 spores ml−1 from a 7-day-old sporulated culture grown
on a PDA slant at 30°C. The contents of each flask were
mixed thoroughly with a sterile inoculating needle after
inoculation for uniform distribution of fungal spores in the
medium. The flasks were incubated at 30°C/37°C. A flask
was harvested, its contents were extracted for enzyme
activity and assayed for phytase activity every 24 h for 8
days.
For tray fermentations, enamel-coated metallic trays
(28 × 24 × 4 cm) containing 50 g or 100 g of wheat bran
and trays (45 × 30 × 4 cm) containing 200 g of wheat bran
moistened with distilled water (ratio 1:1 w/v) were used.
Trays were covered with aluminium foil and autoclaved at
121°C for 40 min, then inoculated with a 10% volume of
a spore suspension containing 106 spores ml−1 from a 7-
day-old sporulated culture grown on a PDA slant at 30°C.
The contents of the trays were mixed before and after
inoculation and were incubated at 30°C.
Factors affecting growth
(1) Biomass estimation:
As it is very difficult to
quantify biomass in solid state fermentations, glucosamine
content was measured and taken as an equivalent value for
biomass amount and growth. This parameter is quite
reliable for biomass determination in solid state fermen-
tation [5].
Glucosamine was measured according to the method of
Nishio et al [33] in which 2 g fermented koji was removed
and dried at 105°C for 24 h. The dried sample (0.5 g) was
soaked in 10 ml of concentrated HCl for 24 h, centrifuged
at 7000 × g for 10 min and 5 ml of the supernatant was
diluted with 2 ml of 8.6 N HCl solution. Then the solution,
in the sealed test tube, was hydrolyzed for 2 h at 100°C
to produce glucosamine. The hydrolyzed solution was then
neutralized with 30% NaOH solution. Glucosamine in the
supernatant of the neutralized solution was measured by the
method of Blix [5].
(2) Effect of moisture level:
To check the influence
of moisture on phytase activity during SSF, 10 g wheat bran
in 250-ml Erlenmeyer flasks was moistened with different
amounts of distilled water. The ratio of wheat bran to dis-
tilled water was varied (1:0.3, 1:0.6, 1:1, 1:1.2, 1:1.5). After
sterilization at 121°C for 40 min these flasks were inocu-
lated with 1 ml spore suspension of A. niger and incubated
at room temperature as described earlier.
Enzyme extraction
After SSF 50 ml of 2% of an aqueous solution of
CaCl2·2H2O was added to each of two flasks, each contain-
ing 10 g fermented wheat bran (or any other agricultural
residue). Flasks were kept on a rotary shaker operated at
200 rpm for 2 h at room temperature for extraction of
enzyme from fermented koji [1,7]. At the end of extraction,
the suspension was squeezed through a double layer of
muslin cloth and it was centrifuged at 5000 × g for 20 min
at 4°C. The clear supernatant was designated as the crude
enzyme preparation.
(3) Effect of phosphate on enzyme production:
To
determine the effect of phosphate on production of phytase
during SSF, 10 g wheat bran in 250-ml Erlenmeyer flasks
was moistened with 10 ml distilled water containing vari-
ous amounts of phosphate in the form of KH2PO4, 0–50 mg
per 100 g of agricultural residue. After sterilization at
121°C for 40 min these flasks were inoculated with a spore
suspension of A. niger and incubated at room temperature
as described earlier.
Phytase assay
Phytase measurements were carried out at 50°C. The reac-
tion mixture consisted of 3 mM sodium phytate buffered
with 100 mM acetate buffer (pH 5.0). Enzymatic reactions
were started by the addition of 50 ␮l of enzyme solution.
After 30 min at 50°C, the liberated inorganic phosphate was
measured by a modification of the ammonium molybdate
method [17]. A freshly prepared solution of acetone: 5 N
H2SO4: 10 mM ammonium molybdate (2:1:1 v/v/v) and
400 ␮l 1 M citric acid was added to the assay mixture.
Absorbance was measured at 370 nm. One unit of phytase
activity (U) was expressed as the amount of enzyme that
liberates 1 ␮mol phosphorus per minute under standard
assay conditions.
(4) Effect of surfactants:
To check the effect of sur-
factants on production of phytase during SSF, 10 g wheat
bran in 250-ml Erlenmeyer flasks was moistened with
10 ml distilled water containing various surfactants: Tween
80, Triton X-100, sodium deoxycholate and NP-40 (0.5%
w/w). After sterilization at 121°C for 40 min these flasks
were inoculated with 1 ml spore suspension and incubated
at room temperature as described earlier.
(5) Extraction using different salts:
The effect of
Each experiment was carried out in triplicate and the
values reported are the mean of three such experiments in
which a maximum of 3–5% variability was observed.
salts on extraction of enzyme from SSF was monitored by
suspending 10 g fermented mouldy bran in 50 ml of a 2%
solution of various salts like CaCl2·2H2O, CaCO3,
Journal of Industrial Microbiology & Biotechnology

Phytase from Aspergillus niger
TN Mandviwala and JM Khire
239
CaSO4·2H2O, NaCl and KCl and it was kept on a shaker
at 200 rpm for 2 h at room temperature. The enzyme was
separated by passing the suspension through a double layer
of muslin cloth and the filtrate was centrifuged at 5000 × g
for 20 min at 4°C.
(6) Effect of metal ions on phytase activity:
The
effect of metal ions on phytase activity was carried out at
50°C for 30 min as described earlier by adding a salt of the
metal ion (1 mM final concentration) to the phytase reaction
mixture viz 100 mM acetate buffer, pH 5.0 containing
3 mM sodium phytate along with control, ie phytase reac-
tion without metal ion.
Scanning electron microscopy
Samples for SEM were fixed with ultraviolet light and were
mounted on brass stubs. Specimens were then coated with
a thin layer of gold (100 Å) in a gold coating unit, model
E 5000, Polaron Equipment Ltd (Cambridge, UK) and were
viewed with an SEM Leica Stereoscan 440 (Leica, Cam-
bridge, UK) at an accelerating voltage of 10 kV, and beam
current 25 Pa.
Figure 2 Effect of temperature on phytase production in SSF. ᭹—᭹
Incubation at 30°C; ᭿—᭿ incubation at 37°C.
a corresponding increase in phytase activity (79.5 U g−1
DMB) till the 7th day of SSF along with a reduction of
phytic acid from wheat bran. Enzyme production was asso-
ciated with increased biomass and a corresponding
reduction of phytic acid.
The effect of temperature of enzyme production is shown
in Figure 2. The enzyme is secreted at both 30°C and 37°C.
The fungus produced only 35 U g−1 DMB phytase activity
at 37°C compared to 79.5 U g−1 DMB at 30°C on the 7th
day.
Results
Time course of phytase production
The time course of phytase production in solid state fer-
mentation using wheat bran as substrate is shown in
Figure 1. The fungus grew rapidly on wheat bran particles
intra and intercellularly as indicated by an increase in
biomass in the form of glucosamine (2.5 mg g−1 DMB) with
Effect of agricultural residues on phytase production
All the substrates except rice husks showed phytase activity
(Table 1). Wheat bran gave 79.5 U g−1 DMB phytase
activity while cowpea meal supported the highest enzyme
production (108 U g−1 DMB). Since wheat bran was very
economical compared to cowpea meal, all fermentation
parameters for SSF were standardized using wheat bran.
Tray fermentation
The yield of enzyme activity (75–77 U g−1 DMB) was com-
parable to that in Erlenmeyer flasks (79.5 U g−1 DMB)
(Table 2).
Table 1 Effect of various substrates on phytase production
Substrate
Activity
(U g−1 DMB)
Wheat bran
79.5
40.6
108.0
50.9
37.3
84.7
47.5
0
Mustard cake
Cowpea meal
Groundnut cake
Coconut cake
Black bean flour
Cotton cake
Figure 1 Phytase production by Aspergillus niger on wheat bran
medium during SSF. ᭹—᭹ Phytase activity (U g−1 DMB); ᭺—᭺
reduction of phytic acid content; ᭿—᭿ glucosamine (mg g−1 DMB).
Rice husk
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Phytase from Aspergillus niger
TN Mandviwala and JM Khire
240
Table 2 Cultivation of Aspergillus niger NCIM 563 in trays
Table 3 Effect of surfactants on phytase production during SSF by
Aspergillus niger NCIM 563
Equipment
Capacity
Substrate quantity
(g)
Activity
(U g−1 DMB)
Surfactant (0.5%)
Phytase activity
(U g−1 DMB)
Flask
Tray
Tray
Tray
250 ml
10
50
100
200
79.5
77.0
77.0
75.0
28 × 24 × 4 cm
28 × 24 × 4 cm
40 × 30 × 4 cm
None
NP 40
Tween 80
79.5
83.0
91.0
Sodium deoxycholate
Triton X-100
92.0
108.0
Extraction using different salt solutions
Salt solutions like 2% CaCl2·2H2O, CaCO3, CaSO4·2H2O,
NaCl and KCl can be used for extraction of the enzyme
from fermented wheat bran from SSF. Compared to 2%
CaCl2·2H2O however, other salts recovered only 82–85%
enzyme activity.
100 g wheat bran in SSF, only 70 U g−1 DMB phytase
activity was produced (Figure 3).
Effect of surfactants
The effects of surfactants on phytase production during SSF
of wheat bran are shown in Table 3. The system with sur-
factants produced more enzyme than the control, which was
not supplemented with any surfactant. Triton X-100 sup-
ported a 30% increase in phytase production (108 U g−1
DMB) whereas Tween 80 and sodium deoxycholate
increased phytase production by 14% (90 U g−1 DMB).
Effect of moisture on phytase production
The effect of initial moisture content of the substrate on
enzyme yield is shown in Figure 3. Maximum phytase
activity was obtained when wheat bran was moistened with
distilled water in a 1:1 ratio (79.5 U g−1 DMB). Above this
ratio enzyme production was decreased as the porosity of
the medium was decreased.
Enzymatic properties
Effect of phosphate in the growth medium
Phytase assay was performed from pH 2.0 to 9.0 using a
variety of buffers at 100 mM in the reaction mixture at
50°C for 30 min, using 3 mM sodium phytate as a substrate.
Glycine-HCl (pH 2.0–5.0), acetate buffer (pH 4.0–6.0) and
Tris-HCl (pH 7.0–9.0) were used. The enzyme has an opti-
mum pH of 5.0 and was virtually inactive above pH 7.5.
However, the enzyme was quite stable towards the lower
pH range (Figure 4). The effect of pH on enzyme stability
was tested in the range of 2.0–9.0 at 4°C. After 24 h the
enzyme was fairly stable from pH 2.5–5.5, while 86% of
the phytase activity was retained for 24 h at pH 3.5
(Figure 4).
Enzyme production increased as the phosphate concen-
tration in SSF increased till 10 mg phosphate per 100 g
wheat bran (125.5 U g−1 DMB). Above this concentration
phytase activity reduced sharply. At 50 mg phosphate per
The enzyme’s optimum temperature was 50°C as shown
in Figure 5. The enzyme was incubated at various tempera-
tures for 15, 30, 45 and 60 min. At 55°C the enzyme
retained 75% of its original activity after 60 min and at
60°C it lost 84% of its activity after 15 min (Figure 5).
Figure 3 Effect of moisture level and concentration of phosphate on
phytase production in SSF. ᭹—᭹ Moisture; ᭺—᭺ phosphate.
Figure 4 Effect of pH on phytase activity (᭹—᭹) and stability (᭿—᭿).
Journal of Industrial Microbiology & Biotechnology

Phytase from Aspergillus niger
TN Mandviwala and JM Khire
241
fermentation. Nair and Duvnjak [28] reported use of Rhi-
zopus oligosporus NRRL 2990, Aspergillus niger NRC
5765, A. carbonarius NRC 401121, A. ficuum and Sac-
charomyces cerevisiae in an SSF process for reduction of
the phytic acid content in canola meal. However, among
the reports on phytase by fungi using the SSF technique,
the present strain of A. niger, NCIM 563, produced a high
activity thermostable phytase (108.5 U g−1 DMB) with
cowpea meal as substrate compared to A. ficuum (4 U g−1
solid state culture) [7] and A. carbonarius (2.5 U g−1 solid
state culture) [1] when canola meal was used as a substrate
for SSF. A low level of phytase appeared in the early stages
of incubation and enzyme levels reached a maximum by
the 7th day of fermentation. A prolonged incubation time
beyond this period did not help to further increase the yield
(Figure 1). The duration of incubation is generally depen-
dent on the growth rate and enzyme production pattern of
the strain [34]. Phytic acid was completely hydrolyzed after
7 days of fermentation (Figure 1).
The culture grew and produced phytase (35 U g−1 DMB)
at 37°C, demonstrating the thermotolerant nature of the
fungus (Figure 2).
Figure 5 Effect of temperature on enzyme activity (̅—̅) and stability
at 50°C (᭹—᭹), 55°C (᭿—᭿), 58°C (̆—̆) and 60°C (᭺—᭺).
There have been reports on the use of 2% CaCl2·2H2O
as an extractant [1,7] of phytase activity from solid sub-
strates used for the fermentation. But we have observed that
various other salt solutions like CaCO3, CaSO4·2H2O, NaCl
and KCl at 2% can also be used to extract phytase with a
recovery of 80–85%. However, 2% CaCl2·2H2O proved to
be the best salt solution amongst the ones tested.
Effect of metal ions on phytase activity
The effect of metal ions was studied by adding a metal
ion at 1 mM final concentration in a reaction mixture using
100 mM acetate buffer, pH 5.0 at 50°C for 30 min using
3 mM sodium phytate as a substrate. The enzyme retained
55% of its activity in the presence of 1 mM Zn2+ while all
other metal ions (Ca2+, Mn2+, Mg2+, Ni2+ and Fe2+) did not
show any activation or inhibition.
The result of tray fermentation was encouraging for
large-scale production of phytase. Similar trials for large-
scale production of ␣-amylase from a thermophilic Bacillus
coagulans in enamel trays have been reported [3]. Phytase
production in enamel trays was comparable with that in
culture flasks, indicating that scaling up does not result in a
reduction of phytase titers. Such tray fermentors have been
described as simple yet efficient in operation even though
the need for a large area is one of their drawbacks [24,25].
The moisture content in a solid state fermentation is a
crucial factor that determines the success of the process
[9]. The importance of moisture level in SSF media and its
influence on microbial growth and product biosynthesis
may be attributed to the impact of moisture on the physical
properties of the solid substrate [9,30,36]. A higher than
optimum moisture level causes decreased porosity, alter-
ation in wheat bran particle structure, lower oxygen transfer
and enhanced formation of aerial mycelia [9,36]. Similarly,
a moisture level lower than optimum leads to a higher water
tension, lower degree of swelling and reduced solubility of
the nutrients of the solid substrate [9]. We have seen that
maximum phytase activity was produced when the ratio of
wheat bran to distilled water was 1:1. Similar results were
reported during SSF of canola meal by A. ficuum [7].
The optimal amount of phosphorus in the growth
medium should be determined [41]. We have observed that
10 mg % phosphate during SSF of wheat bran yielded the
highest enzyme activity (125.5 U g−1 DMB) (Figure 3).
Similar effects of phosphate on phytase production by A.
ficuum in liquid medium were also reported [41]. In
addition, Han et al [15] obtained a similar trend for phytase
production by A. ficuum on semisolid substrate using
Scanning electron microscopy
A. niger grew rapidly on wheat bran and formed a dense
mat of vegetative mycelium which sporulated (Figure 6).
The biomass formed was directly related to enzyme activity
secreted into the medium.
Discussion
Phytases from bacteria, yeasts, fungi and plant sources have
been produced by solid state fermentation as well as liquid
Figure 6 Scanning electron micrograph of wheat bran degradation by
Aspergillus niger during SSF after 3 days of fermentation. Marker, mag-
nigication (× 250).
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Phytase from Aspergillus niger
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242
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Enzymatic properties
Most isolated phytases are active within the pH range 4.5–
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[23]. However, the phytases from Enterobacter, mungbean
and Lilium longiflorum (pollen) have pH optima around 7.5.
The enzymes isolated from animal tissues also have alka-
line optimum pH values. This wide range of pH optima
could be reflected in the molecular structure or the stereo
specificity of the enzyme from different sources. Phytases
show high activity in the temperature range of 50–70°C but
optimum temperatures for the enzyme are mainly between
45° and 60°C [23]. The optimum temperature of the spelt
phytase is 45°C which is the lowest optimal temperature
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Scanning electron microscopy
The degree of substrate transformation in SSF depends
upon the capability of fungal mycelia to penetrate deep into
the intracellular and intercellular spaces [29]. In the present
study we have observed that A. niger attacks wheat bran
particles rapidly and forms a full mat growth with spores on
wheat bran particles which was necessary for high phytase
activity as biomass was related to phytase activity.
Further work on purification and characterization of phy-
tase enzyme is in progress.
22 Kim YO, HK Kim, KS Bae, JH Yu and TK Oh. 1998. Purification
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We are grateful to SR Sainkar, Special Instruments Lab,
National Chemical Laboratory, Pune, India, for his help in
carrying out the SEM studies. Financial assistance to TN
Mandviwala (SRF) from CSIR, New Delhi, India is grate-
fully acknowledged.
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