Brazilian Journal of Microbiology 44, 1, 317-323 (2013) Copyright © 2013, Sociedade Brasileira de Microbiologia

ISSN 1678-4405 www.sbmicrobiologia.org.br

Research Paper

Isolation, morphological and molecular characterization of phytate-hydrolysing fungi

by 18S rDNA sequence analysis

Iti Gontia-Mishra, Dhanshree Deshmukh, Niraj Tripathi, Khushboo Bardiya-Bhurat,

Keerti Tantwai, Sharad Tiwari
Biotechnology Centre, Jawaharlal Nehru Agricultural University, Jabalpur, Madhya Pradesh, India.

Submitted: February 26, 2012; Approved: July 2, 2012.

Abstract

Phytate is the primary storage form of phosphate in plants. Monogastric animals like poultry, pigs
and fishes have very low or no phytase activities in their digestive tracts therefore, are incapable to ef-
ficiently utilize phytate phosphorus from the feed. Phytase from microbial sources are supplemented
to feedstuff of these to increase the uptake of phytate phosphorus. In the present work efforts were
made to isolate and characterize proficient phytase producing fungi from soil. Phytase producing
fungi were isolated using phytate specific medium. Fungal isolates were selected according to their
higher phytase activities. These isolates were further characterized and identified by morphological
and microscopic analysis and confirmed by amplification of 18S rRNA gene, using specific primers.
This gene was subsequently sequenced and phylogenetic affiliations were assigned. Fungal isolates
were identified as various species of Aspergillus. Phytases from these fungi could be utilized as a feed
additive in poultry and swine industries.

Key words: phytase, phytate, fungi, 18S rRNA gene, Aspergillus.

environmental pollution. Furthermore, phytic acid is also

Introduction considered as an anti-nutritional factor, since it causes min-
In many plants, phytic acid (myo-inositol 1,2,3,4,5,6 eral deficiency due to efficient chelation of metal ions
hexakisphosphate) is one of the main storage forms of (Fe3+, Zn2+, Ca2+, Mg2+, K+) and forming complexes with
phosphate, accounting for 60-80% of total phosphorous (P) proteins, thus affecting their digestion (Urbano et al.,
in grains, legumes and oilseeds (Sharma and Gole, 1978; 2000). Microbial phytase produced by fermentation as a
Wang et al., 2007). Cereal grains and oilseed meals are ma- feed additive is widely used to manage the nutritional and
jor ingredients of animal feed. The amount of phosphorus environmental problems caused by phytate.
in cereal grains and oilseed meals would meet the require-
ment of optimal growth of animals if all the phosphorus Commercial production of phytase as feed additives
from phytate were available to animals. Phytate phospho- is mostly focused on fungi and yeasts, as they are the most
rus is not available to monogastric animals (such as poultry, prolific extracellular producers of this enzyme (Farhat et
piggery and fish) due to lack of adequeate levels of phytase al., 2008). Most of the naturally occurring phytases having
enzyme in their gastrointestinal tracts (Brinch-Pedersen et high thermostability and a broad pH range were identified
al., 2002; Singh and Satayanarayana, 2010). Phosphate from fungi (Simon and Igbasan, 2002). Due to immense in-
supplementation is required for optimal growth of animals. dustrial and environmental implication of phytases there is
Phytase catalyzes the dephosphorylation of phytate to an ongoing interest in isolation of new fungal strain produc-
inositol and orthophosphate (Wodzinski and Ullah, 1996; ing phytase and optimization of this enzyme. In the present
Hamada et al., 2005). Moreover, in the regions with intense work efforts were made for isolation, morphological and
animal production, a large amount of undigested and ex- molecular characterization of proficient phytase producing
creted phosphate (phytate) contributes significantly to the fungal strains.

Send correspondence to S. Tiwari. Biotechnology Centre, Jawaharlal Nehru Agricultural University, 482004 Jabalpur, Madhya Pradesh, India. E-mail:
[email protected].
318 Gontia-Mishra et al.

Materials and Methods (2.35 g of ammonium vanadate in 1 L of 2% (v/v) nitric

acid solution), and 165 mL of 65% nitric acid, finally di-
Sample collection luted to 1L with water]. The absorbance was measured at
Samples were collected in sterile polythene bags from 415 nm. Distilled water was used as a blank. One unit of
agricultural land, vermicompost and food based sources phytase activity was defined as 1 mmol of phosphate pro-
such as poultry and pig wastes, and fishery pond located at duced per min per mL of culture filtrate under the assay
Jawaharlal Nehru Agricultural University Campus, Jabal- condition (pH 5.5, temperature 37 °C and substrate concen-
pur, Madhya Pradesh, India (23°17’ N; 72°98’ E). tration, sodium phytate [C6H6O24P6Na12] at 0.0051 mol/L).
Standard curve was prepared using potassium dihydrogen
Isolation of phytase producing fungi phosphate (KH2PO4) in the range 0-1000 mmol.
Soil samples (0.2 g) were suspended in 10 mL of
0.9% saline solution and kept on incubator shaker with pH and temperature optima of crude phytase
150 rpm for 2 h. Soil suspension (1 mL) was inoculated into enzyme
100 mL of phytate specific medium (PSM) containing To determine the pH optimum curve, the enzyme was
1.5% glucose, 0.5% (NH4)2SO4, 0.05% KCl, 0.01% incubated with sodium phytate prepared in 0.2 M Sodium
MgSO4.7H2O, 0.01% NaCl, 0.01% CaCl2.2H2O, 0.001% acetate buffer, pH 3.0, 3.6, 4.2, 4.8, 5.5; 0.2 M citrate
FeSO4.7H20, 0.001% MnSO4.H20, pH 6.5 with 0.5% so- buffer, pH 6.0 and 6.6 and 0.2 M Tris, pH 7.0, 7.5 and 8.5
dium phytate (Hosseinkhani et al., 2009). Medium was for 1 h at 37 °C and released phosphate ions were assayed.
sterilized by autoclaving (15 psi, 121 °C, 20 min), with the The temperature optimum was determined by incubating
exception of sodium phytate, which was sterilized by mem- the enzyme with substrate, prepared in 0.2 M sodium ace-
brane filtration (Millipore, 0.45 mm) and added aseptically tate pH 5.5 at different temperatures for 1 h and the phytase
to cooled autoclaved media. The samples were kept in incu- activity was assayed.
bator shaker with shaking at 150 rpm at 30 °C for 10 days.
Morphological characterization
Fungal cultures were re-inoculated in fresh PSM medium
to ensure enrichment of phytase producing fungi and incu- The morphological identification of isolates
bated at 30 °C, 165 rpm for 7 days. (DD1-DD3; IG1-IG5) was conducted using four different
Fungal cultures obtained through the enrichment pro- types of media viz. Czapek dox agar (CDA), Czapek yeast
cess were inoculated in PSM agar medium containing cal- agar (CYA), Czapek yeast 20% sucrose agar (CY2S) and
cium phytate (0.5%) as sole source of phosphorus. Plates Malt yeast agar (MYA). Creatine sucrose agar (CREA) was
were kept for incubation at 30 °C for 5 days. After incuba- used to study acid or base production by fungi (Samson et
tion zone of clearing around the fungal growth on PSM agar al., 2007). All media were incubated at 28 °C for 7 days.
plates were observed. The zone of clearing around the fun- Colonies on each medium were compared for their diame-
gal growth is indicator of phytase production. The samples ters, overall colors, colors of conidia, reverse colors, tex-
which showed clear zone were considered as positive sam- ture, zonation and sporulation. All the isolates were also
ples. Counterstaining for confirmation of phytase activity subjected to microscopic analysis for their characterization
was performed for the positive isolates according to the and identification.
method of Bae et al. (1999).
Identification of fungi using 18S rRNA gene analysis
Phytase enzyme activity assay Fungal mycelium or spores were cultured on potato
Isolates that produced clear zones on screening me- dextrose agar medium (Himedia, India). The plates were
dium were tested for phytase production in PSM broth with incubated at 30 °C for 2 to 3 days. The fungal mycelium
sodium phytate. A spore suspension of 1 x 107/ mL was in- was used for DNA isolation. DNA was extracted using
oculated in 100 mL of PSM medium in a 500 mL Erlen- method described by Hunt et al. (2004).
meyer flask and incubated at 30 °C with 200 rpm shaking
PCR amplification of 18S rRNA gene
for 7 days. Cultures (2 mL) were centrifuged and the super-
natant was used for phytase activity assays. The enzyme ac- PCR amplification of fungal small-subunit rDNA
tivity was estimated according to the method described by (18S rRNA gene) was carried out using the primer set EF4
Engelen et al. (1994). The incubation mixture (2 mL) con- and EF3 (Smit et al., 1999). The EF4 and EF3 primers am-
tained 1 mL of the culture filtrate, 1mL of substrate solution plified a 1.5-kb section of the 18S rRNA gene. Primer se-
[10 mM sodium phytate as substrate and 0.2 M sodium ace- quences were as follows: EF4
tate buffer (pH 5.5)] and incubated at 37 °C for 1 h. The re- (5’-GGAAGGG[G/A]TGTATTTATTAG-3’) and EF3
action was terminated by the addition of 1mL of the colour (5’-TCCTCTAAATGACCAAGTTTG-3’). PCR amplifi-
developing reagent [250 mL of ammonium hepta-molyb- cation was performed in a 25 mL reaction containing 2.5 U
date solution (10% of ammonium molybdate in 0.25% am- of Taq DNA polymerase (Sigma), a 10 X dilution of the
monia solution), 250 mL of ammonium vanadate solution manufacturer’s buffer (Sigma), 200 mM concentrations of
Phytate-hydrolysing fungi 319

each deoxynucleoside triphosphate (dNTPs), and 20 pM of DD 3. Counterstaining approach was carried out to
primers EF4 and EF3 and 50 ng of genomic DNA. The re- overcome the selection of false positive isolates for phytase
action conditions were as follows: initial denaturation at production on PSM medium (Fredrikson et al., 2002;
94 °C for 4 min, 40 amplification cycles of denaturation at Chadha et al., 2004). The formation of clear zone is attrib-
94 °C for 1 min, annealing at 48 °C for 1 min and primer ex- uted to the production of various acids (acetic acid, maleic
tension at 72 °C for 3 min; followed by a final extension at acid, etc.), which lowers the pH of the medium and hence
72 °C for 10 min. PCR amplifications were carried out us- increase the solubility of calcium phytate (Bae et al., 1999).
ing a Thermo-Hybaid PCR thermal cycler (Thermo Fisher
Scientific USA). Aliquots of the PCR products (5 mL) were Phytase activity and its optimization
analyzed in 1% (w/v) agarose gels (Sigma, USA) by hori- Phytase activity was determined by measuring the
zontal gel electrophoresis. DNAs were visualized by UV amount of liberated inorganic phosphate and its reaction
excitation after staining with ethidium bromide (0.5 mg/L). with colour reagent. It was carried out for fungal isolates ir-
The PCR product was purified using Bangalore Genie PCR respective of clear zone formation on PSM medium. It was
purification kit following the manufacturer’s instruction. found that fungal isolates which did not form clear zone on
The 18S rRNA nucleotide sequence was determined by PSM, showed negligible phytase activities in liquid me-
PCR-direct sequencing done by Chromous Biotech Pvt. dium. Isolate IG 3 and IG 1 showed highest phytase
Ltd., Bangalore, India. activities which were 0.46 U mL-1 and 0.39 U mL-1, respec-
Phylogenetic analysis of the 18S rRNA gene se- tively. Enzyme activities as well as size of zone of clear-
quences was performed with CLC DNA workbench ver- ance for different isolates are shown in Table 1.
sion 6. The phylogenetic trees were inferred using the Optimization of phytase activity for isolate IG 1 and IG 3
neighbour-joining method (Saitau and Nei, 1987) and boot- were carried out to determine their optimum pH and tem-
strap analyses were performed. The evolutionary distances perature. Phytase activity for isolates IG 1 and IG 3 was op-
were computed using the Maximum Composite Likelihood timum at pH 5.5 as depicted from Figure 1a and optimum
method (Tamura et al., 2004). temperature was 37 °C and 40 °C respectively as shown in
Figure 1b. Phytase often has a low-pH optimum range
Results and Discussion (pH 4.5-6.0) with a rapid drop in activity at pH value above
6.0 (Marlida et al., 2010). It is needed to be determined that
Isolation of phytase producing fungi from soil phytase activity for the fungal isolates is extracellular or
Twenty-five soil samples were collected from differ- cell-associated.
ent locations for the isolation of phytase producing fungi.
Out of these only fourteen samples showed fungal growth Morphological and microscopic characterization of
in phytate specific screening medium containing 1% so- fungi
dium phytate. The fungal cultures obtained from these soil Fungal isolates were grown in different growth me-
samples were re-inoculated in PSM broth for enrichment of dium (CDA, CYA, MYA and CY2S) and colonies on each
these phytate utilizing cultures. For further confirmation of medium were recorded for their diameters, overall colors,
their phytase producing trait, fungal cultures were inocu- colors of conidia, reverse colors, texture, zonation and
lated on PSM agar medium containing 0.5% calcium phy- sporulation as shown in Table 2. Isolates IG 5, DD2 showed
tate. Eight fungal isolates produced zone of clearing on similar results to IG 1 and similarly, IG 3 and DD1 showed
PSM agar medium. Counterstaining was also performed to comparable results on different culture media so only a sin-
visualize the zone of clearing. These fungal isolates were gle representatives from them were taken into consider-
designated as IG 1, IG 2, IG 3, IG 4, IG 5, DD 1, DD 2 and ation and shown in Table 2. On the basis of morphological

Table 1 - Phytase activities for different fungal isolates.

S No. Isolates Enzyme activity (U mL-1) mean ± SD Size of clear zone (mm) mean ± SD Source of isolation (Soil)

1 IG 1 0.39 ± 0.007 28 ± 0.2 Agricultural land

2 IG 2 0.22 ± 0.002 19 ± 0.1 Vermicompost
3 IG 3 0.46 ± 0.005 32 ± 0.2 Poultry waste
4 IG 4 0.18 ± 0.002 12 ± 0.3 Pig waste
5 IG 5 0.20 ± 0.004 17 ± 0.1 Poultry waste
6 DD 1 0.32 ± 0.005 22 ± 0.6 Fish pond
7 DD 2 0.26 ± 0.006 19 ± 0.2 Agricultural land
8 DD 3 0.21 ± 0.002 14 ± 0.3 Poultry waste
320 Gontia-Mishra et al.

Figure 1 - Phytase activities for isolates IG 1 (A. niger) and IG 3 (A. awamori) at different pH (a) and temperature (b).

characters and growth pattern on different media, isolates structures (Balajee, 2009). The Isolates IG 3 and DD1
IG 1, IG 5, DD 2 were suggested to be Aspergillus niger showed mycelial characters similar to those of isolates IG
(Sharma and Pandey, 2010). Likewise, isolates IG 2 and IG 1, IG 5 and DD 2 but conidia showed some ornamentation
4 were reported to be A. fumigatus and A. terreus, respec- and were distinctly rough, hence these two isolates could be
tively whereas isolates DD1 and IG 3 showed similarity to subspecies of A. niger.
A. awamori (McClenny, 2005; Zain et al., 2009; Perrone et
al., 2011). Growth abilities of isolates were tested on Molecular characterization of fungi
CREA medium. CREA is the semi-selective media useful The PCR amplification of 18S rRNA gene was done by
for classification of various fungal cultures (Samson et al., using gene specific primers. An amplification product of
2007). On CREA, characteristics of colonial growth, pro- 1.5kb was obtained for all the isolates as shown in Figure 2.
duction of acid (turning of the medium from purple to yel- The nuclear small subunit ribosomal DNA (18S rDNA) was
low) can be used as diagnostic features. Isolate IG 1, IG 3, selected for characterization and identification of fungi
IG 4, IG 5 and DD 1, DD 2 showed moderate growth and firstly, because established universal fungal primers are
good acid having production resulting in large yellowish available based on the conserved regions of 18S rDNA, mak-
halo around the colonies on CREA medium. Similar results ing it possible to obtain the PCR products from most of the
for A. niger were reported by Samson et al. (2007), showing fungi. Secondly, the large numbers of 18S rDNA sequences
this as a characteristic feature for distinguishing biseriate are available in GenBank which makes similarity searches
species (A. niger, A. awamori and A. terreus) from uniseri- convenient. Several workers have also reported characteriza-
ate species. IG 2 showed poor growth and limited acid pro- tion of fungi based on 18S rRNA gene sequence analysis
duction on CREA medium as characteristic feature of (Smit et al., 1999; Borneman and Hartin, 2000).
uniseriate species. On the basis of 18S rRNA gene similarity isolates IG
1, IG 5 and DD 2 showed 99% sequence homology with
Microscopic characterization for definitive identifi-
Aspergillus niger (FJ262990) (Yang et al., 2012), IG 2 and
cation of the isolates was carried out. In case of isolate IG 2
DD3 showed 98% sequence homology with Aspergillus
the microscopic analysis showed that the mycelium was
fumigatus (AF648063) (Wu et al., 2003). The 18S rRNA
wide, septate and hyaline with acute angle branching. Coni-
partial sequence placed the isolates IG 3 and DD1 within
dial head was uniseriate and columnar. Isolates IG 1, IG 5
and DD 2, also showed wide, septate and hyaline mycelium
with acute angle branching but their conidial head was
biseriate and radiate and conidia were attached in chains.
Similar results were reported for A. fumigatus and A. niger,
respectively by McClenney (2005), thus isolates IG1, IG 5,
DD2 could be predicted as A. niger and IG 2 as A.
fumigatus. In microscopic analysis of IG 4, mycelium was
found to be wide, septate and conidiophores were long, co-
lumnar and hyaline. Conidial head were globose to slightly
elliptical and biseriate. Additionally, hyaline accessory
conidia were also visible on the hyphae. A. terreus is the Figure 2 - PCR amplification of 18S rRNA gene for fungal isolates Lane
only member of the genus Aspergillus that produces such M: 1 kb DNA ladder; Lane 1-7: fungal isolates IG 1-IG 5, DD1-DD2.
Table 2 - Morphological colony characteristics and sporulation pattern of fungal isolates on different culture media.

Isolate Media Colony diam (mm) Colony characters Zonation Sporulation Reaction on CREA Identified as Reference
Texture Surface colour Reverse colour
IG 1 CDA 47.33 ± 3.05 Velvety White with dusty yellow White Slightly Radially Moderate Moderate growth Aspergillus niger Sharma and
sporulating area furrowed and good acid pro- Pandey, 2010
duction (large yel-
Phytate-hydrolysing fungi

CYA 47.00 ± 1.00 Powdery White periphery with black Yellowish Radially furrowed Heavy
spores low halo around
colony)
MYA 59.00 ± 3.60 Powdery White periphery with black Cream Radially furrowed Moderate
spores at the centre
CY2S 71.00 ± 2.65 Powdery White periphery with black Cream Heavily wrinkled Heavy
spores
IG 2 CDA 34.33 ± 1.15 Velvety White with grayish green Cream Concentric zones Poor Poor growth and A. fumigatus McClenny, 2005
concentric sporulating rings low acid produc-
CYA Powdery Cream periphery grayish Yellowish Radially furrowed Heavy tion
52.00 ± 1.73
green sporulating area
MYA 47.33 ± 2.08 Powdery Cream with green periphery Yellowish Radially furrowed Moderate
and grayish green
sporulating area
CY2S 73.66 ± 3.21 Powdery Cream periphery grayish Cream Wrinkled and radi- Heavy
green sporulating area ally furrowed
IG 3 CDA 55.66 ± 0.58 Velvety White with brownish White irregularly fur- Moderate Moderate growth A. awamori Perrone et al., 2011
sporulating area rowed and good acid pro-
CYA Powdery White periphery with dark Cream Heavily radially Heavy duction (large yel-
43.33 ± 1.15
brown sporulating area furrowed low halo around
colony)
MYA 57.66 ± 2.08 Velvety White periphery with dark Cream Heavily radially Moderate
brown sporulating area furrowed
CY2S 67.33 ± 1.53 Velvety White periphery with dark Cream Wrinkled Moderate
brown sporulating area
IG 4 CDA 45.33 ± 1.15 Floccose Green periphery with green Yellow Radially furrowed Poor Moderate growth A. terreus Zain et al., 2009
sporulating area and good acid pro-
CYA Velvety White with dark green Light orange Radially furrowed Poor duction (large yel-
44.33 ± 1.53
sporulating area low halo around
colony)
MYA 55.66 ± 2.31 Powdery Green periphery with dark Peach Radially furrowed Moderate
green sporulating area
CY2S 75.00 ± 1.00 Powdery Green periphery with dark Light orange Irregularly fur- Heavy
green sporulating area rowed
321
322 Gontia-Mishra et al.

Figure 3 - Phylogenetic tree showing the relationships of the isolates to closely related fungi. The numbers at branching points refer to bootstrap values,
based on 100 replicates.

Aspergillus genera with 99% sequence homology with Conclusion

Aspergillus awamori (HQ393870) (Srinivasan et al.,
2012). Similarly, isolate IG 4 showed maximum sequence Our finding suggests that phytase production by A.
similarity with Aspergillus terreus (JN831364) (Yin et al., niger strain IG 1 and A. awamori strain IG 3 have signifi-
2012). Partial 18S rRNA sequences of all the isolates were cant values which can be exploited for industrial produc-
submitted to NCBI Genbank under the following accession tion of phytase. Moreover, this enzyme can be used in the
numbers: IG 1, JQ012799; IG 2, JQ012800; IG 3, animal feed industry for improving the nutritional status of
JQ012801; IG 4, JQ012802, IG 5, JQ012803; DD1, feed. The native fungal communities of soil which degrade
JN624277; DD2, JN624278 and DD3, JN624279. Phylo- phytate from manures and soil with subsequent release of
genetic relationship of the fungal isolates with other fungi orthophosphate making it available to plants, thus enhance
is shown in Figure 3. the benefits of manure-derived fertilizer and in combating
environmental pollution due to phytate. Additionally, phy
A. niger is well known for its phytase activity. Phy- gene from these strains could be used to develop transgenic
tase activity from A. niger has been extensively studied and plants (maize, sorghum and oat) which would in turn uti-
reported by several workers (Vats and Banerjee, 2005; lized as animal feed.
Gunashree and Venkateswaran, 2009; Soni et al., 2010).
Similarly, phytase activity from Aspergillus fumigatus has
Acknowledgments
previously been reported (Mullaney et al., 2000; Rodriguez
et al., 2000). Isolate IG 3 showed close similarity with A. IG is grateful to Department of Biotechnology, Min-
awamori and has considerably high phytase activity. A. istry of Science and Technology, Government of India,
awamori and A. terreus are less extensively studied for New Delhi for financial assistance.
phytase activity. To best of our knowledge this is the sec-
ond report of phytase production from A. awamori and A.
terreus. Report of phytase production from A. awamori and
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