Phytase From Some Strains of Thermophilic Blue-green Algae.
Khanuengkan Klanbut,1,* Yuwadee Peerapornpisarn,1 Chartchai Khanongnuch,2 and Masaharu Ishii,3
Applied Algal Research Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang
Mai, 50200, Thailand, 2Department of Biotechnology, Faculty of Agro-industry, Chiang Mai University, Chiang
Mai, 50200, Thailand, and 3Department of Biotechnology, The University of Tokyo, Tokyo, Tokyo 113-8657,
Four isolates of thermotolerant Blue-green algae including Synechococcus lividus
Copeland strain SKP50, S. lividus strain DSK74, S. bigranulatus Skuja and
Chroococcidiopsis thermalis Geitler, isolated from the hot spring in northern Thailand, were
cultivated at 50oC under illumination appoximately 2500 lux in the phosphate-free medium
containing 0.1% (w/v) calcium phytate as a sole phosphorus. The phytase activity was found
in intracellular fraction of Synechococcus lividus strain SKP50, S. lividus strain DSK74 and
S. bigranulatus with the maximum level at 1.83, 1.68 and 1.77 mUml-1 respectively, after
cultivation for 18 days, while 0.99 mUml-1 was detected from Chroococcidiopsis thermalis at
20 days, while the phytase activity were not found in culture broth. The intracellular phytase
was found at the starting period of algal growth characteristics and decreased simultaneously
with the increasing of growth. The phytase activity in culture broth was found in trace amount
in all isolates. The crude intracellular phytase showed the optimal pH at 600C and stable up
Heterotrophic cultures may provide an alternative means for the large-scale production
of algal products. Four isolates of thermotolerant Blue-green algae were cultivated for 30 days
in darkness at 50oC in the phosphate-free medium containing 0.1% (w/v) calcium phytate
supplemented with 0.5% (w/v) glucose and 0.02% (w/v) casamino acids as their sole carbon
and energy source and in the presented or absented of 1%(w/v) Na2CO3 every times that
collected samples. The growth rate was considerably slower than that obtained under
phototrophic conditions.The highest phytase activity was found in intracellular fraction of
Synechococcus lividus strain DSK74 in the absented of 1%(w/v) Na2CO3 with the maximum
level at 0.1635 mUml-1 , after cultivation for 18 days.
Phytate is one of antinutritional factor (ANF) found in many cereals especially the
cereals used in feed ingredient (Yoon et al. 1996). Due to monogastrics including pigs and
poultry lack the enzyme needed to digest phytate in feed, leading to a low phosphorus
availability condition and result in high phosphorus content in excreted feces (Pandey et al.
2001). This causes an environmental problem, because phytate in manure can pose a serious
phosphorus pollution problem contributing to the eutrophication of surface waters of the
world in areas where monogastric livestock production is intensive (Pen et al. 1993; Volfova
et al. 1994). Thus, phytase (myo-inositol hexakisphosphate phosphohydrolase; E.C.220.127.116.11),
the enzyme hydrolyze phytic acid to myo-inositol and inorganic phosphate, are expected to
solve the problem described above. Furthermore, phytase supplemented feed can directly
reduce the antinutritional effect for monogastrics, leading to an improvement of animal
performances. Because of great industrial importance of phytase, there is ongoing interest to
isolate the new organisms for producing novel and efficient phytases. (Kim et al. 1998).
The phytase enzyme was found in various kinds of microorganisms including fungi,
yeast and bacteria (Pandey et al. 2001). Recently, the commercial phytase was obtained from
Aspergillus ficumn (Gibson et al. 1987) and Bacillus subtilis (Powar et al. 1982; Shimizu
1992). Due to the elevated high temperature in feed producing process, the high
thermostability character of phytase is desirable.
Heterotrophic culture may provide a cost-effective, large-scale alternative method of
cultivation for some microalgae that utilize organic carbon substances as their sole carbon and
energy source(Chen 1996). However, to date, the very few reports of such process for
microalgal cultivation have mostly been on lab-scale work. For example, the unicellular
cyanobacterium Synechocystis sp. PCC6803 can grow heterotrophically in complete darkness
in BG11 with 5 mM glucose and 5µM DCMU (Sigma) (Kong et al.2003). Two facultative
cyanobacteria, Aphanocapsa sp. Strain 6714 grown on Allens medium with 0.2% glucose and
Chlorogloea fritschii No.1411/1a grown on medium C with 0.1M sucrose were wrapped with
foil to exclude light and maintained and grown at 340C (Espardellier et al. 1978)
In this paper, we reported about phytase from selected thermophilic blue-green algae
isolated from hot springs in northern Thailand, including Synechococcus lividus Copeland
SKP50, S. lividus DSK74, S. bigranulatus Skuja and Chroococcidiopsis thermalis Geitler.
These strains have capable of growth at 500C or higher, which may be use as an important
source of new thermostable phytase. And examines some aspects of phytase after growth
under heterotrophic conditions.
MATERIALS AND METHODS
Four thermophilic algal strains including Synechococcus lividus Copeland strain
SKP50, S. lividus strain DSK74, S. bigranulatus Skuja and Chroococcidiopsis thermalis
Geitler, were isolated from hot springs in northern Thailand and maintained in BG11
medium. (Panyoo W. 2002). Then cultivated in steriled inorganic phosphorus omitted BG11
(IPO-BG11) medium containing 0.1% (w/v) calcium phytate as a sole phosphorus and carbon
source at 500C under fluorescence light (Hai et al. 1999). To avoid an effects of phosphorus
from seed culture, the culture supernatant was aseptically removed after centrifugation with
6000 rpm for 10 min and algal cells were washed with 0.85% (w/v) NaCl before inoculation.
Growth of S. lividus SKP50, S. lividus DSK74 and S. bigranulatus Skuja were monitored by
measuring an optical density at 560 nm. Due to the natural change in morphology during
cultivation, growth of C. thermalis was monitored by measurement of cell dry weight
(Panyoo W. 2002)
Enzyme preparation and assay
Cultures were sampling and centrifuged with 10,000 rpm at 40C for 10 min and the
supernatant was separated and used as the extracellular fraction. To obtain the cell-free
extract, algal cell was suspended in 5 ml of 0.1M Tris-HCl buffer pH7.0 and disrupted by
ultra-sonication (60 kHz) at 40C for 2 min (30 x 4 sec). Phytase activity was determined by
measuring the increasing rate of inorganic orthophosphate (Pi) using the ascorbic acid method
as described by Fiske and Subbarow (1925). A reaction mixture containing 100 l enzyme
solution, 400 l 2 mM sodium phytate in 0.1 M Tris-HCl buffer pH 7.0 and 2mM CaCl2, was
incubated at 500C for 30 min and terminated by 500 l of 15% trichloroacetic acid (TCA).
The released inorganic phosphate was measured by incubation with 4 ml of color reagent
(1:1:1:2 ratio of 6 N H2SO4 : 2.5% ammonium molybdate : 10% ascorbic acid : H2O) at 500C
for 30 min. Subsequently, the absorbance at 820 nm was measured. One unit of phytase
activity was defined as the amount of enzyme required to liberate 1 mole of phosphorus per
minute under the assay condition.
Optimal pH and thermostability
The enzyme solution obtained as described above was determined for phytase activity
at various temperature including 30, 40, 50 and 60oC. For thermostability test, the enzyme
solution in 0.1M Tris-HCl buffer pH 7.0 was incubated at various temperatures for 1 hour and
the remaining activity was determined at 50oC as described above.
Heterotrophic culture condition
Four isolates of thermophilic Blue-green algae were cultivated for 30 days in darkness
at 50 C in the phosphate-free medium containing 0.1% (w/v) calcium phytate supplemented
with 0.5% (w/v) glucose and 0.02% (w/v) casamino acids (Vaara et al. 1979) and in the
presented or absented of 1%(w/v) Na2CO3 every times that collected samples. Measuring an
optical density and cell dry weight as described above.
Enzyme preparation and assay
As described above.
Growth characteristic of all four strains cultivated in IPO-BG11 medium were
obtained as showed in Figure1. Three isolates of Synechococcus spp. and C. thermalis Geitler
showed the similar growth pattern. Comparison to growth of S. spp. obtained from general
BG-11 medium, optical density at 560 nm in IPO-BG11 medium exhibited 12 times lower
since only 1.0 value of OD560 obtained from IPO-BG11, but those from medium BG11
medium reached approximately 11.0 - 12.0 of OD560 value at the same cultivation time.
However, after 30 days, algal growth obtained from both media still continuously increased.
In case of C. thermalis, dry weight from BG11 medium was 7.5 times higher that from IOP-
Figure 1. Growth curve in IOP-BG11 and BG11 medium of Synechococcus spp. (A) and
Chroococcidiopsis thermalis Geitler (B) : S50, S. lividus Copeland SKP50; S74, S. lividus
DSK74; S55, S. bigranulatus Skuja ; C, Chroococcidiopsis thermalis Geitler
Consideration for relationship between growth and the detectable phytase from those
isolates as showed in Figure 2, the phytase activity was found in intracellular fraction since
after one week of cultivation and reach the maximum level at 18 days in case of S. spp. Then,
the activity was decreased, while algal growth was gradually increasing.
Phytase activity (mU ml -1)
0 10 20 30 40 50 60
Cultivation time (days)
Figure 2. Intracellular activity of phytase from the selected algal isolates : S50,
Synechococcus lividus Copeland SKP50; S74, S. lividus DSK74; S55, S. bigranulatus Skuja ;
C, Chroococcidiopsis thermalis Geitler
The optimal temperature and thermostability of crude intracellular phytase from
S. lividus Copeland strain SKP50 was preliminary studied and the results was showed in
Figure 4. The enzyme showed the optimal temperature at 600C and the enzyme stability was
found up to 600C.
Relative activity (%)
40 45 50 55 60 65 70
Thermostability of SKP 50 Temperature optimization of SKP 50
Figure 3. Optimum temperature and thermostability of intracellular from Synechococcus
lividus Copeland SKP50
Growth characteristic of all four strains cultivated in heterotrophic condition was
considerably slower than that obtained under phototrophic conditions. Three isolates of
S. spp. and C. thermalis showed the similar growth pattern like in phototrophic condition.
Figure 4. Growth curve in heterotrophic conditions of Synechococcus spp. and
Chroococcidiopsis thermalis Geitler : S5 0, S. lividus Copeland SKP50; S74, S. lividus
DSK74; S55, S. bigranulatus Skuja ; C, Chroococcidiopsis thermalis Geitler : Na, in the
presented of 1%(w/v) Na2CO3; H2O, in the presented of H2O instead
Phytase activity (mU ml )
0 5 10 15 20 25 30 35
Cultivation time (days)
Figure 5. Intracellular activity of phytase from 4 isolates in heterotrophic conditions:
S50, S. lividus Copeland SKP50; S74, S. lividus DSK74; S55, S. bigranulatus Skuja ; C,
Chroococcidiopsis thermalis Geitler : Na, in the presented of 1%(w/v) Na2CO3; H2O, in the
presented of H2O instead
The phytase activity in heterotrophic conditions was found in intracellular fraction too.
The relationship between growth and phytase activity was similar to in IOP-BG11 that
showed in Figure 5. However, after one week of cultivation and reach the maximum level at
18 days in case of S. lividus strain DSK74 in the absented of 1%(w/v) Na2CO3 with the
maximum level at 0.1635 mUml-1.
According to the relationships between algal growth and the detectable phytase
activity, we can suggested that at the initial phase of growth, the algal strains needed phytase
for hydrolyzing of phytate to release inorganic phosphate which was essential for growth.
However, the phytase level found in all isolates could be detected in only 8-10 days period,
cultivated day 7-18 of Synechococcus spp. and 12-20 of C. thermalis Geitler, and started to
decrease gradually. It could be suggested that all algal strains tested in this experiment needed
phytase just for initiation of growth and the enzyme was not required any more after some
inorganic phosphates were enzymatic released from calcium phytate supplemented in IOP-
To explain the reason why the phytase activity was found only in IOP-BG11 medium,
this may be caused by an induction effect from phytate as found in B. subtilis (Powar and
Jaganathan 1982), Bacillus sp. DS11 (Kim et al. 1998) and also in Klebsiella sp. No.PG-2
(Shah and Parekh 1990). However, an induction effect for phytase from the selected strains
may be stimulated by the phosphorus limited condition occurred in IPO-BG11, that forced the
algal strains to synthesize the enzyme phytase for surviving. Moreover, from our result, there
was no phytase activity found in the extracellular fraction. That was different from the other
phytases found from various fungi and bacteria that most phytases were extracellular such as
phytase from Aspegillus oryzae (Shimizu 1993), Bacillus sp. DS11 (Kim et al. 1998) and
Enterobacter sp.(Yoon et al. 1996). However, phytase from E. coli was found in the
periplasmic fraction (Greiner et al. 1993) and that from Klebsiella oxytoca MO-3 was also
reported as an intracellular enzyme (Jareonkitmonkol et al. 1997)
Due to the thermostability is one important property for the enzyme applied in feed
industry and the optimal temperature phytase from S. lividus Copeland SKP50 was 600C and
having the thermostability up to 600C. This was almost the same with phytase from A. ficuum
NRRL3135 which was stable at 60oC for 2 hours with 30% activity loss (Ullah 1988).
However, the phytase activity found from our selected strains is very low, that have to be
further more studied for an optimization or may be also an improvement in the molecular
level. From this preliminary result, we concluded that the thermostable phytase was found in
intracellular fraction from the selected isolates of thermophilic blue-green algae and the
optimization for enzyme production is in progress.
Furthermore, the heterotrophic growth rate of this organisms were considerably
slower than that obtained under phototrophic conditions, the enzyme activities had less than
too. Three isolates of S. spp. and C. thermalis showed the similar growth pattern like in
phototrophic condition. However, the phytase level found in all isolates could be detected in
only 12-15 days period, cultivated day 6-18, and started to decrease gradually. The phytase
activity was found in intracellular fraction too. After one week of cultivation and reach the
maximum level at 18 days in case of S. lividus strain DSK74 in the absented of 1%(w/v)
Na2CO3 with the maximum level at 0.1635 mUml-1. Although, Na2CO3 gave a prefer carbon
source to cyanobacteria even in phototrophic or heterotrophic conditions, but in this case S.
lividus DSK74 didn’t want that excess. We concluded that this strain can growth under this
condition with only organic carbon and energy source.
This work was partially supported by Faculty of Science and Graduated School, Chiang
Mai University, Chiang Mai.
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