Effects of Chemical Parameters on Spirulina platensis Biomass
Production: Optimized Method for Phycocyanin Extraction
The micro alga, Spirulina is a rich source of protein, which is
used as a protein supplement for humans, chicks and also in aquaculture.
Among the cultures, CS-1 registered maximum biomass production and S-20
showed highest biomass production among the local isolates. Optimum temperature
of 35°C was the best for maximum biomass production of S. platensis
cultures. Among the cultures CS-1 culture, put forth maximum biomass production
at 35°C. The biomass production of all S. platensis cultures
was maximum at pH 10.0. Among the cultures, CS-1 recorded maximum biomass
at pH 10.0. S. platensis culture S-20 showed highest biomass production
among the local isolates. S. platensis cultures were grown under
different light wave lengths ranging from 340-700 nm and observed that
it grows best in red light but later on there was no significant difference
between the biomass produced under red and normal white lights. S.
platensis culture CS-1 has shown the highest chlorophyll, carotenoids
and phycocyanin and protein contents. When the extracted protein was resolved
on a 15% SDS-PAGE gel, the cultures have polypeptide subunits ranging
from the molecular weights 20 to 95 kDa. The liquid nitrogen method was
found to be the best by extraction higher quantity of phycocyanin from
all S. platensis cultures. Among the cultures, S. platensis
culture CS-1 recorded the highest phycocyanin content and among the local
isolates SM-2 showed the highest pigment content. SDS-PAGE analysis of
phycocyanin pigment revealed two characteristic bands with a molecular
weights of 14.3 and 20.1 kDa approximately for α and β subunits.
The micro algae, Spirulina seemed to be a good protein source
(60-70%) and is comparable with the milk proteins (Shelef and Soeder,
1980); further research revealed that it is a rich source of vitamins,
essential amino acids, minerals and β-carotene. It also find its
immense use as anti cancer formulations, diabetes control wound treatment
and to promote skin metabolism (Surekha Rani and Uma Bala, 2006). This
is widely exploited in the manufacturing of beauty products such as anti-wrinkling,
anti-pimple creams, facemasks and high protein shampoos. It has
been commercially cultivated for its bluish green pigment, called phycocyanin,
which can be used as a natural colorant for food, cosmetics etc (Liang
et al., 2004). Though several thousand algal forms are available
in nature, only a few are amenable to technology. This is based on their
ability to grow in synthetic media, case of separation and stability to
drying and importance of their chemical constituents and finally the cost
effectiveness of the whole system (Venkatraman, 1983). In the light of
the above facts, the present study was undertaken to determine the chemical
parameters of S. platensis with response to organic and inorganic
MATERIALS AND METHODS
Five cultures of S. platensis were used for the present study
(CS-1, SM-2, S-10, S-20 and Sp). Three S. platensis cultures viz,
SM-2, S-10 and S-20 were isolated from field soils of paddy breeding station
and lands of TNAU, Coimbatore and the biomass production and biochemical
constituents were compared with standard cultures CS-1 and SP obtained
from algal laboratory, Madurai. The collected soil samples were inoculated
into the sterile Zarrouk`s Medium (ZM) (Hung et al., 2002) in 100
mL of 250 mL Erlen Mayer flasks and inundated in laboratory growth chamber.
During the experimental period, the light intensity of 3000 lux with 16/8
h alternate light and dark cycles at 32°C was maintained. The flasks
were examined for the algal growth periodically. They were then observed
under microscope. The medium normally used for S. platensis cultures
was Zarrouk ‘s medium with NaHCO3 as the main carbon
source.(Binaghi et al., 2003) Other carbon sources viz., Na2CO3,
D-glucose, Mannitol, (NH4)2CO3, CaCO3,
sucrose and urea were used as the sole carbon source. These compounds
were added to the media on equimolar concentration basis. The flasks were
incubated for 30 days.
To know the effect of temperature on growth, the S. platensis
was exposed with different temperatures viz, 15, 20, 25, 30, 35 and 40°C.
The incubation conditions were same as mentioned earlier. The growth of
S. platensis cultures was tested at 6.0, 8.0, 10.0, 12.0 and 14.0
pH levels. The solution was adjusted to acidic pH with 1 N HCl and to
alkaline pH was with 1 N NaOH. The incubation conditions were same as
mentioned earlier. The growth of S. platensis was tested under
different wavelengths of light sources viz., red (640-740 nm), green (520-570
nm) and blue (450-520 nm) with white light (350-740 nm) as the control
(Gevorgiz and Golovnya, 2002). The different wavelengths of light were
maintained using different colored electric bulbs. The incubation conditions
were same as mentioned earlier and the biomass was estimated after10,
20 and 30 days after inoculation. The cells were subjected to sonication
incubated a solicitor. The cultures were filtered through pre weighed
Whatman No. 42 filter paper. The filter papers with the cyanobacterial
cultures were oven dried at 80°C for 24 h and weighed. The biomass
production was calculated based on the different incubated weight and
expressed as mg dry weight per mL of the culture suspension.
The biochemical constituents were estimated at a regular interval of
10 days after inoculation up to 30 days by following the standard procedures.
The total chlorophyll-a content of the S. platensis cultures was
estimated as per the method of Tailing and Driver (1961). The amount of
carotenoid pigment present in different S. platensis cultures was
estimated by following the method of Siegelman and Kycia (1976). Four
different methods (Liquid nitrogen, Freezing and thawing, Sonication and
Using lyses buffer and lysozyme methods) were followed for efficient extraction
of phycocyanin from S. platensis. The cultures were taken at a
regular interval of 10 days i.e., 10, 20, 30 days after inoculation. The
total protein content of the S. platensis cultures was estimated
by following the method of Lowry et al. (1951). The separation
of protein was done by SDS-PAGE following the method described by Maniatis
et al. (1989).
The effect of carbon sources on the biomass of S. platensis cultures
was estimated at three regular intervals, i.e., 10, 20 and 30 Days after
Inoculation (DAI) and the data are presented in Table 1.
Among the different carbon sources, Na2CO3 significantly
enhanced the biomass production while the least biomass production was
observed with CaCO3. In general, the biomass produced by all
the cultures was significantly higher when NaH2CO3
of the medium was replaced with Na2CO3 irrespective
of the inoculation periods. There was also significant difference between
the biomass produced when (NH4)2CO3 and
CaCO3 are added to the growth medium as sole carbon sources.
Maximum biomass was recorded at 30 days after inoculation. Among the cultures,
CS-1 produced the highest biomass (22.50 mg mL-1) while S-10
registered a least biomass (14.15 mg mL-1) at 30 DAI when Na2CO3
was used as a sole carbon source. The biomass produced by the cultures,
SM-2 and S-10 under different carbon source was on par with each other
at all three intervals. It was also found that S. platensis was
unable to utilize the organic carbon source like D-glucose, mannitol sucrose,
||Effect of carbon source on biomass production of S. platensis
The biomass production of S. platensis cultures was evaluated
to different temperatures, ranging from 20-40°C at three regular intervals
i.e., 10, 20 and 30 days after inoculation. In general, all the cultures
produced maximum biomass at 30 and 40°C (Table 2) It was noticed that
when the laboratory cultures were kept at 45°C for up to 24 h, they
were unable to grow but growth was resumed when the cultures were brought
back to 35°C. The highest biomass (17.22 mg mL-1) was recorded
by CS-1 culture at 35°C on 30 DAI and the least biomass (15.98 mg
mL-1) was recorded in the case of S-10 at same temperature
The biomass produced by S. platensis cultures was calculated at
different pH levels ranging from 6.0-12.0 at three regular intervals,
i.e., 10, 20 and 30 days after inoculation and the results are presented
in Table 3. In general, all the cultures produced highest biomass at pH
10.0 of 30 DAI. There was no significant difference between the biomass
produced at 8.0 and pH 12.0 levels. Least biomass was recorded when the
pH was maintained at 6.0. Maximum biomass (16.92 mg mL-1) and
the minimum biomass (9.65 mg mL-1) were recorded in the case
of CS-1 and S-10 respectively at 30 DAI when the pH was 10.0.
The biomass produced by S. platensis cultures was estimated under
different light wavelengths at three regular intervals i.e., 10, 20 and
30 DAI and the results are shown in Table 4. The cultures were grown under
different light sources, viz; red, blue and green along with normal white
light as control. In general, biomass produced by all the cultures was
maximum under red light at 30 DAI and the least biomass was recorded when
the cultures were grown under green light. There was no significant difference
between the biomass produced by CS-1, S-20 and S. platensis cultures
at 30 DAI under red light. Among the cultures, S. platensis
has shown highest biomass (20.89 mg mL-1) and the least biomass
(13.96 mg mL-1) by SP and SM-2 respectively at 30 DAI under
red light. However, there is not much variation in biomass production
between red and white lights.
||Effect of temperature on biomass production of S. platensis
||Effect of pH and biomass production of S. platensis cultures
The chlorophyll-a content of S. platensis cultures was estimated
at three regular intervals i.e., 10, 20 and 30 DAI and the data are presented
in Table 5. In general increase in chlorophyll-a content of S. platensis
cultures was observed as the inoculation period increases. Highest chlorophyll
content (9.723 μg mL-1) was recorded in CS-1, which is on
par with a local isolate, S-20 and the least chlorophyll content (8.526
μg mL-1) were observed in S-10.
||Effect of light wavelength on biomass production of S. platensis
||Chlorophyll-a content of S. platensis cultures
||Carotenoids content of S. platensis cultures
The carotenoid content of S. platensis cultures was measured at three
regular intervals i.e., 10, 20 and 30 days after inoculation and the results
are presented in Table 6. In general, the carotenoid content increased gradually
for all cultures with increase in the inoculation period. Highest carotenoids
content (2.813 μg mg-1) was recorded in CS-1 followed by
2.775 μg mg-1 in the culture SP. The least carotenoids content
(2.262 μg mg-1) was observed in case of SM-2 at 30 DAI.
The phycocyanin produced by S. platensis cultures was estimated
at 10, 20 and 30 days after inoculation using different methods, viz.,
liquid nitrogen, freezing and thawing, sonication and lysozyme methods
and the data are given in Table 7. In general, highest phycocyanin was
extracted from all the cultures when liquid nitrogen method was used.
Least phycocyanin was extracted when sonication method was used. Among
all the cultures, CS-1 culture has shown the highest phycocyanin content
(110.20 μg mg-1) at 30 DAI by liquid nitrogen method followed
by Sp, Sm-2, S-10 and the least phycocyanin content (65.12 μg mg-1)
was shown by S-20. When the pure phycocyanin pigment was resolved on 15%
gel, these were two bands observed, one having the molecular weight greater
than 14.3 kDa and another band giving the molecular weight greater than
||Phycocyanin content (μg mg-1 dry wt) of S. platensis
||Protein content of S. platensis cultures
The total protein content of different S. platensis cultures was
estimated at 10, 20 and 30 days after inoculation and the data are given
in Table 8. In general, protein content increased as the inoculation period
increased. However, the highest protein content (642.12 μg mg-1)
was recorded in the case of CS-1 culture and the least protein content (593.70
μg mg-1) was recorded in the case of S-10 at 30 DAI. The
total protein was isolated from different cultures of S. platensis.
The results have clearly indicated that the total protein content differ
widely among all the cultures with the molecular weight ranging from nearly
20 kDa to more than 95 kDa. All the cultures produced almost similar bands.
S. platensis has commercial importance due to overall nutritional
qualities, especially high protein and vitamin contents, particularly
B12. Various strains of S. platensis have been grown in various
nutrient media in order to have high yield with low cost inorganic nutrients.
Zarrouk`s medium has almost universally been adopted for S. platensis
before Venkatraman (1983) replaced the costly chemical of Zarrouk`s medium
(Zm) with commercial fertilizer (NPK 15:15:15). Chandgothia and Srivastava
(1994) replaced the A5 nutrient solution by 20% soil extract and 0.2%
NaCl for growing S. subsalsa. In case of S. labyrinthoformis,
when the 70% medium was substituted with groundnut shell ash extract (GSAE)
the growth was on par with Zarrouk`s medium, while the lost input was
reduced by 73%. Sharma and Srivastava (1997) experimented with S. subsalsa
and observed that BGSE added Zarrouk`s medium enhanced 40% density of
In the present study, an attempt was made to grow S. platensis
under different source of carbon, which are added to the medium on the
basis of equimolar concentration. Different carbon source that are used
in the experiment include ammonium carbonate, calcium carbonate, glucose,
mannitol, sodium carbonate, sucrose and urea, while the growth medium
with sodium bicarbonate act as a control. Maximum biomass production was
observed when sodium bicarbonate in the normal Zarrouk`s medium was replaced
with sodium carbonate, which is a cheaper chemical. This result is in
agreement with Shelef and Soeder (1980), who stated that carbonate and
bicarbonate source of carbon are best for the maximum biomass production
of S. platensis. S. platensis has completely failed to show any
growth in the medium, when NaHCO3 was replaced with any organic
source of carbon and within ten days after inoculation, the death of the
culture was observed. This might be due to the reason that photoautotrophs
cannot show any growth in the medium with organic carbon as a sole carbon
source (Ciferri, 1983) and another reason might be that the insufficient
quantity of the carbon needed for specific growth of S. platensis,
if supplied in organic form on equimolar concentration. This is in accordance
with the findings of Joardan (1998) who estimated that 0.5 kg of sugar
is needed to produce one kg biomass of S. platensis, when sugar
is added as sole carbon source. He also suggested the use of cheaper sugar
cane juice in large-scale cultivation. Chen and Zhang (1997) also observed
maximized growth when Zarrouk`s medium was supplemented with glucose (2
g L-1). This confirms the fact that glucose can be used as
an additional carbon source to improve the growth, but not as a sole carbon
source when added on equimolar concentration basis. This is also in tune
with the findings of Snoog (1980), Torre et al. (2003) and Binaghi
et al. (2003) who observed that 2 kg of glucose or 3.5 kg of acetic
acid are needed to produce 1.0 kg of Chlorella dry matter.
Temperature is undoubtedly the most fundamental factor for all living
organisms, which affects all metabolic activities. The optimum temperature
for growth of S. platensis is 30-35°C. However, in tropical
regions in summer; the temperature may go beyond 40°C. The study on
effect of high temperature on photochemical activity of S. platensis
in such situations will help in optimizing its productivity. A detailed
study on the response of S. platensis M-2 strain to temperature
was performed by Torzillo and Vonshak (1994). They observed 28 and 23%
of the optimum growth at the extreme 10°C minimum and 50°C maximum
In the present investigation S. platensis cultures were grown
at different temperatures and the highest biomass was obtained when S.
platensis was grown at 35°C. This could be due to the increased
activity of metabolic enzymes at that temperature, ultimately leading
to higher biomass production; this is in conformity with the findings
of Vonshak (1997). There was no growth when the cultures were grown above
40°C or blow 20°C. But the culture resumes its growth when once
it has been brought to the normal conditions. This might be due to some
mechanism, which must have taken place before the original photosynthetic
activity was reached (Vonshak, 1997).
Cyanobacteria are ubiquitous and able to grow in aside range of pH from
acidic to alkaline (pH 4-12). However S. platensis prefers only
alkaline pH. This assumes significance in the context of minimizing the
microbial contaminations, both in vitro and in vivo conditions.
In the present study, the biomass production of S. platensis has
been evaluated under different pH levels. Maximum growth of S. platensis
was observed at a pH of 10.0. The reason could be attributed to optimal
activity of all the enzymes needed for photosynthesis and respiration,
at this pH, however, at high acidic and alkaline pH, there was a decreased
activity of the photosynthetic enzyme RUBP-carboxylase and an increased
activity of respiratory enzymes glucose 6-phosphate dehydrogenate and
isocitrate dehydrogenate (Kaushik and Sharma, 1997), leading to the reduction
in the biomass production. These are the key enzymes in providing energy,
carbon skeleton and pyridine nucleotide biosyntheses. But some specific
Spirulina strains can adapt themselves to salinity conditions,
when grown in a medium containing 70 g L-1 Nacl (Zotina et
al., 2000). One mechanism proposed for the ability of cyanobacteria
to liquor salinity is the formation of internal osmoticum by the accumulation
of inorganic ions or organic solutes like carbohydrates polyols and quaternary
Light is the most important factor affecting photosynthetic organisms.
Due to the prokaryotic nature of S. platensis, light does not affect
the differentiation and development processes. Nevertheless, S. platensis,
like many other algal grown photoautotrophically depends on light as its
main energy source. Light considered in terms of photoperiod, quality
and intensity, is of paramount importance to micro algae. It is obviously
significant as energy source for photosynthesis, but the fact that light
fluctuate tremendously in both space (depth and latitude) and time (daily
and seasonally) suggests that light may often be a limiting factor for
In the present study, S. platensis growth was tested under different
wavelengths of light using a mineral medium and bicarbonate as the only
carbon source. The different light sources include red (λmax 640-740
nm), blue (λmax 450-420), green (λmax 520-570 nm) and white
light (λmax 350-740 nm). It was found that the biomass production
under red and white lights was significant initially, but later it was
non significant. The reason could be attributed to the maximum absorption
of chlorophyll initially under red light, but after some time the red
light is converted into white light, which has inhibitory effect on photosynthesis
resulting in reduced biomass. Similar results were obtained by Vonshak
et al. (1996) who started that the growth of S. platensis
became saturated at a range of 150-200 μmol m-2 sec-1
and this is about 10-15% of the total solar radiance at 400-700 nm. This
value is highly dependent on growth and correlates with the chlorophyll
to biomass concentration. Almost similar results were drawn by many workers
(Jones and Myres, 1963; Venkatraman, 1983).
The photo systems in cyanobacteria contain only chlorophyll a and not
chlorophyll b. Cyanobacteria, though it is considered as a prokaryote,
it is similar in performing photosynthesis as eukaryotes (Carr and Whitton,
1982). They contain photosynthetic pigments like chlorophyll a, phycobilin
proteins and carotenoids in their filaments. Phycobilin proteins located
in the phycobilinsomes may account for about 40-50% of the total protein
in cyanobacteria (Goedheeri, 1976).
Carotenoids are important components of photosynthetic apparatus of vegetative
cells serving as additional pigment (Giovannoni et al., 1988).
They protect chlorophyll molecules against photo destruction and oxidation
by molecular oxygen (Krinsky, 1979). When both S. platensis is
trichomes and isolated thylakoids were exposed to elevated temperatures
only above 60°C for 10 min in the dark, there was a significant degradation
and bleaching of phycocyanin and allophycocyanin, but not that of chlorophyll
a (Babu et al., 1992). An individual chlorophyll molecule absorbs
only two or three photons per second even under direct solar illumination.
No living organism could grow if its reaction center did not have light
harvesting antenna molecules excess.
In the present study the chlorophyll and carotenoid content of S.
platensis cultures were evaluated at 10, 20 and 30 days after inoculation
and both CS-1, SP cultures were found to be on par with each other and
contents increased with the increase in the incubation period. Among the
local isolates, S-20 has shown highest chlorophyll content and carotenoid
content and was within the range of the value prescribed for S. platensis
Phycocyanin is gaining importance as natural pigments in food, drug and
cosmetic industries, as an alternative to currently used synthetic colour.
The organisms of commercial importance are S. platensis and phormidium
(Glazer, 1981). The blue, water-soluble pigment, which may be up to 10%
of the dry weight of S. platensis, stimulates the immune system
and is also used for the treatment of ulcers and haemorrhoidal bleeding
(Richmond, 1986). It is also plays a major role in immuno diagnostics
apart from its use as food colour (Glazer and Stryer, 1984). Phycocyanin
has been extensively studied due to their involvement in photosynthesis
as major accessory pigment (Myers and Kratz, 1955; Goedheeri, 1976). The
stability of this pigment even at acidic pH of 4.5 has now been exploited
in food products and soft drinks (Venkatraman, 1983).
In the present investigation, four different extraction procedures were
employed for better extraction of phycocyanin pigment from S. platensis
culture. Out of these four methods, highest phycocyanin was extracted
with liquid nitrogen method followed by freezing and thawing method and
lysozyme method, which were on par with each other and the least phycocyanin
was extracted with sonication method. The reason could probably due to
the efficient breakage of cell wall with liquid nitrogen; releasing maximum
amount of the pigment and the cell wall might not be broken completely
with sonication leading to reduce phycocyanin with that method. Similar
results were obtained by Chen and Zang (1997) who reported that freezing
at -20°C; repeated thawings for one hour followed by sonication has
increased the phycocyanin content.
The extracted and purified phycocyanin pigment of S. platensis
cultures, when run on 15% SDS-PAGE gel, typically resolved into two bands
of molecular weights with 14.3 and 20.1 kDa approximately, which might
correspond to the α and β subunits of the pigment. This result
was in tune with Boussiba and Richmond (1980) who observed that the molecular
weight of α subunits and β subunit are 20.5 and 23.5 kDa for
C-phycocyanin and 18 and 20 kDa, respectively for allophycocyanin in S.
Algae as a source of protein have a long history. Micro algae are no
more cheap protein sources. But fairly high value low volume biological
material (Benemann, 1990). S. platensis has been the subject of
a number of basic and applied investigations (Richmond, 1986). This photosynthetic
microorganism can be harvested, processed and used as a natural food for
millennia. Protein Efficiency Ratio (PER) value of S. platensis has
been reported to be higher than vegetable, cereals and soya proteins.
The value of the digestibility coefficient, biological values etc. are
duly marginally lower than casein. Supplementation of S. platensis
with cereals like rice and wheat on isoproteinic levels improved the protein
quality (Anasuya Devi and Venkatraman, 1983). Detailed haematological
and histological tests on chronic toxicity of S. platensis fed
rats (Patterson, 1996) revealed no abnormalities. In the seventies there
were Protein Advisory Guidelines (PAG) of the united nations when micro
algae were considered as a protein source and lots of efforts were made
to meet the safety requirements of a Single Cell Protein (SEP).
In the present study the protein content of S. cultures was measured
at 10, 20 and 30 days after inoculation and it were observed that the
protein content has increase with the increase in inoculation period.
The highest protein content was observed in case of CS-1 cultures and
among the local isolates; SM-2 has shown highest protein content of nearly
60%. These results are in accordance with Bhattacharjee (1970), who stated
that S. platensis contains 60-70% protein and about 50, 000 kg
of protein per hectare could be produced annually.
When the total proteins were extracted from the whole cells of S.
platensis and resolved on a 15% SDS-PAGE gel, it showed a wide range
of polypeptide subunits, the molecular weight of which ranged from 20
to 95 kDa. It is in corroboration with the finding of Chadgothia and Srivastava
(1994) who stated that the total protein content of S. platensis
cultures varied widely when resolved on SDS-PAGE gel. The consistency
in the protein bands of S. platensis cultures either grown in laboratory
or an open condition was further reported by them.
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