Int. J. Biosci.
2015
International Journal of Biosciences | IJB |
ISSN: 2220-6655 (Print), 2222-5234 (Online)
http://www.innspub.net
Vol. 6, No. 5, p. 63-69, 2015
RESEARCH PAPER
OPEN ACCESS
Investigating the effect of different concentrations of nitrate
and phosphate on the quantity of mycosporine like amino acids
production and growth in Spirulina platensis
Sara Saadatmand*, Morteza Zamani
Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
Key words: Nitrate, phosphate, mycosporine like amino acids, chlorophyll a, carotenoids.
http://dx.doi.org/10.12692/ijb/6.5.63-69
Article published on March 09, 2015
Abstract
The present study invested on the effect of different concentrations of NaNO 3 (1.5, 2, 2.5 gl-1) and K2Hpo4 (0.04,
0.05, 0.06 gl-1) on chlorophyll a, carotenoids, fresh and dry weight measures, and the quality of mass absorption
in Spirulina platensis alga. Findings revealed a significant increase in the amount of chlorophyll a and
carotenoids in cultivation setting by increasing NaNO3 and K2HPO4, especially at 2.5gl-1NaNo3 and 0.06 gl-1
K2Hpo4 concentrations. More findings also revealed that nitrate or phosphate supplemented growth medium had
reducing effects on fresh weight; but, adversely affected the dry weight. Here, there was an increase in
concentration of NaNO3 and K2HPO4, especially for 2.5 gl-1nitrateand 0.05 gl-1phosphate concentration samples.
It should also be mentioned that the highest dry mass measures were obtained in 2.5 gl-1 NaNO3, though mass
absorption in Spirulina platensis was at the highest rate in 2 gl-1 nitrate and 0.06 gl-1 phosphate concentration
samples. Supplementary findings also indicated that quantities of mycosporine like amino acids (MAAs)
increased significantly by increasing nitrate and phosphate concentrations. Collectively, it could be concluded
that the altered composition of cultivation medium, especially macro elements like nitrate and phosphate, may
affect the composition of quasi-mycosporine like amino acids by changing nitrogen assimilation, photosynthetic
pigments content, which leads to probably higher photosynthesis and stimulating shikimate pathway.
* Corresponding
Author: Sara Saadatmand  [email protected]
63 Saadatmand and Zamani
Int. J. Biosci.
2015
Introduction
Spirulina
(Yadav et al., 2011) and play different roles in a cell
twisted
such as; protecting it against UV rays, antioxidant
mycosporine string bacteria with no pre-specified, if
molecules and osmotic regularizes (Yadav et al., 2011;
existed, width partitions (Desikachary, 1995). It is
Rastogi et al., 2010). mycosporine like amino acids,
also the richest known green food in the world with
as mentioned before, need nutritious materials to be
no cholesterol and just a little amount of fat and
produced. As a result, a decreasing nitrogen rate and
carbohydrate. The existing pigments in Spirulina
its absence in the environment where alive beings
improve the body immune system and incorporate to
live, leads to a drop in amino acids production.
its health care (Barsanti et al, 2006). The growing
Subsequently, a fall in NO3concentration decreases
importance of Spirulina has led to its growing
the amino acids production as well (Klisch and Donat,
production with commercial purposes in the last 20
2008). In this study we investigated the effect of the
years. Although a few countries use some methods to
different concentrations of NaNO3 and K2Hpo4 on
grow
it
cyanobacteria
directly
in
is
lakes
an
orderly
the
the photosynthetic pigments, fresh and dry weight
commercial algae is usually produced in huge pools
(Belay,
2002),
and the quality of MAAs mass absorption in Spirulina
and under controlled circumstances. Nowadays, it has
platensis alga for estimated MAAs production in the
reached to a mass production of 57000 tons all over
different conditions.
the world (FAO, 2006). The alga could be found at
different areas with fresh and sea water, and also used
Materials and methods
as a nourishing food for goldfish and the ones kept as
Preparing the pure samples
a pet. Although its nourishing level depends on the
Spirulina platensis cyanobacteria was prepared from
circumstances in which it grows (Belay, 2002).
fishery investigation institution, agriculture ministry,
Iran.
Nitrogen is one of the most important and highly
effective mediums used to improve Spirulina algae
Spirulina cultivation and care
growth. Here, the photosynthesis rate strongly
BG11 environment was applied to cultivate Spirulina
depends on the nitrogen rate. As a result, lack of
platensis. Cultivation mediums were kept in the
nitrogen decreases photosynthesis and accordingly
cultivation setting at 25˚C to 30˚C, while being
chlorophyll synthesis. It also is the second prevalent
radiated by 2500 incubation light for 12 hours a day.
element in Arthrospira sp. biomes (Faintuch, 1989).
For the other half a day, they were kept in darkness.
cyanobacteria use mineral nitrogen, and specifically
Three different concentrations of nitrate (1.5, 2, 2.5 gl-
nitrates and ammonium, to improve its growth.
1NaNo3)
Although they could grow up in organic nitrogen too
K2HPO4) were investigated as six experimental
(Faintuch,
groups. Each treatment was repeated six times with
1989).
Many
studies
conducted
investigating the effect of different nitrogen sources in
S.
platensis
cultivation
setting,
among
and phosphate (0.06, 0.05, 0.04 gl-1
the incubation period of 25 days.
which,
Faintuch (1989) proposed nitrate as a source
Growth analysis
producing
Samples were studied for fresh and dry weight. The
the
best
results
in
biology
mass
production.
sum weigh of the fresh alga equals: [wet filter paper
weight plus the whole alga-wet filter paper weight]
Phosphor is another important food resource for alga
and the sum weigh of the dry alga equals: [dry filter
which determines its primary production (Mastert &
paper weight plus the whole alga-dry filter paper
Grobbelaar, 1987). Indeed, the phosphor main role is
weight].
keeping mass production at a high level for
microalgae (Vonshark, 1997). Quasi-mycosporine like
To measure the dry weight, samples were kept inside
amino acids are classified as secondary metabolites
the laboratory environment for 24 hours and then
64 Saadatmand and Zamani
Int. J. Biosci.
2015
in nitrate application phase and especially at 2.5gl-1
they were weighing.
nitrate concentration, comparing to the groups where
Chlorophyll a and Carotenoids analysis
phosphate was applied (figure 2). Based on figure 3,
Chlorophyll a and Carotenoids were measured based
mass absorption also improved by increasing nitrate
on the method proposed by Lichtenthaler and
and phosphate concentration as well. Accordingly, as
wellburn (1983). To this end, 0.03g alga was
nitrate and phosphate concentration increased,
homogenized in 10ml of %80 acetone. Then, 20ml of
cyanobacteria biomass increased too, but its water
the smooth solution was investigated by visible UV
absorption potential decreased. cyanobacteria mass
spectrophotometer for its absorption rate at 470 and
weight growth in the environment also led to an
663
increase
wavelengths,
concentration
was
and
the
reported
cyanobacteria).
The
were
measure
used
to
final
substance
based
following
on
( μg / g
e q u a t i on s
Chlorophyll a
and
Carotenoids:
in
chlorophyll
a
and
carotenoids
concentration. As there was an increase in nitrate and
phosphate
carotenoids
chlorophyll
concentration,
also
a
and
chlorophyll
increased
a
and
significantly.
For
KH2PO4sample
at
0.06gl-1
concentration, the growth was faster comparing to the
other samples (figures 4&5). Mycosporine like amino
acids absorption diagram also proved that, as
comparing to the other samples, the 0.06gl-1
K2HPO4sample increased mycosporine like amino
Mycosporine
like
amino
acids
extraction
by
acids concentration.
spectroscopy
2ml of mature cyanobacteria cells was collected by a
centrifuge at 3000g for 10 minutes. Then, 2ml of
100% pure methanol was added to the solution and
incubated at 4 Co for 24 hours. The mixture then was
centrifuged at 5000g for 15 more minutes and the
supernatant was used as the extracted methanol
substance. And finally, the methanol substance
spectrum was measured at 250-500 nanometers by
UV/ visible spectrophotometers (S i n h a
et
al,
1 9 9 5 ).
Fig. 1.
Fresh weight-NaNO3 and K2HPO4different
samples for Spirulina platensis atP≤0.05.
Data analysis
The collected data was analyzed by one way ANOVA
and Duncan testing method using SPSS (ver. 18).
Results
According to figure 1, as the concentration of nitrate
and phosphate increased in cultivation setting,
comparing to the control group samples, the fresh
weight
of
Spirulina
cyanobacteria
decreased
significantly. Although the dry weight measurement
brought about exact opposite results; due to that, an
increase in nitrate and phosphate concentration led to
an increase in dry weight too. This was also apparent
65 Saadatmand and Zamani
Fig. 2. Dry weight- NaNO3 and K2HPO4 different
samples for Spirulina platensis.
Discussion
Nourishment conditions is one of the key elements
Int. J. Biosci.
affecting
growth
environments
and
(Vanshak
2015
production
and
in
microalga
Richmond,
1988;
study were in the same line with the research carried
out by Mastert and Grobbelaar(1987),proving that the
Faintuch et al., 1991). Variations in nitrogen source
highest Spirulina growth was presented at
and its quantity in cultivation setting restrict the
1
2.5gl-
microalga growth and changes the pigments and their
nitrogen in Spirulina production (Raoof et al., 2006;
chemical compounds (Mostert and Grobbelaar, 1987).
Mastert and Grobbelaar, 1987).
concentration of KNO3, and showing the key role of
Based on the results, as long as NaNO3and
K2HPO4concentrations
increase
in
cultivation
setting, chlorophyll a and carotenoids concentrations
increase significantly. Chlorophyll concentration in
S.platensis also increases by increasing nitrogen
concentration in cultivation setting (Rangel – Yagui et
al., 2004; Madkour et al., 2012; Raoof et al., 2006;
Rodriques et al., 2010). Findings are also at the same
line with that of the other previously conducted
studies working on the same research project.
S.platensis chlorophyll magnitudes depend highly on
Fig. 3. Mass absorption- NaNO3 and K2HPO4
combination of the cultivation setting, and specially
different samples for Spirulina platensis.
type and concentration of nitrogen (Rodriques et al.,
2010;
Rangel
–
Yaguietal,
2004).
Increasing
phosphate concentration led to a significant growth in
chlorophyll a production by S. platensis (Celekli et al,
2009; Raoof et al, 2006Lodi et al. 2005). Based on
the results brought about in some studies, the
produced carotenoid extent in S.platensis wasn’t
affected significantly by variant growth conditions
(Madkour et al., 2012).Although Madkour and et al.
(2012) proved that in a dim light condition together
with nitrogen, the carotenoid extent increased,
incorporating to prove the present study results
regarding carotenoid production growth in the
mentioned conditions. Our findings also showed that
the dry weight and mass absorption in Spirulina pla
tensis alga developed significantly as a result of an
increase in NaNO3 and K2HPO4 concentration. A
majority of the dry weight was reached at 2.5 gl1concentration
of NaNO3. cyanobacteria biomes
production is a highly complicated process with a
number of effective elements that for a proper
growth, needs some key combinations as nitrogen
and phosphor (Madkour et al., 2012). The assimilated
nitrogen at first is used by micro-organisms as a
resource to help the cell growth, and then the rest is
being saved in the form of organic materials such as
protein (Rodrigues et al., 2010).Results of the present
66 Saadatmand and Zamani
Fig. 4. Chlorophyll a- NaNO3 and K2HPO4 different
samples for Spirulina platensis.
The high biomes extent is being identified by
pigments and specially chlorophylls (Rangel –Yagui
et al, 2004). Biomes production degree was highly
improved by using cyanobacterias at 2 and 2.5 nitrate
concentrations (Celekli and Yaruzatmaca, 2009;
Raoofetal. 2006). And also 2.5 and 3 gl-1nitrate
concentrations increased biomes production output
comparing to 2gl-1concentration. Bulgakov and Evichl
(1999) found out that a 10 to 5 nitrate to phosphate
proportion increases cyanobacteria
production,
although the optimum ratio differs from one
condition to another. For example, S. platensis,
compared to Scenedemus obliguus, prefers a smaller
proportion of nitrate to phosphate (Celekli et al.,
Int. J. Biosci.
2015
2008). The optimum nitrate concentration (2.5 gl-1)
MMA production. On the other hand, the existence of
that increases biomes production by the help of S.
ammonium
platensis might be due to the proportion of nitrate to
environments increases MAA production (Klisch and
phosphate (Bulgakov and Levich, 1999; Radmann et
Donat, 2008). Findings of the present study also
al.,2007; Madhyastha and Vatsala, 2007).
proved the same fact once again. mycosporine like
in
red
algae
and
cyanobacteria
amino acids biosynthesis is done through shikimic
acid path. It depends on glycolytic and phosphate
pentose path to produce the necessary raw materials
of this path; accordingly, a growing photosynthesis
rate could increase mycosporine like amino acids
production too (Ghosh et al., 2012). Chlorophyll a is a
touchstone
for
intensity
of
microalgae
cells
photosynthesis; as a result, increasing chlorophyll a
could be used as the optimum condition for the
Fig. 5. Carotenoids- NaNO3 and K2HPO4 different
samples for Spirulina platensis.
intensity of microalgae cells photosynthesis (Chen et
al., 2011). Accordingly, increasing chlorophyll a,
which brings about a higher photosynthesis rate,
increases mycosporine like amino acids production.
MAAs, together with playing a protective role against
UV
harmful
rays
and
neutralization
of
their
antioxidant effect, also control osmotic pressure,
because in organisms of the beings living in salt-rich
sea water, a high concentration of MAAs was found
(Yakovleva et al., 2004). And because NaNO3 and
K2HPO4 salt concentrations in the experiment
environment increased, the mycosporine like amino
acids amino acids grew up too.
Fig. 6. Mycosporine like amino acids- NaNO3 and
K2HPO4 different samples for Spirulina platensis.
References
Barsanti L, Gualtieri P. 2006. Algae: Anatomy,
Those cyanobacterias which at the same time are
Biochemistry and Biotechnology. CRC press, London,
exposed to UV and background active photosynthesis
1-158 p.
radiation (PAR; 400-700nm) have some different
http://dx.doi.org/10.1111/j.1529-8817.2007.00335.x.
defensive mechanisms helping them to reduce the
direct or indirect effects of UV beams. One of these
Belay A. 2002. The potential application of
defensive methods is synthesizing the compositions
Spirulina as a nutritional and therapeutic supplement
such as mycosporine like amino acids
in health Management. The Journal of the American
like that
absorb UV rays (Sinha et al., 2007). Findings of the
Nutraceutical Association 5, 26-50.
present research shed some light on the fact that as
nitrate
and
phosphate
concentrations
increase,
Bulgakov
NG,
Levich
AP.
1999.
The
mycosporine like amino acids concentration also
nitrogen:phosphorus ratio as a factor regulating
increase as well. There are some elements affecting on
phytoplankton structure. Archiv for Hydrobiologie
MMA production. To name some, we can refer to food
146, 3–22.
resources, lack of nitrogen in the environment, as well
as downturn of NO3 concentration, which undermine
67 Saadatmand and Zamani
Celekli A, Yavuzatmaca M. 2009. Predictive
Int. J. Biosci.
modeling
of
2015
biomass
production
by
2012.
Production
and
nutritive
value
of
Spirulinaplatensis as function of nitrate and NaCl
Spirulinaplatensis in reduced cost media. Egyptian
concentrations. Bioresource Technology 100, 1847–
Journal of Aquatic Research 38, 51–57.
1851.
http://dx.doi.org/10.1016/j.ejar.2012.09.00.3
http://dx.doi.org/10.1016/j.biortech.
Chen X, Yvonne Goh Q, Tan W, Hossain I,
Chen WN, Lau R. 2011. Lumostatic strategy for
microalgae cultivation utilizing image analysis and
chlorophyll
a
content
as
design
parameters.
Bioresource Technology 102, 6005–6012.
and energetics of photosynthetic microorganisms in
photobioreactors: application to Spirulina growth.
Advances in Biochemical Engineering/Biotechnology.
59, 155–194.
of agricultural research. New Dehli.Fao. 2006.
Fishstat software.
producao de biomass a apartir de tres cianobacterias
empregando distintas fontes nitrogenadas. Master of
Science Thesis, University of Sao Paulo, Brazil.
Chisti
Y,
Banerjee
U.C.
2012.
30, 1425–1431.
http://dx.doi.org/10.1016/j.biotechadv.2012.03.001.
Klisch M, Donat H. 2008. Mycosporine like amino
acids and marine toxins-the common and the
Marine
Drugs.
ISSN
1660-3397.
http://dx.doi.org/10.3390/md20080008.
of
219–233.
http://dx.doi.org/10.1016/0144-4565(87)90061-8.
microalga Spirulina platensis in open raceway ponds.
Aquaculture 265, 118–126.
http://dx.doi.org/10.1016/j.aquaculture.2007.02.00.
2005.
Fed-batch
Arthrospira
mixotrophic
(Spirulina)
platensis
(Cyanophyceae) with carbon source pulse feeding.
Annals of Microbiology 55, 181–185.
Madkour FF, El-WahabKamil A, Nasr SH.
68 Saadatmand and Zamani
Raoofa B, Kaushika BD, Prasannab R. 2006.
Formulation
of
a
low-cost
medium for
mass
production of Spirulina. Biomass and Bioenergy 30,
Rangel-Yagui CO, Godoy Danesi ED, Monteiro
de Carvalho JC. 2004. Chlorophyll production
from Spirulina platensis: cultivation with urea
addition
by
fed-batch
process.
Bioresource
Technology 92, 133–141.
Rastogi RP, Sinha RP, Singh SR, Hader DP.
2010.
Photoprotective
compunds
from
marine
organism. Journal of Industrial Microbiology 37, 537-
Lodi A, Binaghi L, Faveri DD, Carvalho JCM,
cultivation
quality in outdoor mass algal cultures. Biomass 13,
537–542.
Production of shikimic acid. Biotechnology Advances
A.
of nitrogen and phosphorus on algal growth and
1
Faintuch BL. 1989. An_anlise comparativa da
Converti
24, 301–305.
Optimization of the repeated batch cultivation of
Desikachary TV. 1995. Cyanophyta. Indian council
different.
photophysical conditions. Biomolecular Engineering
Radmann EM, Reinehr CO, Costa JAV. 2007.
http://dx.doi.org/10.1007/BFb0102299.
S,
production in Spirulina fussiformis in different
Mostert ES, Grobbelaar JU. 1987. The influence
Cornet JF, Dussap CG, Gros JB. 1998. Kinetics
Ghosh
Madhyastha HK, Vatsala TM. 2007. Pigment
558.
http://dx.doi.org/10.1007/s10295-010-0718-5.
Rodrigues MS, Ferreira LS, Converti A, Sato S,
Carvalho JCM. 2010. Fed-batch cultivation of
Arthrospira (Spirulina) platensis: Potassium nitrate
and ammonium chloride as simultaneous nitrogen
sources. Bioresource Technology 101, 4491–4498.
Int. J. Biosci.
2015
http://dx.doi.org/10.1016/j.biortech.2010.01.054.
Vonshak A, Richmond A. 1988. Mass production
of blue-green alga Spirulina – an overview. Biomass
Sinha RP, Kumar HD, Kumar A, Hader DP.
15, 233–247.
1995. Effects of UV-B irradiation oh growth, survival,
pigmentation and nitrogen metabolism enzymes in
Yadav S, Sihna RP, Tyagi MB, Kumar A. 2011.
cyanobacteria. Acta protozoologica 34, 187-192.
Cyanobacterial Secindary Metabolites. International
Journal
Vonshark
(Arthospira):
A, Ltd. 1997. Spirulina platensis
Physiology,
Cell
Biology
of
Pharma
and
Bio
Sciences
2,
http://dx.doi.org/10.1111/j.13652672.1992.tb01858.x.
and
Biotrchnology. Journal of Applied Ohycology 9, 295-
Yakovlera IM, Hidaka M. 2004. Diel fluctuations
296.
of mycosporine- like amino acides (MAAs) in shallow
http://dx.doi.org/10.1016/0144-4565(88)900.59-5.
waters scleractinian corals. Marine Biology 145, 863873.
69 Saadatmand and Zamani