ABSTRACT The use of Spirulina as a nutraceutical has been popularized owing to its high essential amino acid, vitamin, carotenoid, chlorophyll content, antioxidant and antiinflamatory properties. This organism can also bioaccumulate and biosorb essential and non essential heavy metals. These properties have been exploited in this study using the organism, Spirulina platensis ARM 728. The fortification of the biomass in different concentrations of Selenium (10 ppm, 40 ppm and 100 ppm) and Zinc (1 ppm, 5 ppm and 10 ppm) was carried out and an increased content of proteins, chlorophyll, carotenoids, SOD, CAT and total antioxidant activity was seen. The biosorption and desorption capacity of the organism for antimony at 80 ppm was also seen with fair results. Keywords: antioxidant properties, bioaccumulation, biosorption, heavy metals, Spirulina fortification.

1. Comparioson of Dry weight, protein, chlorophyll and carotenoid
content
0
2
4
6
8
10
12
14
Control
Se10
Se40
Se100
Zn1
Zn5
Zn10
dry weight
protein
chlorophyll
carotenoids
Comparison of CAT and SOD activity
0
1
2
3
4
5
6
7
Control
Se10
Se40
Se100
Zn1
Zn5
Zn10
CAT
SOD
EFFECT OF HEAVY METALS ON Spirulina platensis ARM 728
Sanaa Farooqui and Krutika Desai
Department of Microbiology, Mithibai College, Vile Parle (W), Mumbai – 400056
INTRODUCTION:
Spirulina (Arthrospira) is widely used as a nutraceutical.
Spirulina has found applications as an excellent source of
amino acids, vitamins, β- carotene, photosynthetic
pigments and other bioactive and antioxidant compounds.
Its extracts show therapeutic ability and anti-cancer
properties (Abd El-Baky, et al., 2008). The organism has
high tolerance levels to various heavy metals like Se, Sb,
Zn, Cu etc. It has the ability to bioaccumulate and biosorb
these metals. Essential metals like Se4+
, Zn2+
, Mg2+
have,
up to certain concentrations, a stimulatory effect on the
growth of the alga in terms of protein, chlorophyll,
carotenoid content and enhancement of the activities of
various antioxidant enzymes like SOD, CAT etc. This
property has lead to the production of fortified biomass rich
in essential heavy metals. Another attribute of the
organism is its ability to biosorb large amounts of heavy
metals via surface receptors in live or dead form. This
property finds applications in remediation, environmental
monitoring and precious metal recovery.
MATERIALS AND METHODS:
The following experiments were carried out
MIC of Cu2+
, Sb3+
, Se4+
and Zn2+
.
Growth profile and protein profiling in presence of heavy
metals.
Growth in varying concentrations of Zn and Se and
checking for increase in
>Dry weight, protein content (Lowry, et al., 1951)
and chlorophyll content.
>Total carotenoids.
>Total antioxidant activity via ABTS and DPPH
methods.
> Activity of enzymes SOD and CAT.
Biosorption and desorption of antimony at its MIC.
CONCLUSION:
Spirulina platensis has high Co tolerance. Growth in metal stress
conditions causes certain changes in the alga which allow it to adapt itself
to the stress, by production of stress proteins, increased activity of stress
related antioxidant enzymes, pigments and total antioxidant property
increase (Chen, et al., 2008). The fortification of the biomass with Se4+
(40
ppm) and Zn2+
(5 ppm) brought about an increase in protein content by 1.6
and 1.43 times respectively. An increase in the SOD, CAT and antioxidant
activity by 1.6, 2.21 and 1.24 times with Se4+
and an increase by 1.96,
2.95 and 1.6 times with Zn2+
fortification was seen. This data shows that
the malleable growth conditions of the alga can be exploited to produce
Se4+
and Zn2+
rich biomass with enhanced nutraceutical quality.
The studies with Sb3+
showed that the metal is biosorbed to a fair extent
and does not desorb easily. This factor is important to consider when
using Spirulina as a food and animal feed supplement.
REFERENCES:
•Abd El-Baky, H.H., El Baz, F.K., El-Baroty, G.S., (2008). Characterization of nutraceutical compounds in blue green
alga Spirulina maxima. J. Medicinal Plants Research. 2(10): 292-300.
•Chen, T.F., Zheng, W.J., Wong, Y.S., Yang, F., (2008). Selenium- induced changes in activities of antioxidant
enzymes and content of photosynthetic pigments in Spirulina platensis. J. Integrative Plant Biology. 50(1): 40-48.
•Li, Z., Guo, S., Li, L., (2003). Bioeffects of selenite on the growth of Spirulina platensis and its biotransformation.
Bioresource Technology. 89: 171-176.
•Lowry, O., Rosebrough, N., Farr, L., Randall, R., (1951). Protein measurement with the Folin Phenol reagent. J. Biol.
Chem. 193: 265-275.
ACKNOWLEDGEMENTS:
I would like to thank my guide, Dr. Krutika Desai and the teaching and non-teaching staff of Microbiology Dept. Mithibai
College. I also want to express my gratitude to the Pharmatech Dept. NM college, Chemistry Dept. CB Patel College
and IIT, Powai for letting me use their instruments. And I would like to thank my family friends and colleagues.
RESULTS AND DISCUSSION:
The MIC of the following heavy metal, on the basis of
decreasing chlorophyll content and absorbance after 10
days of incubation
Comparison of the biomass quality of Spirulina with and without fortification
with heavy metals- Selenium and Zinc
The organism was grown in modified Zourruk’s media with heavy metal Se4+
in 10,
40 and 100 ppm concentration and Zn2+
in 1, 5 and 10 ppm concentration. During
the exponential growth phase i.e. after 12 days, the biomass was harvested and
analyzed for dry weight, protein content, chlorophyll content, carotenoids, total
antioxidant activity (ABTS and DPPH methods), SOD and CAT activities. These
were compared with a control growing in the absence of heavy metals. The
following were the results:
Figure 1: Comparison of Dry weight, protein, chlorophyll and carotenoids
content
The cells from Zn2+
1 and 5 ppm and Se4+
10 and 40 ppm showed an increase in dry
weight, protein content and chlorophyll content as compared to the control (Fig 1).
The increase in proteins could be attributed to the role of zinc in protein synthesis
and formation of selanocysteines. The increase in dry weight could be due to the
increase in the overall bioactive components produced in the organism under stress.
Figure 2: Comparison of total
antioxidant activity with ABTS method
Figure 3: Comparison of CAT and SOD
activities
The amount of biomass (in mg) required to scavenge 50 % of the free radicals
ABTS+
(IC50) under experimental conditions is lesser in the test samples indicating
greater antioxidant activities in Zn2+
5, Se4+
10 and Se4+
40 ppm. The increase in the
antioxidant activity is due to the increase in the composition of carotenoids,
chlorophyll, phycobillins and also due to the metals themselves. The increase in the
SOD and CAT activities could be as a response to the stress conditions. It has been
shown that cell damage prevention due to stress is avoided by a combination of
CAT-SOD activities. Se4+
has also shown to have a protective action over catalase.
Under higher concentrations of heavy metals, the cells showed reduced
growth. This could be due to intolerance to metals at that concentration
leading to cell wall degradation, inhibition of enzyme system,
photosynthetic respiration and chlorophyll content. These results tallied to
some extent with work done by (Li, et al., 2003) and (Chen, et al., 2008).
Metal
MIC
Sb3+
Cu2+
Se4+
Zn2+
80 ppm 1 ppm >250 ppm 10 ppm
The protein profile (PAGE-silver staining) of the alga
under metal stress confirmed the production of many low
molecular weight proteins
Cu
1ppm
Se 100
ppm
Zn 10
ppm
Control Se 40
ppm
Sb 80
ppm
Zn 5
ppm Control
Biosorption and desorption of Antimony
Contaminated sediments and waters contain high levels of Sb3+
ranging
from 8.5–90.4 ppm. Thus the biosorption was checked at 80 ppm.
Maximum biosorption was seen after 10 minutes (31.93 % ). A slight
decline in rate was seen after 20 minutes which again increased after 20
minutes. The recovery of the heavy metal after desorption was quite low,
highest being at 10 minutes (12.6 % ), probably owing to the higher amount
of the metal biosorbed by the organism after 10 minutes. Thus it is difficult
to separate the heavy metal from the biosorption sites. The results also
indicate that a large amount of heavy metal tends to bioaccumulate inside
the organism which is obviously harmful in the long run.
Time(minutes) 0
Biosorbed (%)
Desorbed (%)
28.82 28.98 31.9326.40327.9727.3
10 20 40 605
4.68 3.97 12.6 0.82 8.8
Table 2: percentage Sb 3+
biosorption and desorption
Comparison of the total antioxidant activity using
ABTS method
0
0.5
1
1.5
2
Control
Se10
Se40
Se100
Zn1
Zn5
Zn10
Inhibitionconcentration(IC50)
IC50
I
12.3