JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL SCIENCES

Chakravarthy Kavitha, Senthil Chinnasamy, Sailendra Bhaskar,Ramasamy Rengasamy.

Effect of sodium alginate on growth and lipid production of Botryococcus braunii kutzing
for biodiesel production. Journal of pharmaceutical and biomedical sciences (J Pharm Biomed Sci.)
2013 October; 35(35): 1802-1807.

The online version of this article, along with updated information and services, is located on
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Journal of Pharmaceutical and Biomedical Sciences (J Pharm Biomed Sci.), Member journal.
Committee of Publication ethics (COPE) and Journal donation project (JDP).

ISSN NO- 2230 7885

CODEN JPBSCT
NLM Title: J Pharm Biomed Sci.

Chakravarthy Kavitha, Senthil Chinnasamy, Sailendra Bhaskar,Ramasamy Rengasamy.

Research article
Effect of sodium alginate on growth and lipid production of Botryococcus
braunii kutzing for biodiesel production
Chakravarthy Kavitha*1, Senthil Chinnasamy2, Sailendra Bhaskar2,
Ramasamy Rengasamy1.
Affiliation:1Centre

for Advanced Studies in Botany, University

of Madras, Guindy campus, Chennai-600025,
Tamilnadu, India.
2Aban
Infrastructure
Private
Limited
(Biotechnology
Division),
Chennai
60008,
Tamilnadu, India.
The name of the Department and Institution to
which the work should be attributed:Department of Pharmacology,
Centre for Advanced Studies in Botany, University
of Madras, Guindy campus, Chennai-600025,
Tamilnadu, India.
Authors contributions:
The major work was done by the Corresponding
author, rest guided and supported for this work.

*Corresponding author:

C. Kavitha.
CAS in Botany, University of Madras,
Guindy campus, Chennai-600025.Tamilnadu, India.
Telephone: 8754886646.
Tel/Fax: +914422353309/22352494.

Abstract:
Botryococcus braunii is a green colonial
microalgae commonly found in fresh water,
lakes and ponds. Due to its high lipid and
hydrocarbon content, it is widely recommended
for biodiesel production. The present study
deals with the growth, lipid content, and fatty
acid methyl ester (FAME) of Botryococcus
braunii AP102 grown at different concentration
of sodium alginate viz. 4mg, 8mg, 12mg, 16mg,
and 20mg amended with Chu-13 medium and
compared with control in order to find out the
efficacy of sodium alginate in the growth and
lipid production of B. braunii. The maximum
growth and lipid production was obtained at
16mg concentration and it is most effective
when compared to control. Thus sodium
alginate helps greatly in promoting the growth
of B.braunii for biodiesel production.

Key Words: Botryococcus braunii; Fatty acid;

Lipid; Mass cultivation; Sodium alginate.

Article citation:Chakravarthy Kavitha, Senthil Chinnasamy, Sailendra Bhaskar,Ramasamy Rengasamy. Effect of sodium
alginate on growth and lipid production of Botryococcus braunii kutzing for biodiesel production. Journal of
pharmaceutical and biomedical sciences (J Pharm Biomed Sci.) 2013 October; 35(35):1802-1807. Available at
http://www.jpbms.info

INTRODUCTION

icroalgae are a diverse group of

prokaryotic
and
eukaryotic
photosynthetic microorganisms that
grow rapidly due to their simple structure. They
can be employed potentially for biofuel production
in an economically effective and environmentally
sustainable manner and has the number of
advantages, including higher photosynthetic
efficiency, higher biomass production, and higher
growth rates, as compared to other energy crops1.
Microalgae with high oil productivities are desired
for producing biodiesel. Depending on the species,
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microalgae produce different kinds of lipids,

hydrocarbons, and other complex oils2,3.
The genus, Botryococcus, Kutzing has drawn the
attention towards scientists in recent years due to
its considerable amount of hydrocarbon4. It is one
of the renewable sources for the production of bio
fuel. This alga is characterized by a conspicuous
ability to synthesize and accumulate wide variety
of lipids. These lipid substances include numerous
hydrocarbons, i.e. highly reduced compounds
comprising only carbon and hydrogen as
elements5,6 and a number of specific ether lipids7,8.

ISSN NO- 2230 7885

CODEN JPBSCT
NLM Title: J Pharm Biomed Sci.

Chakravarthy Kavitha, Senthil Chinnasamy, Sailendra Bhaskar,Ramasamy Rengasamy.

Alginic acid and its salts (alginates) occur mainly

in marine brown algae (Pheophyta) comprising
most of their polysaccharides and averaging 40%
of the dry weight9. Alginate occurs in the cell walls
of seaweeds as a mixed salt with the major cations
being Na, Ca, Mg, and K together with a number of
minor metal counterions10. Ishii et al.11 observed
that alginate oligosaccharides, produced by
enzymatic degradation of alginic acid mainly
extracted from brown algae, significantly
stimulated hyphal growth and elongation of
arbuscular mycorrhizal (AM) fungi and triggered
their infectivity on trifoliate orange seedlings.
Alginates, as well as agars and carrageenans, are
characterized by unique functional properties.
Thickening and gelling are the most important of
their functions. Over the past decade, alginates
have become widely adopted as dietetic food
hydrocolloids. Sodium alginate was introduced
into the US Pharmacopoeia as early as 1938.
Sodium salt of alginic acid is well water-soluble;
due to its high molecular weight (from 100 to 500
kDa, depending on the raw material), it forms
viscous solutions. In the food industry, sodium
alginate is used as thickeners and stabilizers in the
production of fruit candy, sweets, soft drinks, and
in juice settling12. Several investigators have
concluded that the alginate oligomers have the
growth promoting activity in higher plants and
microbes.
In the present study, different concentrations of
sodium alginate were amended with chu-13
medium in order to find out its efficacy in growth
and lipid production of B.braunii and mass
cultivation of B.braunii using the selected
concentration of alginate in an open raceway pond
for biodiesel production.

MATERIALS AND METHODS

Microalgae
The Botryococcus braunii culture AP102
(Accession no: JQ585724) was obtained from Algal
culture collection, CAS in botany, University of
Madras, Chennai. It was grown in modified CHU13 medium (Composition : KNO3- 371mg, K2HPO480mg, MgSO4.7H2O-200mg, CaCl2.6H2O-107mg,
Ferric Citrate
- 20mg, Micronutrients; H3BO3 2.86g/L, MnCl2.4H2O -1.81g/L, ZnSO4.7H2O 0.22g/L, Na2MoO.2H2O - 0.39g/L, CuSo4.5H2O0.08g/L, Co (NO3)2.6H2O- 0.05g/ L) amended with
different concentrations of sodium alginate viz.,
4mg, 8mg, 12mg, 16mg, and 20mg under
laboratory conditions kept under 30E m-2 s-1
light intensity, 12/12 light dark cycle and at
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241C. The study was carried out for a period of

30days and all the growth parameters were
analyzed and recorded at every 5 days interval.
Determination of pigment13
Five ml of culture sample was taken and
centrifuged at 5000 rpm for 10 min and the
supernatant was discarded. The algal pellet was
then added with 5mL of 80% acetone and
homogenized in a sonicator. Then it was covered
with black paper and kept overnight at 4C. The
sample was then centrifuged at 5000rpm for 10
minutes. The supernatant was collected and the
optical density was measured at 644.8 , 661.6
and 470 in Milton Roy UV - Visible
spectrophotometer.
Extraction and estimation of total protein14
Five ml of algal culture was taken and centrifuged
at 5000 rpm for 10 minutes. The pellet was
homogenized in 5ml of 0.1M sodium phosphate
buffer at pH 7.0 in a sonicator and then
centrifuged at 5000 rpm for 10 minutes. The
supernatant was taken for the estimation of total
protein. To 0.2 ml of sample protein 5ml of
CBB reagent (100 mg of CBBG-250 dissolved in
50ml of 93% ethanol. To this 100 ml of 85%
Phosphoric acid was added and diluted to 1000 ml
with glass distilled water) was added and mixed
thoroughly. The absorbance was read at 595
nm against a reagent blank . The amount of
protein was calculate by using a standard graph
with Bovine Serum albumin ranging from 10 to
100 g ml-1.
Extraction
and
estimation
of
total
carbohydrate15
Five ml of algal culture was taken and centrifuged
at 5000 rpm for 10 minutes. The pellet was
homogenized with 5ml
of
0.1M sodium
phosphate buffer at pH 6.8 in a sonicator and
then centrifuged at 5000 rpm for 10 minutes .
The supernatant was collected for the estimation
of carbohydrate. To the 1ml of sample 1ml of 5%
phenol and 5ml of H2SO4 was added and mixed
thoroughly. The solution was allowed to stand at
room temperature for 30 minutes. The Optical
Density was read at 490nm. Standard graph was
prepared with different concentrations of Dglucose ranging from 10 to 100gml-1.
Extraction and estimation of total lipid 16
Five ml of culture was taken and centrifuged at
5000 rpm for 5minutes. The pellet was

ISSN NO- 2230 7885

CODEN JPBSCT
NLM Title: J Pharm Biomed Sci.

Chakravarthy Kavitha, Senthil Chinnasamy, Sailendra Bhaskar,Ramasamy Rengasamy.

homogenized in a sonicator with 6ml of

chloroform: methanol (2:1). It was then
transferred to a separating funnel and added with
2ml of 0.9% NaCl solution and mixed well. This
mixture was left undisturbed for overnight. Then
from the lower chloroform phase, 0.5ml was
collected in a clean vial and the solvent was
allowed to evaporate at room temperature and the
pellet was collected. To the pellet 0.5ml of
concentrated sulfuric acid was added and mixed
well. The tubes were closed with glass marbles
kept in a boiling water bath for 10min and
allowed to cool at room temperature. To 0.2ml of
sample 5ml of vanillin reagent (0.2g vanillin in
80ml 0f orthophosphoric acid and 20ml of distilled
water) was added and mixed well. It was allowed
to stand for 30minutes and the colour developed
was read at 520nm. Standard graph was prepared
using cholesterol ranging from 5.0 to 50g/ml and
the values are expressed as gml-1.
Mass cultivation of Botryococcus braunii in open
raceway pond
The Botryococcus braunii was mass cultivated in
an open raceway pond in Modified CFTRI medium
(Urea -0.4 g-L-1, DAP - 0.016 gL-1, Potash - 0.234
gL-1, NaHCO3 1 gL-1, MgSO4 - 0.5 gL-1) amended
with optimized sodium alginate concentration at
pH 7.5 for a period of 15 days. The experiment
was conducted in an open raceway pond of 2000L
capacity. An initial inoculum of 200L of B.braunii
grown in mini raceway pond was transferred to
1800L of Modified CFTRI medium .The culture was
mixed using paddle wheel fixed to the pond in
order to prevent settling and also to increase the
Carbon dioxide concentration. The experiment
was conducted for a period of 15days and at every
5days interval the pigments, total protein,
carbohydrates and lipids were analyzed. The
biomass was harvested for further study.
Harvesting
The biomass was harvested on 15th day through
auto flocculation, shade dried for 3 days and
pulverized using mixer- grinder. A known quantity
of dry biomass was taken and extracted for total
lipid using Chloroform: Methanol 2:1 in Soxhlet
apparatus.
Total lipid extraction and fatty acid analysis
Ten grams of dried algal sample was taken and
extracted using chloroform: methanol (2:1) and
the lipid content was quantified gravimetrically.
The lipid sample was dissolved in benzene and 5%
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methanolic hydrogen chloride (95ml of chilled

methanol and 5ml of acetyl chloride) and vortexed
thoroughly. The mixture was refluxed for 3hour at
65 C and 5% NaCl was added. The FAME (Fatty
Acid Methyl Ester) was extracted using hexane
and the hexane layer was washed with 2%
potassium bicarbonate and dried over anhydrous
sodium sulfate. The FAME was analyzed using GCMS (Perkin-Elmer, Turbomass Gold, Mass
Spectrometer) equipped with FID using SPB-1
(Poly-dimethysiloxane) capillary column (30m
0.32mm ID 1m film thickness) with a temperature
programming 130-280 C at a rate of 2 C/min. The
FAME was identified by comparing their
fragmentation pattern with authentic standards
(Sigma) and also with NIST library17.

RESULTS
Effect of sodium alginate on Chlorophyll a, b,
and carotenoids production of B.braunii under
laboratory conditions:
The effect of sodium alginate on pigment content
of B. braunii AP102 strain was studied. The alga
when grown at different concentrations of sodium
alginate showed maximum growth at the
concentration of 16mg. The maximum amount of
chlorophyll a 20.09mgL-1 chlorophyll b 11.03mgL1 and Carotenoids 8.20mgL-1 were observed at
16mg concentration on 20th day whereas in
control the maximum amount of chlorophyll a
9.62mgL-1, Chlorophyll b 6.53mgL-1 and
carotenoids 5.173mgL-1 was observed on 10th, 25th
and 25th day respectively (Figure 1, 2 &3). The
concentration above 20mg retards the growth of
microalgae and hence the experiment was
restricted up to 20mg. Yokose18 reported that
alginate has the ability to promote the growth rate
of Nanochloropsis oculata during the exponential
phase and showed increased cell density in
stationary phase.
Effect of sodium alginate on protein,
carbohydrate and lipid production of B.braunii
under laboratory conditions:
The total protein, carbohydrate and lipid content
of B.braunii were also found to be increased when
the medium is amended with sodium alginate. The
highest protein content of 14.17mgL-1 was
recorded at 12mg concentration on 25th day
which was higher than that of control 10mgL-1 on
15th day. The carbohydrate showed maximum of
26.27mgL-1 at the concentration of 12mg on 20th
day where as in control only 14.43mgL-1 was
obtained on 20th day. The maximum lipid

ISSN NO- 2230 7885

CODEN JPBSCT
NLM Title: J Pharm Biomed Sci.

Chakravarthy Kavitha, Senthil Chinnasamy, Sailendra Bhaskar,Ramasamy Rengasamy.

production of 200mgL-1 at 16mg concentration

was observed on 20th day which was higher when
compared to control 69.80mgL-1 on 25th day
(Figure 4, 5 & 6). The lipid content was slightly
decreased at 20mg concentration. This reveals

that the sodium alginate has the optimum

concentration to enhance the lipid production of
B.braunii.

Figure 1. Effect of sodium alginate on chlorophyll a production

of B.braunii

Figure 2. Effect of sodium alginate on chlorophyll b production

of B.braunii

Figure 3. Effect of sodium alginate on carotenoids production of

B.braunii

Figure 4. Effect of sodium alginate on protein production of

B.braunii

Figure 5. Effect of sodium alginate on carbohydrate production

of B.braunii

Figure 6. Effect of sodium alginate on lipid production of

B.braunii

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ISSN NO- 2230 7885

CODEN JPBSCT
NLM Title: J Pharm Biomed Sci.

Chakravarthy Kavitha, Senthil Chinnasamy, Sailendra Bhaskar,Ramasamy Rengasamy.

Effect of sodium alginate on the growth of B.braunii under open raceway pond:
The B.braunii when grown with different concentration of Sodium alginate under laboratory conditions
revealed best growth and lipid production at the concentration of 16mg. Hence the above concentration
of sodium alginate was selected for mass cultivation. The B.braunii was mass cultivated in an open
raceway pond with modified CFTRI medium amended with sodium alginate. The algal biomass was
harvested, dried and analyzed for total lipid, total carbohydrate, total protein and total ash content. The
lipid content in the B.braunii dry biomass shows up to 18% (w/w). The carbohydrate and protein content
showed 40% and 17% (w/w) respectively. The results were shown in (Table1).
Table 1. Biochemical Composition of B. braunii dry biomass
Parameters
Open raceway
pond
Total protein (%)
17
Total carbohydrate (%)
40
Total lipid (%)
18
Total ash (%)
25

Fatty acid methyl ester (FAME) analysis

The fatty acid composition of B.braunii biomass was
analyzed through GC-MS. The fame analysis revealed
the presence of oleic, palmitic, lauric, stearic, linoleic,
linolenic, elaidic, lignoceric, margaric and myristic acid
(Table 2). Among that Oleic acid (C18:1) and Palmitic
acid (C16:0) were found to be the major fatty acids from the B.braunii oil. The oleic acid contains 41.63%
and the palmitic acid contains 19.12%. Similar observations were made in B.braunii by Rai et al. 19 in
which the oleic acid constitutes 38.5% and palmitic acid 17.6%. Dayanandha et al.20 reported that the
lipid content in organism would be 22% (w/w) of those the oleic acid and palmitic acid acquires the
major percentage of 22.3% and 40.6% respectively. The biomass to biodiesel conversion showed 22%
(w/w) in this study.
Table 2. Fatty acid profile of B. braunii AP102

DISCUSSION

Fatty Acid

Alginates are natural polysaccharide which is abundant

in nature. Alginates have wider spread application in
the food and beverage, pharmaceutical and
bioengineering industries21. Alginates are the major
components of brown seaweed cell walls made up of Dglucuronic acid and D-mannuronic acid units22. The
brown alga sargassum is the main source of sodium
alginate and it is eco friendly in nature23. In the present
study different concentration of sodium alginate was
tested to enhance the growth and lipid production of
B.braunii. A number of studies have been reported
regarding the growth promoting effect of alginates.
Mollah24 reported the growth enhancing effect of
gamma irradiated sodium alginate on red amaranth
with a total dose of 37.5 k G y at dose rate 3.5kGy/h;
applied in 150 ppm solutions.
Yokose et al.18 achieved maximum growth
promoting effect of Nanochloropsis oculata at
20mgml-1
concentration
with
alginate
oligosaccharides. Yokose et al.25 also achieved
maximum growth promoting effect of alginate
oligosaccharides in Chaetoceros gracilis at a
concentration of 125g ml-1. Naeem et al.26
achieved best plant growth promoting effect of
Mentha arvensis L at the concentration of 100 mgL1 of irradiated sodium alginate. Interestingly,
oligo-alginates also stimulate the growth of marine
and fresh water green microalgae by enhancing
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Oleic acid (18:1)

Palmitic acid (16:0)
E-11-Hexadecenoic acid (18:1)
Palmitelaidic acid (16:1)
Stearic acid (18:0)
Linoleic acid (18:2)
Cyclohexane (C12)
Heptadecane (C17)
Myristic acid (14:0)
Homo-- linolenic acid (20:3)
Eicosatrienoic acid(20:4)
Lignoceric acid (24:0)
Pentadecanoic acid (15:0)
Elaidic acid (9-18:1)
Margaric acid (17:0)
Lauric acid (12:0)
2-Pentadecanone,6,10,14-trimethyl
6-Octen-1-ol, 3,7-dimethyl acetate
1,3-Cyclohexanediamine
(1R,4S)-1,7,7Trimethylbicycloheptan-2-yl 3Chlorobenzoate
2-Methylthio-5-nitro anisole
Ethyl 4,8,12-trimethyl-tridecanoat

Wt
%
41.63
19.12
10.08
7.08
5.81
2.71
2.63
1.44
1.36
0.26
1.04
1.87
0.84
0.57
0.57
0.36
0.83
0.28
0.21
0.84

0.26
0.24

nitrogen and carbon assimilation providing

antimicrobial effect and, increase the content of
fatty acids which is useful for biodiesel
production22.

CONCLUSION
Alginate oligosaccharides enhances the growth of
many terrestrial plants and microalgae helping in

ISSN NO- 2230 7885

CODEN JPBSCT
NLM Title: J Pharm Biomed Sci.

Chakravarthy Kavitha, Senthil Chinnasamy, Sailendra Bhaskar,Ramasamy Rengasamy.

nitrogen assimilation, enhancing photosynthesis,

basal metabolism and cell division and also
increases the fatty acid content providing
protection against pathogen due to its
antimicrobial activity. In this study, the growth
and lipid content of Botryococcus braunii was
evaluated using different concentration of sodium
alginate under laboratory conditions. The best
growth and lipid production was achieved at the
concentration of 16mgL-1. Thus the sodium
alginate plays a vital role in the growth and lipid
production of green alga B.braunii for biodiesel
production.

ACKNOWLEDGEMENTS
The authors are thankful to the management of
Aban Infrastructure Pvt., Ltd., Chennai for the
financial support provided for this study.

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Copyright 2013 Chakravarthy Kavitha, Senthil Chinnasamy, Sailendra Bhaskar, Ramasamy Rengasamy. This is an open access
article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in
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