A STUDY ON THE NUTRITIONAL PARAMETERS

OF SEAWEEDS IN COASTAL TAMILNADU

A THESIS

Submitted by

V.DHANALAKSHMI
[Reg.No.D05BT010]

in fulfillment for the award of the degree

of
DOCTOR OF PHILOSOPHY

FACULTY OF SCIENCE AND HUMANITIES

(INDUSTRIAL BIOTECHNOLOGY)
BHARATH UNIVERSITY
CHENNAI 600 073, INDIA

MAY 2011
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BONAFIDE CERTIFICATE

Certified that this thesis titled “A STUDY ON THE

NUTRITIONAL PARAMETERS OF SEAWEEDS IN COASTAL

TAMILNADU” is a bonafide work of Mrs.V.Dhanalakshmi who carried

out research under my supervision. Certified further that to the best of our

knowledge the work reported herein does not form part of any other thesis or

dissertation on the basis of which a degree or award was conferred on an

earlier occasion of thesis of any other candidate.

SUPERVISOR

Signature :

Name in block letters : Dr.L.JEYANTHI REBECCA

Academic Designation : Professor and Head

Department : Industrial BioTechnology

University/College/Organisation : BHARATH UNIVERSITY,

With address Selaiyur, Chennai 600 073.

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DECLARATION

I V.Dhanalakshmi declare that the thesis entitled “A STUDY ON

THE NUTRITIONAL PARAMETERS OF SEAWEEDS IN COASTAL

TAMILNADU” is the bonafide research work of mine, which was carried

out under the supervision of Dr. L. JEYANTHI REBECCA. I declare

further that to the best of my knowledge the work reported herein does not

form part of any other thesis or dissertation on the basis of which a degree or

award was conferred on an earlier occasion on this or any other candidate.

Date : Signature of the candidate

Place : (V.Dhanalakshmi)
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ABSTRACT

Tamil Nadu has a geographical extent of 1,30,058 sqm. It can be

divided into two broad divisions namely, the eastern coastal plains and hills of
north and east, which is endowed with the varied coastal habitat like
mangroves, mud flats, seaweeds, seagrass beds, salt marshes, mud flats, sand
dunes etc. The coast of Tamil Nadu bears luxuriant growth of seaweeds. More
than 200 species of seaweeds have been found in this area. Indian seaweed
industries depend on this coastline for raw materials for the production of agar
and sodium alginate. The species of red algae namely, Gellidiella sp,
Oracilaria sp, Gracilaria sp, etc., and the species of brown algae namely,
Sargassum, Turbinaria are harvested for agar production. Seaweeds are rich
in minerals, vitamins, trace elements and bioactive compounds. Seaweeds are
consumed in the form of soups as well as salads. The intake of seaweeds in
the diet is said to prevent hair loss in men and women. It is also consumed by
pregnant and lactating mothers because of their rich iron content. They are
called the medical food of the 21 st century.

The main aim of this study is to evaluate the various nutritional

parameters of seaweeds present in the Tamil Nadu coastline. The samples
were collected from Kanyakumari, Pulicat, Kovalam, Ennore and Kalpakkam
by random sampling method. The basic nutritional parameters proteins and
carbohydrates were estimated by Bradford and Anthrone method respectively.
The protein content was highest in Sargassum spp. (1mg/g). The other
seaweeds such as Gracilaria spp., Ulva spp., Padina spp., Chaetomorpha
spp., and Hypnea spp., contained protein in the range of 0.8mg/g to 0.5mg/g.
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The protein content was lowest in Amphiroa spp., (0.1mg/g). Similarly

carbohydrate content was highest in Gracilaria spp., (100mg/g) and lowest in
Halimeda spp., (21mg/g). The percentage of trace elements present in
seaweeds such as Sargassum spp., Ulva spp., and Gracilaria spp., were
analyzed using SEM analysis. The oxygen content is high when compared to
other elements in Ulva spp., whereas carbon content is more in Gracilaria
spp., and Sargassum spp.. The chemical structure of Sargassum spp., Hypnea
spp.,Ulva spp., and Gracilaria spp., were analysed using FTIR spectroscopy.

There is an increasing demand of biodiversity from natural

resources for therapeutic drugs. The potential contribution of marine
organisms to the discovery of new bioactive molecules is increasingly
challenging. The macroalgae have a significant attraction as natural source of
bioactive molecules with a broad range of biological activities, such as
antibiotics, antivirals, antitumorals, antioxidant and anti-inflammatories.
Evidence of phycochemical and pharmacological studies on algae is available
in the literature with special reference to terpenoids and steroids . Algae are
the source of amino acids, terpenoids, phlorotannins, steroids, phenolic
compounds, halogenated ketones and alkanes and cyclic polysulphides.

Many algal species are known to have bactericidal and bacteriostatic

substances. Ethanol, ethanol and chloroform extract (1:1) and methanol
extracts of eight marine algae were evaluated for antibacterial activity against
Aeromonas hydrophila, Edwardsiella tenda, Escherichia coli, Pseudomonas
aeruginosa, Salmonella typhi and S.aureus. Ulva showed more antibacterial
activity than other species.Out of the three solvents used for the extraction of
bio-active materials,ethanol was found to be the best solvent.
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ACKNOWLEDGEMENT

My Sincere thanks to our Honorable Founder of Bharath University,

Chennai, Tamilnadu, India, Dr.S. JAGATHRAKSHAGAN, for his sincere

endeavour in educating us in his Premier Institution. I would like to express

my deep gratitude to our beloved Chairman Er. J. SUNDEEP ANAND, for

his kind words and enthusiastic motivation which has inspired me a lot in

completing my thesis. I would like to express my gratitude to our Vice

Chancellor Dr. K.P.THOOYAMANI, who is responsible for moulding my

thoughts in completing my research.

I wish to express my deep sense of gratitude to my Guide

Dr.L.JEYANTHI REBECCA Professor and Head, Department of Industrial

Biotechnology, Bharath University, for her guidance and support given to me

throughout the research work. I shall cherish my association with her for her

constant encouragement accessibility and valuable suggestions.

I would like to place my graceful thanks to all my colleagues,

S.Sharmila, G.Susithra, Merina Paul and Maharshi, and other staff

members of Department of Industrial Biotechnology, Bharath University, for

their kind help whenever needed. My sincere appreciation is to my beloved

family members without their cooperation and tolerance it would have been

impossible for me to complete this research work.

(V.Dhanalakshmi)
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TABLE OF CONTENTS

CHAPTER No. TITLE PAGE No.

ABSTRACT iv
LIST OF TABLES xi
LIST OF FIGURES xii
LIST OF ABBREVIATIONS xv

1 INTRODUCTION 1
1.1 SEAWEEDS 1
1.2 CLASSIFICATION 3
1.2.1 Green Algae (Chlorophyta) 4
1.2.2 Brown Algae (Phaeophyta) 9
1.2.3 Red Algae (Rhodophyta) 12

2 ESTIMATION OF PROTEIN 16
2.1 SEAWEEDS 16
2.2 SEAWEEDS AS FOOD 16
2.3 SEAWEEDS AS A SOURCE OF AGAR 17
2.4 SEAWEEDS AS FERTILIZER 18
2.5 SEAWEEDS MISCELLANEOUS USES 19
2.6 MATERIALS AND METHODS 19
2.6.1 Collection of samples 19
2.6.2 Seaweed collection procedure 21
2.6.3 Preservation of samples 23
2.6.4 Dry preservation Method. 24
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CHAPTER No. TITLE PAGE No.

2.6.5 Identification of samples based on

morphological criteria 24
2.6.6 Identification of Samples 25
2.6.7 Analysis of Proteins 25
2.6.8 Sample preparation 25
2.6.9 Procedure 26
2.7 RESULTS AND DISCUSSIONS 29

3 ESTIMATION OF CARBOHYDRATE 36
3.1 SEAWEEDS 36
3.1.1 Chlorophyta 36
3.1.2 Phaeophyta 38
3.1.3 Rhodophyta 40
3.2 SEAWEEDS AS POTENTIAL
NUTRIENT SUPPLIERS 42
3.3 SEAWEEDS FOR MEDICAL PURPOSE 45
3.4 ANALYSIS OF CARBOHYDRATE 46
3.4.1 Reagents / chemicals used 46
3.4.2 Sample preparation 46
3.4.3 Procedure 47
3.5 RESULTS AND DISCUSSIONS 48

4 ESTIMATION OF TRACE ELEMENTS 55

4.1 SCANNING ELECTRON MICROSCOPE 55
4.2 ENERGY DISPERSIVE X-RAY
SPECTROSCOPY 56
4.2.1 Equipment 57
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CHAPTER No. TITLE PAGE No.

4.3 ANALYSIS OF TRACE ELEMENTS IN

SEAWEEDS 58
4.4 RESULTS AND DISCUSSIONS 58

5 FTIR SPECTROSCOPIC ANALYSIS

OF SEAWEEDS 69
5.1 FT-IR SPECTROSCOPY 69
5.2 RESULTS AND DISCUSSIONS 74

6 ANTIBACTERIAL EFFECT OF SEAWEEDS 79

6.1 ANTIBACTERIAL ACTIVITY OF
SEAWEEDS AGAINST FISH
PATHOGENS 80
6.1.1 Antimicrobial activity of seaweeds
Gracillaria, Padina and Sargassum spp.on
clinical and phytopathogens 85
6.1.2 An assessment of the antioxidant and
antimicrobial activity of six species of
edible Irish seaweeds 86
6.1.3 Components and Antimicrobial Activity
of Polysaccharides extracted from
Thai brown seaweeds 88
6.1.4 Antimicrobial activity of seaweeds
extracts against multi resistant
pathogens 89
6.1.5 Antifungal activity of seaweeds 90
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CHAPTER No. TITLE PAGE No.

6.2 MATERIALS AND METHODS 91

6.2.1 Preparation of extracts 91
6.2.2 Test microorganisms used 91
6.2.3 Plate assay method 92
6.3 RESULTS AND DISCUSSIONS 93

7. SUMMARY 105
7.1 PROTEIN ESTIMATION OF
SEAWEEDS 106
7.2 ESTIMATION OF CARBOHYDRATES
FROM SEAWEEDS 109
7.3 ESTIMATION OF TRACE ELEMENTS
FROM SEAWEEDS 111
7.4 ANALYSIS OF CHEMICAL STRUCTURE
OF SEAWEEDS 112
7.5 ANTIMICROBIAL ACTIVITY OF
SEAWEEDS 113

8. CONCLUSION 116

REFERENCES 117

APPENDIX 122

LIST OF PUBLICATIONS 124

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LIST OF TABLES

TABLE No. TITLE PAGE No.

4.1(A) & (B) EDAX ZAF Quantification of ULVA spp. 61

4.2 (A) &(B) EDAX ZAF Quantification of
Gracilaria spp. 64
4.3(A) &(B) EDAX ZAF Quantification of
Sargassum spp. 67
5.1 Typical Infrared Absorption Frequencies 73
5.2 Typical Functional Class and their
Characteristic Absorption 74
6.1 Antibacterial Activity of Extracts of Algae 94
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LISTOF FIGURES

FIGURE No. TITLE PAGE No.

1.1 Ulva spp. 5

1.2 Enteromorpha spp. 7
1.3 Chaetomorpha spp. 8
1.4 Stoechospermum spp. 10
1.5 Padina spp. 11
1.6 Amphiroa spp. 13
1.7 Gracilaria spp. 14
2.1 Comparison of protein content in different
seaweeds from various locales. 30
2.2 Protein content of seaweeds from Covelong 31
2.3 Protein content of different seaweeds from
Pulicat 32
2.4 Protein content of seaweeds from Muttam. 32
2.5 Protein content of different seaweeds from
Kanyakumari 33
2.6 Comparision of protein content of Gracilaria spp
from different locations. 33
2.7 Comparision of protein content in Ulva spp from
different areas 34
2.8 Comparision of protein content of Enteromorpha spp
from differentareas 34
2.9 Comparision of protein content in Sargassum spp
from different areas 35
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FIGURE No. TITLE PAGE No.

2.9(a) Comparision of Protein content in Valeneopsin spp

from different areas 35
3.1 Carbohydrate content of different seaweed species
from Pulicat 49
3.2 Carbohydrate content of different seaweed species
from Covelong 50
3.3 Carbohydrate content from different seaweed species
from Muttam 50
3.4 Carbohydrate content in different seaweed species
from Kanyakumari 51
3.5 Carbohydrate content in Ulva spp. from different
locales 51
3.6 Carbohydrate content in Gracilaria spp. from
different locales 52
3.7 Carbohydrate content in Enteromorpha spp. from
different locales 52
3.8 Carbohydrate content in Amphiroa spp. from
different locales 53
3.9 Carbohydrate content in Valeneopsin spp. from
different locales 53
3.9(a) Carbohydrate content in Hypnea spp. from different
locales 54
4.1(A)&(B) SEM-EDAX analysis of Ulva spp. 61
4.2(A)&(B) SEM-EDAX analysis of Gracilaria spp. 64
4.3(A)&(B) SEM-EDAX analysis of Sargassum spp 67.
5.1 FTIR vibrational stretching of Ulva spp. 75
5.2 FTIR vibrational stretching of Sargassum spp. 76
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FIGURE No. TITLE PAGE No.

5.3 FTIR vibrational stretching of Gracilaria spp. 77

5.4 FTIR vibrational stretching of Hypnea spp. 78
6.1 Antibacterial activity of Amphiroa spp.,
Gracilaria spp. and Centroceiod spp. against
Aeromonas hydrophila 95
6.2 Antibacterial activity of Ulva spp., Centroceiod spp.,
Padina spp., Stoechospermum spp.against
Edwardsiella tarda 95
6.3 Antibacterial activity of Ulva spp. and
Stoechospermum spp. against
Escherichia coli 96
6.4 Antibacterial activity of Stoechospermum spp.,
Padina spp. and Chaetomorpha spp.against
Pseudomonas aeruginosa 97
6.5 Antibacterial activity of Centroceiod spp.,
Ulva spp., Enteromorpha spp., Stoechospermum spp.,
Gracilaria spp., Amphiroa spp.and
Chaetomorpha spp.against Pseudomonas
fluorescens 98
6.6 Antibacterial activity of Stoechospermum spp.,
Chaetomorpha spp.,Centroceiod spp.,Ulva spp.,
Gracilaria spp.against Salmonella typhi 99
6.7 Antibacterial activity of Ulva spp.,
Stoechospermum spp.and Padina spp. against
Staphylococcus aureus 100
 xv

LIST OF ABBREVIATIONS

BSA Bovine serum albumin

d.w Dry weight

FTIR Fourier transform infrared

SEM Scanning Electron Microscope

Std Standard
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CHAPTER 1

INTRODUCTION

1.1 SEAWEEDS

The coast of Tamil Nadu bears luxuriant growth of seaweeds. In

coastal waters they grow almost like grass in large areas, extending over

hundreds of kilometers. Marine algae, popularly known as seaweeds are of

immense industrial, human and agricultural value since time immemorial

especially in the orients.

Seaweeds or benthic marine algae are the group of plants that live

either in marine or brackish water environment. Like the land plants,

seaweeds contain photosynthetic pigments and with the help of sunlight and

nutrient present in the seawater, they photosynthesize and produce food.

Seaweeds are found in the coastal region between high tide to low tide and in

the sub tidal region up to a depth where 0.01% availability of photosynthetic

light.

Seaweeds are large macro algae that grow in a saltwater or marine

environment. Seaweeds are plants that lack true stems, roots and leaves. They

possess a blade that is leaf like, a stipe that is stem like and a holdfast that

resembles a root. Seaweeds contain photosynthetic pigments and use sunlight

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to produce food and oxygen from carbon dioxide and water. They are simpler

than the land plants mainly because they absorb the nutrients that they require

from the surrounding water. Some large seaweed such as kelps have root like

parts called holdfasts, but these only serve to attach them to rock. Most

seaweed has to be attached to something in order to survive and only a few

will grow while drifting loose in the sea. Certain seaweeds tend to group

together in bands or stripes that run roughly parallel to coast. Seaweeds live in

the region between the high and low tide levels (intertidal zone) and the low

tide mark (sub tidal zone). The intertidal and sub tidal zones are further

subdivided into bands. Many types of seaweeds may also be found in more

than one band. The ecological niches utilized by seaweeds are wide ranging.

At the highest level are those that inhabit the zone that is only wetted by the

tops of sea spray, the deepest living are those that are attached to the sea- bed

under several meters of water. In some parts of the world, the area colonized

by littoral seaweeds can extend for several miles away from the shore.

The limiting factor in such cases is the availability of sufficient

sunlight to support photosynthesis. The deepest living seaweeds are the

various kelps. A number of species have adapted to the specialized

environment of tidal rock pools. In this niche seaweeds are able to withstand

rapidly changing temperature and salinity and even occasional drying. The

collection of seaweeds in the field is done during the low tide. It is necessary

to go for collection one or two hours before the time of low tide as per tide

tables. This will give more time for seaweed collection and to observe

seaweeds in the natural habitat. Plant pigments, light, exposure, depth,

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temperature, tides and shore characteristics combine to create different

environment that determine the distribution and variety among seaweeds.

When seaweeds break down, they enrich waters by adding dissolved and

particulate organic matter to it. This is used by a number of micro organisms

and many species of marine invertebrates. Various parts of algae can be

described as follows:

Thallus: the algal body

Lamina: a flattened structure that is somewhat leaf like

Sorus: spore cluster

On Focus, airbladders: float assist organ (on blade)

On Kelp, floats: float-assist organ (between lamina and stipe)

Stipe: a stem like structure, may be absent

Holdfast: specialized basal structure providing attachment to a

surface, often a rock or another alga.

The stipe and blade are collectively known as fronds.

1.2 CLASSIFICATION

The criteria to distinguish the different algal groups are based on the

different biochemical, physiological and electron microscopic studies. These

are mainly based on the photosynthetic pigments, storage food products, cell

wall component, fine structure of the cell and flagella.

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Accordingly, algae are classified into three main groups namely,

Green (Chlorophyta), Brown (Phaeophyta) and Red (Rhodophyta). Seaweeds

are similar in form with the higher vascular plants but the structure and

function of the parts significantly differ from the higher plants. Seaweeds do

not have true roots, stem or leaves and whole body of the plant is called the

thallus that consists of the hold fast, stipe and blade. The hold fast resembles

the root of the higher plants but its function is for attachment and not for

nutrient absorption. The stipe resembles the stem of the higher plants but its

main function is for support of the blade for photosynthesis and for absorption

of nutrients from surrounding sea water. The blade may ressemble leaves of

the higher plants and have variable forms namely, smooth, perforated,

segmented, dented etc. The important functions of the blade are

photosynthesis and absorption of nutrients (Agadi, 1973).

1.2.1 Green Algae (Chlorophyta)

Morphology: They are found in the fresh and marine habitats. They

range from unicellular to multicellular, microscopic to macroscopic forms.

Their thalli vary from free filaments to definitely shaped forms. The

photosynthetic portion of the thalli may be moderately to highly calcified

appearing in variety of forms as fan shaped segments, feather like or star

shaped branches with teeth or pinnules, clavate or globose branchlets.

Pigments: They possess photosynthetic pigments such as

chlorophyll a & b, contained in the special cell structure known as

chromatophores cell wall of this group composed of an outer layer of pectin

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and an inner layer of cellulose. The photosynthetic product of this group is

starch.

Reproduction: Green algae can produce sexually and asexually by

forming flagellate spores and sometimes non-flagellate spores. The vegetative

propagation is achieved by fragmentation. Alternation of gametophytic and

sporophytic generation occurs in this group.

Some of the green algae and their characteristics are described

below.

Figure 1.1 Ulva

Order : Ulvales

Family : Ulvaceae

Genus : Ulva
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Characteristics: Plants 1-15 cm. tall, the base of the blade cuneate,

above expanding irregularly lobed, generally irregularly or sometimes

pinnately divided into ligulate or linear lobes which may become several

decimeters long; in section the cells of the midline region much taller than

those of the margin, the thallus much thicker, the margins entire to irregularly

ruffled and crenate with a somewhat paler central portion.

Distribution: Gujarat, Maharashtra, Goa, Karnataka, Kerala,

Lakshadweep.

Ecological status: Open coast (intertidal), Estuaries and mangroves.

Uses: Food, animal feed, medicine

Reproduction in Ulva: Ulva usually multiply by means of fragments

which are accidentally produced from a thallus. Vegetative multiplication also

takes place by means of the proliferation of perennial holdfast. Asexual

reproduction takes place by means of quadriflagellate zoospores.

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Figure 1.2 Enteromorpha

Order : Ulvales

Family : Ulvaceae

Genus : Enteromorpha

Characteristics :Plants generally gregarious, attached, bright to dark

green, 3dm tall, tubular, more or less compressed or collapsed, above

expanded, 220 mm wide, below long, tapering and characteristically with

several branches from the gradually contracted stalk like base which are

similar to the principal blade. The walls not thickened, in section vertically

elongate, the whole membrane 13-20 micron thick.

Distribution: Bombay, Malvan, Ratnagiri (Maharashtra), Goa

Ecological status: Intertidal zone, Mangrove swamps, Estuaries

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Uses: It is used as vegetable and also in the Form of salad, jam and

power, animal feed and medicine

Figure 1.3 Chaetomorpha

Order : Cladophorales

Family : Cladophoraceae

Genus : Chaetomorpha

Characteristics: Chaetomorpha is one of the more delicate forms of

green algae. It resembles straight green or yellowish hair, sometimes white

towards the ends of the filaments if spores or gametes have been released;

filaments are unbranched, usually between 5 and 30 cm (2 - 12 inch) long,

and frequently grow in groups of hundreds or thousands of individuals in

sandy area on rocks or around tide pool; Chaetomorpha is fast and great way

to reduce phosphates and nitrates as well as other nutrients. It’s sometimes

referred to as spaghetti or billow pad macro algae due to the way it grows.

Distribution: Gujarat, Malvan, Ratnagiri, (Maharashtra), Goa,

Karwar, Honawar, Bhatkal (Karnataka)

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Ecological status: Intertidal (supralittoral)

Uses: Food, animal feed and agricultural

1.2.2 Brown Algae (Phaeophyta)

Morphology: Brown algae are exclusively marine forms. They have

different forms from simple, freely branched filaments to highly differentiated

forms. They can be distinguished into blades, stipes, and holdfast.

Pigments: Photosynthetic pigments of the brown algae are

chlorophyll, carotene, xanthophylls and fuxoxanthin(pigment responsible for

brown color).the cell wall composed of an outer layer of algin and an inner

layer of cellulose. The photosynthetic products of the brown algae are

laminarian and mannitol.

Reproduction: This group reproduces sexually and asexually.

Several species of this group reproduce vegetatively by fragmentation.

Members of this group reproduce biflagellate neutral spores found within one

celled or many celled reproductive organs. The sexual reproduction is through

union of flagellated male and female gametes or union of flagellated male and

large non-flagellated female gametes. Alternation of gametophytic and

sporophytic generations occurs in this group except in the members of

fucales.

Some of the brown algae and their characteristics are described

below:
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Figure 1.4 Stoechospermum

Order : Dictyotales

Family : Dictyotaceae

Genus : Stoechospermum

Characteristics :Rigorously forking plants that may reach a length of

40 cm; usually the plants are 20 -30 cm long and 8 - 11 mm broad; thallus

flat, erect, spathulate, dichotomously branched respectively, without a midrib;

margin entire; apex bifid or flatly truncate; fertile plants are easily identified

on the marginal dark lines of crowded sporangia.

Distribution: Gujarat, Ratnagiri, Malvan, (Maharashtra), Goa,

Karwar, Honawar, Bhatkal, (Karnataka).

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Ecological status: Intertidal, zone

Uses: Used as a source of alginate, fertilizer

Figure 1.5 Padina

Order : Dictyotales

Family : Dictyotaceae

Genus : Padina

Characteristics :Thalli flabelliform, usually divided into several

small lobes, regularly and distinctly concentrically zonate; easily recognized

due to dark double lines of sporangia; enclosing a line of colourless hairs in

between; blades composed of two layers of cells, in the young apical involute

portion, fan-shaped fronds, entire when young, but dissected when older.

Distinctive, flattened fan-shaped thallus with concentric markings and rolled

edges. Attaches to rock by rhizoids branched only in one plane, with thin

fronds, often lacerated from edge to base.

 12

Distribution: Gujarat, Malvan, Ratnagiri, Goa, Karwar, Honawar,

Bhatkal, (Karnataka) Lakshadweep

Ecological status: Mangrove swamps (attached to mud)/Intertidal

Uses: Extraction of alginate, fertilizer

1.2.3 Red Algae (Rhodophyta)

Morphology: Except for few species they are exclusively marine.

They vary in size and shape. They are either epiphytes grow as crust on the

rocks or shells as a large fleshy, branched or blade like thalli.

Pigments: They contain chlorophyll a & b, carotene, phycoerythrin

(pigment responsible for red color).The cell wall of this group composed of

an outer layer of pectin and an inner layer of cellulose. The photosynthetic

product of this group is Floridian starch.

Reproduction: This group seldom reproduces asexually. All the

members of this group produce one or more kinds of non-flagellated spores

that are either asexual or sexual in nature. Sexual reproduction is very

complicated involving several structures after fusion of gametes. some

members of this group exhibit biphasic alternation of generation in which

sexual generation (gametophyte) alternates with asexual (tetrasporophyte)

generation, while others are triphasic with three generation or somatic phases

(gametophyte, carosporophyte, tetrasporophyte) successively following one

another.
 13

Some of the red algae and their characteristics are described below

Figure 1.6 Amphiroa

Order : Corallinales

Family : Corallinaceae

Genus :Amphiroa

Characteristics: The thallus is multiaxial and has long and short cells

in the intergenicular region; grows in the lower mid-littoral zone and favors

sheltered areas; thallus is articulate, attaining a height of 4-6 cm; fresh

specimens have a light purple colour; branching is dichotomous; structurally

the thallus is multiaxial, meristematic cells at the apical region are covered by

a single layer of cover cells having a diameter of 6-8 µm intergenicular

medulla consists of long and short cells; cortex varies from slightly to well

developed cortical cells are circular to squarish; single layer of cover cells,
 14

4-8 µm in diameter; Distribution: Gujarat, Malvan, Ratnagiri, (Maharashtra),

Goa, Karwar. Honawar, Bhatkal, (Karnataka), Lakshadweep.

Ecological status: Intertidal zone.

Figure 1.7 Gracilaria

Order : Gracilariales

Family : Gracilariaceae

Genus : Gracilaria

Characteristics: Plants bushy, 1-3 dm tall, with age often becoming

free, texture firmly fleshy, color dull purplish, greyish or greenish translucent,

branches repeatedly dividing, alternately or occasionally dichotomously

branched with numerous lateral proliferations, tapering to the ultimate branch

lets, cortex of 2-3 layers of small cells, tetra sporangia numerous, scattered
 15

over the branch lets, oval, from the surface, cystocarps very prominent, often

numerous.

Distribution: Okha, (Gujarat) Bombay, (Maharashtra) Goa.

Ecological status: Mangrove swamps, brackish water.

In this study seaweed samples were collected from different areas of

Tamil Nadu coastline. The places include Pulicat Lake, Covelong beach,

Muttam, Kanyakumari, Kalpakkam, Ennore and Cape comeron,

Kanyakumari. The collection of seaweeds from the intertidal area was done

during the low tide. The present study thus attempts to evaluate the following

nutritional parameters such as

Estimation of protein content in different seaweeds using

Bradford assay.

Estimation of carbohydrate using Anthrone method.

Identification and estimation of total percentage of various trace

elements present in seaweeds using Scanning electron

microscopic studies.

Study of chemical structure of seaweeds using FTIR analysis.

Study of antibacterial activity of seaweeds against pathogenic

microorganisms using plate assay method.

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CHAPTER 2

ESTIMATION OF PROTEIN

2.1 SEAWEEDS

Tremendous increase in research on the chemistry of seaweeds have

been done in recent years due to the need for compounds possessing

pharmaceutical applications and other varied potential economic properties.A

variety of species have been assayed for their activity and a number of

biodynamic molecules, often with toxic properties and unique structural

features, have been isolated. Since marine organisms live in a significantly

different environment from those of terrestrial organisms, it is reasonable to

justify that their secondary metabolites will differ considerably. Marine

natural product chemistry has seen 25 years of fruitful research (Muhammad

Shaiq Ali and Viqar Uddin Ahmad; 1998). The success of research in this

area would be assured as most marine natural products have no counterparts

in the terrestrial world; provided the novelty and complexity of compounds

discovered are the only criteria.

2.2 SEAWEEDS AS FOOD

Seaweeds are used in many maritime countries as a source of food,

for industrial applications and as a fertilizer. The present uses of seaweeds are
 17

as human foods, cosmetics, fertilizers, and for the extraction of industrial

gums and chemicals. They have the potential to be used as a source of long-

and short-chain chemicals with medicinal and industrial uses. Macroalgal

polysaccharides are used in the food, cosmetics, paint, crop, textile, paper,

rubber and building industries.

The seaweeds are also used as food in the regions of Far East and

Australia. The inhabitants of the Hawaii Island consume large quantities of

seaweeds. The natives of New Zealand use certain green seaweeds in

preparation of salad and soups. The people of China and Japan consume the

seaweeds on large scale. The people living on the sea coasts in these countries

commonly use fresh seaweeds as food. The most important food species in

Japan are Nori (Porphyra species), Kombu (Laminaria species), and Wakame

(Undaria pinnatifida). In Japan Porphyra tenera happens to be one of the

most important edible algae and a product by the name of Amanori and

Asakusa- Nori are made from it (Amin Ismail and Tan Siew Hong, 2002)

2.3 SEAWEEDS AS A SOURCE OF AGAR

The best agar is manufactured from Gelidium of Rhodophyceae,

which is also called vegetative agar. Japan produces the largest quantity of

agar. It produces 95% of the world production. Agar is also obtained from

several other marine algae, the yield of agar, setting temperature and gel

strength of the product from ten species belonging to Gelidium, Sarconema,

Hypnea and Gracilaria were obtained.

 18

Japan is the chief agar producing country and it exports agar to most

of the countries of the world. The agar is used in several ways. It is employed

in the preparation of ice cream, jellies, desserts etc., in sizing the textiles and

clearing many liquids. It is also used in preparing shaving creams, cosmetics

and shoe polishes. The agar has constantly been used in biological

laboratories for media preparation.

2.4 SEAWEEDS AS FERTILIZER

Seaweeds are used in different parts of the world as fertilizer for

various land crops. In India, freshly collected and coast ashore seaweeds are

used as manure for coconut plantation either directly or in the form of

compost in coastal areas of Tamil Nadu and Kerala. Seaweed manure has

been found superior to farm yard manure. Due to the presence of potassium

chloride (KCl) in seaweeds, they are used as fertilizers in many countries,

such as Japan, France, United States, England and South India.

Seaweeds are as a store-house of the important potash, ionic

sulphates, trace elements and growth substances, besides having every other

element and radical required by plants. Seaweed manure seems to increase

resistance to disease. Most of the nutrients including nitrogen compounds are

in ionic form and a quick absorption by crops takes place and relatively little

is left to be broken down by soil microflora, thus preventing acid conditions

of the soil arising from the fermentation. In general the minerals diffuse out

from the seaweed thallus rapidly.

 19

Yet another feature is that seaweed manure holds water and air at

the same time and improves the soil in both respects. Like other manures

seaweeds have a similar role but also contribute the required potassium,

sulphur, phosphorus and calcium. The liquid seaweed fertilizer obtained from

seaweed extract is used as foliar spray for inducing faster growth and yield in

leafy and fleshy vegetables, fruits, orchards and horticultural plants.

2.5 SEAWEEDS; MISCELLANEOUS USES

The use of seaweed extract in cosmetics is a major international

trend at present. The elements contained in seaweeds act in harmony with the

human body, helping to achieve, beauty and relaxation. In cosmetology, it is

important to know the biochemical composition and potential use of

cosmetics. The extract can be used in two ways: either as an agent in

preparation of products or as therapeutic agent itself. Alginates of different

viscosity serve as a thickening and dispersing agents in cream, jellies, liquid

emulsions, lotions, compact powders, toothpaste, soaps and alums etc. By

burning seaweeds on the sea coast, the alkalies are prepared from seaweed

ashes. These alkalies are employed in the manufacture of soaps and alums.

2.6 MATERIALS AND METHOD

2.6.1 Collection of samples

Seaweeds or marine macroalgae form a conspicuous biomass in the

coastal region of the tropics. They are the primary producers in aquatic

habitats supporting rich food chains and they oxygenate the aquatic
 20

ecosystem. Seaweeds can be found around the seashore in large amounts,

clinging to solid substrates like corals, rocks or shells. In this study, seaweed

samples were collected from different areas of the Tamil Nadu coast line. The

places include Pulicat Lake, Covelong beach, Muttam, Kanyakumari

Kalpakkam, Ennore, Cape comeron, Kanyakumari. The collection of

seaweeds from the intertidal area was done during the low tide. It is necessary

to go for collection one or two hours before the time of low tide as per tide

tables. This gives more time for seaweed collection and to observe seaweeds

in the natural habitat. It is important to make notes on the description of the

site location, topography, associated flora and fauna and other related

parameters. Material necessary for seaweed collection are as follows:

Polyethylene bags

Knife or scalpel

Labeling materials (pen/pencil, labels, marker pens etc.)

Rubber bands

Field note book

Samples were selected by Random sampling method as per

requirement. This was done by selecting sampling points in the area.

Sampling points were chosen in such a manner that every species of the study

area had a good chance being selected. This type of sampling is usually done

in the area where the intertidal expanse is very narrow with steep gradient and

also in the area where distribution is patchy. It is also employed for qualitative

estimation of the seaweed. Collected material were be kept in the

 21

polyethylene bags and labeled for further preservation and identification at the

later stage in the laboratory.

2.6.2 Seaweed collection procedure

The collection of seaweeds in the field is done during the low tide. It

is necessary to go for collection one or two hours before the time of low tide

as per tide tables. This will give more time for seaweed collection and to

observe seaweeds in the natural habitat. It is important to make notes on the

description of the site location, topography, associated flora and fauna and

other related parameters. Although, there are a number of methods to collect

seaweeds, we consider here two methods which are practical and easy to

study.

Line transect/belt transect method and random sampling method.

a) Line transect or belt transect method

A line or belt transect is laid perpendicular to the coast from high

tide to the low tide with the help of a long rope. Sampling points along the

rope can be marked depending on the gradient and the expanse of the

intertidal area. In case the intertidal area is small, sampling points can be

marked at 5 m intervals along the rope and if intertidal area is quite large the

sampling point can be marked at 10 or 20 m along the rope.

 22

2
A quadrant measuring 0.25 m area is placed at the sampling

points in triplicate covering an area of 5 m 2 on either side of the

sampling points.

Seaweed species present within the quadrant are collected

(collect complete plant as far as possible along with the hold

fast).

Seaweed specimen can be removed by hand but that specimen

which is closely adhering to the substrate such as crustose and

mat forming seaweeds can be removed with the help of knife or

scalpel. The specimen that grows close to the rocks can be

removed with the rocks using geologist's pick or any other

similar tools.

The entire collected specimen should be counted species wise

and the number of individuals in each species is found for

quantitative assessment of abundance, density, frequency,

species richness, species diversity, percentage cover etc. with

statistical consideration.

The entire collected specimen from the quadrant should be

weighed to estimate standing crop biomass.

Collected material should be kept in the plastic bags/containers

with proper labeling for further preservation and identification at

the later stage in the laboratory.

 23

b) Random sampling method

Samples can be selected at random as per requirement. This can be

done by selecting sampling points in the area and using quadrant. Sampling

points should be selected in such a manner that every species of the study area

has a good chance of being selected. This type of sampling is usually done in

the area where the intertidal expanse is very narrow with steep gradient. It is

also employed for qualitative estimation of the seaweed.

2.6.3 Preservation of samples

The samples were preserved by Wet preservation method. The steps

are as follows (Agadi 1976):

All the adhering materials such as sand particles and other debris

were removed from the seaweeds before preservation.

A solution of 5 -10 % formaldehyde in seawater was prepared to

preserve the seaweed samples.

Before adding the preservative, water from the polyethylene

bags was drained and sufficient preservative was added.

Polyethylene bags were tied with rubber bands properly to

prevent leakage during transportation.

All the bags were properly labeled with date of collection,

locality and time and transport to the laboratory for further

identification.
 24

2.6.4 Dry preservation method

The steps are as follows:

The samples were washed with water to remove debris

They were dried in open air till they lost enough moisture to be

stored.

The samples were then packed into air tight polythene bags.

All the bags were labeled with date of collection, locality and

time and transport to the laboratory for further identification.

2.6.5 Identification of samples based on morphological criteria

The identification is based on simple morphological criteria and

reproductive structures, type of life history, cross sectional anatomical details,

type of growth, cytology and ultra structural criteria and increasingly

molecular evidence. Colour and morphological differences between different

genera/ species and taxonomic characteristic are required to be carefully

studied. The important criteria used to distinguish the different algal groups

based on the recent biochemical, physiological and electron microscopic

studies are:

Photosynthetic pigments,

Storage food products,

Cell wall component,

 25

Fine structure of the cell and

Flagella.

2.6.6 Identification of Samples

The species were sent for identification at the following places:

Centre for Advanced studies in Botany, University of Madras, Guindy,

Chennai-5. Krishnamurthy Institute of Algology, Anna Nagar, Chennai. The

identified samples were used for further bio chemical analysis.

2.6.7 Analysis of Proteins

The protein analysis was done by Bradford method. The list of all

chemicals and solutions prepared and their composition are given in the

Appendix.

2.6.8 Sample preparation

1 g of oven dried sample was taken and was soaked in 3ml of

distilled water overnight.

To the soaked sample, 5 mL of TCA was added in parts till a

paste was obtained.

The paste was taken in centrifuge tubes and centrifuged at

5000 rpm for 10 min.

The supernatant was collected and the pellet was discarded.

The supernatant was stored for further analysis.

 26

2.6.9 Procedure

The protein estimation was done using Bradford protein assay

method (Bradford, 1976).

Principle: The Bradford dye-binding assay is a colorimetric assay

for measuring total protein concentration which involves the binding of

Coomassie Brilliant blue to protein. Both hydrophobic and ionic interactions

stabilize the anionic form of the dye, causing a visible color change. The

assay is useful since the extinction coefficient of a dye-albumin complex

solution is constant over a 10-fold concentration range.

Method: Firstly, to prepare a standard graph, a stock solution of

BSA was prepared in distilled water with a concentration of 0.1 mg/ml. From

this, 0.1 mL to 1 mL aliquots were taken in different tubes and the volume

was made up to 1 mL with distilled water. To each of the tubes, 5 mL of

Bradford Reagent was added and incubated in the dark for 10 min at room

temperature. The UV Visible Spectrophotometer was switched on to stabilize

for a few minutes. It was set to auto zero with a blank (100 µL water + 5 mL

Bradford reagent).

The O.D was measured at 595 nm in UV-Visible spectrophotometer

and a standard graph was drawn with concentration in X axis and O.D value

in the Y axis. Then from the supernatant collected, 100 µL of seaweed sample

was taken in a test tube, to which 5 mL of Bradford reagent was added and

kept for incubation in dark for about 10 min. The optical density values were
 27

used to calculate the protein concentration by extrapolation of the standard

BSA graph.

2.7 RESULTS AND DISCUSSIONS

Analysis of Proteins

The samples were analysed for their protein content by Bradford

Method (Bradford 1976). The varying protein content of different samples

from various locales is shown in Figure 2.1. Of the various samples collected,

Sargassum spp., a brown seaweed from Cape comeron, Kanyakumari,

showed the highest protein content of 950 µg/g whereas the same species

collected from Muttam, Kanyakumari showed a lower concentration of

protein of about 550 µg/g (Figure 2.1).

Two different varieties of Gracilaria spp.,a red seaweed, were

collected from Covelong. while the protein content in one species was about

850 µg/g, the other species of the other species was around 100 µg/g. The

species of Gracilaria spp.with higher protein content collected from other

locales showed variations ranging between (100-200) µg/g as compared to

that of the sample from Covelong. The Gracilaria spp. collected from Pulicat

had a protein content of about 750 µg/g whereas that of Muttam,

Kanyakumari and Cape comeron, Kanyakumari had a protein concentration of

600 µg/g and 500 µg/g respectively (Figures 2.4 and 2.5). In another similar

study by Eswaran et al (2002) the total protein content in Gracilaria spp. was

determined to be as high as 1070 µg/g (d.w).

 28

Another Rhodophyte, Hypnea spp., showed protein content of

480 µg/g. Similar amount of protein content was estimated in Calagossa spp.,

a red algae collected from Cape comeron, Kanyakumari. The protein content

was determined to be 700 µg/g. Yet another red seaweed, Centroceras spp.,

collected from Cape comeron, Kanyakumari had a protein content of

405 µg/g (Figure 2.5). The protein content of Amphiroa spp., a red seaweed,

collected from Muttam, Kanyakumari and covelong was the same (100 µg/g)

(Figures 2.4 & 2.5).

Ulva, a Chlorophytae, was found abundantly in most of the places.

Two different species of Ulva were collected viz., Ulva lactuca and Ulva

fasciata. The U. lactuca from Pulicat had a protein content of about 350 µg/g

whereas the one collected from Cape comeron, Kanyakumari showed

200 µg/g (Figure 2.7). Compared to this species, the other species U. fasciata

from Covelong had about 600 µg/g of protein whereas that of Pulicat had a

protein content of 650 µg/g (Figure 2.1). It has been found in many other

studies that the nutritional contents of macroalgae depend not only on season

and geography. Fleurence (1999), Fleurence et al (1999) found that the total

protein content in U. lactuca to lie between 19.29% and 18.22%, and the total

protein content of Ulva sp. to vary between 18% and 26%.

Other green algae such as Enteromorpha spp. and Chaetomorpha

spp. were collected from different locations.The protein content in

Enteromorpha spp. was determined to be 200 µg/g from Pulicat, 400 µg/g

from Muttam, Kanyakumari and 280 µg/g from Cape comeron, Kanyakumari
 29

respectively. In a similar study by Mathers and Montgomery (1997), the total

protein content in Enteromorpha spp. was found varying between 16.04% and

16.14%. .

The protein content of Stoechospermum spp., a brown algae

collected from Cape comeron, Kanyakumari was determined to be 475 µg/g.

Another Phaeophyte, Padina spp. showed higher protein content of about

765 µg/g. Valeneopsin spp. was collected from two different locales. The

protein content was low; it was determined to be 150 µg/g and 200 µg/g in

samples collected from Muttam, Kanyakumari and Cape comeron,

Kanyakumari respectively (Figure 2.9(a)).

Comparing the different samples collected from Cape comeron

Kanyakumari it is observed that the protein content determined varied

between as low as 100 µg/g in Ulva to a high of 950 µg/g in Sargassum spp.

Of all the samples collected from Cape comeron, Kanyakumari, the protein

content in Phaeophytes seems promising as that of Sargassum spp. and

Padina spp. The different variety of Rhodophytae collected showed variations

from 405 µg/g in Centroceras spp., 480 µg/g in Hypnea spp., 500 µg/g in

Gracilaria spp. to a high of 700 µg/g in Calagossa spp. (Figure 2.1 and

Figure 2.5).

Among the various species collected from Pulicat, Gracilaria

spp.showed the highest quantity of protein of about 750 µg/g whereas the

other Chlorophytae showed comparatively lesser quantities, as that of

650 µg/g in Ulva fasciata and 200 µg/g in Ulva lactuca (Figure 2.7).
 30

1000

900 Gracilaria1(Kovalam)
Gracilaria1(Pulicat)
800 Gracilaria1(Muttam)
Gracilaria1(Kanyakumari)
700 Gracilaria2(Covelong)
Protein content ( g/g)

Ulva fasciata(Kovalam)
600 Ulva fasciata(Pulicat)
Ulva lactuca(Pulicat)
500 Ulva lactuca(Kanyakumari)

400
Enteromorpha (Pulicat)
Enteromorpha (Muttam)
300
Enteromorpha(Kanyakumari)

200
Sargassum(Muttam)
Sargassum(Kanyakumari)
100

Valeneopsin(Muttam)
0
Valeneopsin(Kanyakumari)
)

)
)

t)

)
)

i)

)
i)
m

ng
m
lam

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ng
a

ar
ar
ta

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ta

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lo

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m
ut
va

ut
ut
Pu

ve
ve

ku
ku
(M

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(M

ov
Ko

Co
a(
Co

ya
ya

Amphiroa(Covelong)
1

in

C
iat
1(

a
ia

a(
an
an
2(

a(
ps
ph
ia

ar

sc

iro
ia

ph
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Amphiroa(Muttam)
c il

ar

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m
ca

or
c il

len
ra

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su

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Am
ra

ro
tu
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ra

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Chaetomorpha(Covelong)
Ul

to
as
lac
G

te
G

ae
En

rg
va

Ch
Sa

Chaetomorpha(Muttam)
Ul

Species & locations

Figure 2.1 Comparison of protein content in different seaweeds from

various locales

In the samples collected from Covelong, Chennai, the highest

protein content was found in Gracilaria spp. (850 µg/g) and the lowest was

found in Amphiroa spp. (100 µg/g) Ulva spp. collected from the same place

showed a relatively higher protein content of 600 µg/g whereas

Chaetomorpha spp. was around 300 µg/g.

Among the various samples collected from Muttam, Kanyakumari,

Chaetomorpha spp. showed the highest protein content of 750 µg/g and the

lowest was that of Amphiroa spp. of about 100 µg/g. The Gracilaria spp.

showed a protein content of 600 µg/g. The Sargassum spp. collected has a
 31

protein content of 550 µg/g and that of Enteromorpha spp. has about 400 µg/g

of protein content. This study shows that the protein content of seaweeds

varies from place to place.

900

800

700
Protein content( g/g)

600

500
Series1
400

300

200

100

0
ria
g

ri a
a

a
a
in
iro

at
ph

ila

la
fl

ci
ph

or

ci
c
a

s
om

ra

ra

fa
le
Am

G
wn

a
et

lv
ha
o

U
Br

Species

Figure 2.2 Protein content of seaweeds from Covelong*

 32

800

700

600
Protein content ( g/g)

500

400 Series1

300

200

100

0
Gracilaria Enteromorpha Ulva fasciata Ulva Lactuca

Species

Figure 2.3 Protein content of different seaweeds from Pulicat.

800

700

600
Protein content ( g/g)

500

400 Series1

300

200

100

0
Amphiroa Enteromorpha Gracilaria Sargassum Valeneopsin Chaetomopha
Species

Figure 2.4 Protein content of seaweeds from Muttam, Kanyakumari

 33

1000

900

800

Protein content ( g/g) 700

600

500 Series1

400

300

200

100

va
)
a

na

pa

ri a

m
ia
ia

sa
in
s

a
um
(w
ra

ne

ph
id
em

su
ps

Ul
di

er

ila
ce

go
va

m
yp

yr

or
as
eo

ul
Pa

on

ac
ro

er

la

Sp
Ul

m
H

rg
Ca
len
nt

sp

lp

Ca

Gr

ro
Sa
Ce

Co
o

Va

te
ch

En
oe
St

Species

Figure 2.5 Protein content of different seaweeds from Cape comeron,

Kanyakumari

900

800

700

600
Protein content ( g/g )

500

Series1

400

300

200

100

0
Gracilaria(Kovalam) Gracilaria(Pulicat) Gracilaria(Muttam) Gracilaria(Kanyakumari)
Species & locations

Figure 2.6 Comparision of protein content of Gracilaria spp. from

different locations
 34

700

600

500
Protein content ( g/g )

400

Series1

300

200

100

0
Ulva fasciata(Kovalam) Ulva fasciata(Pulicat) Ulva Lactuca(Pulicat) Ulva lactuca(Kanyakumari)
Species & locations

Figure 2.7 Comparision of protein content in Ulva spp. from different

areas

450

400

350

300
Protein content ( g/g )

250

Series1

200

150

100

50

0
Enteromorpha (Pulicat) Enteromorpha (Muttam) Enteromorpha(Kanyakumari)
Species & locations

Figure 2.8 Comparision of protein content of Enteromorpha spp. from

different areas
 35

1000

900

800

700
Protein content ( g/g )

600

500 Series1

400

300

200

100

0
Sargassum(Muttam) Sargassum(Kanyakumari)
Species & locations

Figure 2.9 Comparision of protein content in Sargassum spp. from

different areas

250

200
Protein content ( g/g )

150

Series1

100

50

0
Valeneopsin(Muttam) Valeneopsin(Kanyakumari)
Species & locations

Figure 2.9(a) Comparision of Protein content in Valeneopsin spp. from

different areas
 36

CHAPTER 3

ESTIMATION OF CARBHOHYDRATE

3.1 SEAWEEDS

Seaweeds or marine macro algae are the group of plants that live

either in marine or brackish water environment. They contain photosynthetic

pigments and with the help of sunlight and nutrient present in the seawater,

they photosynthesize and produce food. They are found in the coastal region

between high tide to low tide and in the sub-tidal region up to a depth where

0.01 % photosynthetic light is available. Plant pigments, light, exposure,

depth, temperature, tides and the shore characteristic combine to create

different environment that determine the distribution and variety among

seaweeds.

3.1.1 Chlorophyta

Green algae are found in the fresh and marine habitats. They range

from unicellular to multi-cellular, microscopic to macroscopic forms. Their

thalli vary from free filaments to definitely shaped forms. The photosynthetic

portion of the thalli may be moderately to highly calcified appearing in

variety of forms as fan shaped segments, feather like or star-shaped branches

with teeth or pinnules and clavate or globose branchlets.The cell has thick and
 37

stratified cell wall consisting of an inner cellulose and outer pectin layer. The

pectin layer is impregnated with calcium carbonate in all Dasycladales and in

many Siphonales (Agadi, 1976).

The majority of the Chlorophyceae have uninucleate cell and

multinucleate condition occurs in Cladophorales and Siphonales during the

formation of reproductive units. In some cases, cell division occurs in plane

parallel to the surface and result in a distromatic or pleurostromatic

paranchymatous thallus. Protoplast usually possesses a conspicuous central

vacuole often traversed by cytoplasmic strands. It possesses photosynthetic

pigments such as chlorophyll a & b, contained in the special cell structure

known as chromatophores. The chloroplast are found in varying shapes and

sizes. It has double membrane envelope and no chloroplast endoplasmic

reticulum is present. In many forms pyrenoids are present in the chloroplast,

which are the major sites of starch formation. The pyrenoids of green algae

are variably regarded as masses of reserve protein and as special organelles of

the cell. The photosynthetic product of this group is starch.

Reproduction in Chlorophyceae shows great diversity. Green algae

can produce sexually and asexually by forming flagellate and sometimes non-

flagellate spores. The vegetative propagation is achieved through

fragmentation. Sexual reproduction may be by isogamous, anisogamous or

oogamous type. The simple mode of reproduction is by isogamy i.e. fusion of

similar gametes. In anisogamy, both the gametes are flagellated but of

different size, while in oogamy the male gamete is flagellated and fuses with

large non-motile female gamete to form zygote. A large number of

 38

Chlorophyceae are haploid and reduction occurs in the germinating zygote.

All the oogamous type shows similar life cycles.

Homologous alternation of two identical phases is known to occur

in number of Ulvalaceae and Cladophoraceae, while all of Siphonales appear

to be deployed. Asexual reproduction by zoospores (motile) or aplanospores

(non-motile) produced by vegetative cells. In many cases, the cells producing

zoospores are not differentiated from vegetative cells and specialized

sporangia are rare. The zoospores are formed either singly or in some

numbers from the cells. The zoospores are naked and posses a more or less

marked colourless beak at the anterior end from which flagella (two or four)

arise. Aplanospores occur in both forms normally producing zoospores and as

permanent development in many genera derived from zoosporic ancestor.

Alternation of gametophytic and sporophytic generation occurs in this group.

3.1.2 Phaeophyta

Brown algae are exclusively marine forms. They have different

forms from simple, freely branched filaments to highly differentiated forms.

Branches are erect arising from prostrate basal filaments held together by

mucilage forming a compact pseudo-parenchymatous aggregation of

filaments into prostrate crust or erect branched axis or leaf like blades

exhibiting the haplostrichous condition. Many species have large massive

thalli with special air bladder, vesicles or float to make them buoyant. The

cell wall is two layered. Outer layer is mucilagenous and sticky due to the
 39

presence of alginate. The inner layer is of cellulose (micro fibrils). The cell is

uninucleate with one or two nucleoli (Agadi, 1976).

The nuclei of Phaeophyta are usually large and possess a large and

readily stained nucleolus with a delicate network having little chromatic

material. The chromosomal organization is much advanced. The chromo

centers on the chromosome of unknown function are characteristic of

Phaeophyta. Cytoplasm contains organelles like mitochondria, golgibodies,

endoplasmic reticulum, chromatophores, vacuoles and fucosan vesicles. The

chromatophores are invariably parietal. The photosynthetic cells in the

majority of brown algae contain numerous small discoid chromatophores.

Chromatophores show movement to changes in the intensity and direction of

illumination. Brown algae vary in colouration from olive-yellow to deep

brown. The colouration is due to the accessory carotenoid pigment and

fucoxanthin. The amount of fucoxanthin varies in different species of brown

algae. Dictyota, Ectocarpus, Laminaria etc. are rich in fucoxanthin, while

species of Fucus are poor in fucoxanthin. Most of the littoral brown algae are

rich in xanthophylls and fucoxanthin. The algae rich in fucoxanthin exhibit a

much higher rate of photosynthesis in blue light than the algae with poor

fucoxathin. The other photosynthetic pigments of the brown algae are

chlorophyll a & c, -carotene and xanthophylls. The photosynthetic products

of the brown algae are laminarian and manitol. Laminarian is dextrin like

polysaccharide, a food reserve; arise from the simple sugar of photosynthesis.

Manitol appears to not to be widely distributed and presence of such

alcohols may account for extreme scarcity of free sugars as they undergo
 40

immediate conversion into alcohol and polysaccharides.This group

reproduces sexually and asexually. Several species of this group reproduce

vegetatively by fragmentation. Members of this group produce biflagellate

neutral spores found with in one celled or many celled reproductive organs.

The asexual reproduction is by the formation of zoospores in the

unilocular or pleurilocular sporangia except in Dictyotales, Tilopteridales and

Fucales. Unilocular sporangia produce haploid gamatophytic stage, while,

pleurilocular sporangia produce diploid phase. The zoospores are asymmetric

or bean shaped with two lateral or sub apical flagella. Zoospores are formed

in the single celled unilocular sporangia by meiosis and gives rise to

gametophytes. Sexual reproduction is by isogamy, anisogamy and oogamy. In

oogamous type of reproduction, the male sex organ (antheridium) and the

female sex organ (oogonium) may be produced on the same plant or on

different plants. Alternation of gametophytic and sporophytic generations

occurs in this group except in the members of Fucales.

3.1.3 Rhodophyta

Except for few species, Rhodophyta are exclusively marine. They

vary in size and shape. They are either epiphytes, grows as crust on the rocks

or shells as a large fleshy, branched or blade like thalli. The thallus is

basically filamentous, simple or branched, free or compacted to form

pseudoparenchyma with uni or multiaxial construction. They inhabit intertidal

to subtidal to deeper waters (Agadi, 1976).

 41

Cells are eukaryotic. Inner cell wall is of cellulose and outer cell

walls with amorphous matrix of mucopolysaccharides (i.e. agar, porphyron,

furcellaran, and carrageenan). Cells are uninucleate/multinucleate with a large

centric vacuole. The cross wall separating neighbouring cells exhibit a distinct

feature - the pit connection or pit plug. The cytoplasm exhibits a high degree

of viscosity and there is often a very firm adhesion to the wall which

penetrates to the inner most layer of the membrane.

The cells of Rhodophyta are always uninucleate except in the older

cells that are multinucleate. The nuclei exhibit a prominent nucleolus and a

well developed network with numerous chromatin grains. The chloroplast

varies from single, axial, stellate in primitive taxa to parietal and discoid

forms in non advance taxa. The colouration of Rhodophyta is due to water-

soluble pigments, the red phycoerythrin and blue phycocyanin.

Other pigments present are chlorophyll a & b, carotene etc. The

photosynthetic product of this group is floridian starch. Phycoerythrin

pigment is found to be in the greater quantity in seaweeds of deeper water and

freely illuminated forms which also show increase ratio of phycoerythrin to

chlorophyll. The accessory pigments that resemble those found in

Myxophyceae are of proteins and show characteristic similar to those of

globulin.

Red algae carry on apparently more photosynthesis in feeble light

than brown and green algae. This group seldom reproduces asexually. All the

members of this group produce one or more kinds of non-flagellated spores

 42

that are either sexual or asexual in nature. Sexual reproduction is very

complicated involving several structures after fusion of gametes.

The male structure called antheridium produces single spermatangia

which give rise to nonmotile spermatia. The female structure is a swollen

oogonium, which usually bears a long drawn out receptive trichogyne. The

zygote is formed either by direct division as in Bangiales or after production

of filamentous outgrowth called oogonimoblast which give rise to number of

sporangia each forming naked spore. Reduction occurs either at the first

division of the zygote nucleus or is postponed and takes place in special

tetrasporangia borne on individual distinct forms.

Some members of this group exhibit biphasic alternation of

generation in which sexual generation (gametophyte) alternates with asexual

(tetrasporophyte) generation, while others are triphasic with three generation

or somatic phases (gametophyte, caropsporophyte, tetrasporophyte)

successively following one another.

3.2 SEAWEEDS AS POTENTIAL NUTRIENT SUPPLIERS

Seaweeds, which have traditionally been used by the Western food

industry for their polysaccharide extractives alginate, carrageenan and agar

also contain compounds with potential nutritional benefits. Seaweeds have

recently been approved in France for human consumption (as vegetables and

condiments), thus opening new opportunities for the food industry (Fleurence,

1999).
 43

The seaweeds are also used as food in the regions of Far East and

Australia. The inhabitants of the Hawaii Island consume large quantities of

seaweeds. The indigenous people of China use large quantities of Durvillea

antarctica and some species of Ulva. The natives of New Zealand use certain

green seaweeds in preparation of salad and soups. The people of China and

Japan consume the seaweeds on large scale. The people living on the

seacoasts in these countries commonly use fresh seaweeds as food. The most

important food species in Japan are Nori (Porphyra species), Kombu

(Laminaria species), and Wakame (Undaria pinnatifida). In Japan Porphyra

tenera happens to be one of the most important edible algae and a product by

the name of Amanori and Asakusa- Nori are made from it.

The use of kelps ("kombu" in Japan; "haidai" in China) dates back

to at least the 5th century in China. The main species used is Laminaria

japonica (Laminariales), but 8-11 other species are used also, mainly in Japan

(Fleurence , 1999).

Plants are dried after harvesting and either cut into strips or

powdered. In Japan, Kombu is used in the preparation of fish, meat dishes,

and soups and also as a vegetable with rice. Powdered kombu is employed

either in sauces and soups or is added to rice in the same way as curry. Some

kinds are used in making an infusion similar to tea.

Another kelp, Undaria pinnatifida (Laminariales), is widely used in

Japan (where it is known as "wakame") and China ("qundai-cai") as food. In

Japan this species is a more important crop than Laminaria both in value and
 44

production.The harvested algae are dried after washing in freshwater. After

re-soaking the plant material is used as an additive to soups (wakame soup is

served with virtually every meal in Japan); toasted (Yaki-wakame); used half

re-soaked, with boiled rice; and coated in sugar and tinned (Ito-wakame).

Nori is a red algae, Porphyra sp. (Bangiophyceae). Nori is sold in

sheets that may be toasted to give a green colour and then flaked and added to

sauces, soups and broths. Sometimes it is just soaked and eaten. Small, dry

nori sheets are used to wrap cold rice balls, which make a popular lunch-time

snack for Japanese children. The food value of Nori lies in its high protein

content (25-35% of dry weight), vitamins and mineral salts, especially iodine.

Its vitamin C content is about 1.5 times that of oranges and 75% of the protein

and carbohydrates are digestible by humans, which is very high for seaweeds.

Seaweeds are the richest source of vitamins. The vitamins A, B and

E are found abundantly in seaweeds. The vitamin B essentially required for

the development of human body is found in great abundance in almost all

Phaeophyceae. The cod liver oil is the rich source of vitamin A, which is

acquired from seaweeds. Vitamin E is equally important for human beings

which are found in many types of seaweed (Fleurence, 1999).

Some countries have even industries to process seaweeds into

suitable cattle feed. The manufacture of cattle feed from seaweeds are made

principally from brown algae and the processed food is fed to cattle, poultry

and even pigs. It has seen recorded that dried seaweeds served as cattle food
 45

have enhanced the milk-yielding and egg-laying capacity of cattle and poultry

respectively.

3.3 SEAWEEDS FOR MEDICAL PURPOSE

There are several medicinal properties of seaweeds. Algae rich in

iodine such as Asparagopsis taxiformis, Sarconema sp. can be used for

controlling goitre disease caused by enlargement of thyroid glands. Many

bioactive compounds can be obtained from seaweeds. Several diseases caused

by vitamin deficiency such as vitex, asthma, tooth decay etc., may be

eradicated, if flour of the seaweeds is added to the food. Iodine is the most

important element to enable the thyroid glands to secrete the tyrosine which

contains 60% iodine. It controls the general development of the animal.

Seaweeds are the best source of iodine for human beings. Several

important seaweed medicinal preparations are prepared in various countries,

i.e., Kelpeck is prepared from kelps in Chicago; Burbank vegetable tablets are

seaweed preparations from United States. Kelpamalt is a seaweed medicinal

preparation from New York (U.S.A.); Isokelp is prepared in California;

Parakelp and Manamar are other medicinal seaweed American preparations.

An antibiotic drug Chlorellum is also obtained from algae. About forty-five

elements are found in a seaweed Macrocystis pyrifera. In addition to these

elements vitamins are also found. No other food contains such a great

abundance of minerals and vitamins.

 46

Some countries have even industries to process seaweeds into

suitable cattle feed. The manufacture of cattle feed from seaweeds are made

principally from brown algae and the processed food is fed to cattle, poultry

and even pigs. It has seen recorded that dried seaweeds served as cattle food

have enhanced the milk-yielding and egg-laying capacity of cattle and poultry

respectively

3.4 ANALYSIS OF CARBHOHYDRATE

3.4.1 Reagents / chemicals used

The list of all chemicals and solutions prepared and their

composition are given in the Appendix. The chemicals used in this analysis

were Anthrone Reagent, HCl, H2 SO4, std glucose and distilled water

3.4.2 Sample preparation

0.1 g of sample was taken in a test tube to which 5 mL of 2.5 N

HCl was added.

The mixture was kept in boiling water bath for 3 hour

The contents were centrifuged at 10,000 rpm for 10 min.

Supernatant was collected for further analysis discarding the

pellet.
 47

3.4.3 Procedure

The method used was Colorimetric estimation by Anthrone reagent

(Yemm and Willis, 1954).

Principle: The carbohydrates were first hydrolyzed to simple

sugars using dilute hydrochloric acid. In the hot acidic medium
glucose is dehydrated to hydroxymethyl furfural. This compound
forms a green coloured medium with absorption maximum at
620 nm.

First, the glucose was standardized with the Anthrone

Reagent.100 mg of glucose was dissolved in 100 mL of water to
serve as Stock solution.10 mL of the stock was diluted to serve
as the working standard. Aliquots were prepared by taking
0.2, 0.4, 0.6, 0.8 and 1 mL from the working standard. 1 mL of
distilled water served as the sample blank. The volume was
made unto 1ml in all the test tubes with distilled water. Then
5 mL of ice cold Anthrone reagent was added to each of the
tubes and incubated for 10 min. The green colour was read at
620 nm. The standard graph was plotted with the concentration
in X-axis and OD in Y-axis.

100 µL of sample was taken in a test tube to which 5 mL of

Anthrone reagent was added. The green colour was read at
620 nm in a colorimeter. Optical density was plotted in the
standard graph.

Carbohydrate content was obtained by intrapolation of the

standard graph.
 48

3.5 RESULTS AND DISCUSSIONS

The carbohydrate analysis of various samples was done by Anthrone

Method (Yemm and Willis, 1954). The carbohydrate content was estimated to

be highest in Gracilaria spp.from Cape comeron, Kanyakumari (Figure 3.6).

It was about 100 mg/g i.e., about 100g/kg. In a similar study, the carbohydrate

content of a certain species of Gracilaria spp. was estimated around

43.07 g/kg (Dere et al 2003). The lowest carbohydrate content was observed

in Halimeda spp. from Cape comeron, Kanyakumari. It was about 21 mg/g.

Two varieties of Ulva were collected. Ulva from Covelong,

Chennai, had a protein content of about 43 mg/g whereas the same species

from Cape comeron Kanyakumari had about 39 mg/g. The other species of

Ulva from Cape comeron Kanyakumari had about 60 mg/g of carbohydrate

content whereas the one collected from Pulicat had about 90.5 mg/g. Similar

results were obtained in another study wheras the carbohydrate content in

Ulva spp. was found to be 63.04 g/kg (Dere et al, 2003).

In another study the carbohydrate content was obtained for different

species of Enteromorpha spp. The contents varied in the range of 10 g/kg to

25 g/kg (Dere et al, 2003). In our study, Enteromorpha spp. from two places

were analysed. The contents were obtained as 39 g/kg from Pulicat and

46 g/kg from Cape comeron Kanyakumari. Amphiroa spp., a red seaweed was

collected from two different locations; Muttam, Kanyakumari and Covelong,

Chennai. The estimated carbohydrate contents were relatively similar;

30 mg/g and 27 mg/g respectively. Valeneopsin spp., collected from two

 49

places Muttam, Kanyakumari and Cape comeron Kanyakumari showed slight

variations of 25 mg/g to 34 mg/g respectively. Centroceras spp., a

Rhodophytae, had a carbohydrate content of about 29 mg/g which is lesser

compared to other red algae collected. Chaetomorpha, a green algae had a

carbohydrate content of about 38 mg/g.

100

90

80

70
Carbohydrate content (mg/g)

60

50 Series1

40

30

20

10

0
Ulva Gracilaria Enteromorpha
Species

Figure 3.1 Carbohydrate content of different seaweed species from

Pulicat
 50

50

45

40

35
Carbohydrate content (mg/g)

30

25 Series1

20

15

10

0
Gracilaria Ulva1 Ulva1 Amphiroa
Species

Figure 3.2 Carbohydrate content of different seaweed species from

Covelong*

45

40

35
Carbohydrate content(mg/g)

30

25

Series1

20

15

10

0
Chaetomorpha Gracilaria Valeneopsin Sargassum Amphiroa
Species

Figure 3.3 Carbohydrate content from different seaweed species from

Muttam, Kanyakumari
 51

120

100 Centroceras
Hypnea
Ulva
Padina
80
Stoechospermum
Concentration (mg/g)

Valeneopsin
Colpomenia
Caulerpa
60
Halimeda
Spyridia
Gracilaria
40 Sargassum
Ulva
Enteromorpha
Gracilaria
20 Amphiroa

0
a

va

va
na

ia
a

ia

ia

a
s

m
ia
sin

a
um
e

ed
rp

iro
ra

ph
id

ar

ar
en

su

Ul
Ul

di
pn

op

le
ce

yr
rm

lim

cil

cil

ph
or
Pa

as
om
Hy

au

Sp
ne
ro

ra

ra
om
pe

Am
Ha

rg
lp

C
nt

G
le
os

Sa

r
Co
Ce

Va

te
ch

En
oe
St

Species

Figure 3.4 Carbohydrate content in different seaweed species from

Cape comeron, Kanyakumari

100

90

80

70
Carbohydrate content (mg/g)

60

50 Series1

40

30

20

10

0
Ulva2(Pulicat) Ulva1(Covelong) Ulva1 (Kanyakumari) Ulva2(Kanyakumari)

Species & locations

Figure 3.5 Carbohydrate content in Ulva spp. from different locales

 52

120

100

Carbohydrate content (mg/g)

80

60 Series1

40

20

0
)
)

i)
i)
at
ng

ar
ar
ic

ta
lo

m
m
l

ut
Pu
ve

ku

ku
(M
Co

2(

ya
ya
ia

an
an
1(

ia
ar

K
K
ria

r
cil

ila

2(
1(
ila

ra

ria
ria
ra
G
ac

la
G

il a
Gr

ci
ac

ra
Gr

G
Species

Figure 3.6 Carbohydrate content in Gracilaria spp. from different locales

48

46

44
Carbohydrate content (mg/g)

42

Series1

40

38

36

34
Enteromorpha (Pulicat) Enteromorpha(Kanyakumari)
Species & locations

Figure 3.7 Carbohydrate content in Enteromorpha spp. from different

locales
 53

30.5

30

29.5

29
Carbohydrate content (mg/g)

28.5

28 Series1

27.5

27

26.5

26

25.5
Amphiroa (Muttam) Amphiroa (Covelong)
Species & locations

Figure 3.8 Carbohydrate content in Amphiroa spp. from different locales

40

35

30
Carbohydrate content (mg/g)

25

20 Series1

15

10

0
Valeneopsin (Muttam) Valeneopsin(Kanyakumari)
Species & locations

Figure 3.9 Carbohydrate content in Valeneopsin spp. from different

locales
 54

70

60

50
Carbohydrate content (mg/g)

40

Series1

30

20

10

0
Hypnea (Kanyakumari) Hypnea (Ennore) Hypnea (Kalpakkam)
Species & locations

Figure 3.9(a) Carbohydrate content in Hypnea spp. from different locales

 55

CHAPTER 4

ESTIMATION OF TRACE ELEMENTS

Seaweeds have been used since ancient times as food, fodder,

fertilizer and as a source of medicine. Trace elements like zinc, cadmium,

copper, manganese, iron, cobolt, nickel etc. are present in seaweeds. Both

macro and micro minerals present in seaweeds can be analyzed using

SEM/EDAX studies. The concentrations of five major and twenty eight trace

elements in thirty five marine algae collected from the coast of China were

determined by instrumental neutron activation analysis (Xiolin Hou and

Xiaojun Yan, 1998).

Trace elements like arsenic, copper, molybdenium, manganese,

zinc, cobalt, antimony, selenium, and iron were analsed in algal samples such

as Ascophyllum nodosum and Laminaria hyperborean. The variation in the

content of trace elements has been studied during a period of one year and

samples being taken every second month. Considerable differences in the

content of trace elements was found between the Laminariaceae and

Fucaceae (Lunde, 1970).

In this study three algal samples such as Ulva spp.,Gracilaria spp.,

Sargassum spp. were studied for their trace elemnts and mineral contents such
 56

as carbon, nitrogen, oxygen, zinc, sodium, magnesium, aluminium, silicon,

phosphorus, sulphur, calcium, barium, cobalt, nickel, arsenium, and lead were

analysed using SEM/ EDAX analysis.

4.1 SCANNING ELECTRON MICROSCOPE

A Scanning electron microscope (SEM) can be used for visualizing

high magnified images of almost all materials. SEM in combination with

EDAX (Energy dispersive X-ray spectroscopy) is highly helpful to find out

the different elements present in different parts of sample.

Biological materials can be examined by SEM with special sample

preparation. For the sample which is dry enough and durable, it is necessary

to cover the sample with a thin metal layer (eg. Gold or carbon). This coating

is to prevent charging of electrons at the sample.

SEM is also used for studying surfaces and structures of fibres,

surface coatings and print in paper and wood. Inorganic elements can be

analysed chemically and it is possible to see their distribution in the material.

4.2 ENERGY DISPERSIVE X-RAY SPECTROSCOPY

EDX or EDS is an analytical technique used for the elemental

analysis or chemical charaterisation of the sample. It is one of the variants of

X-ray fluorescence spectroscopy which relies on investigation of a sample

through interaction between electromagnetic radiation and matter. The

analysed X-rays emitted by the matter in response to being hit with charged
 57

particle. The fundamental principle behind charaterisation of element is that

each element as a unique atomic structure emitting X-rays that are

characteristic of an elements atomic structure.

The emission of characteristic X-rays from a specimen can be

stimulated by focusing a high energy beam of charged particles such as

electrons or protons, or a beam of X-rays is focused in to the sample being

studied. At rest an atom within the sample contains ground state (unexcited)

electrons in discrete energy levels or electron shell bound to the nucleus. The

incident beam may excite an electron in an inner shell, ejecting it from the

shell by creating an electron hole. An electron from an outer, higher energy

shell then fills the hole, and the difference in energy between the higher

energy shell and lower energy shell may be released in the form of an X-ray.

The number and energy of the X-rays emitted from a specimen can be

measured by an energy dispersive spectrometer.

As the energy of the X-rays are characteristic of the difference in

energy between the two shells, and of the atomic structure of the element

from which they were emitted, and helps in identifying the elemental

composition of the specimen to be measured.

4.2.1 Equipment

There are four primary components of the EDS set up.

The beam source

The X-ray detector

 58

The pulse processor and

The analyser

The equipment measures the number of emitted X-rays. EDS system

are most commonly found on scanning electron microscope (SEM-EDS) and

electron micro probes, even though a number of free standing EDS system

exists. A detector is connected to SEM to convert X-ray energy in to voltage

signals, and is sent to a pulse processor, which measures the signals and

passes them on to analyzer for data display and analysis.

Accuracy of EDS spectrum can be affected by many factors. EDS

detectors can not detect elements with atomic number less than 4. Over

voltage settings in EDS alter the peak sizes. The accuracy of the spectrum can

also be affected by the nature of the sample. X-rays can be generated by any

atom in the sample that is sufficiently excited by the incoming beam. The

X-ray escaped from the sample is being available to detect and measure trace

elements and it mainly depends on the energy of the X-ray and the amount

and density of the material it has to pass through. This can result in reduced

accuracy inhomogenous and rough samples.

4.3 ANALYSIS OF TRACE ELEMENTS IN SEAWEEDS

Trace elements like arsenic, copper, molybdenium, manganese,

zinc, cobalt, antimony, selenium, and iron were analysed in algal samples

such as Ascophyllum nodosum and Laminaria hyperborean. The variation in

the content of trace elements has been studied during a period of one year and
 59

samples being taken every second month. Considerable differences in the

content of trace elements was found between the Laminariaceae and

Fucaceae (Lunde, 1970).

Cadmium and lead concentrations were determined in some algal

species living in the southern most coast of Argentina. Algal species of the

genera Lessonia, Macrosystis and Gigartina of commercial interest were

collected from harvest area and analyzed. Accumulation of lead and cadmium

was evident in other common brown seaweeds from the industrial site. High

values of aluminium ranging between 300 and 3000 mgAl/kg (dry basis) were

recorded in the industrialized area. Almost all of the species studied,

Colpomenia sinuso from Gulf Nuevo exhibited highest values of aluminium

(Muse et al, 1995).

Most of the trace elements present in the algal biomass are heavy

metals and algae have been reported strongly active in heavy metals

concentration (Whitton, 1984, Forsberg et al, 1988). While some trace

elements are considered toxic viz., As, Br, Cd, Hg, Pb, Sb, others are

considered essential (Cu, Zn) or necessary to human body (Cr, Se) but

become health hazardous when their intake values are exceeded.

Edible brown and red seaweed could be used as a food supplement

to help to meet the recommended daily intake of some essential minerals and
trace elements. Mineral content was determined in several brown edible sea
vegetables such as Fucus vesiculosus, Laminaria digitata, Undaria
pinnatiafida and also in red algae Chondrus crispus and Porphyra tenera.
 60

Atomic absorption spectrophotometry of the ashes indicated that marine

seaweeds contained higher amounts of both macro minerals (8.083-17,875
mg/100g; sodium, potassium, calcium, magnesium) and trace elements
(5.1 - 15.2 mg/100g; iron, zinc, manganese, copper), than those reported for
edible plants (Ruperez, 2002).

Trace element concentrations varied among different species from

the same locality and also varied according to the position on the shore.
Concentrations of zinc, cadmium, copper, manganese, iron, cobalt, nickel and
molybdenum are examined in brown algae, Fucus serratus and Fucus
vesiculosus from Cardigan bay, Irish sea and Great Britain. In both species a
seasonal variation in metal content was observed. Zinc, cadmium, copper,
iron, nickel and cobalt concentrations were highes and lowest in the autumn,
probably it reflects the level of metabolic activities and climatic factor (Fuge
and James, 1973).

4.4 RESULTS AND DISCUSSIONS

In this study, the three algal samples such as Ulva spp. Gracilaria
spp.and Sargassum spp.were studied for their tace element content using
SEM/EDAX analysis. The Percentage of oxgen was found to be high ( 48%)
in Ulva spp (Figure 4.1A and 4.1B) which is followed by Sargassum spp.,
44% (Figure 4.3A and 4.3B) and Gracilaria spp., 38% (Figure 4.2A and
4.2B). The total percentage of carbon was found to be more in Gracilaria spp.
41% (Figure 4.2A and Figure 4.2B) when compared to Ulva spp., 27%
(Figure 4.1A and Figure 4.1B) and Sargassum spp. 38.5% (Figure 4.3A and
Figure 4.3B) respectively.Mineral content was determined in seaweeds such
as Ulva spp. Gracilaria spp.and Sargassum spp.
 61

Figure 4.1a SEM-EDAX analysis of Ulva Spp.

Table 4.1a. EDAX ZAF quantification of Ulva Spp.

Element Wt % At %
C.K. 33.41 41.79
N.K 09.07 09.72
O.K. 45.97 43.17
ZnL 00.14 00.03
NaK 00.41 00.26
MgK 02.39 01.48
AlK 00.58 00.32
SiK 01.01 00.54
P.K 00.19 00.09
S.K 02.50 01.17
CdL 00.06 00.01
CaK 03.13 01.17
BaL 00.20 00.02
CoK 00.15 00.04
Nik 00.12 00.03
AsK 00.69 00.14
PbL 00.00 00.00
 62

Figure 4.1b. SEM-EDAX analysis of Ulva Spp.

Table 4.1b. EDAX ZAF quantification of Ulva Spp.

Element Wt % At %
C.K. 20.78 29.12
N.K 05.52 06.64
O.K. 50.74 53.38
ZnL 00.00 00.00
NaK 00.19 00.14
MgK 02.47 01.71
AlK 01.75 01.09
SiK 03.78 02.26
P.K 00.23 00.13
S.K 01.58 00.83
CdL 00.24 00.04
CaK 09.50 03.99
BaL 00.46 00.06
FeK 01.38 00.42
CoK 00.21 00.06
NiK 0.013 00.04
AsK 00.18 00.04
PbL 00.86 00.07
 63

SEM analysis of dried powdered seaweeds indicated that marine

seaweeds contain both macro nutrients as well as micro nutrients. The Macro

nutrients such as sodium, calcium and magnesium is present in Ulva spp.

Gracilaria spp. and Sargassum spp. Sodium content is more in Sargassum

spp. 1.6% (Figure 4.3A and Figure 4.3B) when compared to Ulva spp.

0.41% (Figure 4.1A and Figure 4.1B) and Gracilaria spp. 0.31% (Figure

4.2A and Figure 4.2B). Calcium content is more in Gracilaria spp. 7.31%

(Figure 4.2A and Figure 4.2B) than Sargassum spp. 3.52% (Figure 4.3A

and Figure 4.3B) and Ulva spp. 3.13% ( Figure 4.1A and Figure 4.1B).

Figure 4.1A and Figure 4.1B shows that Ulva spp.contain more amount of

magnesium (2.39%) than Sargassum spp. 1.82% (Figure 4.3A and Figure

4.3B) and Gracilaria spp. 1.48% (Figure 4.2A and Figure 4.2B). The macro

nutrient potassium is present only in Sargassum spp. 1.59% where as it is

absent in Gracilaria spp. and Ulva spp.

 64

Figure 4.2a. SEM-EDAX analysis of Gracilaria Spp.

Table 4.2a. EDAX ZAF quantification of Gracilaria Spp.

Element Wt % At %
C.K. 42.29 54.96
N.K 06.84 07.62
O.K. 29.05 28.34
ZnL 00.00 00.00
NaK 00.31 00.21
MgK 01.48 00.93
AlK 00.85 00.49
SiK 01.36 00.75
P.K 00.98 00.50
S.K 04.71 02.29
CdL 00.56 00.08
CaK 07.31 02.84
BaL 00.67 00.08
FeK 01.64 00.46
CoK 00.27 00.07
NiK 00.13 00.04
AsK 01.55 00.32
PbL 00.00 00.00
 65

Figure 4.2b. SEM-EDAX Analysis of Gracilaria Spp.

Table 4.2b EDAX-ZAF Quantification of Gracilaria Spp.

Element Wt % At %
C.K. 40.94 49.30
N.K 05.36 05.54
O.K. 46.64 42.16
ZnL 00.00 00.00
NaK 00.59 00.37
MgK 00.68 00.40
AlK 00.17 00.09
SiK 00.23 00.12
P.K 00.04 00.02
S.K 02.29 01.03
CdL 00.08 00.01
CaK 02.14 00.77
BaL 00.21 00.02
FeK 00.21 00.03
CoK 00.18 00.03
NiK 00.24 00.06
AsK 00.00 00.00
PbL 00.00 00.00
 66

In a similar study mineral content was also determined in several

brown algae (Fucus vsesiculosus, Laminaria digitata, Undaria pinnatifida)

and red algae (Chondrus crispus, Porphyra tenera). Atomic absorption

spectroscopy of the ashes indicated that marine seaweeds contained higher

amounts of both macro minerals (8.083 – 17,875 mg/100g; sodium,

potassium, calcium, magnesium) and trace elements (5.1 – 15.2 mg/100g;

iron, zinc, manganese, copper), than those reported for edible land plants.

(Ruperez, 2002).
 67

Figure 4.3a. SEM-EDAX Analysis of Sargassum Spp.

Table 4.3a EDAX-ZAF Quantification of Sargassum Spp.

Element Wt % At %
C.K. 41.04 30.89
N.K 05.29 03.63
O.K. 40.13 37.36
ZnL 00.00 00.00
NaK 01.62 01.05
MgK 01.84 01.13
AlK 00.21 00.12
SiK 00.21 00.11
P.K 02.65 00.07
S.K 00.97 01.23
CLK 00.03 00.41
CaL 01.39 00.01
K.K 03.32 00.01
BaL 00.00 01.31
FeK 00.09 00.00
CoK 00.04 00.02
NiK 00.08 00.01
AsK 00.00 00.00
PbL 00.33 00.04
 68

Figure 4.3b. SEM-EDAX Analysis of Sargassum Spp.

Table 4.3b EDAX-ZAF Quantification of Sargassum Spp.

Element Wt % At %
C.K. 35.87 43.90
N.K 04.93 03.39
O.K. 43.52 41.71
ZnL 00.00 00.00
NaK 01.63 01.09
MgK 01.72 01.09
AlK 00.27 00.13
SiK 00.37 00.20
P.K 0.11 00.06
S.K 02.25 01.07
CLK 01.19 00.31
CoL 00.08 00.61
K.K 01.73 00.69
CaK 03.11 01.96
BaL 00.24 00.03
FeK 00.40 00.11
CoK 00.23 00.06
NiK 00.33 00.09
AsK 00.00 00.00
PbL 00.00 00.00
 69

Concentrations of trace metals such as iron, zinc, manganese,

copper, cadmium, cobalt, nickel, lead are examined using SEM/EDX analysis.

The red algae Gracilaria spp. (Figure 4.2A and Figure 4.2 B) contains high

concentration of iron (1.64%) than Ulva spp.,1.38% (Figure 4.1A and Figure

4.1B) and is ver low in Sargassum spp., 0.04% (Figure 4.3A and Figure

4.3B). The trace elements cadmium, cobalt, nickel are present in trace

amounts in all the three algal samples. Trace elements such as zinc,

manganese, copper and lead are absent in all the three algal samples.

Fuge and James (1973) examined the concentrations of zinc,

cadmium, copper, manganese, iron, cobalt, nickel and molybdenum are

examined in brown algae, Fucus serratus and Fucus vesiculosus from

Cardigan Bay, Irish sea and Great Britain. In both species a seasonal variation

in metal content was observed. Zinc, cadmium, copper, iron, nickel and cobalt

concentrations were highes and lowest in the autumn, probably it reflects the

level of metabolic activities and climatic factor.

 70

CHAPTER 5

FTIR SPECTROSCOPIC ANALYSIS OF SEAWEEDS

The chemical characterization of algal bodies or colonies requires

the utilization of micro scale analytical tools. Some of these tools include UV
fluorescence, Raman micro spectroscopy and transmission near IR and mid
IR micro spectroscopy. For some years IR spectroscopy has been successfully
applied to the structural characterization of coals and oil shales.More recently
transmission IR micro spectroscopy has been employed for characterizing
individual microscopic components of sedimentary organic matter. So far,
most applications of IR and micro IR techniques to sedimentary organic
matter have been concentrated on the relative abundance of aliphatic C-H, and
acid C=O bands, which were used to indicate the potential for petroleum
generation.

Certain analysis requires numerical derivatization and curve fitting

to unravel the overlapping bands resulting from various coal structures. For
example, in the stretching vibration region between 3000 and 2800 cm-1, the
spetrum has contributions from the three aliphatic components, CH, CH2, and
CH3.The application of this type of data analysis was demonstrated to have
provided greater insight into the aliphatic structures of coals and kerogens.

Vibrational spectroscopy is one of the most useful methods of

investigating molecular interactions as well as structural details. The vibration
 71

frequency obtained is a measure of the force constant between the atoms

constituting a bond, and the constants are related to the bond orders and
electronic distributions between these atoms.Raman spectroscopy has been
extensively employed in investigations of flavins and flavoproteins (Yasuzo
Nishina et al, 2007).

5.1 FTIR SPECTROSCOPY

Fourier transform infrared spectroscopy can be employed to study

the complex structures.This instrument involves fixed and rotating mirrors
that split the incident beam in to two. The beams are recombined after passage
through the sample, but as the two path lengths are different, interference
pattern arise that may be analysed by fourier transform methods.IR
spectroscopy, wavelength is measured in wave numbers (cm-1) it is useful to
divide the infrared region into three sections; near, mid and far infrared; The
infrared, (approx. 400-10 cm-1) lying adjacent to the microwave region, has
low energy and may be used for rotational spectroscopy. The mid infrared
(apprx. 4000-400 cm-1) may be used to study the fundamental vibrations and
associated rotational vibrational structure, whilst the higher energy near IR
(14000-4000 cm -1) can excite overtone or harmonic vibrations. Usually all
molecules will be having vibrations in the form of stretching and bending etc,
the observed energy will be utilized in changing the energy levels associated
with them.

The positions of atoms in a molecule are not fixed; they are subject
to a number of different vibrations. Vibrations fall into the two main
categories of stretching and bending. Change in inter-atomic distance along
bond axis (bond length). In addition to the vibrations mentioned above,
 72

interaction between vibrations can occur (coupling) if the vibrating bonds are
joined to a single, central atom. Vibrational coupling is influenced by a
number of factors;

Strong coupling of stretching vibrations occurs when there is a

common atom between the two vibrating bonds.

Coupling of bending vibrations occurs when there is a common

bond between vibrating groups.

Coupling between a stretching vibration band a bending

vibration occurs if the stretching bond is one side of an angle
varied by bending vibration.

Coupling is greatest when the coupled groups have

approximately equal energies.

no coupling is seen between groups separated by two or more

bonds

Most organic compounds have C-H bonds, a useful rule is that

absorption in the 2850 to 3000 cm-1 is due to sp3 C-H stretching; whereas,
absorption above 3000 cm-1 is from sp 2 C-H stretching or sp C-H stretching if
it is near 3300 cm -1. Absorption bands in the 4000 to 1450 cm-1 region are
usually due to stretching vibrations of diatomic units, and this is sometimes
called the group frequency region. The complexity of infrared spectra in the
1450 to 6000 cm-1 region makes it difficult to assign all the absorption bands
and because of the unique patterns found there it is often called the fingerprint
region.
 73

Table 5.1 Typical Infrared Absorption Frequencies

Stretching Vibrations
Functional Class -1)
Range (cm Intensity Assignment
Alkanes 2850-3000 Str CH3, CH2 & CH
2 or 3 bands
Alkenes 3020-3100 med =C-H &=CH2 (usually sharp)
1630-1680 var C=C(symmetry reduces
intensity)
1900-2000 str C=C asymmetric stretch
Alkynes 3300 str C-H(usually sharp)
2100-2250 var C C (symmetry reduces
intensity)
Alcohols and 3580-3650 var O-H(free),usually sharp
Phenols 3200-3550 str O-H (H-bonded),usually
970-1250 str broad
C-O
Amines 3400-3500(dil.soln.) wk N-H(1° - amines),2 bands
3300-3400 (dil.soln.) wk N-H(2° - amines)
1000-1250 med C-N
Aldehydes & 2690-2840 med C-H(aldehyde C-H)
Ketones (2 bands) str C=O(saturated aldehyde)
1720-1740 str C=O(saturated ketone)
1710-1720 str aryl ketone
1690 str , -unsaturation
1675 str cyclopentanone
1745 str cyclobutanone
1780
Carboxylic Acids 2500-3300(acids) str O-H(very broad)
& Derivatieves Overlap C-H str C=O(H-bonded)
1705-1720(acids) med-str O-C (sometimes 2-peaks)
1210-1320(acids) str C=O
1785-1815 str C=O(2 - bands)
(acyl halides)
1750 &1820 str O-C
(an hydrides)
1040-1100 str C=O
1735-1750 (esters) str O-C( 2-bands)
1000-1300
1630-1695 (amides) str C=O (amide l band)
Nitriles 2240-2260 med C N (sharp)
Isocyanates,losthiocyan 2100-2270 med -N=C=O, -N=C=S
ates, Diimides,Azides -N=C=N-,-N3,C=C=O
&Ketenes
 74

Table 5.2 Typical Functional Class and their Characteristic Absorption

Functional Class Characteristic Absorption

Sulfur Functions
S-H thiols 2550-2600 cm-1 (wk & shp)
S-OR esters 700-900(str)
S-S disulfide 500-540(wk)
C=S thiocarbonyl 1050-1200(str)
S=O sulfoxide 1030-1060(str)
sulfone 1325+25(as) & 1140 +20(s)(both str)
sulfonic acid 1345(str)
sulfonyl chloride 1365 ± 5 (as) & 1180 ± 10 (s) (both str)
sulfate 1350-1450 (str)
Oxidized Nitrogen Functions
=NOH oxime
O-H(stretch) 3550-3600 cm-1(str)
C=N 1665 ±15
N-O 945±15

5.2 RESULTS AND DISCUSSIONS

Vibrational spectroscopy is one of the most useful methods for

investigating molecular interactions as well as structural details. The vibration

frequency obtained is a measure of the force constant between the atoms

constituiting a bond, and the constants are related to the bond orders and

electronic distributions between these atoms.In this study, the seaweed Ulva

spp.,Gracilaria spp., Sargassum spp.and Hypnea spp. were analysed for their

chemical structure using FTIR spectroscopy.

 75

100.0

95

90 ULVA 2142 90 0

85

80 929

75

70

65

60
849
55
790
50
%T
45
620
599
40

35

30

25
2923 1152
20 1549 1430
1254
15

10
3433
5 16 48 1054

0.0
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0
cm-1

Figure 5.1 FTIR vibrational stretching of Ulva spp.

The spectrum shown in Figure 5.1 for Ulva spp. , indicates the

presence of the intense bands in the region 599 cm-1 that is very characteristic

of Phosphate group. The intense bands are also observed at about 1648 cm -1

which are due to the presence of proteins that are assigned to the amide I

vibrations. The bands at 3433 and 1054 cm-1 shows the presence of O-H

functional group and polysaccharides respectively.

 76

100.0

95 2336
2134
90 SARGASSUM 897
876
85

80

75

70

65
813 528
60
782 551
55 765
743
50 721
%T 701 592
45 660
615
40

35

30
2854 1257
25 1319
1161
20

15 2924 1421

10 1638
3412
1034
5

0.0
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0
cm-1

Figure 5.2 FTIR vibrational stretching of Sargassum spp.

The spectrum shown in Figure 5.2 for Sargassum spp. , indicates

the presence of the intense bands in the region 1325–1452 cm -1 that isvery

characteristic of Polysaccharides . The intense bands are also observed at

about 1638 cm -1are due to the presence of proteins that are assigned to the

amide I vibrations. The band at 3412 cm-1 shows the presence of O-H

functional group.
 77

100.0

95
GRACILARIA
90 2328
2125
85
477
80
859
75 892
3774
70 875

65 743 581
774 668
60 696 620
712
55

50 931
%T
45

40

35
1306
30

25
1202
20 2923 1253
1533
15 1074
1155
10 3411 1451
1426
5 1652

0.0
4000.0 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 600 450.0
cm-1

Figure 5.3 FTIR vibrational stretching of Gracilaria spp.

In case of Gracilaria spp.(Figure 5.3) when analysed using FTIR

spectroscopy, intense bands in the region 604 cm-1 indicates the presence of

phosphate group and vibrations at 1101 and 1463 cm-1 shows the presence of

–OH and C-O vibrations in carbhohydrate. The band at 1641 cm-1 indicates

the presence of protein amide I group and vibration at 3445 shows the

presence of –OH (hydroxyl) groups.

 78

Figure 5.4 FTIR vibrational stretching of Hypnea spp.

The FTIR analysis of Hypnea spp. (Figure 5.4), shows the intense

band between 1097 and 1472 cm-1 indicating the presence of polysaccharides.

The vibrational peak at 1640 cm-1 shows the presence of amide I group and

band at 3444 cm-1 indicates the presence of OH functional group.

Similar resuls were obtained by Majda Sekkal et al (1993), in which

the intense bands observed at about 1625 and 1530 cm-1, were are due to the

presence of proteins and they are assingned to the amide 1 and amide 2

vibrations. The presence of the intense bands in the region 1000-1100 cm-1 is

very characteristic of polysaccarides.

 79

CHAPTER 6

ANTIBACTERIAL EFFECT OF SEAWEEDS

Most of the bioactive substances isolated from marine algae are

chemically classified as brominated, aromatics, nitrogen-heterocyclic,

nitrosulphuric-heterocyclic, sterols, dibutanoids, proteins, peptides and

sulphated polysaccharides. The crude extract obtained from various

seaweeds using different solvent is subjected to broad based biological

screening for antifungal, antiviral, antibacterial, antimalarial, antifilarial,

hypoglycaemic and antifertility activity.On other hand, the algae are also used

as food stuff, animal fodder, fertilizer, industrial material such as agar and

minor medicines. In this study the organic solvent extracts of seaweed species

were tested invitro for their antimicrobial activities against various pathogenic

microbes such as Aeromonas hydrophila, Edwardsiella tarda, Psuedomonas

fluorescens, Salmonella typhi, Pseudomonas aeruginosa, Staphylococcus

aureus and Escherichia coli with disc diffusion method.

Dilek Atakan et al (2006) found that diethyl ether was best solvent

for extracting the effective antimicrobial components.A significant difference

in antimicrobial activity was not found between the acetone and methanol

extracts of each alga. Diethyl extracts of Enteromorpha linza, Ulva rigida,

and Gracilaria gracilis showed effective results against all test organisms.
 80

Diethyl extract of Dictyota linearis was ineffective against microorganisms.

The ethanol extract of Dictyota linearis showed an antimicrobial activity

against gram-negative bacteria.This result could be related to the presence of

bioactive metabolites in D.linearis which are soluble in ethanol, but not in

diethyl ether. Ethanolic and diethyl ether fractions appear to be specific,

particularly against the tested gram-positive bacteria. Ethanol extracts of

D.linearis showed antibacterial activity against gram-negative bacteria. The

hexane extract of Gracilaria spp. inhibits only Bacillus subtilis. The diethyl

extract of G.gracilis inhibited S.aureus, E.coli, and P.aeruginosa. Some

studies concerning the effectiveness of extraction methods highlight that

methanol extraction yields higher antimicrobial activity than n-hexane and

ethyl acetate, whereas other reports state that chloroform is better than

methanol and benzene. Using organic solvents always provides a higher

efficiency in extracting compounds for antimicrobial activities compared to

water based methods. Diethyl ether and ethanol extracts of E. linza shows

high and low antimicrobial activities respectively.

6.1 ANTIBACTERIAL ACTIVITY OF SEAWEEDS AGAINST

FISH PATHOGENS

In a study by Choudary (2005), it was found that marine algae

extracts shows species specific activity in inhibiting the growth of virulent

strains of bacteria pathogenic to fish viz, Edwardsiella tarda, Vibrio

alginolyticus, Pseudomonas fluorescens, Pseudomonas aeruginosa and

Aeromonas hydrophila.Alcohol extract of G.corticata, E.compressa and

U.fasciata showed moderate antibacterial activity. Chloroform: Methanol

 81

(1:1) and methanol solvent systems were efficient in extracting the active

compounds. Toluene-methanol (1:3) extracts of species belonging to

Rhodophyceae exhibited broad-spectrum activity when compared to

Chlorophyceae and Phaeophyceae. Enteromorpha intestinalis and G.corticata

were active throughout the year with a peak during the winter season.

Acetone and ethanol extracts of Ulva lactuca and G.corticata showed good

inhibitory activity against Bacillus subtilis. P.fluorescens was moderately

sensitive to algal extracts.

In the studies with algae it was noted that S.aureus was most

susceptible bacterial pathogen followed by Vibrio sp. whereas P.aeruginosa

was most resistant. It was found that G.corticata showed antibacterial activity

only against Salmonella typhi and E.coli whereas methanol and chloroform

extracts had activity against P.aeruginosa. Some of the bacterial strains did

not respond to extracts. This might be due to masking of antibacterial activity

by the presence of some inhibitory compounds or factors in the extract. The

variation of antibacterial activity of extracts might be due to the distribution

of antimicrobial substances which might vary from species to species

The hexane extract of Rhodophyceae Amansia multifida had

displayed layer of zone of inhibition against E.aerogenes, P.aeruginosa,

S.typhi, S.aureus, B. subtilis. Presence of representative halos in

Chlorophyceae hexane extracts was shown only by C.cuperssoides against

S.epidermidis, B. subtilis (Jamal Marasneh et al, 1995). Highest antimicrobial

activity among the three groups was shown by Chlorophyta followed by

Rhodophyta and Phaeophyta. Activity against gram-negative bacteria was less

 82

common than against gram-positive bacteria.The display of antimicrobial

activities was considered to be an indicator for the synthesis of bioactive

secondary metabolites by seaweeds.The use of organic solvents always

provides a higher efficiency in extracting antimicrobial activities compared

with water extraction.

In another study by Mtolera and Semesi (1996), it was found that

the extract of Ulva pertusa was more active against S.aureus and B. subtilis,

but less active against E.coli. Valonia aegrophila was the most active species

against the test organisms whereas the extracts of Halimeda optunia and

Halimeda tuna showed mild activity. Preincubation of the inoculated plates

slowed the growth and gave ample time for the antimicrobial agent to diffuse,

thereby improving sensitivity of the plate diffusion method.A period of

12 hours preincubation storage has been found sufficient. Differences in

activity may be due to different developmental stages, locality etc.

In another work by Kolanjinnathan et al (2009), crude extracts of

seaweeds viz Gracilaria edulis, Calorpha peltada and Hydroclothres spp.

were prepared using the solvent ethanol for screening for their antibacterial

activity against six bacterial pathogens. The test bacterial strains were

Escherichia coli, Enterobacter aerogenes, Staphylococcus aureus,

Pseudomonas aeruginosa, Streptococcus faecalis and Bacillus cereus.

Ethanol extract of Gracilaria edulis inhibited growth of all the test organisms

except Bacillus cereus and Enterobacter aerogenes. Seaweed extract of

Calorpha peltada was found effective against a number of gram negative and

gram positive bacteria such as Escherichia coli, Staphylococcus aureus and

 83

Streptococcus faecalis. Hydroclothres spp. extract inhibited the growth of

Pseudomonas aeruginosa only out of the six tested pathogens. Selective

utilization of marine algae as potential source of pharmaceutical agents has

been increasing in recent years. Many of the seaweeds possess bio-active

components which inhibit the growth of some of the gram positive and gram

negative bacterial pathogens.

The algal extracts were used as a curative and preventive agent for

various diseases. It has been used for the production of antibiotics,

antihelminthics, cough remedies, antihypertensive, antitumor and

antidiarrhoeic substances. The antifouling activity of extracts of nine macro

algae against bacteria, fungi, diatom, has been investigated in relation to

season in bimonthly samples. Of the extracts tested 48.2% were active against

at least one of the fouling organisms and of these extracts 31.2% were

seasonally active with a peak of activity. The green algae Calorpha peltada

contains 1-4 diacetoxy butadiene and fatty esters which possess antibacterial,

anti-ichthyotoxic and anti-hypertensive properties. Antibacterial activity has

been detected in a number of seaweeds collected from the coast of Mandapam

to Cape comeron, Kanyakumari.The highest antibacterial activity was found

in the class Rhodophyceae (80%) followed by the Chlorophyceae (62.5%)

and the Phaeophyceae (61.9%). The maximum antifungal activity was

observed in the red algae 37%, brown algae 33.3% and green algae 8.3%

activity. Staphylococcus aureus was the most susceptible bacterial pathogen

followed by Vibrio spp. (Kolanjinathan et al, 2009).

 84

Alderman and Michel (1992), found that invitro screening of

organic solvent extracts of three marine algae viz.,Gracilaria corticata, Ulva

fasciata and Enteromorpha compressa and five mangroves viz., Aegiceras

corniculatum, Aegialitis rotundifolia, Aglaia cucullata, Cynometra iripa and

Xylocarpus granatum showed species specific activity in inhibiting the

growth of six virulent strains of bacteria pathogenic to fish viz., Edwardsiella

tarda,Vibrio alginolyticus, Pseudomonas fluorescens, Pseudomonas

aeruginosa and Aeromonas hydrophila (2 strains). Three methanol extracts of

C. iripa were active against all the six pathogens, whereas A. corniculatum

and A. cucullata were activeagainst four of the pathogens.

The chromatographic fractionation of active extracts of A.

corniculatum, C. iripa and G. corticata resulted in enriched fractions with

wide spectrum activity and lowered values of minimum inhibitory

concentration. Bacterial diseases are responsible for heavy mortality in wild

and cultured fish. The problems in the farms are usually tackled by preventing

disease outbreaks or by treating the actual disease with drugs or chemicals.

The use of antimicrobial agents has increased significantly in aquaculture

practices. Antibiotics used in both human as well as veterinary medicines

have been tried experimentally to treat bacterial infections of fish. Problems

including solubility, palatability, toxicity, cost, delivery and governmental

restrictions have limited the available antibiotics to a select few, especially in

food fish culture. Decreased efficacy and resistance of pathogens to

antibiotics has necessitated development of new alternatives (Smith et al,

1994).
 85

6.1.1 Antimicrobial activity of seaweeds Gracillaria, Padina and

Sargassum sps. on clinical and phytopathogens

Seaweeds are rich and varied source of bioactive natural products

and have been studied as potential biocidal and pharmaceutical agents. In

recent years, there are numerous of macro algae derived compounds that have

a broad range of biological activities such as antibacterial, antifungal,

antiviral, antineoplastic, antifouling, anti inflammatory, antitumoric, cytotoxic

and antimitotic activities. Presently seaweeds constitute commercially

important marine renewable resources which are providing valuable ideas for

the development of new drugs against cancer, microbial infections and

inflammations. The algae Sargassum spp., Padina spp. and Gracilaria spp.

are used by common people as fertilizers, food additives and animal feed. The

objective of the present study is to bring into limelight the potential activities

of the crude extracts of these algae and to exploit these untapped resources in

various ways for the benefit of the mankind. Because of the evolving

resistance of microorganisms to existing antibiotics, there is an increasing

need for new antibiotics. Since seaweeds offer particularly rich source of

bioactive molecules, the present study was carried out to investigate the

antimicrobial potentiality of the marine algae, Ceteroceiod spp.,

Chaetomorpha spp., Enteromorpha spp., Ulva spp., Padina spp.,

Stoechosperum spp., Amphiroa spp.and Gracilaria spp.

Subba Rangaiah et al (2010) studied on two species of brown algae

namely Sargassum ilicifolium, Padina tetrastromatica and one red algae

Gracilaria corticata collected from different coastal regions for

 86

microbiological testing of the seaweed extracts using agar well diffusion

method. The zone of inhibition was measured for all the different crude algal

extracts (Chloroform, ethanol, methanol and water) against six strains of gram

positive, gram negative bacterial and fungal organisms that cause diseases and

disorders in man, animals and plants. Crude extracts revealed a wide range of

antimicrobial activity against tested pathogens. Seaweed extracts in different

solvents exhibited different antimicrobial activities. In case of Sargassum

ilicifolium, Padina tetrastromatica,of the various solvents used for seaweed

extractions, maximum inhibition was noticed with ethanol extracts and

minimum with chloroform crude extracts while in case of Gracilaria

corticata, maximum inhibition was noticed with methanol and minimum with

chloroform extracts. However, no specific solvent exhibited activity against

all the test organisms effectively.

Further bacterial strains were more sensitive to the extracts when

compared to the fungal organisms. The overall antimicrobial activity assessed

from the above results indicates the presence of active constituents in the

extractions of seaweeds which can be exploited for the production of lead

molecules which are of use in pharmaceutical industry.

6.1.2 An assessment of the antioxidant and antimicrobial activity of

six species of edible Irish seaweeds

Seaweeds belong to a group of plants known as algae. Seaweeds are

classified as Rhodophyta (red algae), Phaeophyta (brown algae) or

Chlorophyta (green algae) depending on their nutrient and chemical

 87

composition. Like other plants, seaweeds contain various inorganic and

organic substances which can benefit human health (Kuda et al, 2002).

Seaweeds are considered as a source of bioactive compounds as they are able

to produce a great variety of secondary metabolites characterised by a broad

spectrum of biological activities. Compounds with antioxidant, antiviral,

antifungal and antimicrobial activities have been detected in brown, red and

green algae (Bansemir et al, 2006). The environment in which seaweeds grow

is harsh as they are exposed to a combination of light and high oxygen

concentrations. These factors can lead to the formation of free radicals and

other strong oxidising agents but seaweeds seldom suffered serious

photodynamic damage during metabolism. This fact implies that seaweed

cells have some protective mechanisms and compounds. Phenolic compounds

can act as antioxidants by chelating metal ions, preventing radical formation

and improving the antioxidant endogenous system.

The term “phenolic compound” describes several hundred

molecules found in edible plants that possess on their structure a benzenic

ring substituted by, at least, one hydroxyl group (Manach et al, 2004). These

phenolic compounds are commonly found in plants, including seaweeds.

Polyphenols represent a diverse class of compounds including flavonoids (i.e.

flavones, flavonols, flavanones, flavononols, chalcones and flavan-3-ols),

lignins, tocopherols, tannins and phenolic acids (Shukla et al, 1997). Interest

in new sources of natural antioxidants and antimicrobials has increased in

recent years in order to reduce the use of synthetic forms such as Butylated

Hydroxyanisole (BHA) and Butylated Hydroxytoluene (BHT). Natural

 88

antioxidants from plant origin can react rapidly with these free radicals and

retard or alleviate the extent of oxidative deterioration. Furthermore,

antioxidants from natural sources can also increase the shelf life of foods.

Therefore, the consumption of antioxidant and/or addition of antioxidant to

food materials could protect the body as well as the foods against these

events. Many marine plants, including seaweeds, often carry significantly less

macro and microepibionts on their thalli compared to co-occurring biofilms

on inanimate substrata (Hellio et al, 2001, Lam and Harder, 2007). Seaweeds

are a plentiful renewable natural resource in Ireland; Laminaria digitata,

Laminaria saccharina, Himanthalia elongata, Palmaria palmata, Chondrus

crispus and Enteromorpha spirulina are common species of seaweeds found

in abundance around the Irish coastline. Many researchers have reported on

the antioxidant and antimicrobial activity of seaweeds.

6.1.3 Components and Antimicrobial Activity of Polysaccharides

Extracted from Thai Brown Seaweeds

Species of brown seaweed are well known to contain large amounts

of cell-wall polysaccharides, most of which are the sulphated polysaccharide,

fucoidan which is not found in terrestrial plants. Fucoidan has a substantial

component of L-fucose and sulfate ester groups and has a wide range of

pharmacological and biomedicinal properties. There have been several studies

on the diverse bioactivities, molecular weights, structural parameters and

physiological characteristics of seaweed polysaccharides. Recently, aqueous

crude extract of the brown seaweed, Hydroclathrus clathratus, from Hong

Kong, has been reported to have high antiviral activity against the herpes
 89

simplex virus (HSV) and low cytotoxicity to Vero and HEp-2 cells. The

antiviral activities of the sulfated polysaccharides extracted from Sargassum

latifolium have also been reported by (Attachai Kantachumpo and Anong

Chirapart, 2010), who found that the activity was dependent on the degree of

sulfation and the molecular weight. Most of the seaweed polysaccharides

isolated using the acid extraction method are crude fucoidans.The

polysaccharide yield extracted from brown seaweed species depended on the

algal species and the extraction method as well as on environmental factors.

There have been reports on the antimicrobial activity of marine algae in

Thaila.The crude fucoidan extracted from Sargassum polycystum has been

observed to reduce the impact of white spot syndrome virus (WSSV)

infection in Penaeus monodon (Chotigeat et al, 2004).

6.1.4 Antimicrobial activity of seaweeds extracts against multi

resistant pathogens

As a consequence of an increasing demand in screening for new

therapeutic drugs from natural products, there is a greater interest towards

marine organisms. Several marine organisms produce bioactive metabolites in

response to ecological pressures such as competition for space, maintenance

of unfouled surfaces, deterrence of predation and the ability to successfully

reproduce (Konig et al, 1994). Seaweeds provide a rich source of structurally

diverse secondary metabolites. These secondary metabolites offers defence

against herbivores, fouling organisms and pathogens; they also play a role in

reproduction, protection from UV radiation and as allelopathic agents

(Vineela and Elizabeth, 2005, Tuney et al, 2006) was the pioneer to observe
 90

the antimicrobial potentials of seaweeds. Many algal species have been shown

to have bactericidal or bacteriostatic substances. The bactericidal agents found

in algae include aminoacids, terpenoids, phlorotannins, acrylic acid, phenolic

compounds, steroids, halogenated ketones and alkanes, cyclic polysulphides

and fatty.

There is an increasing demand of biodiversity from natural

resources for therapeutic drugs. The potential contribution of marine

organisms to the discovery of new bioactive molecules is increasingly

challenging (Sponga et al, 1999, Skulberg, 2000). The macroalgae have a

significant attraction as natural source of bioactive molecules with a broad

range of biological activities, such as antibiotics, antivirals, antitumorals,

antioxidant and anti-inflammatories (Scheuer, 1990, Sreenivasa Rao, 1995,

Fleurence, 1999). Evidence of phycochemical and pharmacological studies on

algae is available in the literature with special reference to terpenoids and

steroids (Parameswaran, 1944 and Patterson, 1968). Algae are the source of

amino acids, terpenoids, phlorotannins, steroids, phenolic compounds,

halogenated ketones and alkanes and cyclic polysulphides (Taskin et al,

2007).

6.1.5 Antifungal activity of seaweeds

Seaweeds are rich and varied source of bioactive natural products

and have been studied as potential biocidal and pharmaceutical agents. They

are used in traditional remedies in many parts of the world. Extracted

substances from seaweeds have antibacterial actions and other properties

 91

include antifungal activities and growth inhibition of plants (Scheuer, 1990).

Seaweeds are also known to aid and stimulate growth of vegetables, fruits and

also protect them from different pathogens and physiological hazards. It is an

established fact that the sea is full of innumerable wealth viz., minerals,

vitamins etc. Marine algae are rich in protein. It is, therefore, essential to

study their chemical composition considering them as a source of protein or a

supplementary food and feed.

6.2 MATERIALS AND METHOD

6.2.1 Preparation of Extracts

The algae after drying were weighed and then chopped. The

chopped samples were finely powdered using mixer grinder. The finely

powdered samples were weighed and 5 grams of it were dissolved in various

organic solvents, such as 80% ethanol, methanol and chloroform. It was kept

for 48 hours at room temperature and mixed at regular intervals. After

48 hours the sample dissolved in each solvent was filtered using Whatman

No1 filter paper to separate the filtrate for further use in antimicrobial testing

of algal samples.

6.2.2 Test Microorganisms Used:

Antibacterial activity was tested against the pathogenic strains of

Aeromonas hydrophila, Edwardsiella tarda, Escherichia coli, Pseudomonas

aeruginosa, Pseudomonas fluorescens, Salmonella typhi, and Staphylococcus

 92

aureus. Loopful samples were inoculated in sterile nutrient broth and kept

overnight at 37 oC for growth.

6.2.3 Plate Assay Method

Antibacterial activity was assayed using the agar well diffusion test

technique. Muller Hinton Agar Medium (MHA) was prepared at a pH of 7.4

and then the medium was sterilized by autoclaving at 121 oC and 15 lbs

pressure for 15 minutes. About 20 mL of the sterilized media was poured into

sterile petri dish and was allowed to solidify at room temperature. A sterile

cotton swab was used for spreading the test microorganism evenly from the

24 hours incubated broth on the MHA plates. Similarly swabbing was done

separately for each test microorganism on the MHA plates and left for few

minutes to allow complete absorption of the inoculum. In each of these plates

5 mm diameter, three wells were made using an appropriate size sterilized

cork borer.

Different concentrations of each algal extract were added to the

respective wells on the MHA plates. Concentrations ranging from 50 µL,

75 µL and 100 µL respectively were placed in the wells and allowed to

diffuse at room temperature for 30 minutes. The extract loaded plates were

kept for incubation at 37 oC for 24 hours. After incubation, a clear zone was

observed around the well which was the evidence for the presence of

antibacterial active compounds in the algal extracts. Diameters of the zone of

inhibition were measured in millimetres (including the diameter of the well).

 93

6.3 RESULTS AND DISCUSSIONS

Seaweeds are rich in varied source of bioactive natural products and

have been studied as potencial biochemical and pharmaceutical agents. The

main objective of the work was to evaluate and compare the ability of

different macro algal species from southwest coast of India to produce

bioactive compounds of potential therapeutic interests. The production of

antimicrobial activities was considered to be an effective indicator of the

capability of the seaweeds to synthesize bioactive secondary metabolites.

Different extracts of Centroceiod spp., Stoechospermum spp. ,

Padina spp., Chaetomorpha spp., Ulva spp., Gracilaria spp. and Amphiroa

spp.were tested for their antimicrobial activity against seven strains of

microorganisms Aeromonas hdrophila, Edwardsiella tarda, Escherichia coli,

Pseudomonas aeruginosa, pseudomonas fluorescens, Salmonella typhi and

Staphylococcus aurues., by agar well diffusion method. The results of

antimicrobial activity against tested pathogens were tabulated in the

Table 6.1.
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Table 6.1 Antibacterial Activity of extracts of algae

Test Organism(Zone of inhibition mm)

Species Extracts
AH ET EC PA PF ST SA
Centroceiod Ethanol - - - - 7 - -
Ethanol+Chloroform - - - - 7 - -
Methanol 3 4 - - - 5 -
Chaetomorpha Ethanol - - - - 5 - -
Ethanol+Chloroform - - - - - - -
Methanol - - - 5 6 8 -
Enteromorpha Ethanol - - - - 8 - -
Ethanol+Chloroform - - - - - - -
Methanol - - - - - - -
Ulva Ethanol - - 7 - 9 6 4
Ethanol+Chloroform - 6 12 - 3 12 5
Methanol - 4 - - 5 - 5
Padina Ethanol - - - 5 - - -
Ethanol+Chloroform - 4 - - - - -
Methanol - - - - - - 8
Stoechospermu Ethanol - - 8 4 5 3 8
m
Ethanol+Chloroform - 5 - - 4 4 3
Methanol - - - - - - -
Amphiroa Ethanol - - - - 6 - -
Ethanol+Chloroform - - - - - - -
Methanol 9 - - - 7 - -
Gracilaria Ethanol - - - - 7 - -
Ethanol+Chloroform - - - - 4 - -
Methanol 7 - - - 4 5 -
 95

Methanol Extract Methanol Extract

Amphiroa and Gracilaria Centroceiod

Figure 6.1 Antibacterial activity of Amphiroa spp. , Gracilaria spp. and

Centroceiod spp. against Aeromonas hydrophila

In case of Amphiroa spp., methanol was the best solvent showing

high activity (Zone: 9 mm) against the pathogenic microorganism Aeromonas

hydrophila whereas low activity was shown by the algae Centroceiod spp.

(Zone: 3 mm). Ethanol : chloroform (1:1) and ethanol extracts of both algae

were not active against Aeromonas hydrophila Figure 6.1.

Methanol Extract Methanol Extract Ethanol + Chloroform

Figure 6.2 Antibacterial activity of Ulva spp, Centroceiod spp, Padina

spp, Stoechospermum spp. against Edwardsiella tarda

 96

In case of Ulva spp., ethanol : chloroform (1:1) was outstanding

solvent and peak activity was demonstrated against Edwardsiella tarda

(Zone: 6 mm).Methanol extracts of Ulva spp. (Zone: 4 mm) and Centroceiod

spp. (Zone: 4 mm) showed inhibitory action against the same microorganism.

Ethanol extracts of both algae were inactive against Edwardsiella tarda

Figure 6.2.

Ethanol + chloroform Ethanol Extract Ethanol Extract

Figure 6.3 Antibacterial activity of Ulva spp. and Stoechospermum spp.

against Escherichia coli

In case of the marine algae Stoechospermum spp., ethanol the first-

rated solvent illustrated maximum activity (Zone: 12 mm) against Escherichia

coli. Only the ethanol : chloroform (1:1) extract of Ulva spp. (Zone: 8 mm)

exhibited inhibition whereas the other algae showed no inhibition against the

same microorganism. In comparison with ethanol and ethanol : chloroform

(1:1) extracts, methanol extract of all the algae did not show any activity

Figure 6.3.
 97

Ethanol Extract Ethanol Extract Methanol Extract

Figure 6.4 Antibacterial activity of Stoechospermum spp., Padina spp.

and Chaetomorpha spp. against Pseudomonas aeruginosa

The ethanol extract of Padina spp. (Zone: 5 mm) and methanol

extract of Chaetomorpha spp. (Zone: 5 mm) showed zone of inhibition

against the microorganism Pseudomonas aeruginosa. A major difference in

activity was not set up between the ethanol and methanol extract. In contrast

to ethanol and methanol extracts, ethanol : chloroform (1:1) extract did not

display any activity Figure 6.4.

 98

Ethanol+chloroform Ethanol+chloroform

Ethanol+chloroform Ethanol Extract

Ethanol Extract Methanol Extract

Figure 6.5 Antibacterial activity of Centroceiod spp., Ulva spp.,

Enteromorpha spp., Stoechospermum spp., Gracilaria spp.,

Amphiroa spp. and Chaetomorpha spp. against Pseudomonas

fluorescens
 99

The ethanol extract of all the seaweeds used in this study showed

inhibitory action against Pseudomonas fluorescens except the algae Padina

spp.The ethanol extract of Ulva spp. showed highest activity (Zone: 9 mm)

whereas the algae Chaetomorpha spp. (Zone: 5 mm) and Stoechospermum

spp.(Zone: 5 mm) has showed lowest inhibition. A significant difference in

activity was not found between the ethanol : chloroform (1:1) and methanol

extracts of each algae, whereas the algae Centroceiod spp. (Zone: 7 mm)

revealed maximum activity in the ethanol : chloroform (1:1) extracts and the

Amphiroa spp. (Zone: 7 mm) was the most excellent species among the

methanol extract Figure 6.5.

Ethanol Extract Methanol Extract

Methanol Extract Ethanol+Chloroform

Figure 6.6 Antibacterial activity of Stoechospermum spp.,

Chaetomorpha spp., Centroceiod spp., Ulva spp., Gracilaria

spp. against Salmonella typhi

 100

Among all the algae the ethanol extract of Stoechospermum spp.

exhibited maximum inhibition activity (Zone: 9 mm) against the test organism

Salmonella typhi. Among methanol extracts of all seaweeds, the antibacterial

activity was more evident in Chaetomorpha spp. (Zone: 8 mm) and mild

activity was shown by Stoechospermum spp. (Zone: 4 mm). The ethanol :

chloroform (1:1) extract of Ulva spp. alone showed inhibition (Zone: 3 mm)

against Salmonella typhi Figure 6.6.

Ethanol Extract Ethanol Extract Methanol Extract

Ulva spp. Stoechospermum spp. Ulva spp.

Methanol Extract Ethanol + chloroform Ethanol + chloroform

Padina spp. Ulva spp. Stoechospermum spp.

Figure 6.7 Antibacterial activity of Ulva spp., Stoechospermum spp.,

and Padina spp. against Staphylococcus aureus

 101

The ethanol extracts of Stoechospermum spp. (Zone: 8 mm) and

methanol extract of Padina spp. (Zone: 8 mm) showed inhibition against the

test organism Staphylococcus aurues and were found to be outstanding

species amongst ethanol and methanol extract respectively. Within ethanol :

chloroform (1:1) extracts only Stoechospermum (Zone: 3mm) illustrated a

mild activity Figure 6.7.

Seaweed extracts in different solvents exhibited different

antimicrobial activities.Various solvents were used for seaweed extractions,

maximum inhibition was noticed with ethanol extracts and minimum with

chloroform crude extracts. However, no specific solvent exhibited activity

against all the test organisms effectively. Results also highlighted that

P.fluorescens was the most sensitive organism. S.aureus and S.typhi was

moderately sensitive to algal extracts while A.hydrophila and P.aeruginosa

were mainly the resistant pathogens

In the present study, it was observed that ethanol+ chloroform was

the best organic solvent for extracting the effective antibacterial material from

the algae species used in this experiment. The result exhibited by chloroform

was less than that exhibited by ethanol and methanol. The best halo-zone

produced was in the extract of Ulva in the ethanol+chloroform extract. The

large diameter of zone inhibition represent the high sensitivity against

microorganisms, small diameter of zone inhibition represent the less

sensitivity against microorganisms and no diameter of zone inhibition

represent the resistance against microorganisms.

 102

In a similar work done by Choudry et al (2005), the invitro

screening of organic solvent extracts of three marine algae viz., Gracilaria

cortica, Ulva fasciata and Enteromorpha compressa showed species activity

in inhibiting the growth of six virulent strains of bacteria pathogenic to fish

.,Edwardsiella tarda,Vibrio aglinolyticus, Pseudomonas fluorescens,

Pseudomonas aeruginosa and Aeromonas hydrophila. Methanol solvent

system were efficient in extracting the active compounds.Gracilaria corticata

showed inhibitory activity against Vibrio alginolyticus, Pseudomonas

flurorescens. The marine algae Enteromorpha compressa is active against

Edwarsiella tarda whereas the algae Ulva fasciata shows positive against

Pseudomonas flurorescens.

The cell extracts and active constituents of various algae have been

shown to have invitro antibacterial activity against gram positivebacteria and

gram negative bacteria. Inci Tuney et al (2006) demonstrated that methanol,

acetone, diethyl ether and ethanol extracts of eleven seaweed species from the

coast of Urla against bacteria such as Candida species, Enterococcus feacalis,

Staphylococcus aurues, Sreptococcus epidermidis, Pseudomonas aureuginosa

and Escherichia coli with the disc diffusion method. Diethyl ether was the

best solution for extracting effective antimicrobial materials from the algal

species with the exception of D.linearis for which ethanol was the most

effective extraction solution. Diethyl eher extracts of fresh Cystoseira

mediterranea, Enteromorpha linza, Ulva rigida, Gracilaria gracilis and

Ectocarpus siliculosus showed effective results against all test organisms. A

 103

significant difference in antimicrobial activity was not observed between the

acetone and methanol extracts of each aigae.

Lima-Filtho et al (2002) found that the hexane extract of Gracilaria

species inhibits only Bacillus subtilis. In contrast, the result of our present

study shows that, the ehanol, methanol and ehanol + chloroform extract of

Gracilaria species showed inhibitory action against Pseudomonas

flurorescens.

Perez et al (1990) found that the extract of Ulva lactuca had no

antimicrobial activity. In constrast, our results showed that the ethanol,

ethanol + chloroform and methanol extract of Uiva spp. inhibits the growth of

test organisms such as Edwardsiella tarda, Escherichia coli, Pseudomonas

fluorescens, Salmonella typhi and Staphylococcus aurues.

Choudry et al (2005) discussed that the ehanol extract of Padina

pavonica showed weak activity against Candida, Enterococcus feacalis,

Pseudomonas aeruginosa and Escherichia coli. Similarly, in this study, the

ethanol extract of Padina species showed weak activity against Pseudomonas

aeruginosa and also the ethanol + chloroform of the same algae shows less

zone of inhibition against Edwardsiella tarda (Figure 6.2 & Figure 6.4).

Gonzalez Delval (2001) also demonstrated the antibacterial and

antifungal activities of the methanol extract of Entermorpha compressa.

According to our results, the extract of ethanol + chloroform and methanol of

the algae Enteromorpha species showed no inhibition against all the test
 104

organisms whereas the ethanol extract of the same algae showed inhibition

only against Pseudomonas flurorescens. `

Because of the evolving resistance of microorganisms to existing

antibiotics, there is an increasing need for new antibiotics. Since seaweeds

offer particularly rich source of bioactive molecules against micro organisms

that cause diseases and disorders in man, animals and plants. Crude extracts

revealed a wide range of antimicrobial activity against tested pathogens.

Seaweed extracts in different solvents exhibited different antimicrobial

activities. The overall antimicrobial activity assessed from the above results

indicates the presence of active constituents in the extractions of seaweeds

which can be exploited for the production of lead molecules which are of use

in pharmaceutical industry.
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CHAPTER 7

SUMMARY

Seaweeds or marine macro algae are the group of plants that live

either in marine or brackish water environment. They contain photosynthetic

pigments and with the help of sunlight and nutrient present in the seawater,

they photosynthesize and produce food. They are found in the coastal region

between high tide to low tide and in the sub-tidal region up to a depth where

0.01 % photosynthetic light is available. Plant pigments, light, exposure,

depth, temperature, tides and the shore characteristic combine to create

different environment that determine the distribution and variety among

seaweeds. Accordingly, algae are classified into three main groups i.e. green

(Chlorophyta), brown (Phaeophyta) and red (Rhodophyta).

In the present investigation, the samples were collected from North

eastern coast of TamilNadu viz Pulicat lake and Ennore, Eastern coast of

TamilNadu viz Kalpakkam and Southern coast of TamilNadu viz Covelong,

Muttam, Kanyakumari and Cape comerin. The basic nutritional parameters,

viz proteins and carbohydrates of various seaweeds of coastal TamilNadu

were estimated. The presence of trace elements was studied using SEM \

EDAX analysis. The chemical structure of the seaweed was analysed using
 106

FTIR analysis. Antibacterial activity of seaweeds against pathogenic

microorganisms was assayed using plate assay method.

In this study, the seaweed samples were collected from different

areas of the Tamil Nadu coast line. The places included Pulicat Lake,

Covelong beach Chennai, Muttam, Kanyakumari, Kalpakkam, Ennore, Cape

comeron Kanyakumari. The collection of seaweeds from the intertidal area

was done during the low tide.

7.1 PROTEIN ESTIMATION OF SEAWEEDS

The samples were analysed for their protein content by Bradford

Method (Bradford, 1976). The varying protein content in different samples

from various locales was compared in Figure 2.1. Of the various samples

collected, Sargassum spp., a brown seaweed from Cape comeron

Kanyakumari, showed the highest protein content as that of 950 µg/g whereas

the same species collected from Muttam, Kanyakumari showed a lower

concentration of protein of about 550 µg/g (Figure 2.1).

Two different varieties of Gracilaria spp., a red seaweed, were

collected from Covelong Chennai. While the protein content in one species

was about 850 µg/g, that of the other species was around 100 µg/g. The

species of Gracilaria spp. with higher protein content collected from other

locales showed variations ranging between (100-200) µg/g in the protein

content as compared to that of the sample from Covelong Chennai. The

Gracilaria spp. collected from Pulicat had a protein content of about 750 µg/g
 107

whereas that of Muttam, Kanyakumari and Cape comeron, Kanyakumari had

a protein concentration of 600 µg/g and 500 µg/g respectively (Figure 2.4 &

Figure 2.5). In another similar study by Eswaran et al (2002) the total protein

content in Gracilaria spp. was determined as high as, 1070 µg/g.

Another Rhodophyte, Hypnea, showed protein content of 480 µg/g.

Similar amount of protein content was estimated in Calagossa spp., a red

algae collected from Cape comeron, Kanyakumari. The protein content was

determined to be 700 µg/g. Yet another red seaweed, Centroceras spp.,

collected from Cape comeron, Kanyakumari had a protein content of

405 µg/g. (Figure 2.5) Amphiroa spp., a red seaweed, had the same protein

content of 100 µg/g even though it was collected separately from Muttam,

Kanyakumari and Covelong, Chennai (Figure 2.4 & Figure 2.5).

Ulva, a Chlorophyta, was found abundantly in most of the places.

Two different species of Ulva were collected viz., Ulva lactuca and Ulva

fasciata. The U. lactuca from Pulicat, Chennai had a protein content of about

350 µg/g whereas the one collected from Cape comeron, Kanyakumari

showed 200 µg/g (Figure 2.7). Compared to this species, the other species

U. fasciata from Covelong, Chennai had about 600 µg/g of protein whereas

that of Pulicat, Chennai had a protein content of 650 µg/g (Figure 2.1). In a

similar study by Dere et al (2003), the total protein content was estimated as

277.58 g/kg (d.w). There is a wide range of variation in the protein content.

The possible reason might be the variation in the growth conditions and the

available nutrients. It has been found in many other studies that the nutritional

contents of macroalgae depended not only on season and geography

 108

(Fleurence (1999), Fleurence et al (1999), Haroon et al (2000), but also on

the nutrient content of the environment. Mathers and Montgomery (1997)

found total protein content in U. lactuca to lie between 19.29% and 18.22%,

whereas Fleurence et al (1999) found the total protein content of Ulva spp. to

vary between 18% and 26%.

Other green algae such as Enteromorpha spp.and Chaetomorpha

spp. were collected from different locations.The protein content in

Enteromorpha spp. was determined as 200 µg/g from Pulicat, Chennai,

400 µg/g from Muttam, Kanyakumari and 280 µg/g from Cape comerin,

Kanyakumari. In a similar study by Mathers and Montgomery (1997), the

total protein content in Enteromorpha spp. was found varying between

16.04% and 16.14%. According to our study, the highest protein content in

Ulva spp. was determined to be 650 µg/g collected from Pulicat, Chennai.

The protein content of Stoechospermum spp., a brown algae

collected from Cape comeron, Kanyakumari was determined to be 475 µg/g.

Another Phaeophyte, Padina spp. showed higher protein content of about

765 µg/g. In the marine algae Valeneopsin spp. which was collected from two

different locales, the protein content was low and it was determined to be

150 µg/g and 200 µg/g in samples collected from Muttam, Kanyakumari and

Cape comeron, Kanyakumari respectively (Figure 2.9(a)).

Comparing the different samples collected from Cape comeron,

Kanyakumari, it was observed that the protein content determined

varied between as low as 100 µg/g in Ulva spp. to a high of 950 µg/g in
 109

Sargassum spp. Of all the samples collected from Cape comeron,

Kanyakumari, the protein content in Phaeophytes seems promising as that of

Sargassum spp. and Padina spp. The different variety of Rhodophyta

collected showed variations from 405 µg/g in Centroceras spp., 480 µg/g in

Hypnea spp., 500 µg/g in Gracilaria spp. to a high of 700 µg/g in

Chaetomorpha spp. (Figure 2.1).

Among the various species collected from Pulicat, Chennai,

Gracilaria spp. showed the highest quantity of protein of about 750 µg/g

where as the other Chlororophyta showed comparatively lesser quantities, as

that of 650 µg/g in Ulva fasciata and 200 µg/g in Ulva lactuca (Figure 2.7).

In the samples collected from Covelong, Chennai, the highest protein content

was found in Gracilaria spp. as that of 850 µg/g and the lowest was found in

Amphiroa spp, Ulva spp. collected from the same place showed a relatively

higher protein content of 600 µg/g where as that of Chaetomorpha spp. was

around 300 µg/g. Among the various samples collected from Muttam,

Kanyakumari Chaetomorpha spp. showed the highest protein content of

750 µg/g and the lowest was that of Amphiroa spp. of about 100 µg/g. The

Gracilaria spp. showed a protein content of 600 µg/g. The Sargassum spp.

collected has a protein content of 550 µg/g and that of Enteromorpha spp.has

about 400 µg/g of protein content.

7.2 ESTIMATION OF CARBOHYDRATES FROM SEAWEEDS

The carbohydrate analysis of various samples was done by Anthrone

Method (Yemm and Willis, 1954). The carbohydrate content was estimated
 110

to be highest in Gracilaria spp. from Cape comeron, Kanyakumari

(Figure 3.6). It was about 100 mg/g i.e., about 100 g/kg. In a similar study,

the carbohydrate content of a certain species of Gracilaria spp. was estimated

around 43.07 g/kg. (Dere et al 2003). The lowest carbohydrate content was

observed in Halimeda spp. from Cape comeron, Kanyakumari. It was about

21 mg/g.

Two varieties of Ulva were collected. Ulva from Covelong,

Chennai, had a carbhohydrate content of about 43 mg/g whereas the same

species from Cape comeron, Kanyakumari had about 39 mg/g. The other

species of Ulva from Cape comeron, Kanyakumari had about 60 mg/g of

carbohydrate content where as the one collected from Pulicat had about

90.5 mg/g. Similar results were obtained in another study. The carbohydrate

content in Ulva spp. was obtained to be 63.04 g/kg. (Dere et al, 2003)

In a study by Dere et al (2003), the carbohydrate content was

obtained for different species of Enteromorpha. The contents varied in the

range of 10 g/kg to 25 g/kg. In our study, Enteromorpha spp. from two places

were analysed. The contents were obtained as 39 mg/g i.e., 39 g/kg from

Pulicat, Chennai and 46 mg/g i.e., 46 g/kg from Cape comeron, Kanyakumari.

Amphiroa spp., a red seaweed was collected from two different locations;

Muttam, Kanyakumari and Covelong, Chennai. The estimated carbohydrate

contents were relatively similar; 30 mg/g and 27 mg/g respectively.

Valeneopsin spp., collected from two places Muttam, Kanyakumari and Cape

comeron, Kanyakumari showed slight variations of 25 mg/g to 34 mg/g,

respectively. Centroceras spp., a Rhodophyta, had a carbohydrate content of

 111

about 29 mg/g which is lesser when compared to other red algae collected.

Chaetomorpha spp., a green algae had a carbohydrate content of about

38 mg/g.

7.3 ESTIMATION OF TRACE ELEMENTS FROM SEAWEEDS

In Ulva spp. (Figure 4.1A & Figure 4.1B) which was studied for

elemental concentration using SEM, the percentage of oxygen was found to

be high 48% which was followed by carbon 27% and trace amounts of

nitrogen, magnesium, sulphur and calcium were also present. The other

elements such as zinc, nickel, sodium, aluminium, phosphorus, cadmium,

arsenium, cobalt and lead were absent.

In Gracilaria spp. (Figure 4.2A & Figure 4.2B) which was studied

for elemental concentration using SEM, the percentage of carbon was found

to be high such as 41%which is followed by oxygen 38% and trace amounts

of nitrogen, magnesium, sulphur, silicon, cadmium, arsenium and calcium

were also present. The other elements such as zinc, sodium, aluminium,

phosphorus, cobalt and lead were absent.Whereas in case of Sargassum spp.

(Figure 4.3A & Figure 4.3B) the percentage of oxygen was found to be high

such as 44% which is followed by carbon 38.5% and trace amounts of

nitrogen, sodium, magnesium, sulphur, potassium and calcium were also

present. The other elements such as zinc, silicon, aluminium, phosphorus,

cobalt, iron and lead were absent.

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7.4 ANALYSIS OF CHEMICAL STRUCTURE OF SEAWEED

The seaweed Ulva spp., Gracilariai spp., Sargassum spp. and

Hypneai spp. were analysed for their chemical structure using FTIR
spectroscopy. The spectrum shown in Figure 5.1 for Ulva spp., indicating the
presence of the intense bands in the region 590 cm -1 is very characteristic of
Phosphate group. The intense bands are also observed at about 1645 cm -1
which are due to the presence of proteins and they are assigned to the amide I
vibrations. The bands at 3425 and 1054 cm -1 showed the presence of O-H
functional group and polysaccharides, respectively.

The spectrum shown in Figure 5.2 for Sargassum spp., indicated

the presence of the intense bands in the region 1325 – 1452 cm -1 which is very
characteristic of polysaccharides. The intense bands are also observed at about
1635 cm-1 they are due to the presence of proteins. Incase of Gracilaria spp.,
shown in Figure 5.3, intense bands in the region 604 cm-1 indicates the
presence of phosphate group and vibrations at 1101 and 1463 cm-1 shows the
presence of –OH and C-O vibrations in carbhohydrate. The band at 1641 cm -1
indicates the presence of protein amide I group and vibration at 3445 shows
the presence of –OH groups.

In case of Hypnea spp., shown in Figure 5.4, the intense band

between 1097 and 1472 cm -1 showed the presence of polysaccharides. The
vibrational peak at 1640 cm-1 shows the presence of amide I group and band at
3444 cm indicates the presence of OH functional group.This was supported by
Majda Sekkal et al (1993), in which the intense bands are observed at about
1625 and 1530 cm-1, which were due to the presence of proteins and they
 113

were assingned to the amide I and amide II vibrations, respectively. The

presence of the intense bands in the region 1000-1100 cm -1 is very
characteristic of polysaccharide I and II.

7.5 ANTIMICROBIAL ACTIVITY OF SEAWEEDS

Bacterial infection causes high rate of mortality in human

population and aquaculture organisms. Escherichia coli, Staphylococcus
aureus and Pseudomonas aeruginosa cause diseases like mastitis, abortion
and upper respiratory complications, while Salmonella sp. causes diarrhea and
typhoid fever (Jawetz et al, 1985, Leven 1987). P. aeruginosa is an important
and prevalent pathogen among burned patients capable of causing life-
threatening illness (Boyd, 1955). Preventing disease outbreaks or treating the
disease with drugs or chemicals tackles these problems. Nowadays, the use of
antibiotics has increased significantly due to heavy infections and the
pathogenic bacteria becoming resistant to drugs are common due to
indiscriminate use of antibiotics. It becomes a greater problem of giving
treatment against resistant pathogenic bacteria (Sieradzki et al, 1999).

The main objective of the work was to evaluate and compare the
ability of different macro algal species from South-East coast of India to
produce bioactive compounds of potential therapeutic interests. The
production of antimicrobial activities was considered to be an effective
indicator of the capability of the seaweeds to synthesize bioactive secondary
metabolites. Different extracts of Centroceiod spp., Stoechospermum spp.,
Padina spp., Chaetomorpha spp., Ulv spp., Gracilaria spp. and Amphiroa
spp.were tested for their antimicrobial activity against seven strains of
 114

microorganisms, by agar well diffusion method. The results of antimicrobial

activity against tested pathogens were tabulated in the Table 6.1. In case of
Amphiroa spp., methanol was the best solvent showing high activity (Zone:
9 mm) against the pathogenic microorganism Aeromonas hydrophila whereas
low activity was shown by the algae Centroceiod spp. (Zone: 3 mm). Ethanol
: chloroform (1:1) and ethanol extracts of both algae were not active against
Aeromonas hydrophila Figure 6.1.

In case of Ulva spp., ethanol : chloroform (1:1) was outstanding

solvent and peak activity was demonstrated against Edwardsiella tarda
(Zone: 6 mm).Methanol extracts of Ulva spp. (Zone: 4 mm) and Centroceiod
spp. (Zone: 4 mm) showed inhibitory action against the same microorganism.
Ethanol extracts of both algae were inactive against Edwardsiella tarda
Figure 6.2.

In case of the marine algae Stoechospermum spp., ethanol the first-

rated solvent illustrated maximum activity (Zone: 12 mm) against Escherichia
coli. Only the ethanol : chloroform (1:1) extract of Ulva spp. (Zone: 8 mm)
exhibited inhibition whereas the other algae showed no inhibition against the
same microorganism. In comparison with ethanol and ethanol : chloroform
(1:1) extracts, methanol extract of all the algae did not show any activity
Figure 6.3. The ethanol extract of Padina spp. (Zone: 5 mm) and methanol
extract of Chaetomorpha spp. (Zone: 5 mm) showed zone of inhibition
against the microorganism Pseudomonas aeruginosa. A major difference in
activity was not set up between the ethanol and methanol extract. In contrast
to ethanol and methanol extracts, ethanol : chloroform (1:1) extract did not
display any activity Figure 6.4.
 115

The ethanol extract of all the seaweeds used in this study showed
inhibitory action against Pseudomonas fluorescens except the algae Padina
spp.The ethanol extract of Ulva spp. showed highest activity (Zone: 9 mm)
whereas the algae Chaetomorpha spp. (Zone: 5 mm) and Stoechospermum
spp. (Zone: 5 mm) has showed lowest inhibition. A significant difference in
activity was not found between the ethanol : chloroform (1:1) and methanol
extracts of each algae, whereas the algae Centroceiod spp. (Zone: 7 mm)
revealed maximum activity in the ethanol : chloroform (1:1) extracts and the
Amphiroa spp. (Zone: 7 mm) was the most excellent species among the
methanol extract Figure 6.5.

Among all the algae the ethanol extract of Stoechospermum

spp.exhibited maximum inhibition activity (Zone: 9 mm) against the test
organism Salmonella typhi. Among methanol extracts of all seaweeds, the
antibacterial activity was more evident in Chaetomorpha spp. (Zone: 8 mm)
and mild activity was shown by Stoechospermum spp. (Zone: 4 mm). The
ethanol : chloroform (1:1) extract of Ulva spp. alone showed inhibition (Zone:
3 mm) against Salmonella typhi Figure 6.6

The ethanol extracts of Stoechospermum spp. (Zone: 8 mm) and

methanol extract of Padina spp. (Zone: 8 mm) showed inhibition against the
test organism Staphylococcus aurues and were found to be outstanding
species amongst ethanol and methanol extract respectively. Within ethanol :
chloroform (1:1) extracts only Stoechospermum (Zone: 3mm) illustrated a
mild activity Figure 6.7.
 116

CHAPTER 8

CONCLUSION

From this study, it was found that the seaweed Sargassum spp.

collected from Cape comeron, Kanyakumari had highest protein and

Gracilaria spp. had the highest carbhohydrate content when compared to

other seaweeds. Ulva spp. and Stoechospermum spp. was found to be having

greatest antibacterial activity against pathogenic microbes. Ethanol was found

to be the best solvent for extracting bioactive compounds from seaweeds.The

present work will be useful to identify the seaweeds having the highest

nutritional value, in order to recommend it as a valuable nutritional

supplement. However there is a need to study more about certain anti

nutritional factors in seaweeds which will be useful to sudstantiate the above

statement.We are currently investigating about the presence of other

nutraceuticals apart from analyzing the antimicrobial agents or bioactive

compounds present in seaweeds.

 117

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 122

APPENDIX

1. Bradford reagent

0.1 mg of Coomasie Brilliant blue stain

50 mL of 95% ethanol (95 mL ethanol made unto 100 mL with

distilled water)

100 mL of 85% ortho-phosphoric acid (85 mL ortho-phosphoric

made upto 100 mL distilled water)

All the ingredients are placed in reagent bottle and kept in shaker for

over night.

2. Anthrone Reagent

700 mg of Anthrone

95 % of ice cold H2 SO4 (95 mL H2 SO4 made upto 100 mL with

distilled water)

3. Phosphate buffer

15.21g potassium dihydrogen phosphate in 97.5 mL distilled

water

24.14g dipotassium hydrogen phosphate in 152.5 mL distilled

water

pH 7 is maintained.
 123

4. 2.5 N HCl

91.25 mL of HCl

250 mL of distilled water

5. 0.1 N H2 SO4

2.452 mL of H2 SO4

500 mL of distilled water

 124

LIST OF PUBLICATIONS

PUBLICATIONS

1. Dhanalakshmi V., Jeyanthi Rebecca L., Revathi G. and Sharmila S.,

(2010). Evaluation of Basic Nutritional parameters of seaweeds in
Coastal Tamil Nadu. International Journal of Biotechnology and
Biochemistry, Vol.6, Number 6, pp. 921-928.

2. Jeyanthi Rebecca L., Sonidas, Dhanalakshmi V. and Anbuselvi S.,

(2010). Effect of ExogenousSpermidine onsalinity Tolerance with
respect to Seed Germination. International Journal of Applied
agricultural Research, Vol.5, No.2, pp.163-169.

3. Debiprasad, Sanjeev Mishra, Anushman Mishra, Sharmila S.,

Dhanalakshmi V., Anbuselvi S. and Jeyanthi Rebecca L. (2011).
Phytoremediation of mercury, Aluminium and Chromium using
Rathanas sativa and Zeamays, International Journal of Biotechnology
and Bioengineering Research, Vol.2, pp.277-286.

PAPER PRESENTATIONS

1. Biosorption of Heavy metals at Helix’05 organized by department of

Industrial Biotechnology, Shri Andal Alagar College of Engineering on
29 th and 30th September’05.

2. Industrial Enzyme Laccase production through Solid State

Fermentation and Textile dye Decolourization by Ganoderma Spp. at
CrossLinx’06 organized by CLRI, Anna University on 24th and 25th
February’06.

3. Concentration, Enumeration and Detection of Polio virus in Water and

detecting whether wild type is replaced by vaccinated by RT-PCR-
RFLP technique at Biohorizon’06 organized by IIT Delhi on 10th and
11 th March’06.

4. Evaluation of Basic Nutritional parameters of Seaweeds in Coastal

Tamil Nadu at BICSUB’08 organized by Sathyabama University on 7th
– 9 th August’08.

5. Evaluation of in vitro Antibacterial property of Seaweeds of South

Coast of Tamil Nadu at Greenomics’10 organized by Hindustan
College of Arts and Science on 30th and 31st August’10.