INTRODUCTION

Cyanobacteria are an ancient group of photosynthetic prokaryotic organisms and are thought
to be the first organisms to carry out oxygenic photosynthesis. Blue-green algae make up the
division Cyanophyta in the kingdom Monera, which is made up of about 1,500 species (Sp.)
of prokaryotic organisms. Cyanobacteria get their name from the bluish pigment
phycocyanin, which they use to capture light for photosynthesis. However, not all "bluegreen" bacteria are blue; some common forms are red or pink from the pigment
phycoerythrin. They also contain chlorophyll a, the same photosynthetic pigment that plants
use. In fact the chloroplast in plants is a symbiotic cyanobacterium, taken up by a green algal
ancestor of the plants sometime in the Precambrian. Cyanobacteria were originally
considered as algae because of their microscopic morphology, pigmentation and oxygen
evolving photosynthesis in which photosystems, PSII and PSI are connected in series but due
to their cellular organization which is characterized by the lack of membrane-bound
organelles such as a true nucleus, a chloroplast or a mitochondrion, and resembles that found
in bacteria, they are now considered as prokaryotes.
These organisms can inhabit a range of habitats including freshwater, marine and soil
environments, as well as extreme habitats such as hot spring waters, and Arctic and Antarctic
environments (Sompong., 2005 and Taton., 2006). These bacteria are often found growing on
greenhouse glass, or around sinks and drains and are found to play a major role in the
nitrogen, carbon, and oxygen dynamics of many aquatic environments. Though
cyanobacteria do not have a great diversity of form, and though they are microscopic, they
are rich in chemical diversity. Cyanobacteria have a range of organization. They can range
from unicellular, to filamentous, to colonial. As primary colonizers they have an important
role of adding organic matter to the soil. The cells of a colony are undifferentiated from each
other. Some colonial forms of cyanobacteria have been known to form mats down on the
surface of the soil, which helps to prevent erosion. The filamentous organization is composed
of a chain of cells and their enveloping sheath; this organization can be modified or lost as
the environment changes (Bold, 1985).
1

Algae including cyanobacterial frequently live in extreme environments of light, salinity, and
temperature. In order to adapt to these extreme conditions, most algae produce a high variety
of secondary metabolites that often have potent biological activities. Most algae are relatively
easy to cultivate or produce at industrial scale. Thus, the production of algal-derived
biologically active compounds may be tuned by the selection of appropriate cultivation
conditions, making these algae true natural bioreactors.
Cyanobacteria produce a large number of compounds with varying bioactivities.
Cyanobacteria have the appeal of being a raw unprocessed food rich in Car, chlorophyll,
aminoacids, minerals and many other bioactive components with antioxidant potential.
Cyanobacteria are rich source of structurally novel and biologically active metabolites. The
metabolites produced by these organisms may be potential bioactive compounds of interest in
the pharmaceutical industry. The aquatic microorganisms are known to produce certain
bioactive molecules which interact with other organism in the environment, inhibiting
bacterial or fungal growth (antibiotic activity) or modulating the development of other
organisms which are found in the vicinity of that plant. In the field of research involving
bioactive substances of plant origin, a greater number of important advances have
occurred in cyanobacterial biotechnology in the recent years. Worldwide attention is drawn
towards cyanobacterial for their possible use in mariculture, food, feed, fuel, fertilizer,
colourant, production of various secondary metabolites including vitamins, toxins, enzymes,
pharmaceuticals,

pharmacological

probes

and

pollution

abatement.

Only a

few

cyanobacterial strains (including Spirulina) have been well-characterized or exploited

commercialy. Because cyanobacteria are largely unexplored, they represent a rich
opportunity for discovery; the expected rate of rediscovery is far lower than for other betterstudied groups of organisms. Cyanobacteria produce a wide variety of bioactive compounds,
which include lipopeptides, amino acids, fatty acids, macrolides and amides. Cyanobacterial
lipopeptides include different compounds like cytotoxic, antitumor, antiviral, antibiotics and
the remaining activities include antimalarial, antimycotics, multi-drug resistance reversers,
antifeedant, herbicides and immunosuppressive agents (Burja et al., 2001); besides the
immune effect, blue green algae improves metabolism. Blue green algae have a cholesterollowering effect in animals and humans. Freshwater cyanobacteria are acknowledged
synthesizers of biologically active and structurally diverse secondary metabolites.
2

Over the past decade, cyanobacteria have been recognized as a major source of active natural
products with potential therapeutic applications in the treatment of cancer and HIV related
diseases. A variety of fine chemicals such as pigments, vitamins and enzymes with varied
applications can be obtained on a commercially viable scale from cyanobacteria. A number
of cyanobacteria are rich in vitamins and many can excrete them into the surrounding
environment. Some marine cyanobacteria are potential source for large scale production of
vitamins of commercial interest such as vitamins of the B-complex group and vitamin E
(Borowitzka, 1988). The Cars and phycobiliproteins, characteristic of cyanobacteria have
high commercial value. They are used as natural food colourants, as food additives to
enhance the colour of the flesh of Salmonid fish and to improve the health and fertility of
cattle.
Cyanobacterial metabolites are also now being explored as important sources of
pharmacologically active compounds useful in diagnostics or pigments as fluorescent probes
and as nutraceuticals and food/feed supplements. Free radicals have been implicated in the
causation of several diseases such as liver chirrhosis, atherosclerosis, cancer, diabetes, etc.
and compounds that can scavenge free radicals have great potential in ameliorating these
disease processes. Antioxidants thus play an important role to protect the human body against
damage by ROS. The commonly used synthetic antioxidants such as butyl hydroxyl anisole
and butyl hydroxy toluene have potential health risks and toxicity. Therefore, these need to
be replaced with natural antioxidants.
Further , microbial resistance to antibiotics is a world-wide problem in human and veterinary
medicine. It is generally accepted that the main risk factor for the increase in the antibiotic
resistance is an extensive use of antibiotics. In fact, for the last 50 years, high levels of
antibiotics are commonly used for treatment and prevention of infectious diseases in humans
and animals. This led to emergence and dissemination of resistant bacteria and resistance
genes in wild populations . The antimicrobial agents used in animal care are also significant,
both in increasing resistance in animal pathogens, and in transmission of resistant bacteria
from animals to humans. Hence the need of the hour is a search for novel antibacterial
compounds with therapeutic potential for which the pathogens may not have resistance (Patil
et al., 2001). Due to the significant role of cyanobacteria, it was considered worthwhile to
3

examine antimicrobial and antioxidant activities for possible biotechnological and

pharmaceutical applications. There is a growing interest in natural additives as potential
antimicrobials and antioxidants. Diseases that remain most challenging in todays healthcare
system tend to be complex involving multiple mechanisms, targets and drugs for effective
disease management. In contrast to current combination therapies, however, plant based
drugs contain a mixture of multiple components thereby saving considerable time and
expense.
The present study was conducted to evaluate the possible antibacterial, activity of various
cyanobacterial extracts. cyanobacterial strains were screened and selected strains were
analyzed for their phycoconstituents. After suitable growth, cyanobacterial culture was
harvested using three different solvents namely Methanol, Hexane and Ethyl acetate.
Extracts, thus prepared, were evaluated for their antibacterial. potential by agar-well
diffusion assay against bacterial sp. of clinical significance. The antioxidant potential of the
methanolic cyanobacterial extract was also carried out as it showed greater extractability of
the antioxidant compounds.

OBJECTIVES
Standardization of growth curve of cyanobacterial strains.
Phytochemical analysis of crude extracts of some selected cyanobacterial strains.
Antibacterial. activity of partially purified cyanobacterial metabolites. Bacterial
bioassays for the observation and quantification of possible antibacterial. effects from
cyanobacterial extracts.
Antioxidant potential of methanolic extract of cyanobacterial strains.

REVIEW OF LITERATURE
Cyanobacteria also known as blue-green bacteria, blue-green algae, and Cyanophyta, is a
phylum of bacteria that obtain their energy through photosynthesis. The name
"cyanobacteria" comes from the color of the bacteria (Greek: (kyans) = blue).
The ability of cyanobacteria to perform oxygenic photosynthesis is thought to have converted
the early reducing atmosphere into an oxidizing one, which dramatically changed the
composition of life forms on Earth by stimulating biodiversity and leading to the nearextinction of oxygen-intolerant organisms. According to endosymbiotic theory, chloroplasts
in plants and eukaryotic algae have evolved from cyanobacterial ancestors via
endosymbiosis.
Cyanobacteria include unicellular and colonial sp. Colonies may form filaments, sheets or
even hollow balls. Some filamentous colonies show the ability to differentiate into several
different cell types: vegetative cells, the normal, photosynthetic cells that are formed under
favorable growing conditions; akinetes, the climate-resistant spores that may form when
environmental conditions become harsh; and thick-walled heterocysts, which contain the
enzyme nitrogenase, vital for nitrogen fixation. Heterocysts may also form under the
appropriate environmental conditions (anoxic) when fixed nitrogen is scarce. Heterocystforming sp. are specialized for nitrogen fixation and are able to fix nitrogen gas into
ammonia (NH3), nitrites.
Historically, Bacteria were first classified as plants constituting the class Schizomycetes,
which along with the Schizophyceae (blue green algae/Cyanobacteria) formed the phylum
Schizophyta. (C. Von Ngeli et al., 1857) then in the phylum Monera in the kingdom Protista
by Haeckel in 1866, comprising Protogens, Protamaeba, Vampyrella, Protomonae and
Vibrio, but not Nostoc and other cyanobacteria, which were classified with algae(Haeckel,
Ernst, 1867) later reclassified as the Prokaryotes by Chatton.)
The cyanobacteria were traditionally classified by morphology into five sections, referred to
by the numerals I-V. The first three Chroococcales, Pleurocapsales, and Oscillatoriales
are not supported by phylogenetic studies. However, the latter two Nostocales and
Stigonematales are monophyletic, and make up the heterocystous cyanobacteria. The
5

members of Chroococales are unicellular and usually aggregate in colonies. The classic
taxonomic criterion has been the cell morphology and the plane of cell division. In
Pleurocapsales, the cells have the ability to form internal spores (baeocytes). The rest of the
sections include filamentous sp.. In Oscillatoriales, the cells are uniseriately arranged and do
not form specialized cells (akinetes and heterocysts). In Nostocales and Stigonematales the
cells have the ability to develop heterocysts in certain conditions. Stigonematales, unlike
Nostocales, includes sp. with truly branched trichomes.
The majority of cyanobacteria are aerobic photoautotrophs. Their life processes require only
water, carbon dioxide, inorganic substances and light. Photosynthesis is their principal mode
of energy metabolism. The prominent habitats of cyanobacteria are limnic and marine
environments. They flourish in water that is salty, brackish or fresh, in cold and hot springs,
and in environments where no other microalgae can exist. Most marine forms (Humm and
Wicks, 1980) grow along the shore as benthic vegetation in the zone between the high and
low tide marks. The cyanobacteria comprise a large component of marine plankton with
global distribution (Wille, 1904; Gallon et al., 1996). A number of freshwater sp. are also
able to withstand relatively high concentrations of sodium chloride. It appears that many
cyanobacteria isolated from coastal environments tolerate saline environments (i.e. are
halotolerant) rather than require salinity (i.e. are halophilic). As frequent colonisers of
euryhaline (very saline) environments, cyanobacteria are found in salt works and salt
marshes, and are capable of growth at combined salt concentrations as high as 3-4 molar
mass (Reed et al., 1984). Freshwater localities with diverse trophic states are the prominent
habitats for cyanobacteria. Numerous sp. characteristically inhabit, and can occasionally
dominate, both near-surface epilimnic and deep, euphotic, hypolimnic waters of lakes
(Whitton, 1973). Others colonise surfaces by attaching to rocks or sediments, sometimes
forming mats that may tear loose and float to the surface.
Cyanobacteria have been identified as one of the most promising groups of organisms from
which were isolated novel, biologically active natural products(Burja et al., 2001). A number
of significant progresses have occurred in cyanobacterial biotechnology in the recent years.
Cyanobacteria are structurally diverse and widely distributed throughout the world and have
proven their potential in several fields, particularly as new therapeutic agents for a variety of
6

diseases. Rapid restorations of cyanobacterial activity under favorable conditions are

characteristics of cyanobacteria (Pankaratova., 1987). In order to adapt in extreme conditions
cyanobacteria produce some metabolites.

Figure 2.1: Secondary metabolites of cyanobacteria (Yadav et al., 2011)

Cyanobacterial secondary metabolites represent a vast diversity of structures and have been
isolated from a number of cyanobacterial genera from different geographical locations.
During the last two decades, cyanobacterial secondary metabolites have attracted the
attention of researchers mainly due to two reasons; (i) acute toxicity of toxins produced by
several bloom forming cyanobacteria in freshwater system and their harmful effect on
animals and human health, and (ii) potential therapeutic use of several secondary metabolites
(Namikoshi and Rinehart., 1996; Burja et al., 2001; Dittmannand Wiegand., 2006).
Cyanobacteria are able to produce wide range of active substances belongs to groups,
alkaloids, phenols and of polyketides, peptides which exhibits diverse biological activities.
PC is a protein-bound pigment found in blue-green algae. PC monomers are themselves
made up of two distinguishable protein subunits designated a and b, which contain at least
three covalently attached bilin chromophores, open chain tetrapyrroles with no metal
complexes (Duerring et al., 1991). These prosthetic groups account for about 4% of the algae
mass, indicating the presence of about sixteen chromophoric groups per unit molecular
weight (Eocha and Phycobilins., 1962). It occurs in four different structural forms,
7

monomeric, trimeric, hexameric and decameric (Coll et al., 1987) and is the most abundant
pigment in blue-green algae, accounting for more than 20% of algal dry weight (Richmond,
1990). The chemical structure of the bilin chromophores in c-PC, (open chain tetrapyrroles)
are very close to that of bilirubin. (Stocker et al., 1987) reported that bilirubin is an
antioxidant of possible physiological importance because it could scavenge peroxy radicals
by donating a hydrogen atom attached to the C-10 bridge of the tetrapyrrole molecule to form
a carbon-centered radical with resonance stabilization extending over the entire bilirubin
molecule. Carotenoid (Car), because of its value as antioxidants, food-supplements and in
animal-feeding, Car are among the most attractive target compounds of marine microalgae.
In addition to cancer, Car research has been extended to other diseases or dysfunctions, e.g.
ischemic heart disease, cerebrovascular disease, autoimmune disease, infectious disease,
reproductive dysfunction, age-related macular degeneration (AMD), cataracts, diabetes
mellitus, and aging/senescence. LDL/HDL cholesterol, which is related to arteriosclerosis, is
improved by Car.
All cyanobacteria produce an amazing diversity of secondary metabolites. One of the most
important groups of these metabolites are phenolic compounds. Phenolics are characterized
by at least one aromatic ring (C6) bearing one or more hydroxyl groups. They are mainly
synthetized from cinnamic acid, which is formed from phenylalanine by the action of Lphenyloalanine ammonia-lyase PAL (EC 4.3.1.5), the branch point enzyme between primary
(shikimate pathway) and secondary (phenylopropanoid) metabolism (Rice-evans et al.,
1997). The significance of this route can be supported by the fact that, in normal growth
conditions, 20% of carbon fixed by plants flows through this pathway (Diaz et al., 2001)
(Fig.2.2) Phenols are divided into several different groups, distinguished by the number of
constitutive carbon atoms in conjunction with the structure of the basic phenolic skeleton
(simple phe-nols, benzoic acids, phenylopropanoids and flavonoids) (Chaudiere et al., 1999).
Phenolics have various functions in plants. An enhancement of phenylopropanoid
metabolism and the amount of phenolic compounds can be observed under different
environmental factors and stress conditions (Grace et al., 2000). The synthesis of isoflavones
and some other flavonoids is induced when plants are infected or injured (Ruiz et al., 2003),
or under low temperatures and low nutrient conditions. Most of them have antimicrobial
activity.
8

Fig. 2.2: Biosynthesis pathways leading to formation of main groups of phenolic

compounds (according to Ryan et al., 1999)
The incidence of multidrug resistant human pathogenic strains due to indiscriminate use of
commercial antimicrobial drugs has been increasing in recent years (Alieora and Afolayan.,
2006). On the other hand oxidative stress induced by the imbalance between formation and
neutralization of peroxidants is the major cause of todays diseases. Free radical generation
as a result of oxidative stress cause adverse effect on the vital biomolecules of life (Hazra et
al.,2008). Antioxidative enzymes protect human cells from damaging effect of free radicals
but failed sometimes due to the disruption by various pathological processes (Niki et al.,
1994). In this condition antioxidant supplementation are vital to combat oxidative damage.
This has forced scientist in the search of new compounds with antimicrobial and antioxidant
potential from various natural sources including plants and microbes to fight against
pathogens with novel strategies. The secondary metabolites from cyanobacteria include a
9

range of compounds showing animal toxicity and antibacterial., anticoagulant, antifungal,

anti-inflammatory, antimalarial, antiprotozoal, antituberculosis, antiviral, antitumor and
cytotoxic activities. More than 800 secondary metabolic compounds belonging to several
classes of substances including enzyme inhibitors, photosynthesis inhibitors, antimicrobial,
antimitotic, immunosuppresses and antitumor depsipeptides have been isolated and classified
and named on the basis of chemical structure, bioassay method and their toxicological targets
(Burja et al., 2001; Tan., 2007; Pearson et al., 2010). The pharmacological activity of several
classes of cyanobacteria has been identified and studied in last decades (Umemura et al.,
2003;Takamatsu et al., 2003; Mayer et al., 2003). Cyanolichens (in symbiosis with lichens)
have

very

potential

applications

in

medicines

(Sunderaraman

et

al.,

1996),

pharmaceuticals(Gustafson et. al, 1989), enzymes, diagnostics, fuel and waste treatment, and
recycling process (Subramanian et al.,1994). Cyanobacteria are considered as the new
therapeutic agents for a variety of diseases (Harada et al., 2002; Romanos et al.,2002).The
antioxidant capacity of bioactive compounds has been related to the prevention of several
diseases including cancer, coronary heart diseases, inflammatory disorders, neurological
degeneration, and aging (Wollgast and Anklam 2000 ; Madhavi et al. 1996). Bioactive
compounds have also been isolated previously from other marine organisms including
crustaceans, fish, and their by-products . In some of these matrices, interesting functional
compounds were isolated previously (Kadam and Prabhasankar 2010 ; Kim and Wijesekara
2010). Most algae cultured under optimum condition contained about 4% of total chlorophyll
on dry weight basis. Chlorophyll provides a chelating agent activity which can be used in
ointment, treatment for pharmaceutical benefits especially liver recovery and ulcer
treatment.Besides that, it repairs cells, increases hemoglobin in blood and faster the cell
growth (Puotinen, 2009).
Due to the significant role of cyanobacteria, it was considered worthwhile to examine
antimicrobial and antioxidant activities for possible biotechnological and pharmaceutical
applications. There is a growing interest in natural additives as potential antimicrobials and
antioxidants. Cyanobacteria are photoautotrophic microorganisms and are source of potential
new bioactive substances which contribute in reducing of the number of bacteria, fungi,
viruses, and other microorganisms. The ability of the Cyanobacteria to produce antimicrobial

10

substance may be noticed not only as a defensive instrument for the strains but also it is good
source of new bioactive compounds (Khavari-Neiad andYazdi., 2005).
The search for cyanobacteria with antimicrobial activity has gained importance in recent
years due to growing worldwide concern about an alarming increase in the emergence of
antibiotic resistance and further increase in the rate of infection by these antibiotic-resistant
microorganisms. Various strains of cyanobacteria are known to produce intracellular and
extracellular metabolites with diverse biological activities such as antibacterial. and
antifungal (Falch et al., 1995).The commercial application of microalgae derived compounds
has received a very little attention in the area of pharmaceuticals, antibiotics and other
biologically active compounds. Hence screening of Cyanobacteria for antibiotics and other
pharmacologically active compounds has received interest as a potential source for new
drugs (Browitzka., 1995).Usage of commercial antibiotics for human and fish disease
treatment produces undesirable side effects. Various strains of Cyanobacteria are known to
produce intracellular and extracellular metabolites with diverse biological activities such as
antialgal, antibacterial. and antiviral activity (Thajuddin and Subramanian., 2005).
The commonly applied methods of antimicrobial activity are based on the agar diffusion and
results were shown as visible zones of growth inhibition. Al-Wathnaniet al. (2012) analyzed
bioactive natural compounds from algae such as cyanobacteria and determined their activity
against human pathogenic bacteria and yeast. In their study, they found that all the algal sp.
extracts which were extracted with acetone/methanol/di-ethyl-ether showed strong inhibition
against S. sonnei,
Emergence concerns have been raised to establish structural and functional properties of the
bioactive compounds described in algal crude extracts, up to date, over 2,400 bioactive
metabolites have been isolated and identified from a diverse group of algal communities
(Faulkner, 2001). Antimicrobial effects from cyanobacterial aqueous and organic solvent
extracts are visualized in bioassays using selected micro-organisms as test organisms
(Kellamet al. 1988; Frankmolleet al. 1992; Falchet al. 1995). Methods commonly applied
are based on the agar diffusion principle using pourplate or spread-plate techniques.
Freshwater cyanobacteria are acknowledged synthesizers of biologically active and
structurally diverse secondary metabolites. The antimicrobial substances involved may target
various kinds of micro-organisms, prokaryotes as well as eukaryotes.
11

It has also been reported that the antibacterial. activity is due to different chemical agents
present in the extract,

including flavonoids and triterpenoids and other compounds of

phenolic nature or free hydroxyl group, classified as active antimicrobial compounds (Rojas
et al,1992). Many authors had found antibacterial. activities of microalgae due to fatty
acids (Viso et al., 1987). To test the antibacterial. potential of the cyanobacterial strain
,different solvents (hexane, ethyl acetate and methanol) based on their polarity were used to
extract the compounds and the antibacterial. activity was determined by disc diffusion assay.
The percent yield varies with the type of solvents and cyanobacterial sp. used. Antimicrobial
effects are shown as visible zones of growth inhibition. Bacterial bioassays comprise
different test bacteria. Micrococcus luteus, Bacillussubtilis, B. cereus and Escherichia coli
are commonly used to detect antibiotic residues in food (Crosby 1991; McGill and Hardy
1992).
Recently, there has been an increasing interest in cyanobacteria as a potential source for new
drugs (Glombitza and Koch 1989; Schwartz et al. 1990).The cyanobacterium Spirulina
platensis is rich in nutrients, such as proteins, vitamins, minerals, carbohydrates, and linolenic acid. It is gaining more and more attention, not only for the foods aspects but also
for the development of potential pharmaceuticals (Quoc&Pascaud 1996). This alga is being
widely studied, not only for nutritional reasons but also for its reported medicinal properties;
thus, several studies have shown that Spirulina or its extracts could prevent or inhibit cancer
in humans and animals, and recent works have indicated that this sp. has immuno-promoting
effects (Hirahashi et al. 2002; Subhashini et al. 2004). Spirulina platensis was also reported
to present antimicrobial activity (Ozdemir et al. 2004) as well as to inhibit the the replication
of several viruses, such as Herpes simplex and HIV-1 (Ayehunie et al. 1998; HernndezCorona et al. 2002). Spirulina contains a whole spectrum of natural mixed carotene and
xanthophyll phytopigments which, together with PC, seem to be related to its antioxidant
activity (Pineiro Estrada et al. 2001).
One potential commercial application of microalgae derived compounds that has, as yet,
received little attention is the area of pharmaceuticals, antibiotics and other biologically
active compounds. Both cell extracts and extracts of the growth media of various unicellular
algae (e.g. Chlorella vulgaris, Chlamydomonas pyrenoidosa) have been proved to have
antibacterial. activity in vitro against both Gram-positive and Gram-negative bacteria. It has
12

also been reported that a wide range of in vitro active antifungal activities are obtained from
extracts of green algae, diatoms and dinoflagellates. Microalgae, such as Ochromonas
antibacterial., Prymnesiumparvum, and a number of blue-green algae produce toxins that
may have potential pharmaceutical applications (Borowitzka and Borowitzka, 1992).Recent
studies reported that Spirulina platensis could be used as a matrix for the production of
selenium-containing compounds and proved to be successful in transforming inorganic
selenium to organic selenium in vivo when cultivated in selenium-rich medium (Li et al.
2003). The antioxidant activities of selenium-containing PC and its different aggregates
(monomer, trimer, and hexamer) against free radicals of superoxide, hydrogen peroxide, and
2,2-diphenyl-1-picryl-hydrazyl (DPPH) were found to be variable (Huang et al. 2007).
Cyanobacteria being photoautotrophs have the ability to photosynthetically transform simple,
labelled compounds such as

14

CO2, 13CO2, 33H2O,

15

NO3 into complex organic compounds.

Isotopicallylabelled cyanobacterial metabolites such as sugars, lipids and amino acids are
commercially available (Patterson, 1996). Rania and Taha (2008) tested antibacterial. and
antifungal activity of three cyanobacteria (Anabaenaoryzae, Tolypothrixceytonica and
Spirulinaplatensis)

and

two

green

microalgae

(Chlorella

pyrenoidosa

and

Scenedesmusquadricauda) against human and plant pathogen microorganisms by using agar

well diffusion method. In their study, it was found that S. platensisand A. oryzae had the
highest antibacterial. and antifungal activity towards the tested bacteria and fungi. However,
A. oryzae methanol extract (5 mg/well) showed lower antimicrobial activity against E. coli,
S. aureus and C. albicans when compared to our study by using disc diffusion method (1.5
mg/disc). The activity differences in these two studies may derive from the different
cyanobacteria and microorganism strains that were used and the methods used in
determination of antimicrobial activity.
The marine environment, which contains a vast array of organisms with unique biological
properties, is one of the most underutilized biological resources. Sethubathi and Prabu (2010)
determined antimicrobial activity of three marine cyanobacterial sp. namely Oscillatoria
antibacterial., Phormidium antibacterial. and Lyngbya majuscule isolated from
Adirampattinam coast against the human pathogenic bacteria such as Streptococcus mutants,
Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis and
Klebsiellapneumoniae. In their study, Oscillatoria antibacterial. showed the maximum
13

inhibition against pathogen strain compared to other sp. and the minimum inhibition activity
was observed in the extract of L. majuscule.
In another study, fifteen cyanobacteria belonging to Anabaena, Nostoc, Scytonemaand
Microcystissp. isolated from various aquatic and terrestrial habitats were screened for
antimicrobial activity by using disc diffusion method (Yadavet al., 2012). In the study,
ethanolic extracts of Anabaena BT2, Nostoc Brf02, Nostoc Brf 04 and ScytonemaBr1 at a
concentration of 500 g/disc showed prominent zone of inhibition against two gram-negative
bacteria (Pseudomonas sb1 and sb3).
Microbial resistance to antibiotics is a world-wide problem in human and veterinary
medicine. It is generally accepted that the main risk factor for the increase in the antibiotic
resistance is an extensive use of antibiotics. In fact, for the last 50 years, high levels of
antibiotics are commonly used for treatment and prevention of infectious diseases in humans
and animals. This led to emergence and dissemination of resistant bacteria and resistance
genes in wild populations (Bogaard and Stobberingh, 2000). The antimicrobial agents used in
animal care are also significant, both in increasing resistance in animal pathogens, and in
transmission of resistant bacteria from animals to humans. Hence the need of the hour is a
search for novel antibacterial. compounds with therapeutic potential for which the pathogens
may not have resistance (Patil et al., 2001).
Several products of algal origin such as alginate, carrragenean and agar as
phycocolloids have been used for decades in medicine and pharmacy. Since algae
have been used in traditional medicine for a long time (Fitton, 2006), and also some
algal

substances

have

bacteriostatic

and

bactericidal

activity,

they

have

been

extensively studied by several researchers (Nora et. al., 2003; Salvador et. al.,2007). This
study will also hopefully expose new frontiers on the current applications of the
cyanobacterial extract. Selection of crude extracts

for screening programs

has the

potential of being more successful in the initial steps than the screening of pure
compounds isolated from natural products.
No attempt has been made to investigate the antioxidant potential of the extracts of
cyanobacteria, however, little information is available on the antibacterial. activity of
14

cyanobacteria. Cyanobacteria are largely unexplored and represent a rich opportunity for
discovery. Therefore, the present study was aimed to investigate the phycochemical and
biological properties of cyanobacteria. This screening study will also help the selection of
crude cyanobacterial extracts having bioactivity for further isolation and purification of the
compound of pharmacological interest.

15

MATERIAL AND METHODS

Place of work:
The present study entitled Growth Characteristics and Biological Potential of Different
Crude Extracts of Selected Cynobacterial Strains was carried out in the Department of
Plant Tissue Culture and Biotechnology, Integral University, Lucknow, during January to
June-2016.
Experimental Organisms:
Cyanobacteria used as experimental organisms which are morphologically different in
characterstics- P. boryanum sp., Nostoc sp., Phormidium sp. were used in the study as test
organism. Axenic culture of cyanobacteria were maintained in the Department of
Microbiology and Biotechnology, Integral University, Lko.
Growth conditions:
The pure culture of cyanobacteria was maintained in the culture room at 27 2 C. For
regular experiments. Cultures were grown in BG-11 medium for rest of the cultures under the
light intensity of 2400 lux and 12/12h light and dark photoperiod
Growth Media:
Composition of modified BG-11 medium
Macronutrients

g/L

NaNO3

1.5

K2HPO4

0.04

Mg SO4.7H2O

0.075

Na2MoO4.2H2O

0.39

Citric acid

0.006

Ferric ammonium citrate

0.006

EDTA (disodium magnesium salt)

0.001

Na2CO3

0.02

Micronutrients

g/L

H3BO3

2.86

MnCl2.4H2O

1.81

ZnSO4.7H2O

0.222
16

CaCl2.2H2O

0.036

CuSO4.5H2O

0.079

Co(NO3)2.6H2O

0.494

Glasswares:
The following glasswares were used in the present study. All the glasswares were of
Borosil brand. Petri plates, Pipettes, Test tubes, Flasks of different volumes. Algal cultures
were grown in flasks of 1000 ml capacity containing 750 ml medium. For experimental use
flask of 100-250 ml capacity containing 50-75 ml of medium were used. Non absorbent
cotton was used to plug the flasks.
Chemicals:
All the chemicals were purchased from a standard company like Sigma, SDS fine, Merk,
BDH

etc.

Sterilization of medium;
All the glasswares and culture media were sterilized in an autoclave at 15 lb inch2 pressure
and at 121 0C temperature for 15 min. Before autoclaving, the desired quantities of media
have been poured in to suitable sized glass wares properly plugged with cotton.
Incubation and maintenance of cultures:
Required inoculums of cyanobacteria were transferred in fresh mineral media in a sterilized
inoculation chamber. Culture were incubated and grown photoautotrophically in culture
room maintained at 27 1 0C, illuminated with white fluorescent tubes providing an intensity
of 50 mol m-2 s-1 and a photoperiod of 8/16 h light and dark. The batch cultures were
regularly shaken twice a day. Log phase cultures were used as inoculum as well as
throughout experimental studies. For large scale use, cyanobacteria were also grown in a
fermenter containing mineral medium.
Growth measurement:
Growth was measured in terms of chlorophyll estimation on alternate days at regular
intervals for 10 days.

17

Phycochemical analysis:
Polyphenol
Total Phenol content was calculated by measuring the optical density at 750 nm by the
method of Singleton and Rossi (1965).
Estimation of protein
The amount of Protein was estimated by measuring the optical density at 650 nm by the
method developed by Lowry et al. (1951)
Chlorophyll-a and Car
Chl-a and Car was estimated by measuring the optical density at 663 nm and 450 nm
respectively by the method given by Myers and Kratz (1955).
Phycocyanin
PC content was extracted and supernatant was obtained after centrifugation and absorbance
was recorded spectrophotometrically at 620 nm as per the method of Blumwald and Tel-Or
(1982).
Carbohydrates
A known volume of cyanobacterial suspension was centrifuged and washed with distilled
water. Soluble carbohydrate in the pellets and in the medium was determined by using
anthrone reagent (Mc Cready et al., 1950).
Alkaloids
The presence of alkaloids was detected by adding 1 % HCl to the 2 ml methanolic extract
and boiled the sample. Addition of 5-6 drops of Mayers reagent develops orange/ red/brown
precipitate indicating the presence of alkaloids.
Flavonoids
The dried methanolic extract of various cyanobacterial strains obtained from heat percolation
method by using soxhlet apparatus were dissolved in 2 ml methanol, and mixed horoughly.
18

The flavonoid spots were separated by TLC using chloroform and methanol (4:1) solvent
mixture. The colour of the spots were visualized and noticed under ultraviolet (UV-254nm)
light (Wagner et.al., 1996).
Tannins
The water extract of dried mass of cyanobacteria was mixed with 2ml of 0.1 M FeCl 3 in 0.1
M HCl and 0.008 M K4Fe(CN)6.3H2O. The absorbance is measured with a
spectrophotometer at 395 nm wavelength within 10 min.
Preparation of extracts

Extraction was according to Doan et al. (2000). Cyanobacterial strains harvested after twenty
days of growth were dried with the help of dryer at room temperature. The biomass (1 g dry
wt.) was extracted twice with 100 ml hexane and methanol separately and centrifuged
(20,000 g, 30 min). The crude extracts were then filtered through Whatman No. 1 filter paper
and extracts were subsequently dried at room temperature.

Test Microorganisms
In the present study Staphylococcus epidermidis was used for testing antibacterial. activity.
This strain was obtained from the National Chemical Laboratory (NCL), Pune, India.

Antibiotic sensitivity testing

Antibiotic sensitivity of bacterial strain was determined by the standard disc diffusion
method of Bauret al. (1966) against a number of antibiotics such as Azythromycin,
Erythromycin, Amoxicillin and chloramphenicol having potency of 10 g per disc.

Antibacterial assay
For antibacterial. testing fresh inoculum of S. epidermidis was prepared and incubated at
37C for 18 h. The cell suspension in each case was swabbed onto MH agar with the help of
a sterile cotton swab and after that the Whatman filter paper discs (No. 1) impregnated with
10 L of each extract prepared in DMSO (20 mg/ml), was placed on the plates (DMSO was

19

used as a control) and incubated at 37C for 18 h. Antibacterial. potential was assessed based
on the inhibition zone size (millimetres).

RESULT
Growth measurement
Growth pattern of various cyanobacteria was monitored at regular intervals for 10 days in

Growth (mg ml-1Chl a)

liquid medium by estimating the chlorophyll content.

1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0

Time (days)

10

12

Fig. 4.1: Growth of Phormidium sp. measured in terms of Chl-a at regular intervals
upto 12 days. Values are means SE (n=3).
Growth pattern of Phormidium sp. as given in figure 4.1 shows two clear phases. Lag phase
is upto two days of incubation and further there is continuous increase in the growth of the
cyanobacterium. The exponential growth was observed from 4th day of incubation and
reached upto 8th day. Further the increase in growth is towards the stationary phase.

20

Growth ( mg ml-1 Chla )

1.8
1.6
1.4
1.2
1
0.8
0.6
0.4

0.2
0
0

10

12

Time (days)

Fig. 4.2: Growth of Nostoc sp. measured in terms of Chl-a at regular intervals upto 12
days. Values are means SE (n=3).
Growth pattern of N. muscorum as given in figure 4.2 depicts two clear phases. Lag phase is
upto two days of incubation and further there is continouos growth in the cyanobacterium.
The exponential growth was observed from second day of incubation and reached upto 6 th
day.

Further

the

increase

in

growth

is

towards

the

stationary

phase.

Growth (mg ml-1 chl a)

1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0

10

12

Time (days)

Fig. 4.3: Growth pattern of P. boryanum sp. measured in terms of Chl-a at regular
intervals upto 12 days. Values are means SE (n=3).

21

The result of Growth pattern of P. boryanum presented in the figure 4.3 clearly demonstrates
lag phase for four days and further growth was exponential.
Specific growth rate and Productivity
There was a variation in the strains with respect to their specific growth rate. N.muscorum
showed highest specific growth rate followed by P.boryanum whereas, lowest specific
growth rate was observed in Phormidium sp. Similar pattern was also observed in their
productivity measured in terms of dry mass. N.muscorum exhibit highest biomass production
followed by P.boryanum whereas, lowest productivity was observed in Phormidium sp.
Table 4.1: Specific growth rate of cyanobacterial strains
Microorganisms

Specific growth rate mmax(day-1)

N.muscorum

0.14 .01

P.boryanum

0.13 .01

Phormidium

0.11 .01

The data refer to mean value of three replicates SE.

mmax = maximum specific growth rate.

Table 4.2: Biomass production (dry mass) of cyanobacterial strains

Microorganisms

Productivity
P288 (mg dry mass L-1 day-1)

N.muscorum

0.14 .01

P.boryanum

0.13 .01

Phormidium

0.11 .01

P288 = productivity in terms of dry mass measured after 288 h = 12 days.

22

Phycochemical analysis
Phycochemical analysis of methanolic extract of selected cyanobacterial strains namely
Phormidium, P. boryanum, Nostoc sp. for major phycoconstituents was undertaken. (Table
4.3)
Table 4.3: Pigments in methanolic extracts of different cyanobacterial strains

Test

Car

PC

organisms
N.muscorum

++

P.boryanum

++

Phormidium

Note: + = Slightly present, ++ = moderately present

Table 4.4: Preliminary qualitative evaluation of bioactive compounds in methanolic
extract of different cyanobacterial strains.
Test

Tannins Flavonoids Alkaloids Total

organisms

Carbohydrate Protein

phenols

N.muscorum

++

P.boryanum

++

++

Phormidium

antibacterial.

Note: + = Slightly present, ++ = moderately present

23

Antibacterial assay
In this study, three cyanobacterial strains (N.muscorum, P.boryanum and Phormidium) were
taken and extracted using three different solvents namely Methanol, Hexane, Ethyl acetate.
Antibacterial. activities of methanolic extracts of different cyanobacterial strains is evident
from Plate 1, plate 2 and plate 3. The results indicates that methanol extract of Phormidium
sp.

show a high inhibitory activity (7mm) against S.epidermidis, further a moderate

inhibitory activity (5mm) was observed against methanol extract of P.boryanum and low
activity was observed in methanolic extract of N.muscorum for S.epidermidis. A low activity
was observed in Hexane extract of Phormidium against S.epidermidis followed by moderate
activity in N.muscorum comparatively high activity was observed in case of P.boryanum.
The ethyl acetate extracts of the Cyanobacteria showed very less inhibitory activity against
the respective bacterial sp. In this study, Methanol was the best solvent for extracting the
effective antibacterial. activity from the respective cyanobacterial cultures.

Plate 1

Plate 2

24

Plate 3
Fig.4.4: Antibacterial activity of methanolic extract of Nostoc Sp., P.boryanum and
Phormidium respectively against S.epidermidis.
10
9

Zone of Inhibition (mm)

8
7
6
5
4
3
2
1

0
Methanol Hexane

Ethyl
Acetate

Extracts

Fig. 4.5: Antibacterial activity of methanol, hexane and ethyl acetate extract of
phormidium sp. against S.epidermidis. Values are means SE with n=3.

25

10
9

Zone of Inhibition (mm)

8
7
6
5
4
3
2
1
0
Methanol Hexane

Ethyl
Acetate

Extracts

Fig. 4.6: Antibacterial activity of methanol, hexane and ethyl acetate extract of
P.boryanum. sp. against S.epidermidis. Values are means SE with n=3.
10
9

Zone of Inhibition (mm)

8
7

6
5
4
3
2
1
0

Methanol Hexane

Ethyl
Acetate

Extracts

Fig. 4.7: Antibacterial activity of methanol, hexane and ethyl acetate extract of Nostoc
sp. against S.epidermidis. Values are means SE with n=3.
26

In the present work we screened cyanobacteria for the antioxidant potential by using DPPH.
The DPPH radical scavenging capacity of the methanol extract was determined. DPPH
radicals have an absorption maximum at 515 nm, which disappears with reduction by an
antioxidant compound. P.boryanum displayed greatest antioxidant potential (30.2 %
inhibition of DPPH) than the positive control ascorbic acid (25 % inhibition) at 5 mg ml -1
followed by Nostoc antibacterial. (25.6%). A moderate radical scavenging activity was
shown by Phormidium antibacterial. (21.8%).

Table 4.5: Antioxidant activity of various cyanobacterial strains

Cyanobacterial Strain

% inhibition

Phormidium sp.

21.8

Nostoc sp.

25.6

P. boryanum

30.2

27

DISCUSSION
The results of the present study show growth characteristics of three cyanobacterial strains,
preliminary phycoanalysis of their extract and their antibacterial. and free radical scavenging
potential.
Growth measurement
The strains were examined for their growth patterns in terms of Chl-a content (Fig. 4.1, 4.2
and 4.3). All the growth phases showed variation among the strains. Cyanobacteria are
photosynthetic microorganisms with diverse morphology and therefore, the variation in
growth pattern could be explained on the basis of their morphological, nutritional or
metabolic differences (Imai et al., 2009).

Specific growth rate and Productivity

There was a variation in the strains of cyanobacteria with respect to their specific growth
rate and productivity ( Table 4.1 and 4.2). In present study Nostoc sp. showed highest
specific growth rate followed by P.boryanum whereas, low specific growth rate was
observed in Phormidium sp. Similar pattern was observed in their productivity measured in
terms of dry mass. Similar to this Tiwari et al (2001) also observed the variation in the dry
mass of some cyanobacterial strains isolated from different habitats of Uttar Pradesh under
laboratory conditions. P. boryanum exhibited greater productivity followed by Nostoc sp.
whereas, least productivity was observed in oscilltoria sp. Greater productivity indicates the
high cell density of the culture. Cultures with high density have several advantages in
industrial application as they reduce the expense of extraction and other downstream
operations ( Yu et al, 2009).

Phycochemical analysis
Microorganisms synthesize primary and secondary metabolites in different phases during the
growth and some of the metabolites from different cyanobacteria revealed the presence of
Car, PC, tannins, alkaloids, protein, carbohydrate and flavonoids in the methanolic extracts
of different cyanobacterial strains ( Table 4.3 and 4.4). The efforts made by Singh and
28

Chowdhary (2010) to identify antimicrobial agents revealed several promising compounds in

alga Pithophora.. The ability to produce antimicrobial agents may be significant not only as a
defensive instrument for algae but also as a good source of new bioactive compounds from a
pharmaceutical point of view ( Suhail et al, 2011). It has also been reported that the
antibacterial. activity is due to different chemical agents present in the extract, including
flavonoids and triterpenoids and other compounds of phenolic nature or free hydroxyl group,
classified as active antimicrobial compounds (Rojas et al, 1992).
Antibacterial assay
The antibacterial. potential of the cyanobacterial strains was determined by agar disc
diffusion method and results are presented in Fig. 4.4. Disc diffusion assay showed that S.
epidermidis was sensitive to methanol extract of all the strains of cyanobacteria. The
maximum sensitivity measured in terms of zone of inhibition was noticed against the
methanolic extract of Phormidium sp (7mm) followed by P.boryanum (5mm). However,
Nostoc sp. showed inhibitory zone of 2 mm against the test bacteria. As compared to the
methanolic extract, hexane extract showed less antibacterial. potential whereas, the ethyl
acetate extract showed almost no sensitivity against the bacteria S. epidermidis. The
antibacterial. activity of the extract could be due to the presence of different chemical agents
that may include flavonoids and triterpenoids and other compounds of phenolic nature or free
hydroxyl group (Yu et al, 2009). Further, the observed differences in the extent of
antibacterial. action of cyanobacterial extract could be attributed to the diversity of
compounds synthesized during the adoption. Zeeshan et al., reported the presence of certain
metabolites such as tannin, alkaloids, protein and flavonoids in the extract of cyanobacteria.
Antioxidant capacity
Biological combustion involved in the respiration process and during the process of Aging
produces harmful intermediates called reactive oxygen species (ROS). Excess ROS in the
body can lead to cumulative damage in proteins, lipids, and DNA, resulting in so-called
oxidative stress. Oxidative stress, has been suggested to be the cause of aging and various
diseases in humans (Badithe et al) . Hence, there is a need of novel antioxidant compound
which can be derived from Cyanobacteria, Plants, Algae and other microbes. In the present
29

work The DPPH radical scavenging capacity of the methanol extract was determined (Table
4.5). Since, it has been shown that the methanol has greater extractability of the antioxidant
compounds we have used the methanol extract of different cyanobacteria for assessing the
antioxidant potential. In the present study P.boryanum displayed greatest antioxidant
potential (30.2 % inhibition of DPPH) followed by Nostoc sp. (25.6%). A moderate radical
scavenging activity was shown by Phormidium sp. (21.8%). These results are in accordance
with a study conducted by Abd El-Baky et al (2008), where pronounced antioxidant activity
was displayed by a crude extracts of spirulina maxima. Spirulina and its antioxidant activity
is well documented by Abd El-Baky (2003), Khan et al (2005) and Athukorela et al (2006).
Total phenolic, PC, Car and Chl-a present in the organic extract might explain their high
antioxidant activity.

30

CONCLUSION
Cyanobacteria are natural inhabitants of fresh, brackish and marine waters, producing a
diverse range of secondary metabolites. All these metabolites make this prokaryotic group
successful for the survival in vastly different habitats thereby interacting and dominating over
microbes to mammals. Several cyanobacterial secondary metabolites have significant
pharmaceutical potentials comprising a rich source of natural cytotoxic compounds.
Production of antimicrobial, anticancer, antiviral and enzyme inhibiting compounds may be
utilized for the benefit of mankind that is conspicuous aims of biomedical research. The
cyanobacteria used in this study are possible sources of antibacterial. and antioxidant
compounds since their extracts were showing antibacterial. activity against disease causing
bacteria S.epidermidis. These observations prompt the necessity for further studies of the
used cyanobacterial sp., focusing on the isolation and structure eluciadation of their
antibacterial. and antioxidant compound(s) so that the potentiality of the cyanobacteria could
be employed as therapeutic agents in managing disease associated with free radicals and also
as additives in the food or cosmetic industries.

31

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