INTRODUCTION

1.1 MARINE ECOSYSTEM

The marine environment harbours huge biodiversity and is a potential source

of marine microorganisms with extensive applications ( Beygmoradi and Homaei,

2017). The earth is mainly surrounded by the ocean (Baharum et al., 2010). The

oceanic ecosystem has been characterized based on the biotic and abiotic components.

Biotic components include microorganisms, predators, parasites, competitors and

mates. Abiotic components include physical and chemical characteristics (Turkoglu et

al., 2018).

The fundamental sources for growth and survival of plants and animals are

abundantly available in the marine environment. The major zone is the sea surface in

which the temperature, salinity, and turbidity of the seawater exhibit significant

variations. The temperature varies with the depth of the ocean. Accumulation of

nutrients will be comparatively high in the estuaries which is connected to the rivers.

The marine ecosystem is considerably different between the on-shore and off-shore

regions (Balasubramanian, 2011).

In the oceanic habitat, a wide variety of organisms survive with various

response and features, ranging from microorganism to microscopic diatoms to fishes

and these organisms are interconnected via the feeding mechanism (Shady et al.,

2012). The marine ecosystem has a huge potential for innovating antimicrobial

compounds. It hosts a rich biodiversity and supports economic activities which are

considerably useful to society (Parraga et al., 2013). The marine microorganism is

also essentially important in degradation of organic matter. The importance of a large

number of marine microorganisms is that they represent genetic diversity. The sea

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water contains 1 million microorganisms per milliliter, which includes several

thousands of microbial types (Oliver et al., 2011).

Fig:1.1 Microbe from the three universes of life interacts with the structure of the ocean

ecosystem and carbon cycle (Worden et al., 2015)

Marine microbes have a vital role in biochemical cycles, which is highly

important for the functioning of the marine ecosystem by maintaining the balance

between produced and fixed carbon dioxide. Phototrophic bacteria, diatoms, pico, and

nanophytoplankton are responsible for the production of 50% oxygen on the earth.

The marine ecosystem is a sustainable source of seafood which regulates the

aquaculture process and is also involved in bioremediation and waste management.

Global changes in the marine ecosystem will certainly affect the activities of the

marine microorganisms (Oliver et al., 2013).

1.2 MARINE BACTERIA

Marine bacteria are defined by bacteria living in a marine environment

ranging from pelagic to benthic ecosystems, in which salinity plays a vital role

(Lozupone and Knight, 2007). The essential contribution of marine microbial

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communities is in global biomass, nitrogen cycling, and biodiversity (Zinger et al.,

2011). The prokaryotic microbes lead a key role in Fe cycling to release the energy

and matter and to maintain ecosystem functioning (Jorgensen, 2000, Philippe et al.,

1999). Marine microbes are involved in the fundamental aspects of controlling the

environment based on biogeochemistry (Roman, 2012). Marine microbes are free-

living which supports research which exhibits the biogeographic pattern (Martiny et

al., 2006 ). Approaches towards the marine microbes were significantly changed by

researchers to isolate the various potential bioactive products (Jha and Xu, 2004).

Gram-negative bacteria can survive better in the rough oceanic environment

because of the cell membrane Lipopolysaccharides (LPS). The outer membrane LPS

triggers the immune response which leads to infection and even causes death (Anwar

and Choi, 2014; Wandersman and Delepelaire, 2004). Marine bacteria are found to be

a great source of novel bioactive compounds, with more than a thousand compounds

being isolated and screened for the potential to produce contemporary medicine

(Jaiganesh and Kumar, 2012).

1.2.1 Iron Metabolism

Metabolic activity of bacterium requires iron for its growth and survival. Iron

exists in two forms, either as Ferrous Fe II or insoluble ferric Fe III, but in the oceanic

environment, it is present in the ferric condition due to the neutral to alkaline pH

(Neilands, 1995). Although the bacterium requires an easily soluble ferrous form,

survival in the aerobic environment was a difficult task. However, in the aerobic

condition under certain biological pH, ferrous is oxidized to ferric iron (Neilands,

1995). Further, to overcome this condition of low iron availability and to sustain in

the aerobic condition, bacteria developed an interesting mechanism to assimilate the

iron from any given environment by secreting iron-carrying or binding molecules,

literally coined as siderophores (Neilands, 1984).

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1.3 SIDEROPHORE

Siderophore, literally coined as an iron chelating compound (Neilands, 1952),

are low -molecular weight ligands (20-2000 Da) produced to hydrolyze and

metabolize iron by bacteria, fungi and plants (Hider and Kong, 2010). The iron

ligation groups were tentatively classified into three major chemical types:

Hydroxamate, Catecholate, and Hydroxycarboxylates; However, other sideropshore

structures, including oxazoline, thiazoline, hydroxypyridinone, α- and β- hydroxy

acids and α-keto acid components, have also been resolved (Raymond and Dertz,

2004). Siderophore peptide backbone is usually made up of several analogs of

nonprotein amino acids, including modified and D-amino acids (Vassiliadis et al.,

2007). Some bacteria produce one type of siderophore while many produce multiple

types of siderophore that enable microbes to grow in different environments

(Boukhalfa and Crumbliss, 2002).

1.3.1 Siderophore Overview

Most of the organisms require iron as an essential element in a variety of

metabolic and cellular pathways due to its unique chemical properties. Iron-

containing cofactors such as iron-sulfur clusters or heme groups are found to be

present in over 100 enzymes that act in processes of primary and secondary

metabolism. Its ability to coordinate and trigger oxygen and to get ideal oxidation

chemistry for involvement in electron transport and metabolic processes makes iron

most suitable for catalyzing broad spectrum of a redox reaction (Miethke and

Marahiel, 2007; Hider and Kong, 2010).

Fe (II) is soluble in aqueous solution at neutral pH and, if the reductive state is

maintained, is, therefore, sufficiently available for living cells. In general,

omnipresent divalent metal transporters can be taken over ( Cartron, 2005; Ballouche

et al., 2009; Miethke and Marahiel, 2007). In bacteria (Cartron et al., 2006) and fungi

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(Howard, 1999; Knight et al., 2002; Haas and Keel, 2003), specific Fe (II)) uptake

systems are known. However, Fe (II) is oxidized to Fe (III)) in most microbial

habitats either spontaneously by reacting in host organisms with molecular oxygen or

enzymatically during assimilation and circulation. Fe (III) forms ferric oxide complex

in the environment. Fe (III)) forms complexes of ferric oxide hydrate in the presence

of oxygen and water with neutral to basic pH. These complexes are very stable,

resulting in a free concentration of Fe (III) (Raymond et al., 2003; Neilands, 1995).

Using multiples of membrane-bound iron siderophore receptors, iron-

coordinated siderophore (III) are accumulated by microorganisms through facilitative

transport. Iron is predominantly removed from siderophore by a redox-mediated

process, the affinity of iron (II) siderophore is much lower than that of iron (III) (Xiao

and Kisaalita, 1998). Some siderophores may be secreted to deprive competing for

iron organisms, thus influencing the ecology of the environment that the secreting

colony occupies (Hamdan et al., 1991; Leong, 1986; Joyce, 1997). The correlation

between siderophore production and virulence is strong for some microorganism

(Fernandez et al., 1998). The fundamental role of siderophore is involved in the

acquisition of high affinity and receptor-dependent ferric ion transport. Siderophore

production regulation is based on iron concentration in the environment (Crosa,

1997).

A distinct characteristic of biotechnological implementation for bacterial and

fungal siderophore was explored including, in sagriculture, medicine, pharmacology,

bioremediation, biodegradation, and the food industry (Saha et al., 2015). The

different applications found in the medical and pharmacological approach-based

literature range from iron overload to drug delivery systems and vaccines, more

recently. To gain a deeper understanding of future biotechnological innovations,

further research in siderophore production and its metabolic significance needs to be

carried out (Luis, 2017).

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1.3.2 Types of siderophores

Microbial products, especially secondary metabolites can be considered as

siderophores when they possess characteristics such as iron chelating property. The

iron level plays a vital role in the biosynthesis of siderophores and has the capability

to transport across the membrane (Winkelmann, 2002). When including secondary

metabolites, different and complex chemical structures within the siderophore are

typical, preventing their unambiguous and universal classification. Even though

siderophore biosynthesis and structural characteristics are diverse, a classification

scheme will be arbitrary to some extent. Metrics may include source organisms

(bacteria, fungi, plants), backbone character (peptide or nonpeptide, cyclic or open-

chain), or chelating group character (Drechsel and Winkelmann, 1997). Given the

considerable structural variation found in siderophores, their common feature is to

form high thermodynamic stability six-coordinate complexes with iron (III). Ligating

groups contain hydroxamate, catecholate, α-hydroxycarboxylic acids and α-keto

carboxylic acids of oxygen atoms (Roosenberg et al., 2000).

Additionally, siderophore with various binding groups of Fe (III) ions, such as

salicylic acid, oxazoline, and thiazoline nitrogen, and even negative nitrogen as in the

case of maduraferrin were isolated (Ali et al., 2011).

Extensive reviews are available on the structural variety of

siderophores(Sajeed et al., 2013). Major types of siderophores are Catecholate,

Hyroxymates, carboxylate, and mixed ligands.

1.3.3 Sources of Siderophores

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 Most of the microorganisms produce a wide range of siderophores (iron-

chelators) in iron-deficient marine ecosystems. Four significant environmental

habitats occur naturally: soil and surface water, marine water, plant tissue (pathogens)

and animal tissue (pathogen). For bacteria and fungi, the soil is a rich source of

habitat. Generally, gram-positive species are those belonging to the Bacillus,

Arthobacter, Actinomycetales and Nocardia genera. Almost all of these microbes

produce and secrete ferrioxamines that promote growth not only of the producing

organisms but also for other microbial population that can avail it. Aspergillus and

Penicillium, which mainly produce ferrichromes are soil fungi. This group of

siderophore is made up of cyclic hexapeptides and therefore is highly resistant to

environmental degradation due to the wide range of hydrolytic enzymes present in the

soil ( Winkelmann, 2007). The pH value of soils containing decaying plant materials

are as low as 3-4. Because of the extreme acid stability of these molecules, organisms

producing hydroxamate siderophore have an advantage under such conditions.

Freshwater lakes also contain large Pseudomonas, Azomonas, Aeromonas and

Alcaligenes species populations (Bossier, 1988).

Unlike most freshwater sources, surface sea-water iron levels are extremely

low (1nM to 1 μM in the upper 200 m) and much lower than those of V, Co, Ni, Cu,

and Zn. Almost all of this iron is in the state of iron (III) and complex to organic

ligands ( Rue and Bruland, 1995). The distinct nature of the marine pelagic

atmosphere promotes large diffuse losses and poses a problem with the efficiency of

normal siderophore based iron uptake strategies. Many heterotrophic marine bacteria,

however, produce siderophore, although they have different properties than those

produced by terrestrial organisms. Marine siderophores are active on the surface and

tend to form molecular aggregates (Xu et al., 2002).

1.3.4 Properties of Siderophore

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 High affinity and selectivity for ferric iron binding are important attributes that

a siderophore must meet, in order to exercise its biological function. Since negatively

charged oxygen atoms have extremely tight interactions with Fe3+, it is of no surprise

that catecholates, hydroxamates, and α-hydroxycarboxylates, each of which has two

oxygen donor atoms, are frequent elements of siderophore stimulants (Hider and

Kong, 2010). Therefore, siderophore with such a molecule is often less restrictive in

the complexation of metal ion other than ferric iron (Chaturvedi et al., 2012). The

effect of chelate maximizes the stability of complexes Fe3+ siderophore.

Spontaneously, ferric ions form octahedral Fe (H2O)6 3 + complexes in water. Since

the complete displacement of the six coordinated water molecules by single

hexadentate ligand in terms of entropy is greatly favored, many siderophores have six

possible coordinating sites. Typically, the respective donor atoms are distributed over

three bidentate ligand groups and integrated into a conformally restrained scaffold that

adopts octahedral or pseudo-octahedral geometry around the Fe3+ center (Drechsel and

Winkelmann, 1997).

Metal chelation competes with donor atom protonation and therefore depends on

the environments pH and ligand pKa values. A donor with low pKa values is more

efficient iron chelators in an acidic habitat (pH 3.0 to 5.0), where catecholates and

hydroxamates are still fully protonated. However, at a higher pH, the situation reverses

(Miethke et al., 2007). Siderophore iron affinity is also linked to the coordinated ferric

irons redox potential.

A higher pH dependent -p Fe value will be combined with a more negative redox

potential by approximation (Hider and Kong, 2010).There are two main siderophore

biosynthesis pathways one is run by a large family of modular multienzymes called

non-ribosomal peptide synthetases (NRPSs), the other is increased dramatically over

the past two decades and the enzymology of several NRPS-dependent pathways is

now well understood (Barry and Challis , 2009).

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1.3.5 Application of Siderophore

Microbial ecology is the study of microorganism relationship with their

surrounding environments. Siderophore enhances the growth in artificial media of

unculturable microorganism. The siderophore based approach has made it possible to

cultivate organisms that are only remotely related to microbes previously grown. For

many strains from this habitat, the lack of growth in the laboratory stems from an

inability to produce siderophore autonomously, and the resulting chemical

dependence on other microbes regulates community establishment in the environment

(Onofrio et al., 2010).

Siderophore involved in the growth of plant species to increase the yield by

enhancing the iron uptake by the plant. In agriculture, soil inoculation with

Pseudomonas putida, which produces pseudobactin, increases the growth and yield of

different plants (Kloepper et al., 1980). Powell et al., 1980 demonstrated the presence

of different varieties of hydroxamate siderophore. Siderophore type hydroxamate

present in soil plays an important role in immobilizing the metals in these regards.

Siderophore production activity plays a central role in determining the ability

of various microorganism to improve the development of plants in soil. The

mechanism for fluorescent Pseudomonas to suppress siderophore re- mediated

disease. Siderophore acts against harmful phytopathogen and holds the ability to

substitute hazardous pesticides (Duijif et al., 1993).

In the Medical field, Siderophore uses the Trojan strategy to form complexes

with antibiotics and helps in the selective delivery of antibiotics to the antibiotic-

resistant bacteria. Certain iron overload diseases, for example, Sickle cell anemia

(Silliman et al., 1993) can be treated and Deferiprone and Desferrioxamine combined

chelation therapy of thalassemia major disease can also be treated (Ricchi et al.,

2010).

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 Reversed siderophore also possess an antimalarial activity. The synthetic

ferrichromes antimicrobial activity correlated with their lipophilicity, and this

antimalarial activity was prevented when applied as iron (II) complexes to the

chelators. Synthetic ferrichrome action sites are located in the intraerythrocytic

parasite and not in the serum or on normal components of erythrocytes. The agents

have been effective against all parasite growth. (Shanzer et al., 1991).

1.4 MAJOR SIDEROPHORE-PRODUCING BACTERIA

Ustilago sphaerogena were grown in the iron-deficient medium for the

isolation of hemoprotein and ferrichrome was termed as ferric hydroxamate (Neilands

et al., 1952). Media optimization of siderophore production by using plant growth

promoting bacteria under the iron-limiting condition with temperature and pH

variation among 12 different strains were used to screen the maximum yield for

siderophore. Among the 12 different stains 7 isolates maximum yield of siderophore

to 85% siderophore units S11 isolate are efficient to obtain the maximum yield up to

96% siderophore units (Tailor and Joshi ,2012).

Pseudomonas flurosces and P.putida will produce hydroxamate siderophore

under the iron limiting condition in the modified succinate medium the hydroxymate

siderophore yields up to 87% and 83% siderophore units were recorded. Sunflower oil

was found to be suitable for siderophore production in case of bioreactor. Urea was

considered to be an optimum yield of siderophore production (Sayyed et al., 2005).

Siderophore optimization was statistically designed to Placket-Burman design (PBD)

method (Marathe et al., 2015).

Optimization of siderophore production using Bioreactor for maximum yield

was 68.41% obtained using the modified succinate medium with two statistical

methodologies ( Shaikh et al., 2016). Xylella fastidiosa siderophore-producing

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bacteria were also tested experimentally in the iron-limiting condition in MM9

medium (Stenico et al., 2005) Pseudomonas putida under optimum condition was

standardized by response surface methodology by using MM9 medium with the

different conditions of carbon, nitrogen, and amino acid sources (Murrugappan et al.,

2012).

1.5 VIBRIO CAMPBELLII

Vibrionaceae family are widely distributed in the marine environment

comprising of several species which cause diseases to human beings and animals

(Andrus et al., 1983). Subsequently, Vibrios have a profound ability to produce

bioactive secondary metabolites (Mansson et al., 2011). Like other pathogenic

bacteria, Vibrios also require iron for their growth (Mey et al., 2005).

Vibrio campbellii is a marine gram-negative, a curved rod-shaped bacterium

closely linked to its sister species, Vibrio harveyi. In aquatic organisms, it is an

emerging pathogen (Orata and Hedreyda, 2011). Qurom sensing enables the

bacterium to communicate with autoinducers, a secreted chemical signalling

molecule. Some V.campbellii strains are known not to be luminescent; these strains

are believed to be less virulent than the luminescent strains (Suadee et al., 2007).

1.5.1 Gene Properties

Siderophore is synthesized and secreted in their environment where they

chelate ferric iron to overcome hunger. Once bound, the siderophore receptor

recognizes the ferric iron-siderophore complexes and are transported via ABC

transporters over the membrane using TonB complexes as energy transducers

(Hibbing et al., 2010).

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Fig.1.2:The organization of Vibrionaceae siderophore clusters of biosynthesis and the
corresponding siderophore schematic structure.a)Biosynthesis clusters of
Vibrionaceae and carboxylate and siderophore.b)Catecholatae Vibrionanaceae
and biosynthesis cluster mixed catechol/hydroxamate siderophore c)
Vibrionaceae siderophore with known biosynthesis gene cluster representation of
the schematic 2D structure. (Thode et al., 2018).

Vibrionaceae siderophore biosynthesis and siderophore receptors and

phylogenetic analyses were used to investigate their organization, distribution, origin

and evolution. In literature search nine different siderophore biosynthesis systems

were identified and 13 siderophore receptors in Vibrionaceae was found. Blast

searches identified homologs and mapped the results to the phylogeny of

Vibrionaceae and identified 81 biosynthetic system in 45 species of Vibrionaceae and

16 unclassified strains of Vibrionaceae and 409 receptors in 89 species of

Vibrionaceae and 49 unclassified strains of Vibrionaceae (Thode et al., 2018).

Most taxa are associated with at least one type of siderophore biosynthesis

system, some of which are widely distributed in the family (e.g., aerobactin and

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vibrioferrin), while others (e.g., bisucaberin and vibriobactin) are found in one

lineage. More widespread receptors cognate are found. Three siderophore system in

phylogenetic analysis are piscibactin,vibrioferrin, and aerobactin.

1.5.2 Gene Studies

Protein or fur like proteins which regulates iron uptake in a variety of bacterial

species by fur (ferric uptake regulation) (Raymond et al., 2003). Since Hantke's

discovery in 1981, Fur has been coined as the ubiquitous iron regulator protein as it

was found to be a transcriptional repressor in model bacteria with more than 90

different genes, one of which is from E.coli ( Wexler et al., 2003).

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1.6 AIM & SCOPE OF THE STUDY

Very few siderophores have been isolated from the marine environment

compared to its terrestrial counterpart. Moreover, siderophores are highly efficient

drug-delivery agents, plant-growth promoting agents, biosensors, etc. and also have

high prospects in the field of bioimaging and development of nanocomposites for

treatment of various ailments. Therefore, the present study was chosen to isolate

siderophores from the marine bacteria towards the evaluation of bioactive potential in

medical and/or industrial applications.

1.7 OBJECTIVES

1. Isolation of siderophore-producing bacteria from the marine

environment.

 Sample collection

 Isolation of Total Heterotrophic Bacteria

 Screening for Siderophore-producing bacteria using detection assays

 Biochemical & Molecular identification

2. Identification of appropriate media for Siderophore production.

3. Standardization of optimum growth factors for maximum Siderophore

yield.

4. Isolation and characterization of candidate molecules.

5. Determination of bioactive potential of isolated siderophores.

6. Synthesis of Phenolate siderophore.

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 REVIEW OF LITERATURE

The objective of this review is to peruse the available literature to explain the

importance of iron for bacterial growth and virulence, structure and general contour of

the iron assimilation system, the role of siderophores in a marine ecosystem with

special reference to marine bacteria and the bioactive potential and application

quotient of siderophores.

2.1 Marine Bacteria

Marine microorganisms make up about 70 % of marine biomass. As they act

as decomposers, they are crucial for nutrient recycling in ecosystems. A small

proportion of microorganism is pathogenic, causing disease and even death in plants

and animals. Microbial marine systems drive changes in every global system as

inhabitants of the largest environment on earth (Yinon et al., 2018).

Marine bacteria was collected from water, mud, fish, shark, and one island

during the RV Anton Bruun’s eighth cruise in the Indian Ocean. Marine samples from

the depth ranging from 100 to 3,050 m were collected. A total of 127 isolates were

classified by genus and their source was associated with the physiological response of

the culture to 17 diagnostic tests. The isolates physiological test response, irrespective

of the genus, was more closely correlated with the culture source than the genera

( Johnson et al., 1968).

For the discovery of structurally diverse secondary metabolites, marine

actinobacteria continue to be a rich source. A new hydroxymate siderophore produced

by Amycolatopsis albispora, is a recently described species of this less explored

actinomycete genus. The new siderophore, designated albisporachelin, was isolated

from iron limiting medium and the structure was established by 1D and 2D NMR and

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MS/MS experiments, and application of a modified Marfey’s method. Albisporachelin

is composed of one N-methylated-formylated/hydroxylated L-ornithine (N-Me-fh-L-

Orn), one L-serine (L-Ser), one formylated/hydroxylated L-ornithine (fh-L-Orn) and a

cyclo-N-methylated-hydroxylated L-ornithine (cyclo-N-Me-h-L-Orn) (Wu et al.,

2018).

2.2. Iron Metabolism

Pathogen ability to obtain iron from their host transferrins, ferritin,

haemoglobin, and other iron-containing protein is central to whether they are living or

dying. Animals go into an iron-with holding mode to fight invading bacteria and also

use a protein (Nramp1) to produce reactive oxygen species in an attempt to kill the

pathogen (Ratledge and Dover, 2011).

Fig.2.1: Overview of iron metabolism (Cisternas et al., 2018)

Iron is a critical nutrient for most bacterial species growth and survival. The

mechanisms by which host organisms sequester iron from invading bacteria and how

bacteria acquire iron from their environment have been given much attention.

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However, during the host immune response, iron is released under oxidative stress

conditions such as those found in phagocytic cells (Elaine et al., 2014).

Iron is essential for all organisms, but it poses toxicity problems and poor

solvency. Bacteria have developed different mechanisms to counter the problems of

their iron dependence, enabling them to achieve effective iron homeostasis under a

variety of iron regimes. Highly efficient iron acquisition systems are used in iron-

restricted conditions to scavenge iron from the environment (Simon et al., 2003).

2.3 SIDEROPHORE

2.3.1 Siderophore Overview

Siderophores are low molecular weight high-affinity ferric iron chelators

which are synthesized and secreted by many microorganisms in response to iron

deprivation. These compounds solubilize and bound iron and transport it back into the

microbial cell, usually through specific membrane receptors(Neilands, 1984).

2.3.2 Types of siderophore

Fig.2.2: Types of siderophore (Bacterial siderophore during infection ) (Holden and

Bachman, 2015)

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 The siderophore structure produced by many bacteria shows a great variation.

Generally speaking, siderophores are classified based on coordinating groups that

chelate the Fe (III) ion. Catecholates, hydroxamates, and carboxylates are the most

common coordinating groups (Ali and Vidhale, 2013). A group of siderophores,

including salicylic acid, oxazoline, or thiazoline nitrogen, have chemically distinction

binding group Fe (III). Some siderophores, including pyoverdines, are classified as'

mixed ligands' with coordinating groups that fall into various classes of chemicals.

Electrophoretic mobility, spectrophotometric titration, proton have identified

dissimilar types of siderophores proton nuclear magnetic resonance spectroscopy,

mass spectrometry, acid hydrolysis, and biological activity.

Catecholate

Siderophore displaying phenolate or binding groups of 2,3-dihydroxybenzoate

(DHB) belongs to the siderophore type of catecholate. Catechol, also known as

pyrocatechol or 1,2-dihydroxybenzene, is a molecular formula C6 H4 (OH)2 organic

compound. It is the ortho isomer of the three benzenediols (Page & Tigerstrom, 1988).

This colourless compound occurs naturally in trace amounts. In an iron-limited

medium, Azotobacter vinelandii produces three catecholate siderophores, namely,

tricatecholate protochelin, the dicatecholate azotochelin and the monocatecholate

aminochelin (Cornish and Page, 1995; Wittmann et al., 2001).

Enterobactin

Siderophore type of catecholate is a cyclic trimmer made up of 2,3-

dihydroxy-N-benzoylserine.Three DHB-Ser formed molecules undergo

intermolecular cycling, resulting in enterobactin. Enterobactin was the first

siderophore of tricatechol, isolated from Escherichia coli, Aerobacter aerogenes, and

Salmonella typhimurium (Ward et al., 1999). Enterobactin (Enterochelin) is produced

by the Enterobacteriaceae family, which includes all E .coli having a high affinity for

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iron. S. typhimurium, Klebsiella pneumoniae and Erwinia herbicola are also known to

produce enterobactin. Enterobactin is the strongest siderophore with the ability to

chelate iron even from a very low iron concentration environment (Raymond et al.,

2003). E. Coli enterobactin exceeds the outer membrane size limit by a molecular

weight of 669 kDa. Thus, an active transport mechanism is used to synthesize

enterobactin. In 1998, Cornish and Page observed that hyper-produced catecholate

siderophores offered chemical protection from O2 and Fe catalyzed oxidative damage.

Hydroxamate-type siderophores

Due to their production of many soil fungi (Zahneret et al., 1963 ; Sulivan &

Gara 1992 ; Schalk et al., 2011), ferrichrome-type hydroxamate siderophores are of

particular ecological interest, including symbiotic ectomycorrhizal fungi, and their

ability to mobilize iron in neutral and alkaline soils where other naturally occurring

compounds are ineffective as iron chelators due to competitive conditions (Cline et

al., 1982). Experimental evidence suggests that some plant species may receive iron

from hydroxamate siderophores. Siderophores of hydroxamate are generally produced

by fungi and belong to Zygomycotina (Mucorales), Ascomycotina (Aspergilli,

Penicillia, Neurosporacrassa) and Deuteromycotina (Fusarium dimerum). Mostly

trihydroxamates were hydroxamate siderophores, followed by dihydroxamates. None

of the reported fungal siderophores showed monohydroxamate nature.

Hexadentate, tetradentates and bidentate ligands were formed by hydroxamate

siderophores. A good correlation between groups of hydroxamate and property of

ligand was observed. Hydroxamate ferrichromes are either linear or structurally

crystal. Some derivatives of ferrichrome (ferrioxamines) exhibit antibiotic activity and

have been identified as ferrimycins (Bickel, 1960). The siderophore family of

hydroxamate includes rhodotorulic acid, dimerium acid, alkaline and putribactin.

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A ferrioxamines

A linear trihydroxamates siderophores are produced by Streptomyces and

Nocardia with a molecular formula of C25H48N6O. These are employed therapeutically

in the treatment of thalasemia to bind excess blood iron (Timothy et al., 2014). It is on

the World Health Organization’s List of Essential Medicines, a list of the most

significant medication needed in a basic health system WHO Model List of Essential

Medicines (World Health Organization, 2013).

Ferrichrome

A ferrichromes are large family of hydroxamate siderophores. These are

siderophores of cyclic trihydroxamate. Ferrichromes are cyclic hexapeptide

siderophores composed of three N-acyl-N-hydroxyl-L-ornithine, two variable amino

acids (alanine, serine, or glycine), and a glycine linked by way of peptide bonds. At

may be acetyl, malonyl, trans-β-methylglutaconyl, trans-anhydromevalonyl, and cis-

anhydromevalonyl, five acyl groups. In each ferrichrome produced by Basidomycetes,

the acyl groups are homogeneous, except for the asperchromes synthesized by

Aspergillus ochraceous (Jalal and Van der Helm, 1991; Renshaw et al., 2002; Ali et

al., 2011). Ferrichrome produced by the Ustilago sphaerogena fungus was the initial

siderophore to be isolated and proved to be a factor of growth for other microbes

(Neilands, 1981).

Aerobactin

Aerobactin is an iron-chelating (siderophore) bacterial agent found in E. Coli

with C22H36N4 O13 (Neilands, 1995; Johnson et al., 1988) molecular formula. This

hydroxamate siderophore is an exogeneous siderophore of Pseudomonas (marine

origin), K. pneumonia, A. aerogenes, E. coli and other bacteria (Buyer et al., 1991). It

is also factor of virulence in iron limiting environments.The Pseudomonas sp. under

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iron-limiting conditions produces Aerobactin, a siderophore dihydroxamate

previously found only in the Enterobacteriaceae family.

Carboxylate-type siderophore

Rhizoferrin

A novel siderophore class whose members do not possess hydroxamate or

phenolate ligands but rather binds iron through hydroxyl carboxylate and carboxylates

(Schwyn and Neilands, 1987). Rhizoferrin consists of symmetrically acylated

diaminopropane with citric acid via amine bonds to the citric acid terminal

carboxylate (Drechsel et al., 1992). These siderophores are also found in the realm of

fungi in the bacteria kingdom. Rhizoferrin is the only known fungi-produced

carboxylate siderophore, specifically synthesized by zygomycetes members.It also

includes rhizobacteria, staphyloferrin and rhizopherrin (Stephan et al., 1996).

Interestingly, both fungi and bacteria produce rhizoferrin, only R-rhizoferrin is

produced by fungi, while S-rhizoferrin is produced by a few bacteria (Munzinger et

al., 1999).

Siderophore with mixed ligand

A part from the above siderophores, certain siderophores have the mixed

ligands of lysine, ornithine and histamine derivatives (Sah and Singh, 2015).

2.3.3 Sources of Siderophore

Different types of microbes, including various enteric bacteria, human, animal

and plant pathogenic bacteria and fungi are natural sources of Siderophore. The

detection and determination methodology of Siderophores has been discussed in detail

by Neilands (1984).

21
2.3.4 Properties of Siderophore

In our daily lives, plants and microbes are of enormous importance. Iron is

said to be the fourth most abundant element in the earth's soil crust, yet many plants

face problems with iron uptake because it is found in an insoluble form that severely

restricts this metal's bioavailability. Microorganisms in the soil such as Pseudomonas

sp., Enterobacter sp., and Bacillus sp., are respond to siderophore.

Many of the studies are focused on the microbial iron chelators, called

siderophores, facilitating chemistry. Initial research (the early 1970s) focused on

simple siderophore analogs that included ligands of hydroxamate, catecholate, or

hydroxycarboxylate. The subsequent work focused was increasingly on the transport

of siderophores and their transport of microbial iron. While these are pseudo-

octahedral complexes that are often made up of bidentate ligands, there is chirality in

the metal center that is independent of ligand chirality in principle. In many cases,

chiral recognition of the complex has been shown to occur. In Gram-positive (single

membrane) and Gram-negative (double membrane) bacteria many techniques were

used to elucidate the iron uptake processes (Hider and Kong, 2010).

Siderophore recognition and transportation generally involves receptors that

recognize the iron-siderophore complex's metal chelate portion. A second mechanism

called the siderophore shuttle, less commonly found to date, involves the receptor

binding an aposiderophore (Raymond et al., 2015).

Since the strength of their competing siderophore complexes is one of the

primary ways microbes compete with each other for iron stores, characterizing the

solution thermodynamics of these species is important. Since the acidity of

siderophores varies considerably, only the constant stability does not provide a direct

measure of the relative competitive strength of the binding.

22
 The pM value is therefore compared. The pM, like pH, is a measure of the free

metal ion concentration's negative log, typically calculated at pH 7.4, and the standard

total metal and ligand concentrations. Characterizing the ferric siderophores electronic

structure has done a great deal to help in explaining the high stability of these

complexes.

With the interpretation about what are now called siderocalins, a new phase in

siderophore science has emerged. These proteins initially found as a protein of the

human innate immune system to bind both ferric and aposiderophores to inactivate the

siderophore transport system and thus deny iron to an invasive pathogenic microbe.

Siderocalins can also play a part in the host's iron transport (Wilson et al., 2016 and

Correnti and Strong, 2012).

2.3.5 Application of Siderophore

Varoius studies explore the important roles and applications of siderophores in

various sectors including ecology, agriculture, bioremediation, biosensors, and

medicine.

In the medical field

Siderophores have a large number of applications in the medical sciences

substituted derivatives. The most important application is to defeat drug-resistant

bacteria through selective drug delivery, a Trojan horse strategy. It is the potentially

powerful application that uses siderophores iron transport capabilities to transport

drugs into cells through siderophore drug conjugate. Other important clinical

applications include treatment of diseases such as haemochromatosis, thalassemia,

encephalopathy with dialysis, removal of transuranic elements such as aluminum and

vanadium, and antimalarial activity (Nagoba and Vedpathak 2012).

23
 A new siderophore, bisucaberin, was produced by Alteromonas haloplanktis

strain SB-1123 isolated from deep-sea mud. Bisucaberin made tumor cells susceptible

to cytolysis mediated by peritoneal murine macrophages caused by Protease peptone

and not yet activated by lymphokine. Ferric ion specifically inhibited bisucaberin

activity. In the absence of macrophages, bisucaberin showed direct cytostasis for

tumor cells but did not cause cytolysis. Bisucaberin cytostasis was attributed to the

specific inhibition of tumor DNA synthesis (Kaymeyama et al., 1987).

Antimalarial activity

FR160, a chelator of catechol iron, and tetracyclines or norfloxacin perform in

vitro additive or synergistic activity against a clone of Plasmodium falciparum

resistant to chloroquine. FR160 shows macrolide, ofloxacin, and rifampin antagonistic

effects (Paradines et al., 2002)

In the agricultural field

Microbial siderophore provides the plant with Fe nutrition to improve their

growth metabolism when the bioavailability of iron is low (Crowley, 2006). The

complete process of iron uptake is still unknown.Two possible mechanisms for plants

to obtain Fe from microbial siderophores have been suggested: Microbial

siderophores with high redox potential can be reduced to donate Fe(II) to the plant's

transportation system. It was hypothesized in this mechanism that the microbial

Fe(III)–siderophores are transported to the plant root apoplast where siderophore

reduction can occur (Mengel, 1995).Fe(II) is therefore trapped in the apoplast,

resulting in high levels of Fe in the root (Kosegarten et al., 1999).In another

mechanism microbial siderophores may chelate Fe from the soil and then interact with

phytosiderophores (Masalha et al., 2000). This mechanism depends on several

parameters, i.e. constants of stability and concentrations of microbial and

24
phytosiderophores as well as pH and redox conditions of the root environment

(Crowley, 2006).

It has been suggested that Siderophores be an environmentally friendly

alternative to dangerous pesticides (Schenk et al., 2012). For more than three decades,

it has been known that different species of Pseudomonas can improve plant growth by

producing siderophores (pyoverdines) and/or protecting them from pathogens, and

thus this group of bacteria has been classified as plant-growth promoting bacteria

(Kloepper et al., 1980). It is also possible to use mycorrhizal fungi as biofertilizers to

boost plant growth. It has been shown that mycorrhizal sorghum plants have higher

levels of Fe than non-mycorrhizal plants (Caris et al., 1998). The associations of

ectomycorrhizal fungi in plant nutrition are suggested to be dependent on fungal

siderophores (Van Scholl et al., 2008). Recently, fungi-friendly plant growth activities

promoted by Aspergillus niger, Penicillium citrinum and Trichoderma harzianum

were found to increase the shoot and root lengths of chickpeas (Cicer arietinum)

(Yadav et al., 2011).

There are certain studies on the role of siderophores in biological control by

plant pathogens. Pyoverdine siderophores produced by pseudomonads have been

found to control wilt potato diseases caused by Fusarium oxysporum (Schippers et al.,

1987), in addition to being involved in the biocontrol of Gaeumannomyces graminis

causing deficiencies in the growth of wheat and barley (Voisard et al., 1987). Also,

siderophores produced by A. indica had a high affinity to chelate Fe(III) and thus had

a negative impact on the growth of several fungal pathogens (Verma et al., 2011).

Bioremediation of environmental pollutants

Siderophores are highly effective in solubilizing and increasing the mobility of

a wide range of metals such as Cd, Cu, Ni, Pb, Zn and Th(IV), U(IV) and Pu(IV)

actinides (Schalk et al., 2011). This ability of siderophores depends mainly on their

25
ligand functionality, meaning that siderophores can have a strong affinity or

selectivity with respect to a specific metal other than Fe regarding this metal-

siderophore complex's stability constants (Hernlem et al., 1999). Siderophores thus

become a useful tool for bioremediation, a technique that is cost-effective and

environmentally friendly (Rajkumar et al., 2010). One of the major environmental

issues is petroleum hydrocarbons in marine ecosystems. Microorganisms could play a

significant role in the marine environment remediation of petroleum hydrocarbons

(Das and Chandran, 2011). Petrobactin was the first structurally characterized

siderophore produced by Marinobacter hydrocarbonoclasticus, an oil-degrading

marine bacterium (Barbeau et al., 2002). Hickford et al., (2004) identified another

siderophore isolated from the same oil-degrading marine bacterium called' Petrobactin

sulfonate.

Nuclear fuel processing

Siderophore anionic hydroxamate or catecholate removes of actinides.

Desferrioxamine B forms a stable U(VI) complex where its functional group of

hydroxamate is similar to that of acetohydroxamic acid, a ligand proposed for actinide

complexation (Mullen et al., 2007). Recently, Marshall et al., (2010) reported that low

siderophore concentrations were sufficient to potentially affect the dissolution of

spent nuclear fuel and Siderophores were proposed to remediate radioactive waste and

reprocess nuclear fuel based on these studies.

Optical Biosensor

A biosensor is a biomolecule coupled with an electrical device such as a

transducer, amplifier or noise filter to increase the signal-to-noise ratio that enables

the detection of different types of responses by means of specifically designed system

(Gupta et al., 2008) Pyoverdines are yellow-green water-soluble fluorescent

siderophores which form a strong Fe(III) complex and have a weak or negligible

26
Fe(II) affinity; and the Fe(III) complexes have very high stability constants (about K=

1032) (Kurtz and Crouch, 1991; Barrero et al., 1993).

Biobleaching of pulps (Bleaching processing)

The pulp and paper industry is a primary source of many environmental

problems, including global warming, human toxicity, ecotoxicity, photochemical

oxidation, acidification, nitrification, and solid waste (Singh et al., 2008 ). The main

problem in the production of pulp and paper is the bleaching processes. Some

pollutants are emitted into the air, while others are discharged in wastewater (Bajpai,

2010). Siderophores are considered to be effective agents in the treatment of pulp,

where they can reduce 70% of the chemicals needed to bleach Kraft pulp (Bajpai,

2004), making siderophores environmentally friendly alternatives. Brown-red

microorganisms are considered as one of the major groups of wood-decaying

microorganisms.

Biomarker

A biomarker is basically a measurable indicator whose presence serves as a

sign of a phenomenon, in medicine biomarkers are used to indicate the presence,

severity, and monitoring of diseases, in pharmacology, biomarkers are used to study

drug target identification and drug response. Biomarkers are also applied in other

fields such as biofuels, forensics, and security. Biomarkers that are being employed in

fungal pathology, ecology, and classification include siderophores, fatty acids, amino

acids, carbohydrates and carboxylic acids, the most common ones are ferrichromes,

coprogens, triacetylfusarinine C, palmitic acid, oleic acid, stearic acid, glycine, L-

valine, L-serine, L-isoleucine, erythrose, D-ribose-hexitol, glycerol, a-a-trehalose,

phenylacetic acid, fumaric acid, isocitric acid and L-aspartic acid ( Aliferis et al.,

2010).

27
2.4 MAJOR SIDEROPHORE-PRODUCING BACTERIA

Pseudomonas aeruginosa ATCC 15692 in iron-limiting condition has the

ability to synthesize the two major siderophore molecules pyoverdin Pa and

pyoverdin PaB, and two other compounds has been produced during hydrolysis and

the pyoverdin PaA, during purification procedure PaC were been isolated and

structural analysis was screened through NMR assignment (Pascal et al., 1990).

The most common Fungal Siderophores include Ferrichromes, Coprogens and

Triacetylfusarinine C. Some Siderophores have the ability to remove iron from

mammalian-iron binding proteins (Neilands, 1995).

2.5 VIBRIO CAMPBELLII

The family Vibrionaceae is widespread in the marine environment. Totally 128

species of vibrios are known. For their pathogenicity or symbiotic relationships,

several of them are famed. Despite their ability to interact with eukaryotes, Vibrios

are largely explored because of their ability to produce bioactive secondary

metabolites and studies have been limited to a few species. Most of the compounds

isolated from vibrios are non-ribosomal peptides or their hybrids with examples of

nonribosomal peptide synthetases (NRPS) produced independently of N-containing

compounds.

Vibrios produce compounds with attractive biological activities, including

antibacterial, anticancer, and antivirulence activities. Many of the compounds found

in vibrios have also been isolated from other bacteria that are distantly related. This

cosmopolitan occurrence of metabolites indicates a high incidence of transferring

horizontal genes, raising interesting issues. Some of these molecules have an

ecological function. This account highlights the pending potential of exploring new

28
bioactive compounds bacterial sources and the challenges associated with their

research ( Mansson et al., 2011).

Sandy et al., 2010 reported that produces Vibrio sp. DS40M4 marine

bacterium a new triscatechol amide siderophore, trivanchrobactin and a related new

biscatecholamide compound, divanchrobactin besides previously reported

siderophores, vanchrobactin and anguibactin. Vanchrobactin is consists of L-serine,

D-arginine- and 2,3-dihydroxybenzoic acid, while trivanchrobactin is a linear trimer

of vanchrobactin joined by two serine ester linkages. Anguibactin was found to be

cytotoxic against the P388 Murine leukemia cell line (IC50 < 15 μM).

Vibrio campbellii BAA-1116 (formerly Vibrio harveyi) is a model organism

for quorum sensing producing anguibactin and amphi-enterobactin siderophore. Naka

et al., (2018) examined the mechanisms and specificity of siderophore uptake in V.

campbellii and V. harveyi, and examined the diversity of V. campbellii and V. harveyi

strains in siderophore. Electrospray ionization mass spectrometry and bioassay

revealved through that different V. campbellii and V. harveyi strains produce a series

of amphi-enterobactins with different fatty acid appendages, including several novel

amphi-enterobactins(Naka et al., 2018).

29
2.5.1 Gene Properties

Fig.2.3: KEGG Biosynthesis Pathway

Microorganisms have to tightly regulate enzymes and transport systems for the

use of siderophores that allow for concerted siderophore biosynthesis, secretion, iron

absorption by siderophore and iron release. In bacteria, gene regulation of siderophore

use and iron homeostasis, in general, is mediated by the ferric uptake repressor Fur or

the diphtheria toxin regulator DtxR mainly at the transcriptional level (Hantke et al.,

2003).

The Fur is the global iron regulator for many gram-negative(e.g., enteric

bacteria) and low-GC-content gram-positive(e.g., Bacillus spp.) bacteria. DtxR plays

a comparable role in high -GC (streptomycetes, mycobacteria, and corynebacteria)

30
gram-positive bacteria. DtxR-like proteins generally regulate manganese transport in

bacteria that regulate iron homeostasis by Fur. Although there are no obvious

sequence similarities, in terms of domain organization and metal binding, the

structural aspects of both regulators are very similar.

2.5.2 Gene Studies

Naka et al., (2018) reported that the amphi enterobactin gene cluster Vibrio

campbellii BAA-1116 harbors a gene called fapA, that encodes Fe(III)-siderophore-

specific outer membrane receptors. Also carries this cluster including fapA, V.

campbellii HY01, a pathogenic strain to shrimp.HY01-derived indicator strains show

that the FapA protein located in outer membrane fraction V. campbellii HY01 is

essential for the uptake of both Fe(III)-amphi-enterobactin and enterobactin from E.

coli, but not from V. anguillarum RV22 vanchrobactin from V. anguillarum will take

up Fe(III)-Amphi-enterobactin.V. campbellii HY01 may absorb amphi-enterobactin

via FapA, indicating that amphi-enterobactin production is a common phenotype

among V. campbellii and V. harveyi, whereas earlier work, reported . V. campbellii

strains are produced only by an anguibactin. Their results also indicated that in both

anguibactin and amphi- enterobactin which suggested that biosynthesis, the 2,3-

dihydroxybenzoic acid gene, aebA, located in the Amphi-enterobactin gene cluster,

needed for the synthesis of amphi-enterobactin may is a native siderophore of V.

campbellii and V. harveyi, while V. campbellii acquired the anguibactin system during

evolution.

31
Fig.2.4: Complete genomic studies of Vibrio campbelli ( Dong et al ., 2017)

Dias et al. (2011) isolated the bacterial strain Vibrio sps DS40M4 is a isolated

from the ocean and classified it as V.campbelli using genomic taxonomy. Their

genomic analysis revealed that V.campbelli DS40M4 harbors genes related to the

transport of iron, virulence and environmental fitness, such as those encoding

anguibactin and vanchrobactin. Protein types II, III, IV and VI Secretion system and

proteorhodopsin.

A new luciferase (Lux Vc) from V. campbellii was cloned and expressed in

Escherichia coli and purified to homogeneity. Although the sequences of amino acids

32
and Lux Vc's catalytic reactions are very similar to those of luciferase (Lux Vh) from

V. harveyi, the two enzymes have different affinities towards reduced FMN

(FMNH(-). Suadee et al. (2007) studied using Stop-flow absorbance and

luminescence spectroscopy at 4 º C and pH 8 monitored the catalytic reactions of Lux

Vc and Lux Vh. The measured Kd for the binding of FMNH(-) to Lux Vc at 4 ºC was

1.8 µM, while it was 11 µM for Lux Vh. Another difference between the two enzymes

is that, over a range of temperatures, Lux Vc is more stable than Lux Vh; the tighter

binding of make Lux Vc a more tractable luciferase for further structural and

functional studies than Lux Vh, as well as a more appropriate enzyme for some

applications.

2.5.3 Siderophore Potential

Challis (2005) reported that there are two major pathway for the

biosynthesis of siderophore molecules involved in iron scavenging pathway.

dependent Nonribosomal peptide synthases (NRPS) and independent Nonribosomal

peptide synthases (NRPS). Siderophore will possess the high affinity for iron III, the

biosynthesis of which is regulated by iron levels and function of which is to supply

iron into the cell (Hider and Kong 2009). Complete genome sequence of the

biosynthesis from Agrobacterium tumefaciens C58 gene cluster was analyzed by

Rondon et al. (2004) using stem inoculation method. Petrobactin a catechol

siderophore of Bacillus anthrax involved in iron acquisition and virulence in murine

model of anthrax. (Jung et al., 2007). Seifert et al. 1992 documented the production of

the siderophore enterobactin from Escherichia coliAN311 using Four distinct

fermentation system and The purity of enterobactin was established by HPLC method.

33
 Chemical characteristics of Siderophores of twenty fungi belonging to

Zygomycotina(5 Mucorales), Ascomycotina (7 aspergilli, 6 penicillin, Neuropora) and

Deuteromycottna (Fusarium dimerum) were examined by Dave and Dube (2000).

Siderophores produced by fungi other than Mucorales were all hydroxamates.

Mucorales produced carboxylate siderophores. Catecholate type of siderophores was

not detectable. Hydroxamate siderophores were mostly (9 out of 15) trihydroxamates

while six were dihydroxamates. Monohydroxamate nature was not shown by any of

the 15 test fungal siderophores. In ligand properties,12 out of 15 hydroxamate

siderophores formed hexadentate ligands, while two formed tetradentate and one

bidentate. There was good correlation between number of hydroxamate groups and

ligand property.

Shrimp diseases are frequently reported to be caused by closely related

vibrios, and in many cases they are tentatively but in an exact manner identified as

Vibrio harveyi and related vibrios. 28 biochemically identified V. harveyi-related

strains isolated from diseased shrimps were randomly selected for further

characterization by molecular tools. Twenty-six strains were identified as Vibrio

campbellii and two as V. harveyi by sequence analysis of 16SrRNA and uridylate

kinase genes. Hemolysin-gene-based species-specific multiplex PCR also confirmed

these results. Experimental challenge studies using Artemia as a model showed that

eight isolates were highly pathogenic, three were moderately pathogenic and the

remaining 17 were non-pathogenic (Haldar et al., 2010).

Tailor and Joshi (2012) screened 63 isolates from 12 different sugarcane field

and out of 63 isolates 12 were found to produce siderophore production. Among the

12 isolates 7 were shown to produce more than 85 % siderophore units of 7 isolates S-

11 was found to be most efficient production 96% siderophore units. Sayyed et al.

(2005) reported that two fluroscent Pseudomonas flurosces NCIM 5096 and

34
Pseudomonas putida NCIM 2847 produced maximum yield of 87% and 83%

siderophore units respectively of hydroxamate type siderophore.

Siderophore optimization was statistically designed using Placket-Burman

design (PBD) method (Marathe et al., 2015) Optimization of siderophore production

using Bioreactor was 68.41% obtained using the modified succinate medium with two

statistical methodologies (Shaikh et al., 2016). Xylella fastidiosa siderophore-

producing bacteria were also experiment tested in the iron-limiting condition in MM9

medium (Stenico et al., 2005).

Pseudomonas putida under optimum condition was standardized by response

surface methodology by using MM9 medium with the different condition of carbon,

nitrogen, and amino acid sources (Murrugappan et al., 2012). Alterobactin 1 and

Alterobactin 2 were isolated and purified from the sea water from Alteromonas

luteovionaceae (Richard et al., 1993).

35
 MATERIALS AND METHODS

3.1 COLLECTION OF SEAWATER SAMPLE

3.1.1 Sample Collection

Surface seawater was collected from coastal regions near Kovalam, Tamil

Nadu under the aseptic condition in disposable plastic containers transported to the

laboratory immediately for further processing. Samples were processed for

bacteriological analysis within 4 hours of sample collection. Samples were serially

diluted with sterile seawater and 1ml of diluted samples was transferred to Zobells

Marine Agar for bacterial isolation.

3.1.2 Chemicals

M / S Hi-Media Mumbai, Glaxo Labs Ltd, Mumbai, and Sigma Chemicals Co

obtained all the necessary chemicals. The USA Chemicals of the grade (AR / GR)

were used throughout the experiment. All reagents used for screening were freshly

prepared.

3.1.3 Cleaning of Glassware

In order to remove the trace element and other residual iron, present.The

glasswares was rinsed with distilled water and dried in the oven and was cleaned with

6 M HCL before 24 hours.

3.1.4 Isolation of Heterotrophic Bacteria

The sample was serially diluted to 4 dilutions using filtered sterile seawater

and were plated on four different types of media, i.e. Pseudomonas agar, TCBS agar,

Zobell’s marine agar, and Coliform agar. Triplicates were plated for each agar

36
medium using standard spread plate technique and incubated at room temperature for

24 -48 hours.

3.1.5 Morphological Characteristics of Isolates 1 to 48

Macroscopic properties were determined using colony characterization and

appearance. Gram staining was performed using a Gram staining kit (Hi-Media)

according to the manufacturer’s instructions.

3.1.6 Screening of Siderophore Production

Screening of siderophore production by Neilands Assay and CAS assay of

isolates 1 to 48

Qualitative analysis for detection of siderophore production using Neilands

(Atkin et al., 1970)

Qualitative analysis was carried out with culture supernatant of the test strains,

wherein 0.5ml of culture supernatant was added to 0.5ml of freshly prepared 2%

FeCl3 aqueous solution. The formation of orange colour indicated the siderophore

production potential of the test strain.

Qualitative analysis for detection of siderophore production using CAS assay

(Schwyn and Neilands, 1987).

Qualitative analysis was carried out with culture supernatant of the test strains,

where in 0.5ml of culture supernatant was added to 0.5ml of freshly prepared CAS

aqueous solution. The formation of blue to orange indicates the siderophore

production potential of the test strain.

37
3.1.7 Screening of Siderophore Production of 14 Isolates by Neilands and CAS

Assay

Quantitative analysis for Neilands and Universal CAS assay was used to

detect siderophores produced by 14 isolates. Siderophore production was tested by

using culture supernatant. The composition of Neilands assay and CAS assay was

prepared according to Schwyn and Neilands, 1987.

3.1.8 Colony characterization and Biochemical characterization for selected six

isolates based on Neilands and CAS assay

Colony characterization and Biochemical characterization of the siderophore

producing six isolates was screened using Hi Assorted Kit for Gram-negative and Hi

Bacillus identification Kit.

3.2 MOLECULAR CHARACTERIZATION OF STRAINS WITH

POTENTIAL FOR SIDEROPHORE PRODUCTION

3.2.1 DNA Extraction Protocol

Bacterial genomic DNA was extracted from six siderophore potential strains

by the phenol extraction method.

1. 15 ml of falcon tubes with grown isolates 8,30, 37,42,43, 48 were centrifuged

at 2600 rpm for 20 minutes.

2. The supernatant was stored for further biochemical analysis.
3. To the pellet 250 μl, TE buffer was added and vortexed
4. 2ml new Eppendorf was autoclaved and to it, the above 250 μl mixture was

transferred.1ml STE buffer was added and vortexed. Followed by a short

centrifugation at 8000rpm for 5 minutes at 4oC.

5. The supernatant was then discarded. The above step was repeated again
6. 50 μl lysozyme (10 mg/ml) was added and incubated for 1-hour, Mixing was

done at intervals for every 15 minutes.

38
 7. 25 μl of 10 % SDS and 20 μl of proteinase K (10 mg /ml )were added and

mixed in short vortex and incubated at 37o C for 2 hours and mixing was done

in intervals.
8. An equal volume of Tris-saturated phenol (500 μl) was added and mixed

gently for about 1 minute

9. The Eppendorf were centrifuged at 13000 rpm for 10 minutes at 4oC, The

upper aqueous phase was transferred to fresh labelled Eppendorf and stored at

2oC.

Phenol saturation was processed using 10 X TE buffer. Saturation process was

completed and 30 ml was recovered.

1. 100 μl of 1 X TE buffer (sterile ) was added and made up to 500 μl.

2. 500 μl of saturated phenol was added to the Eppendorf and gently mixed.

3. They were centrifuged again at 13000 rpm for 10 minutes at 4 oC, The upper

aqueous phase was transferred to freshly labelled Eppendorf.

4. The above step was repeated again and 500 μl CHCl3 was added in all the

Eppendorf tubes. Then it was centrifuged at 13000 rpm for 10 minutes at 4oC.

5. The upper layer was transferred to fresh labeled Eppendorf tubes and again

500 μl of CHCl3 was added similar to the previous step.

6. Totally 3 times, the CHCl3 spin was done and finally the upper aqueous layer

400 μl was collected in a fresh Eppendorf and stored at 2oC.

39
3.2.2 Agarose Gel Electrophoresis

1. Agarose 0.8 % in 1 X TAE was prepared and the mixture was heated up to

solubilize the agarose

2. The gel solution was allowed to cool to lukewarm temperature and 4-5 μl EtBr

was added.
3. The gel was poured in a gel tray and a comb with required wells were placed.
4. After the gel was set, the comb and the cellophane tape were removed. The gel

was placed in the tank filled with 1X TAE buffer.

5. 5 μl of the sample was mixed with 1 μl of gel loading dye (approximately 1

drop), mixed well and loaded in the well. The connection was set and the gel

was run at 110 amperes.

6. The gel was removed from the tray and observed under UV light. Bands were

formed.

3.2.3 Polymerase Chain Reaction Protocol

Gene fragment was amplified using the Gene Amp TM PCR system Thermal

cycler with the following primers. Primer details are as follows:

1. PCR ingredients: Reaction mix volume 25 μl

2. Taq polymerase -12.5 μl x 10 samples = 125 μl

3. Forward primers -1.2 μl x 10 samples = 12 μl

4. Reverse primers -1.2 μl x 10 samples = 12 μl

5. Water -8.1 x 10 samples =81 μl

6. Forward primer no: 27 F (5) Slot 5

7. Reverse primer no: 1525 R (1) Slot 1

Table 3.1 Observation of agarose gel electrophoresis 1st set of primers

40
 Before PCR DNA DNA samples DNA band on gel after
Well no Sample no
band on the gel for PCR DNA

1 8 Faint 3 μl No band
2 30 Very Strong 1 μl Strong band
3 37 Moderate 2 μl No band
4 42 Very Strong 1 μl Strong band
5 43 Very Strong 1 μl No band
6 48 Strong 2 μl No band

The samples were taken in the following measurement

1. The primer used for 2nd Run 27 F – Forward primer and 1488 R – Reverse

Primer

2. Sample No=8 + inc

3. Run volume = 25 μl

4. Taq polymerase -12.5 μl x 9 samples = 112 μl

5. Forward primers sample-1.2 μl x 9 samples = 10.8 μl

6. Reverse primers -1.2 μl x 9 samples = 10.8 μl

7. Water -8.1 x 9 samples =72.9 μl

Table 3.2 Observation of agarose gel electrophoresis 2nd set of primers

Well Sample Concentration of

RP Used Band
no no DNA
1 8 3 μl 1488 R(1) Strong
2 30 1 μl 1525 R (1) Strong
3 37 2 μl 1488 R(1) Strong
4 42 1 μl 1525 R (1) Strong
5 43 1.5 μl 1488 R(1) Strong
6 48 1.5 μl 1488 R(1) Strong

3.2.4 Purification of PCR Samples

The pooling of PCR samples was done in 0.5 ml microcentrifuge tubes. The

final sample volume (x) was calculated. Addition of PEG –NaCl (y) was also

41
calculated and was mixed well and kept for incubation at 37 oC for 15 minutes to 2

hours. Then it was centrifuged at 13000 rpm for 10 minutes at 4oC. The supernatant

was then discarded.

Preparation of 80 % ethanol in Milli Q water

100 μl of 80% ethanol was added to the above-derived pellet with Gentle tap.

Centrifugation was followed until the pellet was completely dried. Completion of the

drying process was by placing in an incubator for about 2 hours to a maximum for 24

hours. It was reconstituted with 20 μl double autoclaved MilliQ water. The

verification step was performed by loading 3 μl DNA in 1 % AGE with sample

loading dye. Finally purified PCR product was obtained.

3.2.5 Genomic DNA extraction and PCR amplification of 16S rRNA gene

Genomic DNA for these six bacterial isolates (NIOT/SID/8, NIOT/SID/30,

NIOT/SID/37, NIOT/SID/42, NIOT/SID/43, NIOT/SID/48) was extracted using a

DNA extraction Phenol method. PCR amplification of 16 S rRNA gene was carried

out with the bacterial consent primers (Forward primer no: 27 F Reverse primer no:

1525 R) universal forward and reverse 16S rRNA primer16. The PCR of the genomic

DNA isolate was made up to in a final volume of 25μl. The reaction mixture contains

10X PCR buffer,25 mM MgCl2,10Μm dNTP's,1 U of Taq DNA polymerase,10 pmol

of each forward and reverse oligonucleotide primers and approximately 20 ng of

genomic DNA. The amplification profile consisted of an initial denaturation at 94 o C

for 3 minutes, followed by 35 cycles at 94 o C for 1 minute, 55o C for 1 minute and 72o

C for 1 minute. This was followed by a final extension of 72 o C for 5 minutes. The

samples were held at 4o C until further sequence analysis.

3.2.6 Sequencing Protocol

42
 Single -Pass sequencing was performed using 16 S rRNA universal primers as

given below on each template. The fluorescent-labeled fragments were purified with

an ethanol precipitation method from the distinct terminators. The sample was

subjected to electrophoresis in an ABI 3730xl sequencer Applied Biosystem in

distilled water.

3.2.7 Phylogenetic Analysis

Basic Local Alignment Search Tool (BLAST) was explored for the

identification of microorganism. The 16 S rRNA sequence was identified using the

BLAST discrepancy search tool of the National Center for Biotechnology Information

(NCBI) (Dereeper et al., 2010). The Phylogenetic analysis of the sequence of blast

results was performed followed by multiple sequence alignment. Finally, the

phylogenetic tree was constructed using MEGA 5 (Tamura et al., 2004).

3.3 GROWTH AND MAINTENANCE OF VIBRIO CAMPBELLII

Vibrio campbellii (NIOT/SID/43) was obtained from surface seawater

collected from the Kovalam coastal region, East coast of India. It is motile, curved

rod-shaped, gram-negative marine bacteria. For the preparation of stock culture,

Vibrio campbellii (NIOT/SID/43) were grown in the sterile Zobell Marine Broth

(ZMB) under optimized conditions (8 days at 28°C and pH 5.5) (Soto et al., 2009).

3.3.1 Glycerol Stock

Vibrio campbellii (NIOT/SID/43) stock culture of glycerol was also prepared

and stored at –80o C. Cultures were grown in 50 ml Zobell Marine Broth (ZMB) until

OD 600nm = 0.5 -0.6- and 0.8-ml aliquots were added to 0.2 ml sterile 80 % glycerol in

2ml vials.

43
3.3.2 Preparation of Inoculum

Bacterial cultures were grown in an iron-limited chemically defined Minimal

medium (MM9; i.e. 0.3 g KH2PO4,0.5g l NaCl, 1.0g l NH4Cl, 6.0g l NaOH and

30.24 g l piperazine – N,N’-bis 2-ethanesulphonic acid, PIPES).The solutions were

prepared and supplemented with 30ml 10% (m/v) defferated casaminoacid

(contaminated iron was removed with 3% 8-hydroxyquinoline in chloroform)2.0g l

glucose ,1ml 1M Mgcl2 and 1ml 100mM CaCl2.The salt solution was autoclaved

separately (Muruggapan et al., 2011). After the incubation period, the cultures were

centrifuged at 10,000 rpm for 15 min and the cell-free supernatant was subjected to

screening for siderophore by FeCl3 test, chrome azurol sulphonate (CAS assay) and

CAS agar plate assay (Louden et al., 2011). Furthermore, the supernatant was

subjected to Ferric chloride, Arnow assay to confirm the nature of the siderophore

molecule.

3.4 OPTIMIZATION OF MEDIA AND CONDITIONS FOR

SIDEROPHORE PRODUCTION

3.4.1 Preparation of Various Growth Media

A total of 11 different media (250 ml each) were prepared separately in sterile

conical flasks (500 ml, Erlenmeyer) and was autoclaved at 121oC for 15 minutes.

After sterilization, the broth was cooled and inoculated with 500μl of V. campbellii

strain. The flasks were incubated in shaking incubator at 220rpm at 28°C. The growth

media selected for the experiment are listed below in Table 3.3.

44
Table 3.3: Various growth media used for the optimization of Siderophore
production

S.No Growth Media

1 ZMB (full strength)

2 ZMB (half strength)
Modified Succinate deferrated medium (with and without chloroform
3 extraction)Succinate medium contains grams per liter (6.0 g of K2HPO4, 3.0 g of
KH2 PO4, 0.2 g MgSO4.7H2O,1.0 g of (NH4)2 SO4,4.0g of succinic acid PH 7.0)
Iron deficient low nutrient sea water based liquid medium(IDSM){1mMFe(III)
4
fortified}
MM9 medium (with low glucose 0.08%) [(minimal salt medium) grams per litre
0.3g of KH2 PO4, 0.5g of NaCl, 1.0 g of NH4Cl,6.0g of NaOH,30.24g Piperazine
5 –N-N-bis (2-ethane sulphonic acid (PIPES), 30ml (10%m/v) defeated casamino
acid, 2.0 g of glucose and the iron contaminated iron was removed with 3% 8-
hydroxyquinoline in chloroform,1ml of 1M Mgcl2 and 1ml of 100mM Cacl2 ]
6 MM9 medium (with high glucose strength 1%)
7 BOSS medium
8 MM9 native medium (0.4% glucose)
R2A medium without Iron (0.5g of glucose, 0.5g of soluble starch,0.3g of
9
K2HPO4,0.05g of MgSo4.7H20,0.3g of sodium pyruvate)
10 R2A medium + 0.001%Fe
11 MM9 (+HEPES)

3.4.2 Estimation of growth and biomass production by V. campbellii

V. campbellii strain was inoculated on the respective 11 different growth media

plates (three replicates for each day of incubation) by spread plate technique and

incubated for a maximum of 14 days at 28°C. The plates were analysed on alternate

day of incubation (i.e. 2, 4, 6, 8, 10, 12 and 14 th day). After required incubation, the

colony forming units (CFU) of each plate was counted.

Towards biomass estimation, V. campbellii strain was inoculated in 10 mL

each of the 11 different growth media and incubated for a maximum of 14 days at

45
28°C. The tubes were also analysed every alternate day of incubation (i.e. 2, 4, 6, 8,

10, 12 and 14th day). After required incubation, the Optical Density (OD) was

measured using UV Spectrophotometer (Perkin Elmer; Sutton, 2011). Biomass was

measured directly by dry weight process after washing with distilled water (Stone et

al., 1992).

3.4.3 Estimation of Siderophore production by V. campbellii

The amount of siderophore production by V. campbellii grown on the 11

different growth media was estimated using universal chrome azurol sulfonate (CAS)

assay (Schwyn and Neilands, 1987).

3.4.4 Preparation of CAS assay solution

To a 100 ml volumetric flask, 6ml of 10mM HDTMA solution was added. To

this, 1.5 ml of iron (III) solution containing (1mM Fecl3. 6H 2O, 10mM Hcl) and 2mM

aqueous CAS Solution 7.5 ml was added slowly under stirring. Further, anhydrous

piperazine 4.307g was dissolved in water and 6.25 ml 12M hydrochloric acid was

added carefully. The volumetric flask was rinsed and buffer solution was maintained

at pH 5.6. CAS Solution was made up to 100 ml using sterile distilled water. 5-

sulfosalicylic acid was then added to the above solution as CAS shuttle solution at a

concentration of 4 mM. The solution was stored in polyethylene bottles and

maintained in the dark.

For estimation of siderophore concentration, 0.5ml of CAS solution was added

to 0.5ml of culture supernatant of V. campbellii grown in the various media. The

appearance of colour change from blue to orange indicated the presence of

siderophore. The siderophore concentration was measured using UV-visible

Spectrophotometer (Perkin Elmer) at 630 nm. The amount of siderophore produced

was measured using the following formula:

46
 Ar-As
Siderophore (SU)% =
Ar x100

where Ar = Absorbance of reference at 630 nm (CAS reagent) As = Absorbance of the

sample at 630 nm.

3.4.5 Optimization of growth conditions for siderophore production

Optimization temperature, pH, incubation period, iron and glucose

concentration preferred by these bacteria was determined by growing the cells in

MM9 broth at variable temperature, pH, incubation period, iron and glucose

concentrations. Siderophore production was determined under optimum conditions.

3.4.5.1 Optimization by Response surface methodology

Response surface methodology (RSM) is the second phase optimization of

medium composition and to find out optimum concentration and significant

component by using central composite design applied for the interaction between the

significant components and to determine the optimum levels (Box and Draper, 1987).

The steps involved such as optimum region, the response in the optimum region of

variables and estimation of the optimal condition and verification of data (Giunta et

al., 1996; van Campen et al., 1990, Toropov et al., 1996).CCD used in this study is to

find out the interaction between the significant components and also for determining

their optimum level using Quadratic model (Tanyildizi et al., 2005).

The concentration factors such as temperature, pH, incubation period, glucose,

iron. Concentrations factors were optimized by keeping other variables constant at

zero levels. Each factor has been studied in five different levels (-2,-1, 0, +1,

+2).Table 3. The experimental plan CCD with values are coded in actual forms. The

siderophore production units (SU) were measured in triplicate in 32 experimental

trials run. RSM was assessed by using analysis of variance (ANOVA) to determine

47
the significance of the factor model. Experimental data analyzed using polynomial

equation. Different factors levels were also involved.

3.5 DETECTION OF SIDEROPHORE PRODUCTION

3.5.1 CAS Agar preparation (Louden et al., 2011)

A) Blue Dye

Solution 1was prepared by 0.06 g CAS dissolving in 50ml distilled water.

Solution 2 was prepared by 0.0027 g of FeCl3.6H20 in 10ml of 10Mm HCL.

Solution 3 was prepared by 0.073 g of HDTMA in 40ml of double distilled

water. The solution 1 was mixed with 9 ml of solution 2.Then followed by solution 3,

Finally, prepared solution was blue in color. It was autoclaved and stored in a plastic

container/bottle.

B) Medium Solution

a) MM9 Salt solution stock was prepared by dissolving 15 g KH 2PO4, 25

g NaCl, NH4Cl in 500 ml of DDH2O.

b) 20%glucose stock was prepared by dissolving 20g of glucose in 100ml

DDH2O.

c) Then NaOH stock solution was prepared by dissolving 25g of NaOH

in 150ml DDH2O and pH maintained at 12 .

d) Casamino acid solution was prepared by 3 g of casamino acid

dissolved in 27 ml of DDH2O.

e) Extraction with 3% 8-hydroxyquinoline in chloroform was done to

remove any trace iron. Then it was filtered sterilized.

48
C) CAS agar preparation

MM9 Salt solution 100ml was dissolved in 750 ml ddH2O. Piperazine-N,N’-

bis (2-ethane sulfonic acid) PIPES 32.24g was dissolved PIPES will not dissolve

below pH of 5 by stirring slowly PIPES the pH will drop as PIPES get dissolved.

Then the pH was brought slowly up to 6.8 with the continuous stirring process. If the

pH exceeds 6.8 the solution will turn green. Bacto agar 15g was added and autoclaved

and cool to 50oC. After that it was followed by adding 30 ml of sterile casamino acids

solution. Then 20% glucose solution to was added MM9/PIPES mixture.100ml of

Blue dye solution was slowly added with enough agitation to mix thoroughly. The

plates were poured aspectically.

3.6 PURIFICATION OF SIDEROPHORE

3.6.1 Mass culture and separation

Mass culture of 5 litres was raised and harvested after 8 days of the incubation

period for partial purification of siderophore. The culture was centrifuged at 10000

rpm for 15 minutes. The supernatant was concentrated to 1/10 volume. The crude

extract was separated into phenol-chloroform and aqueous fractions.

Inoculating Vibrio campbelli NIOT/SID/43 strain in predetermined volume

of optimised MM9 medium was followed by harvesting after predetermined days of

incubation to obtain a culture;

1. Centrifuging the said culture in predetermined conditions to obtain

residue and supernatant wherein the said residue is discarded;

2. Concentrating the said supernatant of step(b) to predetermined volume

to obtain crude extract;

3. Partitioning predetermined volume of the said crude extract in to an

organic fraction and an aqueous fraction using predetermined volume

of Phenol: predetermined volume of Chloroform;

49
4. Extracting the said organic fraction of step(d) with distilled water in

predetermined ratio to obtain chloroform extract and aqueous extract

wherein the said aqueous extract is discarded and the said chloroform

extract is concentrated and lyophilized to obtain very small size low

polar phenolate siderophore fraction;

5. Extracting the said aqueous fraction of step(d) with ethyl acetate in

predetermined ratio to obtain ethyl acetate extract and aqueous extract

wherein the said ethyl acetate extract is discarded and the said aqueous

extract is concentrated and lyophilized to obtain polar hydroxamate

siderophore fraction.

Fig : 3.1 Extraction and purification of siderophore

50
 Fig: 3.2 Purification scheme Phenol-chloroform extraction

3.6.2 Thin layer chromatography (Leo Poorvin et al., 2011)

Thin layer chromatography (TLC) was performed on the concentrated

supernatant. Concentrated eluent (5µl) was spotted on silica gel TLC (Whatmann PE

SIL G) and run in the solvent methanol: water (70:30 v/v) after the sample run, the

plates were developed with I2 to identify for the presence organic material present.

Separated plates were developed with FeCl3 (1 % w/v in ethanol) to identify the Fe.

3.6.3 XAD-2 Column chromatography

The phenol-chloroform crude extract (A) acidified was passed through a 30x5

cm column packed with 60g of Amberlite XAD-2 (20-60 mesh) in distilled water.

Before the column was packed, the resin was stored overnight at room temperature for

water absorption. The column was then packed approximately to about 20 cm.

Acidified supernatant was washed with distilled water and collected separately. The

column was washed with the 2-bed volume of distilled water. The column was eluted

with 250 ml methanol. All together 50ml fractions were collected in five sets,

followed by washing with methanol and then distilled water to re-equilibrate the

51
column. All fractions were evaluated for siderophore screening for CAS assay. The

fraction which tested positive was concentrated to give a white powder 64.58 grams.

The concentrated white sample was redissolved in 5ml methanol and stored in -20 oC

for further purification and analysis.

3.6.4 Silica gel column Purification

The XAD-2 eluted fractions were further purified by chromatography on a

2.5x50 cm column packed with Silica gel 60-120. The phenol-chloroform crude

extract (A) was subjected to column chromatography silica gel 60-120 mesh using

hexane and ethyl acetate mixture as an eluting solvent. Approximately 96 fractions

were collected in a 5ml volume and monitored using TLC with methanol sulphuric

acid as the spray reagent. The fraction testing for positive for siderophore content was

pooled, dried by rotary evaporator and redissolved in 3ml chloroform. The amount of

siderophore was evaluated using CAS assay. The concentrated sample 114.8 mg white

powder was loaded again and further purified through the silica gel column to obtain

as pure of a sample as for as possible. Fractions were collected, tested pooled,

evaporated, and stored at -20oC until used for further purification for chemical

characterization.

3.6.5 FTIR analysis of purified siderophore

The Fourier-transform Infra Red (FTIR) spectrum was recorded for the

siderophore produced by V. campbellii isolate. After lyophilization, the siderophore

sample was pelleted with potassium bromide (KBr) and subjected to FTIR

spectroscopy (IR Affinity) for the determination of functional groups. The spectra

were recorded in the range from 4000 to 400 cm-1.

52
3.6.6 Analytical HPLC

Together with the standard DHBA, the purified samples were analyzed using

analytical HPLC (Shimadzu LC-2010 ) C18 column 250 x 4.6mm, 5µm integrated pre-

column as stationary phase with UV -visible, PDA 100) and methanol: water (0.1 %

formic acid) (70:30 ) at injection 20 µl 1 mg /ml minute at 25 º C at 254 nm and 210

nm to identify the catechol component of the siderophore type based on retention

times.

3.6.7 Preparative HPLC

The HPLC semi-preparative Shimadzu was used for siderophore separation.

LunaR 5µm Column C18 (2 LC) 250 x 10mm AP Prominent preparative liquid

chromatography PDA detector, photodiode array 190 -800 nm ) 2.5 ml per minutes

flow rate sample concentration 15mg /ml in DMSO run time 10 minutes, The mobile

phase used was 70:30 acetonitrile: water (0.1% formic acid) The wavelength range of

254nm and 210nm were measured by the purified catechol type siderophore.

3.6.8 Nuclear Magnetic Resonance analysis of purified siderophore

The 1H NMR spectra collected on an instrument equipped with a triple

resonance probe and triple axis gradients. XAD-purified siderophore sample was

dissolved in 0.75ml deuterated dimethyl sulphoxide (DMSO) and injected through a

5-mm one-dimensional NMR tube to record the nuclear magnetic resonance. The

purified sample and standards were also analyzed to investigate the chemical

structural properties. The NMR experiments were performed as standard on a 400

MHz Jeol NMR tetramethylsilane (TMS) spectrophotometer. The sample was

absorbed in a magnetic field struck by radio waves. The ubiquitous nucleus is

irradiated with a radio frequency that causes the nucleus and its magnetic field to

reverberate with relaxation. The spectrophotometer detects the signal and generates it

53
in an interpretable form of peaks. The density of the electron around the proton affects

the peak position. This is meant to refer to the chemical shift.

3.6.9 Mass spectrometry

Liquid chromatography-mass spectrometry (LC-MS) is now a routine

technique for electrospray ionization (ESI) development that provides a simple and

strong inference. It can be applied to a wide range of biological molecules and the use

of tandem MS and stable internal isotope standards makes it possible to develop a

highly sensitive and accurate test. Fast scanning speeds allow a high degree of

multiplexing and many compounds can be measured in a single analytical run.

3.7 SYNTHESIS OF SIDEROPHORE MOLECULE

3.7.1 Experimental Procedure

NH2 O NH2 O
H H
N O
SOCl2, MeOH, 0 C N
H3C OH H3C OCH3
O O
a b

NH2 O
H
BnBr, K2CO3, Acetone N
H3C OCH3 DCC, HOBt, DCM, 0OC
e
HO COOH BnO COOH O
OH OBn
c d b

O
O H2, Pd/C H
H N OCH3
N OCH3 HO N
BnO N H
H OH O CH3 O
OBn O CH3 O
e f

54
 Benzyl bromide (Sigma Aldrich) was freshly distilled before use. Purification

of the products was carried out on a short silica gel column (60-120 mesh, Merck)

using an increasing percentage of ethyl acetate in hexane as eluant. The IR spectra

was recorded on a FTIR spectrometer. The 1H/13C NMR spectra were recorded on a

Jeol 400 MHz instrument as CDCl3/DMSO-d6 solutions with TMS as internal

standard. The chemical shift values are given in (ppm) units relative to TMS.

3.7.2 Benzylation of 2, 3-Dihydroxy Benzoic acid (d)

Benzoic acid (c) (0.500 grams, 3.24 mmol) was stirred in K2CO3 (0.050 grams,

0.361 mmol) in dry acetone (10 ml) for 15 minutes. Then anhydrous benzyl bromide

(0.959 ml, 8.10 mmol) was added dropwise with constant stirring. The mixture was

refluxed for 2 hours and the progress of the reaction was monitored by TLC. After

completion of the reaction, the reaction mixture was filtered, the residue washed with

acetone and the solvent was evaporated in vacuo to give the corresponding benzyl

ethyl ether. The crude product was purified by column chromatography (Hexane %:

ethylacetate %) to yield b (114gms, 40% of yield).

3.7.3 Methylation of Ala-Phe (a)

Thionyl chloride (0.06 ml, 0.821 mmol) was added to a stirred suspension of

Alanine-phenylalanine (0.100 g, 0.423 mmol) in methanol (10 ml) at 0 °C. This was

stirred for 24 h at room temperature and the solvent was removed. The crude product

was recrystallised from EtOAc/EtOH (95:5) to give a white solid of the title

compound (88mg, 35 % yield).

55
3.8 TOXICITY STUDIES
3.8.1 Anticancer activity

MTT Assay (Mosmann et al., 1983)

Procedure:
1. Toxicity testing of VC-6 against cancer cell lines like Breast cancer cell lines

MCF-7, cervical (HeLa) and human embryonic kidney epithelial cells (HEK)

were obtained from National center for cell sciences Pune (NCCS).

2. By thawing has brought the medium and Trypsin was brought to room

temperature. The tissue culture was observed in an inverted microscope for

growth, cell degeneration, pH and turbidity.

3. After the cells become 80% confluent subculturing was done. The mouth of

the bottle was wiped off by using spirit-soaked cotton to remove the adhering

particles.

4. The growth medium was discarded 4-5 ml of DMEM Medium was added

without Fetal Bovine Serum (FBS) and rinsed gently by tilting. The dead cells

and excess FBS were washed out, and the medium was discarded. Trypsin was

added over the cells, incubated at 37º C for 5 minutes for disaggregation.

5. The cells individual were present as suspension. 5ml of 10% DMEM was

added to FBS by using a serological pipette.

6. Passaging was formed with a serological pipette. After passaging the cells

were split into 1:2 and 1:3 ratios for cytotoxicity studies by a plating method.

Anticancer activity of samples on MCF-7, Cervical (Hela) and human

embryonic kidney epithelial cells (HEK) cells were determined by the MTT (3-(4, 5-

dimethyl thiazol-2yl)-2, 5-diphenyl tetrazolium bromide) assay to assess the

cytotoxicity (Horiuchi et al.,1988).

56
 1. Cells (1 × 105 /well) were plated in 0.2 ml of medium/well in 96-well plates.

For MTT assay the medium from the wells was removed carefully after

incubation.

2. Each well was washed with DMEM without FBS for 2-3 times and 200µl of

MTT (5mg/ml) was added.

3. The plates were incubated for 6-7 hrs in 5% CO2 incubator for cytotoxicity.

4. After incubation, 1ml of DMSO (solubilizing reagent) was added to each well

and mixed well by micropipette and was left for 45sec.

5. Presence of viable cells was visualized by the development of purple colour

due to the formation of formazan crystals. The suspension was transferred to

the cuvette of a spectrophotometer and the OD (optical density) values were

read at 595nm by using DMSO as a blank.

6. The concentration required for a 50% inhibition of viability (IC50) was

determined graphically Standard Graph was plotted by taking a concentration

of the drug in X-axis and relative cell viability in Y-axis.

Cell viability (%) = Mean OD/Control OD x 100%

3.8.2 Brime Shrimp Assay (Solis et al., 1992)

Preparation of bioassay

1. Multiwell plates in filtered sterile seawater with the final concentration was

made to volume 5 ml

2. The concentration of extract for each species 1000,100.10 µg of extract per

ml

57
 3. The concentration were obtained by transferring the corresponding volume of

the stock solution in different well evaporation

4. After evaporation, 5 ml of seawater was added to each well with gentle

shaking to ensure that the compounds diffused adequately in the aqueous

solution. The extract was then subjected to the test.

Brimp shrimp hatchability test:

(NIOT/SID/43) Siderophore bioactivity based on the inhibition of hatching of

the cyst.

Brimp shrimp lethality test:

The extracts, fractions and pure isolated compounds (NIOT/SID/43) were

evaluated in a test for lethality to brine shrimp larvae, with minor modifications.

3.8.3 Siderophore Caenorhabditis elegans assay (Dengg et al., 2004)

C.elegans strain was cultured at 20º C on nematode growth medium. Assay for

lethality with C.elegans were performed with complete S- medium (Lewis and

Fleming 1995) and cultured in 24 multiwell plates. VC6 stock solution of the

compounds were made in DMSO. The experiment was carried out for one-day, where

in appropriate aliquots of the drug stock solution were added to S- medium containing

between 80 to 100 synchronized second stage larvae (L2). The final concentration of

the compound was added in the range of 0.1 -0.5 mg/ml and maximal DMSO

concentration amounted to 2%. As a negative control experiment, nematodes were

exposed to complete S-medium containing 2 % DMSO. The animals were incubated

for 24 hours at 20º C. Data was calculated from three independent experiment

58
variability. For handling and examination of the nematodes, a stereo microscope stemi

2000 C (ZEISS Germany) with a magnification of 13 to 100-fold was used.

3.9 BIOIMAGING ASSAY

3.9.1 Preparation of liposome coated siderophore nanocomposite

(Selvadoss et al., 2018)

Liposome 90 mg and cholesterol 45 mg and 0.5mg of crude siderophore were

dissolved in ethanol (approximately 2.0 mg/ml) in a round-bottomed flask containing

1 ml of chloroform. It was then condensed using rotary evaporator to remove the last

trace of chloroform evaporated. Then 2ml distilled water was added to the flask to

hydrate the dry lipid film and the mixture was gently under shaken for10 minutes. It

was then Sonicated for 2 hours in bath sonication (Fan et al., 2012). The suspension

was incubated at room temperature for overnight. A transparent upper suspension

containing liposome siderophore nanocomposite.

3.9.2 Characterization of Nanocomposite Liposome coated siderophore

The optical absorbance Nanocomposite Liposome Coated Siderophore

(NLCS) of was obtained from the UV–vis spectra (UV-1800, Shimadzu) measured in

the range of 300–1100 nm. Fluorescence spectroscopy was employed to study the

emission properties of nanocomposites. Functional groups of NLCS were analyzed

using FT-IR spectroscopy (Shimadzu, IR Affinity-1S) (Fan et al., 2012). Surface

morphology was studied using FE-SEM (Carl Zeiss SUPRA-55 microscope) by

applying an electron accelerating voltage of 5 kV for which the nanocomposite

suspension was dropped over a carbon-coated copper grid and examined after drying.

59
3.9.3 Artemia. nauplii maintenance

OSI brand Artemia nauplii cysts were procured and allowed to hatch by

decapsulation by adding sodium hypochlorite. Approximately, 2 g cysts were

incubated in 1 L of filtered seawater and incubated in aerated condition at 30 ºC for 12

hours under 1500 lux light illumination. Aeration was maintained by a small pipe line

extending to the bottom of the hatching device from an aquarium air pump. Under

these conditions, Artemia nauplii cysts hatched within 12 hours were used for

analysis.

3.9.4 Fluorescent Imaging of NCLS Using Artemia nauplii

Artemia nauplii post larvae were treated with an appropriate concentration of

NCLS for 15 min. After treatment, the organisms were examined under a fluorescence

microscope.

60
 RESULT

4.1 ISOLATION AND CHARACTERIZATION OF SIDEROPHORE-

PRODUCING BACTERIA FROM THE MARINE ENVIRONMENT

In the present study, the sample was collected from the coastal waters of

Kovalam, East coast of India, 48 bacterial isolates were identified using the spread

plate method on four different types of media, i.e. Pseudomonas agar, TCBS agar,

Zobell’s marine agar, and Coliform agar.

Morphological characterization of the 48 bacterial isolates indicated the

presence of 17 gram-positive cocci, 16 gram-positive rods and 15 gram-negative rods

(Table 4.1). Among these, few isolates exhibited a mixed culture of rods and cocci, as

well as few, were coccobacilli isolates. Further, 5 strains (NIOT/SID/27,

NIOT/SID/38, NIOT/SID/39, NIOT/SID/40 and NIOT/SID/43) exhibited

bioluminescence.

Among the 48 isolates screened for siderophore production using qualitative

analysis (Neilands assay and Universal CAS assay), 14 isolates (NIOT/SID/2,

NIOT/SID/8, NIOT/SID/9, NIOT/SID/10, NIOT/SID/14, NIOT/SID/20,

NIOT/SID/25, NIOT/SID/30, NIOT/SID/37, NIOT/SID/42, NIOT/SID/43,

NIOT/SID/46, NIOT/SID/48) tested positive (Table 4.2). Further quantitative analysis

of these 14 isolates indicated six strains (NIOT/SID/8, NIOT/SID/30, NIOT/SID/37,

NIOT/SID/42, NIOT/SID/43, and NIOT/SID/48) to have high siderophore production

potential (Table 4.3). The biochemical and morphological characterization of the six

potential isolates indicated the strains NIOT/SID/8 and NIOT/SID/37 to possibly be

Bacillus sp., NIOT/SID/30 to be Pseudomonas sp. and NIOT/SID/42, NIOT/SID/43

and NIOT/SID/48 to be Vibrio sp. or Photobacterium sp. (Tables 4.4 and 4.5).

61
Table 4.1 Morphological characteristics of the bacterial isolates from

Kovalam, East coast of India

62
Table 4.2. Qualitative screening of 48 bacterial isolates for siderophore
production potential using Neiland’s Assay and universal CAS Assay

S.NO ISOLATE NO NEILANDS ASSAY GRADE CAS ASSAY GRADE

1 NIOT/SID/1 Orange - Blue -
2 NIOT/SID/2 Light brown ++ Orange ++
3 NIOT/SID/3 Orange - Blue -
4 NIOT/SID/4 Orange - Blue -

5 NIOT/SID/5 Orange - Blue -

6 NIOT/SID/6 Orange - Blue -

7 NIOT/SID/7 Orange - Blue -
8 NIOT/SID/8 Reddish orange ++ Orange ++
9 NIOT/SID/9 Reddish orange ++ Orange ++
10 NIOT/SID/10 Reddish orange ++ Orange ++
11 NIOT/SID/11 Dark orange + Blue +
12 NIOT/SID/12 Orange - Blue -
13 NIOT/SID/13 Orange - Blue -
14 NIOT/SID/14 Light Brown + Blue +
15 NIOT/SID/15 Orange - Blue -
16 NIOT/SID/16 Orange - Blue -
17 NIOT/SID/17 Orange - Blue -

18 NIOT/SID/18 Orange - Blue -

19 NIOT/SID/19 Orange - Blue -

20 NIOT/SID/20 Reddish orange +++ Orange +++

21 NIOT/SID/21 Orange - Blue -

22 NIOT/SID/22 Orange - Blue -
23 NIOT/SID/23 Orange - Blue -
24 NIOT/SID/24 Orange - Blue -
25 NIOT/SID/25 Dark orange-red +++ Orange +++
26 NIOT/SID/26 Dark orange-red +++ Orange +++
27 NIOT/SID/27 Dark orange ++ Slight Orange ++
28 NIOT/SID/28 Dark orange ++ Slight Orange ++
29 NIOT/SID/29 Reddish orange +++ Orange +++
30 NIOT/SID/30 Reddish orange +++ Orange +++
31 NIOT/SID/31 Dark orange ++ Slight Orange ++
32 NIOT/SID/32 Dark orange ++ Slight Orange ++
33 NIOT/SID/33 Orange - Blue -
34 NIOT/SID/34 Pale orange - Blue -
35 NIOT/SID/35 Pale orange - Blue -
36 NIOT/SID/36 Pale to Dark orange + Slight Orange +
37 NIOT/SID/37 Dark orange + Slight Orange +

63
 38 NIOT/SID/38 Dark orange ++ Slight Orange ++
39 NIOT/SID/39 Dark orange ++ Slight Orange ++
40 NIOT/SID/40 Dark orange ++ Slight Orange ++
41 NIOT/SID/41 Orange - Blue -
42 NIOT/SID/42 Dark orange ++ Slight Orange ++
43 NIOT/SID/43 Dark orange ++ Slight Orange +++
44 NIOT/SID/44 Orange - Blue -
45 NIOT/SID/45 Orange - Blue -
46 NIOT/SID/46 Dark orange ++ Slight Orange ++
47 NIOT/SID/47 Orange - Blue -
48 NIOT/SID/48 Dark orange ++ Slight Orange ++
Reddish orange , +++; Dark orange, ++; Orange, - (Neilands Assay)
Blue-., Slight orange ++., Orange+++ (CAS Assay)

Table 4.3 Quantitative analysis of selected bacterial strains for determination of

siderophore production potential using Neiland’s Assay and universal
CAS Assay

Isolate No CAS % Neiland’s %

630 nm Hydroxamate (630nm) Catecholate (495 nm)
NIOT/SID/2 57 50.1 49.08
NIOT/SID/8 60 73.29 69.54
NIOT/SID/9 53 49.61 25.59
NIOT/SID/10 50 30.54 31.69
NIOT/SID/14 48 35.69 32.54
NIOT/SID/18 52 49.54 42.61
NIOT/SID/20 51 36.78 32.56
NIOT/SID/25 43 33.59 30.61
NIOT/SID/30 64 72.59 62.54
NIOT/SID/37 62 76.35 61.59
NIOT/SID/42 65 70.05 65.23
NIOT/SID/43 66 80.68 75.76
NIOT/SID/46 61 74.96 76.54
NIOT/SID/48 66 74.98 72.58

Table 4.4 Colony characteristics of six strains with potential for siderophore
production

64
Table 4.5 Biochemical characteristics of six strains with potential for
siderophore production

YC – yellow colonies

4.2 MOLECULAR CHARACTERISATION OF NIOT STRAINS WITH

PRODUCTION OF SIDEROPHORE

4.2.1 Phylogenetic tree

Towards molecular characterization of the potential isolates, Phylogenetic

dendograms were constructed using the neighbour-joining algorithm and maximum

likelihood method and processed using MEGA 5. The conditional tree topology was

evaluated with 1000 bootstrap replicates (Figs. 4.1a to f).

The six potential siderophore producing strains isolated during the current

study were thus deciphered as Bacillus aerophilus (NIOT/SID/8), Psychrobacter

celer (NIOT/SID/30), Pseudoalteromonas nigrifaciens (NIOT/SID/37),

Psychrobacter lutiphocae (NIOT/SID/42), Vibrio campbellii (NIOT/SID/43) and

Pseudoalteromonas shioyasakiensis (NIOT/SID/48; Table 4.6).

65
Fig. 4.1a. Bacillus aerophilus (NIOT/SID/8) 16S ribosomal RNA gene, partial
sequence

Fig. 4.1b. Psychrobacter celer (NIOT/SID/30) 16S ribosomal RNA gene, partial
sequence

Fig. 4.1c. Pseudoalteromonas nigrifaciens (NIOT/SID/37) 16S ribosomal RNA

gene, partial sequence

66
Fig. 4.1d. Psychrobacter lutiphocae (NIOT/SID/42) 16S ribosomal RNA gene,
partial sequence

Fig. 4.1e. Vibrio campbellii strain (NIOT/SID/43) 16S ribosomal RNA gene,
partial sequence

67
Fig. 4.1f. Pseudoalteromonas shioyasakiensis (NIOT/SID/46) 16S ribosomal
RNA gene, partial sequence

Table 4.6 Details of BLAST analysis, percentage of similarity and NCBI

accession number of potential siderophore-producing bacteria
isolated from Kovalam, East coast of India.

S. Sequence Similarity NCBI Accession

Isolate code BLAST Results
No Length % number

1 NIOT/SID/8 788 99 Bacillus aerophilus KU721010

2 NIOT/SID/30 727 99 Psychrobacter celer KU721011

Pseudoalteromonas
3 NIOT/SID/37 700 99 KU721012
nigrifaciens
Psychrobacter
4 NIOT/SID/42 728 99 KU721013
lutiphocae

5 NIOT/SID/43 707 99 Vibrio campbellii KU721014

Pseudoalteromona
6 NIOT/SID/48 657 99 KU721017
s shioyasakiensis

68
4.3 GROWTH AND MAINTENANCE OF V.CAMPBELLII NIOT/SID/43

Among the six isolates with siderophore producing potential, Vibrio

campbellii (NIOT/SID/43) exhibited maximum siderophore production and was hence

taken up for further studies. V. campbellii isolated during the current study exhibited

rich bioluminescence (Figs. 42a to c).

Fig. 4.2a. Bioluminescence exhibited by Vibrio campbellii isolated from

Kovalam, East coast of India

Fig. 4.2b. Sampling plate of Fig. 4.2c. Sampling plate of

V. campbellii in daylight V. campbellii in dark

69
4.4. OPTIMIZATION OF SIDEROPHORE PRODUCTION BY
V. CAMPBELLII (NIOT/SID/43)

4.4.1 Optimization of media for siderophore production by V. campbellii

V. campbellii exhibited significant growth on all the 11 different media

evaluated during the current study (Table 4.7). The maximum growth was observed

on MM9 native medium, followed by MM9 medium with low glucose (0.08%) and

MM9 medium with high glucose strength (1%), while ZMB (half strength) media

recorded the minimum growth of V. campbellii. During the maximum incubation

period of 14 days adopted for this study, significant differences were observed in the

growth of V. campbellii on the different media. The three MM9 media exhibited a

diauxic growth pattern (Fig. 4.3).

Table 4.7: The growth pattern of V. campbellii on various media after

incubation of 0 to 14 days

M1, ZMB (full strength); M2, ZMB (half strength); M3, Modified Succinate deferrated medium; M4,
IDSM{1mMFe(III) fortified}; M5, MM9 medium (with low glucose 0.08%); M6, MM9 medium (with high glucose
strength 1%); M7, BOSS medium; M8, MM9 Native; M9, R2A medium without Iron; M10, R2A medium with
0.001% Iron; M11, MM9 (+HEPES)

70
Fig 4.3 Diauxic growth pattern of V. campbellii grown on three different MM9
media (blue line, M5 (MM9 medium with low glucose 0.08%); red line,
M6 (MM9 medium with high glucose strength 1%); green line, M8
(MM9 Native medium)

Among the different growth media, maximum biomass of V. campbellii was

recorded on MM9 native media after 14 days of incubation (98.685 mg/mL), while

ZMB (half strength) media exhibited minimum biomass (6.42 mg/mL) even after 14

days of incubation (Table 4.8).

Among the 11 growth media evaluated for their role on siderophore

production by V. campbellii, maximum siderophore production was recorded on MM9

native media after 8 days of incubation (Fig. 4.4), while MM9 (+HEPES) media

exhibited minimum siderophore production even after 14 days of incubation. The

maximum siderophore production by V. campbellii on MM9 native medium was

observed during late log phase. The analysis of siderophore production using CAS

assay is depicted in Figure 4.4. On day 0, there was no significant difference in

siderophore production among the 11 media used (p>0.05). However, during all the

other incubation periods, there was a significant difference in siderophore production

in the 11 media (p<0.05).

71
Table 4.8 Increase in biomass concentration of V. campbellii on various media
after incubation of 0 to 14 days

M1, ZMB (full strength); M2, ZMB (half strength); M3, Modified Succinate deferrated medium; M4,
IDSM{1mMFe(III) fortified}; M5, MM9 medium (with low glucose 0.08%); M6, MM9 medium (with high glucose
strength 1%); M7, BOSS medium; M8, MM9 Native; M9, R2A medium without Iron; M10, R2A medium with
0.001% Iron; M11, MM9 (+HEPES)

Fig. 4.4. Differences in siderophore production by V. campbellii grown on 11

different growth media

M1, ZMB (full strength); M2, ZMB (half strength); M3, Modified Succinate deferrated medium; M4,
IDSM{1mMFe(III) fortified}; M5, MM9 medium (with low glucose 0.08%); M6, MM9 medium (with high glucose
strength 1%); M7, BOSS medium; M8, MM9 Native; M9, R2A medium without Iron; M10, R2A medium with
0.001% Iron; M11, MM9 (+HEPES)

72
4.4.2 Response Surface Methodology (RSM) for optimisation of conditions for

maximum siderophore production

Response surface methodology was applied to determine the optimum

concentration and significant components for maximum siderophore yield by using

central composite design. The concentration of factors such as temperature, pH,

incubation period, glucose and Fe III were studied at five different levels (-2,-1, 0, +1,

+2). The concentration of the factors was optimized by keeping other variables

constant at zero levels. The experimental range levels of the variables component by

response surface methodology using central composite design is shown in Table 4.9.

Table 4.9. Experimental range levels of the variables component by response

surface methodology for optimization of siderophore production by
V. campbellii

Variable Symbol Ranges and levels

coded
-2 -1 0 +1 +2

Temperature X1 22 27 32 37 42

pH X2 5 6.25 7.50 8.75 10.0

Incubation period X3 1 4 7 10 13

Glucose (%) X4 0 0.50 1.0 1.50 2.0

Iron µM X5 0 25 50 75 100

Xo= corresponded to the central value, Xi=X1-Xo (i= 1, 2, 3 k)

Statistical significance of the respective model equation was checked using

analysis of variance and the results are summarised in table 4.15. The "Pred R-

Squared" of 0.9191 is in reasonable agreement with the "Adj R-Squared" of 0.9838

i.e. the difference is less than 0.2."Adeq Precision" measures the signal to noise ratio.

73
A ratio greater than 4 is desirable. The ratio of 37.978 indicates an adequate signal.

This model can be used to navigate the design space.

The "Pred R-Squared" of 0.7783 is in reasonable agreement with the "Adj R-

Squared" of 0.9592 i.e. the difference is less than 0.2."Adeq Precision" measures the

signal to noise ratio. A ratio greater than 4 is desirable. The ratio of 20.227 indicates

an adequate signal. This model can be used to navigate the design space.

The equation in terms of coded factors can be used to make predictions about

the response for given levels of each factor. By default, the high levels of the factors

are coded as +1 and the low levels of the factors are coded as -1. The coded equation

is useful for identifying the relative impact of the factors by comparing the factor

coefficients. The above equation indicates a good agreement between the

experimental and predicted values for siderophore production. Actual values

experimental analysis were determined by the model equation as given in Table 4.15.

The 3D response surface graphs represent the regression equation by the

response surface plot. Interaction between four variables and their optimum levels

were found based on Response surface methodology using central composite

design,After 8 days of incubation the maximum siderophore production was obtained

at 37°C, pH 7.0, glucose 1.015 %, Iron 45 µM and eight days of incubation.

74
Table 4.10 The experimental design used in RSM studies by using five
independent variables with six center points showing siderophore
production

Factor 1 Factor2 Factor 3 Factor 4 Factor5 Response 1 Response 2

Run C:Period
E:Fe Siderophore Biomass
of D:Glucose
A: Temperature C B: pH (III) production production
incubation mg/L
M U mg/L
(in days)
1 1 -1 -1 1 1 71.53 77.03
2 1 1 -1 -1 1 87.6 20.415
3 0 0 -2 0 0 111.15 51.05
4 -1 -1 1 1 1 15 90
5 -1 1 -1 -1 -1 83.5 35.27
6 1 1 1 -1 -1 92.95 64
7 0 0 0 0 0 63.23 126.88
8 -1 -1 -1 1 -1 57.83 48.94
9 0 0 0 2 0 26.08 91
10 1 -1 1 1 -1 91.82 70
11 -1 -1 1 -1 -1 41.18 77
12 1 -1 1 -1 1 84.51 54.32
13 0 0 2 0 0 88.63 113.64
14 -1 -1 -1 -1 1 78.44 36.76
15 0 -2 0 0 0 39.37 93
16 1 1 1 1 1 23.77 48
17 -1 1 -1 1 1 28.65 40.09
18 0 0 0 0 0 62.38 122.91
19 2 0 0 0 0 60.36 8.33
20 1 -1 -1 -1 -1 35.83 42.68
21 0 0 0 0 0 62.77 126.03
22 -1 1 1 -1 1 31.46 81.33
23 0 0 0 0 0 66.5 140
24 0 2 0 0 0 12.95 58.33
25 0 0 0 0 0 61.51 132.67
26 0 0 0 0 2 40.95 82.33
27 1 1 -1 1 -1 21.53 36.05
28 -1 1 1 1 -1 3.65 93.67
29 0 0 0 0 0 70.57 140.27
30 -2 0 0 0 0 19.99 36
31 0 0 0 0 -2 44.25 111
32 0 0 0 -2 0 77.53 81
Table 4.11. ANOVA and regression analysis for siderophore production in
V.campbellii

Source SS DF MS F-Value Prob(p)>F

Model 24179.14 20 1208.96 95.19 <0.0001 significant

75
Residual error 139.71 11 12.70

Lack of fit 80.82 6 13.47 1.14 0.4510 not significant

Pure error 58.88 5 11.78

Total 24318.85 31

Std Dev: 3.56, R2 =0.9943, Mean=54.92, Adj R2=0.9838, C.V % 6.49, Pred
R2=0.9191,
PRESS 1968.08, Ad Precision =37.978.

Table 4.12 ANOVA and regression analysis for the siderophore production in
V.campbellii
ANOVA for Response Surface Quadratic model
Analysis of variance table [Partial sum of squares - Type III]
Sum of Mean F p-value
Source Squares df Square Value Prob > F
Model 24179.14 20 1208.96 95.19 < 0.0001 significant
A-A 2616.06 1 2616.06 205.98 < 0.0001
B-B 1012.31 1 1012.31 79.71 < 0.0001
C-C 657.41 1 657.41 51.76 < 0.0001
D-D 4389.94 1 4389.94 345.65 < 0.0001
E-E 8.09 1 8.09 0.64 0.4418
AB 10.00 1 10.00 0.79 0.3939
AC 3413.19 1 3413.19 268.74 < 0.0001
AD 86.54 1 86.54 6.81 0.0242
AE 209.45 1 209.45 16.49 0.0019
BC 212.65 1 212.65 16.74 0.0018
BD 2865.73 1 2865.73 225.64 < 0.0001
BE 175.36 1 175.36 13.81 0.0034
CD 6.29 1 6.29 0.50 0.4963
CE 1267.18 1 1267.18 99.77 < 0.0001
DE 259.45 1 259.45 20.43 0.0009
A^2 837.81 1 837.81 65.97 < 0.0001
B^2 2296.46 1 2296.46 180.81 < 0.0001
C^2 2694.60 1 2694.60 212.16 < 0.0001
D^2 174.18 1 174.18 13.71 0.0035
E^2 658.51 1 658.51 51.85 < 0.0001
Residual 139.71 11 12.70
Lack of Fit 80.82 6 13.47 1.14 0.4510 not significant
Pure Error 58.88 5 11.78

76
 Cor Total 24318.85 31

Std. Dev. 3.56 R-Squared 0.9943

Mean 54.92 Adj R-Squared 0.9838
C.V. % 6.49 Pred R-Squared 0.9191
PRESS 1968.08 Adeq Precision 37.978

Design-Expert ®Software
Factor Coding:Act ual
Si derophore production (U)
Designpoints abovepredictedvalue
111.15

3. 65

X1= A:Temperat ure

X2= B:pH

Act ual Factors

C:Peri odofincubation =1
D:Glucose= -1
E: Fe( II) =-1

91.2111

150
Siderophore production (U )

100

50

-50

1 1
0.5 0.5
0 0
-0.5 -0.5
B: pH A: Temperature (C)
-1 -1

77
Design-Ex pert®Soft ware
FactorC oding: Act ual
Sid erophor eproduc tion (U)
Desig nPoints
111.15

3.65

X1 =A:Temperature
X2 =B:pH

Ac tual F
ac tors
C: Periodof incubati on =1
D: Glucose = -1
E: Fe(II ) =-1

Siderophore production (U)

1

Prediction 91.2111

0.5

90

B: pH
0

60 70
80

-0.5

50

-1
-1 -0.5 0 0.5 1

A: Temperature (C)

Fig. 4.5. Central composites design for optimization of siderophore production

units by V. campbellii

Table 4.13. Coefficients analysis for the siderophore production in V.campbellii

Coefficient Standard 95% CI 95% CI
Factor Estimate df Error Low High VIF
Intercept 63.76 1 1.42 60.63 66.89
A-A 10.44 1 0.73 8.84 12.04 1.00
B-B -6.49 1 0.73 -8.10 -4.89 1.00
C-C -5.23 1 0.73 -6.83 -3.63 1.00
D-D -13.52 1 0.73 -15.13 -11.92 1.00
E-E -0.58 1 0.73 -2.18 1.02 1.00
AB -0.79 1 0.89 -2.75 1.17 1.00
AC 14.61 1 0.89 12.64 16.57 1.00
AD 2.33 1 0.89 0.36 4.29 1.00
AE 3.62 1 0.89 1.66 5.58 1.00
BC -3.65 1 0.89 -5.61 -1.68 1.00
BD -13.38 1 0.89 -15.34 -11.42 1.00
BE -3.31 1 0.89 -5.27 -1.35 1.00
CD -0.63 1 0.89 -2.59 1.33 1.00
CE -8.90 1 0.89 -10.86 -6.94 1.00
DE -4.03 1 0.89 -5.99 -2.07 1.00
A^2 -5.34 1 0.66 -6.79 -3.90 1.02

78
 B^2 -8.85 1 0.66 -10.30 -7.40 1.02
C^2 9.58 1 0.66 8.14 11.03 1.02
D^2 -2.44 1 0.66 -3.89 -0.99 1.02

E^2 -4.74 1 0.66 -6.19 -3.29 1.02

Design-Expert®Soft ware
FactorCoding:Act ual
Biomassproduction (mg/L)
140.27

8.33
X1=A: Temper ature
X2=B: pH

ActualFactors
C:Periodof incubation= 0.254207
D:Glucose= 0.0882394
E: Fe(I I)= -0.177629

101.432

160
B io m a s s p ro d u c tio n (m g /L )

140
120
100
80
60
40
20
0

1 1
0.5 0.5
0 0
-0.5 -0.5
B: pH A: Temperature (C)
-1 -1

79
Design-Expert®Soft ware
FactorCoding:Actual
Bio
ma ss prod
ucti o
n (mg/L)
140.27

8.3
3

X1= A:T
emperatu
re
X2= B:p
H

Actu
al Fac t ors
C:Periodofi nc ubation =0.2542
07
D:Glu
cose= 0 .0
88239 4
E:Fe(II I)=-0.177629

Biomass production (mg/L)

1
120
110 90

100
0.5

110

B: pH
0

Prediction 101.432

-0.5

130
110
100

-1
-1 -0.5 0 0.5 1

A: Temperature (C)

Fig. 4.6 Central composites design for optimization of Biomass production

(mg/L) by V. campbellii

80
 Table 4.14. Coefficients analysis of siderophore production by V.campbellii
Coefficient Standard 95% CI 95% CI
Factor Estimate df Error Low High VIF
Intercept 63.76 1 1.42 60.63 66.89
A-A 10.44 1 0.73 8.84 12.04 1.00
B-B -6.49 1 0.73 -8.10 -4.89 1.00
C-C -5.23 1 0.73 -6.83 -3.63 1.00
D-D -13.52 1 0.73 -15.13 -11.92 1.00
E-E -0.58 1 0.73 -2.18 1.02 1.00
AB -0.79 1 0.89 -2.75 1.17 1.00
AC 14.61 1 0.89 12.64 16.57 1.00
AD 2.33 1 0.89 0.36 4.29 1.00
AE 3.62 1 0.89 1.66 5.58 1.00
BC -3.65 1 0.89 -5.61 -1.68 1.00
BD -13.38 1 0.89 -15.34 -11.42 1.00
BE -3.31 1 0.89 -5.27 -1.35 1.00
CD -0.63 1 0.89 -2.59 1.33 1.00
CE -8.90 1 0.89 -10.86 -6.94 1.00
DE -4.03 1 0.89 -5.99 -2.07 1.00
A^2 -5.34 1 0.66 -6.79 -3.90 1.02
B^2 -8.85 1 0.66 -10.30 -7.40 1.02
C^2 9.58 1 0.66 8.14 11.03 1.02
D^2 -2.44 1 0.66 -3.89 -0.99 1.02
E^2 -4.74 1 0.66 -6.19 -3.29 1.02

Enhancement of siderophore production by Central composite design with

different level of siderophore production in V.campbellii. The final response equation

represents a suitable model for siderophore production.

Table :4.15 Final Equation in Terms of Actual Factors

Siderophore production R1 =
+63.75807
+10.44042 *A

81
 -6.49458 *B
-5.23375 *C
-13.52458 *D
-0.58042 *E
-0.79063 *A* B
+14.60562 *A* C
+2.32562 *A* D
+3.61813 *A* E
-3.64562 *B*C
-13.38313 *B*D
-3.31063 *B*E
-0.62688 *C*D
-8.89937 *C*E
-4.02688 *D*E
-5.34432 * A^2
-8.84807 * B^2
+9.58443 * C^2
-2.43682 * D^2
-4.73807 * E^2

4.5 DETECTION OF SIDEROPHORE PRODUCTION

Siderophore production of V. campbellii was detected by qualitative analysis

of Neilands and CAS assay colour change from yellow to orange for Neiland’s assay,

and blue to orange for CAS assay, Fig. 4.7a & b. The production of siderophore was

also confirmed by plating V. campbellii on CAS agar plates. After 8 days of

incubation at 37°C, the V. campbellii colonies exhibited orange colour, which

confirming its siderophore production (Fig. 4.8).

82
 Positive Negative Positive Negative

Fig. 4.7a Neilands Assay in V.campbellii Fig. 4.7b. CAS Assay in V.campbellii

Control Positive

Fig. 4.8. CAS Agar plate test in V.campbellii

4.6 PURIFICATION OF SIDEROPHORES FROM V. CAMPBELLII

(NIOT/SID/43)

Large amounts of siderophore production was obtained when the growth

conditions were optimized for maximum siderophore production. In order to purify

siderophore from Vibrio campbellii (NIOT/SID/43). The culture was grown in large

volume batch cultures. Cultures were grown under optimized media conditions for a

83
total volume of 5 liters. The bacterial culture was centrifuged and the supernatant was

collected and acidified to pH 2 using concentrated HCl. Acidifying the supernatant

helped the siderophore to bind more readily to the hydrophobic XAD-2 column by

decreasing the solubility in water.

The partitioning of crude extract of V. campbellii (NIOT/SID/43) using

phenol: chloroform yielded 43.4205 gm of chloroform extract (A) and 32.3769 gm of

aqueous extract (B). XAD column chromatography of the extract A resulted in 7

fractions using methanol (250 ml) as the eluant. The siderophore was collected in first

5 fractions and monitored by Reverse Thin Layer chromatography plate (TLC solvent

system Methanol: H2O ) profile which that indicated the chloroform fraction

contained low polar Phenolates, while the aqueous fraction was rich in polar

Hydroxamates (Fig 4.8 a & 4.8 b). There was a possibility that both catechol-type and

hydroxamate-type siderophores were eluted together from the column. In order to

separate these siderophores and to remove other cyclic compounds that might have

been eluted, further purification were carried out.

Fig 4.8 a Chloroform fraction Fig 4.8 b Aqueous fraction

84
 The XAD-2 eluted sample was further purified using a silica gel column;

however, to achieve better separation, The XAD-2 fractions containing siderophore

(fractions 2, 3,4,5, 6) were concentrated using rotary evaporator. A thick brown gum

(C) was obtained (58.5 gms.). The fraction C was column chromatographed (60-120)

using hexane: ethyl acetate as the eluting solvent. 96 fractions around 40 ml volume

each were collected. The fractions were checked for content using Reversed TLC

plate as described earlier. The fractions testing positive for each siderophore type were

pooled (catechol and hydroxamate separately) and evaporated to dryness. The

fractions 52 -94 shows a strongly positive CAS assay. Each sample was then

rechromatographed again to ensure separation of the siderophores and to obtain as

pure of a sample as possible. The same steps were as described above were followed

for the first round of purification through the silica gel column. The sample thus

purified twice through the silica gel column was used for chemical characterization.

The partially purified sample (D) was analyzed by analytical HPLC for method

development and further purified by Preparative HPLC.

cm-1
The FTIR spectrum was recorded in the frequency of 4000-410 using KBr

(Potassium Bromide) pellet. VC6-2 showed characteristic vibrational peaks at

3830.63 and 3732.26 cm-1, which can be assigned to –OH and –NH stretch

respectively. The downfield –NH stretch was indicative of amide –NH (-C=O-NH-)

group. The Infra Red peaks observed at 2976 and 2891 cm-1 are characteristic of –CH

stretch. The peak at 2306 cm-1 was due to CO2. The carbonyl (C=O) asymmetric

stretching were assigned to IR peaks at 1788, 1749 and 1687 cm-1. The peaks at 1788

and 1749 cm-1 were characteristic of ester carbonyl (-C=O-OR) and peak at 1687 cm -1

was due to the presence of (–C=O=NH-) amide carbonyl group.IR peaks at 1533cm -1

85
are attributed to (–C=C-), phenolic stretch. The peak at 1396 cm -1 is due to –OH

bending frequency and at 677 cm-1 was due to =C-H stretch (alkenes). Based on the

above inferences it was concluded that VC6-2 was a phenolic compound with an

amide functional group.

Fig: 4.9 FTIR spectrum of the catecholate siderophore VC-6

OH

C
NHR

Fig: 4.10 Phenolic compound

86
 Fig: 4.111H NMR of the catecholate siderophore VC-6

Table 4.16. 13C NMR (100 MHz), 1H NMR (400 MHz), and HMBC spectral

data of camcholate(1) in DMSO-d6

Position δC δH DEPT HMBC (H C)

DHBA
C1 163.5 C
C2 119.5 C
C3 144.5 C
C4 145.1 C
C5 124.9 6.90 CH C-4,6,7
C6 128.2 6.92 CH C-4, 5, 7
C7 128.4 7.47 CH C-1, 2, 6
Alanine
C1’ 170.4 C

87
C2’ 49.4 4.71 CH C-1’, -CH3
-CH3 18.2 1.48 CH C-1’, 2’
-NH 7.79
Phenyl alanine
C1” 176.6 C
C2” 57.8 4.88 CH C-1”, 3”
C3” 38.8 3.16, 2.91 CH2 C-1”, 2”, 4”
C4” 139.7 C
C5” 127.8 7.16 CH C-4”, 6”
C6” 129.7 7.27 CH C-5”, 7”
C7” 130.7 7.03 CH C-6”, 8”
C8” 129.7 7.18 CH C-4”, 7”
C9” 127.8 7.16 CH C-4”, 8”
-NH 7.89

Fig: 4.12 13C NMR of the catecholate siderophore VC-6

88
 6 6"
O OH
5 1"
7 O

4 H 4"
N 8"
2 1 2' 1' 2"
HO N
3 H 3"
OH O CH3

Fig. 4.13a Structure of Camcholate (IUPAC name: N-[N’-(2,3-dihydroxyl

benzoyl)- alanyl]-phenyl alanine)

Fig :4.13 b. Siderophore molecule from Vibrio campbelli

The purified siderophore molecule is a novel molecule and is being

reported for the first time from V.campbellii The IUPAC name of the molecule is

deduced to be C20H22 N2O6. Considering the novelty of the isolated molecule the name

Camcholate was proposed.

Chemical characterization of purified catecholate siderophore showed with molecular

weight,calculated mass,chemical formal were 341.34, 341.34, C22H45O2 respectively.

The chemically Synthesized catecholate molecular weight was also found to be

similar. From the above results, the siderophore purified from V.campbellii was

identified as a catecholate siderophore. Ion -exchange chromatography and

preparative HPLC yielded a pure compound that contained N- [N’-(2,3-dihydroxyl

89
benzoyl)-alanyl]-phenyl alanine. FTIR-ES-MS of iron -free and ferric form of

camcholate indicated the same elemental composition values as described earlier.

4.7 SYNTHESIS OF CATECHOLATE SIDEROPHORE

Further synthesis of catecholate siderophore were subjected to mass

spectroscopy found to be similar. Ion -exchange chromatography and preparative

HPLC yielded a pure compound with contained N- [N’-(2,3-dihydroxyl benzoyl)-

alanyl]-phenyl alanine. FTIR-ES-MS of iron -free and ferric form of synthesised

camcholate molecule indicated an elemental composition of molecular weight

341.3445, calculated mass: 341.3420, chemical formula C22H45O2. NMR was used to

confirm the molecular structure by the ES/MS data. The proton NMR resonance for

each step were compared to the known data. The chemical shifts and multiplicities for

phenolate extremely reviewed in literature. The connectivity was further elucidated by

2D-NMR technique (COSY, HSQC, HMBC, DEPT) Fig: 4.15 a - d indicating that

camcholate is a novel linear peptide consisting of 2,3 dihydroxybenzoic acid linked to

alanine- phenyl alanine.

Table 4.17. 13C NMR (100 MHz), 1H NMR (400 MHz), and HMBC spectral data

of VC3 in DMSO-d6

S. δH No. of multiplicity J Hydrogen

No. ppm Hydrogens Coupling
constant Hz
1 5.331 1H brs - -OH
2 6.772 2H d 7.6 Ar-H
3 6.851 1H t 7.6 Ar-H
4 7.164 2H t 7.6 Ar-H
brs-broad singlet, d-doublet, t-triplet

δC
S. No. Carbon
ppm
1 114.3 C-6

90
 2 119.7 C-3
3 128.6 C-4, C-5
4 154.5 C-1, C-2

Fig : 4.14. Pyrocatechol

OH

OH
pyrocatechol

Molecular Formula: C6H6O2

Molecular Weight: 110

13
Table 4.18 C NMR (100 MHz), 1H NMR (400 MHz), and HMBC spectral

data of VC 4 in DMSO-d6 NMR Analysis of VC4

S. δH No. of multiplicity J Hydrogen

No. ppm Hydrogens Coupling
constant
Hz
1 0.880 3H t 6.8 -CH3
2 1.286 2H t 6.8 -CH2
3 1.334 2H d 5.2 -CH2
4 1.374 2H brs - -CH2
5 1.412 2H brs - -CH2
6 1.678 6H brs - -(CH3)2
7 7.153 2H d 8.4 Ar-H
8 7.239 2H d 8.4 Ar-H

S. No. δC Carbon
ppm
1 152.1 C-1
2 148.9 C-2
3 148.2 C-3
4 127.9 C-5
5 120.3 C-4, C-6

91
 6 42.5 C-1’
7 33.8 C-2’
8 31.9 C-3’
9 29.7 CH3
10 29.3 CH3
11 22.7 C-4’
12 14.13 C-5’

Fig :4.15 3(methylhexane – 2-ylamino) benzene -1,2-diol

6
HO
1 5 5'

2 4'
4
HO 3'
3

HN 2'
1'

H3C CH3
3-(2-methylhexan-2-ylamino)benzene-1,2-diol

Molecular formula: C13H21NO2

Molecular Weight: 223.1

92
 Fig : 4.16 NMR spectrum d

Fig :4.17 C 13NMR spectrum d

93
Fig :4.18 HRMS Analysis VCP Final

Fig : 4.19 (a)Peptide DQF COSY

94
Fig: 4.19 (b)Peptide DEPT 135

Fig :4.19 (c) Peptide HSQC

95
4.8 TOXICITY STUDIES

Toxicity testing of VC-6 against cancer cell lines like breast cancer cells

(MDA-MB-231-S, MCF-7), cervical (HeLa) and human embryonic kidney epithelial

cells (HEK) indicated no activity even at 100 μg/mL. Further, VC-6 had no activity on

the Artemia nauplii when tested at concentrations ranging from 1 to 100 µg/mL and

against Caenorhabditis elegans when tested at 10 and 100 µg/mL.(Fig :

4.20,4.21,4.22)

Fig: 4.20 Siderophore Artemia cytotoxicity assay

96
 Fig :4.21 Siderophore Caenorhabditis elegans assay

Fig: 4.22 MTT assay VC-6 against cancer cell lines like breast cancer cells

4.9 BIOIMAGING ASSAY

Nanocomposite liposome-coated siderophore have the potential for

comparatively long term imaging, which is used for biomedical application and

detection of aquaculture pathogen. The SEM images show that nanocomposite

liposome-coated siderophore is square in shape and have a comparatively narrow size

distribution (average diameter 17.4 x 12.6 nm, Figure 4.23 a, b, c). Nanocomposites

with a small diameter are suitable for bioimaging because of the ideal contrast agent

10- 50 nm in size. The advantage of siderophore for biomedical imaging is its low

toxicity in cells as shown in figure 4.20, the MTT assay demonstrated that cancer cell

line viability did not decrease obviously after the cells had been incubated with

nanocomposites for 2 hours. The viability of the Artemia nauplii and C.elegans was

also not obviously affected by the nanocomposites. This indicates that the siderophore

may exhibit low toxicity if this material exists in cells for 3 days. In addition, the

fluorescence of Nanocomposite liposome-coated siderophore inside the Artemia

nauplii could be seen at intestine seenFig: 4.24 (a to f) For example, the fluorescence

97
of these nanocomposites in the Artemia could be captured by the in fluorescent

imaging system an in the vivo imaging system. As a control, dihydroxybenzoic acid.

4.23 (a)Control 4.23(b ) Liposome coated control

4.23.(c) Liposome coated sidreophore

Fig: 4.23 (a,b,c) Characterisation of Nanocomposite Liposome coated

siderophore by SEM Analysis

98
 Control Artemia (a) Treated Artemia (b)

Control Artemia merged cells ( c) Treated Artemia merged cells ( d )

99
 Control with DHBA ( e ) Treated with DHBA (f)

Fig: 4.24 (a to f ) Bioimaging Properties Nanocomposite Liposome coated

siderophore

In vivo bio-imaging properties of the fabricated Nanocomposite liposome

coated siderophore were investigated in NCLS. Artemia nauplii. Fig :4.24 (a to f)

show the phase contrast and fluroscent images of the organisms, respectively after

administration of NCLS. It was observed that the nanocomposites emitted brighter

near infrared fluroscence which was remarkably evident in the alimentary canal of

Artemia nauplii and was also found to be distributed throughout the body of the

organisms.

100
 DISCUSSION

5.1 IMPORTANCE OF BIOPROSPECTING MARINE BACTERIA

Marine ecosystem is the largest ecosystem on the Earth, which provides

abundant research resources, yet this domain's potential remains largely uncharted.

Being the planet⸴s largest dynamic habitat, the marine environment provides space for

many living things, inclusive of microbes, to be inhabited (Jayadev and Navami2013).

Microbes are employed in biotechnology application such as bioprospecting,

fermentation, bioremediation, medical and industrial field. An enzyme isolated from

the marine source commonly acts as a biocatalyst in the food, detergent, textile, and

industries.

Microbes associated with the marine sources have a more challenge to

isolate biological active compounds (Zhang and Kim 2010). Because they often have

unique structures, metabolic pathways, sensory and defense mechanism, marine

microbes are of great scientific interest. There are vast majority of marine microbes

yet to be identified. Hence bioprospecting plays a vital role.Various approaches have

been employed to retrieve natural bioactive compounds important in the fields of

medicine, industries, and many other processes (Lindeuist, 2016 and Datta et

al.,2015).

Innovation in the marine microbes for space and essential nutrients is a

powerful selective force that has led to evolution. With the recent development of

biotechnology, the interest and demand for enzymes with new properties have

significantly increased (Zhang and Kim 2010). More than 1000 novel bioactive

compound from marine microbes isolated and being commersialised. Many more are

under screening for their potential activity (Jaiganesh and Kumar 2012). In current

101
study was carried out by bioprospecting of marine bacterium for isolation of

siderophores molecule. Siderophore is iron chelating molecule which acts as a carrier

molecule to transport iron from the nutrient-rich environment to microorganism for

surviving in the rough oceanic environment.

Studies on siderophore production in marine microorganisms and

identification of its structure are very limited (Sandy and Butler 2009). Hence an

attempt was made to isolate and characterize the siderophore produced by isolated

bacteria from Kovalam, Coastal region of Tamil Nadu,India.

5.2 MARINE BACTERIA BETTER SOURCES OF SIDEROPHORE

The literature survey of the terrestrial environment and marine environment,

reveals that compared to terrestrial environment, microorganism from marine

environment have specific genetic structures, and with marine ecosystem. Marine life

ranges from nutrient-rich regions to the nutritionally scattered location where only a

few organisms can survive. The density is due to, high salinity, high pressure, low

temperature, and special lighting conditions. It is noted that there are substantial

differences between the enzymes produced by marine microbes and homologous

enzymes from terrestrial microbes ,subsequent in the compounds and the valuable

products produced by them (Lozupone and Knight 2007).

In particular, a considerable number of drug candidates are marine microbial

enzymes. Marine microorganism, whose enormous genetic and biochemical diversity

is still in their infancy, rich source of novel chemical compounds for the discovery of

more effective drugs (Burkhard Haefner 2003). Hence marine microorganism has a

challenging role which tends to produce secondary metabolites with high bioactive

potential. A total 48 bacterial isolates were identified in this study. Among them

102
Vibrio campbellii was found to be the bacteria it high amount of siderophore

production.

5.3 VIBRIONACEAE IN THE MARINE ENVIRONMENT

In current research, Vibrionaceae has emerged as an unexploited reserve of

biodiversity and this present study investigated the family Vibrionaceae (Vibrios) for

their potential as a new biodiversity reservoir with species appropriate to the marine

ecosystem. According to Bergey's Manual of Systematic Bacteriology belonging to

the Gammaproteobacteria, gram-negative motile rods was commonly found in the

marine environment. They were easily cultivated in Marine agar and Thiosulphate

Citrate Bile Salt (TCBS) agar.

Vibrios in aquaculture world are pathogens especially V.harveyi,

V.parahaemolyticus, V.anguillarium, V.vulnificuous, V.campbellii, etc (Chatterjee and

Haldar 2012). The present work is aimed to isolate siderophore molecule from

V.campbellii, a gram-negative, curved rod-shaped marine bacterium closely related to

its Vibrio harveyi sister species. V. campbellii is also an emerging pathogen in

aquaculture.

5.4 SIDEROPHORES FROM VIBRIONACEAE

Siderophores are isolated from gram-negative bacteria. This is due to the high lipid

bilayer content and affinity towards the oxygen virulence (Fenical and Jensen 1993).

Among the 48 bacteria isolated from the study area, 8 out of the 16gram-negative rods

exhibited slight to high siderophore production potential. Bioluminescent bacteria

have mostly been reported to produce siderophore. Bacteria belonging Vibrionaceae

family are extensive.

103
Overall literature analysis 128 species were identified and for many of them

pathogenicity are unknown. The Vibrios are largely underexplored for their facility to

produce potential secondary metabolites. Further 93 compounds have been isolated

till date (Mansson et al., 2011). Photobactin produced by luminescent bacteria

Photohabdus luminescens is in parallel association with Heterrohabditis

bacteriophora NC1 Nematodes (Ciche et al., 2003).

From Vibrio cholera, a novel siderophore was isolated which belonged to the

catecholamide iron chelating family. The structure of the new siderophore was

determined to be N-[3-(2,3-dihydroxybenzamido)propyl]-1,3- bis [2,3-

dihydroxyphenyl]-tran similar -5-methyl-2-oxazoline -4-carboxamido] propane .The

compound has been given the trivial name vibriobactin. Vibrio cholera was found to

be more responsive than enterobactin to vibriobactin, agrobactin and ferrichrome

(Griffith et al., 1984).

Vibrio nigripulchritudo produced a novel siderophore, Nigribactin. This novel

siderophore shows a high therapeutic potential against Staphylococcus aureus rather

than viability targeting virulence genes (Neilson et al., 2012). Vibrio species has

structural diversity applied among the siderophore. Two different siderophore system,

namely anguibactin and vanchrobactin are from V. anguillarum. A unique structural

type of siderophore catecholate and hydroxymate and thiazole core produce non

ribosomal peptide anguibactin. An unidentified Vibrio recently produces a dimeric

and trimeric version of vanchrobactin (Jalal et al., 1982, Sandy et al., 2010).

104
 Vibrio salmonicidia produce siderophore during iron-limited condition and is

structurally characterized as Bisucaberin. Vulnibactin is a new siderophore molecule

has been isolated from Vibrio vulnificus. Vibrio campbellii BAA-1116 is a model

organism for studying quorum sensing, producing anguibactin and Amphi-

enterobactin siderophores (Naka et al.,2018). Among the five bioluminescent bacteria

isolated in this study V.campbellii exhibited higher siderophore production potential.

This is in agreement with earlier reports which indicate that Vibrionaceae family has

high potential to produce iron chelator molecules to sustain and thrive in rough

oceanic environment.

Siderophore producing bacteria with high percentage yield in open seawater

are competitive and iron binding compounds have been isolated under iron-limiting

conditions analyzed the siderophore-producing bacteria and fungi isolates in the

marine environment of Tanoura Inlet, Japan (Korat et al., 2001, Sugita et al., 2012)

5.5 DIFFERENT TYPES OF SIDEROPHORES FROM VIBRIONACEAE

Vibrionaceae family produce three different types of siderophores such as

hydroxamates, catecholates and carboxylates. Catecholate type siderophores are

Anguibactin, Bis (3,2,3 dihydroxy-benzoyl amino) propyl-amine, Divanchrobactin,

Fluvibactin, Trivanchrobactin, Vanchrobactin, Vibriobactin, Vulnibactin.

Hydroxymate type siderophore is as bisucarberin and aerobactin.Carboxylate type

siderophore is Vibrioferrin (Mansson et al., 2011, Saito et al., 1994).Among three

types of siderophore catecholate type siderophore has been highly reported more.

Importance of catecholate and Phenolate siderophores

Catecholates have been commonly employed in the agricultural field, which act as

growth enhancement factor, and tend to produce anticancer activity. Commonly

105
siderophores act as drug carrier using Trojan Horse strategy. An important resistance

factor of bacterial pathogens is the outer membrane permeability barrier (Mollmann

et al., 2009).

5.6 OPTIMISATION OF SIDEROPHORE PRODUCTION IN VIBRIO

CAMPBELLII

The siderophore production in bacteria is mostly mediated by the nutrient source and

growth conditions, along with the genetic make-up. The growth media provide the

bacteria with most of its nutrient requirements and hence play a major role in

siderophore production, as well. Various growth media have been found to be suitable

for maximum siderophore production by different types of bacteria. For example,

Pseudomonas aeruginosa FP6 exhibited maximum siderophore production in

succinate medium (Sasirekha and Srividya, 2016). Many bacilli have also exhibited

high siderophore production using succinate media (Santos et al., 2014).

Pseudomonas flurosces and P. putida produced hydroxamate siderophore under iron

limiting condition in the modified succinate medium (Sayyed et al., 2005). The

common aspect of all these media is the low iron content in their composition.

In the current study, the growth and siderophore production potential of V. campbellii

was evaluated using 11 different media. V. campbellii is closely related to V. harveyii

and has been found to be pathogenic to aquatic organisms like shrimp (Wang et al.,

2015). Thiosulfate-citrate-bile salts-sucrose (TCBS) agar has been widely used as a

medium for isolation and enumeration of Vibrio species from marine and estuarine

ecosystems. However, it has also been found that TCBS agar is inhibitory to many of

the Vibrio species occurring in the marine environment. Subsequently, a non-

106
inhibitory medium was developed for the selective enumeration and isolation of

Vibrionaceae in seawater (Simidu and Tsukamoto, 1980, Harris et al., 1996). Later,

with increase in pathogenic outbreaks caused by V. harveyi and V. campbellii, various

efficient growth media were developed to facilitate early detection and employment of

preventive measures.

In view of the fact that the siderophore production potential of V. campbellii, along

with biomass production is envisaged, 11 different media with nil to varying

proportions of iron were selected for the present study. Only 3 [MM9 medium (with

low glucose 0.08%), MM9 medium (with high glucose strength 1%) and MM9 native

medium] out of 11media selected exhibited considerable increase in growth and

biomass production during an incubation period of 14 days, with MM9 native

medium recording the highest growth and biomass production. While all the selected

media have been previously used for enumeration of marine bacteria (Zobell et al.,

1935; Reasoner et al., 1985; Klein et al., 1998; Guan et al., 2001; Sayyed et al., 2005;

Muruggapan et al., 2012), the relatively higher growth and biomass production

exhibited by MM9 media could be attributed to their specific glucose concentrations

and diauxic growth. Glucose has been reported to cause diauxic growth in

Pseudomonas fluorescens grown in LB medium (Jonathan and Richard, 2011).

Another important constituent of MM9 media facilitating higher growth could be

casamino acid, which provides a completely hydrolysed protein nitrogen source.

Growth rates of cultured bacteria have been reported to be highest in complex media

and in media supplemented by amino acids (Marr 1991).

107
Subsequently, the maximum siderophore production was also exhibited by MM9

native medium. There are only a few earlier reports that acknowledge the siderophore-

production potential of MM9 medium. Rodriguez et al. (2012) used iron-deficient

MM9 broth to elucidate the siderophore-production in V. harveyi. The siderophore-

producing bacterium, Xylella fastidiosa was also grown in iron-limiting condition in

MM9 medium (Stenico et al., 2005) and Pseudomonas putida was standardized for

siderophore production using MM9 medium with the different concentrations of

carbon, nitrogen, and amino acid sources (Murugappan et al., 2012).

The higher siderophore production by MM9 native medium over the other two media

exhibiting relatively higher growth of V. campbellii (MM9 medium with low glucose

0.08% and MM9 medium with high glucose strength 1%) could also be attributed to

the concentration of glucose in MM9 native medium (0.4%) along with iron

depletion. The mode of action is not known clearly. Also, the relatively higher growth

and biomass production by V. campbellii grown in MM9 native medium could have

possibly resulted in higher siderophore yield. In general, the minimal media, MM9

resemble the marine environment in its composition and this could have enhanced the

biological potential and siderophore production efficiency of V. campbellii (Gram,

1996; Murugappan et al., 2012). Further, the maximum siderophore production during

late log phase by V. campbellii indicates the critical demand for iron during this

particular phase of the growth cycle of the bacterium. Similar observations have

earlier been reported for Pseudomonas putida (Murugappan et al., 2012).

Another interesting observation during the present study was the considerable

yield of siderophore (~ 48% SU) by V. campbellii grown on a BOSS medium, in spite

108
of relatively low growth and biomass production. Klein et al., (1998) has reported the

use of BOSS Medium specifically for luminescent bacteria. In this case, we have not

observed considerable growth or biomass production, but only siderophore production

was relatively high. One possible cause could be that V. campbellii exhibited

synchronous growth in BOSS medium, such that at any given point of time, all the

bacterial cells would be in the same stage of the cell cycle (Harvey, 1972). In such a

scenario, the siderophore production of the total cell population would be depicted

during a given time. Normally, the cells in culture would be in various stages of the

growth phase and therefore, the siderophore produced may be the effort of only a

certain percentage of the population.

5.7 EXTRACTION AND PURIFICATION OF SIDEROPHORE FROM

VIBRIO CAMPBELLII

Small sized phenolate siderophore has been isolated from Vibrio campbellii

for the first time by phenol: chloroform extraction method in this study. This

methodology would facilitate the isolation of small-sized phenolate siderophores,

which otherwise may be missed during normal aqueous separation. Most of the

siderophores isolated from marine bacteria are large-sized, which makes it difficult

for application as potential drug carriers and also for large-scale synthesis. The

isolated phenolate siderophore it has been successfully synthesized in the laboratory

and has exhibited promising bioactive properties. According to the literature analysis

Legiobactin siderophore has been extracted with the culture supernatant of Legionella

pneumophila by Allard et al., (2009) . The supernantant was fractionated using CM

sephadex C-25 and the fraction was further purified by HPLC and NMR

analysis.Siderophores using Alcaligenes faecalis BCCM ID 2374 showed siderophore

109
type hydroxamate and catecholate. They purified these siderophore using Amberlite

XAD-4 column (Sayyed et al., 2005).

5.8 BIOIMAGING POTENTIAL OF SIDEROPHORES

For widely used medical imaging techniques such as fluorescence, magnetic

resonance, ultrasound, and nuclear imaging, liposomes have been used as nanocarrier.

To enhance the bioavailability, stability, and performance of existing fluroscent

compounds, the optical bioimaging potential plays a vital role in the biomedical field.

The present study indicates one of the newest developments in siderophore-based

nanocomposite treated as a food source in Artemia nauplii. The specificity and high

affinity of siderophore caused natural emission of light by cells and tissue components

which elicited radiation of suitable wavelength. This is based on the mechanism of

energy transfer between the two light-sensitive compounds,a donor and receptor in

contrast Fluorescent resonance energy transfer (FRET) in Bioluminescence resonance

energy transfer (BRET) (Li et al., 2019).

Frequently used molecular tools are the luciferase (Luc) protein and the jellyfish

green fluorescent protein (GFP), which can be easily identified in living cells. They

have quite different characteristics that make GFP or Luc helpful for a specific

experimental implementation. Their ability can be enhanced by making them as a

recombinant protein having both fluorescent and bioluminescent properties (Day et

al., 1998) .Barrero et al .(1993) employed Pyoverdin, a natural flurosecent pigment

biosynthesized on controlled pore glass by Pseudomonas fluroscences .

Toxicity assessment is, therefore, a major obstacle to the discovery of compound.

Brime Shrimp assay has been shown to be suitable for rapid toxicity assessment

system. The work elucidates efficacy of Caenorhabditis elegans convenient toxicity

assessment, especially at the crucial early stages of product development. The

110
siderophore compound isolated in this work also highlights C. elegans sensitivity and

platforms for drug discovery based on C.elegans ( Xiaong et al., 2017, Himri et al.,

2013). MTT assay based on quantification of cell viability and proliferation has been

previously used to identify the cytotoxic potential of drugs (Bahuguna et al., 2017).

The siderophore Camcholate isolated in this study can thus be labelled as non-toxic,

considering its inactivity during MTT assay. The positive results obtained during

analysis of bioimaging potential by using Nanocomposite method indicated that the

Nanocomposite Liposomes coated siderophore Camcholate as a promising candidate

for alimentary canal mapping.

5.9. FUTURE APPLICATIONS OF THE SIDEROPHORE MOLECULE.

Vibrio spp. are identified as native flora of marine environments, playing

pivotal roles in biogeochemical cycles. However, many of them are opportunistic

pathogens causing diarrhoea, gastroenteritis, necrotizing fasciitis, and septicaemia in

humans and vibriosis in aquatic animals. With the increase in aquaculture activities,

identifying the aqueous pathogenicity risk in aquaculture ponds has gained high

significance.

The main target of the future application is to screen the species of

Vibrionaceae family causing diseases to fish and artemia, especially Vibrio harveyi,

Vibrio campbellii, Vibrio fischeri, and Vibrio anguillaram. Sudden disturbances in

aquaculture due to pathogenic Vibrio spp. are very common and recurrent disease

problems are on the rise in shrimp aquaculture (Li et al., 2016)

111
Early studies showed these mortality syndromes are mostly caused by Vibrio sp. In

the current approach, siderophore producing gene of Vibrionaceae was mainly

targeted instead of the virulence gene. Rapid methods for detection of Vibrios which

produce siderophore are predominantly applied for this application. Detection

methods was generally carried out using nucleic acid based, biosensor-based and

immunological based method. These detection methods include simple polymerase

chain reaction, Real-Time PCR, Nucleic acid-based amplification, oligonucleotide

DNA Microarray, biosensor-based methods, and Enzyme-Linked Immunosorbent

Assay (Panicker et al., 2004).

Rapid detection methods are vital in the prevention and treatment of aquatic

pathogens. After identification of siderophore producing genes, the respective primers

could be synthesized and used as fluorescent attached probes. Also, the most relevant

genes would be selected and analyzed for interaction between the genes. Gene

sequences for vanchrobactin, angiobactin, trivanchrobactin primer would be primarily

designed and used in this study. In order to bring down the use of antibiotics,

pesticides and other chemicals and to improve the ecological environment of shrimp

farms, research is being focused on the potential use of marine bacteria which produce

siderophores. This would help in improving the water quality by immediate detection

of Vibrio population in water and devising effective preventive measures

112
 SUMMARY & CONCLUSION

More than 500 siderophores have been isolated from a huge number of marine

bacteria till date. With mankind’s ever-increasing search for novel molecules towards

industrial and medical applications, siderophores have gained high importance. These

chelating ligands have immense potential in promoting plant growth, drug-delivery,

treatment of iron-overload, etc. Many of the potential siderophores have been isolated

from bacteria like Pseudomonas, Bacillus, Nocardia, etc. Bacteria belonging to the

family Vibrionaceae have recently gained focus owing to their rich potential in

secreting siderophores. Many of the vibrionales, viz. Vibrio harveyii, V. anguillarium,

V. campbellii. etc. are aquatic pathogens. These bacteria require iron for their growth

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and virulence, and hence produce a wide variety of siderophores. Many of the Vibrio

sp. have been widely explored for siderophore production, however, emerging

aquaculture pathogens like V. campbellii are still largely unexplored.

Screening & isolation

 A total of 48 Marine bacteria were isolated from seawater sample from

Kovalam, Coastal region of Tamil Nadu.

 The isolation was carried out by using the Serial dilution technique on four

different media by spread plate method.

 These were designated as NIOT/SID/1 to NIOT/SID/48. All the isolates were

identified on the basis of morphological characteristics such as colony shape,

size, pigmentation, and color, etc.

 Among the 48 isolates screened for siderophore production using qualitative

analysis (Neilands assay and Universal CAS assay), 14 isolates tested positive.
 Further quantitative analysis of these 14 isolates indicated six strains to have

high siderophore production potential.

 The biochemical and morphological characterization of the six potential

isolates indicated the strains NIOT/SID/8, NIOT/SID /30, NIOT/SID/37

NIOT/SID/42 NIOT/SID/43 NIOT/SID /48.

 The six potential siderophore producing strains was isolated during the

current study Among the six isolates with siderophore producing potential,

Vibrio campbellii (NIOT/SID/43) exhibited maximum siderophore production

and was taken up for further studies.

Optimization and RSM

 The growth and siderophore production potential of V. campbellii on 11

different growth media were evaluated and significant differences were

observed.

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  Among the selected media, only three media, namely, MM9 medium with low

glucose (0.08%), MM9 medium with high glucose strength (1%) and MM9

native medium, exhibited a considerable increase in growth and biomass

production during an incubation period of 0 to 14 days.

 This observation is attributed to the specific glucose concentrations of the

MM9 media, resulting in diauxic growth and also the presence of casamino

acid, a rich source of nitrogen for the bacteria. MM9 native medium exhibited

the highest siderophore production potential, followed by the BOSS medium.

 The siderophore production efficiency of MM9 native medium is attributed to

its specific glucose concentration (0.4%) and iron depletion.

 In the case of a BOSS medium, the considerable yield of siderophore inspite

of relatively low growth and biomass yield is hypothesized to be the result of

synchronous growth.

 The statistical-based optimization offered an efficient and feasible approach. A

32.685 mg/mL % increase in siderophore production was achieved with the

optimized factors Based on Response surface methodology by using central

composite design, the maximum siderophore production was obtained at 37°C,

pH 7.0, glucose 1.015 %, Iron 45 µM after eight days of incubation.

 This set up yielded the maximum siderophore production of 98.685% SU

obtained in the unoptimized protocol (66 mg/ml %). The experimental values

agreed with the predicted values generated by Central Composite Design.

Purification – novel molecule – synthesis

 The present invention relates to a process of producing siderophores. More

particularly the present invention relates to a process of preparing siderophore

fraction of very small size from Vibrio Campbelli NIOT/SID/43.

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  In present work Phenol:chloroform extraction method for producing

Siderophore from Vibrio Campbelli NIOT/SID/43 was followed

 Purification of siderophore was achieved by using Amerbrlite XAD followed

by Silica gel column hexane: ethyl acetate followed by concentration on a

rotary evaporator.

 Vibrio campbellii NIOT/SID/43 yield 64.57 mg/ml of purified siderophore,

respectively. The purified siderophore was subjected to analysis by HPLC

FTIR, NMR, and Mass spectroscopy analysis.

 The purified siderophore of Vibrio campbellii NIOT/SID/43 was partially

identified as Enterobactin (a catecholate type of siderophore). Synthesis of

siderophore molecule yielded 117.24 mg/ml.

Non-toxic nature & Bioimaging potential

 The Catecholate siderophore (VC6) molecule exhibited nontoxic in nature and

it was confirmed with toxicity screening

 VC6 when tested exhibited no activity against human cancer cell line, when

comparing antiproliferative activity and cancer activity, we came to the

conclusion that there are camcholate that did not shows promising potential

chemotherapeutics, especially MCF-7, HeLa, HEK cancer line.

 Toxicity test of (VC6) against the cancer cell lines MCF-7, cervical HeLa and

human embryonic kidney epithelial cells (HEK) specified VC6 was naturally

non-toxic.

 Bioimaging potential of camcholate was found to be effective in Artemia

nauplii

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  In summary, Vibrio campbellii produced siderophore molecule

during iron limiting condition was encapsulated into the

liposomes and used for the alimentary canal mapping.

 Due to the circumstance that the lipophilic siderophore can

be well dispersed in liposomes, the liposome coated

siderophore nanocomposites improved in water solubility, but

also significantly improved the near-infrared fluorescence of

the siderophore.

 No toxicity was detected after incubation of cells with

siderophore. whether alone and also nontoxic with Artemia

nauplii and C.eleagns.

 The artemia alimentary canal can be observed after for 15

minutes incubation either with siderophore coated liposomes,

DHBA coated liposomes, DHBA – liposomes are found toxic

with Artemia nauplii.

 The alimentary canal could be observed directly through near-

infrared fluorescence.

 On the basis of these finding, it believed that the use of near-

infrared fluroscence from liposome coated siderophore

nanocomposites has clinical promise for alimentary canal

mapping.

CONCLUSION

 Siderophores, the iron-chelating compounds, are usually produced to

hydrolyze and metabolize iron by bacteria, fungi, and plants.

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 The siderophore production has mostly been linked to virulence and hence,

most of the marine pathogenic bacteria secrete siderophore molecules.

 Among these, the Vibrionaceae family are focused in current research owing

to their relevance in aquaculture activities.

 Many of the Vibrio sp. have been widely explored for siderophore production,

however, emerging aquaculture pathogens like V. campbellii are still largely

unexplored.

 Detailed research on siderophore production potential of Vibrio sp. would be

highly beneficial to the aquaculture fraternity.

 The present invention is to utilize the prepared Siderophore for strong binding

of Fe 3+ ions in order to facilitate strong iron mobilization in humans for the

treatment of iron diseases, and also as potential drug carriers with promising

bioactive properties.

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