1.

INTRODUCTION

Microorganisms in soil are important because they affect soil structure and
fertility. Soil microorganisms can be classified as bacteria, actinomycetes, fungi, algae
and protozoa. These groups has characteristics that define them and their functions in
soil. Up to billion bacterial cells inhabit each gram of soil in and around plant roots, a
region known as the rhizosphere. An extreme environment contains conditions that are
hard to survive for most known life forms. These conditions may be extremely high or
low temperature or pressure; high or low content of oxygen or carbon dioxide in the
atmosphere; high levels of radiation, activity., or alkalinity; absence of water; water
containing a high concentration of salt or sugar; presence of Sulphur, petroleum, and
other toxic substances. The distribution of extreme environments on earth has varied
through geological time. Humans generally do not inhabit extreme environments. There
are organisms referred to as extremophiles that do live in such conditions and are so well-
adapted that they readily grow and multiply. Among extremely cold, extremely hot,
hyper-saline, places altered by humans, such as mine tailings or oil impacted habitats.

Halophiles are organisms that thrive in high salt concentrations. They are a
type of extremophile organism. The name comes from the Greek word for "salt-loving".
While most halophiles are classified into the Archaea domain, there are
also bacterial halophiles and some eukaryota, such as the alga Dunalillasalina or fungs
Wallemiaichthyophaga. Some well-known species give off a red color from carotenoid
compounds, notably bacteriorhodopsin. Halophiles can be found anywhere with a
concentration of salt five times greater than the salt concentration of the ocean, such as
the Great Salt Lake in Utah, Owens Lake in California, the Dead Sea, and in evaporation
ponds. Halophiles are categorized as slight, moderate, or extreme, by the extent of
their halotolerance. Slight halophiles prefer 0.3 to 0.8 M (1.7 to 4.8%—seawater is 0.6 M
or 3.5%), moderate halophiles 0.8 to 3.4 M (4.7 to 20%), and extreme halophiles 3.4 to
5.1 M (20 to 30%) salt content. Halophiles require sodium chloride (salt) for growth, in
contrast to halotolerant organisms, which do not require salt but can grow under saline
conditions.

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 Halophiles are classified as slight, moderate or extreme, depending on their
requirement for sodium chloride. While most marine organisms are slight halophiles,
moderate and extreme halophiles are generally more specialized microbes inhabiting
hypersaline environments found all over the world in arid, coastal and deep‐sea locations,
underground salt mines and artificial salterns. Halophilic microorganisms include
heterotrophic, phototrophic and methanogenic archaea, photosynthetic, lithotrophic and
heterotrophic bacteria and photosynthetic and heterotrophic eukaryotes. Examples of
extremely halophilic microorganisms include Halobacterium sp. NRC‐1, an
archaeon; Aphanothecehalophytica, a cyanobacterium; and Dunaliellasalina, a green
alga. Common multicellular halophilic eukaryotes include brine shrimp and brine fly
larvae that serve as an important food source for birds. In order to balance the osmotic
stress of hypersaline environments, halophilic microorganisms either accumulate organic
compatible solutes internally, produce acidic proteins to increase solvation, prevent
protein aggregation, precipitation and denaturation, and improve function in high salinity,
or use a combination of strategies. Halophiles and their biomolecules are useful for
applications in biotechnology, medicine and industry. They are metabolically diverse,
ranging from simple fermenters to iron reducers and sulfide oxidizers. Most are
photosynthetic autotrophs. The photosynthesizers in this category are purple because
instead of using chlorophyll to photosynthesize, they use a similar pigment called
bacteriorhodopsin, which uses all light except for purple light, making the cells appear
purple or a pinkish color. They are mostly aerobic, have specialized cell walls, and
carotenoids for ultraviolet protection.

Extreme halophiles act as an ideal source of extremozymes (halozymes) with

extreme stability, and the application of these enzymes as biocatalysts is attractive
because they are stable and active under conditions that were previously regarded as
incompatible with biological materials. Most significantly halophiles secrete a wide
range of extracellular hydrolytic enzymes such as nucleases, proteases, cellulases,
lipases and xylanases of potential commercial values.

Halozymes are distinguished from their homologous proteins by exhibiting

remarkable instability in solutions with low salt concentrations of salt upto 25%

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concentration. This feature of halophilic microorganisms has showed several
biotechnological applications. Halophilic proteases are active and highly stable in high
salt concentrations which inhibit or even denature many conventional enzymes of non-
halophilic organisms. Probably the largest application of proteases is in removing the
protein based stains from clothing.

Halophilic bacteria have been found to perform fermentation, acetogenesis,

sulfate reduction, photography, and methanogenesis. Halophilic microorganism used in
the treatment of saline and hypersaline wastewater and the production of
exopolysaccharide bioplastics and biofuel are being investigated. The unique cellular
enzymatic machinery of halophilic microbes allows them to thrive in extreme saline
environments. That these microorganisms can prosper in hypersaline environments has
been correlated with the elevated acidic amino acid content in their proteins, which
increase the negative protein surface potential. Because these microorganisms effectively
use hydrocarbons as their sole carbon and energy sources, they may prove to be valuable
bioremediation agents for the treatment of saline effluents and hypersaline waters
contaminated with toxic compounds that are resistant to degradation.

Hydrolases constitute a class of enzymes widely distributed in nature bacteria to

higher eukaryotes. The halo tolerance of many enzymes derived from halophilic bacteria
can be exploited wherever enzymatic transformations are required to function under
physical and chemical conditions, such as in the presence of organic solvents and
extremes in temperature and salt content. In recent years, different screening programs
have been performed in saline habitats in order to isolate and characterize novel
enzymatic activities with different properties to those of conventional enzymes. Several
halophilic hydrolases have been described, including amylases, lipases and proteases, and
then used for biotechnological applications. Moreover, the discovery of biopolymer-
degrading enzymes offers a new solution for the treatment of oilfield waste, where high
temperature and salinity are typically found, while providing valuable information about
heterotrophic processes in saline environments.

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 Hypersaline environments are extreme habitats on the planet and have a diverse
microbial population formed by halophilic microorganisms. They are considered to be
actual or potential sources for discovery of bioactive compounds, compatible solutes
including novel and/or extraordinarily enzymes. To date, a number of bioactive
compounds for the use in various fields of biotechnology which show assorted biological
activities ranging from antioxidant, sunscreen and antibiotic actions have been reported.
In addition, some halophilic microorganisms are capable of producing massive amounts
of compatible solutes that are useful as stabilizers for biomolecules or stress-protective
agents.Halophiles are actual sources for producing compatible solutes of biotechnological
interest at high concentrations. Compatible solutes ectoine and hydroxyectoine have
many existing and potential applications. Ectoine and its hydroxyl derivative,
hydroxyectoine, are of particular interest as stabilizers for proteins and protectors of
membranes from desiccation. Thus, these natural compounds were applied in cosmetics
such as skin care products.Hydroxyectoine can stabilize immunotoxins in vitro, and was
found to lessen the side effects of immunotoxins in an animal model when ectoine was
co-administered. It may thus have application as an excipient for cancer therapeutics.

Since the halophilic bacteria can tolerate high salt concentration and low
nutritional requirement, that confer them a significant potential in harsh industrial
processes. Biotechnological applications of halophiles include the production of
compatible solutes, biopolymers and carotenoids; they have also studied for various
environmental bioremediation processes. As they are stable and active at high salt
concentrations, halophilic enzymes can be used in food processing, environmental
bioremediation and biosynthetic processes. Accordingly, discovering novel enzymes,
showing optimal activities at various ranges of salt concentrations, temperatures and pH
values are commercially important.

Extremolphiles are those microorganism which live in extreme environment like

high or low temperature, salt concentration, pH etc. Extremophiles live under conditions
that would kill most other creatures and many cannot survive in the normal global
conditions. On the bases of their habitation these extremophiles can be categorized as:
acidophiles, alkalophiles, halophiles, thermophiles and psychrophiles. Among

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extremophiles halophiles are those which are able to survive in high salt
concentrations. These microorganisms inhabit the worldʼs most saline environments, like
Hypersaline Lake, solar saltern, evaporation pond, salt flats and tidal marine
environments. Halophilic bacteria have the capacity to balance the osmotic pressure of
the environment and resist the denaturing effects of salts. On the basis of their
halotolerance or optimum salt concentration wherein these organisms grow they are
categorized as slight (2-5% NaCl), moderate (5-15% NaCl) or extreme halophiles (15-
30% NaCl).

To survive in higher saline environment halophilic microorganisms use different

types of strategies, they synthesize compatible solutes in cells or possess the transporters
that help them to survive insuch type of harsh condition. Osmoregulatory solutes such as
potassium ion, glutamate, proline, ectoine and betaine have been reported in these
bacteria. Some halophiles produce acidic proteins that have the capacity to function in
high salt concentration.These microorganisms produce different bioactive compounds
like enzymes, pigments, solutes, metabolites and exopolysaccharides which have the
ability towork in such type of saline conditions. These properties can be used for the
development of techniques outlined essentially on the optimal condition of these
biomolecules.

These properties could be exploited for the development of additional bio-

industrial processes based on the optimal conditions of these biomolecules. To cope up
with the high and often changing salinity of their environment, the aerobic halophilic
bacteria, similar to all other microorganisms, need to balance their cytoplasm with the
osmotic pressure exerted by the external medium.

The growing demand for enzymes stable under high salt concentrations has
focused attention on halophiles. Halophiles receive special interest because they can grow
in a wide range of salt concentrations. Halophilic microorganisms are a potential source
of extremozymes called halozymes like proteases, amylases, nucleases, lipases,
cellulases, xylanases, catalases, and esterases, which are capable of functioning under
high concentrations of salt, wide range of pH values, and temperature at which other

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proteins will usually precipitate or denature. The halozymes can be exploited wherever
enzymatic transformations are required to function in the presence of organic solvents
and extremes in temperature and salt con- tent. Halozymes have identical enzymatic
features like their non-halophilic counterpart but exhibit marked differences in their
structural features which make them functional at harsh conditions. These include high
proportion of acidic amino-acidslocatedpredominantlyattheproteinsurfaceand requirement
of high salt concentration for optimum biological functions.

The halophilic enzymes are also stable in the presence of high salt concentrations
due to conglomeration of slightly hydrophobic groups and hydration of the protein
surface due to carboxylic groups present in glutamate and aspartate. Certain enzymes
from halophiles display polyextremophilic features like haloalkaliphilicity, thus showing
activity in high saline and high alkaline condition.

They are mostly moderately saline and fragile ecosystems. In spite of that they are
highly productive and biologically diversified habitats. Because of richness in minerals
and other nutrient content, the mangrove ecosystem harbours diverse microbial
communities which can adapt to the saline condition of this ecosystem. The bacterial
communities in saline environment would be halophilic and halotolerant bacteria of
various functionalities i.e sulphur oxidization, phosphate solubilization, cellulose
degradation, and antibiotic and enzyme production. Many of these microbes possess
unique capability to tolerate the hyper saline condition as well as various heavy metals
and metalloids. Heavy metals are increasingly found in microbial habitats due to natural
and industrial processes, for which microbes have evolved several mechanisms to tolerate
the presence of heavy metals. Due to their high stress tolerance capacity these
microorganismsare very useful for biotechnological applications in terms of
bioremediation and biomineralization. Therefore, there is a need to study the
microorganisms of these soils. Bacterial growth is often accompanied by the production
of extracellular polymeric substance (EPS), which have important ecological and
physiological functions. Increasing interest is being generated in the study of these
molecules because of their wide applications in food, pharmaceutical, petroleum and
other industries.

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 Osmolytes are zwitterionic, non-charged, or anionic and acts without inhibiting
normal metabolism of the cell and at the same time providing osmotic balance under
osmotic conditions. Cytoplasmic accumulation of osmolytes by uptake or by synthesis is
a general phenomenon of osmoadaptation in halophiles. However, uptake is more
energetically favorable than synthesis. Osmolytes prevent water efflux from the cell and
prevent cell death due to plasmolysis. Microorganisms belonging to archaea, bacteria,
yeast, filamentous fungi and algae, relyexclusively on osmolytes for osmoadaptation
except for Halobacteriaceae, Halanaerobiales and the recently discovered bacterium
Salinibacter rubrum, depends on intracellular accumulation of inorganic salts, mainly
KCl.

Different groups of enzymes are produced in halophilic bacteria particularly

protease; these enzymes serve various industrial purposes. Proteases are a single class of
derivative enzymes that catalyze the cleavage of peptide bonds leading to total hydrolysis
of proteins. They are physiologically significant for living organisms given their crucial
applications in both physiological and commercial fields. Proteases are ubiquitous in
various sources, such as microorganisms, plants and animals. Given that the plant and
animal protease cannot meet the current global demands, microbial protease has thus
gained increasing interest. Most commercial proteases are neutral and alkaline. The
increase in the microbial proteases accounts for approximately 60% of the total enzyme
sales in the world. Proteolytic activity with potential industrial application has been
characterized in Halo bacterium spp. Halophilic proteases are less suitable for saline
fermentation, because they require at least 12.5% NaCl to express high activities.
Protease comprises one of the most important groups of industrial enzymes with versatile
applications, such as in cheese-making, meat tenderization, detergents, de hairing ,
baking, waste management and silver recovery.

The capability of osmo-adaptation is a prerequisite of organisms that live in an

environment with changing salinities. Halobacillus halophilus is a moderately halophilic
bacterium that grows between 0.4 and 3 M NaCl by accumulating both chloride and
compatible solutes as osmolytes. Chloride is absolutely essential for growth and,
moreover, was shown to modulate gene expression and activity of enzymes involved in

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osmoadaptation. The synthesis of different compatible solutes is strictly salinity- and
growth phase-dependent. This unique hybrid strategy of H. halophilus will be reviewed
here taking into account the recently published genome sequence. Based on identified
genes we will speculate about possible scenarios of the synthesis of compatible solutes
and the uptake of potassium ion which would complete our knowledge of the fine-tuned
osmoregulation and intracellular osmolyte balance in H. halophilus.

Certain important biotechnological and ecological features are grabbing the

attention of scientists to study halophilic biology more deeply. This study presents
overview of the predominant halophilic organism in the saltpans of Thoothukudi district
with special focus on its cultural and biochemical characteristics.

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 2. AIM AND OBJECTIVE:

2.1 AIM:

To isolate and characterize micro-organisms isolated from saltpans of

Thoothukudi district.

2.2 OBJECTIVE:

 To isolate halotolerant microbes from salt pans.

 Morphological analysis of the isolates.
 Cell structure analysis of the isolate.
 Biochemical analysis of the isolated organism.

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 3. REVIEW OF LITERATURE

Halophiles are extremophile organisms that thrive in environments with very high
concentrations of salt. The name comes from the Greek for "salt-loving". Halophiles are
categorized as slight, moderate or extreme, based on the extent of their halotolerance.
Slight halophiles prefer 0.3 to 0.8 M (1.8 to 4.7% - seawater is 0.6 M or 3.5%), moderate
halophiles 0.8 to 3.4 M (4.7 to 20%), and extreme halophiles 3.4 to 5.1 M (20 to 30%)
NaCl. They include mainly prokaryotic and eukaryotic microorganisms with the capacity
to balance the osmotic pressure of the environment and resist the denaturing effects of
salts. Among halophilic microorganisms are a variety of heterotrophic and
methanogenicarchaea; photosynthetic, lithographic, and heterotrophic bacteria; and
photosynthetic and heterotrophic eukaryotes. Halophilic microorganisms have several
biotechnological applications like -carotene production of fermented foods. In recent
years, uses of halophilic microorganisms have significantly increased. (MohsinAzhar et
al, 2014).

Hydrolases constitute a class of enzymes widely distributed in nature from

bacteria to higher eukaryotes. The growing demand for enzymes stable under high salt
concentrations has focused attention on halophiles. Halophiles receive special interest
because they can grow in a wide range of salt concentrations. Halophilic microorganisms
are a potential source of extremozymes called halozymes like proteases, amylases,
nucleases, lipases, cellulases, xylanases, catalases, and esterases, which are capable of
functioning under high ,wide range of values, and tempera tures at which other
proteins will usually precipitate or dana true. The halozymes can be exploited wherever
enzymatic transformations are required to function in the presence of organic solvents
and extremes in temperature and salt con- tent. Halozymes have identical enzymatic
features like their nonhalophilic counterpart but exhibit marked differences in their
structural features which make them functional at harsh conditions. (Sonika Gupta et al,
2016).

On the bases of their habitation these extremophiles can be categorized as:

acidophiles, alkalophiles, halophiles, thermophiles and psychrophiles. Among
extremophiles halophiles are those which are able to survive in high salt
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concentrations. These microorganisms inhabit the world’s most saline environments, like
hyper saline Lake, solar saltern, evaporation pond, salt flats and tidal marine
environments. Halophilic bacteria have the capacity to balance the osmotic pressure of
the environment and resist the denaturing effects of salts. (Deepa Yadav et al, 2015).

Microbial life can be found over a wide range of extreme conditions (salinity, pH,
temperature, pressure, light intensity, oxygen and nutrient conditions). Hypersaline
environments constitute typical examples of environments with extreme conditions due to
their high salinity, exposure to high and low temperatures, low oxygen conditions and in
some cases, high pH values. Bacteria and Archaea are the most widely distributed
organisms in these environments. Halophiles have developed two different adaptive
strategies to cope with the osmotic pressure induced by the high NaCl concentration of
the normal environments they inhabit. The halobacteria and some extremely halophilic
bacteria accumulate inorganic ions in the cytoplasm (K+, Na+, Clí) to balance the
osmotic pressure of the medium, and they have developed specific proteins that are stable
and active in the presence of salts. In contrast, moderate halophiles accumulate in the
cytoplasm high amounts of specific organic osmolytes, which function as
osmoprotectants, providing osmotic balance without interfering with the normal
metabolism of the cell. (Maria de Lourdes Moreno et al, 2013).

The extracellular hydrolytic activities of halophilic bacteria isolated from

subterranean rock salt. The estimated number of grown colonies was approximately 3000
per gram of a rock salt on the surface of the medium MH containing 10% NaCl. The
investigated strains, randomly selected from the colonies observed on the plate, showed
at least one of the tested extracellular hydrolytic activities and one strain hydrolyzed six
out of seven tested substrates. Our investigations showed that hydrolytic activities for
Tween 80 and casein are predominant among the isolated strains although the NaCl
concentration varies up to 2M. The presence of combined hydrolytic activities in some
isolated strains could be an advantage to use in some biotechnological applications in
various fields of industry or agriculture. To the author’s knowledge, this is the first study
on the extracellular hydrolytic properties of halophilic microorganisms isolated from
subterranean salt crystals at 206m. (Roxana cojoc.et.al. 2009).

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 To replace the chemical NPK fertilizers, nitrogen-fixing, P-solubilizing, and K-
solubilizing (NPK) microbiomes should be utilized as biofertilizers in large-scale crop
production. In the future, the research on PGP mechanisms and its application for
ameliorations of salt stress as well as in other biotechnological application such as
microbe-mediated bioremediations and production of industrially and pharmaceutically
important molecule from microbes harbor in diverse extreme environments will be
increased. The beneficial microbes may be also screened for different probiotic attributes,
which may be used for human health and nutrition. The research on microbiomes from
extreme environments and plant microbiomes is very promising and will have
noteworthy economic and environmental impacts in the future. (Ajar Nath Yadav.et.al.,
2018).

The biotechnological potentials of halophiles as natural source of unique

biomolecules that exhibit distinct biological activities varying from antioxidants,
sunscreens, compatible solutes and hydrolytic enzymes are described. These
biomolecules are valuable and show commercial potential for food, pharmaceutical,
biomedical, industrial and environment. The availability of these halophilic biomolecules
and their advantages in production can be optimized to produce sustainable yields at
industrial scale. The recent availability of various complete genome sequences of
halophiles together with advances in omics technologies would further provide new
opportunities for exploration, discovery and identification of unique properties and/or
novel biomolecules derived from halophiles in the future. (RungaroonWaditee-
Sirisattha. et.al. 2016).

Seven isolates obtained from salt sample were subjected to morphological and
biochemical characterization along with pH and NaCl optimum and it is concluded that
the isolates were found to be three as Gram negative and four as Gram positive, their
colony characteristics ranging from round to irregular, rough to smooth, moist to dry. The
optimum pH was 7 to 7.5 and the NaCl requirement was 20% to 22 %. (M.F.Mansuri.et.
al, 2018).

Extreme halophiles act as an ideal source of extremozymes (halozymes) with

extreme stability, and the application of these enzymes as biocatalysts is attractive

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because they are stable and active under conditions that were previously regarded as
incompatible with biological materials. Most significantly halophiles secrete a wide range
of extracellular hydrolytic enzymes such as nucleases, proteases, amylases, cellulases,
lipases and xylanases of potential commercial values. (M. JothiBasu et al, 2015).

The marine environment is a un trapped source for many useful metabolites and
an assessment of this potential is imperative. Marine microorganisms have recently
appeared as rich sources for the isolation of industrial enzymes. Most of enzymes are
derived from microbes flourishing in ambient temperature, natural pH and other modest
condition. So, searching for extremophiles and their machinery has been attracting
greater attention. Proteases are among the most important class of industrial enzymes,
which constitute more than 65% of the total industrial applications, as well as in
bioremediation process. (Sumithra.V et al, 2015).

Furthermore, a wide variety of biotechnological products such as

bacteriorhodopsins, halorhodopsins, compatible solutes, biopolymers, bio surfactants,
exopolysaccharides, polyhydroxyalkanoates, flavouring agents, isomerases, hydrolyses,
nucleases, amylases, cellulases, proteases, lipases, anti-tumour drugs, and liposomes are
produced by halophiles. In addition, halophilic organisms play important roles in
fermenting fish sauces, modifying food textures and flavours, and in transforming and
degrading waste and organic pollutants. To examine their phenotypic features and their
physiological and biochemical characteristics, as well as their sensitivity to different
antibiotics and to demonstrate the presence or lack of proteolytic activity of halophilic
bacteria in the salt of fiereflikoçhisar Salt Lake. (MeralBirBir et al, 2003).

Halophilic microorganisms have several biotechnological applications like β-

carotene production of fermented foods. In recent years, uses of halophilic
microorganisms have significantly increased. Many enzymes, stabilizers and valuable
compounds from halophiles may present advantages for the development of
biotechnological production processes. Pakistan is renowned for its unique biodiversity.
It is especially endowed with a richness of extremophiles environments. A number of
thermal springs, many glacial areas and salt mines exist in remote areas of Pakistan.
Studies of these areas are far from complete. Many strains of microorganisms have been

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isolated from various sources but still these are not yet validated from Pakistani ecology.
The purpose of this research was to explore any novel extremely halotolerant / halophilic
aerobic or facultative anaerobic bacteria, and to examine their phenotypic, physiological
and biochemical characteristics. It was also aimed to assess the bacterial biodiversity of
halophilic bacteria in Pakistan using biochemical and molecular techniques.
(AneelaRoohi et al, 2012).

Halophiles bacteria and Archaea are unique and diverse group extremophilic
microbes that are well adapted to harsh, hypersaline conditions. They possess the ability
to compete the denaturing effects of salts. The diversity of these microbes in hypersaline
environments is of growing interest. A few hypersaline environments have been
extensively studied using molecular methodologies. Molecular mechanisms of adaptation
to hypersaline conditions have only recently been studied and modern findings of
bacterial and Archaea metabolic activities suggests that these unique microbes possess
genes conferring salt tolerance by synthesizing compatible solutes or osmolytes that can
withstand the denaturing effects of salts. These genes and their regulation have
becomethe focus of recent investigations for the production of transgenic crops and novel
products. (Muhammad Kaleem Sarwar.et. al, 2015).

The unique cellular enzymatic machinery of halophilic microbes allows them to

thrive in extreme saline environments. That these microorganisms can prosper in
hypersaline environments has been correlated with the elevated acidic amino acid content
in their proteins, which increase the negative protein surface potential. Because these
microorganisms effectively use hydrocarbons as their sole carbon and energy sources,
they may prove to be valuable bioremediation agents for the treatment of saline effluents
and hypersaline waters contaminated with toxic compounds that are resistant to
degradation. This review highlights the various strategies adopted by halophiles to
compensate for their saline surroundings and includes descriptions of recent studies that
have used these microorganisms for bioremediation of environments contaminated by
petroleum hydrocarbons. The known halo tolerant dehalogenase-producing microbes,
their dehalogenation mechanisms, and how their proteins are stabilized is also reviewed.
In view of their robustness in saline environments, efforts to document their full potential

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regarding remediation of contaminated hypersaline ecosystems merits further
exploration. (Mohamed Faraj Edbeib.et.al., 2016).

It is generally assumed that hypersaline environments with sodium chloride

concentrations close to satu ration are dominated by halophilic members of the domain
Archaea, while Bacteria are not considered to be relevant in this kind of environment.
Here, we report the high abundance and growth of a new group of hitherto-uncultured
Bacteria in crystallizer ponds (salinity, from 30 to 37%) from multi pond solar salterns. In
the present study, these Bacteria constituted from 5 to 25% of the total prokaryotic
community and were affiliated with the Cytophaga -Flavobacterium-Bacteroides phylum.
Growth was demonstrated in saturated NaCl. A provisional classification of this new
bacterial group as “CandidatusSalinibacter gen. Nov.” is proposed. The perception that
Archaea are the only ecologically relevant prokaryotes in hypersaline aquatic
environments should be revised. (Josefa Anton, et. al., 2000).

Halophilic microorganisms are organisms that grow optimally in the presence of

NaCl at least 0.2M.The applications of halophilic bacteria include food and
pharmaceutical industries, production of enzymes, polymers and various cosmetic
products. The objective of the study was isolation and its characterization of potentially
important microorganisms from saltpans. In this preliminary investigation, the total
microbial counts were studied from the Sali sample at three month intervals for one year.
Totally 9 organisms were studied from the salt sample at three physiological and
biochemical studies. These strains were subjected to screening of potential enzymes such
as; amylase, protease, gelatinase, etc. (Saju K. A, M. Michael Babu et al, 2011).

Salt marshes are costal ecosystems in the upper intertidal zone between land and
open sea water. Soil and water of this area face drastic changes in salinities since the land
is regularly flooded by tides. In addition, water evaporates in summer leading to dryness
and extremely high salinities of up to 3 M NaCl, while extensive rainfalls can decrease
the salinity to fresh water concentrations. Organisms from all kingdoms of life that live in
these areas have to adapt to such changing salinities by various strategies of
osmoadaptation. (Inga Hänelt and Volker Müller et al, 2013).

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 Mangroves are typically tropical fragile coastal ecosystems of inter-tidal zones
of river deltas and back water areas. They are mostly moderately saline and fragile
ecosystems. In spite of that they are highly productive and biologically diversified
habitats. Because of richness in minerals and other nutrient content, the mangrove
ecosystem harbours diverse microbial communities which can adapt to the saline
condition of this ecosystem. The bacterial communities in saline environment would be
halophilic and halotolerant bacteria of various functionalities i.e. Sulphur oxidization,
phosphatesolubilization, cellulose degradation, antibiotic and enzymes production etc.
Many of these microbes possess unique capability to tolerate the hyper saline condition
as well as various heavy metals and metalloids. Heavy metals are increasingly found in
microbial habitats due to natural and industrial processes, for which microbes have
evolved several mechanisms to tolerate the presence of heavy metals. Due to their high
stress tolerance capacity these microorganismsare very useful for biotechnological
applications in terms of bioremediation and bio mineralization. (DebaratiHalder et al,
2016).

Combined hydrolytic activities have been detected in a number of strains. Only

four strains presented the five hydrolytic activities tested (amylase, DNase, lipase,
protease and pullulanase). Besides, 36 strains showed three combined hydrolase activities
and 20 strains were able to produce two extracellular enzymes. All the pullulanolytic
strains characterized displayed amylase and DNase activities. (C. Sanchez-Porro et al,
2013).

Solar salterns or salt pans are artificial, shallow, open ponds used to evaporate
brine for the salt production. The high salt concentrations at these environment represent
a unique group of organisms that survive at high salinities, high temperatures and tolerate
severe solar radiations. (Jamadar et al, 2016).

Based on the growth characteristics, the isolated strains can be considered as

an‘‘Alkaliphilic moderate thermophiles”.It utilizes maximum number of carbon as well
as nitrogen sources used in this study forits growth. Cell densities were found to be more
in broth containing diethyl ether compared to other solvents. (Murugan S et al, 2018).

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 Pakistan is unique in its biodiversity and blessed with diverse extreme
environments. Remote areas of Pakistan are endowed with many thermal springs, salt
mines, and many glacial areas. Khewra salt mine, Pakistan is considered as the largest
salt mine in the world based on area, second only to Wieliczka Salt mine in Poland.
(Sameeni et al, 2009).

Sambhar Lake is a haloalkaline lake and brine samples always showed high pH.
Bacilluslicheniformis prefers alkaline growth range. Since the isolated bacillus strain was
capable of growing in twin extremities of pH and salinity, it can be categorized as halo-
alkaliphilic. Survival strategies, robust biocatalysts and unique metabolic capabilities of
these organisms can be a great source of novel commercial applications. Members of the
order Bacillales shows great metabolic versatility and ability to grow under physico-
chemical extremes, which make them survive in almost every environment on earth from
the stratosphere to the deep subsurface. This ubiquity is, in part, attributed to their ability
to form resilient spores that can be transported over long distances. (Archana Gaur et al,
2015).

There has been considerable interest in how halophilic bacteria protect themselves
from the physical parameters to which they were exposed in hypersaline environments. It
has been well-known that the production of organic compounds, accumulated into the
cytoplasm of halophilic bacteria (i.e. "compatible solutes") is the most important strategy
which allows halophilic bacteria to adapt to the extreme saline environment without
interfering with their cellular metabolism. The accumulation of compatible solutes can
determine the tolerant range of halophiles to salinity. Slightly halophilic bacteria usually
accumulate sugars (e.g. sucrose and/or trehalose), in response to salt stress, while
moderately halophilic bacteria accumulate glucosylglycerol, and extreme halophiles
accumulate ectoine` and quaternary ammonium compounds such as glutamate betaine
and glycine betaine. (Reda Hassan Amashaet al, 2018).

In aquaculture industries the bacterial forms such as Vibrio spp. Cause severe
economic loss in worldwide. The prevention and control of such diseases many remedies
has been proposed use of antibiotics, vaccines and immunostimulants etc. The use
antibiotics will lead the formation of drug resistant forms, it is more difficult to control

17 | P a g e
and eradication of these kind of bacterial forms. Salt fond organisms are defined as
halophilic microbial forms which usually inhabit salt rich environments. Halophilic
microbial forms are economically important because it produce several bioactive
compounds which are useful for many pharmaceutical industries. Among the halophilic
microbial forms, the halophilic bacterial forms are mostly known for its secondary
metabolites such as proteins amino acids, etc. In recent years many reports are available
for the application of halophilic bacterial forms as a probiotic in shrimp aquaculture. The
strain H.salinus has produced several interesting compounds and thus may be good
candidate for the pharmaceutical industry. (P.Mayavu et al, 2014).

The largest family of Archaea, the Halobacteriaceae holds its name as the
extremely halophilic group containing the highest salt requiring microorganisms. High
sodium chloride concentration allows members of Halobacteriaceae to degrade organic
compounds aerobically. (Oren et al, 2006).

The growth of extremely halophilic bacteria requires relatively high NaCl and the
majority of them require magnesium ion for their growth whereas slightly and
moderately-halophilic bacteria do not require magnesium ion for growth. (Grant et al,
2001).

18 | P a g e
 4. MATERIALS AND METHODS

4.1 SAMPLE COLLECTION

Around 60 salt pan water samples were collected into the sterile containers from
the 20 different areas (Alagaapuri, Aarumuganeri, Palayakaayal, Vepalodai,
Tharavakulam, Punnaikaayal, Agaram, Kovankaadu, Pottalkaadu, Uppaathaoodai,
Vempaaru, Pattanaputhur, Manjaneerkaayal, Pullavali, Mudukkukaadu, Overbeach
paalam, Sathya nagar, CG colony, Muthiapuram, Mullakaadu) in Thoothukudi
district.(Shown in Fig.4.1-4.3). The collected samples were stored at 4ºC in the laboratory
until used for isolation of the strains.

4.2 ISOLATION OF BACTERIA

The halophilic media (Himedia, Cat No. M590-500G) was used to isolate the
halophilic bacteria from the sample. The pH of the medium was maintained at above 8.
0.1ml sample was spread plated on to the agar and incubated at 37˚C for 2 weeks.

4.3 IDENTIFICATION OF ISOLATES

Isolates were examined for colony and cell morphology. Colony morphology was
described with special emphasis on pigmentation, colony elevation and opacity. These
characteristics were described from cultures growth at optimum temperature, pH, and salt
concentration. Biochemical tests, like Catalase test, Gelatinase, Amylase, Oxidase,
Citrate, Urease, Indole test, Tryptophan Deaminase, VogesProskauer, Fermentation/
oxidation(lactose, sucrose, dextrose) were carried out. Cell morphology were studied
with staining techniques. (According to Bergey’s manual).

4.4 GRAM STAINING

The gram stain is a differential stain which is used to differentiate bacteria into
two groups Gram positive bacteria and Gram negative bacteria. The technique is based on
the fact that Gram positive cell wall has a stronger attraction for crystal violet when
iodine is applied and therefore retains the crystal violet and therefore will remain purple

19 | P a g e
after decolorizing while Gram negative bacteria will be colourless after decolorizing with
alcohol, counterstaining with Safranin will make them to appear pink.

4.5 IMVIC TEST

4.5.1 INDOLE TEST
Tryptophan is an essential amino acid that can undergo oxidation by the way of
enzymatic activities of bacteria and converted into metabolic products (indole, pyruvic
acid and ammonia) is mediated by the enzyme tryptophanase. The presence of indole is
detected by adding Kovac's reagent which produces cherry red colour. The colour is
produced by the reagent which is composed of p-dimethylaminobenzaldehyde yielding
the cherry red colour. Absence of red coloration demonstrates that the substrate
tryptophan was not hydrolysed and indicates an indole negative.

4.5.2 METHYL RED TEST

All enteric microorganisms ferment glucose and produce organic acids. .The
methyl red indicator which is used in this test, in the pH range of 4 will turn red, which is
the indicative of a positive test. At a pH of 6, still indicating the presence of acid but with
a lower hydrogen ion concentration, the indicator turns yellow and is a negative test.

4.5.3 VOGES PROSKAUER TEST

VogesProskauer test determines the capability of some microorganisms to

produce non-acidic or neutral end products, such as acetyl methyl carbinol, from the
organic acids that results from glucose metabolism. The reagent used in this test, Baritt’s
reagent consists of mixture of alcoholic alpha- napthol and potassium hydroxide
solutions. Detection of acetyl methyl carbinol requires this end product to be oxidized to
a diacetyl compound. This reaction will occur in the presence of alphanapthol catalyst
and a guanidine group that is present in the peptone of MR-VP medium. As a result, a
pink complex is formed, imparting a rose colour to the medium.

4.5.4 CITRATE UTILIZATION TEST

In the absence of fermentable glucose or lactose, some micro-organisms are

capable of using citrate as a carbon sources for energy. This ability depends on the

20 | P a g e
presences of citrate permeas that facilitates transport of citrate in the cell. During this
reaction the medium becomes alkaline, the carbon dioxide that is generated combines
with sodium and water to form sodium carbonate, an alkaline product. The presence of
carbonate changes the Bromothymol blue indicator incorporated into the medium from
green to Prussian blue. After incubation citrate positive culture are identified by the
presence of growth on the surface of slants, which is accompanied by blue coloration.
Citrate negative will show no growth and the medium will remain green.

4.6 CATALASE TEST

3% hydrogen peroxide were added slowly to the Test tube have the isolates. The
emergence of bubbles from the organism was noted. The presence of bubble displayed a
positive test indicating the presence of enzyme catalase. If no gas is produced, this is a
negative reaction.

4.7 GELATIN LIQUEFACTION

Gelatin is a protein produced by hydrolysis of collagen. Hydrolysis of gelatin is

brought about by microorganism capable of producing a proteolytic exoenzyme known as
gelatinase. The degradation of gelatin occurs in the medium by an exoenzyme, it can be
detected by observing liquefaction (i.e. flooding the gelatin agar medium with mercuric
chloride solution and observe the plates for clearing around the line of growth) because
gelatin is also precipitated by chemicals that coagulate proteins while the end products of
degradation (i.e. amino acids) are not precipitated by same chemicals.

4.8 UREASE TEST

Urease test is performed by growing the test organisms on urea broth or agar
medium containing the pH indicator phenol red (pH 6.8). During incubation,
microorganisms possessing urease will produce ammonia that raises the pH of the
medium/broth. As the pH becomes higher, the phenol red changes from a yellow colour
(pH 6.8) to a red or deep pink colour. Failure of the development of a deep pink colour
due to on ammonia production is evidence of a lack of urease production by the
microorganisms.

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4.9 OXIDASE TEST

The oxidase test is a test used in microbiology to determine if a bacterium

produces certain cytochrome c oxidases. It uses disks impregnated with a reagent such as
N, N, N, N-tetra methyl-pphenylenediamine (TMPD) or N, Ndimethyl- p-
phenylenediamine (DMPD), which is also a redox indicator. The reagent is a dark-blue to
maroon color when oxidized, and colorless when reduced. Oxidase-positive bacteria
possess cytochrome oxidase or indophenol oxidase (an iron-containing hemoprotein).
These both catalyze the transport of electrons from donor compounds (NADH) to
electron acceptors (usually oxygen). The test reagent, TMPD dihydrochloride acts as an
artificial electron donor for the enzyme oxidase. The oxidized reagent forms the colored
compound indophenol blue. The cytochrome system is usually only present in aerobic
organisms that are capable of using oxygen as the final hydrogen receptor. The end
product of this metabolism is either water or hydrogen peroxide (broken down by
catalase). Live bacteria cultivated on trypticase soy agar plates may be prepared using
sterile technique with a single-line streak inoculation. The inoculated plates are incubated
at 37°C for 24 - 48 hours to establish colonies. Fresh bacterial preparations should be
used. After colonies have grown on the medium, 2-3 drops of the reagent DMPD are
added to the surface of each organism to be tested. A positive test (OX+) will result in a
color change violet to purple, within 10 30 seconds. A negative test (OX-) will result in a
light-pink or absence of coloration.

4.10 AMYLASE TEST

In this test, isolate was point inoculated on starch agar plates and incubated at
37˚C for two days. After incubation, iodine solution was poured on the agar and
examined for hydrolysis of starch by the production of clear zone around the microbial
growth.

4.11 PROTEASE TEST

In this test, isolate was point inoculated on skim milk agar plates and incubated at
37˚C for 24 hours. After incubation, the agar plates and examined for hydrolysis of
protein by the production of clear zone around the microbial growth.

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4.12 CARBOHYDRATE FERMENTATION TEST

Fermentation of carbohydrates (glucose, sucrose, lactose etc.) are carried out by

microorganism, under anaerobic condition in which a Durham tube is placed in inverted
position to trap the gas bubble formed due to production of gas. The fermentation broth
contains ingredients of nutrient broth, a specific carbohydrate and a pH indicator (phenol
red), which is red at neutral pH (7) and turns yellow at or below a pH of 6.8 due the
production of an organic acid.

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Fig. 4.1 Salt Pan (Kovankadu)

Fig. 4.2 Salt Pan (Vepalodai)

24 | P a g e
Fig. 4.3 Thoothukudi Map

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 5. RESULTS

5.1 COLONY MORPHOLOGY

Inoculated samples showed circular yellow opaque colonies and circular red
colored colonies on the halophilic agar medium after 2 weeks of incubation. (Shown in
Fig.5.1).

5.2 CELL MORPHOLOGY

Under the microscope isolate 1 was seen as pink colour rods. This shows that the
isolate 1 is gram- negative, rod shaped bacterium arranged in long chains (Shown in
Fig.5.2),But isolate 2 shows gram positive coccal shaped bacterium arranged in pairs or
long chains.(Given in Fig.5.14).

5.3 OPTIMIZATION OF GROWTH CONDITIONS

The growth conditions of all the isolated strains were optimized for pH and salt
tolerance. The purpose of optimization of the strains was to find their optimum growth in
different pH. From the results, it was concluded that the halophilic bacteria species grow
best at 7-8 .Similarly NaCl tolerance was checked from these species, most strains, grow
best in the range of 10-20% at temperature 37ºC.

5.4 IMVIC TEST

5.4.1 INDOLE TEST

Both isolates are negative to Indole Test. The substrate tryptophan was not
hydrolysed. So there is no development of cherry red colour after the addition of kovac´s
reagent. (Given in Fig.5.3 and Fig.5.15).

5.4.2 METHYL RED TEST

Both isolates are methyl red positive. The medium changed red colour after the
addition of methyl red indicator, which indicates that the isolates were capable of
fermenting glucose and produce organic acids. (Shown in Fig.5.4 - Fig.5.16).

26 | P a g e
5.4.3 VOGES PROSKAUER TEST

After the addition of Baritt´s reagent, the MR-VP medium inoculated with isolate
1 impacts pink colour, which indicates the presence of non-acidic, neutral end product
that results from glucose metabolism. Hence isolate 1 is positive to voges proskauer test.
(Given in Fig.5.5).But isolate 2 is negative to Voges Proskauer Test. Since there is no
colour change. (Given in Fig. 5.17).

5.4.4 CITRATE UTILIZATION TEST

The isolates changed the colour of Bromothymol blue indicator in the medium to
Prussian blue. This shows that the isolates have citrate permease that facilitates the
transport of citrate in the cell, so that the citrate could be used as carbon source. Hence
the isolates are citrate positive. (Which is depiced in Fig.5.6 and Fig.5.18).

5.5 CATALASE TEST

No bubbles were emerged from the isolates while adding Hydrogen peroxide to
the test tubes, which indicates the isolates are negative for catalase production. (Shown in
Fig.5.7).

5.6 GELATINASE TEST

The isolate 1 form zone around the colonies, which indicates the gelatin, has been
liquefied. This shows the capability of producing gelatinase enzyme by the isolate.
(Given in Fig. 5.8).

5.7 UREASE TEST

As shown in figure 5.9, The isolate 1 impacts pink colouration on urea agar.
Which indicates the urease production by the isolated microorganism.

5.8 OXIDASE TEST

The isolate 1 showed the positive result of violet colour within 10-30 seconds
after the addition of hydrogen peroxide on oxidase disc. (Given in Fig.5.10).

27 | P a g e
5.9 AMYLASE TEST

The isolate 1 showed the hydrolysis of starch by the production of clear zone
around the colonies on starch agar after the addition of iodine solution, which indicates
the production of amylase. (Shown in Fig.5.11).

5.10 PROTEASE TEST

The isolate also showed the clear zone around the growth on skim milk agar,
Which indicates its ability to produce protease. (Given in Fig. 5.12).

5.11 CARBOHYDRATE FERMENTATION TEST

The isolate 1 showed the positive result of yellow coloration on carbohydrate

fermentation medium which indicates the production of an organic acid. (Shown in
Fig.5.13).

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Fig. 5.1 Halophilic Agar – isolate 1 (yellow colonies)
Isolate 2 (red colonies)

Fig. 5.2 Isolate 1 – Gram staining

Fig. 5.3 Indole Test – isolate 1 (negative)

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 Fig. 5.4 Methyl red test – isolate 1 (negative)

Fig. 5.5 Voges proskauer test – isolate 1 (Positive)

30 | P a g e
Fig. 5.6 Citrate utilization test – Isolate 1 (Positive)

Fig. 5.7 Catalase test – isolate 1 (negative)

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Fig. 5.8 Gelatinase test –isolate 1 (positive)

Fig. 5.9 Urease test – isolate 1 (positive)

Fig. 5.10 Oxidase test - isolate 1 (positive)

32 | P a g e
 Fig. 5.11 Amylase test - isolate 1 (positive)

Fig. 5.12 protease test - isolate 1

Fig. 5.13 Carbohydrate fermentation test - isolate 1

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 Fig. 5.14 isolate 2 - Gram staining

Fig. 5. 15 isolate 2 - Indole test (negative)

Fig. 5.16 isolate 2 - Methyl red test (negative)

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Fig. 5. 17 isolate 2 - Voges proskauer test (negative)

Fig. 5.18 isolate 2 - Citrate utilization test (positive)

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 TABLE 5.19 CULTURAL CHARACTERISTICS OF ISOLATES

Characteristics Isolate 1 Isolate 2

Colonial morphology Circular Circular

Colony Flat Flat

Colony density Opaque Translucent

Pigmentation Cream Red

Cell shape Rod Cocci

Cell arrangement Long chains Long Chains or paired

Gram staining Negative Positive

TABLE 5.20 BIOCHEMICAL CHARACTERISTICS OF ISOLATED

BACTERIAL SPECIES

Biochemical test Isolate 1 Isolate 2

Indole test Negative Negative

Methyl red test Negative Negative

Voges proskauer test Positive Negative

Citrate utilization test Positive Positive

Catalase test Negative _

Gelatinase test Positive _

Urease test Positive _

Oxidase test Positive _

Amylase test Positive _

Protease test Positive _

Carbohydrate fermentation test Positive (acid production) _

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 6. DISCUSSION
Thoothukudi is well known for the production of salt, since it is the coastal area of
Tamilnadu. Majority of the area is covered by saltpans and so cultivation of salt is the
major occupation of thoothukudi next to fishing. This ecological conditions drive me to
do research in this area. Microorganisms is omnipresent. Organism present in saltpans,
tolerate the high alkalinity and temperature prevails there.

Predominantly two different types of colonies were isolated from the samples
collected from various areas in and around Thoothukudi. So I have chosen those two
colonies for my study. Both isolates were subjected to different biochemical assays. They
differ vastly in morphological and biochemical aspects. Similarly was observed in Indole,
Methyl red and citrate utilization Test. Both colonies were circular and flat, but differ in
density, pigmentation, shape, arrangement and Gram staining.

Furthur deep analysis of these two isolates are needed to identify its genus and
species. Cream coloured colonies were found to produce many enzymes like Gelatinase,
Urease, Oxidase, Amylase, and Protease, which could be produced commercially, if
detailed study was made.

37 | P a g e
 7. SUMMARY

Predominantly two different types of colonies were isolated from the sample
collected from salt pans. They are red and yellow circular opaque colonies on halophilic
agar, at pH level 7-8, optimum temperature at 37ºC. Cell structure analysis under
microscope showed that the colonies have rod and coccal shaped bacteria, arranged in
long chains.

Biochemical analysis of the isolates showed that they have the ability to presence
certain halo enzymes like Amylase, Protease, Gelatinase, Oxidase.The isolated organisms
are also positive to Voges proskauer test, Citrate Utilization test, Gelatinase test, Urease
test, Amylase test, Protease test, Carbohydrate fermentation test. Hence the role of these
halophiles in the field of life science in vast.

38 | P a g e
 8. CONCLUSION

This study focussed on the microorganisms predominantly present in salt pans in

and around Thoothukudi district. The predominantly organisms were isolated and
characterized both culturally and biochemically. Results of the analysis helps to conclude
that the organisms isolated have the ability to produce many halo enzymes which could
be produced commercially and might be used in further research.

To conclude, characterization of similar enzyme producing halophiles have great

significance in present day biotechnology and hence will enable us to use them for
various biotechnological purposes.

8.1 FUTURE PLAN

 DNA Sequencing of the isolates.
 Enzyme extraction from the isolates.
 Commercial usage of Enzymes

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 9. BIBLIOGRAPHY

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kamekura and Madalinenache (2009). Extracellular hydrolytic enzymes of
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Halophilic microorganism resources and their applications in industrial and
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Gadhi(2018).Isolation and characterization of halophiles from solar salterns of
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 M. JothiBasuV.Indra, J.Hemapriya and S.Vijayanand (2015). Bioprocess
Optimization of Halophilic Protease Production by a Halophilic Bacterial Strain
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 Sumithra.V, S.S.Sudha and Nisha S Jayasankar (2015). Isolation, screening and
assay of halophilic protease from saltpan samples of tuticorin district’’ SIRJ-
MBT Volume 2 Issue 2 (April 2015) ISSN 2349-0101.
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 AneelaRoohi, Iftikhar Ahmed, Muhammad Iqbal and Muhammad Jamil
(2012).PRELIMINARY ISOLATION AND CHARACTERIZATION OF
HALOTOLERANT AND HALOPHILIC BACTERIA FROM SALT MINES OF
KARAK, Pakistan Pak. J. Bot.,44: 365-370, Special Issue March 2012.
 MuhammmadKaleemSarwar, IqraAzam, Tahir Iqbal (2015). Biology and
Applications of Halophilic Bacteria and Archaea: A Review. Electronic Journal
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 Mohamed FarajEdbeibRoswanira Abdul WahabFahrulHuyop (2016).Halophiles:
biology, adaptation, and their role in decontamination of hypersaline
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VALERA, AND RUDOLF AMANN (2000).Extremely Halophilic Bacteria in
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 Inga Hänelt and Volker Müller (2013). Molecular Mechanisms of Adaptation of
the Moderately Halophilic BacteriumHalobacillishalophilus to Its Environment.
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 DebaratiHalder, Pardita Dutta, ArpitaMondal, MaliniBasu (2016).Isolation and
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 10. APPENDICES
1. NUTRIENT AGAR (PH7.0)

 Peptone-5.0g
 Beef extract-3.0g
 Sodium Chloride-5.0g
 Agar Agar-15g
 Distilled Water-1000mL.

2. HALOPHILIC AGAR

 Casein acid hydrolysate -10.000g

 Yeast Extract -10.000g
 Protease peptone -5.000g
 Trisodium Citrate- 3.000g
 Potassium chloride -2.000g
 Magnesium sulphate -25.000g
 Sodium chloride -250.000g
 Agar 20.000g
 Final pH (at 25ºc)-7.2±2.0

3. STARCH AGAR

 Peptone-5g
 Beef Extract-3g
 Soluble Starch-2g
 Agar Agar-15g
 Distilled Water-1000mL
 pH -7

4. IODINE
 Potassium Iodide-20g
 Iodine-10g
 Distilled water-1000mL

44 | P a g e
5. NUTRIENT GELATIN

 Peptone-5g
 Beef Extract-3g
 Gelatin-0.4g
 Agar Agar-15
 Distilled Water-1000mL
 pH-6.8-7.2

6. CARBOHYDRATE FERMENTATION MEDIUM

 Peptone-10g
 D.glucose-5g
 Bromo cresolpurple-0.03g
 Agar Agar-13g
 Distilled Water-1000mL
 pH-1000mL

7. SKIM MILK AGAR

 SM powder-28.000
 Tryptone-5.000
 Yeast extract-2.500
 Dextrose (glucose)-1.000
 Agar-15.000
 Final pH - (at 25ºc) 7.0±15.000

8. OXIDASE REAGENT
 Tetra methyl P-Phenylene diamine hydrochloride-0.1g
 Distilled Water-10mL

9. UREA AGAR
 Peptic digest of animal tissue-1g
 Dextrose-1g

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  Sodium chloride-5g
 Mono potassium phosphate-2g
 Urea-20g
 Phenol red-0.012
 Distilled Water-1000mL
 pH-6.8-7

10. SIMMONS CITRATE AGAR

 Magnesium Sulphate-0.2g
 Ammonium dihydrogen phosphate-1g
 Dipotassium Phosphate-1g
 Sodium citrate-2g
 Sodium chloride-5g
 Bromothymol blue-0.08g
 Agar Agar-15g
 Distilled water-1000Ml
 pH-6.8-7

11. PEPTONE WATER

 Peptone-1g
 Sodium chloride-0.5g
 Distilled Water-100mL
 pH-7.4

12. KOVAC´S INDOLE REAGENT

 Para dimethyl amino benzoldehyde-2g

 Iso amyl alcohol-30mL
 Concentrated Hcl-10mL

13. MR-VP BROTH

 Peptone-0.5g
 Glucose-0.5g

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  Dipotassium Hydrogen phosphate-0.5g
 Distilled Water-100mL
 pH-7.4-7.6

14. METHYL RED SOLUTION

 Methyl red-0.05g
 Ethanol-28mL
 Distilled water-22mL

15. α-NAPTHOL

 α-napthol-5g
 Ethyl alcohol-100mL
 Potassium hydroxide-40g
 Distilled Water-100mL

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