1.

GENERAL INTRODUCTION
The world of Extremophiles is highly diverse. The extremophiles majorly include Halophiles,
Thermophiles, Barophiles, Pscryophiles and Acidophiles under one roof (Austin 1988; Herbert
and Sharp 1992; Singh 2006) Hypersaline habitats are possessed by living beings having a place
with the three Domains of life Archaea, Bacteria, and Eukarya (Oren, 2002).Among these
Halophilic archaea adopted salt concentrations are much higher than the salinity of seawater
(3.5%, w/vol). Apart from salinity, the organisms in these environments face extremes of
temperatures, low oxygen and alkaline conditions. Some microorganisms may even
simultaneously withstand high salt concentrations (200g/l) and high temperatures upto 68°C
(Cayol et al., 1994). Yet organisms have adapted to survive in the extreme conditions (Ventosa,
2006) Halophiles, when they get entrapped in the salt crystals survive for years, and it explain
how they are evolved for thousands of years to survive in such a harsh condition. The cell
morphology physiology, metabolism, genetic regulation, adaptations, as well as biochemical
nature of halophilic cells attracts interests of microbiologists as they can find answers for many
unanswered questions. The halophiles thriving in Kutch ecosystem are not only diverse at
phylogenetic level but also vary in their physiology, metabolism, biochemistry and their genetic
regulation.
Diversity of halophilic archaea in Kutch ecosystem can be immediately indentify when one
observes reddish samples of brine or sediments dregs from this hyper-saline region. Halophilic
members from three domains are expected from unexplored harsh Kutch ecosystem. Many
members of these halophiles are awaiting isolation and characterization. The halophiles thriving
in Kutch ecosystem are not only diverse at phylogenetic level but also vary in their physiology,
metabolism, biochemistry and their genetic regulation.
The Kutch ecosystems one of the unique ecosystem in the World, which also known as the salt
marsh ecosystem which have two major interrelated saline regions as Greater Rann of Kutchh
and Little Rann of Kutchh , which were once a part of the shallow Arabian Sea which have and
average elevation of 15 m above sea level, where salt precipitates to form white desert during
summer, however, only some inner regions remains salty marsh throughout the year. Majority of
the western plains are novel source of microorganisms, due to the adverse conditions like,
extreme temperatures, salinity and drought which made it distinct from other hyper-saline
regions. Both regions remains hyper saline throughout the year but the concentration of salt
varies from time to time as well as place to place, due to native environmental factors. Study of
biodiversity mainly focuses on abundance of individuals in an ecosystem and biodiversity of
hyper-saline regions of Kutch ecosystem will give outline about abundance of halophiles of
different domain, family, genera, species and strains that will be enumerated and characterised.
For microbial community studies from marshy hyper-saline Kutch ecosystem (Little Rann of
Kutch) exploration will mainly be made from salt crystals, saline soil sediment core and brine
samples of different maturity, primarily by cultivation-dependent and cultivation independent
method.
This possibility of different archaeal halophiles in this region has been explored in the present
study. The Rann of Kutch presents unique challenges to the halophilic microorganisms
inhabiting in it, dilutions during rainy season, sea water inflow during high tide, and white salt
crust formation during summer which limit available water, prolonged sunlight, limited oxygen
and high temperature. The rain flood causes the dilution of the salts, leads to decrease in salinity
which affect the growth of halophilic archaea by changing osmotic balance. The fluctuating ionic
composition of the soil and its peculiar ionic composition with high Mg2+ contents and presence
of some heavy metals are also prompting some challenges to halophiles of this region.
Availability of organic components is little in this salt desert due to lack of visible primary
producers, but a dynamic prokaryotic community is thriving in this system which is evident after
examining brines through microscopic observation that confirms some microbial primary
producer population present in this ecosystem. Our knowledge about the types and distributions
of microorganisms in the environment is rudimentary. Because of inaccessibility of the eco-
system, the eco- region has largely been un-explored. Exploration of the life of these eco-systems
would definitely facilitate the understanding the domain of life on evolutionary perspective. At
the same time exploring the microbes of these hypersaline environments will help in
understanding the osmotolerance mechanism(s) being operated in these unique environments
from very dawn of civilization. As novelty of organisms is expected, prospecting gene(s)
responsible for extreme osmotolerance is possible.
This study will focus on isolation, characterisation and evaluation of halophilic archaeal diversity
in hyper- saline regions using culture dependent molecular phylogenetic approach. Systemic
studies of aerobic culturable archaea will reveal novel lineages
Based on conventional method , microbial community analysis discovered only 20%
normophiles with the dominating nearness of 40% halophiles and 40% haloalkliphiles, Among
these halophilic/haloalkaliphilic archaea have been less explored, due to difficulties in isolation
and pure culture maintenance as well. In spite of the possibility of the presence of novel
organisms with high economic and industrial potential, there are less comprehensive information
on the microbial diversity, phylogeny and the enzyme profiling of this region.
The major challenge for Halophilic microorganisms is their survival and also to produce active
and stable enzymes at extream condtions. Therefore the present study has ample of scope to
study various phenomena associated with the production, purification and characterization
enzymes from halophilic and haloalkaliphilic archaea along with their diversity and molecular
phylogeny.
To conquer the extreme limits of culture-dependent strategies in recognizing microbial diversity,
molecular biological methods (16s rRNA conserved regions) have develop into progressively
well known (Muyzer, 1996). This study allows analysis of selected populations within complex
microbial communities.
Thereby, the conventional and advanced techniques should be used to judge the diversity.
Besides, the culture-dependent and culture-independent, commonly known as metagenomic
approaches should coherently be employed, while exploring the diversity and phylogeny.
Moreover, the halophilic archaea produce extracellular, which are of great concern at
commercial level (Antranikian et al., 2005). Owing to the advantage of having higher enzyme
stability under harsh conditions, the enzymes obtained by the halophilic archeaa have wider
application in chemical and bioprocessing industries.
Significantly, proteases enzyme widely used in detergent, food and pharmaceutical industries.
Roughly it is estimated that microbial proteases represent around 40% of the aggregate overall
protein enzymes worldwide (Godfrey and West, 1996). Miobially derived proteases have their
inherent properties and it can be mainly stabile under various extreme conditions, such as high
temperatures, pH, chemical denaturants, surfactants, chelators and solvents which make them
potential candidate at the industrial level. A commercial production of protease by microbial
activity is mainly achieved by the Bacillus sp.
The present study is broadly focused on two aspects, mainly the diversity and the biocatalytic
potentials of halophilic archaea isolated from the saline desert little rann of kutch, Gujarat State,
India. The diversity aspect comprises of the culture-dependent and culture-independent
approaches (16s rrna illumina sequencing approach) of the halophilic archaea along with the
physicochemical composition of the studied site. Concurrently, followed by screening for
various extracellular enzymes and optimization of different physicochemical environmental
conditions, the Proteases were purified and characterized from two halipohilic archaea namely
Halolamina salifodinae J3.3 KU947980 and Haloarchaeon J1.2 KR857505 by hydrophobic
interaction chromatography.
The purpose of this study was to improve the understanding of the Kutch hyper-saline ecology
and its haloarchaeal community.
The principal aim of the present work was to describe the diversity of halophilic archaea and to
identify the unique or unusual features. Members of both Eubacteria and Eukarya disappear at
some point of salinity and archaea start occupying the space of the niche, this encourages to
select archaea rather than other members of halophiles as topic of research. The nutrition for
halophiles in hyper-saline system is limited but it does not signify that hyper-saline habitat may
nutrient poor; here haloarchaeal mechanisms with extreme enzymes make a possible survival.
OBJECTIVES
___________________________________________________________________________
Based on the facts stated above, research work was undertaken to isolate and characterize
extreme halophilic archaea for accessing their diversity saline ecoregion, and to understand
specific mechanisms of haloarchaeal physiology, biochemical and the biocatalytic potentials of
halophilic /Haloalkaliphilic archaea from the saline habitates of little rann of Kutch desert in the
Gujarat State, India.
 The physicochemical characterization of the water and soil samples collected saline
habitates of little rann of Kutch desert in the Gujarat State, India.. This was followed by
the isolation of Halophili and /Haloalkaliphilic archaea
.
 The assessment of diversity of archaea judged on the basis physiological and biological
characteristics the variation on the archaeal population dynamics in the particular
ecosystem by culture-dependent and culture-independent approaches.

 Combination of various conventional microbiological techniques to the advanced

molecular techniques, such as 16S rRNA sequencing using universal primer and 16S
rRNA Amplicon Sequencing on Illumina Platform, followed by sequence homology to
judge the population heterogeneity to investigate the archaeal diversity.

 Screening for the extracellular enzymes on selective media plates, mainly Amylase,
Cellulase , Protease , lipase , Xylase followed by optimization of various
physicochemical environmental parameters affecting production of Protease.

 Purification and characterization of alkaline protease from potential strains with respect
to enzyme catalysis and stability
 Glimpse of work flow

Isolation and characterization of halophilic Molecular Phylogeny and assessment of

/haloalkaliphilic archaea diversity based on
Samples collection and characterization 16s rRNA gene seq by sangers sequencing
Isolation and identification and
Biochemical and sugar utilization 16s rRNA amplicon sequencing on illumina
Antibiotic Profile sequencing
Pigmentation profile

Diversity, Phylogeny, Enzymatic potential of Halophilic/Haloalkaliphilic Archaea

Production , Purification and characterization

Occurrence and Distribution of Extracellular
Enzymes
(Amylase , Cellulase, Protease ,Lipase ,Xylase)
Factors(Nacl,pH) influencing their secretion and
growth
of proteases
Optimization of the factors affecting Proteases
Production
Purification and Characterization of Proteases
The Stability of Alkaline proteases

1.1 Diversity of Halophilic Microorganisms

Diversity of halophilic microorganisms in Kutch ecosystem can be quickly recognized when one
examines samples of brine or sediment from this hyper-saline region under microscope.
Halophilic members from three domains are expected from unexplored harsh Kutch ecosystem.
In ‘universal tree of life’ or universal phylogenetic tree, representatives of halophilic members
present in different branches of all three domains, Archaea, Bacteria and Eukaryota. Many
members of these halophiles are awaiting isolation and characterization. The halophiles thriving
in Kutch ecosystem are not only diverse at phylogenetic level but also vary in their physiology,
metabolism, biochemistry and their genetic regulation. Their entire life cycles of these halophiles
are highly stressed, so its isolation, characterization, classification and studies are very important
to understand regulation at different level.
The hyper-saline or saltern ecosystem encompasses environments with salinity similar to sea
water to halite saturation or beyond and thus provides a habitat for microorganisms which can
adapt to different salt concentrations, all within a small accessible geographical or inaccessible
area like trenches, inland saline lakes, salt deserts etc. Halophiles, when they get entrapped in the
salt crystals survive for years, and it explain how they are evolved for thousands of years to
survive in such a harsh condition. The cell morphology physiology, metabolism, genetic
regulation, adaptations, as well as biochemical nature of halophilic cells attracts interests of
microbiologists as they can find answers for many unanswered questions. Salterns are by no
means the only habitats in which halophilic microorganisms are found, but a community with
special skills can be easily detected from less saline regions. For the microscopists, it is
interesting to see the varied shapes of the microorganisms to macro-organism with high
densities, representing all three domains, inhabiting in hyper-saline region.
Halophilic archaeal microorganisms (haloarchaea) thrive in Kutch hyper- saline region which
resembling other extreme environments worldwide with salt concentrations approaching
saturation, such as marine solar salterns or the Dead Sea (DasSarma and Arora, 2002). Stress
conditions in these environments vary from location to location based on its physiological
parameters, salinity, pH, temperature, evaporation rate chemical nature, dilution rate, etc.
Similarly, these hyper-saline regions have a clear uniformity in major soil characters, chemical
composition, salt and other minerals concentrations. The Kutch ecosystems are very special
ecosystem, which has been studied with different perspectives for years (Dudhagara et al., 2008;
Ghosh et al., 2012) but when samples from the crystallizers were observed microscopically a
fascinating but not very diverse group of prokaryotes can be observed up to saturation point of
salts.
Kutch Hyper-saline Ecosystem
Geographically, Kutch ecosystem is located in the arid North-West region of peninsular India
connected to Pakistan occupying thousands of square kilometres as an extension of the Thar
Desert. The Rann of Kutch have two major interconnected hyper saline regions viz. Little Rann
of Kutch and Great Rann of Kutch (Figure 1.1) with an average elevation of 15 m above sea
level, where salt precipitates to form white desert during summer, however, only some inner
regions remains salty marsh throughout the year. Majority of the western plains are novel source
of microorganisms, due to the adverse conditions like, extreme temperatures, salinity and
drought which made it distinct from other hyper-saline regions. Both regions remains hyper
saline throughout the year but the concentration of salt varies from time to time as well as place
to place (Figure1.2) due to native environmental factors. In the present study, saline samples of
two major divisions of hyper-saline region in Rann of Kutch, Little and Great Rann were studied.
Studies on biodiversity basically aimed to understand individuals in an eco- system after their
characterization and their application if any, in various aspects has been deciphered. Study of
biodiversity mainly focuses on abundance of individuals in an ecosystem and biodiversity of
hyper-saline regions of Kutch ecosystem will give outline about abundance of halophiles of
different domain, family, genera, species and strains that will be enumerated and characterised.
For microbial community studies from marshy hyper-saline Kutch ecosystem (Little and Great
Rann of Kutch) exploration will mainly be made from salt crystals, saline soil sediment core and
brine samples of different maturity, primarily by cultivation-dependent method. Kutch hyper
saline geography covers thousands of square kilometres and majority of hyper-saline regions are
located either through the coastal region of Kutch or wide inland marshes of Little and Great
Rann, mainly around 23º N to 70º E. Majority of inland crystallizers having a salt concentration
near to saturation point (4.5 M to 5.3 M) throughout the year unless rain water disturb them
(Figure 1.2). The colour of these crystallizers varies due to the concentration of salt as well as
native microbial population thriving in them and their colour may vary from muddy to bright red
or colourless. These marshy environments share some common environmental properties
namely; high salinity, high temperature, and shallow subsurface, that might be acted as selective
factors for the establishment of the special microbial communities.
The hypersaline situation of the Rann of Kutch and that of crystallizers may contain dense
microbial communities including members of three domains: Archaea, Eubacteria and
Eukaryota. The red to pink pigmentation of brines (Figure 1.2) and salt crystals from different
regions of Kutch emphasise the presence of living entities in the hyper-saline marsh. From the
colouration of salt water, it is assumed that red halophilic archaea belongs to the family
Halobacteriaceae may dominate in this hyper-saline Rann environment. This halophilic archaea
belongs to Phyla Euryarchaeota which are a diverse and primitive group that can survive in most
extreme saline conditions and observed in hyper-saline regions worldwide. Members of
Halobacteriaceae are normally inhabitants of saline environments, from lower to high saline
regions such as salt lakes, salt ponds, crystallizers, marine salterns, salt sediments etc. Similar
environmental conditions can also be observed in hyper-saline marshes of the Little and Great
Rann of Kutch. The chemical composition and the predominant ions in such harsh environment
depend on the surrounding topography, geology, and general climatic conditions, which can
indirectly pointing the possibility of existence of phylogenetically distinct archaeal
extremophiles. This possibility of different archaeal halophiles in this region has been explored
in the present study.
The Rann of Kutch presents unique challenges to the halophilic microorganisms inhabiting in it,
dilutions during rainy season, sea water inflow during high tide, and white salt crust formation
during summer which limit available water, prolonged sunlight, limited oxygen and high
temperature. The rain flood causes the dilution of the salts, leads to decrease in salinity which
affect the growth of halophilic archaea by changing osmotic balance. The fluctuating ionic
composition of the soil and its peculiar ionic composition with high Mg2+ contents and presence
of some heavy metals are also prompting some challenges to halophiles of this region.
Availability of organic components is little in this salt desert due to lack of visible primary
producers, but a dynamic prokaryotic community is thriving in this system which is evident after
examining brines through microscopic observation that confirms some microbial primary
producer population present in this ecosystem.
Our knowledge about the types and distributions of microorganisms in the environment is
rudimentary. Because of inaccessibility of the eco-system, the eco- region has largely been un-
explored. Exploration of the life of these eco-systems would definitely facilitate the
understanding the domain of life on evolutionary perspective. At the same time exploring the
microbes of these hypersaline environments will help in understanding the osmotolerance
mechanism(s) being operated in these unique environments from very dawn of civilization. As
novelty of organisms is expected, prospecting gene(s) responsible for extreme osmotolerance is
possible. From this study, a few cultivable halophilic phylotypes are expected with special
adaptation mechanisms, acquired through evolution process and will allow some predictions of
metabolic changes in the fluctuating marshy Rann and microbial interaction in this very special
ecosystem. Since there were no previous reports on haloarchaeal diversity of this very peculiar
hyper-saline environment, this study was undertaken to evaluate the haloarchaeal diversity of
Kutch. This study will focus on isolation, characterisation and evaluation of halophilic archaeal
diversity in hyper- saline regions using culture dependent molecular phylogenetic approach.
Systemic studies of aerobic culturable archaea will reveal novel lineages within archaeal domain.
Extremophiles living under extreme environmental conditions are gaining increasing importance
for several biotechnological applications (Davis, 1998) and the analysis of their metabolism
constitutes all possible range and is flexible for future metabolic engineering (Bailey, 1991). No
examples of haloarchaea being directly responsible for a human or plant disease, so this
harmlessness but special intrinsic features, which are assumed to prevent archaea from posing a
threat to vertebrates, are utilized in biotechnological applications. The ability of halophilic
archaea to thrive at high salinity has sparked considerable research of their principles that allow
their proteins function under conditions that ‘salt out’ normal proteins. The halotolerance of
many enzymes derived from haloarchaea can be exploited wherever enzymatic transformations
are required to function at low water activities, e.g. in the presence of high salt conditions. There
is similarity in regulation of cellular process in archaea with bacteria and eukaryotes. The
machinery for transcription, for example, is much simpler in archaea than in eukaryotes, and
therefore also more amenable to analysis. This list of possible biotechnological uses of
haloarchaea is by no means exhaustive and is reviewed in a several articles (Rodriguez-Valera,
1992; Ventosa and Nieto, 1995).
The purpose of this study was to improve the understanding of the Kutch hyper-saline ecology
and its haloarchaeal community. The research into extreme environments has important
implications for biology and may lay the foundations for applications in the biotechnology
industry. Studying the metabolism of haloarchaea may provide insights into different pathways
which they adapt during the process of evolution. The principal aim of the present work was to
describe the diversity of halophilic archaea and to identify the unique or unusual features of their
metabolism. Because most hyper-saline environments are derived from the evaporation of sea
water, encouraged by restricted in-flow, high temperatures, low rainfall, low humidity and high
wind speed, these hyper-saline environments have been significant reservoirs for the long-term
evolution of specifically adapted microorganisms.
Halophiles from the three domains of life have adapted to grow in hyper-saline Kutch
environments but as the concentration of salt increases, the overall diversity of physiological
groups decreases (Oren, 1999), members of both Eubacteria and Eukarya disappear at some
point of salinity and archaea start occupying the space of the niche, this encourages to select
archaea rather than other members of halophiles as topic of research. The nutrition for halophiles
in hyper-saline system is limited but it does not mean that hyper-saline environments are nutrient
poor; here haloarchaeal mechanisms with extreme enzymes make a possible survival.