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Chapter 1
Introduction
1. Introduction
1.1 Extremophiles
An extremophile (from Latin extremus meaning "extreme" and Greek philiā meaning
"love") is an organism that thrives in physically or geochemically extreme
conditions that are detrimental to most life on earth (Rampelotto, 2010). The term
extremophile was first used by MacElroy (MacElory, 1974). Extreme environments,
such as acidic or hot springs, saline and/or alkaline lakes, deserts and the ocean beds
are also found in nature, which are too harsh for normal life to exist. Extreme
environment is a relative term, since environments that are extreme for one organism
may be essential for the survival of another organism. Thermophiles were the first
extremophile to be discovered from the hot springs of Yellowstone National Park,
USA. There are many different classes of extremophiles, each corresponding to the
way its environmental niche differs from mesophilic conditions. Microorganisms
grow at high temperature (55 - 121°C) are called as thermophiles and at low
temperature of –2 to 20°C are called psycrophiles. Microorganism which grow in high
salinity (2–5 M NaCl) are known as halophiles, pH > 8 are alkaliphiles and pH < 3 are
acidophiles (Rothschild and Manicinelli, 2001). Other extremophiles including
oligotrophs (grow in limited nutrition, < 1 mgL-1), metallotolerants (tolerate heavy
metals), piezophiles (grow at high hydrostatic pressure of > 130 M pa) ,
radioresistants (resistance to high level of ionizing radiation most commonly
ultraviolet radiation of > 400 J/m2 UV) and xerophiles (very low water activity, aw <
0.85) (Rothschild and Manicinelli, 2001). The classification of extremophiles and
their habitats are summarized in table 1.1. Extremophiles include representatives of
all three domains (Bacteria, Archaea, and Eukarya). The thermophiles are members
of the Archaea and Bacteria (Rothschild and Mancinelli, 2001).
A class of extremophiles is found in environments with a combination of
extreme conditions such as high temperature and acidity, high alkalinity and pressure,
high salinity and alkalinity. Therefore, these are known as polyextremophiles.
Thermo-acidophiles are combination of thermophiles and acidophiles that prefer
temperature between 70 - 80°C and pH 2-3. The most extreme thermophilic and acidloving species of Picrophilus has been isolated, which has the ability to grow at pH
0.7 equivalent to pH of 1.2 M H2SO4 and temperature of 60ºC (Schleper et al., 1996).
Similarly, different haloalkaliphilic microbes like Alkaliphilic bacillus spp. have the
ability to grow at pH 11 and high concentrations of Na+, Cl-, SO42- and HCO3- (140,
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150, 20 and 70 g L-1 respectively) (Cavicchioli and Thomas, 2003). Extremely
halophilic alkalithermophilic bacteria Natranaerobius thermophilus, showed growth
at pH 9.5, temperature 53°C in the presence of 1.5-3.3 M NaCl (Mesbah et al.,
2007). These organisms have developed adaptive features including accumulation of
salt or organic solutes, protein flexibility, hydrophobicity, increased surface charge
and increased protein core to survive and propagate under extreme conditions
(Galinski, 1993; Kumar and Nussinov, 2001).
Table 1.1 Classification of extremophiles and their growth habitats (Prescott et
al., 1999; Van den Burg, 2003).
Environmental
parameter
Temperature
Class
Growth
Habitats
Examples
Hyperthermophiles
>80°C
Submarine vents
Thermophiles
60-80°C
Terrestrial hot
spring
Thermus
aquaticus,
Pyrolobus fumarii
Geobacillus sp.
Mesophiles
15-45°C
Psycrophiles
<15°C
Polar regions
Psycrobacter
Salinity
Halophiles
2-5 M NaCl
Dunaliella salina
pH
Acidophiles
pH <3
Alkaliphiles
pH >9
Salt brines,
salterns
Sulfide-rich
geothermal zones
Soda lakes
Desiccation
Xerophiles
Anhydrobioti
c
aw < 0.85
Deserts
Radiation
Radioresistant
Oxygen
Anaerobes
> 400 J/m2
UV
Cannot
tolerate O2
Tolerate
some O2
Require O2
Nuclear power
plants
-
Microareophiles
Aerobe
Homo sapiens
Ferroplasma sp.
Natronobacterium,
Bacillus okhensis
Tricosporonoidis
nigresecns, fungi,
lichens
-
Deinococcus
radiodurans
Methanococcus
jannaschii
Clostridium sp.
-
H. sapiens
Pressure
Piezophiles
Pressure
> 100 MPa
Deep marine
trenches
Halomonas salaria
Metals
Metallotolerants
Tolerate high
concentration
of metals
-
Ralstonia sp. CH34
1.2 Thermophiles
Interest in thermophilic microorganisms that live and thrive at high temperatures of up
to or above 90°C has been increasing in recent decades (Singleton and Amelunxen,
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1973). Heat-loving microbes or thermophiles are the best studied among the
extremophiles. Thermophiles reproduce and grow readily at temperatures > 50°C, and
hyperthermophiles favour temperatures above 80°C. Thermophiles have been known
for a long time, but true thermophiles those able to flourish in greater heat were first
discovered about 55 years ago. In the late 1960s, the first extremophile capable of
growing at temperatures greater than 70°C was identified as Thermus aquaticus.
Microorganisms designated as thermophiles that have a minimum
temperature of 45°C, optimum at 55°C and maximum at 70°C. Thermophiles are
classified as true thermophiles, which show optimum growth at 60-70°C and no
growth or very little growth below 45°C (Bergey, 1919). Temperatures of ~100°C
normally denature proteins, nucleic acids, and increase the fluidity of membranes to
lethal levels, but for hyper thermophiles, this temperature is optimum for growth. In
1898, Schillinger proposed the term "thermotolerant" for bacteria, which grew at both
high (> 50°C) and low temperatures (< 37°C) and "thermophilic" for bacteria which
grew at high temperatures (> 60°C), but not at
temperature of human body
(Schillinger, 1898).
Habitat of thermophiles includes natural geothermal areas which are widely
distributed across the globe and primarily associated with tectonically active zones at
which the movements of the Earth’s crust occur. Hot springs are distributed all over
the world and the countries known for their hot springs are Iceland, New Zealand,
Mexico, USA, Chile, Japan, Indonesia, Russia and Brazil. Hot spring microbial
communities have been extensively studied in many areas such as Yellowstone
National Park in the United States (Barns et al., 1994; Hugenholtz et al., 1998;
Reysenbach et al., 2000; Blank et al., 2002; Meyer-Dombard et al., 2005),
Kamchatka hot springs in Russia (Perevalova et al., 2008), the island of the Lesser
Antilles (Kvist et al., 2007), Icelandic hot springs (Perevalova et al., 2008;),
Mt.Unzen hot springs in Japan (Takai and Sako, 1999), Ohwakudani hot springs in
Japan (Kato et al., 2011), Pisciarelli hot springs in Italy (Kvist et al., 2005), Bor
Khlueng hot springs in Thailand (Kanokratana et al., 2004), Wai-o-tapu geothermal
area in New Zealand (Childs et al., 2008), Tengchong hot Springs in China (Song et
al., 2010), and Manikaran Hotspring of Himachal Pradesh, India (Sharma et al.,
2012). The hot springs are of terrestrial, subterranean and marine types. The
worldwide distribution of geothermal hot springs is shown in figure 1(a) and hot
springs located in India are shown in fig 1(b)
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a
b











Bendrutheertha
Chavalpani
Suryakund
Rishi
Taptapani
Bakreshwar
Tulshishyam
Sohna hot spring
Manikaran
Tattapani
Tattapani
Karnataka
Madhya Pradesh
Bihar
Sikkim
Orissa
West Bengal
Gujarat
Gurgaon
Himachal Pradesh
Himachal Pradesh
Jammu & Kashmir
Figure 1.1: Geothermal hot springs. (a) Worldwide distribution of geothermal
springs, adapted from http://geothermal.marin.org/geomap_1.html. (b) Distribution
of geothermal springs in India.
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Thermophiles can grow and reproduce at high temperature due to the
presence of special proteins known as ‘chaperonins’, which are thermostable and
resistant to denaturation and proteolysis. Proteins of thermophiles denatured at high
temperature, are refolded by the chaperonins, thus restoring their native form and
function (Kumar and Nussinov, 2001). The cell membrane of thermophiles consists of
saturated fatty acids that provides a hydrophobic environment for the cell and
maintains the cell rigidity even at elevated temperatures (Herbert and Sharp, 1992).
The Archaea, which comprises most of the hyperthermophiles, have lipids linked with
ether on the cell wall and are more heat resistant than a membrane formed of fatty
acids (De Rosa et al., 1994). The DNA of thermophiles contain reverse DNA gyrase,
which produces positive super coils in the DNA (Lopez, 1999) and raise the melting
point of the DNA (the temperature at which the strands of the double helix separate).
Thermophiles also tolerate high temperature by using increased interaction like
electrostatic, disulphide bridge and hydrophobic interactions in polypeptide chain
(Kumar and Nussinov, 2001).
The most hyperthermophilic organisms are archaea, with Pyrolobus fumarii
(Crenarchaeota), a nitrate reducing chemolithoautotroph, capable of growing at the
highest temperatures of up to 113°C (Rothschild and Manicinelli, 2001). There are
thermophiles among the phototrophic bacteria (Cyanobacteria, purple and green
bacteria), eubacteria (Bacillus, Clostridium, Thiobacillus, Desulfotomaculum,
Thermus, lactic acid bacteria, actinomycetes, spirochetes and numerous other genera)
and the archaea (Pyrococcus, Thermococcus, Thermoplasma, Sulfolobus and the
methanogens). In contrast, the upper limit for eukaryotes is ~60°C, a temperature
suitable for some protozoa, algae and fungi.
1.3 Extremozymes
Enzymes are biocatalytic protein molecules that enhance the rates of biological
reactions by 106 to 1023 fold over the uncatalyzed reactions. A number of enzymes
such as glucose isomerase, amylase, lipase and proteases are used in a variety of food,
beverages and detergent industries, while Taq polymerase, T4 DNA ligase, lysozyme,
ribonuclease and malate dehydrogenase are enzymes used in research laboratories.
Although enzymes can be derived from several sources, such as plants, animal and
micro-organisms; enzymes from microbial sources generally meet industrial demands
of food, textiles and detergents. Extremophiles are potent source of enzymes
(extremozymes) and many other biomolecules with extreme stability. The application
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of these enzymes as biocatalysts is attractive, because they are stable and active under
harsh conditions such as high or low temperature / pH or high salt. Due to their
extreme stability, extremozymes offer new opportunities for biocatalysis and
biotransformation (Van den Burg, 2003). Consequently, much attention has been
given to the microorganisms that are able to thrive in extreme environments. Thus, the
research on extremophiles is rapidly being transformed from an academic science to
an industrially viable technology. A huge amount of money is being invested
worldwide in the development of industrial as well as biomedical applications of
extremozymes. Table 1.2 enlist the different classes of extremophiles and potential
applications of their enzymes.
Biotechnologists have exploited extremophiles as a source of enzymes for the
degradation of polymers such as chitin, cellulose and starch (Sellek and Chaudhuri,
1999). Since the discovery of thermophilc microorganisms and invention of Taq
Polymerase from Thermus aquaticus, large number of thermophilic enzymes has
found industrial application including their importance as source of thermostable
enzymes (proteases, amylase, lipase, xylanase, cellulase, DNA restriction enzymes)
and other products of industrial interest (Bergquist and Morgan, 1992; Rainey et al.,
1994; Maugeri et al., 2001; Lama et al., 2004; Schallmey et al., 2004). The
thermoalkaliphilic catalase, breakdown hydrogen peroxide into oxygen and water,
was isolated from Thermus brockianus, found in Yellowstone National Park. The
thermoalkaliphilic catalase operates over a temperature range from 30°C to 94°C and
a pH range from 6-10. This catalase is extremely stable compared to other catalases at
high temperatures and pH. In a comparative study, the T. brockianus catalase
exhibited a half life of 15 days at 80°C and pH 10, while a catalase derived from
mesophilic Aspergillus niger had a half life of 15 seconds under the same conditions.
The catalase have applications for removal of hydrogen peroxide in industrial
processes such as pulp and paper bleaching, textile bleaching, food pasteurization, and
surface decontamination of food packaging.
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Table 1.2: Classification of extremophiles and applications of extremozymes
(Ven den Burg, 2003)
Class
Thermophiles
Psycrophiles
Halophiles
Alkaliphiles
Acidophiles
Extremozymes
Glucosyl hydrolysis
Chitinase
Xylanase
DNA polymerase
Dehydrogenase
Protease
Amylase
Cellulase
Lipase
Dehydrogenase
Protease
Dehydrogenase
Protease and cellulase
Amylase
Protease
Application
Starch, cellulose, chitin, pectin processing and
textile industries
Chitin modification for food and health product
Paper bleaching
Molecular biology (PCR)
Oxidation reaction.
Detergent, food application and dairy product.
Detergent and bakery
Detergent, feed and textile
Detergent, food and cosmetic
Biosensors
Peptide synthesis.
Biocatalysis in organic media.
Detergent, food and feed.
Starch processing
Feed component
1.4 Thermozymes
The main reason for the search of thermophilic micro-organisms is to obtain
thermostable enzymes useful for industrial applications. Thermophilic microbes as
such do not have many applications, but their enzymes, biomolecules and proteins
have wide industrial applications. There are several advantages in using thermostable
enzymes in industrial processes as compared to thermolabile enzymes. The main
advantage of increasing the temperature of process is that, the rate of reaction is also
increased. As a 10˚C increase in temperature, approximately doubles the reaction rate,
which in turn decrease the amount of enzyme required for the product formation
(Haki and Rakshit, 2003). The thermostable enzymes are able to tolerate higher
temperatures, which give longer half-life stability to the enzyme. It is not only their
thermostability, but also their greater stability under other extreme conditions like
high or low pH, low water availability etc. The use of higher temperature in industrial
processes reduces the risk of contamination caused by other mesophilic microorganisms. The thermostable enzymes (thermozymes) are very useful in the
processing of lower viscosity medium (at higher temperature viscosity is usually
reduced) and also reduces filtration, centrifugation and costs of pumping. The transfer
rates of heat and mass are also improved at high temperature, diffusion rates are
higher and mass transfer is less limiting. As temperature increases, more substrate
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dissolved which can shift the equilibrium to a higher product yield in comparison to
other enzymes of low temperature stability. Thus, thermophilic bacteria are regarded
as a promising source of thermostable enzymes and the enzymes are also often
resistant to and active in the presence of organic solvents and detergents (Jaenicke et
al., 1996).
Thermozymes like proteases, amylases, nucleases, lipases, cellulases,
xylanases, catalases, esterases, DNAase and biopolymers which are capable of
functioning and stable at high temperature have been commercialized in various
industries including food, baking, feed, chemical, pharmaceutical, paper and pulp, and
detergents (Haki and Rakshit, 2003). Hyperthermophilic enzymes can have higher
temperature optimum; for example, activity up to 142°C for amylopullulanase.
Enzymes from thermophiles and extreme thermophiles can replace their mesophilic
counterparts in different industrial processes. For instance, a variety of industries
employ microbial amylolytic enzymes in the enzymatic conversion of starch into
different sugar solutions, representing an important growth area of industrial enzyme
usage. Similarly, thermostable proteases are used in the decomposition of hard-todegrade animal proteins at high temperature. The use of thermostable glutaminase
enables enzyme reaction under high temperature conditions, for food production
processes and prevents contamination by saprophytes.
Thermostable DNA
polymerases, isolated from hyperthermophiles, have led to a tremendous advance in
molecular biology due to their capacity to amplify DNA, in the so-called polymerase
chain reaction (Fujiwara 2002) (Rothschild and Manicinelli, 2001). Various
thermozymes have been isolated and studied as described in table 1.3.
Table 1.3: Different classes of extremozmes from thermophiles.
Thermophiles
Sulfolobus
acidocaldarius
Sulfolobus
solfataricus
Pyrobaculum
aerophilum
Methanopyrus
kandleri
Pyrodictium
T opt
(°C)
75
Enzyme
Habitat
Reference
DNA polymerase
Proteinase
RNA polymerase
Topoisomerase I
Topoisomerase II
Terrestrial
Terrestrial
100
α-Amylase
β-Glycosidase
Proteinase
Elie et al., 1989
Lin and Tang, 1990
Bergquist and Morgan,
1993
Forterre et al., 1989
Nadal et al., 1988
Kengen et al., 1996
Marine
Volkl et al., 1994
98
Topoisomerase type I
Marine
Slesarev et al., 1994
105
DNA polymerase
Marine
Uemori et al., 1993
80
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occultum
Thermococcus
litoralis
Thermoproteus
tenax
Introduction
88
Proteinase
Marine
Klingeberg et al., 1991
88
RNA polymerase
α-Amylase
Cellulase
Xylanase
Terrestrial
Bergquist and Morgan,
1993
Kengen et al., 1996
Bragger et al., 1989
Desulfurococcus
mucosus
88
Marine
Pyrodictium
abyssi
97
Canganella et al., 1994
Bergquist and Morgan,
1993
Andrade, 1996
Pyrococcus
woesei
100
α-Amylase
Pullulanas
Transglucosylase
α-Amylase
Pullulanase
Xylanase
α-Amylase
α-Glycosidase
Marine
Marine
Koch et al., 1991
Leuschner and
Antranikian 1995
Certain enzymes from thermophiles display polyextremophilic features like
thermoalkaliphilicity, and thus showed activity at high temperature and high alkaline
conditions. Thermococcus profundus exhibited maximal amylase activity at 80-100°C
and stable in the range of pH 5.9 to 9.8. Pullulanase from Pyrodictium abyssi showed
highest activity at alkaline pH 9.0, and 100°C.
There is a tremendous demand of thermophilic bacteria because of their
biotechnological importance as sources of thermostable enzymes and other products
of industrial interest. The exploration and characterization of thermophilic microbial
communities continues and expected to stimulate hypotheses about the structure,
dynamics and distributions and phylogenetic relationship of thermophilic microbial
populations from different geographical regions. Hot spring differs from each others
in terms of temperature, chemical composition and geographical location, which
determine the nature of microorganisms. Therefore, exploration of the hot spring
microbial communities all around the world would provide deeper insights in
evolution of species and microflora residing deep inside the earth’s atmosphere.
Unlike earlier microbiological work, recent development of techniques, such as the
16S rRNA approach (Rainey et al., 1994) and genetic fingerprinting by RAPD
(Ronimus et al., 2003) analysis revealed considerably more details and accurate
analysis of microbial diversity and their evolution. Recently, Geobacillus species with
hydrolytic activities have been characterized from Jordanian hot springs (Obeidat et
al., 2012). Nine different thermophilic bacterial isolates representing Bacillus
licheniformis and Aeribacillus pallidus were characterized by molecular tests
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including fatty acid and BOX profiles, and 16S rDNA sequence form Pasinler Hot
spring of Turky (Ahmet et al., 2011).
Thermophilic microorganisms has been isolated from many thermal hot
springs across the world. Various hot spring of India such as Tarabalo (Odisha),
Bakreshwar (West Bengal), Soldhar ( Uttar pradesh) Suryakund (Jharkhand),
Manikaran (Himachal Pradesh), have been explored for thermophiles ( Panda 2011;
Sharma et al., 2012; Panda et al., 2013; Ghati et al., 2013; Verma et al 2014).
Tattapani is one of the volcanic regions of Himachal Pradesh, India, situated in the
snowy mountains of Himalayas. Till date, there is no report on the isolation and
characterization of microorganisms form Tattapani Hot Spring as reviewed by Daniel
(Daniel, 2014). Moreover, thermophilic microbes of Indian hotsprings are not well
studied at molecular level. Considering the wealth of amazing biodiversity of
thermophiles, the present study was undertaken to determine enzymatic characterstics
and the phylogenetic relationship among thermophilic bacteria isolated from water
and soil sediment samples of Tattapani Hot Spring of Himachal Pradesh, India. The
thermophilic bacteria identified from this study can be used for large scale production
of thermozymes by fermentation as well as by recombinant expression of the
thermophilic enzymes. Moreover phylogenetic study of the thermophiles would shed
light onto their evolution and diversity of their habitats.
1.5 Objectives of the research
1) Isolation and identification of thermophilic bacteria from Tattapani, hot
spring Mandi, Himachal Pradesh.
2) Screening and biochemical characterization of thermozymes such as
amylase, cellulase, protease, lipase and glutaminase.
3) Genetic relatedness of thermophilic bacterial isolates and their evolution
in relation to word wide isolated thermophiles.
4) Isolation and cloning of gene encoding for thermophilic glutaminase.
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