CHARACTERIZATION OF INDUSTRIALLY IMPORTANT

ENZYMES FROM THERMOPHILIC BACTERIA FROM HOT

SPRING OF TATTA PANI AJ&K

By

MISBAH AZIZ

(Regd. No. 2015-UKT-001120)

Session 2015-2017

Department of Biotechnology
Faculty of Basic and Applied Sciences
University of Kotli Azad Jammu and Kashmir
 CHARACTERIZATION OF INDUSTRIALLY IMPORTANT
ENZYMES FROM THERMOPHILIC BACTERIA FROM HOT
SPRING OF TATTA PANI AJ&K

BY

MISBAH AZIZ

(Regd. No. 2015-UKT-001120)

A Thesis

Submitted in partial fulfillment of the requirements for the degree of

Master of Philosophy

in

Biotechnology

Session 2015 -17

Department of Biotechnology
Faculty of Basic and Applied Sciences
University of Kotli Azad Jammu and Kashmir

ii
 CERTIFICATION

I hereby undertake that this research is an original one and no part of this thesis falls
under plagiarism. If found otherwise at any stage, I will be responsible for the
consequences.
Student’s Name: Misbah Aziz Signature: ____________________
Registration No. 2015-UKT-001120 Date: _____________________

Certified that the contents and form of thesis entitled " Characterization of
Industrially Important Enzymes from Thermophilic Bacteria from Hot Spring of
Tattapani AJ&K" submitted by Miss Misbah Aziz have been satisfactory for the
requirement of the degree of Master of Philosophy (M. Phil) in Biotechnology.

i. Supervisor: Dr. Simab Kanwal: _________________________

(Assistant Professor)

ii. Co-Supervisor: Raja Zafar Ishaque: _________________________

iii. External Examiner: _________________________

Chairperson
Department of Biotechnology

Dean Director
Faculty of Basic and Applied Sciences Advance Studies & Research

iii
 DEDICATION

“It is my genuine gratefulness and warmest regard that I dedicate this work to my

beloved parents & my family”

for their love, endless

support, encouragement

& sacrifices.

iv
 TABLE OF CONTENTS
Chapter Title Page No

Acknowledgments……………………………………………………………………...x

Abstract………………………………………………………………………………..xi

Chapter 1 ........................................................................................................................ 1

1 1.0 INTRODUCTION ........................................................................................... 1

1.1 THERMOPHILIC BACTERIA AND THEIR OCCURRENCE ..................... 1

1.2 ENZYME PRODUCTION FROM MICROBES ............................................. 2

1.3 INDUSTRIALLY IMPORTANT ENZYMES ................................................ 3

1.4 IMPORTANCE OF AMYLASE ..................................................................... 5

1.5 HOW THERMOPHILIC MICROBES LIVE AND YIELD ENZYMES AT

HIGHER TEMPERATURE? ...................................................................................... 6

1.6 SIGNIFICANCE OF THERMOPHILES ......................................................... 8

1.7 AIMS OF THIS RESEARCH .......................................................................... 8

Chapter 2 ........................................................................................................................ 9

2 2.0 LITERATURE REVIEW ............................................................................... 9

Chapter 3 ...................................................................................................................... 20

3 3.0 MATRIALS AND METHOD ....................................................................... 20

3.1 STUDY AREA ............................................................................................... 20

3.2 SAMPLE COLLECTION .............................................................................. 21

3.3 WATER PROPERTIES ................................................................................. 22

3.3.1 Temperature ............................................................................................ 22

3.3.2 pH & Electrical Conductivity ................................................................. 22

3.4 EQUIPMENT USED ..................................................................................... 23

3.5 ISOLATION OF THERMOPHILIC BACTERIA ......................................... 24

v
 3.6 PRESERVATION OF CULTURE ................................................................ 25

3.7 EXTERNAL STRUCTURE DESCRIPTION ............................................... 25

3.8 BIOCHEMICAL IDENTIFICATION OF THERMOPHILIC BACTERIA . 25

3.8.1 Oxidase test ............................................................................................. 25

3.8.2 Catalase test ............................................................................................ 25

3.8.3 Gram‟s staining ....................................................................................... 26

3.8.4 Molecular Characterization ..................................................................... 26

3.9 GROWTH ANALYSIS OF ISOLATED THERMOPHILIC BACTERIA ... 27

3.10 SCREENING FOR AMYLASE ENZYME ................................................... 27

Chapter 4 ...................................................................................................................... 29

4 4.0 RESULTS AND DISCUSSIONS .................................................................. 29

4.1 ANALYSIS OF TEMPERATURE, pH AND ELECTRICAL

CONDUCTIVITY OF COLLECTED SAMPLE ..................................................... 29

4.2 ISOLATION AND MORPHOLOGICAL STUDIES .................................... 31

4.3 GROWTH OF ISOLATED THERMOPHILIC BACTERIA ........................ 34

4.4 BIOCHEMICAL CHARACTERIZATION ................................................... 34

4.5 MOLECULAR CHARACTERIZATION OF ISOLATED THERMOPHILIC

BACTERIAL STRAINS ........................................................................................... 35

4.6 GROWTH ANALYSIS OF ISOLATED THERMOPHILIC BACTERIA ... 37

4.7 SCREENING FOR AMYLASE ENZYME ................................................... 39

5 CONCLUSION .................................................................................................... 44

REFRENCES .............................................................................................................. 45

vi
 List of Table
S. # Title Page No

Table 1.1: Some Industrially Important Enzymes and their Uses .................................. 4
Table 3.1: Equipment and their model number............................................................. 23
Table 3.2: Media composition of MacConkey agar and Nutrient agar ......................... 24
Table 4.1: Physicochemical Parameters of Collected Sample ...................................... 31
Table 4.2: Colony morphology of isolated strains ........................................................ 32
Table 4.3: Biochemical Characterization ...................................................................... 35

vii
 List of Figure

S. # Title Page No

Figure 3.1: Google map of study area Tatta pani.......................................................... 21

Figure 3.2: Study area (Hot spring Tattapani) .............................................................. 21
Figure 4.1: Collection of Sample .................................................................................. 22
Figure 4.2: Morphology of Bactarial Isolates Growing on Nutrient Ager.................... 33
Figure 4.3: Morphology of Bacterial Isolates Growing on MacConkey Ager ............. 34
Figure 4.4: Gram‟s Staining of Strain M 1 ................................................................... 35
Figure 4.5: Phylogenetic tree of isolated thermophilic bacterial strains ....................... 36
Figure 4.6: Enzyme Activity of All Five Strains, M1, M2 (A), M2 (B), M5, M8....... 43

viii
 LIST OF ABBREVIATION

Electrical conductivity EC

Gram gm

Hour Hr

Kilometers Km

Miles Mi

Milli liter mL

Nutrient agar NA

Optical density OD

ix
 ACKNOWLEDGMENTS
`Firstly, I am heartily thankful to Almighty ALLAH, Who gave me this opportunity to

continue my studies till this level (M. Phil) and to perform this research work.

Secondly, I am very thankful to my parents, brothers and sisters for their kind

support to educate me.

I wish to express my thanks to my supervisor Dr.Semab Kanwal for giving me the

opportunity to work with this fascinating subject. I would like to say thanks to the

department of biotechnology, university of Kotli, and all my respected teachers, Dr.

Sajida Rasheed, Mr. Zafar Ishaque and my class fellows to support me to complete

this work. I would also like to say thanx to my nephew Tallal who helped me a lot

during this work. This thesis is heartily dedicated to my mother who took the lead to

heaven before the completion of this work. May the Almighty ALLAH richly bless all

of you.

Misbah Aziz

x
 ABSTRACT
Thermophiles (the first extremophiles), which may nurture at temperature over

50 °C, have engrossed the concentration of many scientists due to their

biotechnological prospective. Remarkable molecules including unusual enzymes,

anticancer, antialgal and antibiotic compounds are secreted by thermophilic strains.

Thermophilic bacteria can produce thermostable enzymes like α -amylase, cellulases, α

and β- glucosidase, α and β-galactosidase, protease, and xylanase. These enzymes

remain constant at the temperature range of 90 to 105 °C.

This study was conducted to search enzyme constructing thermophiles bacteria

from hot spring of Tattapani Azad Kashmir. The temperature of hot spring was 62 °C

and pH was 8.06. Five thermophiles bacterial strains were isolated and checked for

their temperature range from 37 - 80 °C. One out of five bacterial strain showed

growth starting from 37 °C to 80 °C, and remaining four showed growth from 37 °C to

60 °C. Colony morphology of all these five strains was observed. Different

biochemical tests like catalase test, oxidase test and gram‟s staining were done for all

the five strains by using standard procedures based on Bergey‟s manual. PCR and

phylogenetic analysis based on partial 16S rRNA gene sequence comparison method

was used for the characterization of thermophilic bacteria. All these five bacterial

isolates were tested for amylase activity and were found positive for amylase activity.

xi
 Chapter 1

1 1.0 INTRODUCTION

1.1 THERMOPHILIC BACTERIA AND THEIR OCCURRENCE

Bacteria are omnipresent and extremely diverse species. They can live in

almost all kinds of unreceptive surroundings. According to a report of last two decades

it is exposed that 99 percent of bacteria existing in the atmosphere are quite unfamiliar

or ignored in research laboratory farming and therefore persist as unclear for their

environmental purposes and unemployed for biotechnological uses (Kellenberger,

2001).

Thermophile microorganisms like bacteria generally dwell in hot water springs.

These bacteria can live and continue their life at higher temperatures even more than

70 °C. They have been less discovered due to complications in separation and

preservation of uncontaminated culture. Consequently, their variety and

biotechnological prospective leftovers to discovered from common warm

environments. In the result of growing at higher temperatures and exclusive

macromolecular possessions, thermophilic microbes can have higher metabolic rate,

physically and chemically constant enzymes and lesser progress, however greater end

product produces than comparable mesophilic species (Haki & Rakshit, 2003).

Thermotolerant microbes have gained universal significance because of their

spectacular potential to yield heat labile enzyme that have widespread uses in

medications and productions (Coolbear et al., 1992).

1
 Geothermal regions are considered as the birthplace of thermophilic

microorganisms (Khalil, 2011). Geothermal landscapes are not common ecological

landscapes; they arise in groups, in a rare extensively detached places of the world

where the surroundings are precise for their existence. Because of the definite

landscape of the geothermal resources, warm water springs are accessible in a rare

zones only. The top renowned zones and those best considered are in Japan, Italy, New

Zealand Iceland, Indonesia United States, Central Africa, and Central America

(Baltaci et al., 2017 Boomer et al., 2009; Maugeri et al., 2001;). The striking story of

warm water possessions is the ecology with its variability of the creatures and the

molecular power of its constituents (Genc et al., 2015).

Heat labile bacteria dwell at 45°C-80°C and yield thermostable enzymes.

Enzyme that came from thermophilic bacteria also known as thermophilic enzyme

because these enzymes are thermostable and thermo-active (Fooladi & Sajjadian,

2012).

1.2 ENZYME PRODUCTION FROM MICROBES

Microbes are the utmost significant resources for the construction of enzyme.

Production of desirable enzyme can be enhanced through selecting the accurate

organism. Enzyme production for industrial use is a nonstop process for the separation

and classification of new auspicious strains by utilizing inexpensive carbon and

nitrogen source. Microbes have become progressively significant as creator of

industrial enzymes. Because of their biochemical multiplicity and the comfort with

which enzyme concentrations may be amplified via ecological and genetic

employment, efforts are now being made to substitute enzymes, which, conventionally

2
have been separated from complex eukaryotes. In the biotechnological industries

amylolytic enzymes are very essential because they degrade starch and vastly used in

food, fermentation, textile and paper industries (Pandey et al., 2000; Ashwini et al.,

2011).

Enzyme-based reactions are striking replacements to boring and exclusive

chemical methods. Because of continually growing industrial development, the request

for thermostable enzymes like lipases, amylases, proteases, cellulase, xylanases and

DNA polymerases has also amplified (Prakash et al., 2013). Microorganisms are

significant bases for enzyme production and are favored over animal and plant-based

enzyme production. In industrial procedures, contribution of bacterial enzymes is

higher than 50 percent (Turner et al., 2007).

1.3 INDUSTRIALLY IMPORTANT ENZYMES

Industrially exploitable enzymes are only few, although there are 3000

enzymes discovered so far. These are chiefly extracellular hydrolytic enzymes, which

cut down naturally happening polymers such as starch, proteins, pectin‟s and cellulose

(Vyas & Dixit, 2006).

Among these industrially significant enzymes are the protease: alkaline

protease is used in detergents due to its property of excessive constancy and protease

enzyme is also used in bioindustries like washing powder, food industry, leather

processing, and pharmaceuticals and for biological studies (Vijayalakshmi et al.,

2011). Furthermore, cellulase enzymes presented excessive commercial prospective

for making the glucose feedstock from agricultural cellulosic resources (Pandey et al.,

1999) and in the invention of bioethanol and value-added organic compounds from

3
renewable agricultural remains (Hardiman et al., 2010). Different enzymes have

important uses in bioindustries; for example, protease and amylases are collectively

used in various industries like the food industry, detergent industries, and

pharmaceuticals (Hmidet et al., 2009). Another significant enzyme gelatinase has

increased its importance as targets for drug developments, due to their role in

connective tissue degradation associated with tumor metastasis (Sathya & Ushadevi,

2014).

From the whole world-wide sales, 60 percent contribution is of protease

enzymes (Mala et al., 1998). Cellulose contains glucose units associated with β-1, 4-

glycosidic bonds in a linear mode and it is the most plentiful biological basis of feed

fuel and chemicals. In the recent manufacturing procedures, cellulolytic enzymes are

used in cleansers, for color enhancing and making pliable, pitting of jeans,

pretreatment of cellulosic biomass and industrial wilds (Sukumaran et al., 2005; Jang

and Chang, 2005).

Table 1.1: Some Industrially Important Enzymes and their Uses

ENZYMES USES
Protease Used in detergents and bioindustries like washing powder, food
industry, leather processing, and pharmaceuticals
Cellulase Used in the manufacturing of glucose feedstock from agricultural
cellulosic resources and in the invention of bioethanol
Amylases Used in food industry, detergent industries, and pharmaceuticals
Gelatinase Used in drug development
Xylanases Used in bioconversion of lignocelluloses, prebleaching of Kraft pulp
and fuels

4
1.4 IMPORTANCE OF AMYLASE

Amylase enzyme accounts for 25 percent of the worldwide enzyme market and

it is very significant industrial enzyme. Due to constancy at low pH thermostable α-

amylase has widespread profitable use in starch processing and also used in fermenting

and sugar making, desizing in fabric industries and in cleanser making procedures

because of its property of constancy at high pH (Leveque et al., 2000; Saxena et al.,

2007).

Plant, bacteria, fungi and animal, yield diverse kinds of amylase enzymes. Many

investigators produce amylase enzyme by utilizing Bacillus spp. (Zambare, 2010).

The enzyme α-amylase is used for manufacturing of glucose syrup. For this

procedure the α-amylase is utilized in the initial phase of enzymatic deprivation for

producing a combination of fructose and glucose with greater fructose content. Many

kinds of plants animals and microbes can produce amylase enzyme. The amylases

from microbial sources encounter industrial burdens because it is inexpensive when

manufactured in huge amounts (Lonsane & Ramesh, 1990). Amylase has been

obtained from different kinds of yeast, fungi, actinomycetes and bacteria but, enzymes

obtained through bacterial and fungal sources have conquered uses in manufacturing

areas. Due to its flexibility and massive accessibility microbial amylase is favored over

other sources. Amylase from microbial source has nearly exceeded the artificial

sources in several industries (Pandey et al., 2000). Amylolytic enzymes are

extensively dispersed in bacteria and fungi. They are characterized in to exo-acting,

endo-acting and debranching enzymes. Rare bacterial amylases are exist in

alkalophilic acidophilic, and thermo acidophilic bacteria (Boyer & Ingle, 1972). At the

5
present time, amylase obtained from these resources is enormously utilized in amylase

construction underneath thrilling situations of pH and temperature. There are

numerous hearsays on starch corrupting microbes from diverse resources and relevant

amylase activity (Kathiresan & Manivannan, 2006).

In the worldwide enzyme market thermostable amylase accounts 30 percent

which is used in industries (Munoz et al., 2011).

1.5 HOW THERMOPHILIC MICROBES LIVE AND YIELD ENZYMES AT

HIGHER TEMPERATURE?

Microbes adjust themselves according to the circumstances. In case of heat

labile microbes, plasma membrane has saturated fatty acid that is responsible for

hydrophobic situation of the cell and sustain the cell strictness at high temperatures

(Herbert & Sharp, 1992). Additionally, the hyperthermophilic archae have lipids

associated with ether on the cell wall, which is answerable for heat confrontation (Rosa

et al., 1994). Freshly, tetraether membrane lipids were described in a thermo

acidophilic euryarchaeota Candidatus “Aciduliprofundumboonei”(Schouten et al.,

2008). Moreover to the mechanical alterations of cell membrane and cell wall, the

DNA of thermophilic microbes holds reverse DNA gyrase, which improve the melting

point by creating super coils in the DNA (Lopez, 1999). In Sulfolobussolfataricus a

minor DNA binding protein, Sso7d, not only responsible for heat tolerance of the

DNA but also stimulates the annealing of complementary strands above the melting

point and the ATPase-dependent rescue of the aggregated proteins (Ciaramella et al.,

2002; Guagliardi et al., 2000). Heat-labile microbes have special heat-labile proteins

which repel denaturation and proteolysis (Kumar & Nussinov, 2001). One more reason

6
of thermophilic actinomycetes, archae and bacteria to adjust at higher temperatures is

the amplified electrostatic, disulphide and hydrophobic interactions in their proteins

(Pebone et al., 2008).

Moreover to the above approaches, thermophilic actinomycetes, archea and

bacteria produce definite specific proteins, recognized as „chaperons‟ which aid to

refold the proteins to their inborn form and reestablish their functions (Singh et al.,

2010; Laksanalamai & Robb., 2004).

In Acidianusam bivalens which is hyperthermophilic archaeon, constancy and

folding pattern of ferredoxin by using Guanidine HCI as a chemical denaturant has

been studied (Pernilla et al., 2000). Definite thermophilic enzymes become stable by

definite conformational modifications (Fitter, 2003). In thermophilic microorganisms,

one more reason of thermo stability is the occurrence of definite metals (Vieille &

Zeikus, 2001), inorganic salts (Dodia et al., 2008), and substrate molecules is also

described. The typical Equilibrium of these enzymes established on thermal

performance, has been defined to expose the consequences of temperature on enzyme

action by adjustable active –inactive conversion positions (Daniel et al., 2008).

Definite computation alalogrithms and bioinformatics tools are planned to

foresee the protein inflexibility and firmness in command to increase the thermo

stability, because the claim of extremely thermos table enzymes is growing day by

day. Site-directed mutagenesis can be used for protein maintenance. The focused

development which includes gene shambling through which structure optimization

leads to the mixture of innovative characters, is another dominant protein engineering

technique to enhance the thermo stability (Hayashi et al., 2001).

7
1.6 SIGNIFICANCE OF THERMOPHILES

With the increasing progress of industrial development, claim of thermotolerant

enzymes has enlarged enormously because of its extraordinary thermostability and

viability to the procedures involved (Haki & Rakshit, 2003). One of the noticeable

benefits of doing biotechnological procedures at higher temperatures is decreasing the

hazard of impurity by communal mesophiles. Moreover, an action at the elevated

temperatures has not worthy impact on the bioavailability and solubility of organic

compounds leading to the competent bioremediation (Becker, 1997). Reactions at

elevated temperatures has more advantages of reduced thickness sand therefore

amplified diffusion coefficient of substrates leading to the auspicious equilibrium

movement in endothermic reactions (Kumar & Swati, 2001).

1.7 AIMS

There is a lot of hot water springs present in this universe but the information on micro

fauna found in hot water springs is not enough (Adhikary & Sahu, 1987). There is a lot

of endemic and diverse micro fauna present in hot water spring of Tatta Pani AJK,

which can be used for isolation of some commercial products. Keeping in view the

significance of the region for microbial diversity proposed research is conducted to

attain following objectives:

 Isolation of thermophilic bacteria from Tattapani AJK.

 Characterization of isolated thermophilic bacteria.

 Observation of amylase activity of isolated thermophilic bacteria.

8
 Chapter 2

2 2.0 LITERATURE REVIEW

At elevated temperature farming of thermophiles is precisely and efficiently

beneficial as it minimizes the risk of pollution and viscosity (Turner, 2007).

Thermophiles, psychrophiles, halophiles, and acidophiles are included among the chief

extremophiles (Singh, 2006 and Austain, 1988). On the basis of temperature

acceptance, thermophiles are further subdivided; for example facultative thermophiles

the temperature range is 50-65 °C but they can also nurture at 37 °C with slow rate,

obligate thermophiles can grow between the range of 65-70 °C but can‟t nurture under

40 °C. Tremendously thermophiles can reproduce from 40-70 °C with perfect

temperature of 65 °C and hyperthermophiles can grow over 90° C but their optimum

temperature is 80-115 °C. Due to hurdles in separation and preservation in pure culture

of actinomyces and thermophilic bacteria minimum species have been explored yet

(Brock, 1986). Bacteria establish a huge domain of prokaryotic microorganisms.

Bacteria were amongst the foremost natural life procedures to look on Earth and occur

in maximum of its environments. Bacteria occupy water soil, radioactive waste, acidic

hot springs and the profound portions of Earth‟s crust. Thermophilic bacteria have

different types. e.g. Geobacillus pallidus, Anoxybacillus flavithermus and

Actinomycetes etc. Actinomycetes are the bacteria which can produce several known

enzymes and also used to produce degrading multipart organic materials. From

Maharashtra (Kongan coast) a variety of enzymes such for example amylase,

gelatinase, protease, cellulases, ureases and lectinases, from different strains of

actinomycetes were reported (Gluve and Deshmukh, 2012). Actinomycetes are

9
filamentous grampositive bacteria spontaneously or saprophytically found in diverse

surroundings like warm water, soil and marine water (Santos et al., 2012; Padmadhas

& Ragunathan, 2010; Salami, 2004).

With the discovery of extremophiles researchers have explored an interesting

and demanding platform. Investigators also mesmerized with the ability of

extremophiles to grow under harsh conditions and production of industrially important

compounds like antibiotics and enzyme etc. Microorganisms like actinomycetes and

bacteria can produce these compounds (Turner, 2007).

The most important challenge for the survival of microaerophilic bacteria is the

survival, production of vigorous and constant enzymes at high temperatures. It is also

important to investigate their adaptive features. However, it also clear that the

thermophilic organism is also an important source of novel biotechnological products.

Xylanases, cellulases and amylases are the chief industrially vital enzymes globally

because of their wide application. For example, amylase enzyme is used in fabric,

detergent, fermentation and food manufacturing and bacterial and fungal amylases can

be appreciated in fine chemical and medicinal industries (Ghorai et al., 2009). With the

discovery of thermostable enzymes their application increases at rapid rate because of

lower cost and more stability (Rasoli et al., 2008). Cellulases are also used in various

industrial applications such as grain alcohol fermentation, animal feed production,

starch processing extraction of fruit juices and pulp and paper industry (Ogel et al.,

2001).

One of the prime industrial enzymes is xylanases which have the largest

demand for bioconversion of lignocelluloses, prebleaching of Kraft pulp and fuels

10
(Kim et al., 2000). It is also estimated that enzymes help in dough handing, stability

and bread volume (Bhat, 2000).

Ragunthan & Padhmadas (2013) conducted a study to collect Streptomyces

spp. Rhodococcus spp. from Siruvani forest of Tamilandu and estimated that amylase

production for Streptomyces spp was maximum 56 u/ml whereas for Rhodococcus it

was 50 u/ml on third day of incubation but the optimum temperature was same for

both the strains (40 °C).

Chaudhary & Prabhu (2016) worked on thermophilic actinomycetes for the

invention of technologically imperative enzymes cellulases and amylase. They isolated

five strains of actinomycetes from hot water spring located at Vajreshwari near

Mumbai. Out of five, one strain of actinomycetes produced maximum yield of amylase

at the temperature of 55° C and pH 4.8. For cellulases production, its pH was 9 and

temperature was same as 55 °C.

Zahoor et al. (2012) isolated a bacterium TP1 (Geobacillus pallidus) from the

hot water spring of AJK. At optimum temperature 65 °C and pH 7, the growth of

thermophilic facultatively anaerobic bacterium TP1 (Geobacillus pallidus) was good.

It was used for the hydrolysis of Gelatin and Ortho Nitrophenyl β

Galactopyranosidase.

Jadoon et al. (2014) conducted a study to collect Thermo actinomycete

sacchari from Tatta Pani AJK. On AGS medium growth of actinomycete and amylase

production was found maximum Thermophiles have different classes, e.g. moderate,

extreme and hyperthermophiles. Modest thermophiles have optimum growth rate at 50

to 60°C exciting thermophiles have ideal growth rate at 60 to 80°C and

hyperthermophiles have optimum growth rate at 80 to110°C (Gupta et al., 2014).

11
Thermophiles have been secluded from diverse environmental regions like hot springs

and profound sea of the earth. Different genera like Pyrobaculum Pyrodictium,

Pyrococcus, and Melanopyrus of the domain Archaea have such organisms, which can

grow at maximum temperature of 103 to 110 C; while Ascomycetes and Zygomycetes

classes of fungi have maximum growing temperatures (Busk and Lange. 2013), within

bacteria, Thermotoga maritime and Aquifexpyrophilus show the maximum growing

temperatures of 90 and 95 C correspondingly (Kumar et al.,2014). Thermophile

microbes can be categorized as Gram-positive or Gram-negative, they can occur under

aerobic or anaerobic circumstances, and some of thermophiles have the process of

spore formation. Researchers have focused their lessons to determine latest genus and

species of thermophiles throughout the world because of their amplified significance,

prospective uses and applications in diverse fields (Aanniz et al., 2015; Cihan et al.,

2014; Yoneda et al., 2013).

The knowledge about enzyme producing microbial community present in warm

water springs of AJK and Pakistan is not sufficient. The present study was proposed to

explore thermophilic bacteria from Tattapani AJK and extraction of industrially

important enzymes from them.

The exciting bionetworks are specially made up for those microorganisms who

can live in the temperature that is suitable for highly thermophiles. In these types of

environments, the conditions of pH, temperature, salinity and pressure are very low or

high. Archaea, bacteria and eukarya all these three disciplines of life can have highly

thermophilic microbes. Numerous researches engrossed on their potential as bases of

extremely energetic enzymes „extremozymes‟ and other products like antibiotics,

compatible solutes (Robb et al.,2008).

12
 There are certain complications in growing classification and documentation of

thermophiles (Savas et al., 2009). Expansion of molecular practices such as PCR-

based procedures like rep-PCR and 16S rRNA permit their consistent documentation,

while conservative techniques are uncontrollable and not dependable (Kublanov et

al.,2009).

In 1990‟s the events in the area of thermostable enzymes has intensely

amplified due to the usage and progress of molecular biology procedures allowing

genetic scrutiny and gene transferences for recombinant construction. This also

encouraged separation of several microorganisms from warm atmospheres in order to

contact enzymes that could meaningfully rise the space for enzymatic bioprocess

operations. Taq- polymerase, a heat-labile enzyme was analytically used in polymerase

chain reactions (PCR) for amplification of DNA is one of the initial fruitful

commercialized examples in this area, and many other DNA altering enzymes from

thermophilic resources have been commercialized (Podar et al., 2006; Satyanarayana

et al., 2005; Fujiwara, 2002). One of the most interesting things is to seeking for

industrial enzyme for their usage in practical goods and progressions at a big scale

very oftenely. These enzymes can be beneficial as industrial reagents as they hardly

involve poisonous metal ions for functionality, hereafter generating the opportunity to

use more ecologically responsive processing (Comfort et al., 2004).

Our planet earth has huge number of unknown reservoir of biodiversity in the

microbial world (Verma, 2014). Extremophiles are microbes that can live at exciting

environmental condition (Horikoshi & Grant, 1998). Amongst this heat labile

microbes covers the greater percentage that display best development at temperature of

13
50°C or higher. The enzyme from Thermus aquatics which is an industrially important

enzyme was obtained from a thermophiles (Thermus aquatics) which dwells in hot

springs. The temperature and environmental condition of these hot springs can be from

moderate to high (Sharma, 2013; Mathai, 2015).

Different strains of Actinomycetes like Streptomycetes spp. and Actinomadura

produce an enzyme named Keratinases which is an industrially important enzyme

(Habbeche & Kerouaz et al., 2014). Hydrolysis of keratin is frequently done with the

enzyme Keratinases. There is an excessive request for emerging biotechnological

replacements for reutilizing of keratic wastes, renovating unexploited chicken feather,

hairs, nails and wool to beneficial goods with the assistance of Actinomycetes

keratinases (Dastager & Lee et al., 2008).

Many species of Streptomyces like S. lydicus produce an enzyme pectinase

(Jacob et al., 2008). Pectinases are applied in food manufacturing for abstraction and

elucidation of wines, juices, oils, flavoring compounds and in fabric industry

pectinases are used in manufacturing of linen textile and hemp production. There are

many kinds of pectinases, one of them is Polygalacturonase which is commonly

applied in diverse industries (Janki et al., 2016).

chitnases enzymes are frequently used in industries because these are very

energetic at different Chitinases are one more collection of technologically imperative

enzymes which have capability to hydrolyse chitin. A few kinds of actinomycetes

release heat labile enzyme named chitaneses. These range of pH (Gadre, & Kulkarni

2002). Streptomyces thermoviolaceus and Microbispora spp. are recognized as

14
chitineses manufacturers. Both these strains belong to Actinomycetes and enzyme

chitinases produced by these strains was used to improve chitibiose, a potential

antioxidant which typically have application in biomedical and diet manufacturing

(Bhattacharya et al., 2007). The strains of actinomycetes other than Streptomyces such

as Nocardiopsis parasina produce chitinases which is beneficial in hydrolysis of chitin

oligosaccharides which has potential to apply as antioxidant, antimicrobial, anticancer,

anticoagulant and antitumor agent (Horikoshi,1999). Leather industry produce

different kinds of wastes which is disposed off by using chitinases. Streptomyces spp.

like S. aureofaciens, S. griseoloalbus and S. griseus produce enzyme chitinases which

is the potential antifungal agent and is beneficial against phytopathogenic fungi

(Inamori & Tsujibo et al., 2003).

Hydrolysis of triacylglyceroles to glyceroles and free fatty acids is catalyzed by

the enzymes lipases. Now a days enzyme lipases are used as biocatalyst because they

demonstrate exclusive chemo-, regio-, enantioselectivities, which allows the

construction of innovative medications, agrochemicals, and satisfactory foodstuffs

(Kaushik et al., 2006).

Lipases have huge applications in industries like food, pharmaceuticals,

cleansers, cosmetics, leather, dairy, fabric, and even biodiesel, because several lipases

have capacity to do hydrolytic as well as synthetic reactions (Robles-Medina, 2009;

Hasan et al., 2006).

A large number of extracellular lipases are commercially access able which are

produced by microbes, although lipases are commonly found in animals and plants

(Salihu et al., 2012).

15
 Industries have a big problem about the price of lipase manufacturing

procedures. So, numerous labors are being made to apply wastes as raw supplies for

lipase formation. Now a days lipase and other supplementary products are mainly

manufactured through agronomic remains and it would hold a bulging place in

upcoming biotechnologies, mostly due to its eco friendliness and elasticity to both

developing and developed countries. Numerous remains for example oil cakes, fibrous

residues, and industrial waste have growing consideration as plentiful and inexpensive

renewable feedstock (Salihu et al., 2012; Sahasrabudhe et al., 2012). In biotechnology,

mostly enzymes from thermophiles and alkaliphiles are applied because of their high

constancy at adverse working and /or storing situations (Golaki & Karkhane et al.,

2015).

Several compensations were made for booming out biotechnological and

manufacturing procedures in high temperatures: high solubility of substrates (in

specific for unwell solvable or polymeric molecules) resulting in sophisticated creation

profit, higher feedback taxes, enlarged accessibility of substrates, reduced hazard of

microbial infection, and lesser thickness of reaction mixtures which in turn decreases

the expenses associated to propelling, purification, and centrifugation, and saving an

excessive control rate would be exploited for cooling (Bisht & Panda, 2011; Trincone,

2011).

In 2004, Business Communication Company Inc, reported that international

marketplace for industrial enzymes was projected to totally $2 billion. Additionally,

the yearly growing frequency of manufacturing enzymes is forecast to be between 4

and 5% and with this approach lesser charges of enzymes because of an increased

16
struggle at the marketplace. The manufacturing enzyme marketplace can be divided

into three application sections: (1) technical enzymes, (2) food enzymes, and (3)

animal feed enzymes. In the entire world marketplace technical enzymes are the chief

parts where enzymes applied for cleansers and pulp and paper establish 52 percent in

the market. Important enzymes in this unit are hydrolytic enzymes, categorized as

proteases and amylases, which cover 20 and 25 percent of the total market,

correspondingly. Hydrolases are usually easy to use in bioprocesses, as they typically

do not want co-factors or complex substrates. Likewise, they can be applied at an

initial phase on the voluntarily accessible material found in the forest and agricultural

sectors.

The variety of microorganisms has been conventionally considered by means

of the valuation of existence in specific species by various genotypic and phenotypic

approaches (Curtis and Sloan, 2004; Dahllof, 2002; Farber, 1996). In the 1970, the

term polyphasic classification was recommended, directing at the incorporation of

diverse categories of consensual information and material on phylogeny, phenotype

and genotype of microbes and ultimately at the purpose of distinctive designs precisely

to the isolates (Vandamme et al.,1970). Heat resistant bacteria were further prevalent

and characterized by several species. They have been discovered from different types

of habitats such as telluric and aquatic warm environments, as well as natural and

synthetic surroundings (Stetter, 2006; Ovreas, 2000). These environments comprise of

continental solfataras, profound geothermally heated oil comprising stratifications,

narrow marine, profound sea warm deposits and hydrothermal outlets positioned as for

as 4,000 m beneath sea level (Vieille & Zeikus, 2001).

17
 Irregularly warm atmospheres like those which are produced via sun energy or

the disintegration of organic matter rarely authorize the development of thermophiles

(Cava et al., 2009). In the warm industrial surroundings, thermophiles and

hyperthermophiles have also been found. But a countless diversity of bacteria has been

separated from hot springs. The surroundings of hot springs frequently comprise of

purple and green photosynthetic bacteria, mats of cyanobacteria, thermotogales, and

Aquificals. More variety which dwells in hot springs includes some enterobacteria like

Clostridium, Bacillus and Thiobacillus or thionic bacteria (Stetter, 2006; Curtis &

Sloan, 2004).

For assembling main taxonomic characteristics of prokaryotes, the Bergey‟s

Manual of Systematic Bacteriology was used. It gives material in entire known species

of prokaryotes and holds a lot of explanations to assess the possessions specified

(Madigan et al., 1997). In recognizing bacteria, a few universal features have main

status for defining the main clusters to which the novel isolate was possible to have its

place. Morphological features like rod, coccus, helical or other; Gram positive or

negative reactions and nutrional classification like phototrophy, chemoorganotrophy

and chemolithotrophy are very significant and mostly used. The other significant

characteristics include products of fermentation, antibiotic sensitivity, temperature and

pH necessities, pathogenicity and immunological characteristics (Madigan et al., 1997;

Smibert & Krieg, 1994). Subsequently phenotypic indicators might not be firmly

stated underneath certain ecological or culture situations. For the discrepancy of

microbes only phenotypic methodologies are not used, because these are not as much

reasonable and dependable. The molecular description approaches include DNA built

18
investigation of chromosomal and extra chromosomal genetic material (Farber, 1996).

The evaluation of ribosomal RNA sequences recognized a molecular sequence-based

phylogenetic tree that would be applied to narrate entire creatures and renovate the

antiquity of Lifecycle (Woese et al., 1990; Woese & Fox, 1977). Ribosomal RNA

twisted out to be a brilliant evolutionary chronometer, due to the expected antiquity of

the protein manufacturing procedure. Ribosomal RNA was an antique molecule,

functionally persistent, commonly dispersed and temperately well preserved across

wide phylogenetic detachments (Madigan et al., 1997). Furthermore, there was no

indication of lateral gene transference of rRNA genes among dissimilar species and

therefore rRNA genes can transport accurate evidence concerning evolutionary

associations (Pace, 1997). Numerous other genotypic approaches like; The amplified

rDNA retriction analysis (ARDRA) (Ovreas, 2000) and the denaturing gradient gel

electrophoresis(DGGE) (Muyzer, 1999) were assumed fewer benefits because of the

newest progress in high throughout clone libraries organization schemes as well as

sequencing practices, which made feasible to process further comprehensive

admittance in a comparatively brief period (Abd-El-Haleem, 2002).

19
 Chapter 3

3 3.0 MATRIALS AND METHOD

3.1 STUDY AREA

Tatta Pani is a village in Poonch District, Azad Kashmir. The distance of Tatta

Pani from Rawalakot is 45 kilometers (Km) (28 miles) (mi), distance from Kotli is 26

km (16mi), and the distance from Hajeera is 29 km (18mi). It is located on the series

of Poonch River at the elevation of 2,237 feet. They are situated between the longitude

73o56´41.28” E and latitude 33o36´07.64” N, at an altitude of 1200 m (Zahoor et

al.,2012).

Heat is created because of the of Sulphur existence in Patala shales, resistance

along with the Chief Edge Thrust and profound rotation of swift water below the

surface is the probable reason of warmness that results into hot water springs in Tatta

Pani, Azad Kashmir (Khan et al., 1999).

This village is renowned for its springs of warm water have Sulphur. A tourist

lodge of AJK Tourism & Archaeology Department has situated here for tourists stay.

These Sulphur springs surround the area of 1square km, and are understood to have

therapeutic values. The sulphurous spring water is said to have remedial influences

that delivered from disorders like joint pain, fatigue and skin diseases.

20
 Figure 3.1: Google map of study area Tatta pani

Figure 3.2: Study area (Hot spring Tattapani)

3.2 SAMPLE COLLECTION

The water sample was gathered from inside the source of the warm water

spring Tattapani, Kotli AJK. and experiments were conducted at Department of

Biotechnology, University of Kotli, Azad Jammu and Kashmir Pakistan. Samples

were taken in a disinfected flask and transported to laboratory instantly and spreaded

on agar plates within 60 minutes after collection. Collected water sample was kept in

incubator and 1 ml of water sample was utilized for separating the thermophilic

bacteria via agar plate culture procedure.

21
 Figure 3.3: Collection of Sample
3.3 WATER PROPERTIES

Physico chemical parameters like conductivity, pH and temperature were

determined through applying multi meter.

3.3.1 Temperature

Temperature of the water was measured on the spot of hot spring by using

mercury filled thermometer.

3.3.2 pH & Electrical Conductivity

The collected water sample was occupied in minor beaker and then the probe of the pH

meter was positioned inside the water and saved for some time. The reading was

exposed on the pH meter, but the ultimate value was taken at that time when the

reading on the screen became static.

22
3.4 EQUIPMENT USED

Material and equipment used are given in (Table no. 2)

Table 3.2: Equipment and their model number

Equipment Model

pH meter pH Master

Autoclave HvA-110

Incubator D116.0743

Microscope IM-910

Laminar flow HD-1220

Centrifuge machine Z 216mk

Micropipettes P5000

Shaking incubator 13000irmeco

Ultra- freezer MDF-U33V

Spectrophotometer AE-S-MD90

Material used: petri plates, conical flasks, test tubes, test tube stands, measuring

cylinders, glass slides, filter paper, wire loop, glass beakers etc.

23
3.5 ISOLATION OF THERMOPHILIC BACTERIA

The water sample had been taken from hot water spring. Plates of Nutrient agar

(NA) and MacConkey agar were prepared aseptically, and water sample was spread on

the plates. Isolation of thermophilic bacteria to attain single colony was done by

applying spread and streak plate procedure. These plates were sealed, labeled and kept

at 55°C in incubator. Fast growing bacterial plate was chosen when agar plate was

ready. Bacteria with different shape growing on agar plate were selected and processed

further. By using sterilized procedure, the isolated bacteria on the agar plate were

streaked.

The streaked agar plate was wrapped and reserved at 55 °C in incubator for

growth. Afterward 24 hours (hrs) another streak plate technique and incubation was

done to purify the bacteria.

Ideal temperature for development was determined by incubating the isolates in NA

medium at different temperatures (37- 80 oC) with 5 oC interval.

Table 3.2: Media composition of MacConkey agar and Nutrient agar

Ingredients of MacConkey agar gm/L Ingredients of Nutrient agar gm/L

Peptone 17 Peptone 0.5

Protease peptone 3
Yeast extract 0.3
Lactose monohydrates 10
NaCl 0.5
Bile salt 1.5
Sodium chloride 5 Glucose 0.25
Neutral red 0.03
Agar 1.5
Agar 13.5
Final pH (at 25 °C) 7.4± 0.2
Final pH (at 25 °C) 7.1 ±0

24
3.6 PRESERVATION OF CULTURE

Bacterial culture was preserved at 4°C in nutrient agar. Bacterial cultures was

sub cultured at 15 days interval, and also preserved the cultures in 80 percent glycerol

at minus 20 °C.

3.7 EXTERNAL STRUCTURE DESCRIPTION

The external structural features like shape color and margin of bacterial

colonies were detected from 48 h culture on NA agar plate.

3.8 BIOCHEMICAL IDENTIFICATION OF THERMOPHILIC BACTERIA

Biochemical assessments were done by typical procedure built on Bergey‟s

manual. For identification of thermophilic microbes from hot water spring, several

biochemical identification procedures such as Gram staining, catalase test, oxidase test

had been used.

3.8.1 Oxidase test

In this test filter paper swamped with the stain tetra methyl phenylenediamine

dihydrochloride was used. Put one drop of sterile distilled water on filter paper and

took the bacterial colony with the help of wire loop. Then smeared on filter paper with

platinum loop observed that part of filter paper for alteration in color to dark blue or

purple within 15-20 seconds. Blue color indicates it Oxidase positive and if no color

appears on filter paper it indicates that it is Oxidase negative.

3.8.2 Catalase test

In catalase test 3 percent Hydrogen peroxide is used. Take small bacterial

colony with the help of sterile wire loop on the surface of dehydrated fresh glass slide.

25
Put a droplet of 3 percent Hydrogen peroxide on the glass slide having bacterial colony

on it. If the enzyme catalase is present in the bacterial colony then quick bubble will

produce and there will be no bubble production if there is no enzyme. Oxygen will be

evolved in the form of bubbles within 5-10 seconds and this will be catalase positive.

If there is no bubble production, the result will catalase negative.

3.8.3 Gram‟s staining

Gram negative and positive bacteria are differentiated on the basis of staining

test which is called as Gram‟s staining test. Take a dehydrated fresh glass slide and

put a drop of distilled water at the center of slide and then take a bacterial colony with

the help of sterilized wire loop and make smear with the water drop. After air drying

the smear fix it with heat. Then treat that fixed bacterial colony with primary stain

crystal violet for one minute and then wash with water. After that lay some droplets of

Gram iodine solution on that slide and keep it for 60 seconds, then rinse it with tap

water, after that decolorized it with 95 percent alcohol and again washed it with water

instantly after decolorization. At last used some droplets of counter stain safranin on

slide and after 45 seconds washed with water and observed under microscope.

3.8.4 Molecular Characterization

Thermophilic Bacterial isolates were sent to MACROGEN (Seoul Korea) company for

molecular identification. The bacterial 16S rRNA gene fragments were amplified by

PCR using Applied Biosystems 7500 Real time PCR using primers UF (5‟-

TCCTACGGGAGGCAGCAGT3‟) UR(5‟GGACTACCAGGGTATCTAATCCTGTT-

3‟) by Macrogen, Inc., Seoul, Korea.

26
3.9 GROWTH ANALYSIS OF ISOLATED THERMOPHILIC BACTERIA

Application of spectrophotometer is to measure optical density (OD) which is

used as a measure of the concentration of bacteria in a suspension. Light scattering is

occurred, when evident light is passed through a cell suspension. If sufficient amount

of bacteria or any other material is present in suspension, then more light is scattered.

Sterilized broth media was arranged and used for inoculation at suitable temperature.

The flask having sterilized media should not be too hot while using, it should be easily

handled. Spectrophotometer should be ready to use before time. Wavelength of

spectrophotometer was established at 600 nm. Fresh clean cuvette was taken and

added 3ml of non-inoculated hygienic media inside the cuvette. Then noted the optical

density of blank media. After finding the optical density of that blank media inoculated

that media with different strains in different conical flasks. Instantly next to the

inoculation, 3 ml of the inoculated media was taken and pipette it into a fresh cuvette.

It was placed in the blanked spectrophotometer and noted the OD reading. This

reading was noted at time 0. Repeated the former step at 60-minute pauses till the

absorbance no more rises. Then plotted the readings of Time at the X-axis and OD at

the Y-axis on a graph (Gilbert, P. et al., 1987).

3.10 SCREENING FOR AMYLASE ENZYME

Plate assay technique was used to check extracellular amylase enzyme activity

for whole strains which were isolated during this research work. Plates having starch

nutrient agar were used for determining the activity of enzyme amylase. The

composition of starch nutrient agar; starch 10 gm; yeast extract 3gm; peptone 5gm;

agar 30 gm; NaCl 10 gm and pH 7 in 1 liter.

27
 Nutrient broth (100 ml) was equipped in 250 ml conical flask, autoclaved it at

121 °C for 15 minutes. Later the broth cool down inoculated with one isolate, and

placed it in shaking incubator (rpm 150-200 for bacteria) at 40 °C for 8 to 10 hrs.

Dilutions were made by taking 1ml from this 8-10 hours broth culture with 9ml

sterilized distilled water. This was 10 -0 dilution. Then took 1ml from 10-0 dilution and

added 9ml sterilized distilled water and made 10 -1 dilution. By repeating the same

process dilutions were made upto10-6 and 10-7. Different (10 µL and 50 µL)

concentrations of different dilutions were spread on sterilized starch nutrient agar

plates to get single colonies. At 40 °C starch NA plates were incubated for one day.

After attaining the single colonies, these plates were swamped with Gram‟s iodine

solution. The pure zones were obtained everywhere the single colonies which showed

the production of amylase enzyme (Nayaka and Vidyasagar, 2012). Streak plate

method was also used to check the enzyme activity. Same process was repeated for

each isolate to check amylase activity.

28
 Chapter 4

4 4.0 RESULTS AND DISCUSSIONS

This learning was aimed to separate the enzyme producing thermophilic

microbes from warm water spring Tatta pani Kotli Azad Jammu and Kashmir and

learning was directed in department of biotechnology university of Kotli Azad

Kashmir. Samples were composed in a sterile plastic bottles and instantly brought to

research laboratory. Water samples were stored in incubator. Physico chemical

parameters were analyzed.

4.1 ANALYSIS OF TEMPERATURE, pH AND ELECTRICAL CONDUCTIVITY

OF COLLECTED SAMPLE

As long as, temperature, pH, osmolarity or pressure are considered our planet

anchorages a large number of severe surroundings that are measured as exciting from

anthropocentric point of view. But these unusual biotopes have been fruitfully

occupied by several creatures, mostly extremophilic bacteria and archaea. The

probable biotechnological application of thermophilic bacteria and their thermotolerant

enzymes has directed to widespread separation studies in a varied diversity of

thermophilic atmosphere. Investigators from all over the world have discovered hot

springs among these atmospheres as a latent basis of thermophilic bacteria. Narayan et

al., 2008 from Savasavu hot spring of Fiji discovered aerobic thermophilic bacteria. In

2009, Lu et al. discovered thermophilic anaerobic bacteria from hot springs of

Tengchong Rehai., In 2011, from different hot springs of Jordan, thermophilic

bacteria, i.e., Geobacillus pallidus and Anoxybacillus flavithermus were discovered by

Albatayneh et al. In diverse areas of Pakistan equitably huge quantity of hot springs

29
are described. But the knowledge about microbial community present in warm water

springs of AJK and Pakistan is not sufficient (Zahoor et al., 2012).

In 2012, Zahoor et al. conducted a study to isolate a bacterium TP1 from hot

spring of Tattapani Kotli AJK., and recorded the temperature at the site of hot spring

as 82 °C (source of water), and 62°C at 10 feet far from the source, pH was 7.5 which

is somewhat alkaline, electrical conductivity (EC) value was 1.12 (mS/cm). In 2016,

Zahoor et al. isolated a bacterium TP2 from hot spring of Tattapani AJK, and also

described the comparative values of temperature, pH and electrical conductivity of his

and Khan et al. to create the profile of hot spring. In 1999, Khan et al., recorded the

temperature of hot spring fluctuating from 79-86 °C, almost neutral pH of 6.93 and

electrical conductivity value was ranging from 7020 -9560μS/cm. In 2016, Zahoor et

al., recorded the temperature of warm water spring ranging from 56 to 60 °C, pH

acidic ranging from 1.70 to 1.86 and electrical conductivity value was 462.6 μS/cm.

Temperature of water of hot spring during present study is 62°C, pH 8.06 alkaline and

electrical conductivity value is 96μS/cm. Water characterization results of present

study has showed some variation as compared to previous studies. These changes may

be because of the alteration in time period of this learning, and because of variation in

amount of sulfur and different ions concentrations present in water, or all these

changes may be due to the earthquake that happened in 2005. Results of water

parameters of present study are shown in table below.

30
Table 3.1: Physicochemical Parameters of Collected Sample

Parameters Values
Temperature ( C) 62
pH 8.06

Conductivity μS/cm 96

4.2 ISOLATION AND MORPHOLOGICAL STUDIES

After creating the profile of hot water spring Tattapani AJK, few bacteria were

isolated. Isolation was done by sampling water from bottom of hot spring. To separate

discriminating thermophilic bacterial strains from warm water spring Nutrient agar

was used. For the current learning over-all eight bacterial strains were separeted from

hot spring of Tattaapani AJK. Five out of eight were thermophilic and showed amylase

activity. One bacterial strain showed growth starting from 37 °C to 80 °C and four

bacterial strains showed growth starting from 37 °C to 60 °C in nutrient agar. The

whole five isolates were recognized by morphological, biochemical and cultural

characteristics. Isolated bacterial strains were identified by universal morphological

techniques built on size, shape, margin, colony color, surface and texture. Colony

morphology of thermophilic isolated bacteria shown in figure. Morphological

appearance of strain M1 was large in size, wavy margin, sides uneven, raised from the

center, wet appearance and milky off-white in color. Morphological appearance of

strain M 2(A) was large in size, flat, uneven margins, dry in center and wet on

margins, and white in color. Morphological appearance of strain M 2(B) was round

small, wet, translucent, and yellow in color. Morphological appearance of strain M 5

was medium in size, smooth margins, shiny appearance, elevated colon y milky yellow

31
in color. Morphological appearance of strain M 8 was large in size, smooth margins,

elevated colony milky white in color.

Table 4.2: Colony morphology of isolated strains

Strain Colony Morphology

M1 Large in size, wavy margin, sides uneven, raised from the center, wet

appearance and milky off white in color.

M 2(A) Large in size, flat, uneven margins, dry in center and wet on margins, and

white in color.

M 2(B) Round small, wet, translucent, and yellow in color.

M5 Medium in size, smooth margins, shiny appearance, elevated colony

milky yellow in color.

M8 Large in size, smooth margins, elevated colony milky white in color.

32
Figure 4.1: Morphology of Bactarial Isolates Growing on Nutrient Ager

33
 Figure 4.2: Morphology of Bacterial Isolates Growing on MacConkey Ager
4.3 GROWTH OF ISOLATED THERMOPHILIC BACTERIA

Growth of isolated bacteria was observed at different temperatures starting

from 37 to 80 °C. Strain M 1 showed the growth at temperatures starting from 37 to 80

°C, and the Strains M 2(A), M 2(B), M 5, and M 8 showed the growth at 37 to 60 °C.

4.4 BIOCHEMICAL CHARACTERIZATION

Bacterial isolates were biochemically investigated for the actions of Catalase,

Oxidase, and Gram staining. (Table 6). The trials were done to classify the

thermophilic bacterial isolates rendering to Bergey's Manual of Determinative

bacteriology. Table 6 displays the consequences of catalase test Oxidase test and

Gram‟s staining in which M 1 shown Catalase negative Oxidase negative and Gram

staining positive result. M 2(A) shown Catalase positive Oxidase positive and Gram

staining negative. M 2(B) shown Catalase positive Oxidase negative and Gram

staining negative result. M 5 shown Catalase positive Oxidase negative and Gram

staining negative result. M 8 shown Catalase negative Oxidase negative and Gram

staining positive results.

34
Table 4.3: Biochemical Characterization

Strains Catalase Oxidase Gram’s staining

M1 - - +

M 2(A) + + -

M 2(B) + - -

M5 + - -

M8 - - +

Positive + Negative –

Figure 4.3: Gram’s Staining of Strain M 1

4.5 MOLECULAR CHARACTERIZATION OF ISOLATED THERMOPHILIC

BACTERIAL STRAINS

For the current learning, five thermophilic bacterial strains were acknowledged

on the foundation of 16S rRNA gene sequences. These heat-labile bacterial strains

were separated from warm spring of Tattapani AJK. The results of thermophilic

35
bacterial strains after identification are shown in the figure in the form of phylogenetic

tree.

Figure 4.4: Phylogenetic tree of isolated thermophilic bacterial strains

The strain M1(S11) is closely related to Lysinibacillus, Parageobacillus,

Bacillus and Pseudomonas. Strain M 2A (S12) is closely related to Shigella and

Serratia. Strain M 2B (S13) is closely related to Enterobacteriaceae and Escherichia.

Strain M 5 (S14) and Strain M 8 (S15) are closely related to Kosakonia.

Lycinibacillus, Bacillus and Parageobacillus belongs to phylum Firmricutes.

Serratia belongs to phylum Proteobacteria and family Enterobacteriaceae. Shigella,

Kosakonia and Escherica also belongs to family Enterobacteriaceae. The whole

recognized bacterial strains are assembled together with strictly associated

resemblances. Five known bacterial strains sequences and nine additional sequences of

dissimilar bacterial species were applied as input sequences in the procedure of

alignments. The consequence of blast search specified that M 1 (S11) strain shares

73% resemblance with Lysinibacillus, 68% similarity with Pseudomonas, 65%

36
similarity with Serratia, Shigella and Kosakoia, 64% similarity with Escherichia,

Enterobacteriaceae and Bacillus, 61% similarity with Parageobacillus.

4.5 GROWTH ANALYSIS OF ISOLATED THERMOPHILIC BACTERIA

3
Growth Curve of Bacterial strains
2.5

1.5
OD(600n

0.5
m)

0
0 2 4 6 8 10 12
-0.5

M1 M2A M2B M5 M8

Time (hour)

Analysis of these bacterial strains was done through growth curve. It was

determined by the optical density (OD) at 40 ºC. Spectrophotometer at 600 nm

wavelength was set to measure OD at every 1 hr. interval.

The graph between optical density at Y-axis and time at X-axis was drawn to

show growth curve of isolated bacterial strains (Figure). According to fig; strain M 1

in fresh medium remains almost 2 hrs in lag phase, in which bacteria increase in size

only, because bacteria do not reproduce in this phase. At third hr M 1 strain enter into

acceleration phase. After acceleration phase strain M 1 enter into log phase which lasts

from fourth to sixth hr. In this dynamic phase microbes multiply very rapidly. At the

seventh hr, with the decline in development proportion of microorganisms at the

37
termination of log phase, commencement of declaration phase took place. Stationary

phase was started at eighth hr, during this phase growth of bacteria almost finish

because the substrate becomes insufficient and the metabolic end product prevent the

growth. At ninth hr death phase was started, due to the termination of metabolic rate

and energy assets. The cell dies at an exponential rate. Death of cells occur because of

toxins produced.

In bacterial growth curve optical density plotted versus time (figure) shown M

2(A) Strain inter into the lag phase. When M 2(A) is presented into the new medium it

took 2 h to regulate with the novel situation. This phase is called as lag phase in which

cellular metabolism enhanced and cells increase in size. But the bacteria are not able to

duplicate and thus no rise in cell mass. At third hr M 2(A) strain enter into acceleration

phase through which cells start increasing gradually. Actually, acceleration phase

attaches the lag phase and log phase. After the acceleration phase strain M 2(A) enter

into log phase which lasts from fourth to sixth hr. This is the most active phase for

growth of microorganisms, wherein multiplication occurs rapidly. At the seventh hr,

with the decline in growth rate of microorganisms at the end of log phase,

commencement of declaration phase took place. Stationary phase remained almost two

h(8&9hr). At tenth hr death phase was started. Strain M 2(B) remained in lag phase

almost one hr, and at second hr acceleration phase started. Log phase started from third

hr and remained till fifth hr. Fifth hr was declaration phase. Stationary phase was

remained from fifth to sixth hr. After that almost at seventh hr, death phase was

started. Strain M 5 remained in lag phase almost 2 hrs. At second hr acceleration phase

was started, and then log phase which remained from third to sixth hr. Sixth hr was

declaration phase. After that from sixth to seventh hr was stationary phase. Death
38
phase started from eighth hr. Strain M 8 remained in lag phase almost 2 hr. At second

hr acceleration phase was started, and then log phase which remained from third to

sixth hr. Sixth hr was declaration phase. After that from sixth to eighth hr was

stationary phase. Death phase started from ninth hr.

4.6 SCREENING FOR AMYLASE ENZYME

According to this study, whole thermophilic strains that were found in water after

identification were screened for extracellular amylase enzyme activity according to

defined way and this was gained that Isolates M 1, M 2(A), M 2(B), M 5, M 8 showed

good amylolytic activity. Amylase enzyme activity was verified by the creation of

clear zone around these isolates as shown in figure.

Thermophiles are adapted to dwell at higher temperatures in hot springs.

Microbes from these hot springs can produce distinctive enzymes that can be used in

many industrial procedures which require higher temperatures. Thermostable enzymes

obtained from thermophilic microbes are most extensively applied in industrial

processes (Haki & Rakslit 2003).For the manufacturing procedure of sweeteners from

starch, the pH which is used is near to 7 and 50 °C or more than 50 °C temperature is

applied °to avoid the frying properties and to minimize the thickness of starch pastes.

The risk of polymerization of D- glucose to isomaltose is also reduced due to the

process of hydrolysis done at high temperature (Pandey et al., 2000). Thermostable

amylases like the one obtained from these thermophilic bacteria are looked for this

purpose as their usage would also diminish infection hazard and decrease the reaction

time.

39
Application of such enzymes is, however often restricted by the cost of construction. It

is necessary to have some more research work to enhance the production cost of this

important enzyme from thermophilic bacteria.

40
41
42
Figure 4.5: Enzyme Activity of All Five Strains, M1, M2 (A), M2 (B), M5, M8.

43
 5 CONCLUSION

One of the major requirements for the synthesis of agitated food is α-amylases.

Applications of amylase enzyme is not only growing everyday in food and starch

productions but many other industries like pulp and paper, textile etc. are in search of

this enzyme (Gupta et al., 2001). With the rise in its submission range, the request is

for the enzyme with specificity. From the current learning it can be decided that five

thermophilic strains (M.1, M.2(A), M.2(B), M.5, M.8) were successfully isolated from

hot spring of Tatta Pani AJK. All these five thermostable strains produce amylase

enzyme. All these five thermophilic strains can be significantly used in industries due

to its industrially vital thermostable amylase enzyme.

44
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