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1
AEROBIC THERMOPHILIC BACTERIA FROM THE
SAVUSAVU HOT SPRINGS, FIJI ISLANDS
By
Vinay Vikash Narayan
A thesis submitted in partial fulfillment of the
requirements for the degree of
Master of Science
Division of Biology
School of Biological, Chemical and Environmental Sciences
Faculty of Science and Technology
University of the South Pacific
July 2007.
2
©Vinay Vikash Narayan
\
3
DECLARATION OF ORIGINALITY
I, Vinay Vikash Narayan, declare that this thesis is my own work and that, to the
best of my knowledge, it contains no material previously published, or substantially
overlapping with material submitted for the award of any other degree at any institution,
except where due acknowledgment is made in the text and in the references.
…………………………………..
Student Researcher
……………………………….
Date
…………………………………
Principal Supervisor
Dr. Dhana Rao
………………………………..
Date
4
ACKNOWLEDGEMENT
A sincere thanks goes to my principal supervisor, Dr. Dhana Rao for her immense
help in all walks of my research. Also, I would like to thank Dr. M. Hatha of Cochin
University of Science and Technology (India) for all the assistance provided in the
formulation and planning of my research. Hearty gratitude is also extended towards Mr.
Abhineshwar Vinay Prasad, laboratory technician at the Division of Biology, for helping
me with all the technical aspects of my research here in Fiji.
I would also like to acknowledge Professor Hugh Morgan, Ms. Lynn Parker, Mr.
Adrian Bayer, Mr. Anderas Reukert, Ms. Naomi Crawford and Ms. Rochelle Soo of the
Thermophile Research Unit at the University of Waikato for the immeasurable support in
carrying out the molecular analysis of my samples, provision of comparison cultures and
for making my stay in New Zealand a very educational one. Further gratitude is also
extended towards Mr. and Mrs. Mukesh Chand of Sevarekareka, Savusavu, for providing
me with accommodation every time that I went for my fieldwork at the Savusavu Hot
Springs.
Furthermore, I would like to thank my family and friends, especially Ms. Sujlesh
Sharma, for their support and understanding, for helping me up when my wings could not
remember how to fly and for providing me with words of encouragement/inspiration in
times when my motivation ran low.
5
I would like to express sincere gratitude to the University of the South of the
South Pacific Research Committee for approving and financially supporting my research.
This gratitude is further extended towards the Ministry of Food Safety and Agriculture,
New Zealand Quarantine Services and the Fiji Immigration and Fiji Quarantine Services
for allowing my safe passage along with my samples between Fiji and New Zealand.
All these words cannot express completely how grateful I am to all of you, but
without you, I would not have managed to successfully complete this endeavor.
6
ABSTRACT
Surveys of SavuSavu Hot Springs, Fiji’s largest and most active hot springs have
been restricted to mostly geological descriptions.
In the current investigation, a
microbiological study was conducted to determine the presence of aerobic thermophilic
bacteria. Samples were collected over four sampling periods between September 2005
and March 2006. 104 thermophilic bacterial isolates from these hot springs were
characterized by staining, biochemical, molecular tests and the ability to produce
extracellular hydrolytic enzymes. DNA was extracted using CTAB/chloroform-phenol
double extraction method and subjected to 16S rDNA PCR with primers EUB A (R) and
EUB B (F). Products were restriction digested using EcoRI and HaeIII, run on 2.5%
TBE-Agarose gel and analyzed using UV AlphaImager. Majority of the isolates were
Gram positive, produced endospores, and were motile. Catalase and oxidase activity
were prominent and most isolates could utilize glucose in an oxidative manner. Amylase,
lipase and gelatinase activities were also observed. Using Thermus and Bacillus strains
as positive controls, 58% of the isolates were identified as Anoxybacillus flavithermus,
19% as Geobacillus stearothermophilus/Bacillus licheniformis, 10% as Thermus TG153
and 10% as Thermus TG206. Four of the isolates were unique in their molecular patterns
suggesting that there may be novel bacteria in the Savusavu hot springs.
7
TABLE OF CONTENTS
Acknowledgements…………………………………………………….…..…….4
Abstract………………………………………………………………...………...6
Table of contents………………………………………………..………………..7
List of tables………………………………...…………………………………..11
List of figures……………………………………………………………………12
Abbreviations…………………………………………………………….……...15
Chapter 1: Introduction and Literature Review…………………………….16
1.1
Temperature as a limiting factor………………………………………...16
1.2
Thermophiles and evolution…………………………………………….17
1.3
Formation of thermal environments on Earth……………………… … 19
1.4
Importance of thermophiles……………………………………………..20
1.5
Thermozymes……………………………………………………………23
1.6
A young branch of microbiology..……………………………………….24
1.7
Aims.………………………………..……………………………………26
Chapter 2: Materials and Methods……………………………………………28
2.1
Sampling………………………………………………………………...28
2.2
Handling and Transport………………………………………………….28
2.3
Analysis of Geothermal environment…………………………………….29
2.4
Bacteriological analysis of Hot Pool water…………………………..…..29
2.5
Total Plate Counts (TPC)…………………………………………….…..29
8
2.6
Staining………………………………………………………………..…30
2.7
Biochemical testing…………………………………………………...…31
2.8
Determination of hydrolytic enzyme production……………………..…33
2.9
Overview of DNA Analysis…………………………...……………..…..34
2.10
16S rDNA Analysis……………………………………………………..36
2.10.1
DNA extraction…..……………………………………………….....36
2.10.2
DNA quantification…..…………………………………………...…37
2.10.3
Polymerase chain reaction (PCR)…………………………..……….38
2.10.4
Bacterial 16S rDNA PCR…….……….……………………………..39
2.10.5
Randomly Amplified Polymorphic DNA (RAPD) PCR….....…...….40
2.10.6
Restriction endonuclease digestion……………….………..…..…….41
2.10.7
Electrophoresis………………………………….…………..…..……42
2.10.7.1
Agarose gel electrophoresis…….…………………………..….…..42
Chapter 3: Results……………………….………………………………..…….44
3.1
Bacteriological analysis and tolerance limit tests……..…………..….….44
3.2
Growth temperature limits……..…………………………………..….…44
3.3
Sodium chloride/halophily limits………………………………..….……45
3.4
Growth at varied pH levels in Nutrient Broth…………………..…..……45
3.5
A: Biochemical characterization. ……………………………………….46
3.6
B: Extracellular hydrolytic enzyme screening ……………………….…52
3.7
C: DNA analysis……………………………………………………..…..55
9
Chapter 4: Discussion…………………………………………..………...…….66
4.1
The Savusavu Hot Springs………………………………………………66
4.2
Bacteriological Analysis and tolerance limit tests……..………………..68
4.3
Staining, biochemical and exoenzyme activity………………………….69
4.4
Molecular analysis of thermophilic bacterial isolates………………….70
4.5
Limitations and recommendations……………………………………..74
Chapter 5: Conclusion………………………………………………..…….…..76
Appendices……………………………………………………….……………..77
Appendix A: Growth Media……………………………..……….………..…..77
1. Nutrient Agar (NA)
2. Nutrient Broth (NB)
3. Medium 74
4. Medium 878
5. Motility Medium
6. Oxferm Media
7. Nutrient Starch Agar (NSA)
8. Nutrient Gelatine Agar (NGA)
9. Nutrient Tributyrin Agar (NTA)
10
Appendix B: Stains…………………………………………………………….79
1. Grams Crystal Violet
2. Grams Iodine
3. Safranin
4. Malachite Green
5. Nigrosin
6. Mercuric chloride
7. Lugols Iodine
Appendix C: 16S rDNA PCR product confirmation gels………………….…….80
Appendix D: Maps of Fiji showing Savusavu…………………………………...85
Appendix E: Phase Contrast Microscopy of the “Uniques”……………………..87
Appendix F: a. Additional pictures of Savusavu hot springs……………………88
b. Standard 1Kb Plus DNA Ladder………………………………..95
c. Temperature mapping of research site…………………………..96
References……………………………………………………………………….97
11
LIST OF TABLES
Table 1: List of PCR primer numbers, their sequences and the analysis technique used
Table 2: Recipe for preparation of template thermophilic bacterial DNA for 16S rDNA
PCR using primers EUB A and EUB B
Table 3: List of PCR components for RAPD analysis of template thermophilic bacterial
DNA
Table 4: List of PCR primers used for RAPD analysis and their sequences
Table 5: Recipe for preparation of 16S rDNA-PCR product for restriction endonuclease
digestion reaction
Table 6: List of restriction endonucleases, their corresponding buffers, incubation
temperature and cutting sites used for digestion of 16S rDNA-PCR products
12
LIST OF FIGURES
Figure 1: 060502(2): 16S r DNA PCR of isolates 70oC 1-19
Figure 2: 060502(1): 16S r DNA PCR of isolates 70oC 20-45
Figure 3: 060607: EcoRI/React 3 of isolates 70oC 8-42
Figure 4: 060613: Hae III/ React 2 of isolates 70oC 8-42
Figure 5: 060615gA: 16S r DNA PCR of isolates 70oC 38-55
Figure 6: 060615Gb: 16S r DNA PCR of isolates 70oC 57-66
Figure 7: 060619G1B: 16S r DNA PCR of isolates 70oC 58, 60 and positives
Figure 8: 060628Gel 1bF: 16S r DNA PCR of isolates 70oC 8-66
Figure 9: 060628Gel 1Good: 16S r DNA PCR of isolates 70oC 8-55
Figure 10: 060630GelA EcoRI/React 3 of isolates 70oC 38-55
Figure 11: 060630GelB EcoRI/React 3 of isolates 70oC 8-66
Figure 12: 30706HaeIII/React 2 of isolates 70oC 38-54
Figure 13: 130706HaeIII/React 2: 16S r DNA PCR of isolates 70oC 57-66
Figure 14: EcoR1/React 3;full 200706 of isolates 75oC 4-62
Figure 15: EcoR1/React 3 B 060705 of isolates 70oC 8-66
Figure 16: HaeIII/React 2: UNIQUE 210706 isolates 70oC 8-60
Figure 17: Figure 17: RAPD ENLARGED0607074 of isolates 70oC8, 35, 35, 45 and 60
Figure 18: RAPD 060713 isolates 70oC 8, 35, 37, 45, 55 and 6
Figure 19: RAPD NEW 060711 of positive controls
Figure 20: RAPD OPR13 180706 of isolates 70oC 35, 37 and positives
Figure 21: RAPD T3 060710 of isolates 70oC 8, 35, 37, 45, 55, 60 and positives
Figure 22: TRIAL GEL 4 060621 of isolates 75 oC 19, 58 and 60
Figure 23: UNIQUE ECOR1/React 3 210706 of isolates 70oC 8, 35, 37, 45, 55, 60
13
Figure 24: 16S PCR GA 200706 of isolates 75oC 4-18
Figure 25: 16S PCR GB 200706 75oC 19-161
Figure 26: 060613 Hae III/React 2 of isolates of 70oC 8-42
Figure 27: (A) Fiji islands map
(B) Detailed map of the Fiji islands showing Savusavu
Figure 28: Phase contract microscopy of unique thermophilic bacterial isolate 70 oC 8
Figure 32: Picture showing both, springs number 1(denser steam) and 5
Figure 33: Picture showing the runoff, spring number 6 (arrow) and spring number 1
Figure 34: Picture showing spring number 1
Figure 36: Picture showing wide view showing position of spring number 5 (steam) and
pathway leading to other springs
Figure 37: Picture showing runoff from the hot springs that flows down and links up with
the sea
Figure 38: Picture showing algae type 1 found in the runoff stream/spring 5 at a water
temperature of 49oC
Figure 39: Picture showing algae type 2 found in the runoff stream/spring 5 at a water
temperature of 61oC
Figure 40: Picture showing cyanobacterial mat community found at 53 oC
14
Figure 43: Picture showing spring number 1 (uppermost), 7 (middle) and 3 (lowermost)
15
ABBREVIATIONS
bp
base pair
BPB
bromophenol blue
CTAB
hexadecyltrimethylammonium bromide
DNTP
deoxy-nucleotide triphosphates (dATP, dCTP, dGTP, dTTP)
DSM
Deutsche Sammlug von Mikroorganismen (und
Zellkulturen)
EDTA
ethylene diamine tetra-acetic acid
GLB
gel loading buffer
Kb
kilobase
Milli-Q
Millipore Cooperation
ηm
nanometers
PVP
polyvinyl-pyrrolidone
RAPD
randomly amplified polymorphic DNA
SDS
sodium-dodecyl sulphate
TAE
tris-acetic acid EDTA buffer
TBE
tris-borate EDTA buffer
TE
tris-EDTA buffer
UV
ultra violet
w/v
weight per volume
µM
micro molar
pH
hydrogen ion concentration in gram atoms per liter
16
CHAPTER 1.0: INTRODUCTION AND LITERATURE REVIEW
Microorganisms are exceptional in their ability to adapt to a wide variety of
environmental stresses (Rivers & Amelunxen, 1973).
One of the more extreme
environments is that of elevated temperature. It seems surprising to find organisms
existing at temperatures that preclude the life processes, that is, at temperatures, which in
vitro can cause the destruction and denaturation of many macromolecules necessary for
life (Babel et al., 1972).
1.1 Temperature as a limiting factor
MacElroy (1974) formulated the term “extremophile” over a quarter of a century
ago.
Extremophiles are organisms that grow and thrive in extreme environmental
conditions, e.g., extremes of pH, temperature, salinity, radiation, pressure and oxygen
tension. Although the word extremophile has been interpreted in a number of ways, it
has understandably become associated with environments that are regarded as extreme to
mammals; thus, the definition is traditionally an anthropocentric one (Baird and Irwin,
2004).
As temperature is the major determinant of life on Earth, all living things have
their minimum, maximum and optimum temperatures for growth and other functions.
The range of temperatures at which they exist may depend, among other factors, on the
species and on the ancestral history of the individual (Daniel and Cowan, 2000). Many
investigators have examined growth of organisms at high temperatures with varied and
interesting results.
17
It has been determined that the realm of extremophiles is remarkably diverse and
expands through all three domains of life: bacteria, archaea and eukaryotes (Aravella et.
al., 1998). Those extremophiles that are adapted to temperature extremes include the
thermophiles (thrive at temperatures above 45oC), the hyperthermophiles (comprising
archaea and bacteria that grow at temperatures above 80oC and fail to grow at
temperatures below 60oC) and the cold tolerant psychrophiles (Prescott et al., 1999).
Many criteria have been described by different scientists as to the temperature
limit definitions of thermophiles. In his physiological classification of bacteria, Giltner
(1916) designated as thermophilic those microorganisms that have a minimum
temperature of 45°C, optimum 55°C and maximum 70°C. According to Hewlett (1902),
there is a group of so-called thermophilic bacteria that thrive best at a temperature of
60°C to 70°C.
1.2 Thermophiles and Evolution
Observations of thermophilic growth have been dated from early times (Brock,
1967). Brock (1967) has speculated that thermophiles may have retained characteristics
of primordial life forms. Thus, a study of the physiology and biochemistry of the
thermophilic bacteria may lead to a better understanding of how living systems have
evolved and developed.
18
Arrhenius (1927) suggested that thermophiles or thermophilic microbes had their
origin on the planet Venus and were carried to Earth by radiation pressure from the sun in
a few days.
Tanaka et al. (1971) suggests that from the evidence available, it is
conclusive that mesophiles originated from thermophiles. The most compelling support
for this hypothesis comes from the argument that evolution proceeded from an
environment considerably warmer than present today.
Another scientific, and probably the most intriguing reason for the rapid increase
and interest in thermophile research is the theory of “Panspermia”. Panspermia is the
theory that microbes in space bring life to planets like Earth, or the process whereby this
happens.
In all the varied forms of the theory, light pressure (Arrhenius’s radio-
panspermia), meteorites (ballistic panspermia) or comets (Hoyle and Wickramasinghes
modern panspermia) transport the microbes.
More simply, it is the theory of life
travelling between worlds.
Upon the beginning of life, Earth was a hotter planet due to the greenhouse effect
of the carbondioxide rich atmosphere. Therefore it is logical to infer that the first
organisms were accustomed to high temperatures and adapted to cooler temperatures as
Earth cooled (Postgate, 1994). Thus, thermophiles are likely to be our closet link to the
very first organisms.
19
There are further speculations that the early Earth’s atmosphere did not contain
oxygen until 2 billion years ago. Because life started on Earth 3.5 billion years ago, life
was exclusively anaerobic for at least 1.5 billion years (Stevens, 1994).
As stated above, many surface, subsurface and deep-sea thermophiles are
anaerobic as well. It appears that the unique heat resistance and anaerobic nature of
many hyperthermophiles could be traits of the earliest organisms and such environments
of temperature range from 40 to 70oC that favour thermophiles common on Earth’s
surface.
1.3 Formation of Thermal Environments on Earth
According to Brock (1978), there are four distinct processes that could form these
thermal environments: solar heating, combustion processes, radioactive decay and
geothermal activity.
Solar heating most commonly is seen in desert surfaces and shallow water pools,
which causes significant increases in their temperature.
Combustion processes are
exhibited in compost piles as biological degradation of organic matter generates
temperatures as high as 60oC (Carpenter-Boggs et al., 1998). Radioactive decay, in itself,
raises soil temperature when leakages from storage tanks or improper radioactive
disposal adds radioactive isotopes into the soil that start “decaying” through fusion
processes and generates excess heat enough to increase the temperature of the
surrounding soil significantly.
20
For scientists interested in thermophiles/extremophiles, geothermal heating is of
most significance. Geothermal heating occurs when tectonic plate movements force
magma to rise up to the mantle of the Earth heating up subsurface water trapped in the
vicinity. This subsurface groundwater, being superheated by the magma, is forced to the
surface (Earths crust) by thermal expansion, where the water forms geysers, hot springs
and thermal mud pools (Herbert and Sharp, 1993). Most of the hot springs have a
temperature range above 50oC. However, above 75oC, life is generally confined to the
bacteria and archaea (Daniel and Cowan, 2000), with only the members of archaea
capable of growth above 95oC.
Thermophiles are most commonly isolated from the surface hot springs because
these are the most common and readily accessible thermophilic habitat on Earth. The
origin of hot springs can be volcanic, like the ones in Yellowstone National Park, Iceland
and Japan, or from high heat flow that is not associated with volcanism such as the ones
found in the Big Sur coast in California (http://www.answers.com).
Normally, the
highest water temperature present in hot springs are that of boiling water, but much
milder temperature springs are also found in other parts of the world.
1.4 Importance of Thermophiles
The higher water temperature of hot springs confers the water additional
solubility properties, so such waters normally have low amounts of dissolved gases and
higher amounts of minerals that have leached off the rocks deeper in the earth’s crust
(http://en.wikipedia.org). Thermophiles survive and flourish in these environments by
21
metabolizing the matter that is present in the water.
As a result, many types of
thermophiles, such as anaerobic, obligately thermophilic, facultatively thermophilic and
chemo-heterotrophic thermophiles like sulfur metabolizing bacteria can be found in
certain hot springs (Brock, 1978).
Temperatures exceeding 40oC cause a wide range of problems for living
organisms, from the denaturation of biomolecules/proteins to the loss of solubility of
gases and the increase in the fluidity of cell membranes (Stetter, 1999).
Since
temperatures of this nature denature most proteins and nucleic acids, as well as many
low-molecular weight compounds, any organism existing under these conditions must
possess unusual mechanisms that ensure its survival. Thermophilic bacteria are one such
organism. These thermophiles have particularly stable proteins, DNA and cell
membranes (Baird and Irwin, 2004).
Thermophiles are among the best-studied
extremophiles. The two basic motivations spurring the search of thermophiles are (1)
unravelling the molecular basis of their adaptation to high temperatures and (2) exploiting
the unique secondary metabolites for different industrial and modern biotechnological
processes (Aravillia et al., 1998).
Since thermophiles thrive at such high temperatures, it could only mean that they
have adapted at molecular level for these high temperature environments. The major
biomolecule that is affected at such temperatures is the proteins (Jaenicke and Bohm,
1998). Proteins are a major component of nearly all cellular structures. The enzymes or
biocatalysts that enable all cellular reaction to occur are proteins. At temperatures higher
than 40oC, proteins start to denature and lose functionality.
22
Thus, the modifications to protein structure for survival at extremes of
temperature has been thoroughly reviewed, with most research focused on thermophilic
enzymes (thermozymes) (Adams, 1993, Burg et al., 1998). This is because the enzymes
from thermophiles generally have the same activity and stability as their mesophilic
counterparts, but are better adapted to maintain functionality at much more extreme
conditions.
Since the discovery of Thermus aquaticus (optimal growth temperature between
50 and 80oC, with a pH ideal between 7.5 and 8.0) by Brock in 1965, a lot of research has
taken place on thermophiles and their enzymes, especially because of the tremendous
success of DNA polymerase from T.aquaticus (Taq polymerase) in genetic engineering
operations. The unique biochemistry of T.aquaticus became essential worldwide in the
polymerase chain reaction (PCR) method (Brock, 1994).
Numerous thermophilic
restriction endonucleases are now commercialised. Most of them, isolated from Bacillus
and Thermus strains, are optimally active in the range of 50°C to 65°C.
From thereon, many new microorganisms have been isolated and identified such
as Bacillus acidocaldarius, Thermomicrobium roseum, Pseudomonas furiosus, Thermus
thermophilus, Thermus scodoductus, Thermus neopolitana (to name a few).
Interestingly, all of them had some enzymes that have found various uses in all aspects of
modern industries (Bruinns et al., 2001).
23
1.5 Thermozymes
Recent developments show that thermophiles are a good source of novel catalysts
that are of great industrial importance.
The potential biotechnological use of
thermophilic bacteria and their thermostable enzymes has lead to the extensive isolation
studies in a wide variety of thermophilic environments (Rainey et al., 1993). However, in
recent years, much interest has been focused on the characterization of these
microorganisms, and in particular, their enzymes.
Thermophilic enzymes have shown great potential and commercial success in
industrial processes, as they are highly thermostable, resistant to denaturation (e.g. in
organic solvents) and are optimally active at high temperatures (Zeikus et al., 1998).
These include bio-catalysis in fine chemical applications as these enzymes produce
optically pure compounds (Demirijian et al., 2001).
Most importantly, the use of
thermostable DNA polymerases in biotechnology was a revolution.
The whole organisms are also being used in various industries (such as the
fermentation industries) to increase product yields, decrease the processing time and
contamination rates. This is because of the high stability of other cell components of
thermophiles apart from their proteins, such as their cell membranes, DNA and RNA
(Russell & Hamamoto, 1998; Premuzic & Lin, 1999; Selek & Chaudhuri, 1999;
Cavicchioli & Thomas, 2000; Gerday et al., 2000; Abe & Horikoshi, 2001).
24
1.6 A young branch of microbiology
Much work has been done on thermophiles and extremophiles in other parts of the
world.
However most of it has focused on looking for specific type or types of
thermophiles with culture-independent methods as a major practice.
For example,
Chung et al. (2000) worked on the hot springs in Iceland and found two new species of
thermophilic bacteria growing at 80oC belonging to the genus Thermus. Similarly, Poli
with her co-workers (2005) isolated a novel bacterium, Anoxybacillus amylolyticus from
Mount Rittman, Antarctica geothermal soil.
This suggests that hot springs and
geothermally heated soils can be found in the most unexpected of places. The rapid
increase of research in this field over the past thirty years has yielded many new species
of thermophiles, opening new windows of environmental and industrial applications that
these exceptional organisms may offer.
Hudson et al. (1988) conducted one of the most extensive works on terrestrial hot
springs. They worked on the numerical classification of 131 Thermus strains that were
isolated from the hot springs in New Zealand, Iceland, Yellowstone National Park
(USA), New Mexico, Japan, USSR, Fiji and the United Kingdom.
Of all the hot springs on Earth, the most extensively studied are the ones in
Yellowstone National Park. This is because it was here that the first thermophile was
isolated and also because it has a large variety of hot springs with varied hydro-chemical
properties (Brock and Freeze, 1969).
25
Many types of hot springs all around the world have been studied with great
success. Most of these have been acidic, alkaliphilic, near neutral springs and solfataras.
Geothermal surveys were carried out by the Land and Mineral Resources Department in
Fiji, and revealed that there are numerous hot springs located throughout Vanua Levu and
Viti Levu, with varied temperatures and levels of activity (Autar, 1996).
However, the only account of any work or sampling that has been done here in
Fiji was in 1986 by Hudson and his colleagues (Hudson, et al., 1986). They isolated a
Thermus strain from the Savusavu beach (Vanua Levu, Fiji) and compared it with
various isolates from New Zealand and Icelandic hot pools.
Using numerical
classification, Hudson and colleagues tried to get a cluster map of the isolates to test if
they fell into the two validly named species of Thermus, T.aquaticus and T.ruber. They
found that the isolate from Fiji did not fall into any of the three major clusters in their
results. They concluded that different thermal regions have different insular phenotypes.
Therefore, until a comprehensive range of thermal areas have been studied, the full
taxonomic structure of the genus cannot be known (Hudson, et al., 1986).
Thus, by sampling at the Savusavu Hot Springs (a.k.a Nakama Hot Springs), this
research looks into furthering the description of the thermophilic microfauna on culture
dependant techniques.
26
1.7
•
The aims of this research project are to:
To culture and isolate aerobic thermophilic bacteria from the soil/water samples
collected from the Savusavu Hot Springs (a.k.a. Nakama Hot Springs)
•
To characterize the bacterial isolates obtained on the basis of certain
morphological and biochemical tests
•
To employ techniques of screening the thermophilic bacterial isolates for the
presence of extracellular hydrolytic enzyme production
•
To carry out comparative genetic analysis in order to correctly identify the aerobic
thermophilic bacteria
27
Enlarged map of the Savusavu sea front showing the location of the Savusavu Hot Spring
28
CHAPTER 2.0: MATERIALS AND METHODOLOGY
2.1 Sampling
Description of the Sampling Site:
Samples were collected from the Savusavu Hot Springs, located in Nakama,
Savusavu, Vanua Levu, Fiji. This is a major site of geothermal springs in Fiji and has
been acknowledged as the most active (Healy, 1960). The water temperature is above
100oC at the hottest points and the conditions are ideal for the growth of obligate
thermophiles.
Permission had to be sought from the Savusavu town clerk, Mr. Dharmendra
Prasad, to carry out the sampling from this site.
2.2 Handling and transportation
Sample Collection:
The water/soil samples were collected in pre-sterilised 500ml glass Duran Schott
bottles. The samples were transported to the laboratory on the same day and kept in
water baths at a temperature of 65oC until further analysis. Four collections (quarterly
sampling) were conducted in order to study seasonal variation.
29
2.3 Analysis of the Geothermal Environment
For the Savusavu Hot Springs, multiple temperature readings were recorded for
all the springs and the immediate surroundings, followed down to the runoff to establish
an outline of temperature fluctuations. This has been mapped out in the appendix
(appendix F (c)). Along with temperature, pH readings were also recorded to establish
the overall classification of the hot spring.
2.4 Bacteriological Analysis of hot pool water
All laboratory aspects of bacteriological and biochemical characterization have
been adapted from Harley (2005).
2.5 Total plate counts (TPC)
Samples were serially diluted, wherever necessary, and plated onto nutrient agar
(NA). The plates were incubated at 65oC for 48 to 96 hours. Plates were observed for
development of bacterial colonies, which were counted and expressed as total
thermophilic bacterial load per ml of soil suspension per sample.
Samples were also incubated at 37oC, 45-50oC for estimating the load of any
mesophilic and facultative/ moderate thermophiles. A further step included the incubation
of samples at 55oC, 65oC, 75oC, 85oC and 90oC. The plates from 65oC were observed
after incubation of 24-48 hours and representative isolates were selected and maintained
on nutrient agar slants for further characterisation.
30
All staining, biochemical testing and screening for enzymatic activity were
performed in triplicates for each isolate.
2.6 Staining
2.6.1 Gram Staining
Smears were prepared of thermophilic bacterial cultures (less than 20 hours old)
and heat fixed. Slides were flooded with crystal violet solution for a minute, rinsed with
water, and counter stained with Gram’s iodine for another minute. Decolourisation was
done using 95% ethanol immediately followed by water. Final staining was done with
safranin for a minute, which was finally rinsed off with water and slides were observed
under oil immersion using an Olympus C65 light microscope.
2.6.2 Endospore Staining
Smears were also prepared for endospore staining. Slides were flooded with
malachite green and heated on a hot plate to allow the stain to steam for three minutes.
After cooling to room temperature, slides were rinsed with water, counter-stained with
safranin for a minute and rinsed off with water. Slides were then observed under oil
immersion using an Olympus C65 light microscope.
31
2.7 Biochemical Testing
2.7.1 Motility Test
Motility medium (0.3% sterile Nutrient Agar) was prepared and dispensed into
test tubes. 0.1% tetrazoleum chloride was also added to the motility medium to act as an
indicator. Cultures were stab inoculated into the medium and incubated at 65oC for 24
hours. Growth along the stab path was regarded as a negative result whereas diffuse
growth was regarded as positive.
2.7.2 Kovac’s Oxidase Test
This test indicates the presence of the enzyme cytochrome oxidase.
The
cytochrome enzyme is able to oxidise the substrate tetramethylene paraphenylene
diamine dihydrochloride forming a purple coloured end product.
Filter paper
impregnated with a solution of 1% tetramethylene paraphenylene diamine dihydro
chloride was used to perform this test.
A small amount of 24-hour-old culture was aseptically transferred and scratched
over the filter paper impregnated with the reagent. A change in colour to deep purple
within 10 seconds was considered a positive oxidase test. A delayed or no colour change
was considered indicative of a negative test.
32
2.7.3 Oxidation Fermentation Test (O/F TEST)
Oxidation fermentation test was used to check for the oxidation/fermentation of
glucose that is incorporated in the O/F basal medium. After preparation and sterilization
of the O/F basal medium, 1% glucose solution (aqueous, filter sterilized) was added in to
the medium and transferred to sterile test tubes and slants prepared. Cultures were
inoculated by stabbing the butt and streaking the slant and incubated at 65oC for 24 hours.
The pH indicator (bromothymol blue) was incorporated in the medium. A color
change from green to yellow throughout the medium indicated that the culture was
fermentative. A color change in the slant only indicated that the culture was oxidative.
2.7.4 Catalase Test
The enzyme “Catalase” catalyses the liberation of oxygen and water from
hydrogen peroxide, a metabolic end product, which is toxic to bacteria. A small amount
of culture was transferred to a clean slide with a sterile loop and 3% hydrogen peroxide
was placed on to the surface of the culture. Effervescence was recorded as positive
reaction for catalase resulting from the breakdown of hydrogen peroxide with the
evolution of oxygen bubbles.
33
2.8 Determination of hydrolytic enzyme production of the isolates
2.8.1 Amylase production:
Nutrient starch agar (nutrient agar with 0.2% starch) was used to test the
elaboration of hydrolytic enzyme amylase. The bacterial isolates were spot inoculated in
the plates. After incubation at 65oC for 24-48 hours, the plates were flooded with
Lugol’s iodine. Clear zones around the colony indicated a positive test resulting from the
utilization of starch by the isolate. The areas where starch was present were indicated by
the development of blue colour, on addition of Gram’s iodine.
2.8.2 Gelatinase Production:
Nutrient gelatine agar (nutrient agar with 0.4% gelatine) plates were used to
detect the production of gelatinase. The cultures, after spot inoculation, were incubated
at 65 oC for 24-48 hours. After incubation the plates were flooded with mercuric chloride
solution and allowed to stand for 5 to 10 minutes. Extreme care was taken to avoid any
skin contact with mercuric chloride since it is very toxic. Clear zones around the culture
indicated gelatinase production resulting in the utilization of gelatine. Areas where
gelatine was found would turn opaque due to denaturation of gelatine by mercuric
chloride solution.
34
2.8.3 Lipase production:
Tributyrin agar (Nutrient agar with 10% tributyrin) was used to detect lipase
activity. The cultures, after spot inoculation, were incubated at 65oC for 24 - 72 hours.
After incubation plates were observed for change in opacity of the medium around the
cultures. Such cultures were considered positive for lipase production.
With the objective of also evaluating the growth conditions of the bacteria,
several parameters were also tested: increase in media sodium chloride concentrations,
pH tolerance, type of media (solid/liquid), and temperature tolerance.
Mixed cultures were grown in media containing varied amounts of sodium
chloride (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0 and 5.0% NaCl). To
test limits of pH tolerance, pH of the media was altered to pH ranging from 1-10. All of
these cultures were incubated at temperatures of 35oC, 45oC, 55oC, 60oC, 65oC, 70oC,
75oC, 80oC, 85oC and 90oC in both, nutrient agar and nutrient broth to detect the
temperature limits of growth and the form of media preferred.
2.9 Overview of 16S rDNA Analysis
The choice of methods for the isolation of DNA depends on the degree of purity
of the DNA required for the analysis to be performed. Some DNA analysis, (e.g., those
using restriction enzymes) require DNA of high purity in relatively large amounts. In
contrast, analysis based on polymerase chain reaction (PCR) only requires very small
35
amounts of DNA whose quality can be crude (Rapley & Walker, 1998). This is because
even if the sample is crude, the primers will only attach to the specific DNA sequence
and only that fragment will be synthesized. The entire remaining crude DNA will be
broken down and used to synthesize the new fragment.
For the molecular analysis of the thermophilic bacterial isolates from the
Savusavu Hot Springs, DNA extraction was performed using the chloroform/isoamyl
alcohol method (Dempster et al., 1999). DNA was then quantified and the concentration
determined using the Nanodrop ND 1000 Spectrophotometer.
Following this, DNA was subjected to 16S rDNA PCR (Polymerase Chain
Reaction). The PCR products were run on 1% TAE-Agarose gel electrophoresis for
confirmation of product formation.
The gels were photographed using the UV
Transilluminator and a Polaroid Camera.
The 16S rDNA PCR products were then
exposed to restriction digestion and again run on 0.8% TBE-Agarose gels and
photographed as for the TAE-Agarose gel.
Comparison cultures of thermophiles from New Zealand hot pools and the freezedried culture stock were obtained from the Thermophile Research Unit Culture
Collection, University of Waikato (Hamilton, New Zealand) and were used as positives.
36
2.10 16S rDNA Analysis
This work was done in collaboration with Professor Hugh Morgan at the
University of Waikato Thermophile Research Unit in Hamilton, New Zealand.
Thermophile cultures were transported at room temperature in nutrient agar
slants, and were sub-cultured by inoculation into Thermus 162 broth medium (Medium
878), which was then incubated at 60oC (which was 5oC less than the actual temperature
of culturing and isolation). After 36-48 hours, the broth cultures were streaked onto
Thermophilus medium plates (Medium 74) and again incubated at 65oC for 18-24 hours.
2.10.1 DNA Extraction
The method used was slightly modified from Dempster et al., (1999). Mass
culturing of the cells were done in 15ml broth tubes of medium 878 and incubated at
65oC for 18-24 hours. It was then centrifuged at 13.2 x 1000rpm in an Eppendorf
Centrifuge
to
produce
cell
pellets.
DNA
extraction
was
done
using
the
chloroform/isoamyl alcohol method. 0.5ml of CTAB (100mM Tris-HCl, 1.4M NaCl,
20mM EDTA, 2% w/v CTAB, 1% w/v PVP [mol. weight 360,000], pH 8.0 and 0.4% w/v
2-mercaptoethanol) buffer was added to the cell pellets and centrifuged at 13.2 x 1000
rpm for 5 minutes. When centrifugation was complete, the tubes were removed and
incubated in a water bath at 100oC for 20 minutes. Equal volume of chloroform/isoamyl
alcohol mixture (24:1) was added to cooled tubes and placed on a rotator mixer for
another 20 minutes, then quickly centrifuged again at 13.2 x 1000 rpm for 15 minutes.
The uppermost phase was transferred into a new 2ml sterile Eppendorf tube, to
which 0.5ml of 5.0 M NaCl and 1 volume isopropanol was added, then inverted several
37
times to mix and incubated at -70oC for at least 1 hour. After the freeze treatment, the
tubes were centrifuged for 30 minutes at 13.2 x 100 rpm. The supernatant was decanted
to waste and the pellet was washed with 80% ethanol quickly followed by a 20-second
centrifuge step.
The supernatant was discarded and tubes were incubated in an inverted position at
room temperature. After all traces of ethanol disappeared, the DNA pellet was resuspended in 20µl of sterile PCR grade water and stored at -20oC for later determination
of concentration and purity.
2.10.2 DNA Concentration And Purity Determination
The concentration of the nucleic acids was determined by the absorbance of UV
light at 260ηm wavelength using a spectrophotometer: the ND 1000 Nanodrop
(Nanodrop, DE, USA) instrument. The purity of the nucleic acid was determined by the
ratio of readings taken at 260ηm and 280ηm wavelengths (both in the UV range) as
proteins absorb at 280ηm.
1µl of DNA suspension was used for the instrumental
analysis. A 260nm/280nm ratio of approximately 1.8 and 2.0 was considered to represent
DNA and RNA respectively (http://mullinslab.ucsf.edu.).
38
2.10.3 Polymerase Chain Reaction (PCR)
All of the PCR’s were undertaken using the Eppendorf MasterCycler Gradient
Thermocycler (Eppendorf, A.G, Hamburg, Germany).
All PCR reagents (Taq
polymerase, 10x PCR buffer [100mM Tris-Hcl, 500mM KCl, pH 8] and 25mM MgCl2 )
were obtained from Biolab, New Zealand (Rosche Diagnostics).
All PCR runs contained a negative control consisting of sterile PCR grade water
obtained from the DNA Sequencing Lab at the Thermophile Research Unit (University of
Waikato, NZ) replacing the template DNA.
Particular runs also contained positive controls whereby standard DNA was used
to ensure that the PCR was functioning correctly. The primers utilized are listed in the
table below. Stock solutions of PCR primers were stored at 60µM concentration in 1x
TAE Buffer (10mM Tris, 1Mmedta, pH 8.0) at -20oC. Commonly, a master mix of all the
PCR components was prepared and dispensed into 0.5 ml sterile PCR tubes prior to the
addition of template DNA. The master mix components added are listed in table 1.
Table 1: List of PCR Primer numbers, their sequences and the analysis technique
used
Primer Number
Purpose
Purpose
Sequence (5’(5’-3’)
OPR 13
OPR 12
EUB A (F)
EUB B (R)
RR69 (F)
RR77 (R)
RAPD
RAPD
Eubacterial 16S rDNA
Eubacterial 16S rDNA
Bacterial 16S rDNA (27F)
16S rDNA (1522R)
GGACGACAAG
ACAGGTGCGT
P.S: EUB B is the reverse primer
AGATTTCGATCCTGGCTCAG
AAGGAGGTGATCCARCCGCA
EUB A is the forward primer
39
2.10.4 Bacterial 16S rDNA PCR
Near full length bacterial 16S rDNA (approximately 1,522 bp) was amplified
using the primers EUB A (R) and EUB B (F), and RR69 (F)/RR77(R). As stated earlier,
a master mix of the PCR components was made (excluding Taq polymerase and template
DNA) and dispensed into 0.5ml sterile PCR tubes (see table 2). Template DNA and the
Taq polymerase were then added and amplification initiated.
Table 2: Recipe for preparation of template thermophilic bacterial DNA for 16S
rDNA PCR using primers EUB A and EUB B
PCR Component
Water
MgCl2 (25mM)
10X PCR buffer (No MgCl2 )
dNTP (2mM)
Eub B (10(M)
Eub A (10(M)
Taq Polymerase (1U/(l)
Template DNA
Volume (µ
µl)
To 25
2.5
2.5
2.5
1.0
1.0
0.75
1.0
The thermocycling conditions that were used on the Eppendorf Master Cycler
involved an initial denaturation at 94oC for 2 minutes. This was followed by 35 cycles of
denaturation at 94oC for 30 seconds, annealing at 50oC for 30 seconds and extension at
72oC for 2 minutes, followed by a final extension at 72oC for 5 minutes.
40
2.10.5 Randomly Amplified Polymorphic DNA (RAPD) PCR
A modified version of the RAPD assay developed by Romnius et al. (1997) was
used in this research. RAPD assays were undertaken in 25µl volume reactions containing
the items listed in table 3.
Table 3: List of PCR components for RAPD analysis of template thermophilic
bacterial DNA
PCR component
Volume (µ
µl)
Water
MgCl2
10X PCR buffer (No MgCl2 )
dNTP(2mM)
To 25
2.5
2.5
2.5
5.0
1.25
Primer (10µM)
Taq polymerase (1U/µl )
Template DNA
≈20.0
Table 4: List of PCR primers used for RAPD analysis and their sequences
Primer Number
OPR 13
OPR 12
Purpose
RAPD
RAPD
Sequence (5’-3’)
GGACGACAAG
ACAGGTGCGT
Template DNA was amplified by a RAPD PCR programme involving an initial
denaturing temperature of 94oC for 1 minute and 30 seconds; followed by 40 cycles of
94oC for 30 seconds, primer annealing at 36oC for 30 seconds, and primer extension at
72oC for 2 minutes followed by an additional final extension at 72oC for 4 minutes.
41
2.10.6 Restriction Endonuclease Digests
Typically, a master mix of all the components was made (as stated in table 5) and
dispensed into sterile 0.5ml tubes and then the PCR product (16S rDNA) were added.
Table 5: Recipe for preparation of 16S rDNA-PCR product for restriction
endonuclease digestion reaction
Component
Volume (µ
µl)
Restriction Endonuclease buffer (10X)
2.0
Restriction Endonuclease (10U/µl)
0.4
DNA-PCR product
2.0
Milli-Q water
Up to 20.0
Reactions were undertaken by placing the final mixtures in a hot air incubator at
37oC for 12-18 hours, then 10µl of 3X SDS GLB (30% glycerol, 3% SDS, 0.025%BPB
and 1mM EDTA) was added to stop the reaction; following which the tubes were
incubated at 65oC for 20 minutes prior to loading into a 2.5% TBE Agarose gel. The
restriction endonucleases used are listed in Table 6 below.
Table 6: List of restriction endonucleases, their corresponding buffers, incubation
temperature and cutting sites used for digestion of 16S rDNA-PCR products
Restriction
Corresponding
Endonuclease
buffer
EcoRI
React 3
Cut site
Incubation
Supplier
(oC)
GAATTC
37
Invitrogen,
CA,
USA
Hae III
React 2
GCG C
37
Invitrogen,
USA
CA,
42
2.10.7 ELECTROPHORESIS
2.10.7.1 Agarose Gel Electrophoresis
The electrophoresis of DNA fragments through 0.8-3% agarose gels was used to
separate PCR products and restriction endonuclease DNA digests. Appropriate amount
of agarose powder was added to either; 1X TBE buffer (1L of 5X TBE buffer stock
solution contained: 54g Tris, 27.5g boric acid and 20ml of 0.5M EDTA, pH 8.0); or 1X
TAE buffer (1L of 50X TAE buffer stock contained: 242g Tris, 57.1ml glacial acetic
acid, 100ml of 0.5M EDTA, pH 8.0) and boiled until all of the agarose powder dissolved.
It was ensured that the agarose-buffer solution was weighed before and after boiling, and
adding sterile PCR Grade water made up for the difference in the weight.
TAE buffer was used for PCR product separation and TBE buffer was used for
restriction endonuclease digest products. When the agarose solution had cooled down to
approximately 55oC, it was poured into the gel electrophoresis platform, the comb was
placed into position and allowed to set. Once set, the appropriate buffer was then added
to ensure adequate recirculation between the anode and the cathode reservoirs.
A 6X Gel Loading Buffer (GLB) (0.04% bromophenol blue, 30% glycerol) was
added to the samples prior to the loading of samples into the gels. However, a 3X GLB
containing SDS (sodiumdodecylsulphate) replaced the 6X GLB for the restriction digest
samples. All of the agarose gel electrophoresis runs included a 1kb plus size standard
containing approximately 1µg of DNA. The profile of the DNA ladder is included in the
43
appendix (appendix F (b)). The TAE buffer gels were run at 67V whereas the TBE gels
were run at 100V. Following electrophoresis, gels were stained with 0.5mg/L of ethidium
bromide solution for 30-40 minutes and then de-stained with sterile distilled water for the
same amount of time. The DNA was visualized and photographed under UV (260nm)
light with an AlphaImager System (AlphaInnotech, CA, USA).
44
CHAPTER 3: RESULTS
3.1 Bacteriological Analysis and tolerance limit test results.
Estimation of thermophilic bacterial load:
•
September 2005: 960 cfu/ml
•
November 2005: 1260 cfu/ml
•
January 2006: 12200 cfu/ml
•
March 2006: 21600 cfu/ml
3.2 Growth temperature limit results:
Incubation temperature
o
Observation
35 C
No growth observed in NA/NB
45 oC
-Very low turbidity observed in NB
-Only 11 colonies on NA plates
o
55 C
-Turbidity greater than that of NB in 45 oC
-46 colonies grew on NA plates
o
65 C
-Very high turbidity observed in NB
-63 colonies on NA plates
o
70 C
-Turbidity as intense as of above in NB
75 oC
-Turbidity same as above in NB
o
80 C
-Quite turbid but less than that of 75 oC NB
o
85 C
-Light turbidity, slightly more than that of NB in 55
o
C, but less than that of 65 oC
o
90 C
- NB turbidity equivalent to that of 45 oC
- 9 colonies grew on NA plates
**All of the above tests were carried out simultaneously in triplicates for bacteriological
analysis and in quadruplicates for growth temperature and pH limits.
45
3.3 Sodium chloride/halophily limits.
NaCl concentration
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
2.0
3.0
4.0
5.0
Observation
Weak growth
Weak growth
Slightly more turbid
Same as for above
Same as for above
Good turbidity
High turbidity
High turbidity
High turbidity
High turbidity
High turbidity
Weak growth, low turbidity
Near zero turbidity
No growth
3.4 Growth at varied pH levels in Nutrient Broth.
pH of Nutrient Broth
1
2
3
4
5
6
7
8
9
10
Observation
No growth
No growth
No growth
No growth
Turbid
Turbid
Turbid
Turbid
No growth
No growth
46
3.5 A: Biochemical characterization
Isolate
BATCH 1
70oC:1
70oC:9
70oC:10
70oC:11
70oC:12
70oC:13
70oC:14
70oC:15
70oC:16
70oC:19
70oC:20
70oC:21
70oC:24
70oC:25
70oC:36
70oC:40
70oC:41
70oC:42
70oC:43
70oC:46
70oC:47
70oC:48
70oC:49
70oC:51
70oC:52
70oC:53
70oC:54
70oC:55
70oC:56
70oC:57
70oC:58
70oC:60
70oC:61
70oC:64
70oC:65
70oC:66
Gram Reaction
Endospore
Oxidation
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+/−
+
+
+
+
+
+
+
+
+
+
+/−
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
−
−
−
−
−
+
+
+
+
−
+
−
−
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Fermentation
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
47
Isolate
Gram Reaction
BATCH 1 contd…
70oC:69
+
70oC:70
+
o
70 C:71
+
70oC: 73
+
o
70 C:79
+
o
70 C:80
+
70oC:81
+
o
70 C:96
+
70oC:97
+
o
70 C:103
+
o
70 C:104
+
70oC:118
+
o
70 C:120
+
o
70 C:121
+
70oC:122
+
o
70 C:123
+
o
70 C:126
+
o
70 C:146
+
o
75 C:9
+
75oC:10
+
75oC:11
+
o
75 C:12
+
o
75 C:129
+
75oC:138
+
BATCH 2
70oC:61
−
o
70 C:64
−
75oC:72
−
75oC:75
−
o
75 C:108
−
o
75 C:129
−
o
75 C:138
−
75oC:145
−
75oC:146
−
o
75 C:161
−
BATCH 3
70oC:38
−
70oC:44
−
o
70 C:45
−
o
70 C:46
−
Endospore
+
+
−
+
+
+
+
+
−
+
+
+
−
−
−
−
−
−
−
+
−
−
−
−
Oxidation
Fermentation
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
−
−
−
−
−
−
−
−
+
+
+
+
+
+
+
+
+
+
−
−
+
−
+
+
+
+
+
48
Isolate
Gram Reaction
BATCH 3 contd...
70oC:47
−
75oC:9
−
75oC:10
−
o
75 C:11
−
o
75 C:12
−/+
o
75 C:162
−
BATCH 4
70oC:5
+
o
70 C:27
+
o
70 C:28
+
70oC:29
+
o
70 C:32
+
70oC:33
+
o
70 C:34
+
o
70 C:45
+
70oC:50
+
o
70 C:88
+
70oC:92
+
o
70 C:93
+
70oC:115
+
o
70 C:117
+
o
75 C:20
+
o
75 C:69
+
o
75 C:71
+
75oC:72
+
o
75 C:75
+
o
75 C:108
+
BATCH 5
70oC:8
+
o
70 C:35
+
o
70 C:37
−
o
75 C:19
+/−
Endospore
Oxidation
Fermentation
−
−
+
−
−
−
+
+
+
+
+
+
+
−
−
−
+
+
+
+
+
+
+
+
+
−
−
−
−
−
−
−
−
+
+
+
+
+
+
+
−
+
−
−
+
+/−
+/−
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
49
Isolate
BATCH 1
70oC:1
70oC:9
70oC:10
70oC:11
70oC:12
70oC:13
70oC:14
70oC:15
70oC:16
70oC:19
70oC:20
70oC:21
70oC:24
70oC:25
70oC:36
70oC:40
70oC:41
70oC:42
70oC:43
70oC:46
70oC:47
70oC:48
70oC:49
70oC:51
70oC:52
70oC:53
70oC:54
70oC:55
70oC:56
70oC:57
70oC:58
70oC:60
70oC:61
70oC:64
70oC:65
70oC:66
70oC:69
70oC:70
70oC:71
70oC: 73
Motile
Catalase Activity
Oxidase Activity
+
+
+
+
+
+
+
+/−
+/−
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
−
−
−
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
−
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
50
Isolate
Batch 1 contd...
70oC:79
70oC:80
70oC:81
70oC:96
70oC:107
70oC:104
70oC:118
70oC:120
70oC:121
70oC:122
70oC:123
70oC:126
70oC:146
75oC:9
75oC:10
75oC:11
75oC:12
75oC:129
75oC:138
BATCH 2
70oC:61
70oC:64
75oC:72
75oC:75
75oC:108
75oC:129
75oC:138
75oC:145
75oC:146
75oC:161
BATCH 3
70oC:38
70oC:44
70oC:45
70oC:46
70oC:47
75oC:9
75oC:10
75oC:11
75oC:12
Motile
Catalase Activity
Oxidase Activity
+/−
+
+
−
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+/−
+
+
+
+
+
+
+
+
+/−
+
+
+
+
+
+
+
+
+
+/−
+/−
−
−
+/−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
+
+
+
+
+
+
+
+
+
+
−
−
−
−
−
−
−
−
−
+
+
+
+
+
+
+
+
+
−
−
−
−
−
−
−
−
−
51
75oC:162
Isolate
BATCH 4
70oC:5
70oC:27
70oC:28
70oC:29
70oC:32
70oC:33
70oC:34
70oC:45
70oC:50
70oC:88
70oC:92
70oC:93
70oC:115
70oC:117
75oC:20
75oC:69
75oC:71
75oC:72
75oC:75
75oC:108
BATCH 5
70oC:8
70oC:35
70oC:37
75oC:19
−
Motile
+
Catalase Activity
−
Oxidase Activity
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+/−
+/−
+/−
+
+
+
+
+
+
−
−
+
+
+
+
−
+
−
−
−
+
−
−
+
+
−
+
−
+
52
3.6 B: Extracellular hydrolytic enzyme screening
Isolate
BATCH 1
70oC:1
70oC:9
70oC:10
70oC:11
70oC:12
70oC:13
70oC:14
70oC:15
70oC:16
70oC:19
70oC:20
70oC:21
70oC:24
70oC:25
70oC:36
70oC:40
70oC:41
70oC:42
70oC:43
70oC:46
70oC:47
70oC:48
70oC:49
70oC:51
70oC:52
70oC:53
70oC:54
70oC:55
70oC:56
70oC:57
70oC:58
70oC:60
70oC:61
70oC:64
70oC:65
70oC:66
70oC:69
Amylase Activity
Gelatinase activity
Lipase Activity
+
+
+
+
+
+
+
+
+
+
+
+
+
+/−
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+/−
+
+
−
−
+
+
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
53
Isolate
BATCH 1 contd…
70oC:70
70oC:71
70oC: 73
70oC:79
70oC:80
70oC:81
70oC:96
70oC:107
70oC:104
70oC:118
70oC:120
70oC:121
70oC:122
70oC:123
70oC:126
70oC:146
75oC:9
75oC:10
75oC:11
75oC:12
75oC:129
75oC:138
BATCH 2
70oC:61
70oC:64
75oC:72
75oC:75
75oC:108
75oC:129
75oC:138
75oC:145
75oC:146
75oC:161
BATCH 3
70oC:38
70oC:44
70oC:45
70oC:46
70oC:47
75oC:9
Amylase Activity
Gelatinase activity
Lipase Activity
+
+
+
+
+
+
+
+
+
+
+
+
+
−
−
+
+
+
+
+
−
+
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
+
+
+
+
+
−
−
−
−
+
+
+
+
+
+
+
+
+
−
−
−
−
−
−
−
−
−
−
−
−
−
+
+
+
+
−
−
+
+
+
+
+
+
+
+
+
+
+
54
Isolate
BATCH 3 contd...
75oC:10
75oC:11
75oC:12
75oC:162
BATCH 4
70oC:5
70oC:27
70oC:28
70oC:29
70oC:32
70oC:33
70oC:34
70oC:45
70oC:50
70oC:88
70oC:92
70oC:93
70oC:115
70oC:117
75oC:20
75oC:69
75oC:71
75oC:72
75oC:75
75oC:108
BATCH 5
70oC:8
70oC:35
70oC:37
75oC:19
Amylase Activity
Gelatinase activity
Lipase Activity
−
−
−
−
+
+
+
+
+
+
−
−
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
−
−
−
−
−
−
−
−
+
−
−
−
−
−
−
−
−
+
+
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
+
+
+
+
−
−
−
−
+
−
+
−
55
3.7 C: DNA analysis
16S rDNA PCR, Restriction digestion and RAPD gels photographed under UV
(260nm) light with an AlphaImager System (AlphaInnotech, CA, USA).
Figure 3: 060607: EcoRI/React 3
1
2
3
4
5
6
7
8
9
10 11
12
13 14
15 16
7: 70oC: 14
8: 70oC: 15
9: Standard DNA ladder
10: 70oC: 19
11: 70oC: 20
12: 70oC: 21
2: CN
3: 70oC: 8
4: 70oC: 10
5: 70oC: 11
6: 70oC: 12
Figure 4: 060613: Hae III/ React 2
1 2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20
17 18
19 20
13: -VE Control
14: 70oC: 21
15: 70oC: 40
16: 70oC: 41
17: 70oC: 42
18: 70oC: 35
56
7: 70oC: 14
8: 70oC: 15
10: 70oC: 19
11: 70oC: 20
12: 70oC: 21
13: 70oC: 24
2: CN
3: 70oC: 8
4: 70oC: 10
5: 70oC: 11
6: 70oC: 12
14: 70oC: 40
15: 70oC: 41
16: 70oC: 42
17: 70oC: 35
18: -VE Control
19
Figure 10: 060630GelA EcoRI/React 3
1
2
3
4
5
6
7
8
9
10
11 12
13 14
15
8: 70oC: 38
9: 70oC: 43
10: 70oC: 44
11: 70oC: 45
12: 70oC: 46
13: 70oC: 47
14: 70oC: 48
1: DNA ladder
2: CN
3: RT 41 A
4: TG 275
5: TG 206
6: TG 153
7: TG 8
Figure 11: 060630GelB EcoRI/React 3
1 2
3
4
5 6
7
8
9
10 11 12 13 14 15 16 17 18 19 20
16 17
18
19 20
15: 70oC: 49
16: 70oC: 52
17: 70oC: 53
18: 70oC: 54
19: 70oC: 55
20: DNA ladder
57
1: DNA ladder
2: CN
3: RT 41 A
4: TG 275
5: TG 206
6: TG 153
13: 70oC: 8
14: 70oC: 35
15: 70oC: 60
16: 70oC: 37
17: DNA ladder
7: TG 8
8: 70oC: 57
9: 70oC: 61
10: 70oC: 64
11: 70oC: 65
12: 70oC: 66
Figure 12: 130706HaeIII/React 2
1
2
3
1: DNA ladder
2:CN
3:RT 41 A
4: TG 275
5: TG 206
6:TG 153
7: TG 8
4
5
6
7
8
9
10
11 12
13 14
8: 70oC: 38
9: 70oC: 43
10: 70oC: 44
11: 70oC: 45
12: 70oC: 46
13: 70oC: 47
14: DNA ladder
15 16
17 18
19 20
15: 70oC: 48
16: 70oC: 49
17: 70oC: 52
18: 70oC: 53
19: 70oC: 54
58
Figure 13: 130706HaeIII/React 2: 16S r DNA PCR
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15
1: DNA ladder
2: CN
3: RT 41 A
4: TG 275
5: TG 206
6: TG 153
16 17 18 19 20
7: TG 8
8: 70oC: 57
9: 70oC: 61
10: 70oC: 64
11: 70oC: 65
12: 70oC: 66
13: DNA ladder
14: DNA ladder
15: 70oC: 8
16: 70oC: 35
17: 70oC: 37
18: 70oC: 60
Figure 14: EcoR1/React 3;full 200706
1
2
3
4
5
6
7
8
9
10 11 12
13 14 15
21 22 23 24 25 26 27 28 29 30 31 32 33 34
16 17 18 19 20
35 36 37 38 39
40
59
1: RT 41 A
2: TG 206
3: TG 8
4: DNA ladder
5: CN
6: AM
7: FG
8: 75oC: 4
9: 75oC: 9
10: 75oC: 10
11: 75oC: 11
12: 75oC: 12
13: 75oC: 13
14: 75oC: 14
15: DNA ladder
16: 75oC: 15
17: 75oC: 16
18: 75oC: 17
19: 75oC: 18
20: 75oC: 19
21: RT 41 A
22:TG 206
23: TG 8
24: DNA ladder
25: CN
26: AM
27: FG
28: 75oC: 20
29: 75oC: 69
30: 75oC: 71
31: 75oC: 72
32: 75oC: 75
33: 75oC: 108
34: DNA ladder
35: 75oC: 129
36: 75oC: 138
37: 75oC: 145
38: 75oC: 146
39: 75oC: 161
40: 75oC: 162
Figure 15: EcoR1/React 3 B 060705
1
2
3
1:DNA ladder
2: CN
3: RT 41 A
4: TG 275
5: TG 206
6: TG 153
4
5
6
7
8
9 10 11 12
13 14 15 16 17 18 19 20
7: TG 8
8: 70oC: 57
9: 70oC: 61
10: 70oC: 64
11: 70oC: 65
12: 70oC: 66
13: 70oC: 8
14: 70oC: 35
15: 70oC: 60
16: 70oC: 37
17: DNA ladder
60
Figure 16: HaeIII/React 2: UNIQUE 210706
1
2
3
4
5
6
7
8
9
1: RT 41 A
2:TG 275
3:TG 205
4:TG 153
5:TG 8
10
11 12 13
14
11: 70oC: 45
12: 70oC: 55
13: 70oC: 60
14: DNA ladder
6:DNA ladder
7:CN
8: 70oC: 8
9: 70oC: 35
10: 70oC: 37
Figure 17: RAPD ENLARGED0607074
1
2
3
1: DNA ladder
2: -ve Control
3: CN
4: TG 206
4
5
6
7
8
9
10
11
5: TG 153
6: 70oC: 8
7: 70oC: 60
8: 70oC: 35
12
13
14
9: 70oC: 37
10: 70oC: 45
11: 70oC: 35
12: DNA ladder
61
Figure 18 : RAPD 060713
1
2
3
4
5
6
7
8
1: DNA ladder
2:CN
3:AM
4:FG
5:TG 206
9
10
11
6: TG 153
7: 70oC: 8
8: 70oC: 35
9: 70oC: 37
10: DNA ladder
12
13
14
11: 70oC: 45
12: 70oC: 55
13: 70oC: 6
Figure 19 : RAPD NEW 060711
1
2
1: DNA ladder
2: -ve control
3
4
5
6
7
8
3: CN A
4: CN B
5: AM
6: FG
8: DNA ladder
62
Figure 20 : RAPD OPR13 180706
1
2
3
4
5
6
1:
2: DNA ladder
4: CN
7
8
5: AM
6: 70oC: 35
7: 70oC: 37
9
10
8: TG 206
10: DNA ladder
Figure 21 : RAPD T3 060710
1
2
3
1: DNA ladder
2: CN
3: TG 206
4: TG 153
4
5
6
7
8
9 10
11 12
13 14
5: 70oC: 8
6: 70oC: 35
7: 70oC: 37
8: 70oC: 45
9: 70oC: 55
10: 70oC: 60
11: DNA ladder
12: DNA ladder
63
Figure 22: TRIAL GEL 060621
1
2
3
4
5
6
7
8
4: 75oC: 58
5: 75oC: 60
6: Tn
1: DNA ladder
2: CN
3: 75oC: 19
7: CP Rod
Figure 23: UNIQUE ECOR1 210706
1
2
3
1: RT 41 A
2: TG 206
3: TG 8
4: DNA ladder
5: CN
4
5
6
7
8
9 10 11 12 13 14
6:AM
7:FG
8: 70oC: 8
9: 70oC: 35
10: 70oC: 37
11: 70oC: 45
12: 70oC: 55
13: 70oC: 60
14: DNA ladder
64
Figure 26: 060613 Hae III/React 2
1
2
3
4
5
6
7
8
9
10 11
1: DNA ladder
2: CN
3: 70oC: 8
4: 70oC: 10
5: 70oC: 11
6: 70oC: 12
12 13 14 15
7: 70oC: 14
8: 70oC: 15
9: DNA ladder
10: 70oC: 19
11: 70oC: 20
12: 70oC: 21
16 17 18
19 20
13: 70oC: 24
14: 70oC: 40
15: 70oC: 41
16: 70oC: 42
17: 70oC: 35
From all the above gels, it can be clearly highlighted that four of the isolates
showed unique patterns. The DNA band patterns that these isolates showed and other
characteristics are listed below.
70oC 8:
•
16S rDNA PCR: EUB A/EUB B: 2 bands at 850bp and 300bp respectively
•
Restriction digestion: EcoRI/React 3: 3 bands at 650bp, 500bp and 300bp
•
Restriction digestion: HaeIII/React 2: 3 bands at 900bp, 800bp and 550bp
•
Gram positive, no endospore detected, oxidative pathway, immotile, catalase and
oxidase negative, positive for amylase and lipase production, negative for
gelatinase activity
65
70oC 35:
•
16S rDNA PCR: EUB A/EUB B: 2 bands at 400bp and 200bp
•
Restriction digestion: EcoRI/React 3: 2 bands at 400bp and 300bp
•
Restriction digestion: HaeIII/React 2: 2 bands at 800bp and 300bp
•
Gram positive, endospore present, both oxidative/fermentative, immotile, catalase
negative and oxidase positive, positive for amylase activity and negative for
gelatinase and lipase activity
70oC 37:
•
16S rDNA PCR: EUB A/EUB B: 3 bands at 850bp, 400bp and 200bp
•
Restriction digestion: EcoRI/React 3: 3 bands at 1650bp, 1200bp and 650bp
•
Restriction digestion: HaeIII/React 2: 3 bands at 850bp, 650bp and 400bp
•
Gram negative, endospore absent, oxidative/fermentative metabolism, immotile,
catalase positive, oxidase negative, positive for amylase and lipase activity
75oC 19:
•
16S rDNA PCR: EUB A/EUB B: 2 bands at 850bp and 650bp
•
Restriction digestion: EcoRI/React 3: No product formed
•
Restriction digestion: HaeIII/React 2: No product formed
•
Mixed Gram reaction, no endospore production observed, oxidative metabolism,
immotile, catalase and oxidase positive, positive for amylase activity and negative
for both, gelatinase and lipase activity.
66
CHAPTER 4.0: DISCUSSION
4.1 The Savusavu Hot Springs
The Savusavu Hot Springs are the most extensive and active hot springs in Fiji.
What makes these hot springs unique is the fact that there are hot spots scattered for
about a kilometer along the north coast of Savusavu peninsula towards the eastern bay
and there are also the dominant hot springs located on land, approximately 165 meters
from the shore. These inland hot springs are actually the ones that are best known of all
the hot springs in Savusavu.
The Savusavu Hot Spring is located a short distance east of the Savusavu Hot
Spring Hotel alongside a stream, and just a distance of approximately 165 meters back
from the shore. The other less active, but spectacular hot springs are scattered all along
the beach for a distance of approximately 1.5 kilometers up to the main wharf. However,
only the Savusavu Hot Spring site was the subject of study for this research.
The Savusavu Hot Springs are located on a hollow depression on the ground,
stretching out for approximately 20 meters across the bank of a small stream located on
its left. Just in front of these springs is a large soccer field and located further on the right
of the spring is the Khemendra Bhartiya Primary School.
Although 12 hot spots were identified within the Savusavu Hot Springs, most of
them were quite small, in both, size and activity. Only six of them were large enough and
67
active enough to be of significance to be sampled. These are clearly illustrated in the
diagram attached in appendix F (a).
Another observation made that was quite significant is that the Savusavu Hot
Spring shows both, daily temporal and seasonal variation in activity. During September,
the springs are less active, and by November, only the springs numbered 1 and 5 are
active, and their activity is very low. This condition prevails until late February. By the
beginning of March, all the springs start coming back to life, and by April, the springs are
at their peak activity. That is, all the springs are at their best, and record temperatures
slightly higher than temperatures recorded in the previous months.
Based on daily observations, in the morning, at 9 a.m, spring number 1 is very
active, along with springs numbered 2, 3, 4 and 7. By midday, the activity of the springs
changes dramatically. Spring number 2 becomes nearly dry, along with that of springs 3
and 7. At this hour, spring number 4 is at a minimal level of activity. In the afternoon, 3
p.m., the activity of spring number 1 also decreases quite significantly, while that of
spring number 5 starts increasing. By the evening, 6.30 p.m., all the springs suddenly
come back to life, but the activity of springs numbered 1 and 5 is the greatest, with the
latter being dominant. Although similar observations had been made before (Healy,
1960), no scientific reasoning has been explained for this trend observed at the Savusavu
Hot Springs.
68
From the measurements of the pH of the Savusavu Hot Springs, it was determined
that this was a neutral to slightly alkaline springs (6.5-7.5). The temperature varied
according to the activity of the hot springs, ranging from 66 oC -102 oC.
4.2 Bacteriological Analysis and tolerance limit test results.
As stated in the description above, the Savusavu Hot Springs exhibit a seasonal
activity, with a corresponding seasonal variation in the microbial load in the soil/water of
the springs. The results (Chapter 3: 3.1) of estimation of the thermophilic bacterial load
clearly show a fluctuation in the colony forming units per milliliter of the samples
collected over the 4 different sampling periods.
Temperature tests (Chapter 3: 3.2) showed that no growth was observed at 35oC,
whereas at 90oC, it was conclusive that the cultured microbes were just surviving. Thus,
it can be said that the growth limits of the isolates obtained was from 45-85oC. It also
shows that there are facultative, obligate and hyperthermophilic bacteria present in the
Savusavu Hot Springs.
These isolates could tolerate sodium chloride up to 3.0% and have a pH range of
5-8 (Chapter 3: 3.3, 3.4). Thus, they do have the ability to tolerate quite high salt
concentrations, probably a fact relating to the location of the hot spring near the sea.
69
4.3 Staining, Biochemical and Exoenzyme Activity.
As outlined in the methods section, various staining, biochemical and exoenzyme
screening were done on all the thermophilic isolates obtained from the Savusavu Hot
Springs.
From the staining procedures (section 3.5), it was evident that majority of the
isolates were Gram positive (80 Gram positive, 20 Gram negative and 4 Gram variable),
produced endospores (53 positive and 51 negative), and were motile (74 isolates were
motile).
Furthermore, oxidase activity was also observed and 62 of the thermophilic
bacterial isolates from the Savusavu Hot Springs were found to be able to utilize glucose
in an oxidative manner. 28 of the remaining isolates utilized glucose in a fermentative
manner and 15 could use the glucose in both, oxidative and fermentative manner. The
catalase test recorded the largest number of positives. That is, of the 104 isolates tested
for catalase activity, 90 were positive (Section 3.6).
It was more difficult to carry out the extracellular enzyme screening tests because
of difficulty in carrying out very neat spot inoculations and problems associated with
moisture accumulation within the nutrient plates. However, interesting results were
obtained for the amylase, lipase and gelatinase tests. 85 isolates showed amylase activity,
2 were amylase variable whereas 17 did not produce extracellular amylase. In contrast to
the amylase test, lipase activity was seen to be very recessive in these thermophilic
70
bacterial isolates. This is confirmed in the results table B, which shows that out of the 104
isolates screened for lipase activity, there were only 12 positives and 92 negatives.
Similar result was also seen for the extracellular gelatinase production. Only 24 of the
bacterial isolates showed gelatinase activity, while the remaining 80 did not produce any
extracellular gelatinase.
All these results show that there is limited input of lipids and gelatin in the
Savusavu Hot Springs. Therefore, these bacteria do not portray strong evidence for the
production of extracellular hydrolytic enzymes that can breakdown these compounds.
Many of these isolates must also have alternate pathways to get rid of the hydrogen
peroxide produced by metabolic reactions, as oxidase activity was observed to be at its
minimum.
4.4 Molecular analysis of the thermophilic bacterial isolates
The cultures were inoculated into Thermus broth medium 162 medium (also
known as Medium 878) and then streaked onto Medium 74 (Thermophilus medium) agar
plates. Upon DNA extraction, purity and concentration determination as described earlier
in the methodology, 16S rDNA PCRs were carried out using the primers EUB A (R) and
EUB B (F). Running 1% TAE-agarose gels tested the product formation from this PCR.
Upon confirmation of product formation and initial comparisons with the positives, the
extracted DNA was subjected to restriction digestion using EcoRI and Hae III.
71
The restriction digestion products were run on 0.8% TBE-agarose gels and stained
using 0.5mg/l ethidium bromide before using the Polaroid camera to take the pictures of
the gels.
Most of the results obtained have been attached in Appendix A. The comparative
cultures that were used were as follows:
1. CN- Anoxybacillus flavithermus
2. AM- Geobacillus stearothermophilus
3. FG- Bacillus licheniformis
4. RT 41 A
5. Thermus TG 275
Isolates previously obtained from various
6. Thermus TG 206
hot pools in New Zealand and stored in
7. Thermus TG 153
the culture collection at the TRU as
8. Thermus TG 8
freeze-dried ampoules
In summary, of all the isolates that were analyzed, 60 showed DNA patterns
similar to that of Anoxybacillus flavithermus, 10 were similar to Thermus TG 206,
another 10 were similar to Thermus TG 153 and 20 were similar to that of Geobacillus
stearothermophilus. However, it was very interesting to note that four of the thermophilic
bacterial isolates that were obtained from the Savusavu Hot Springs did not match DNA
patterns to any of the comparative cultures used, and they also showed very “different”
72
DNA patterns in both, 16S rDNA PCR with EUB A and EUB B, and with restriction
digestion by EcoRI and Hae III.
These were the isolates labeled as 70 oC 8, 70oC 35, 70oC 37 and 75oC 19. Their
unique DNA band patterns can be clearly seen in the results section C, where they have
been clearly listed and their microscopy pictures are attached in appendix E as Phase
Contrast microscopy pictures (Figures 28-31).
Complete 16S rDNA analysis with primers RR 69 (F) and RR 77(R) was also
attempted but without success. RAPD PCR with both, OPR 12 and OPR 13 also failed.
The primers OPR 12 and OPR 13 were able to amplify the DNA from positive controls
but not for the 4 isolates listed in Figures 28-31.
All these data may imply that these isolates could be “novel” thermophile species.
This is because it not uncommon for scientists to identify novel thermophiles. Chen et al.
(2004) discovered two novel thermophilic bacteria while working on the Lu-shan Hot
Springs in the central region of Taiwan. It was confirmed by phylogenetic analysis of the
16S rRNA gene, DNA-DNA hybridization, biochemical features and fatty acid
composition. The name Rubrobacter taiwanensis sp. nov. was proposed for this novel
species.
In addition, Jessica Simbahan and her team (Simbahan et al., 2004) also isolated a
novel Gram-positive thermophilic bacterium from a geothermal pool located in Coso Hot
Springs in the Mojave Desert, California, USA. Based on similar analysis carried out by
73
the Chen team (2004), the name Alicyclobacillus vulcanalis sp. nov. was suggested by the
team for their bacterium. Similarly, Aguiar et al. (2004) isolated from terrestrial hot
springs at Furnas, São Miguel Island, Azores, Portugal, a species of bacterium that was
thought belonged to the recently described genus Sulfurihydrogenibium: and further
proposed that Az-Fu1 (T) represented a new species, Sulfurihydrogenibium azorense.
Sokolova and his colleagues (Sokolova et al., 2004) also isolated from a hot
spring at Norris Basin, Yellowstone National Park a new anaerobic, thermophilic,
facultatively carboxydotrophic bacterium, strain Nor1 (T), which they proposed to be
assigned to a new genus, Thermosinus gen. nov. and the type species Thermosinus
carboxydivorans sp. nov. (Patel et al., 1986) obtained seven isolates from New Zealand
hot pools, of which five were similar to Thermoproteus. Three of these five isolates were
obligate heterotrophs that had never before been reported.
The molecular analysis reveals that all of the identified thermophiles have been found
previously in other hot springs around the world. However, all of these have great
significance. Geobacillus stearothermophilus spores are commonly included with packs
of materials being autoclaved in industries. Death of the spore form indicates the
autoclave is functioning properly and sterilizations carried out are successful. Recently, it
has also been shown that Geobacillus stearothermophilus has very high Cadmium ion
adsorption potential and can be used for metal mobilization in the environment (such as
contamination of drinking water). It can also be used to improve waste treatment of metal
polluted water and soil (Hetzer et al., 2006). Thermus TG153 and TG206 have significant
74
casein and tributyrin hydrolytic properties. Anoxybacillus flavithermus has been the
subject of study for its gelatinases, which are used in gelatin processing (Rivers &
Amelunxen, 1973). In addition, the presence of four strains with significantly variant
DNA patterns indicate that there might be novel bacteria present.
This study shows that there are industrially important thermophilic bacteria present in the
Savusavu Hot Springs and further work should be done to create complete microbial
community profile of the thermophiles in this hot springs.
4.5 Limitations and Recommendations
Upon the completion of the bench work for this research, certain limitations were
realized that had considerable implications. The first and foremost limitation was that
most of the culturing was done using nutrient broth. Many of the other species present
may not have survived in the media because of competition by the more successful
species. Thus, various selective media could have been used to reduce the probability of
this happening. Secondly, apart from the aerobic thermophilic bacteria, there may be an
array of anaerobic bacteria and algae that also colonize the hot springs, however this
study did not test for their presence. Furthermore, there are many thermophilic bacteria
that are still non culturable under laboratory conditions, so these also have not been
accounted for.
Therefore, from all the work that has been done in this research, there may be a
need to carry out further detailed research. Further research may include other thorough
biochemical and molecular techniques to try and correctly identify the four cultures that
75
have earned themselves the temporary status of being “unique” amongst all the
thermophilic bacterial isolates that were subjected to 16S rDNA PCR and restriction
digestions with EcoRI and HaeIII, RR66 and RR77.
The presence of foreshore inter-tidal hot springs also indicates the opportunity for
new research into the thermophilic halophiles that may be present in this rapidly
changing environment. Presence of algae was also observed at temperatures of 49oC and
61oC. Like the foreshore thermophilic microbes, these may also be researched.
Last but not the least, since this research focused only on the aerobic thermophilic
bacteria from the Savusavu Hot Springs, there is still a lot of information missing on the
anaerobic thermophilic bacteria that may be present in this environment. There is a need
to carry out more research to tap into this important group of bacteria, as they also have a
lot of significance in the food, canning and fermentation industries.
76
CHAPTER 5: CONCLUSION
After all the biochemical and molecular analysis were done on the bacterial thermophilic
isolates, it can be concluded that 60 of the isolates had DNA patterns similar to that of
Anoxybacillus flavithermus (Results table: Batch 1; Blue prints), 10 were similar to the
Thermus isolate form New Zealand coded TG 153 (Results table: Batch 2; Violet print),
10 were similar to another Thermus isolate from New Zealand coded TG 206 (Results
table:
Batch
3;
Light
Blue
print),
20
were
similar
to
Geobacillus
stearothermophilus/Bacillus licheniformis (Results table: Batch 4; Dark Red Print) and 4
of the isolates were deemed unique amongst all of isolates listed above (Results table:
Batch 5; Sea Green print).
All of the isolates obtained from the Savusavu Hot Springs have shown
significant extracellular enzyme activity, of which nearly all have great industrial
applications, and the presence of four “unique” isolates suggests that there may be novel
bacteria present in the Savusavu Hot Springs.
77
APPENDICES
APPENDIX A: Growth media
1. Nutrient Agar (NA)
•
Peptic digest of animal tissue 5.0
•
Beef extract 3.0
•
Agar 15.0
•
Final pH 6.8
Mix thoroughly and adjust the pH to 6.8 and then autoclave at 121oC for 15 minutes.
2. Nutrient Broth (NB)
•
Beef extract: 3.0g
•
Peptone: 5.0g
•
Distilled water: 100.0ml
3. Medium 74
• Yeast extract: 4.0g
• Polypeptone: 8.0g
• NaCl: 2.0g
• Distilled water: up to 1000.0ml
4. Medium 878
• Yeast extract: 1.00g
• Tryptone: 1.00g
• Agar: 28.0g
• Nitriloacetic acid: 100.0mg
• CaSO4.2H2O: 40.0MG
• MgCl2. 6H2O: 200.0mg
• 0.01M Fe citrate: 0.5ml
• Phosphate buffer (see below): 100.0ml
- KH2 PO4: 5.44g
- Na2HPO4. 12H2O: 43.00G
- Distilled water: up to 1000.00ml
•
-
Trace element solution (see below): 0.5ml
H2SO4: 0.5ML
MnSO4.H2O: 2.28g
ZnSO4.7H2O: 0.5g
H3BO3: 0.5g
CuSo4.5H2O: 25.0g
Na2MoO4.2H2O: 25.0g
78
-
CoCl2. 6H2O: 45.0g
Distilled water: up to 1000.0ml
Adjust the pH to 7.2 with NaOH. Autoclave at 121oC for 25 minutes. Autoclave the
phosphate buffer separately and then add to the medium.
5. Motility medium
•
•
0.3% NA gel
0.1% tetrazoleum chloride indicator
6. Oxferm medium
• NA media: 4.0g in 150.0ml. Mix and autoclave at 121 oC for 15 minutes.
• Prepare 10% glucose solution: 10.0g glucose in 100.0ml distilled water.
Mix, filter then sterilize at 121 oC for 15 minutes.
After autoclaving, mix the above 2 preparations by adding 50.0ml of the 10% glucose
solution to the 150.0ml of NA media. Mix thoroughly and prepare slants,
7. Nutrient Starch Agar (NSA)
•
•
•
NA: 10.0g
Add 1.0g starch to the above, mix.
8. Nutrient Gelatin Agar (NGA)
• NA: 10.0G
• Distilled water: 500.0ml
• Add 2.0g of gelatin to the above. Mix and autoclave at 121 oC for 25
minutes.
9. Nutrient Tributyrin Agar/Lipid Agar
• Prepare separately: NA: 10.0g, distilled water: 450.0ml
• Prepare separately: 10% tributyrin solution
- tributyrin: 10.0g in 100.0ml distilled water
Autoclave them separately. Then add 50.0ml of the tributyrin solution to the
450.0ml NA, mix thoroughly and plate out.
79
APPENDIX B: STAINS
1. Gram’s stain:
a. Crystal violet
-Solution A: Crystal violet solution:
Dissolve 2.0g of crystal violet in 20ml of 95% ethanol.
-Solution B: Oxalate solution
Dissolve 0.8g of oxalate in 80.0ml of deionised/distilled/sterile water.
Working crystal violet solution: Mix the above two solutions and store in a
glass-stoppered bottle.
b. Gram’s iodine
Dissolve 2.0g of potassium iodide in 5.0ml of deionised/distilled/sterile
water. Then add and dissolve 1.0g of iodine crystals to it and bring the
final volume up to 300.0ml with deionised/distilled/sterile water. Mix well
and store in an amber glass bottle.
c. Gram’s decolouriser
Is simply 95% ethanol stored in a glass-stoppered bottle.
d. Safranin;
Dissolve 0.5g of safranin in 100ml of distilled/deionised/sterile water.
Filter using a gravity filter apparatus to remove undissolved dyes.
Stock safranin: Add 10.0ml of safranin (2.5% solution in 95% ethanol) to
100.0ml of deionised/distilled/sterile water. Mix well and store in a glass
stoppered bottle.
2. Endospore stain;
a. Malachite green
Dissolve 5.0g of malachite green in 100ml distilled/deionised/sterile
water. Filter using a gravity filter apparatus to remove any undissolved
dyes.
b. Safranin-as above for Gram’s stain
3. Indirect stain: Nigrosin
4. Mercuric chloride
•
HgCl2: mol. wt: 271.52: 10.0mg
•
80
APPENDIX C: 16S rDNA PCR product confirmation and initial
comparison agarose gels
Figure 1:060502(2): 16S r DNA PCR
1
2
3
4
5
6
7
8
9 10
11 12 13 14 15 16 17 18 19
20
7: 70oC: 6
8: 70oC: 8
9: 70oC: 9
10: 70oC: 10
12: 70oC: 11
2: -VE Control
3:CN
4:70oC: 1
5: 70oC: 4
6: 70oC: 5
13: 70oC: 12
14: 70oC: 13
15: 70oC: 14
16: 70oC: 15
17: 70oC: 16
18: 70oC: 19
Figure 2: 060502(1) : 16S r DNA PCR
1
2
3
4
5
1:
3: -VE Control
4:CN
5: 70oC: 20
6
7
8
9
10 11 12 13 14 15
6: 70oC: 21
7: 70oC: 24
8: 70oC: 30
9: 70oC: 35
10: 70oC: 36
16 17 18
19
11: 70oC: 37
12: 70oC: 40
13: 70oC: 41
14: 70oC: 42
81
Figure 5: 060615gA: 16S r DNA PCR
1 2 3
4
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
7: 70oC: 45
8: 70oC: 46
9: 70oC: 47
10: 70oC: 48
11: 70oC: 49
2: -VE Control
3: CN
4: 70oC: 38
5: 70oC: 43
6: 70oC: 44
13: 70oC: 52
14: 70oC: 53
15: 70oC: 54
16: 70oC: 55
Figure 6: 060615Gb: 16S r DNA PCR
1 2
3 4
5
6 7
1:
3: -VE Control
4: CN
8
9 10 11 12 13 14 15 16 17 18 19 20
5: 70oC: 57
6: 70oC: 58
7: 70oC: 60
8: 70oC: 61
9: 70oC: 64
10: 70oC: 65
11: 70oC: 66
12: Ta
82
13: CP Rod
15:
16:
17:
18:
19:
20:
Figure 7: 060619G1B: 16S r DNA PCR
1
2
3
4
2: -VE Control
3: CN
5
6
7
8
4: E.coli
5: 70oC: 58
6: 70oC: 60
7: Ta
8: CP Rod
Figure 8: 060628Gel 1bF: 16S r DNA PCR
1 2
3
4
1:
2: DNA ladder
3: CN
4: RT 41 A
13: 70oC: 66
14: 70oC: 8
5 6
7
8
9 10 11 12 13 14 15 16 17 18 19 20
5: TG 275
6: TG 206
7: TG 153
8: TG 8
15: 70oC: 35
16: 70oC: 60
9: 70oC: 57
10: 70oC: 61
11: 70oC: 64
12: 70oC: 65
17: DNA ladder
83
Figure 9: 060628Gel 1Good: 16S r DNA PCR
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15 16 17 18 19 20
8: 70oC: 38
9: 70oC: 43
10: 70oC: 44
11: 70oC: 45
12: 70oC: 46
13: 70oC: 47
14: 70oC: 48
2: CN
3: RT 41A
4: TG 275
5: TG 206
6: TG 153
7: TG 8
15: 70oC: 49
16: 70oC: 52
17: 70oC: 53
18: 70oC: 54
19: 70oC: 55
Figure 24:16S PCR GA 200706
1
2
3
1: RT 41 A
2: TG 206
3:TG 153
4: TG 8
4
5
6
7
8
9
10 11 12
13 14 15
5: DNA ladder
6: CN
7:AM
8: FG
16
17 18
19 20
9: 75oC: 4
10: 75oC: 9
11: 75oC: 10
12: 75oC: 11
84
13: 75oC: 12
14: 75oC: 13
15: 75oC: 14
16: 75oC: 15
17: 75oC: 16
18: 75oC: 17
19: 75oC: 18
Figure 25: 16S PCR GB 200706
1
2
3
1: RT 41 A
2: TG 206
3: TG 153
4: TG 8
5: DNA ladder
6: CN
7:AM
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20
8: FG
9: 75oC: 19
10: 75oC: 20
11: 75oC: 69
12: 75oC: 71
13: 75oC: 72
14: 75oC: 75
15: 75oC: 108
16: 75oC: 129
17: 75oC: 138
18: 75oC: 145
19: 75oC: 146
20: 75oC: 16
85
APPENDIX D: Research related pictures
Figure 27 (A): Fiji islands map
Source: Taken from http:// www.oceania-maps.com/fiji.htm
86
Figure 27 (B): Detailed map of the Fiji islands showing Savusavu
Source: Taken from http:// www.goodnoni.biz/fijimap.html
87
APPENDIX E: Phase contrast microscopy pictures of the unique
isolates from the Savusavu Hot Springs
Figure 28:
o
©Vinay Vikash Narayan 70 C 8
Figure 30 (A):
©Vinay Vikash Narayan 70oC 35
Figure 31 (A):
Figure 29:
Figure 30 (B):
Figure 31 (B):
88
APPENDIX F: (a): Additional pictures of the Savusavu Hot Springs
Figure 32: Picture showing both, springs number 1(denser steam) and 5.
Figure 33: Picture showing the runoff, spring number 6 (arrow) and spring number 1 (steaming)
89
Figure 34: Spring number 1
Figure 35: Spring number: 5
90
Figure 36: Wide view: showing position of spring number 5 (steam) and pathway leading to other springs
.
Figure 37: The above runoff from the hot springs flows down and links up with the sea.
91
Figure 38: Algae type 1 found in the runoff stream/spring 5 at a water temperature of 49oC
Figure 39: Algae type 2 found in the runoff stream/spring 5 at a water temperature of 61oC
92
Figure 40: Cyanobacterial mat community found at 53oC
93
Figure 43: Picture showing spring number 1 (uppermost), 7 (middle) and 3 (lowermost)
94
Hot spots along the intertidal zone of Savusavu foreshore adjacent to the Savusavu
Hot Springs
95
APPENDIX F: (b): Standard 1Kb Plus DNA ladder
•
1 Kb Plus DNA ladder, 0.7µg/lane
•
0.9% Agarose gel stained with ethidium bromide
•
The bands smaller than 1000 bp are derived from
lambda DNA.
(Adapted from the attached notes from the 1 Kb plus DNA Ladder kit)
96
APPENDIX F: (c) Temperature mapping of research site (Savusavu Hot
Springs)
Key:
Bench
Bure/shelter
SPRING
1. A.
B.
C.
2.
3.
4.
5. A
B
C
D
E
6.
7.
8.
9.
TEMPERATURE (OC)
100
97
90
95
74
69
98
100
102
98
97
95
66
95
104
97
REFERENCES
Abe, F. and Horikoshi, K. (2001). The biotechnological potential of piezophiles Trends
Biotech. 19: 102-108.
Adams, M. W. W. (1993). Enzymes and proteins from organisms that grow near and
above 100°C. Annu. Rev. Microbiol. 47: 627-658.
Aguiar, P., Beveridge, T. J. and Reysenbach, A. L. (2004). Sulfurihydrogenibium
azorense, sp. nov., a thermophilic hydrogen-oxidizing microaerophile from
terrestrial hot springs in the Azores. Int. J. Syst. Evol. Microbiol. 54 (Pt 1): 33-39.
Aravillia, R. N., Qunxina, S. and Garrett, R. A. (1998). Archea and the new age of
microorganisms. Trends Ecol. & Evol. 13: 190-194.
Arrhenius, S. (1927). Die thermophilen Bakterien unde der Sralungsdruck der
Sonne. 2. Phy. Chem. (Liepzig) 130: 516-519.
Autar, R. K. (1996). Fiji geothermal resource assessment and development
programme. Proceedings: 21st Workshop on Geothermal Reservoir Engineering.
Stanford University. Stanford, California. Jan. 22-24. SGP-TP 151.
Babel, W., Rosenthal, H. A. and Rapaport, S. (1972). A unified hypothesis on the
causes of the cardinal temperatures of microorganisms; the temperature minimum
of Bacillus stearothermophilus. Acta. Biol. Med. Gem. 28: 565-576.
98
Baird, A. W., and Irwin, J. A. (2004). Extremophiles and their application to
veterinary medicine. Ir. Vet. J. 57 (6): 348-354.
Brock, T. D. (1994). Life at high temperatures. Yellowstone Association for Natural
Science, History and Education, Inc. Wyoming.
Brock, T. D. (1978). Thermophilic microorganisms and life at high temperatures.
Springer-Verlag, New York.
Brock, T. D. (1967). Life at high temperatures. Science. 158: 1012-1019.
Brock, T. D., and Darland, G. K. (1970). Limits of microbial existence; temperature
and pH. Science. 169: 1316-1318.
Brock, T. D. and Freeze, H. (1969). Thermus aquaticus gen. N. and sp. N., a
nonsporulating extreme thermophile. J. Bacteriol. 98: 289-297.
Bruinns, M. E., Janssen, A. E. M., and Boom, R.M. (2001). Thermozymes and their
applications: A review of recent literature and patents. Applied Biochemistry and
Biotechnology. 90 (2): 155-186.
99
Burg, B. V. D., Vriemd, G., Veltman, O. R., Venema, G. and Eijsink, V. G. H. (1998).
Engineering an enzyme to resist boiling. Proc. Natl. Acad. Sci. USA. 95: 2056-60.
Byrd, J., Davidson, B., Haskins, A., Henderson, D., Irwin, K., Ougouag, R., Shotton, E.,
Shotton, J. and Stanton, A. (1998). The microbiology of Thermus aquaticus isolated
from the Vulcan Hot Springs: a review: retrieved on 21st Dec. 2006 from the Internet
source: http://www.cascadehs.csd.k12.id.us/advbio/9899/final%20pages/finalDNA.html.
Carpenter-Boggs, L, Kennedy, A. C. and Reganold, J. P. (1998). Use of Phospholipid
Fatty Acids and Carbon Source Utilization Patterns To Track Microbial Community
Succession in Developing Compost. App. Env. Microbiol. 64 (10) : 4062-4064.
Cavicchioli, R. and Thomas, T. (2000) Extremophiles in encyclopedia of microbiology,
2nd ed. Academic Press, San Diego
Chen, M. Y., Shih-Hsiung Wu, Guang-Huey L., Chun-Ping L., Yung-Ting L., WenChang C. and San-San T. (2004). Rubrobacter taiwanensis sp. nov., a novel,
thermophilic, radiation-resistant species isolated from hot springs. Evol Microbiol.
54 (Pt 5): 1849-1855.
Chung, A.P., Rainey, F. A., Valente, M., Nobre, M. F. and MS da Costa. (2000).
Thermus igniterrae sp. nov., and Thermus antranikianii sp. nov., two new species
from Iceland. Int. Jour. Syst. Evol. Microbiol. 50: 209-217.
100
Daniel, R. M. and Cowan, D. A. (2000). Biomolecular stability and life at high
temperatures. Cell. & Mol. Life Sciences 57: 250-264.
Dempster, E. L., K. V. Pryor, D. Francis, J. E. Young, andH. J. Rogers. (1999). Rapid
DNA extraction from ferns for PCR-based analyses. Biotechniques 27:66-68.
Demirijian, D. C., Moris-Varas, F. and Cassidy, C. S. (2001). Enzymes from
Extremophiles. Curr. Opin. Chem. Biol. 5: 144-151.
Gerday, C., Aittaleb M., Bentahir, M., Chessa, J. P., Claverie, P., Collins, T., D'Amico,
S., Dumont, J., Garsoux, G., Georlette, D., Hoyoux, A., Lonhienne, T., Meuwis, M. A.
and Feller, G. (2000). Cold-adapted enzymes: from fundamentals to biotechnology.
TIBTECH 18: 103-107.
Giltner, W. (1916). Laboratory manual in general microbiology. John Wiley and
Sons, New York.
Harley, J. P. (2005). Laboratory exercises in microbiology, 6th ed.
Companies, Inc., New York.
McGraw-Hill
101
Healy, J. (1960). The Hot Springs and Geothermal Resources of Fiji. R. Owen,
Government Printer. Wellington, New Zealand. New Zealand Dept. of Scientific and
Industrial Research. Bulletin 136.
Herbert, R. A. and Sharp, R. J. (ed) (1993). Molecular Biology and Biotechnology of
Extremophiles. Chapman & Hall, New York
Hetzer. A., Daughney. C. J. and Morgan, H. W. 2006. Cadmium Ion Biosorption by the
Thermophilic Bacteria Geobacillus sterothermophilus and G. thermocatenulatus.
Appl. Environ. Microbiol. 72: (6) 4020-4027.
Hewlett, A. T. (1902). A manual of bacteriology. J. and A. Churchill, London.
Hudson, J. A., Morgan, H. W. and Daniel, R. M. (1988). Numerical classification of
Thermus isolates from globally distributed hot springs. Syst. Appl. Microbiol. 11:
250-256.
Hudson, J. A., Morgan, H. W. and Daniel, R. M. (1986). Numerical classification of
Thermus isolates from Icelandic Hot Springs. Syst. Appl. Microbiol. 9: 218-223.
Jaenicke, R. and Böhm, G. (1998) The stability of proteins in extreme environments
Curr. Opin. Struct. Biol. 8: 738-48.
102
MacEroy, R. D. (1974). Some comments on the evolution of extremophiles.
Biosystems 6: 74-75
Patel, B. K. C., Jasperse-herse, P. M., Morgan, H. W. and Daniel, R. M. (1986). Isolation
of anaerobic, extremely thermophilic, sulfur metabolizing archeabacteria from New
Zealand hot springs. N. Z. J. Mar. Freshwater Res. 20: 39-445.
Poli, A. et al. (2005). Anoxybacillus amylolyticus sp. nov., a thermophilic amylase
producing bacterium isolated from Mount Rittmann (Antarctica).
Syst. Appl.
Microbiol. 11: 250-256.
Postgate, J. (1994). The Outer Reaches of Life. Cambrodge University Press, Cambridge.
Premuzic, E. T. and Lin, M. S. (1999). Induced biochemical conversions of heavy
crude oils. Journal of Petroleum Sc. Engin. 22: 171-80.
Prescott, L. M., Harley, J. P. and Klein, D. A. (1999). Microbiology, Int./4th ed.
WCB/McGraw-Hill Companies, USA.
Rainey, F. A. et al. (1993). Phylogenetic analysis of anaerobic thermophilic bacteria:
aid for their reclassification. J. Bacteriol. 175 (15). 4772-4779.
103
Rapley, R and Walker, J. M. (1998). Polymerase chain reaction: molecular
biomethods handbook. Humana Press Inc., New Jersey.
Rath, C. C., and Subramanyam, V. R. (1994). Enzyme activity of bacteria from hot
springs. Microbes for better living. Ed. R. Shankaran. 583-585.
Rivers, S. and Amelunxen, R. E. (1973). Proteins form thermophilic microorganisms.
Bacteriol. Rev. 37 (3): 320-342
Rossie, M. et al. (2003). Meeting review: extremophiles 2002. Jour. of Bact. 185 (13):
3683-3689.
Ronimus, R. S., L. E. Parker, and H. W. Morgan. (1997). The utilization of RAPD-PCR
to identifying thermophilic and mesophilic Bacillus species. FEMS Microbiol. Lett.
147: 75-79.
Russell, N. and Hamamoto, T. (1998). Psychrophiles in extremophiles: microbial life
in extreme environments. Wiley-Liss, New York
Sellek, G. A. and Chaudhuri, J. B. (1999). Biocatalysis in organic media using enzymes
from extremophiles. Enzyme Microb. Tech. 25: 471-482.
104
Simbahan, J., Drijber, R. and Blum, P. (2004). Alicyclobacillus vulcanalis sp. nov.,
thermophilic, acidophilic bacterium isolated from Coso Hot Springs, California,
USA. Int. J. Syst. Evol. Microbiol. 54 (Pt 5): 1703-1707.
Sokolova, T. G. et al. (2004). Thermosinus carboxydivorans gen. nov., a new aerobic,
thermophilic carbon-monoxide-oxidizing, hydrogenogenic bacterium form a hot
pool of Yellowstone National Park Int. J. Syst. Evol. Microbiol. 54 (Pt 6): 2353-2359.
Stetter, K. O. (1999). Extremophiles and their adaptation to hot environments. FEBS
Lett. 452: 22-25.
Stevens, T. (1994). Subsurface microbiology and the evolution of the biosphere, in
Penny, A. and Hadleman, D. (ed). The microbiology of the terrestrial deep subsurface.
CRC Lewis Publishers, Boca Raton. 215-219.
Tanaka, M. M., Haniu, M., Yasunobu, K. T., Himes, R. H. and Akagi, J. M. (1971). The
primary structure of the Clostridium tatarivorium ferrodoxin, a heat stable
ferrodoxin. J. Biol. Chem. 246: 3953-3960.
Zeikus, J. G., Vielle, C. and Savchenko, A. (1998). Thermozymes: biotechnology and
structure-function relationships. Extremophiles. 2: 179-83.
105
Internet References:
1. Retrieved on 11th April, 2007 from http://www.microbeworld.org/know/hot.aspx
2. Retrieved on 13th April, 2007 from
http://mullinslab.ucsf.edu/protocols/html/QUANTITATION_OF_DNA_AND_R
NA_AND.htm.
3. Retrieved on 3rd May, 2007 from http://www.answers.com/topic/spring-water2?cat=technology

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