Wolkite University

Department of Biotechnology

College of Natural and Computational Science

Isolation, Identification and Characterization of Thermostable Amylase

Producing Bacteria from Woliso Hot Spring, Woliso, Oromia, Ethiopia

Senior Project Submitted to Wolkite University College of Natural and Computational

Science Department of Biotechnology for Partial Fulfillment of Bachelor of Science
Degree in Biotechnology.

By:

Firdos Abdulreshid ID No NCSR /268/07

Mekdes Tekleyohannes ID No NCSR/434/07
Merhun Degefu ID No NCSR/448/07
Mulatu Mokonon ID No NCSR/476/07
Olana Assefa ID No NCSR/514/07
Tajer Abdo ID No NCSR/581/07
Tegegn Tanto ID No NCSR/600/07

Advisor Mr. Belay T. (Msc. In Biotechnology)

Wolkite, Ethiopia

June, 2018
 Acknowledgements
First of all, we would like to give special thanks go to our family for their kind
understanding, warmth and wonderful job as both financial backing and for taking care
of us physically and encouraging us through all the obstacles. Meanwhile, we express
our cordial thanks, whole hearted gratitude, profound regards and the sincerest
appreciation to our advisor Mr. Belay Tilahun for his time, unreserved guidance and
useful comments on our work starting from proposal development to completion of this
senior project work. We would also like to acknowledge Mis. Meseret Guta from Jimma
University, department of microbiology for her unlimited support during bacterial
identification. We would also like to thank Biotechnology laboratory technicians; Mr.
Sembeto Beri and Biology department laboratory technician Mr. Enkossa for his
technical and material support. Finally, we extend our deepest appreciation to all staff
members of biotechnology department for their help in accomplishing our project.
Isolation, Identification and Characterization of Thermostable Amylase
Producing Bacteria from Woliso Hot Spring, Woliso, Oromia, Ethiopia
Firdos Abdulreshid1, Mekdes Tekleyohannes, 1 Merhun Degefu1, Mulatu Mokonon1, Olana
Assefa1, Tajer Abdo1, Tegegn Tanto1 and Belay Tilahun2
1
Wolkite University 4th year Biotechnology Department graduate students
2
Wolkite University, Department of Biotechnology, Email: [email protected]

Abstract
Extremophilic microorganisms are microorganisms that are stable over a wide range of
harsh environmental conditions. Thermophile microorganisms live at 45°C–80°C and
produce enzyme which are thermostable. Thermostable microbial amylases are more
simplified and economical than other sources. The present research was aimed to isolate
and characterize thermostable amylase producing bacterial isolates from Woliso hot
spring of Negash Lodge. So, thirty samples were collected from the source; then, then
from each sample 1ml was transfer on starch agar medium and incubated at 53°C for
48 hrs. Then 3 to 5 colonies were selected based on their colonial characteristics.
Totally 18 isolates were isolated and 10 isolates were secerned based on their ability to
hydrolyze starch. Different biochemical tests were done to identify isolates at genus
and species level i.e. KOH test oxidase test, indole test, H2S test, VP test, citrate test,
catalase test and methyl red test based on Bergey’s Manual of systematic bacteriology.
Amylase were produced by submerged liquid medium with 1% soluble starch at pH 7
and 53˚C for 2 days in shaker incubator with 100 rpm. The optimum parameters where
enzymatic activity of crude extract active was determined by measuring hydrolysis of
starch at 660nm using spectrophotometry. The data were analyzed by SPSS version 20
software. The results revealed that three enzymes from Bacillus spp. (SED2),
Pseudomonas spp. (SED5) and Bacillus spp. (SED7) have maximum activity at 4%
starch, 65˚C and pH of 8.5. Lactobacillus spp. (W20-0) and Bacillus cereus (SED12-1)
at 8% starch, 75 and 65˚C, 8.5 and 7.5pH respectively. Bacillus subtilis (W26-2)
Bacillus licheniformis (W20-1), Lactobacillus spp. (SED10) at 6%starch; 35, 65, 45˚C;
6.5, 4.5, 4.5 pH., Bacillus spp.(SED8-3) &Bacillus spp. (W9) at 4% & 2%, 55˚C, 8.5
&4.5 pH., and have maximum activity at 4% starch, 65˚C and pH of 8.5 and at 8%
starch, 75 and 65˚C, 8.5 and 7.5pH respectively., Lactobacillus spp. (SED10) at
6%starch; 35, 65, 45˚C; 6.5, 4.5, 4.5 pH. Bacillus spp. (SED8-3) & Bacillus spp. (W9)
at 4% & 2%, 55˚C, 8.5 &4.5 pH. The amylase produced by bacterial isolates from
Woliso hot spring was thermostable and can performed from 65oC up to 75oC. The
optimum hydrolysis conditions required for amylase activity were optimized. For most
of the enzymes the optimum temperature was observed to be 65oC; whereas optimum
substrate concentration and pH for most of them were 4% starch and 8.5pH
respectively. They were active at a various starch conditions (2%-10%), pH of 4.5-8.5,
and temperature of 35-75°C.Generally, the bacteria isolated were thermophilic and
produced thermostable amylase. Therefore, Woliso hot spring could be the source of
thermophilic microorganisms and thermozymes as well.
Keywords: Hot spring, optimization, thermostable amylase, thermophilic
microorganisms.

ii
Table of contents
Contents
Acknowledgements .....................................................................................................................i
Abstract ......................................................................................................................................ii
Table of contents ....................................................................................................................... iii
List of figures .............................................................................................................................. v
List of Table ............................................................................................................................... vi
List of abbreviations ................................................................................................................. vii
1. Introduction ....................................................................................................................... 1
1.1. Statement of problem ............................................................................................... 3
1.2. Objective of the study ............................................................................................... 4
General objective .............................................................................................................. 4
Specific objective ............................................................................................................... 4
1.3. Significance of the study............................................................................................ 5
2. Literature Review .............................................................................................................. 6
2.1. Thermophilic Microorganisms................................................................................... 6
2.1.1. Thermophilic fungi................................................................................................. 6
2.1.2. Thermophilic Bacteria ........................................................................................... 7
2.2. Source of Thermophilic Bacteria ............................................................................... 7
2.2.1. Production method ........................................................................................... 8
2.3. Amylase Producing Thermophilic Bacteria................................................................ 8
2.4. Microbial Source of Thermostable Amylase ............................................................. 9
2.5. Microbial Enzyme production ................................................................................. 10
2.5.1. Process parameters for amylase production .................................................. 10
2.6. Applications of Amylase .......................................................................................... 11
3. Materials and Methods ................................................................................................... 13
3.1. Sample collection and Description of the Study sites ............................................. 13
3.2. Isolation of pure culture .......................................................................................... 13
3.3. Characterization of thermophilic isolates ............................................................... 14
3.1.1. Biochemical characterization of isolates ......................................................... 14
3.4. Crude Thermostable Amylase production............................................................... 16
3.5. Enzyme activity Assay .............................................................................................. 16
3.6. Enzymatic starch hydrolysis condition optimization ............................................... 16
3.9.1. Substrate concentration optimization ............................................................ 16
3.9.2. Optimum pH determination ............................................................................ 17
3.9.3. Optimum temperature determination ............................................................ 17

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 3.7. Data analysis ............................................................................................................ 17
4. Results and discussion ..................................................................................................... 18
4.1. Physical characteristics Sample ........................................................................... 18
4.2. Isolation of pure culture ...................................................................................... 18
4.1.6.1. Biochemical characterization ...................................................................... 18
5. Starch Hydrolysis test .............................................................................................. 20
4.1.6.2. Salt Tolerance Test ...................................................................................... 21
5.1. Crude Thermostable Amylase production............................................................... 22
5.1.1. Enzyme activity Assay .......................................................................................... 22
5.1.2. Substrate concentration optimization ................................................................ 22
5.1.3. Optimum pH determination ................................................................................ 23
5.1.4. Optimum temperature determination ................................................................ 24
6. Conclusion ....................................................................................................................... 26
7. Recommendations........................................................................................................... 27
8. References ....................................................................................................................... 28

iv
List of figures
Pages
Figure 1 Isolation of pure culture................................................................................. 13
Figure 2 Clear zone observed around amylase-producing bacteria ............................. 20
Figure 3 Effect of salt concentration on bacterial isolates ........................................... 21
Figure 4 profile plot 2 showing estimated marginal plot ............................................. 22
Figure 5 Optimum pH value for amylase activity ....................................................... 24

v
List of Table
Table 1 Morphological characteristics of bacterial isolates from water and Sediment
samples of Woliso hot water spring ............................................................................. 18
Table 2 Biochemical characteristics of the bacterial isolates isolated from Woliso hot
spring............................................................................................................................ 19
Table 3 Effect of salt concentration on bacterial isolates ............................................ 21
Table 4 Substrate concentration optimization for amylase activity ............................. 23
Table 5 Effect of pH on amylase activity .................................................................... 23
Table 6 Effect of temperatures on amylase activity..................................................... 25

vi
List of abbreviations
ANOVA: Analysis of Variance

KOH test: Potassium Hydroxide test

MR Reagent: methyl red reagent

NA Medium: nutrient agar medium

nm: nanometer

OD: Optical Density

PH: power of hydrogen

Rpm: revolution per minute

SED: Sediment

SIM test: Sulphide, Indole and Motility test

SmF: Submerged Fermentation

SPSS: Statistical Package for Social Science

VP test: voges-proskauer test

W: Water

vii
 1. Introduction
Thermophilic microorganisms have the characteristics of adapting to survive in harsh
environmental conditions (Horikoshi et al., 1998). Existence of life at high
temperatures is quiet fascinating. At elevated temperatures, only thermophilic
microorganisms are capable of growth and survival. Thermophilic bacteria are
microbes that mostly inhabit hot springs, live and survive in temperatures above 42°C.
(Wajeeha et al., 2011). The discovery of thermophilic bacteria capable of carrying out
life processes in the boiling hot springs of Yellowstone National Park has become a
foundation of developments in medicine and biotechnology. Then, thermophiles have
been isolated in geothermal features of all over the world (Moracci et al., 2001).
Thermophiles have been isolated from different ecological zones (e.g., hot springs and
deep sea) of the earth. Thermophiles can be categorized into moderate thermophiles
(growth optimum, 50–60°C), extreme thermophiles (growth optimum, 60–80°C), and
hyperthermophiles (growth optimum, 80–110°C) (Gupta et al., 2014). Thermophilic
microorganisms can be classified as Gram-positive or Gram-negative, they can exist
under aerobic or anaerobic conditions, and some of them can form spores. The
microorganisms with the highest growth temperatures (103–110°C) are members of the
genera Pyrobaculum, Pyrodictium, Pyrococcus, and Melanopyrus belonging to
Archaea; within Fungi, the Ascomycetes and Zygomycetes classes have high growth
temperatures (Busk et al., 2013; Kumar et al., 2014).

Due to their increased importance, potential applications, and roles in different fields,
scientists have concentrated their studies to discover new genus and species across the
world (Yonedaet al., 2013; Cihanet al., 2014; Aannizet al., 2015)

The production of microbial thermophilic amylase from bacteria is dependent on the

type of strain, composition of medium, method of cultivation, cell growth, nutrients
requirements, incubation period, pH, temperature, metal ions and thermostability.
Thermostability is a desired characteristic of most of the industrial enzymes.
Thermostable enzymes are isolated from thermophilic organisms had found a number
of commercial applications because of their stability are at high temperature (100-
110°C) where enzymatic liquefaction and gelatinization of starch have been performed
(Reddy, 2003). Thermophilic amylase enzymes are biological catalysts which are an
indispensable component of biological reaction (Haki et al., 2003). These enzymes are
now being used in various sectors of industry. They are used in detergents; paper
industry, textile industry, food industry and many others industrial applications.
Thermophilic amylase enzymes have been in use since ancient times and they have
been used in saccharification of starch, production of beverages like beer, treatment of
digestive disorders and production of cheese from milk (Thota, 2015). Thermostable
amylolytic enzymes have been currently investigated to improve industrial process of
starch degradation and were of great interest for the production of valuable products
like glucose, crystalline dextrose, dextrose syrup, maltose and maltodextrins (Aquino
et al.,2003).

Ethiopia have difrente echological areas having from highst altitude tolowst altitudinal
varation which gives achanse of getting different ecologies including extrim
environments like hot springs live volcano etc. Woliso hot spring is one of the extrim
environment where thermophiles will be found which is located at Negash Lodge of
Woliso and surrounding area is one of the hot springs in Oromia region of Ethiopia.
Woliso hot spring is one of the potential source of thermotolerant amylase producing
microorganisms. The microbial lode of the hot spring has been not yet fully explored.

2
 1.1.Statement of problem
For industrial applications, enzymes must be stable under process conditions. However,
amylases that are derived from plants and animals are not sufficient enough to be used
at industrial scale. In addition to their production in less quantity, enzymes from animal
and plants are not thermostable. These make them less stable for industrial applications.
Hence, thermostable microbial enzymes play an important role in different industries
due to their stability at harsh environmental conditions, such as extreme temperatures.
Even though, its uses at different areas of industries hold a great position, the
thermostable amylase produced by thermophilic bacteria for industrial application in
Ethiopia has not yet been fully explored. Several researchers have reported
thermophilic bacteria from diverse environmental habitats such as geothermal sites and
hot springs around the world. Here in our country we have diversified ecological areas
having extreme conditions. From those woliso hot spring is one of the area having this
future. However, the thermophilic microbes of this site and their enzymes have not been
reported till date. So, isolation and characterization of microbes from this site is
important to know the diversity of thermophilic bacteria that can produce thermostable
amylase.

3
 1.2.Objective of the study

General objective
✓ The main objective of study is to isolate, identify and characterize
thermostable amylase producing bacteria from Woliso hot spring.

Specific objective
✓ To isolate thermophilic bacteria from Woliso hot spring
✓ To identify thermophilic bacteria from Woliso hot spring
✓ To characterize thermostable bacterial isolates from Woliso hot spring
✓ To screen amylase producing bacterial isolates.
✓ To extract crude thermostable amylase from bacteria by submerged
fermentation.
✓ To assess the enzymatic activity of crude thermostable amylase enzymes
on starch substrate.
✓ To determine optimum conditions for enzymatic starch hydrolysis.

4
 1.3.Significance of the study
Nowadays industry has been minimizing the use of chemicals or replaced by enzymes
to solve environmental problems associated with those chemicals. But most of
industries have operated at high temperature. Consequently, thermostable enzymes are
most significantly applicable because thermophilic process is more stable, faster, needs
lower costs. They have higher stability to organic solvents, acidic and alkaline pH and
detergents. As a result, thermostable amylases are of great significance in industrially
viable technology and have a number of commercial applications due to their overall
inherent stability. So, this research will increase thermostable amylase enzymes for
industrial use as amylase is one of the most important industrial enzymes, having
applications in different industrial processes such as brewing, baking, textiles,
pharmaceuticals, starch processing, and detergents. Therefore, in this regard searching
enzymes from such environment will fill the gap to feed different industries.

5
 2. Literature Review
2.1.Thermophilic Microorganisms
Thermophilic microorganism is a microorganism that thrives at relatively high
temperatures, between 45 and 80 °C. Thermophiles, meaning heat-loving organisms,
are organisms with an optimum growth temperature of 50 °C or more, a maximum of
up to 70 °C or more, and a minimum of about 40 degrees C, but these are only
approximate. Some extreme thermophiles (hyperthermophiles) require a very high
temperature (80 °C to 105 °C) for growth. Their membranes and proteins are unusually
stable at these extremely high temperatures. Thermophile bacteria live at 45°C–80°C
and produce enzyme which are thermostable. Enzymes from thermophile bacteria are
also known as thermophile enzyme because they are thermostable and thermo-active.
The thermophile enzyme, like protease, lipase and amylase that are thermostable
happened because those were resistant to high temperature and pH. Thus, many
important biotechnological processes utilize thermophilic enzymes because of their
ability to withstand intense heat (Gustina and Ruth, 2018).

Originally, studies did not focus on the survival of thermophilic organisms even though
the existence of thermophilic organisms was well known. There were two types of
observations that led to the discovery and cultivation of thermophilic organisms.
Firstly, it was discovered by standard bacterial culture procedures mainly in the canning
industry that led to the cause of many problems by heat tolerant, spore forming bacteria.
The second type of research resulted from ecological studies of organisms living in
geothermal habitats. Later, because of careful observations, organisms living at high
temperatures were discovered. Narayan et al. (2008) conducted a study to determine
the presence of aerobic thermophilic bacteria in Savusavu hot springs in Fiji. The
existence of thermophilic organisms had though been known from the early years of
bacteriology (Michael et al., 2009).

2.1.1. Thermophilic fungi

Thermophilic fungi are a small assemblage in eukaryota that have a unique mechanism
of growing at elevated temperature extending up to 60 to 62°C. During the last four
decades many species of thermophilic fungi sporulating at 45oC have been reported.
Much is known about the occurrence of thermophilic fungi from various types of soils
and in habitats where decomposition of plant material takes place. These include:
composts, piles of hays, stored grains, wood chip piles, nesting material of birds and

6
animals, snuff, and municipal refuse, and other accumulations of organic matter
wherein the warm, humid, and aerobic environment provides the basic physiological
conditions for their development. In these habitats thermophiles may occur either as
resting propagules or as active mycelia depending on the availability of nutrients and
favourable environmental conditions (Lee et al., 2014; Salar and Aneja, 2007).

2.1.2. Thermophilic Bacteria

Thermophilic bacteria are those that grow at temperatures above the maximum
temperature for the great majority of bacteria, especially the pathogenic forms. The
maximum temperature for the pathogenic bacteria is about 45°C. Their optimum
temperature is about 37.50C. The true thermophiles show no growth, or only very
feeble growth, below 40 to 450C. Their development requires temperatures above
500C., and some are able to develop at a temperature of 800C., though most abundant
growth is shown at 60 to 70°0. A group of facultative thermophilic bacteria has been
discovered which develop at room temperature, about 200C., and have their optimum
temperature at about 500C., and their maximum temperature at about 600C. In this group
belong some of the well-known spore-forming soil organisms. Bacillus sp. was tested
for thermo resistance within the temperature range of 95–135°C and various exposure
times. The highest thermo resistance was found with B.licheniformisspores which
survived the temperature of 135°C. The results indicate that some Bacillus spores may
survive in milk even after the heat treatment (Bergey, 1919; Melzock et al., 2004).

2.2.Source of Thermophilic Bacteria

Thermophilic microbes are naturally found in shallow and deep marine hydrothermal
vent environments, heated beach sediments, continental solfataric areas, geysers and
hot springs. The temperatures and pressures of these habitats vary considerably. The
majority of these systems are characterized by extremely low oxygen concentrations.
Consequently, most of the known species of thermophiles are classified as obligate or
facultative anaerobes, though aerobic and micro aerophilic isolates are also known. The
main aquatic environments are hot springs and hydrothermal vents; hydrothermal vents
being the only marine thermophilic environment (Kumar and Swati, 2001).
Hot springs and geothermal vents are found in several parts of the world; some
thermophilic prokaryotes (bacteria and achaea) are specially adapted to grow in these
environments. A zonation of microorganisms was found according to their temperature
optima (Khalil A. 2011).

7
Many of the prokaryotes that grow in the most extreme environments are achaea- a
group that is clearly distinguishable from both the present-day bacteria and the
eukaryotes. Members of the genus Sulfolobus (achaea) are among the best-studied
hyperthermophiles. They are commonly found in geothermal environments, with a
maximum growth temperature of about 85-90oC, optimum of about 80oC and minimum
of about 60oC. They also have a low pH optimum (pH 2-3) so they are termed
thermoacidophiles. Sulfolobus species gain their energy by oxidizing the sulphur
granules around hot springs, generating sulphuric acid and thereby lowering the pH

2.2.1. Production method

There are mainly two methods which are used for production of amylase on a
commercial scale. These are: submerged fermentation and solid-State fermentation.
The latter is a fairly new method while the former is a traditional method of enzyme
production from microbes which has been in use for a longer period of time.
Submerged fermentation employs free flowing liquid substrates, such as molasses and
broths. The products yielded in fermentation are secreted into the fermentation broth.
The substrates are utilized quite rapidly; hence the substrates need to be constantly
replenished. This fermentation technique is suitable for microorganisms such as
bacteria that require high moisture content for their growth. SmF is primarily used for
the extraction of secondary metabolites that need to be used in liquid form (Couto and
Sanroman, 2006).

2.3.Amylase Producing Thermophilic Bacteria

Thermophiles are a large category of microorganisms that show optimum growth at
temperatures of 50°C or higher. These microbes thrive in various environments in both
marine and terrestrial habitats. The ability of microorganisms to proliferate under
extreme conditions is of widespread importance in microbial physiology, ecological
cycle, industry and evolution (Horikoshi et al., 1998).

Thermophiles play a complex role in ecosystem maintenance, particularly with respect

to carbon cycle and biomass deconstruction. These ecological processes are carried out
by various thermophilic microorganisms (Lawrence, 2011). Temperature is one of the
most important factors controlling the activities and evolution of organisms, and limited
species diversity found in extreme environments suggests that many organisms lack the
capacity for successful adaptation to these environments (Busk et al., 2013).

8
High-temperature environments are of special interest, in that they reveal the extremes
to which evolution has been pushed and their distinct strategies in carbon cycling
(Brown et al., 1985).

2.4.Microbial Source of Thermostable Amylase

Amylases are universally distributed throughout the animal, plant and microbial
kingdoms. Over the past few decades, considerable research has been undertaken with
the extracellular amylase being produced by a wide variety of microorganisms
(Setyorini, 2006). The major advantage of using microorganisms for the production of
amylases is the economical bulk production capacity and microbes are easy to
manipulate to obtain enzymes of desired characteristics. The microbial enzyme meets
the industrial demand and a large number of them are available commercially. Amylase
has been derived from several fungi, yeasts, bacteria and actinomycetes, however,
enzymes from fungal and bacterial sources have dominated applications in industrial
sectors. Fungal sources are mostly terrestrial isolates such as Aspergillus species.
Amylases from plant and microbial sources are employed for centuries as food
additives (Mabel et al., 2006). Fungal and bacterial amylases are mainly used for
industrial applications due to their cost effectiveness, consistency, less time and space
requirement for production and ease of process optimization and modification (Ritu et
al.,2017).

A wide range of bacterial species has been isolated for amylase secretion. Most
are Bacillus species (B. subtilis, B. stearothermophilus, B. amyloliquefaciens, B.
licheniformis, B. coagulans, B. polymyxa, B. mesentericus, B. vulgaris, B.
megaterium, B. cereus, B. halodurans, and Bacillus sp.), but amylases from Rhodother
musmarinus, Corynebacterium gigantea, Chromohalobacter sp., Geobacillus
thermoleovorans, Lactobacillus fermentum, Lactobacillus manihotivorans,
and Pseudomonas stutzeri have also been isolated (Hussain et al.,2013). The genus
Bacillus produces large variety of extracellular enzymes, of which amylases and
proteases are of significant industrial importance. Thermostable amylases were
produced by Bacillus spp., Bacillus licheniformis, Bacillus halodurans and B.
amyloliquefaciens (Archanaet and Tulasi, 2011).

9
 2.5.Microbial Enzyme production
Enzymes are prepared by inoculating a microbial strain in to nutrient broth and
incubating in shaker for 24 hrs. The inoculums is then transferred aseptically to
production medium (Bacteriological peptone, MgSO4.7H2O; KCl; Starch and
incubating it in incubator shaker at 180 rpm for 14 hrs. This production is carried
out in shake flask fermentation using production media (Singh et al., 2015).

2.5.1. Process parameters for amylase production

2.5.1.1.Temperature
Process parameters like temperature, pH, aeration, oxygen transfer and moisture should
be controlled conveniently. The optimum process control parameters vary depending
on the microbial source, desired end product, method of fermentation employed and
many other such factors. The optimum temperatures for strains of B. licheniformis and
B. subtilis29 studied for growth and amylase production by Reddy et al and they found
it to be 45°C to 46 °C and 50 °C, respectively. The optimum temperature for production
of the enzyme by Bacillus sp. isolated from dhal industry waste was found to be 60°C.
The amylase produced by Clostridium acetobutylicum was incubated at temperatures
ranging from 30-60°C. The optimal activity was found to be at 45°C. Rhodothermus
marinus, a thermophilic marine microorganism exhibited optimal yield of thermostable
α-Amylase at 61°C (Ajita and Thirupathihalli,2014, Reddy et al., 2003). Thermococcus
α-Amylases are optimally active at temperatures close to 80°C. Pyrococcus enzymes
are optimally active around 100°C (Upgade, et al., 2011).

2.5.1.2.pH
Enzymes are pH sensitive and hence care must be taken to control the pH of the
production process. Pyrococcus furiosus produces α-Amylase which shows activity at
an optimum pH of 6.5–7.5 (Haki and Rakshit, 2003). Thermophilic microorganisms
such as P. woesei and Thermococcus profundus exhibited optimal production of α-
Amylase at pH value of 5.0. Bacillus amyloliquefaciens produces the enzyme with an
optimum pH of 7.0 (Ramachandran, 2004, Reddy et al., 2004).

2.5.1.3.Substrate
Optimum substrate concentration is a critical factor for the stability of enzyme
produced. Bacillus subtilis was studied for production of α-Amylase using solid state
fermentation for 48 hours at 37°C with wheat bran as substrate. The specific activity of

10
amylase was found to be 13.14 µmol/mg/min at 40° (Ajita and Thirupathihalli, 2014;
Gangadharan et al, 2006).

Investigation of production of α-Amylase by Bacillus cereus MTCC 1305 revealed that

among different carbon sources that were studied, glucose (0.04 g/g) showed enhanced
enzyme production (122±5) U/g) (Ajita and Thirupathihalli, 2014; Sivaramakrishnan
et al.,2007).

2.6.Applications of Amylase
Starch degrading amylase enzymes are most important in the biotechnology industries
with huge application; most widely used in industries such as food, fermentation, starch
processing, textile and paper. Amylase has a great deal of application in starch
saccharification. The amylolytic enzymes find a wide spectrum of applications in food
industry for production of glucose syrups, high fructose corn syrups, maltose syrups,
reduction of viscosity of sugar syrups, reduction of haze formation in juices,
solubilization and saccharification of starch for alcohol fermentation in brewing
industries, and also find a wide range of application in baking, paper, textile and
detergent industry (Naidu and Saranraj, 2013; Ritu et al., 2017).
Alternative energy source that is widely studied is bioethanol, a renewable energy
which is more environmentally friendly and reliable. Generally, bioethanol is produced
from carbohydrate materials, either from simple or complex carbohydrates. Starchy
material is the most widely used raw material for bioethanol. In the bioethanol
production process, starch in carbohydrates is firstly converted into glucose, and then
the glucose is fermented to form bioethanol. Conversion of starch into glucose can be
done chemically or enzymatically using amylase enzyme group. Amylase is supposed
to be active and stable at high temperatures such as in the processed gelatinization (100-
110°C) and liquefaction for the efficiency of the process, (Ritu et al.,2017)

Thermostable amylase is used as a thinning agent, which brings about reduction in

viscosity and partial hydrolysis of starch. Amylases convert starch in to fermentable
sugars. They act up on starch which is used as a raw material to manufacture ethyl
alcohol. In the presence of amylases, the starch is first converted in to fermentable
sugars. The use of bacterial enzyme partly replaces mal in brewing industry, thus
making the process more economically significant (Kiro, 2012; Sang-Lang et al.,
2011).

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12
 3. Materials and Methods
3.1.Sample collection and Description of the Study sites
Woliso is the capital of South West Shoa administrative zone. The town is located at a
distance of 114 kilometers south west of Addis Ababa, along the Addis Ababa-Jimma
route. The coordinates of the town are 8°32′N latitude and 37°58′E longitude. It is
characterized by temperate type of climate with daily temperature ranging from 180c
and 270C and is located 1900m above sea level. The hot springs located at Negash
Lodge of woliso and surrounding area is one of the hot springs in Oromia region of
Ethiopia. The present study will be conducted at the laboratory of Biotechnology
Department, Wolkite University from April to June 2018. Total of 30 samples (i.e. 15
water samples and 15 sediment samples) were collected from Woliso hot spring
aseptically in falcon tubes and the samples were transported in icebox aseptically to
Wolkite University, Biotechnology laboratory, for processing and microbial isolation.
Sample processing, laboratory isolation, identification and preservation of bacteria
were carried out in Wolkite University, Biotechnology laboratory. The temperature and
pH of sample were recorded during the time of sampling.

3.2.Isolation of pure culture

After the samples were transported to laboratory, Isolation of thermophilic bacteria
from the culture were done by serial dilution up to 10-5 or 10-6 dilution. 0.1 ml of each
dilution were inoculated by spread plate technique into the prepared Starch Agar
Medium (which consists of nutrient agar-yeast extract, peptic digest of animal tissue,
meat extract, peptone, agar and soluble starch) (Figure1).

Figure 1 Isolation of pure culture

Then, the inoculated plates were incubated at 53oC for 48hr and the growths of
thermophilic bacteria were observed. Representative 3-5 colonies of bacterial isolates
were randomly picked from countable MRS agar plates. Each bacterial isolate was

13
purified by repeated streak-plating on SAM agar for three times. The pure isolates were
maintained on nutrient broth containing 10%glycerol at -4oC and sub-cultured every
four weeks until required for characterization and kept at -80oC for long term
preservation (Abebe, 2016; Gustina Ruth, 2018).

3.3.Characterization of thermophilic isolates

3.1.1. Biochemical characterization of isolates

The morphological characterization of bacterial isolates was determined according to
their cultural characteristics using microscope (colony size, colony color, colony
texture) and Biochemical characterizations will be conducted. These were oxidase test,
indole test, H2S test, VP test, citrate test, catalase test and methyl red test based on
Bergey’s Manual of systematic bacteriology. The preliminary selection test to be used
for characterization of the bacterial isolates will be graham staining (Giffel et al., 1995)

3.1.1.1. Oxidase test

Oxidase test were undergone by soaking a filter paper in a solution of 1% kovac’s
oxidase reagent, followed by smearing colonies on the filter paper with a clean loop
(Vashit et al., 2013).

3.1.1.2. Voges-Proskauer (VP) test

Suspended colony from pure culture were investigated in VP medium and incubated at
53oc for 48 hr., then added 0.2ml of 40% of KOH and 0.5 ml of alpha naphtol solution.
VP Positive result will show Pink or red color at the surface of the medium and VP
Negative result has shown yellow or copper color at the surface of the medium
(Macfaddin, 2000)

3.1.1.3. Citrate test

Small number of bacteria was inoculated in citrate medium (sodium citrate, an
ammonium salt and the indicator bromothymol blue) and incubated at53oC for 48hr.
The positive result has developed color change from green to blue and the negative
result shown no color change on citrate medium (Macfaddin, 2000).

3.1.1.4. Catalase test

The bacteria were spread on starch agar plate and it was incubated for 48hr under
appropriate condition. Then bacteria were collected from colony and applied on
microscopic slide. After that one drop of hydrogen peroxide was added. Then the

14
positive result was formation of oxygen in the form of bubble and in negative result no
bubble was formed (Paik, 1980).

3.1.1.5. Methyl red test

The methyl red test was used to detect the ability of an organism to produce and
maintain acid end products from glucose fermentation. 3-5 drops of MR reagent were
added to bacterial isolates grown on starch agar medium. The Positive result was pink
or red in color and the negative was yellowish-orange (Murray et al., 2003).

3.1.1.6. Indole test

The indole test screens for the ability of an organism to degrade the amino acid
tryptophan and produce indole. Tryptophan was an essential amino acid, which was
oxidized by some bacteria resulting in the formation of indole, pynivic acid and
ammonia. The indole which was produced was detected by adding KOVAC’s reagent
which produced cherry red colored ring (Vashit et al., 2013).

3.1.1.7. Hydrogen sulfide test

The ability of the microorganisms to produce H2S was tested by using H2S production
test. SIM medium can be used for the test. Colonies were grown on the SIM medium at
530c. If H2S production is present, black color was appeared (Vashit et al., 2013).

3.2. Screening thermotolerant Amylase-producing bacteria

Amylase producing bacteria were screened based on their ability to degrade starch in
the medium. Single bacterial isolate was grown on starch containing nutrient agar
medium at 53°C for 48hr., and then was examined by flooding,
1% iodine solution on starch agar plate. The presence of blue color around the growth
media indicated negative result, and formation of clear zone around the bacterial colony
indicated a positive amylase-producing bacterium (Win et al., 2015).

3.3. Salt Tolerance Test

Salt tolerance of the bacterial isolate were tested by inoculating each isolate into
nutrient broth in a test tube with varying NaCl concentrations (0.5,1.5,2.5,3.5,4.5). The
nutrient broth without NaCl was used as negative control and bacterial growth was
assessed by measuring its OD (Subra k. mukhopadhyay et al., 2013).

15
3.4. Crude Thermostable Amylase production
Positive amylase producer isolates were transferred in to production medium for
production of thermostable amylase. The production medium contains soluble starch
(10 g/L), peptone (5 g/L), (NH4)2 SO4 (2 g/L), KH2PO4 (1 g/L), K2HPO4, (2 g/L), MgCl2
(0.01 g/L) preparedat7 pH. 2ml of inoculum was taken in to 250 ml Erlenmeyer flask
containing 40ml of production medium. After two days of incubation at 530c and
agitation with shaker at 100 rpm. After incubation, fermented broths were centrifuged
at 4000 rpm for 25 minutes in a centrifuge. As amylase is extracellular enzyme, it is
released to the medium as a supernatant (Ekka and Namdeo, 2018). Hence, the
supernatant was taken and served as a crude amylase for further enzyme activity. This
crude enzyme extract was used in amylase enzyme activity measurement and
characterization (Abebe, 2016; Alemayehu and Temam, 2017).

3.5. Enzyme activity Assay

The activity of the amylase was determined by using starch as the substrate. The
reducing sugar released as an enzymatic reaction product was measured. A sample tube
containing 1 ml of 1% starch was incubated for 5 minutes at 5°C. After five minutes 1
ml of amylase and 1ml of 0.85% NaCl was added and the incubation was continued for
30 minutes. The enzyme activity was stopped by adding 1 ml of 0.7% NH2SO4 and by
putting in water bath at 100oC. A control tube was also prepared using the same
procedure in the absence of amylase. The absorbance of the samples was measured at
660 nm and enzyme activity was determined (Deljou and Arezi, 2016; Singh et al.,
2016).

3.6. Enzymatic starch hydrolysis condition optimization

3.9.1. Substrate concentration optimization

Starch was used as a substrate. Substrate solutions with varying concentration of starch
that include 2%, 4%, 6%, 8%, 10%(w/v) were produced. The starch solution was then
dissolved with each enzyme and incubated in oven at 53°C for 45 minutes. The
variation in enzymatic activity due to substrate concentration was measured using
spectrophotometer (Subra et al., 2013; Suman and Ramesh, 2010).

16
 3.9.2. Optimum pH determination
Each enzyme isolates and substrate at the optimum concentration was dissolved in 5 ml
of various buffers (pH 4.0 to 10.0). For pH adjustment Tris-HCl and borax NaOH buffer
were used. Then after incubation for 45 minutes, the enzyme activity variation at
different pH level was measured using spectrophotometer (Singh et al., 2016; Subra et
al., 2013; Suman and Ramesh, 2010).

3.9.3. Optimum temperature determination

This procedure was performed using spectrophotometer at various temperatures of 35,
45, 55, 65 and 75°C for 45 minutes, under optimum pH and substrate concentration.
The temperature that resulted in the highest reducing sugar was selected as the optimum
temperature for enzymatic reaction (Singh et al., 2016; Suman and Ramesh, 2010).

3.7. Data analysis

The data in this study were analyzed using SPSS version 20. Means and standard
deviations were calculated using analysis of variance (ANOVA) to analyze the
significant differences between the means using Duncan’s multiple range test (p <
0.05). The triplicates mean values of tests were analyzed. Significant difference was
defined as p < 0.05 (Fentahun and Kumari, 2017).

17
 4. Results and discussion

4.1.Physical characteristics Sample

At the source, different parameters of the samples (such as temperature, pH, and salt
concentration) were measured and recorded. The pH range was between 8.02 up to
8.68, temperature range was between 470c up to 57 0c, and the amount of salt was
recorded as 0.017g. Then, the samples were transported to Wolkite University
Biotechnology Laboratory.

4.2.Isolation of pure culture

From 30 samples collected and processed a total of 18 bacterial isolates were isolated
and purified. Purified isolates that are found to be Gram-positive and Gram-negative
with diverse cultural characteristics with regard to color (white, gray pink or whitish),
shape (circular or irregular), edge (irregular or smooth) and elevation (flat or raised).
Furthermore, the sizes of the colonies ranged from medium to large and motility motile
and nonmotile as summarized in table 1.

Morphological characteristics of bacterial isolates from water and Sediment

Table 1
samples of Woliso hot water spring
Colony Bacterial Colony Morphology
code isolates
Shape Color Texture Size Motility Elevation
Sed-2 Bacillus spp. Grey Smooth Medium None
W-26-2 Bacillus subtilis Grey Rough Large Motile Rise
Sed-8-3 Bacillus spp. Regular Grey Smooth Small Motile
W-20-0 Lactobacillus Grey Smooth Large None Rise
spp.
Sed-12-1 Bacillus cereus White Smooth Large Motile Flat
Sed-7-1 Bacillus Grey Rough Medium Motile Rise
licheniformis
Sed-7 Bacillus spp. Grey Smooth Small Motile Flat
Sed -5 Pseudomonas Grey Rough Small Motile Flat
spp.
Sed-10 Lactobacillus Grey Rough Small None Rise
spp.
W-9 Bacillus spp. Whitish Smooth Small None
brown

4.1.6.1. Biochemical characterization

Among 10 isolates, 8 isolates were positive for gram test which was done using
3%KOH showing that they were gram positive, whereas the rest 2 isolates were gram
negative. The result indicated that the cell of isolates was not disrupted by KOH and
the cell of the two-gram negative isolates was disrupted. The mechanism was based on
the sticky and viscous formation. Negative isolates had formed viscous. For catalase

18
 reaction, four isolates were positive. This result depicted that the isolates were aerobic.
The remaining 6 isolates were negative indicating they were not aerobic. The positive
isolates had formed bubble, while the negatives had not. Nine isolates were positive for
methyl red, forming red and pink color. This confirmed that the isolates have the ability
to form acid up on fermentation. All were shown negative reaction to VP test. They
were not turned to red. For citrate test, all isolates except one were developed blue color.
They were recorded as a positive citrate reaction. This made sure that the isolates able
to utilize citrate as a carbon source and ammonium as a nitrogen source as presented in
Table 2.

Table 2 Biochemical characteristics of the bacterial isolates isolated from Woliso hot
spring
Sample Bacterial Biochemical tests
code Isolates Gra Starch Citra Methyl VP Catala H2S Oxidase Indole
m hydrol te red test tes se test test test test
test ysis t
Sed-2 Bacillus spp. - + + +(pink) - + - + +
W-26-2 Bacillus subtilis + ++ + +(red) - + - + +
Sed-8-3 Bacillus spp. + + + +(pink) - - - - +
W-20-1 Lactobacillus spp. + + +
W-20-0 Bacillus cereus + + + +(pink) - - - + -
Sed-12-1 Bacillus + + + +(pink) - - - + +
licheniformis
Sed-7 Bacillus spp. - +++ - -(yellow) - + - + +
Sed-5 Pseudomonas spp. - ++ + +(red) - - - + +
Sed10 Lactobacillus spp. + +++ + +(pink) - - - Delayed -
+
W-9 Bacillus spp. + ++ + + (red) - + - Delayed _
+

According to Bergey”s Manual (Whitman, 2009)., the 10 bacterial isolates were

identified to genus and species level by using the previous biochemical tests. So
bacterial isolates obtained from Woliso hot spring were identified under genus Bacillus,
lactobacillus and pseudomonas. From the identified bacterial isolates 70% were under
genus Bacillus, 20% were Lactobacillus spp. And the rest one isolate was Pseudomonas
Spp. The bacterial isolates were identified as Bacillus Spp. Bacillus subtilis
Lactobacillus Spp. Bacillus cereus Bacillus licheniformis Pseudomonas Spp.
Lactobacillus spp.

19
 5. Starch Hydrolysis test
After the plates were flooded with IKI solution (iodine, 1 g; potassium iodide, 2 g;
distilled water, 100 ml). A clear zone around a colony was recorded as positive reaction.
The clear zone indicates the level of starch hydrolysis capacity of isolates. Potential
isolates can produce large clear zone and indicates complete hydrolysis of starch.

Figure 2 Clear zone observed around amylase-producing bacteria

A clear zone around a colony on starch (after addition of iodine) gave an indication
amylase producing bacteria. The isolate showing the maximum zone of hydrolysis was
selected for amylase production because it is an indication for the bacteria to produce
amylase based on its hydrolysis potential (Amin and Zusfahair, 2012; Singh et al.,
2015). Similarly, in our study, from 18 bacterial isolates 10 isolates were selected as a
strong positive for the reaction. This procedure showed a positive result for the
Aspergillus strain as Singh et al., 2015 indicates. The formation of clear zone observed
was due to the fact that the amylase produced during the growth of the organisms has
hydrolysed the starch around the colony. The unhydrolysed part was appeared as blue
black indicating negative activity of amylase producer bacteria.

20
 4.1.6.2. Salt Tolerance Test
Selected isolates have different salt tolerance for different sate concentration some have
low salt tolerance capacity and others have high salt tolerance capacity as it is indicated
in table 3.

Table 3 Effect of salt concentration on bacterial isolates

Sample Bacterial Isolates Salt Concentration p
code 0.5 1.5 2.5 3.5 4.5
Sed-10 Lactobacillus spp. 2.02±0.95 1.59±0.30 1.85±0.45 2.02±0.02 1.73±0.45 0.146
W-9 Bacillus spp. 1.91±1.22 1.66±0.47 2.07±0.13 2.04±0.19 1.67±0.18 0.174
Sed-12 Bacillus cereus. 2.09±0.91 1.72±0.54 1.75±0.23 1.70±0.44 1.61±0.34 0.149
W-20-1 Bacillus licheniformis. 1.06±0.07 2.03±0.29 1.77±0.17 1.96±0.03 1.57±0.10 0.000
Sed-7 Bacillus spp. 2.30±0.05 1.74±0.25 1.82±0.45 2.00±0.18 1.65±0.09 0.000
W-20-0 Lactobacillus spp. 2.10±0.06 1.44±0.22 1.83±0.23 1.98±0.17 1.59±0.06 0.000
W-26-2 Bacillus Subtilis. 1.92±0.44 1.55±0.16 2.01±0.09 2.05±0.24 1.71±0.08 0.000
Sed-2 Bacillus spp. 2.26±0.25 1.71±0.22 1.70±0.21 1.64±0.20 1.59±0.28 0.000
Sed-8-3 Bacillus spp. 2.24±0.46 1.59±0.11 1.72±0.07 1.94±0.28 1.69±0.16 0.000
Sed-5 Pseudomonas spp. 2.21±0.02 1.87±0.11 1.88±0.13 2.09±0.04 1.56±0.28 0.000

The isolates were tested for salt tolerance at different NaCl concentrations (0.5, 1.5,
2.5, 3.5, and 4.5). Above 50% of them were shown maximum performance, at 0.5%;
40% were at 3.5%; 20% were performed well at all concentrations. Most were
performed less at 1.5% and the growth of three isolates was decreased at 4.5%.
Generally, the result revealed that the isolates could be survived at saline environments.

SALT TOLERANCE
0.5 1.5 2.5 3.5 4.5

2.5

1.5

0.5

0
SED-10 W-9 SED-12 W-20-1 SED-7 W-20-0 W-26-2 SED-2 SED-8-3 SED-5

Figure 3 Effect of salt concentration on bacterial isolates

21
 5.1.Crude Thermostable Amylase production
5.1.1. Enzyme activity Assay
The amylase activity assessed by measuring OD value and the observed result was as
the basis of level of productivity of the amylase, an isolate producing a maximum of
amylase activity was Sed-5 (Pseudomonas spp.) and W-20-0 (Lactobacillus spp.) used
for detailed investigation as indicated to Figure 4. Screening of the amylase producing
organisms was carried out on the starch agar. Based on the enzyme activity assay, all
were positive. However, SED5(Pseudomonas spp.) involved the highest activity and
W20-0 (Lactobacillus spp.) shown the second maximum enzyme activity next to
SED5(Pseudomonas spp.).

Figure 4 profile plot 2 showing estimated marginal plot

5.1.2. Substrate concentration optimization

Starch which was used as a substrate was prepared with concentrations; 2%, 4%, 6%,
8% and 10%, (w/v) at pH7. Substrate concentration variation was measured using
spectrophotometer summarized in the table 4.

22
 Table 4 Substrate concentration optimization for amylase activity
Sample Bacterial Isolates Starch concentration p
code 2 4 6 8 10
Sed-10 Lactobacillus spp. 1.99± 0.02 2.62±0.47 2.84±0.18 2.51±0.35 2.48±0.27 0.000
W-9 Bacillus spp. 2.63±0.24 2.59±0.28 2.54±0.12 2.40±0.59 2.45±0.54 0.670
Sed-12-1 Bacillus cereus. 2.25±0.83 2.75±0.19 2.56±0.41 2.52±0.49 2.53±0.13 0.100
W-20-1 Bacillus licheniformis. 2.68±0.15 2.53±0.41 2.72±0.16 2.53±0.33 2.34±0.46 0.038
Sed-7 Bacillus spp. 2.59±0.014 2.76±0.03 2.62±0.08 2.6±0.07 2.51±0.14 0.000
W-20-0 Lactobacillus spp. 2.44±0.43 2.57±0.39 2.48±0.33 2.66±0.08 2.55±0.22 0.266
W-26-2 Bacillus Subtilis. 2.50±0.724 2.55±0.36 2.86±0.33 2.44±0.62 2.44±0.39 0.152
Sed-2 Bacillus spp. 2.21±0.03 2.44±0.04 2.56±0.57 2.64±0.31 2.50±0.22 0.015
Sed-8-3 Bacillus spp. 2.48±0.63 2.65±0.29 2.59±0.23 2.09±2.17 2.43±1.01 0.613
Sed-5 Pseudomonas spp. 2.65±0.03 2.75±0.06 2.71±0.74 2.58±0.08 2.46±0.05 0.152

Following the positive activity of the amylase, the enzymatic hydrolysis process
parameters such as optimum substrate concentration, pH, and temperature optimization
were determined. For substrate optimization, starch at different concentration (2%, 4%,
6%, 8% and 10%) was used. Out of 10 enzymes, 40% of them had utilized 4% starch;
30% had used 6% starch for their maximum growth. 20% were seen to have optimum
activity at 8% starch, where as 10% had used 2% starch as the optimum substrate.

5.1.3. Optimum pH determination

Substrate at the optimum concentration was prepared at various pH ranges (i.e., at pH
of 4.5, 5.5, 6.5, 7.5 and 8.5). The pH was adjusted using NaOH and Tris-HCl. The
enzyme activity variation at different pH level was measured as below in table 5.

Table 5 Effect of pH on amylase activity

Sample Bacterial isolates pH P
code
4.5 5.5 6.5 7.5 8.5
Sed-8-3 Bacillus subtilis 2.47±0.163 2.04±0.07 2.16±0.38 2.02±0.01 2.89±0.05 0.000
Sed-7 Bacillus spp. 0.94±0.006 0.53±0.009 0.42±0.009 0.22±0.29 1.39±0.03 0.000
Sed-5 Pseudomonas spp. 0.52±0.54 1.44±0.11 1.88±0.02 1.50±0.03 2.51±0.01 0.000
Sed-12-1 Bacillus cereus 0.23±0.04 0.23±0.10 0.15±0.11 0.46±1.12 0.16±0.01 0.389
W-20-0 Lactobacillus spp. 2.48±0.02 2.76±0.23 2.55±1.08 2.75±0.01 2.80±0.01 0.254
W-20-1 Bacillus licheniformis 1.66±0.01 1.22±0.02 1.17±0.13 0.98±0.02 1.58±0.01 0.000
Sed-10 Lactobacillus spp. 1.76±0.03 1.34±0.6 1.69±0.37 1.18±0.03 1.86±0.17 0.001
W-26-2 Bacillus subtilis 1.57±0.09 1.64±0.16 1.96±0.07 1.65±0.03 1.86±0.17 0.000
Sed-2 Bacillus spp. 0.87±0.25 0.87±0.005 1.40±0.004 1.21±0.15 2.45±0.08 0.000
W-9 Bacillus spp. 2.19±0.005 1.92±0.04 1.86±0.08 1.75±0.03 1.70±0.03 0.000

Regarding pH optimization, optimum substrate concentrations at 530c were adjusted at

different pH levels (4.5, 5.5, 6.5, 7.5 and 8.5) using Tris-HCl and NaOH. Reddy et al.,
2003 stated amylases are generally stable over a wide range of pH from 4-11. Our study

23
was supported by Reddy et al., 2003 stated, the crude amylase was stable over a pH
range of 4.5 to 8.5. However, their optimum pH was varied at acidic, neutral and basic
pH. Five (5) of them were developed maximum hydrolysis activity at 8.5 pH. This result
indicated that they have maximum activity at alkaline environments than they do at
acidic conditions. Three of them were observed to show optimum activity at pH of 4.5
revealing their optimum activity was at acidic environment than basic. For the
remaining two neutral pH of about 6.5 and 7.5 were determined as optimum. This
investigation depicted that the two enzymes’ activity was highest at neutral
environments than at alkaline and acidic conditions. Another most important parameter
for the enzyme activity was temperature (Reddy et al., 2003; Sundaram et al., 2014).

pH range
3
2.5
2
1.5
1
0.5
0

4.5 5.5 6.5 7.5 8.5

Figure 5 Optimum pH value for amylase activity

5.1.4. Optimum temperature determination

This procedure will be performed using spectrophotometer at various temperatures of
30, 35, 40, 45, 50, 55, 60, 65, and 70°C for 45 minutes, under optimum pH and substrate
concentration (Table 6). The temperature that resulted in the highest reducing sugar will
be the optimum temperature for enzymatic reaction.

24
 Table 6 Effect of temperatures on amylase activity
Sample Bacterial isolate Temperature p
code 35 45 55 65 75
W-26-2 Bacillus subtilis 1.08±0.004 1.04±0.007 1.01±0.01 1.01±0.005 0.39±0.0062 0.000
Sed-10 Lactobacillus spp. 0.09±0.02 0.89±0.007 0.86±0.01 0.86±0.004 0.30±0.01 0.000
Sed-8-3 Bacillus spp. 0.27±0.002 0.37±0.06 0.38±0.01 0.38±0.01 0.31±0.02 0.000
W-9 Bacillus spp. 2.45±0.13 2.49±0.01 2.51±0.02 2.45±0.01 2.05±0.01 0.000
Sed-7 Bacillus spp. 0.28±0.01 0.33±0.007 0.32±0.01 0.43±0.005 0.18±0.01 0.000
Sed-2 Bacillus spp. 0.29±0.004 0.28±0.007 0.32±0.01 0.37±0.01 0.20±0.01 0.000
Sed-5 Pseudomonas spp. 0.15±0.02 0.36±0.005 0.37±0.004 0.45±0.01 0.17±0.01 0.000
Sed-12-1 Bacillus cereus 0.33±0.005 0.32±0.001 0.41±0.01 0.42±0.01 0.33±0.006 0.000
W-20-0 Lactobacillus spp. 0.19±0.006 0.21±0.01 0.20±0.01 0.19±0.01 0.94±2.88 0.367
W-20-1 Bacillus lichenoformis 1.05±0.08 1.07±0.01 1.09±0.02 1.08±0.03 1.30±0.009 0.000

In this study, to find the optimum temperature for amylase activity, at the optimum pH
and substrate concentration, temperature was varied at 350c, 450c, 550c, 650c and 750c.
Even though, Ved Pal et al., 2016 reported that thermostable amylase was stable at
broad range temperature (50-800c) and the same is true for amylase in this work, their
optimum temperature was measured as below. 40% were performed best at 650c; 20%
at 550c; 20% at 750c; 10% at 350c, and 10% at 450c. Bacterial species, such as B. subtilis,
B. megaterium, B. amyloliquefaciens and B. licheniformis which produce α-amylase
enzymes that can withstand a temperature of 70oC have been reported previously
(Hussain et al., 2013). Likewise, in the present work, Bacillus licheniformis produced
thermostable amylase that can withstand a temperature of 750c. In addition,
Lactobacillus which was isolated from W-20-0 produced amylase at the same
temperature. Generally, this result confirmed that about 90% of the amylases were
shown maximum starch hydrolysis activity at a temperature of greater than 45 0c. That
means almost they were themostable and the bacteria isolated from the Woliso hot
spring were thermophilic and they had been the source of thermozymes.

25
 6. Conclusion
The amylase produced by bacterial isolates from Woliso hot spring was thermostable
and can performed from 65 oC up to 75 oC. The optimum hydrolysis conditions required
for amylase activity were optimized. For most of the enzymes the optimum temperature
was observed to be 65oC; whereas optimum substrate concentration and pH for most of
them were 4% starch and 8.5pH respectively. Despite these optimum conditions, the
enzymes were active over a wide range of conditions. They were active at a various
starch conditions (2%-10%), pH of 4.5-8.5, and temperature of 35-75°C.Generally, the
bacteria isolated were thermophilic and produced thermostable amylase. Therefore,
Woliso hot spring could be the source of thermophilic microorganisms and
thermozymes as well.

26
 7. Recommendations
Since this is one of the biological resources of our country it is necessary to study and
explore further microbiological aspects of the Woliso hot spring using molecular
technique of characterization and identification of the thermophiles and thermostable
amylase producing microorganisms and it is better to go for large scale thermostable
amylase production.

27
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