Wolkite University

College of Natural and Computational Science

Department of Biotechnology

Proposal: Isolation and Characterization of Thermostable Amylase Producing

Bacteria from Woliso Hot Spring

Senior Project proposal Submitted to Wolkite University College of Natural and

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

By:

1. Firdos Abdulreshid ID No NCSR /268/07

2. Mekdes Tekleyohannes ID No NCSR/434/07
3. Merhun Degefu ID No NCSR/448/07
4. Mulatu Mokonon ID No NCSR/476/07
5. Olana Assefa ID No NCSR/514/07
6. Tajer Abdo ID No NCSR/581/07
7. Tegegn Tanto ID No NCSR/600/07

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

Wolkite, Ethiopia
 February, 2018

Contents

List of Tables iv

List of abbreviations v

1. Introduction -1-

1.1. Back ground of the study -1-

1.2. Statement of problem -3-

1.3. Objective of the study -4-

1.3.1. General objective -4-

1.3.2. Specific objective -4-

1.4. Significance of the study -5-

2. Materials and Methods -6-

2.1. Description of the study area -6-

2.2. Sample collection -6-

2.3. Isolation of pure culture -6-

3. Characterization of thermophilic isolates -6-

3.1 Morphological characterization -6-

3.2 Biochemical characterization of isolates -6-

3.2.1. Urease test -7-

3.2.2. Voges-Proskauer (VP) test -7-

3.2.3. Citrate test -7-

3.2.4. Catalase test -7-

3.2.5. Methyl red test -7-

3.3 Screening thermotolerant Amylase-producing bacteria -8-

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 3.4 Crude Thermostable Amylase production -8-

3.5 Enzyme activity Assay -8-

3.6. Enzymatic starch hydrolysis condition optimization -9-

3.6.1. Substrate concentration optimization -9-

3.6.2. Optimum pH determination -9-

3.6.3. Optimum temperature determination -9-

4. Expected outcome - 10 -

5. Beneficiaries - 11 -

6. Work Plan - 12 -

7. Budget plan - 12 -

7.1. Stationary - 12 -

7.2. Miscellaneous Expenses - 13 -

7.3. Transport Expenses - 13 -

7.4. Personal Expenses Error! Bookmark not defined.

7.5. Chemicals and Reagents Expense - 14 -

7.6. Budget Summary - 13 -

8. References - 16 -

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List of Tables
Page ……………………………………………………………. Table

- - 12 Table 1 Work plan activities

- - 12 Table 2 Stationary costs
- - 13 Table 3: Miscellaneous costs
- - 13 Table 4: Transport costs
- - 13 Table 5: Total budget summary
- - 14 Table 6: Chemicals and reagents

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5
List of abbreviations
MR Reagent: methyl red reagent

NA Medium: nutrient agar medium

nm: nanometer

PH: power of hydrogen

rpm: revolution per minute

VP test: voges-proskauer test

WKU: Wolkite University

ml: milliliter

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 1. Introduction

1.1. Back ground of the study

Thermophilic microorganisms have the adaptability to survive in high environmental
conditions (Horikoshi et al., 1998). 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).
Thermophiles have been isolated from different ecological zones (e.g., hot springs
and deep sea) of the earth The organisms 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), while, in case of bacteria,
Thermotoga maritime and Aquifex pyrophilus exhibit the highest growth
temperatures of 90 and 95°C, respectively (Kumar 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. 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 (Yoneda et al., 2013; Cihan et al., 2014; Aanniz et al., 2015)

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).

Thermophilic amylase enzymes are biological catalysts which are an indispensible

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 used 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).

Among the many thermophilic amylase enzymes that were widely used α-Amylase
has been in increasing demand due to its crucial role of starch hydrolysis and the
applications of this hydrolytic action. Researchers have elaborated the types of
amylases and their roles in enzymatic reactions (Yang and Liu, 2004). Amylases
accounts for about 30% of the world’s enzyme production. Recently they are being
used for biofilm removal and degradation of extracellular polymeric substances (Abu
et al., 2014).

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). 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).

Woliso hot spring is one of the potential area where thermophilic bacteria which
produces amylase. The hot springs located at Negash Lodge of Woliso and
surrounding area is one of the hot springs in Oromia region of Ethiopia; which has
been not yet fully explored in details microbiologically.

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 1.2. 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.

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 1.3. Objective of the study

1.3.1. General objective

✔ The main objective of study is to isolate and characterize thermostable amylase
producing bacteria from Woliso hot spring.

1.3.2. Specific objective

✔ To isolate thermophilic bacteria from Woliso hot spring
✔ To characterize thermostable bacterial isolates from Woliso hot spring
✔ To screen Amylase producing bacterial isolates.
✔ Crude amylase production from isolated thermostable bacteria by
submerged fermentation.
✔ To assess activity of the produced crude thermostable amylase
enzymes on starch substrate.
✔ To determine optimum conditions for enzymatic starch hydrolysis.

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 1.4. 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.

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 2. Materials and Methods
2.1. Description of the study area
Woliso is the capital of South West Shewa 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; which has been not yet fully explored in details microbiologically.
The present study will be conducted at the laboratory of Biotechnology Department,
Wolkite University (WKU) from April to June 2018.

2.2. Sample collection

Total of 30 samples (15 water samples and 15 sediment samples) will be collected
from woliso hot spring aseptically using 500 ml sterile thermal glass containers which
keep the temperature of the samples constant (Khalil et al., 1998). The temperature
and pH of sample will be recorded during the time of sampling. Then the sample will
be transported to laboratory of Biotechnology Department, Wolkite University.

2.3. Isolation of pure culture

Isolation of thermophilic bacteria from the source will be done by serial dilution up
to 10-5 or 10-6 dilution. 0.1 ml of each dilution will be pipetted out and inoculated into
the prepared Starch Agar Medium. Then, the inoculated plates will be incubated at
47/57oC for 24hr and the growths of thermophilic bacteria will be observed. Then
after incubation, the selected colonies (10-20) will be sub-cultured on Starch Agar
Medium three times to purify the isolates (Abebe, 2016).

3. Characterization of thermophilic isolates

3.1 Morphological characterization
The morphological characterization of bacterial isolates will be determined according
to their cultural characteristics using microscope (colony size, colony color, colony

6
texture) based on Bergey’s Manual of systematic bacteriology. The preliminary
selection test to be used for characterisation of the bacterial isolates will be graham
staining (Giffel et al., 1995)

3.2 Biochemical characterization of isolates

Biochemical characterizations will be conducted. These will be urease test, VP test,
citrate test, catalase test and methyl red test.

3.2.1. Urease test

Urease medium (yeast extract, monopotassium phosphate, disodium phosphate,
urea, and phenol red indicator) will be streak-inoculated with suspended colony
from pure cultures of bacterial isolates, covering the entire agar surface with a heavy
inoculum. It will be incubated aerobically at 35°C for 24 hours and the positive result
will show bright pink (fuchsia) colour on the agar broth whereas the negative result
show no colour change in agar slant/broth. Rapid positive and slow positive test
results will be recorded while negative agar tubes will be inspected daily for pink
color formation for up to six days (Brink, 2013).

3.2.2. Voges-Proskauer (VP) test

Suspended colony from pure culture will be investigated in VP medium and
incubated at 37oc 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 will show yellow or copper color at the surface of the
medium (Macfaddin, 2000)

3.2.3. Citrate test

Small number of bacteria inoculated in citrate medium (sodium citrate, an
ammonium salt and the indicator bromothymol blue) and incubated at 50oC for 48hr.
The positive result will develop color change from green to blue and the negative
result shows no color change on citrate medium (Macfaddin, 2000).

3.2.4. Catalase test

The bacteria will be spread on starch agar plate and it will be incubated for 24hr
under appropriate condition. Then bacteria will be collected from colony and will be
applied on microscopic slide. After that one drop of hydrogen peroxide will be

7
added. Then the positive result will be formation of oxygen in the form of bubble
and in negative result no bubble will be formed (Paik, 1980).

3.2.5. Methyl red test

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

3.3 Screening thermotolerant Amylase-producing bacteria

Amylase producer bacteria will be screened based on their ability to degrade starch
in the medium. Single bacterial isolate will be grown on starch containing nutrient
agar medium at 50°C for 24 hr., and then will be examined by flooding,
1% iodine solution on starch agar plate. The presence of blue color around the
growth media will indicate negative result, and formation of clear zone around the
bacterial colony will indicate a positive amylase-producing bacterium ( Win et al.,
2015).

3.4 Crude Thermostable Amylase production

A loop full of thermophilic amylase positive isolates bacterial culture will be
transferred from starch-nutrient agar slants to fermentation 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) at pH 7. The flask will be placed on a shaker incubator at 200
rpm and 37°C for 24 hours. After incubation, fermented broth will be centrifuged at
8000 rpm for 20 minutes in a centrifuge. The supernatant containing cell free extract
will be removed and served as crude enzyme extract. This crude enzyme extract will
be used in amylase enzyme activity measurement and characterization (Alemayehu
and Temam, 2017). The enzyme activity will be determined by sampling at specific
time intervals (Abebe, 2016).

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 3.5 Enzyme activity Assay
The activity of the amylase will be determined by using starch as the substrate.
The reducing sugar released as an enzymatic reaction product will be measured.
A sample tube containing 0.5 ml of 1% starch will be incubated for 5 minutes at
37°C. After five minutes 0.5 ml of amylase and 0.5 ml of 0.85% NaCl will be added
and the incubation will be continued for 30 minutes. The enzyme activity will be
stopped by adding 1 ml of 10% sodium tungstate and 1 ml of 0.7 NH 2SO4. A
control tube will be also prepared using the same procedure in the absence of
amylase. The sample and control solution will be filtered and the reducing sugar
will be measured in the filtrate obtained.

The reducing sugar will be measured by preparing a series of 5 ml test tube, each
will be filled with 0.1 ml of sample, control and standard solution of glucose at the
concentrations of 100, 200, 300, 400, and 500 mg/ml. 0.2 ml of alkaline cupric
tartrate will be added in each tube and held at 100°C for 30 minutes. This mixture
will be then cooled and 0.2 ml arsenomolybdate followed by 7.5 ml of distilled
water added. The absorbance of the samples will be measured at 660 nm and the
reducing sugar will be calculated using the following formula:

The number of reducing sugar produced by each ml of enzyme = reducing sugar in

sample - reducing sugar control (mg/ml).

3.6. Enzymatic starch hydrolysis condition optimization

3.6.1. Substrate concentration optimization

Starch will be used as a substrate. About 100 g of starch will be dried to a
maximum of 16% water content. One gram of the substrate will be dissolved in 50
ml phosphate buffer pH 7. Starch solution will be incubated in the oven at 70°C
for 15 minutes and then the solution will be transferred to an incubator at the
temperature of 45°C. Substrate concentration variation will be performed using
the same procedure as the enzyme activity test, with concentrations of 2%, 4%,
6%, 8%, 10%, 12% and 14% (w/v).

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 3.6.2. Optimum pH determination
Substrate at the optimum concentration will be dissolved in 5 ml of various buffers
(pH 4.0 to 10.0). For pH of 4.0 and 5.0, sodium-acetate buffer will be used, for pH of
6.0 and 7.0 sodium phosphate buffer will be used, for pH of 8.0 and 9.0 Tris-HCl will
be used, whereas for pH of 10 borax NaOH buffer will be used. This procedure will
be performed under the same procedure as the amylase activity test with various pH
conditions.

3.6.3. Optimum temperature determination

This procedure will be performed under the same procedure as the amylase activity
test, using optimum pH and substrate concentration, and various temperatures of
30, 35, 40, 45, 50, 55, 60, 65, and 70°C for 45 minutes. The temperature that
resulted in the highest reducing sugar will be the optimum temperature for
enzymatic reaction.

4. Expected outcome
At the end of this experiment, it is expected to come up with the following outputs:

❖ Identification of thermostable amylase producing bacterial isolates

❖ Production of thermostable amylase enzyme and
❖ determining the optimum operation condition of crude amylase.

10
 5. Beneficiaries
Upon the success of our research the following agents will benefited: Brewery-
bioconversion of starch; textile industries- for desizing process; food industry- for
processed products or food; environment- by degrading starchy residues; pulp and
paper industries- modification of starch of coated paper, smoothen and strengthen
paper; and in detergent industries- to enhance the ability of detergents and making
them environmentally safe. It is also used to researchers and other students for
some labratory experiments and research works.

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 6. Work Plan
To undertake the research in well-organized manner it is planned and indicated as
follows. It assumed that the whole research will be completed within three months.

Table 1 Work plan activities

No Activities Implementation time Remark

1 Developing project proposal and March –April, 2018

submission to department

2 Sample collection, media April, 2018

preparation, culturing and
transferring

3 Isolation of pure culture April, 2018

4 Characterization of thermophilic April, 2018

isolates

5 Screening thermotolerant Amylase- April,2018

producing bacteria

6 Crude Thermostable Amylase April, 2018

production

7 Enzyme activity Assay May, 2018

8 Enzymatic starch hydrolysis May, 2018

condition optimization

9 Literature review and progress of May,2018

report

10 Data interpretation and thesis June,2018

writing

11 Thesis submission and defense June, 2018

7. Budget plan
7.1. Stationary
Table 2 Stationary costs

No Item Unit Quantity Unit price Total

(Birr)

1 Flash disc(4GB) Pcs 1 280.00 280.00

12
 2 CD-R Pcs 10 6.00 60.00

3 CD-RW Pcs 4 20.00 80.00

4 Pen Numbe 3 3.00 19.00

r

5 Marker Pcs 5 10.00 50.00

6 Printing paper Packet 5 100.00 500.00

7 Photocopy paper Packet 4 90.00 360.00

8 Note pad small Pcs 4 15.00 60.00

Sub total 1399

7.2. Miscellaneous Expenses

Table 3: Miscellaneous costs

No Item Total

1 Binding work 500

2 Photocopy 400

Sub total 900

7.3. Transport Expenses

Table 4: Transport costs
No Travelers Departure Destination No of trips Transport Cost / Total
trip cost

1 Three WKU main Woliso 3(2x2) Bus 35 420.00

Campus

7.4. Budget Summary

Table 5: Total budget summary

No Description of expenses Amount

1 Stationary 1399

2 Miscellaneous expenses 900

13
3 Transport expenses 420

4 Personal expenses 6850

Total 9569

7.5. Chemicals and Reagents

Table 6: Chemicals and reagents

No Chemicals and reagents Quantity

1 Soluble starch 500g

2 Peptone 250g

3 (NH4)2SO4 100g

4 KH2PO4 50g

5 K2HPO4 100g

6 MgCl2 1g

7 Iodine solution

9 Deionized water 1000ml

10 Potassium hydroxide 10g

11 5% Alpha-naphthol& ethanol 10g

12 Methyl red indicator 2g

13 Glucose broth 100g

14 MR/VP broth 100g

15 3% hydrogen peroxide 2g

16 Sodium citrate 2g

17 Ammonium salt 5g

18 Bromothymol blue 2g

19 Squarts urea broth 100g

20 Monopotassium phosphate 5g

21 Disodium phosphate 2g

22 Phenol red indicator 2g

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23 Urea 100g

24 Iodine 10g

25 Crystal violet 2g

26 Ethanol – acetone 10g

27 Yeast extract 50g

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