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Enzyme activity screening of thermophilic bacteria isolated from Dusun Tua Hot
Spring, Malaysia

Conference Paper  in  AIP Conference Proceedings · April 2018

DOI: 10.1063/1.5027985

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Enzyme activity screening of thermophilic bacteria isolated from Dusun Tua Hot
Spring, Malaysia
Marwan Msarah, Izyanti Ibrahim, and Wan Syaidatul Aqma

Citation: AIP Conference Proceedings 1940, 020070 (2018); doi: 10.1063/1.5027985

View online: https://doi.org/10.1063/1.5027985
View Table of Contents: http://aip.scitation.org/toc/apc/1940/1
Published by the American Institute of Physics
 Enzyme Activity Screening of Thermophilic Bacteria
Isolated from Dusun Tua Hot Spring, Malaysia
Marwan Msaraha), Izyanti Ibrahimb) and Wan Syaidatul Aqmac)
School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia,
43600 Bangi, Selangor, Malaysia.
a)
marwan_masar21@yahoo.com
b)
izzy@ukm.edu.my
c)
Corresponding author: syaidatul@ukm.edu.my

Abstract. Thermophilic bacteria have biotechnological importance due to the availability of unique enzymes which are
stable in extreme circumstances. The aim of this study includes to isolate thermophilic bacteria from hot spring and
screen for important enzyme activities. Water samples from the Dusun Tua Hot Spring were collected and the
physiochemical characterisation of water was measured. Eight thermophilic bacteria were isolated and determined to
have at least three strong enzyme activity including protease, lipase, amylase, cellulase, pectinase and xylanase. The
results showed that HuluC2 displayed all the enzyme activities and can be further studied.

INTRODUCTION
Thermophiles are the organisms which are adapted to live at high temperatures and these high temperature
environments are widely distributed on the earth. The availability of thermophiles to grow at temperatures above
60oC is associated with extremely thermostable macromolecules present in them and have been reported in most
boiling water environments, where they normally reproduce [1].
Thermophilic microorganisms have been found to be good potential and a sources of thermostable enzymes.
Thermostable enzymes acquired from these thermophilic microorganisms are used in most of industrial applications
such as food, pulp, papers, feeds, starch, pharmaceutical, textile, detergents, waste management industries and used
as biocatalysis, biotransformation and biodegradation due to their extreme stability in elevated high temperatures [2–
4]. Thermophilic bacteria are more thermostable as well as more resistant than their mesophilic counterparts to
organic solvents, detergents, low and high pH and other denaturing agents [5].
Thermostable enzymes were the result of discovery of thermophilic microorganisms from hot water springs in
1960s [1]. It was discovered that many bacteria in hot springs not only survived but also thrive at higher
temperatures. Undoubtedly, the most famous and well-studied geothermal area is Yellowstone National Park
(Wyoming, USA), where the first thermostable enzyme Taq polymerase was obtained from Thermus aquaticus in
1974 and considerable of the pioneering microbiological work in Yellowstone was carried out by Thomas Brock and
co-workers [1], and it was followed by more thermostable enzymes by emerging industrial demands for new
enzymes that are more stable under harsh conditions [1, 6].
Thermophilic bacteria, having growth at high temperature and specific macromolecular properties, possess high
metabolic rates, physically and chemically stable enzymes [1]. Thermophilic processes appear to be more stable,
rapid and less expensive and facilitate reactant activity and product recovery [7], advantage of conducting
biotechnological processes at elevated temperatures is reducing the risk of contamination by common mesophiles.
Allowing a higher operation temperature has also a significant influence on the bioavailability and solubility of
organic compounds and thereby provides efficient bioremediation [7].
In the Malay Peninsula, about 60 hot springs have been discovered so far and 75% of them are in easily
accessible areas [8], and are turned into recreational resorts with hotels, hot spas, and swimming pools. None of

The 2017 UKM FST Postgraduate Colloquium

AIP Conf. Proc. 1940, 020070-1–020070-5; https://doi.org/10.1063/1.5027985
Published by AIP Publishing. 978-0-7354-1632-1/$30.00

020070-1
these hot springs has been utilized for use as a source for thermostable enzymes [8]. Therefore, in this study we
focused on the screening of extracellular degrading enzymes, such as protease, lipase, amylase, cellulase, pectinase
and xylanase.

MATERIALS AND METHODS

Samples Collection

A total of three samples were collected from the Dusun Tua Hot Spring (3°08'21.2"N 101°50'10.9"E), water
samples collected by using three 1 L sterile bottles for each hot spring and the physiochemical characterisation of
water measured on site using YSI 556 multi probe system according to the manual. The sampling was conducted on
11 May 2016. The water samples collected from different sources around the hot spring then transported to the
laboratory of Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia for bacteriological analysis.

Isolation of Bacteria
Isolation of thermophiles bacteria was performed according to [9], through serial dilution method. Thermophilic
bacteria were isolated and cultured on thermus agar (ATCC medium 697) containing 0.5% NaCl, 0.5% peptone,
0.4% beef extract, 0.2% yeast extract and 2% agar [10], the pH of the medium was adjusted to 7.0 before
autoclaving plates and then incubated at 45°C for 24 to 48 h. Isolation of pure culture was done using streak plate
method. Different isolates were identified by the colony morphology and microscopic examination [11]. A loop full
of the overnight culture grown on thermus agar plates for 18 h at 45°C was stained with Gram staining and then
examined using light microscope.

Screening for Extracellular Enzymes

The thermophilic microbial isolates were grown on thermus agar plates and incubated at 45°C for 24 h and
subjected to various preliminary tests. The screening for protease activity was performed according to the method
described by [12], on skim milk agar containing 8 g/L nutrient broth, 10 g/L skim milk and 17 g/L agar agar, then
incubated for 36 hours at 45ºC. The presence of protease activity was confirmed by the appearance of a clear halo
around the well indicating degradation of casein milk. Screening for lipase activity was performed according to the
method described by [13], on a medium containing 8 g/L nutrient broth, 0.25 g/L CaCl22H2O, 9 g/L agar agar
dissolved in 500 mL deionized water and 5 mL of Tween 20 dissolved in 500 mL deionized water autoclaved
separately at 121oC for 15 min and add to the medium, then the medium with the cultures incubated for two days at
45ºC. Opaque halos occur around the colonies indicate the presence of lipase activity.
The screening of the amylase activity was performed according to [12], on a medium containing 1 g/L yeast
extract, 5 g/L soluble starch and 17 g/L agar agar. Ingredients were dissolved in deionized water and sterilized by
autoclaved at 121oC for 15 min and incubated for 1–2 days at 45ºC. The presence of amylase activity was
confirmed by the appearance of a clear halo around the well after the staining with lugol.
The presence of xylanase activity was preformed according to [12], on a medium containing 1 g/L yeast extract,
5 g/L xylene and 17 g/L agar agar then incubated for 3–4 days at 45ºC. Xylanase activity was confirmed by the
appearance of a clear zone around the tested strain following the staining with Congo Red.
Screening for cellulase activity was preformed according to [12], on a medium containing 1 g/L yeast extract, 5
g/L CMC salt and 17 g/L agar agar then incubated for 3–4 days at 45ºC. Cellulase activity resulted in the appearance
of a clear halo around the tested strain after treatment with iodine. Identification of strains displaying pectinase
activity was performed according to [12], on a medium containing 1 g/L yeast extract, 2 g/L ammonium sulfate, 6
g/L Na2HPO4, 3 g/L KH2PO4, 5 g/L pectin and 17 g/L agar agar. Ingredients were dissolved in deionized water and
sterilized by autoclaved at 121oC for 15 min and incubated for 3–4 days at 45ºC. Colonies with clear zones indicated
pectinase activity.

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 RESULTS AND DISCUSSIONS

Physicochemical Parameters of Water Samples

The physicochemical parameters of samples collected from three different ponds at the Dusun Tua Hot Spring
are shown in Table 1. Basically, the pH is in the acidic to neutral pH, with temperature range between 42- 63°C,
almost similar conductivity and salinity. The dissolved oxygen and saturation is highest for the sample at the edge of
hot spring and lowest in the stream.

TABLE 1. The initial physical and chemical properties of water samples from the Dusun Tua Hot Spring.
Isolate pH Temperature Conductivity Salinity Dissolved Saturation
(°C) ms/cm2 oxygen DO (mg/L)
DO (%)
Pond 1 6.93 45 0.278 0.13 226.3 13.20
(Edge of the hot-spring)
Pond 2 7.70 63.54 0.282 0.12 36.3 1.62
(Hot Spring stream source)
Pond 3 6.73 42.69 0.281 0.13 51.4 3.16
(Biofilm forming)

Isolation of Thermophiles

Eight thermophilic bacterial isolates were acquired based on their difference in morphology (Table 2). The total
count of bacteria after 24 h incubation is also indicated. These bacteria were confirmed as thermophiles by growing
on thermus agar medium. Microscopic analysis showed all eight isolates were Gram negative bacilli (Fig. 1.).
Several thermophilic bacteria have been isolated from hot springs and bacillus are commonly found despite of the
location [14, 15].

TABLE 2. Total count of bacteria after 24h incubation, Gram reaction and colony morphology.
Isolates CFU/ml Colony edge Colony Surface Color
HuluA1 3.16 ×106 Irregullar Flat, Dry Off -white
HuluA2 3.27 ×106 Irregullar Flat, Dry Off -white
HuluB1 3.32 × 106 Irregullar Flat, Moist Off -white
HuluB2 3.27 × 106 Irregullar Flat, Moist Off -white
HuluB3 3.16 × 106 Irregullar Flat, Dry Off -white
HuluC1 3.18 × 106 Irregullar Flat, Moist Off -white
HuluC2 3.6 ×106 Irregullar Flat, Moist Yellowish
HuluC3 3.16 ×106 Irregullar Flat, Dry Off -white

(a) (b)

FIGURE 1. Microscope view of the thermophilic bacteria a) HuluA1 b) HuluC3. Magnification 1000×.

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 Screening of the Enzymes

Hot spring can be the home to bacteria with useful enzymes. Table 3 shows the results of extracellular enzymes
activity for the 8 samples when incubated at 45°C. The enzyme activity is reflected by the width of the clearing
zones [16]. Figure 2 shows representative diameters of halos from three different enzyme activities. In this study, all
thermophilic bacteria that were isolated showed at least three strong enzyme activities. One isolate, HuluC2
displayed all enzyme activities and can be further investigated for their potential biotechnological purposes.
Thermophilic bacteria have tremendous potential in future microbial and enzyme technology because of their unique
property to work at high temperature.

TABLE 3. Detection of extracellular enzymes activity.

Samples Protease Lipase Amylase Cellulase Pectinase Xylanase
HuluA1 Weak Strong Strong Strong Weak Strong
HuluA2 Strong Strong Weak Strong Weak Weak
HuluB1 Strong Strong Strong Strong Strong Weak
HuluB2 Weak Strong Strong Strong Strong Strong
HuluB3 Strong Strong Strong Strong Weak Strong
HuluC1 Weak Strong Strong Strong Weak Weak
HuluC2 Strong Strong Strong Strong Strong Strong
HuluC3 Weak Strong Strong Strong Weak Weak
Note: Weak: Diameter < 2cm, Strong: Diameter > 2 cm.

(a) (b) (c)

FIGURE 2. Determination of (a) protease, (b) xylanase and (c) pectinase activity.

CONCLUSION

Eight isolates have been isolated from Dusun Tua Hot Spring with potential to be further investigated for the
protease, lipase, amylase, cellulase, pectinase and/or xylanase activities. One isolate in particular HuluC3 displayed
all the enzyme actitvities and can be further investigated for the biotechnological application.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge Universiti Kebangsaan Malaysia (UKM) for partly supporting this research
project.

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