Geomicrobiology Journal

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Isolation and Characterization of Thermophilic

Bacteria from Gavmesh Goli Hot Spring in
Sabalan Geothermal Field, Iran: Thermomonas
hydrothermalis and Bacillus altitudinis Isolates as a
Potential Source of Thermostable Protease

Pantea Abdollahi , Maryam Ghane & Laleh Babaeekhou

To cite this article: Pantea Abdollahi , Maryam Ghane & Laleh Babaeekhou (2020): Isolation and
Characterization of Thermophilic Bacteria from Gavmesh Goli Hot Spring in Sabalan Geothermal
Field, Iran: Thermomonas�hydrothermalis and Bacillus�altitudinis Isolates as a Potential Source of
Thermostable Protease, Geomicrobiology Journal, DOI: 10.1080/01490451.2020.1812774

To link to this article: https://doi.org/10.1080/01490451.2020.1812774

Published online: 03 Sep 2020.

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GEOMICROBIOLOGY JOURNAL
https://doi.org/10.1080/01490451.2020.1812774

Isolation and Characterization of Thermophilic Bacteria from Gavmesh Goli Hot

Spring in Sabalan Geothermal Field, Iran: Thermomonas hydrothermalis and
Bacillus altitudinis Isolates as a Potential Source of Thermostable Protease
Pantea Abdollahi, Maryam Ghane , and Laleh Babaeekhou
Department of Biology, Islamshahr Branch, Islamic Azad University, Islamshahr, Iran

ABSTRACT ARTICLE HISTORY

Thermophilic bacteria have attracted great attention due to their ability to produce thermostable Received 25 April 2020
enzymes. The aim of this study was the isolation and characterization of thermophilic bacteria Accepted 17 August 2020
from Gavmesh Goli hot spring in Sareyn, North West of Iran. Of 10 water samples collected, 36
KEYWORDS
thermophilic bacteria were obtained. The thermophilic bacteria were tested for their ability to pro-
Bacillus altitudinis; hot
duce hydrolase enzymes. All the isolates were potentially protease producers. Lipase, DNase, and springs; Thermomonas
amylase activities were confirmed in 34 (94.4%), 8 (22.2%), and 3 (8.3%) isolates, respectively. Five hydrothermalis; thermo-
isolates with higher levels of enzyme activity were selected for further studies. Morphological, bio- stable protease
chemical, and molecular analysis by 16S rRNA gene sequencing revealed that four isolates (DH15,
DH16, DH20, and DH29) could be identified as Thermomonas hydrothermalis and one (PA10)
Bacillus altitudinis. The protease produced by these isolates was optimally active at 50–55
C, pH
8–8.5, and 0–0.5 M NaCl. In this first time study, we isolated T. hydrothermalis and B. altitudinis
from Iranian hot springs and demonstrated the characteristics of T. hydrothermalis protease.
Accordingly, due to the valuable potential of these bacteria such as the production of protease
with high temperature and pH stability, these isolates can be introduced as promising candidates
for industrial applications.

Introduction polypeptides. They have wide industrial applications includ-

Extremophilic microorganisms live in extreme ecosystems
with high/low temperatures/pH and high pressure and salin-
ity which include all three domains of life, Bacteria,
Archaea, and Eukarya (Aanniz et al. 2015). Thermophilic
bacteria mainly reside in hot springs and can survive at tem-
peratures of 45–80
C. Due to their specific properties such
as growth at high temperatures and unique macromolecular
characteristics, thermophiles can possess strong metabolism,
chemically and physically stable enzymes, and lower growth
but higher productivity than similar mesophilic species
(Mehta et al. 2016).
Thermophiles are intensively studied due to their poten-
tial to produce thermostable enzymes such as proteases,
amylases, lipases, xylanases, pectinases, chitinases, cellulases,
and DNA polymerases. These enzymes exhibit unique prop-
erties that may be suitable for accomplishing biotechno-
logical processes at elevated temperatures (Singh et al. 2010).
Furthermore, it has been reported that they are stable
against detergents, solvents, and acidic and alkaline pH
(Mohammad et al. 2017).
Among these industrially important enzymes are pro-
teases which account for about 60% of the total global
enzyme sale (Mamo and Assefa 2018). Proteases catalyze the
hydrolysis of peptide bonds present in proteins and
ing leather industry, pharmaceutical industry, food industry
and manufacture of protein hydrolysates, and waste process-
ing industry. Proteases produced by thermophilic bacteria
are of importance because they can catalyze the reactions at
higher temperatures, resulting in increased solubility of the
reactants and products, which in turn accelerate the reaction
rates. Furthermore, a higher processing temperature can
reduce the risk of microbial contamination by mesophilic
bacteria (Abusham et al. 2009).
Investigation of new sources of enzymes useful for the
industry is one of the most important fields of research in
the biotechnology sector of enzymes. Recently, heat-resistant
proteases have been studied, but most are not yet available
in the market (Sadiq et al. 2016).
Hot springs occur in a few widely separated locations of
the world and can be observed in areas of active volcanism,
or areas that have inactive volcanoes. There are several hot
springs with temperatures ranging from 28 to 82
C within
the Sabalan geothermal field in Ardebil Province, northwest
of Iran (Haeri et al. 2011). The hot springs in this area have
not yet been investigated in terms of microbial composition
and biotechnological prospects. Therefore, the goal of this
study is to screen for a new thermophilic bacterium with
high biotechnological and environmental potential. This is a

CONTACT Maryam Ghane ghane@iiau.ac.ir, maryamghaneh@yahoo.com Department of Biology, Islamshahr Branch, Islamic Azad University, Sayyad Shirazi
St., P.O. Box: 33135/369, Islamshahr, Iran
ß 2020 Informa UK Limited, trading as Taylor & Francis Group
2 P. ABDOLLAHI ET AL.
first-time study that focuses on culturable thermophilic bac-
teria and investigates their enzyme production from
Gavmesh Goli hot spring in Iran.
Materials and methods
Sample collection
In this study, sampling was done from hot mineral water of
Gavmesh Goli hot spring located in Sareyn city (38
080
60.00ʺ N, 48
030 60.00ʺ E) in the northwest of Iran. The
temperature and pH of the hot spring were 46
C and 6.58,
respectively. Ten water samples were collected from 20 cm
under the surface using a 100 mL thermal glass container.
The samples were transported to the microbiology labora-
tory and immediately analyzed.
Bacterial isolation and conventional identification
The amount of 50 mL of each sample was streaked on
Nutrient agar plates (NA) (Merk, Germany). To prevent
drying of the medium during incubation, plates were placed
in plastic bags and incubated for 48 h at 50
C. Bacterial col-
onies with various morphologies were selected and subcul-
tured on NA plates to obtain pure cultures. Purified
colonies were stoked in Nutrient broth (NB) (Merk,
Germany) containing 15% of glycerol at 80
C for further
studies. Characterization of each isolate was carried out by
examination of colony morphology, size, color, and gram
staining. Biochemical tests such as catalase and oxidase,
nitrate reduction, citrate utilization, and acid production
from glucose, sucrose, maltose, sorbitol, and mannitol were
assayed according to the methods described previously
(Johan et al. 2003). The ability of the isolates to grow at dif-
ferent temperatures was assayed by incubating the inocu-
lated NB medium (pH 7) at 40, 50, 60, 70, and 80
C for
24 h. The effect of different concentrations of NaCl on the
growth of the isolates was evaluated in the NB medium (pH
7) containing 0.5–15% (w/v) NaCl at 50
C for 24 h.
Optimum pH for growth of the isolates was determined by
incubating the inoculated NB medium with pH values rang-
ing from 5 to 12 at 50
C for 24 h.
Screening for hydrolytic enzymes
Amylase activity
The starch hydrolysis method was used to evaluate the
amylase activity of the isolates. Bacteria were cultured in
starch agar (1.5% yeast extract, 0.5% peptone, 1% soluble
starch, 1.5% agar) and incubated at 50
C for 24–48 h. After
incubation, a few drops of iodine solution (0.3% I2–0.6% KI)
were added to the plate and the results of discoloration were
examined. The presence of a clear zone around the growth
indicated the presence of amylase activity; however, the blue
color around the growth indicated no amylase production
(Mohammad et al. 2017).
Cellulase activity
Luria–Bertani (LB) agar medium (Merck, Germany) contain-
ing 1% carboxymethyl cellulose substrate (CMC) (Sigma-
Aldrich) was used to determine the cellulase activity of the
isolates. After incubation of inoculated plate at 50
C for
24–48 h, a few drops of Congo-red solution (1% w/v) were
added and kept at room temperature for 15 min. Then, 1 M
NaCl was added to the medium to detect the enzymatic
activity. Clear zones around growing bacterial colonies indi-
cated cellulose hydrolysis and growth without clearing
around the colonies was evaluated as negative (Patagundi
et al. 2014).
Lipase activity
The lipase activity of the isolates was investigated on tween
80 medium composed of (g/L): peptone, 10; agar-agar, 20;
NaCl, 5; CaCl2.2H2O, 0.1; tween 80, 10 mL (v/v). The bac-
teria were spot-inoculated on tween 80 medium and incu-
bated at 50
C for 24–48 h. Lipase activity was confirmed by
opaque zones around the colonies; however, no opaque zone
formation was evaluated as negative (Kumar et al. 2012).
Deoxyribonuclease activity test
DNase Test Agar plates (Merck, Germany) were inoculated
with the bacterial strains and incubated at 50
C for 24 h.
After incubation, hydrochloric acid was added to the plates
and then placed on a dark background and examined. A
clear zone around the growth was taken as DNase activity
(Pimenta et al. 2008).
Protease activity
To screen the extracellular protease activity, each isolate was
spotted in Thermus Medium Modified Agar (TMMA) con-
sisting of 0.01% MgSO4.7H2O, 0.1% K2HPO4, 0.35%
(NH4)2SO4, 0.1% NaCl, 0.05% Yeast extract, 0.05% peptone,
2% agar, and 2% skim milk. After the incubation period at
50
C for 24–48 h, a clear zone formed around the colony
indicated the ability of the isolate to produce extracellular
protease (Ginting et al. 2020). To calculate the level of
enzyme activity (LEA), the diameter of the zone of proteoly-
sis divided by the diameter of the bacterial colony (in milli-
meters) and LEA was expressed high (LEA >2), medium
(LEA 1.31–1.99), and low (LEA <1.3). Strains with higher
levels of protease activity were used for further studies.
Molecular identification of the isolates
PCR amplification
DNA extraction was performed using the DNA extraction
kit (Sinaclon, Iran) according to the manufacturers’ proto-
col. The 16S rRNA genes of strains were amplified by PCR
using universal primers Uni-27F (AGAGTTTGATCCTGGC
TCAG) and Uni-1492R (GGTTACCTTGTTACGACTT) as
described previously (Lane 1991). The PCR reaction was
carried out in a final volume of 25 mL containing 2.5 mL of
PCR buffer (10 mM Tris, 50 mM KCl, pH: 8), 1 mL of each

primer (10 mM), 1 mL of MgCl2 (25 mM), 1 mL of dNTP
(50 mM), 2.5 mL extracted DNA, 2 U of Taq DNA
Polymerase (Sinaclon, Iran). The PCR amplification was per-
formed under the following conditions: initial denaturation
at 95
C for 5 min followed by denaturation at 95
C for
30 s, annealing at 60
C for 50 s and extension at 72
C for
90 s (35 cycles), and a final extension at 72
C for 10 min.
The PCR products were analyzed on 1% agarose gel (Sigma-
Aldrich) containing 0.5 mg/mL ethidium bromide
(CinnaGen, Iran). For the final approval of the isolates, the
PCR product of the 16S rRNA gene was purified and sent
to Microgene Company (Korea) for sequencing.
Sequencing and phylogeny analysis
As the PCR products of the 16S rRNA gene were approxi-
mately 900 bp in length, two-way sequencing was performed
and the Consensus sequence was obtained using DNAstar
software (version 14.1). The Consensus sequence using
Mega software (version 6) was compared with related
sequences recorded in the NCBI database, and the dendro-
gram was plotted based on the nucleotide sequence similar-
ity. The evolutionary history was inferred using the
Neighbor-Joining method. The evolutionary distances were
computed using the Maximum Composite Likelihood
method and are in the units of the number of base substitu-
tions per site (Tamura et al. 2004).
Quantitative determination of protease activity
Strains with high levels of protease activity obtained in the
agar plate method were subjected to quantitative protease
assay. The protease activity of the bacterial cell-free extract
was estimated by the method described by Cupp-Enyard
with slight modifications (Cupp-Enyard 2008). Bacterial
strains were cultured in protease production medium con-
taining 1 g glucose, 10 g peptone, 0.2 g yeast extract, 0.1 g
CaCl2, 0.1 g MgSO4, and 0.5 g K2HPO4; pH 7 and incubated
for 48 h at 50
C with agitation (180 rpm). Then the culture
medium was centrifuged at 8000 rpm for 10 min and cell-
free supernatant was used for enzyme assay. 1.0 mL of
supernatant was added to 5 mL of the substrate containing
0.65% casein in phosphate buffer (pH 7) and incubated at
50
C for 10 min. In the next step, the reaction was stopped
by adding 5 mL of trichloroacetic acid (TCA) (110 mM).
Finally, 2 mL of the mixture was added to 5 mL of Na2CO3
(500 mM) and 1 mL of Folin–Ciocalteau (0.5 M) reagent
(Sigma, Aldrich) and the optical density was recorded at
660 nm. The protease activity was determined in terms of
Unit/mL by comparison of the liberated tyrosine from each
sample with a tyrosine standard curve.
Preliminary characterization of thermostable protease
Protease activity was investigated at different pH ranging
from 5 to 12 using reaction buffer including 0.1 M sodium
acetate buffer (pH 4–5), 0.1 M sodium phosphate buffer (pH
6.0–7.0), and 0.1 M Tris-HCl buffer (pH 8.0–12.0). The
GEOMICROBIOLOGY JOURNAL 3
effect of salt on enzyme activity was investigated using vary-
ing concentrations of NaCl (0–4 M) in the reaction mixture.
To study the effect of temperature on the enzymatic activity,
the enzyme reaction mixture was incubated at different tem-
peratures of 30–80
C, and the enzymatic activity was meas-
ured according to the standard method (Bouacem
et al. 2015).
To examine thermal stability, the enzyme solution was
pre-incubated for 20 min at various temperatures between
30 and 80
C and then proteolytic activity was analyzed
under standard assay conditions. The maximum temperature
at which 100% of the enzyme activity remained was deter-
mined to emphasize the maximum temperature value of
enzyme to catalysis. Stability at varying pH values (5–12)
was analyzed by incubating enzyme solution at 50
C for
20 min and the maximum pH at which 100% of enzyme
activity remained was recorded.
Correlation between growth and extracellular
protease activity
The bacterial strain was inoculated in protease production
medium and incubated for 48 h at 50
C with agitation
(180 rpm). The fermentation broth was taken every 4 h and
the absorbance was measured by spectrophotometer at
600 nm to draw the growth curve of the strain. Protease
activity was performed by standard method on a cell-free
extract of culture samples.
Analysis of results
The results were analyzed by SPSS software (Version 20)
using Tukey’s test. All experiments were performed in tripli-
cate and the data expressed as the mean ± SD. A p-value of
<0.05 was considered to be statistically significant.
Results
Isolation and identification of thermophilic isolates
In total, 36 strains with different colony morphology were
isolated from water samples and subjected to conventional
tests to characterize the isolates. From the 36 isolated
strains, 18 (50%) had white, 17 (47.2%) had cream and one
(4.1%) had yellow colonies on NA (Table 1). All the isolates
were able to grow at 40 and 50
C, 23 (63.9%) grow at 60
C
and 8 (22.2%) grow at 70
C. None of the isolates was cap-
able of growing at 80
C. All the isolates could grow in the
presence of 3% and 5% NaCl, and 7 (19.4%) grow at 10%
NaCl. None of the isolates was able to grow at 15% NaCl.
Our finding indicated the dominance of the Bacillus genus
represented by 32 isolates. All the isolates identified as
Bacillus were found to be gram-positive, aerobic, endospore-
forming rods, and motile. They had white, cream, and yel-
low-colored colonies and produced central or sub-terminally
ellipsoidal to oval endospores in non-swollen sporangia.
Also, four rod-shaped, aerobic, non-motile, gram-negative
bacteria with positive oxidase and catalase reaction and
4 P. ABDOLLAHI ET AL.

Table 1. Conventional analysis results of thermophilic isolates (n ¼ 36). Primary characterization of protease produced by
Colony Gram Endospore the isolates
Isolate staining staining Catalase Oxidase Motility formation
PA1 White þ þ þ þ þ Since this study aimed to screen thermophilic bacteria cap-
PA2 Cream þ þ þ þ þ able of producing stable enzymes, the isolates were grown in
PA3 White þ þ þ þ þ
PA4 Cream þ þ þ þ þ culture media, and the crude enzymes were partially charac-
PA5 Yellow þ þ  þ þ terized. The primary characterization of the protease pro-
PA6 Cream þ þ þ þ þ duced by thermophilic bacteria showed that their optimum
PA7 Cream þ þ þ þ þ
PA8 White þ þ þ þ þ salt requirement was in the range of 0.5–1 M. They were
PA9 Cream þ þ þ þ þ optimally active at pH 8–8.5 and their optimal temperature
PA10 White þ þ þ þ þ was in the range of 50–55
C (Table 3). Furthermore, all the
DH1 White þ þ þ þ þ
DH2 White þ þ þ þ þ proteases were stable at 50
C and pH 8–8.5. Among the iso-
DH3 White þ þ þ þ þ lated strains, strain PA10 with the highest protease activity
DH4 Cream þ þ þ þ þ
DH5 Cream þ þ  þ þ
was further studied.
DH6 White þ þ þ þ þ The effect of various temperatures on the protease activ-
DH7 White þ þ þ þ þ ity of strain PA10 is presented in Figure 3(a). The optimum
DH8 White þ þ þ þ þ
DH9 White þ þ þ þ þ
temperature for enzyme activity was found to be 50
C,
DH10 Cream þ þ  þ þ which was not significantly different from its activity at
DH11 White þ þ þ þ þ 55
C. The protease produced by strain PA10 was active in
DH12 Cream þ þ þ þ þ
DH13 White þ þ þ þ þ higher temperature and 80% of activity was retained at
DH14 White þ þ þ þ þ 60
C. It was active in a wide range of pH values (Figure
DH17 Cream þ þ  þ þ 3(b)). The optimum pH was found at pH 8 and 8.5
DH18 Cream þ þ þ þ þ
DH19 White þ þ þ þ þ (p < 0.05), and 20% of the initial activity was retained at pH
DH21 White þ þ þ þ þ 11. The effect of varying concentrations of NaCl ranging
DH22 cream þ þ þ þ þ from 0 to 4 M on protease activity is shown in Figure 3(c).
DH23 White þ þ  þ þ
DH24 Cream þ þ þ þ þ The optimum activity was found at 0 and 0.5 M NaCl
DH25 White þ þ þ þ þ (p < 0.05). However, 20% of the activity remained at
DH15 Cream – þ þ  
DH16 Cream – þ þ  
3 M NaCl.
DH20 Cream – þ þ   The stability of the enzyme solution under various condi-
DH29 Cream – þ þ   tions was also determined (Figure 4). To examine thermal
All the isolates were rod-shaped. stability, samples were pre-incubated for 30 and 60 min at
various temperatures between 30 and 80
C, and then pro-
teolytic activity was analyzed under standard assay condi-
optimum temperature for growth at 50
C belonged to the tions. More than 80% of initial activity retained after 30 min
genus Thermomonas were identified. The physiological char- of incubation at 55
C Figure 4(a). Stability at varying pH
acteristics of thermophilic isolates are shown in Figure 1. values (4–12) was analyzed by incubating enzyme solution
at 50
C for 1 h. As shown in Figure 4(b), the protease was
stable in a wide range of pH (6–10) and 58% of initial activ-
Production of thermostable enzymes
ity retained at pH 10.
All the isolates (100%) were potentially protease producers.
Lipase, DNase, and amylase activities were confirmed in 34
(94.4%), 8 (22.2%), and 3 (8.3%) isolates, respectively. Correlation between growth and extracellular
protease activity
However, none of the isolates produced cellulases. Five iso-
lates with a higher level of protease activity were selected for As shown in Figure 5, protease activity increased along with
further experiments. The biochemical and hydrolytic activ- bacterial growth, and the maximum activity was observed
ities of five selected isolates are summarized in Table 2. during the stationary phase in the culture supernatant of
PA10 cells. The highest increase in protease activity was
obtained between 16 and 24 h of incubation, and the max-
Molecular identification of selected isolates
imum activity was recorded after 36 h incubation at 50
C.
The 16S rDNA sequences of selected isolates were aligned Further increases in incubation time caused a reduction in
with their closely related sequences by the Basic Local protease activities.
Alignment Search Tool (BLAST) program. The sequence
analysis indicated that the isolates DH15, DH16, DH20, and
Discussion
DH29 fell within the species Thermomonas hydrothermalis
with a similarity between 98.13 and 99.93%. However, strain The earth we are living on has a variety of ecological niches
PA10 was related to Bacillus altitudinis (99.93% similarity). that many of their inhabiting microorganisms and their bio-
The phylogenetic tree based on 16S rDNA sequences align- technological potentials are still unknown. Hot springs are
ment is represented in Figure 2. ideal habitats for most of the thermophilic microorganisms.
 GEOMICROBIOLOGY JOURNAL 5

Figure 1. Physiological features of 36 thermophilic isolates.

Table 2. Biochemical and physiological characteristics of selected isolates.

In Iran, there are several hot springs renowned for their
Thermophilic strains
medicinal properties. However, their microbial communities
Characteristics and their potential biotechnological applications are still
DH15 DH16 DH20 DH29 PA10
unknown. In recent study, we isolated 36 thermophilic bac-
Acid from:
Glucose þ   þ þ
teria using culture-dependent methods. All strains could
Sucrose  þ  þ þ grow optimally at 50
C; so, according to Perry and Staley
Maltose þ þ  þ  (1997), and Souza and Martins (2001) they can be classified
Sorbitol     
Mannitol   þ  þ
as thermophilic bacteria. Based on salt requirements, and
Optimal temperature (
C) 50 50 50 50 50 following Kushner classification, all the isolates were halo-
Optimal pH 7.5 8 8 7.5 8 tolerants (Kushner and Kamekura 1988). In this study,
Optimum NaCl (%) 2 2 2 3 5
Nitrate reduction þ þ þ þ þ
most of the isolates were classified into the Bacillus genus.
Citrate utilization     þ Similar to our results, in previous studies across the world,
Urease þ þ þ þ  Bacillus is reported as the dominant strain in hot springs.
protease þ þ þ þ þ
Amylase –    þ
For example, 97.5% of the strains recovered from
Lipase þ þ þ þ þ Moroccan hot springs were known as Bacillus (Aanniz
Cellulase      et al. 2015). Maugeri et al. (2001) also isolated 87 thermo-
DNase þ    
philic bacteria from Italy, which were mostly categorized in
the Bacillus genus, and thermophilic Bacillus was the dom-
These environments are often nutrient-poor with a moderate inant strain from Jordanian hot springs (Abou-Shanab
to high temperature. In these conditions, microorganisms 2007). The ecological conditions of the hot springs are
have to develop strategies to overcome these stresses. Exo- known as the moderate to high temperature and nutrient-
enzymes production by thermophilic bacteria is one of the poor. Strains belonging to the genus Bacillus are well
most important strategies for utilizing any organic matter, adapted to the hot environments and oligotrophic condi-
which is available in these nutrient-poor environments, this tions. Therefore, they can colonize and grow in environ-
makes them an excellent source of various enzymes used in ments like hot springs, salt marshes, and desert soils
the industry. (Khiyami et al. 2012).
6 P. ABDOLLAHI ET AL.

Figure 2. A neighbor-joining phylogenetic tree based on 16S rRNA gene sequences of selected isolates. The tree contains the closest type strain for each isolate.
The optimal tree with the sum of branch length ¼ 0.02324416 is shown. The percentage of replicate trees in which the associated taxa clustered together in the
bootstrap test (1000 replicates) is shown next to the branches (Kumar et al. 2018).

Table 3. Characterization of thermophilic proteases.

Production
level Thermal
Isolates (U mL1) pH optima NaCl optima (M) Temprature optima (
C) stability (
C)a pH stabilitya
DH15 0.82 8.5 0.5 55 50 8
DH16 0.92 8 0.5 55 50 8
DH20 0.8 8.5 0.5 50 50 8.5
DH29 1.2 8 1 50 55 8
PA10 1.56 8 1 50 50 8
a
Stability for 20 min.

(a) (b) (c)

120 120 120

100 100 100

Relave acvity (%)

Relave acvity (%)

Relave acvity (%)

80 80 80

60 60 60

40 40 40

20 20 20

0 0 0
30 40 50 60 70 80 5 6 7 8 9 10 11 12 0 0.5 1 1.5 2 2.5 3 3.5 4

Temperature (°C) pH NaCl (M)

Figure 3. (a) Effect of temperature (at pH 8), (b) pH (at 50
C), and (c) varying concentrations of NaCl on protease activity of strain PA10.

It has been documented that thermophilic bacteria, pro- and genetic potency for using accessible organic matter, via
duce valuable heat-resistant enzymes that can hydrolyze pro- exo-enzymes production (Berrada et al. 2012). This ability
teins, lipids, polysaccharides, and other nutrients (Rey et al. makes them survive and adapt to such ecosystems with low
2004). In our study, all the isolates exhibited protease activ- organic content and enables them to absorb any food avail-
ities. Also, 94.4, 22.2, and 8.3% of isolates were lipase, able (Aanniz et al. 2015).
DNase, and amylase producers, respectively. Our finding Thermostable enzymes are of great biotechnological inter-
showed that the isolates may have developed physiological est due to their ability to minimize microbial contamination
 GEOMICROBIOLOGY JOURNAL 7

(a) (b)
120 120

100 100

Relave acvity (%)

Relave acvity (%)

80 80

60 60

40 40

20 20

0 0
30 40 50 60 70 4 5 6 7 8 9 10 11 12
Temperature °C pH
30 min 60 min

Figure 4. (a) Effect of temperature on enzyme stability (pH 8), (b) effect of pH on enzyme stability (1 h at 50
C).

Enzyme activity
Bacterial growth
1.8 2
1.6 1.8

Cell density (OD 600 nm)

Enzyme acvity (U mL-1)

1.4 1.6
1.2 1.4
1.2
1
1
0.8
0.8
0.6 0.6
0.4 0.4
0.2 0.2
0 0
0 4 8 12 16 20 24 28 32 36 40 44
Incubation Time (h)
Figure 5. Correlation between bacterial growth and extracellular protease activity in strain PA10.

during industrial processes and working for long periods distribution of both substrate and particularly enzyme mole-
(Wang et al. 2012). Among thermophilic isolates obtained in cules. Therefore, we evaluated the protease activity of
this study, five isolates showed the highest protease activ- selected strains at different temperature and pH conditions.
ities. Analysis of 16S rRNA gene sequencing indicated that Based on our results, the optimum temperature and pH val-
the selected isolates were related to the Bacillus and ues for protease produced by T. hydrothermalis strains were
Thermomonas genera. The genus Thermomonas is a Gram- determined at 50–55
C and pH 8.0–8.5. Therefore, protease
negative, rod-shape, thermophilic bacterium that belongs to obtained from these isolates can be classified as moderately
the family Xanthomonadaceae (Busse et al. 2002). The inter- thermo-active alkaline proteases. On the other hand, the
esting finding in this study is the isolation of strains from T. temperature and pH stability of the protease enzyme is an
hydrothermalis species; there are no previous reports con- essential matter particularly in industrial applications (Raval
cerning the isolation of these strains from hot springs in et al. 2014). Thermal and pH stability characteristics of T.
Iran. Furthermore, few reports regarding these strains hydrothermalis protease are highlights of the present study.
are available. Considering all the above, the protease enzyme derived from
T. hydrothermalis was first isolated from hot springs in T. hydrothermalis can have many uses in different industrial
Portugal (Alves et al. 2003). It was described as rod-shape, sectors. Given that there are not many reports from these
Gram-negative, and none motile cells with a colony diam- isolates, this makes it an attractive strain for biotechno-
eter of 0.5–2.0 mm. There are a few detailed enzyme profiles logical and environmental applications.
available for T. hydrothermalis strains. Positive amylase, pro- Another protease producing isolates from Iranian hot
tease, lipase, and cellulase activities have been reported for spring was strain PA10, which showed the highest similarity
these strains which is consistent with our study (Baltaci to Bacillus altitudinis. B. altitudinis is a thermophilic bacter-
et al. 2017; Mohammad et al. 2017). However, no DNase ium that first isolated from cryogenic tubes used for collect-
activity is reported from T. hydrothermalis strains. This is ing air samples from high altitudes (Shivaji et al. 2006). The
the first study describing positive DNase activity from T. organism had also been isolated from hot springs of the
hydrothermalis strains. Uzon caldera in Russia (Rozanov et al. 2018). However, no
It is also worth mentioning that the activity of the report about the isolation of this strain from Iranian hot
enzymes is markedly affected by pH because substrate bind- springs is available. Our study confirmed the previous stud-
ing and catalysis are often dependent on the charge ies concerning the capability of B. altitudinis for the
8 P. ABDOLLAHI ET AL.
production of protease and amylase (Kumar et al. 2011; Li
et al. 2015). However, no documented report about lipase
production by this strain is available.
Further investigation revealed that the protease from
strain PA10 had high-temperature and pH stability
(Figure 4). Similar results are found in protease produced by
Bacillus sp. which retains 80% of its activity after 30 min at
60
C (Nascimento and Martins 2004). The protease from,
Brevibacillus sp. PLI-1 keeps more than 70% of its activity
after 60 min at 70
C and pH 8 (Wang et al. 2012). These
characteristics make them suitable for various industrial
applications, especially in detergent and leather industries.
In conclusion, this work reports the isolation and identi-
fication of cultured thermophilic bacteria belonging to
Thermomonas and Bacillus genera from Iranian hot springs.
These strains showed the ability to produce varying hydro-
lytic enzymes, including protease, lipase, amylase, and
DNase. Temperature and pH stability of protease from these
thermophilic isolates make them useful for biotechnological
applications. This is the first report from the isolation of B.
altitudinis and T. hydrothermalis strains from Iranian hot
springs. This study highlights Sareyn Hot Springs as an
important reservoir of thermophilic microorganisms harbor-
ing industrially important enzymes. The obtained promising
results can be exploited for the production of thermo-
stable enzymes.
Disclosure statement
No potential conflict of interest was reported by the author(s).
ORCID
Maryam Ghane http://orcid.org/0000-0003-0305-0241
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