Exploration, isolation, and genus identification of hydrolytic-

producing bacteria from hot spring in Riau and Sumatera Barat

Province
Saryono1*, Finna Piska1, Usman Pato2, Riryn Novianty1, Nova Wahyu Pratiwi 3, Aulia
Ardhi4
1
Department of Chemistry, Faculty of Mathematics and Natural Sciences Universitas Riau
2
Department of Agriculture, Faculty of Agriculture Universitas Riau
3
Department of Biology, Faculty of Mathematics and Natural Sciences Universitas Riau
4
Department of Food and Agricultural Product Technology, Faculty of Agricultural Technology, Universitas Gadjah Mada

Corresponding author’s email: saryono@lecturer.unri.ac.id

ABSTRACT.

Aims: Central Sumatra, including Riau and Sumatera Barat, is an area which is rich in hot springs. This study aimed
to explore, isolate, and identify thermophilic bacteria that live in hot springs in some regions of Riau and Sumatera
Barat Province.
Methodology and results: Those isolates were incubated at 45, 50, and 55 °C and isolated using pour plate method
prior to morphological characterization. Biochemistry tests were due to the isolates for known its genera. There were
110 bacteria obtained consisting of 3 genera, including Thermus, Pseudomonas, and Bacillus. The isolates were
tested its ability producing amylase, lipase, cellulose, and inulinase on starch, olive oil, CMC, and inulin (from tuber
Dahlia), respectively. The clear zone were observed for known its ability for producing the enzymes.
Conclusion: All isolates were able to produce cellulase at all three incubation temperatures used, otherwise, lipase
was not produced at all temperatures. Only a few isolates can produce amylase and inulinase.

Keywords: bacteria, Central Sumatera, exploration, hot spring, identification

INTRODUCTION
Thermophilic bacteria can be isolated from hot
spring, which now becomes of great interest because
they can produce enzymes that resistant to high
temperature and extreme pH. More recently, many
studies are devoted to the comprehension of the
molecular basis of the bacterial adaptation to high
temperature (Akmar et al., 2011). Hot spring is an
extreme environment; however, it gives optimized
growth for thermophilic bacteria. Thermophilic
bacteria can released thermostable enzymes from it.
Msarah et.al (2017) proved thermophilic bacteria can
be ability produced enzymes with physically and
chemically are stable.
Thermophilic bacterial are reported to produce
thermostable enzymes such as cellulose, amylase,
lipase, inulinase, and protease (Azhar et.al., 2013;
Mohammad et.al, 2017). Cellulose is a complex
polymer formed glucose with β-1,4, by the
hydrolyzed with synergetic action of a mixture of
cellulose. Amylose can hydrolyzed by amylase in α-
1,4 glycosidic produced maltose, maltotriose,
maltotetraose, maltopentaose, and maltoheptaose are
linking by 2-10 glucopyranosyl residues via α-1,4
bonds formed glucose (Otzurk et.al., 2014). In the
other hand, inulin is a complex polymer formed
fructose by linked β-1,2. Inulin can hydrolysis into
fructose by enzymes called inulinase (exo-inulinase
and endo-inulinase) (Saryono et al., 2015). Lipases
are enzymes called triacylglycerol acyl-hydrolase for
released fatty acids and glycerol from triacylglycerol
by process hydrolyzed (Mehta et al., 2017). However,
protease has the ability for hydrolysis protein of
casein or skim milk.
Lorenita et. al (2013) have isolated fungal from tuber
Dahlia with 4 genus, are following: Monillia,
Aureobasidium, Moniliella, and Sporothrix. The
secondary metabolite of the fungal were identify by
Saryono et.al (2015), which its activity and
molecular characterization. The fungal has activity as
antimicrobial against S.aureus, E.coli. and
C.albicans. Therefore, we isolated bacterial from h ot
springs in Riau and Sumatera Barat, that are
manifestations of geological activity and have
extreme environments. The temperature of those hot
springs is about 70 - 90°C. In this research, six hot
springs are explored as follows: Sungai Pinang
Kuantan Singingi and Pawan Rokan Hulu (Riau
Province); Padang Ganting Batusangkar; Rimbo
Panti Pasaman; Bukik Kili and Bukik Gadang Solok.
Thus, from these hot springs, a large number of
thermophilic bacterial can be obtained with different
characterizations. Thermophilic bacterial obtained are
carried out hydrolysis tests on various media to
produce thermostable enzymes which can later be
commercial and have high potential in the
biotechnology industry.
Methods and Material
a. Location and sampling preparation
Samples of this study were taken from hot
springs in Rokan Hulu, West Sumatera, and Teluk
Kuantan province. The location of the hot springs can
be seen in table 1. Sampling was done using
purposive sampling method, with there were three
spots in one location. Every one point, the sample
was taken 100 mL for 2 bottles with a volume of 250
mL. After that, the sample was stored in the freezer
to prevent microbial growth during storage (Salano et
al., 2017).
b. Isolation and purification
At each site, 1 mL of each sample
micropipette and tip sterile was transferred to
Nutrient Agar (NA) in a petridish using micropipette
and sterile tips. The inoculum was then incubated at
45°, 50°, and 55°C for two days. The grown colonies
were purified on NA medium by quadrant streak
method.
c. Staining gram and acid-resistant test.
Staining gram was applied to colonies that
had grown after two days resulting to identification
of the form of cells; basil and coccus, and the stain;
positive or negative. In acid-resistant coloration test,
each of actinomycetes and bacterial isolates was
added on glass preparations, spilled with
carbolfuchsin and heated for 5 minutes. It was
washed and soaked in 96% alcohol for 15 minutes,
rinsed with flowing water, soaked with methylene
blue for 2 minutes, and rinsed back with flowing
water. The observation was taken under a
microscope. The positive test was showed by red
cells while the negative test was showed by blue or
colorless.
d. Morphological characterization of bacterial
Characterization of grown colonies in NA
medium was such as pinpoint/punctiform, shape, and
elevation. The growth of isolates was observed such
as filiform, echinulate, beaded, arborescent, plumose,
papillate, and villose. The patterns of colony isolates
were observed such as filiform, echinulate, beaded,
effuse, spreading, plumose, and rhizoid. Observations
were also made on broth media to observe the growth
of isolates based on O2 requirements, with anaerobic
facultative patterns (uniform turbidity and flocculent
growth), aerobic (pellicle), microaerophilic (ring),
and anaerobes (sediment).
e. Biochemical assay
Some biochemical assays were carried out in
order to obtain the ability of the isolates to produce
catalase; hydrolysis amylum, casein and gelatin; and
methylene blue reduction test.
Catalase test
The isolates were put in preparation test and solution
of 3% H2O2 was dropped onto the isolate. The
positive test showed if gas formed on the preparation.
Gelatin hydrolysis test
Gelatin media were made with the following
composition of 15 g gelatin, 15 g agar, 4 g peptone, 1
g yeast extract in 1 L of demineralized water. The
patterns of growth were observed, such as filiform,
echinulate, beaded, arborescent, plumose, papillate,
and villose. The gelatin hydrolysis test was observed
by incubating the media containing the isolate into
the freezer for 1 h. The test was positive if the media
melted.
Methylene blue reduction test
The isolates inoculated into NB media were added
with 3 mL of 10% methylene blue solution. The
homogenized mixture was stored for 60 min at 37°C
and then observed the reduction of blue color of the
media. The color change from blue into clear
indicated that the isolates were able to reduce
methylene blue (Suhartati and Depi, 2014).
f. Hydrolytic Enzyme-Producing Test:
Hydrolytic enzymes produced by bacterial
isolates which were confirmed in this study included
cellulase, inulinase, amylase, lipase, and protease
using a clear zone method. The isolates were grown
and incubated for 3-4 days at room temperature in
each selective media and added with a few drops of
iodine solution. A clear zone formed indicated that
the isolate was able to produce certain hydrolytic
enzymes. The ratio of clear zone diameter and colony
isolates was calculated using the formula proposed by
Hardianty et al. (2010).
Cellulase Production Media: The fungal ability on
degrading cellulose was confirmed on CMC media
containing 10 g carboxymethyl cellulose, 10 g
K2HPO4, 10 g MgSO4, 0,05 g KCl, 2 g (NH4)2SO4,
0.1 mg FeSO4 and 20 g agar (Saryono et al., 2018).
Inulinase Production Media: The fungal isolates
were grown on inulin selective media composed of
1,5 g NaNO3, 2 g (NH4)2SO4, 1 g KH2PO4, 0,5 g
MgSO4.7H2O, 0,1 g FeSO4.7H2O, 10 g inulin and 18
g agar (Saryono et al, 2018).
Amylase Production Media: The composition of
selective amylase media used was 1,5 g NaNO 3, 2 g
(NH4)2SO4, 1 g KH2PO4, 0,5 g MgSO4.7H2O, 0,1 g
FeSO4.7H2O, 10 g starch and 18 g agar (Saryono et
al., 2018).
Lipase Production Media: Lipid selective media
were made with the following composition of 5 g
peptone, 1 g KH2PO4, 1 g FeSO4.7H2O, 1 g
MgSO4.7H2O, 10 g NH4NO3, 10 g sucrose, 10 g olive
oil and 15 g agar (Saryono et al., 2018).
Protease Production Media: The fungal isolates
were grown on skim milk agar composed of 20 g
skim milk and 7,5 g agar (Saryono et al., 2018).
Results and Discussion
The number of bacterial isolates obtained
from this study was 110 isolates. Each isolate
obtained was grouped based on the results of the
gram and macroscopic staining. From the grouping
obtained 3 groups of isolates, can be seen in table 2.
All the groups of bacterial isolates were tested to
determine the genus of 110 isolates.

Table 1. The number of bacterial isolates from each location and temperature.
Temperature
Location
45°C 50°C 55°C
Sungai Pinang 17 6 3
Rimbo Panti 10 13 2
Padang Gantiang 5 6 3
Pawan 6 6 5
Bukik Gadang 6 6 1
Bukik Kili 6 6 3
The biochemical test results of 110 isolates were with gram-negative (Figure 1). In table 3 it can be
grouped into 3 groups as in Table 2. The 3 groups seen that the results of biochemical tests in each
were classified into Bacillus (B1), Thermus (B2), and genus showed almost the same results. No genera
Pseudomonas (C1) which had different could perform gelatin hydrolysis, reduction for
morphologies. Bacillus had gram-positive basil methylene blue, and acid resistant. In catalase test,
morphological features, Thermus was the bubbles produced by each isolate were different
morphologically featured with gram-negative bacilli. even with the same genus.
Meanwhile, Pseudomonas had a form of coccus cell

Table 2. Macroscopic and microscopic morphology of the isolates

Genus Deep NA Slant NA Broth Staining gram
-
B1 Echinulate Echinulate Uniform turbidity
basil
-
C1 Beaded Echinulate Uniform turbidity
coccus
+
B2 Beaded Echinulate Uniform turbidity
basil

Table 3. Biochemical assay of three company isolates

Methylen blue Resistance of Catalase hydrolized
Isolate Gelatin Genus
reduction test acid test
B1 - - - +++ Thermus
C1 - - - ++ Pseudomonas
B2 - - - +++ Bacillus
 A B
Figure 1. Microscopic view of three genera: Thermus (A), with pink color on the cell, irregularly located, and the basil tended to
be short; Bacillus, (B) had basil cell shape that tended to be elongated, purple, and irregularly located; Pseudomonas (C) had the
shape of a cocci cell, pink, and its random location.
Table 4, 5, and 6 showed results of hydrolytic
enzyme-producing tests of all bacterial isolates from
various locations with the isolation temperatures of
45, 50, and 55°C. All isolates hardly produced clear
zones on the agar lipid media. On the contrary,
almost all isolates produced clear zones on cellulose
media and agar starch. This showed that each isolate
has a different ability to use carbon sources from
each medium. The resulting clear zone indicated that
the bacteria had the potential to produce enzymes
from the degradation amylum, cellulose, inulin, and
lipid results.
Discussion
In this study, there were 53 isolates of gram-positive
Bacillus, 37 isolates were gram-negative bacilli, and
21 of them were gram-negative cocci. The
morphological properties of all these bacterial
isolates were differing, with the ability to hydrolyze
different starches, casein, and H2O2. The negative
results in the methylene blue reduction test in the
table above probably because the number of bacterial
cells was not proportional to the volume of
methylene blue solution added to the media. This test
of methylene blue reduction according to Suhartati
and Depi (2014) served to see the presence of
contaminants in the media. Negative results on
gelatin hydrolysis could be due to the excess
composition of the gelatin media and the inability of
bacterial isolates to hydrolyze collagen. Hemraj et al.
(2013) stated that gelatin is a derivative of collagen,
which does not dissolve in warm water or ordinary
temperature, but dissolves in cold water. Therefore,
C
in this test, the media was stored in the refrigerator to
see the ability of bacteria to melt gelatin in cold
temperatures. The ability of bacterial isolates to
produce catalase was indicated by the presence of
bubbles when a 3% hydrogen peroxide solution is
added. Catalase is produced in cells, degrading H2O2
to be O2 and H2O (Hemraj et al., 2013). The positive
results in the acid resistance test showed that
bacterial cell wall contained a thick lipid layer, while
the negative results showed that bacterial cell wall
did not or contained lipids slightly. Gram-negative
bacteria usually showed resistance to the acidic
environment and gave positive in acid resistant test
(Mahmudah et al., 2016).
These bacteria are known to have extensive habitats
in which the temperature was quite extreme. Akmar
et al. (2011) stated that bacteria from hot springs had
optimal growth temperatures at above 50 °C. The
possibility of obtaining these bacteria was probably
because the bacteria had other optimal temperatures
to grow, such as at 45 and 50 °C. In accordance with
Pitri et al. (2015), the optimal temperature for the
growth of thermophilic bacteria was above 45°C.
Muharni (2009) also stated that the optimal
temperature of Bacillus was in the range of 30, 60,
and 72°C.
Results of enzymes production

Table 5. Clear Zone Obtained from Bacterial Isolates at 45 °C

Amylase Cellulase Lipase Inulinase
Origin of
Genus LBKURCC Isolate Ratio Ratio Ratio Ratio
Location Activity Activity Activity Activity
(mm) (mm) (mm) (mm)
Pseudomonas 197 Sungai Pinang 10.515 +++++ 17.131 +++++ - - - -
Bacillus 198 Sungai Pinang 0.882 + - - - - - -
Thermus 199 Sungai Pinang 19.966 +++++ - - - - - -
Thermus 200 Sungai Pinang 7.186 +++++ 4.389 +++++ - - - -
Bacillus 201 Sungai Pinang - - 5.132 +++++ - - - -
Pseudomonas 202 Sungai Pinang 18.821 +++++ 23.751 +++++ - - - -
Bacillus 203 Sungai Pinang - - 17.927 +++++ - - 1.671 ++
Thermus 204 Sungai Pinang 2.515 +++ 11.250 +++++ - - - -
Bacillus 205 Sungai Pinang - - 15.388 +++++ - - - -
Bacillus 206 Sungai Pinang 1.597 ++ 26.094 +++++ - - - -
Bacillus 207 Sungai Pinang 0.059 + - - - - - -
Pseudomonas 208 Sungai Pinang 0.127 + 6.721 +++++ - - - -
Pseudomonas 209 Sungai Pinang 0.190 + 15.897 +++++ - - - -
Pseudomonas 210 Sungai Pinang 0.273 + 19.412 +++++ - - - -
Bacillus 211 Sungai Pinang 0.154 + 10.515 +++++ - - - -
Bacillus 212 Sungai Pinang - - 3.596 ++++ - - - -
Bacillus 213 Sungai Pinang 0.544 + 52.425 +++++ - - 4.284 +++++
Bacillus 214 Rimbo Panti 8.431 +++++ - - - - 3.982 ++++
Bacillus 215 Rimbo Panti 0.890 + 18.851 +++++ - - - -
Bacillus 216 Rimbo Panti 0.019 + - - - - 3.402 ++++
Bacillus 217 Rimbo Panti 8.218 +++++ 20.649 +++++ - - - -
Bacillus 218 Rimbo Panti - - 24.228 +++++ - - 0.813 +
Bacillus 219 Rimbo Panti 0.192 + 1.248 ++ - - 3.983 ++++
Bacillus 220 Rimbo Panti 3.558 ++++ 11.250 +++++ - - 0.463 +
Bacillus 221 Rimbo Panti 2.587 +++ 16.165 +++++ - - 1.024 ++
Bacillus 222 Rimbo Panti 6.216 +++++ - - - - - -
Bacillus 223 Rimbo Panti 2.606 +++ - - - - 4.134 +++++
Bacillus 224 Padang Ganting 1.325 ++ 3.596 ++++ - - - -
Bacillus 225 Padang Ganting 2.587 +++ 2.391 +++ - - - -
 Bacillus 226 Padang Ganting - - 2.237 +++ - - - -
Bacillus 227 Padang Ganting - - 7.606 +++++ - - - -
Pseudomonas 228 Padang Ganting - - 6.662 +++++ - - - -
Pseudomonas 229 Pawan 3.558 11.250 +++++ - - - -
Bacillus 230 Pawan 7.015 +++++ 8.819 +++++ - - - -
Pseudomonas 231 Pawan 8.431 +++++ 0.084 + - - 0.191 +
Bacillus 232 Pawan - - 14 +++++ - - 3.491 ++++
Thermus 233 Pawan 8.371 +++++ - - 6.342 +++++ 0.602 +
Pseudomonas 234 Pawan - - 2.587 +++ - - - -
Thermus 235 Bukik Gadang - - - - - - - -
Thermus 236 Bukik Gadang - - 17.302 +++++ - - 2.00 ++
Thermus 237 Bukik Gadang 5.132 +++++ 5.624 +++++ - - - -
Bacillus 238 Bukik Gadang - - 0.558 + - - - -
Thermus 239 Bukik Gadang 8.037 +++++ - - - - - -
Thermus 240 Bukik Gadang - - 1.981 ++ - - 2.742 +++
Bacillus 241 Bukik Kili - - 0.662 + - - 2.862 +++
Bacillus 242 Bukik Kili 4.984 +++++ 3.868 ++++ - - - -
Thermus 243 Bukik Kili 2.165 ++ 7.315 +++++ - - - -
Pseudomonas 244 Bukik Kili - - 8.493 +++++ - - - -
Bacillus 245 Bukik Kili 7.917 +++++ 1.028 + - - - -
Thermus 246 Bukik Kili 0.562 + 53.934 +++++ - - - -

Table 6. Clear Zone Obtained from Bacterial Isolates at 50 °C

Amylase Cellulase Lipase Inulinase
Origin of Isolate
Genus LBKURCC Ratio Ratio Ratio Ratio
Location Activity Activity Activity Activity
(mm) (mm) (mm) (mm)
Bacillus 154 Sungai Pinang 14.561 +++++ 4.936 +++++ - - 3.416 ++++
Thermus 155 Sungai Pinang 10.720 +++++ 20.660 +++++ - - - -
Thermus 156 Sungai Pinang 13.503 +++++ 24.379 +++++ - - - -
Thermus 157 Sungai Pinang - - 14.320 +++++ - - - -
Bacillus 158 Sungai Pinang 6.045 +++++ 10.947 +++++ - - - -
Bacillus 159 Sungai Pinang 11.894 +++++ 19.793 +++++ - - - -
Pseudomonas 160 Rimbo Panti 2.513 +++ 7.078 +++++ - - - -
Pseudomonas 161 Rimbo Panti 6.049 +++++ - - - - - -
Bacillus 162 Rimbo Panti 0.407 + 4.342 +++++ - - 11.674 +++++
Thermus 163 Rimbo Panti - - 5.650 +++++ - - - -
Thermus 164 Rimbo Panti 10.441 +++++ 9.017 +++++ - - - -
Pseudomonas 165 Rimbo Panti - - 6.958 +++++ - - - -
 Thermus 166 Rimbo Panti 11.309 +++++ 10.587 +++++ - - - -
Pseudomonas 167 Rimbo Panti 3.870 ++++ - - - - - -
Thermus 168 Rimbo Panti 5.624 +++++ 20.855 +++++ - - 5.135 +++++
Bacillus 169 Rimbo Panti 9.135 +++++ 2.554 +++ - - 4.575 +++++
Thermus 170 Rimbo Panti - - - - - - 0.001 +
Thermus 171 Rimbo Panti 8.562 +++++ 8.753 +++++ - - - -
Thermus 172 Rimbo Panti 4.389 +++++ 8.951 +++++ - - - -
Pseudomonas 173 Padang Ganting - - 19.416 +++++ - - 0.827 +
Thermus 174 Padang Ganting 0.406 + 13.362 +++++ - - - -
Pseudomonas 175 Padang Ganting - - 11.080 +++++ - - - -
Thermus 176 Padang Ganting 7.258 +++++ 8.884 +++++ - - - -
Thermus 177 Padang Ganting 0.303 + 11.085 +++++ - - - -
Thermus 178 Padang Ganting 10.373 +++++ - - - - - -
Bacillus 179 Pawan 7.680 +++++ - - - - - -
Bacillus 180 Pawan 6.785 +++++ 8.819 +++++ - - - -
Bacillus 181 Pawan 10.029 +++++ 6.610 +++++ - - - -
Bacillus 182 Pawan 9.135 +++++ 9.165 +++++ - - - -
Thermus 183 Pawan 9.456 +++++ 0.253 - - - - -
Bacillus 184 Pawan 9.683 +++++ 9.824 +++++ - - - -
Thermus 185 Bukik Gadang 11.970 +++++ 10,22 +++++ - - - -
Pseudomonas 186 Bukik Gadang 11.524 +++++ 0.083 + - - - -
Pseudomonas 187 Bukik Gadang 6.109 +++++ 17.940 +++++ - - - -
Thermus 188 Bukik Gadang - - - - - - - -
Pseudomonas 189 Bukik Gadang 9.618 +++++ 8.620 +++++ - - - -
Pseudomonas 190 Bukik Gadang 13.355 +++++ 8.294 +++++ - - 9.074 +++++
Bacillus 191 Bukik Kili 8.175 +++++ 9.686 +++++ - - - -
Bacillus 192 Bukik Kili 13.598 +++++ 13.120 +++++ - - - -
Bacillus 193 Bukik Kili 4.150 ++++ 3.422 ++++ - - - -
Thermus 194 Bukik Kili - - 28.730 +++++ - - - -
Thermus 195 Bukik Kili - - 7.489 +++++ - - - -
Bacillus 196 Bukik Kili 9.748 +++++ - - - - - -

Table 7. Clear Zone Obtained from Bacterial Isolates at 55 °C

Amylase Cellulase Lipase Inulinase
Origin of
Genus LBKURCC Ratio Ratio Ratio Ratio
Isolate Location Activity Activity Activity Activity
(mm) (mm) (mm) (mm)
 Pseudomonas 247 Sungai Pinang - - 22.520 +++++ - - - -
Bacillus 248 Sungai Pinang - - 4.050 +++++ - - - -
Bacillus 249 Sungai Pinang - - 23.350 +++++ - - - -
Thermus 250 Rimbo Panti - - 16.680 +++++ - - - -
Bacillus 251 Rimbo Panti - - 10.802 +++++ - - - -
Bacillus 252 Padang Ganting - - 19.590 +++++ - - - -
Thermus 253 Padang Ganting - - 20.950 +++++ - - - -
Thermus 254 Padang Ganting - - 10.220 +++++ - - - -
Bacillus 255 Pawan - - 18.290 +++++ - - - -
Bacillus 256 Pawan - - 23.350 +++++ - - - -
Bacillus 257 Pawan - - 12.730 +++++ - - - -
Bacillus 258 Pawan - - 9.960 +++++ - - - -
Thermus 259 Pawan - - 10.890 +++++ - - - -
Bacillus 260 Bukik Gadang - - 20.605 +++++ - - - -
Bacillus 261 Bukik Kili - - - - - - - -
Bacillus 262 Bukik Kili - - 37.090 +++++ - - - -
Bacillus 263 Bukik Kili - - 11.520 +++++ - - - -
Bacillus 264 Bukik Kili - - - - - - - -
Information:
1. + (very low)
2. ++ (low)
3. +++ (medium)
4. ++++ (high)
5. +++++ (very high)

Some isolates of Pseudomonas were obtained with the
characteristics of coccus and gram-negative cells, and yellow colonies.
Mahmudah et al. (2016) isolated Pseudomonas from Lejja hot springs in
Sopeng Regency. Besides, Saiki (2014) obtained Thermus sp. with the color
of yellow colonies, basil cell forms, and gram-negative. Other experiences
came from Akmar et al. (2011) which obtained bacteria with positive and
negative bacillus cell forms from Ulu Legong hot springs while Muharni
(2009) succeeded in isolating bacteria with positive bacillus cell forms with
the genus Bacillus from the hot springs of Lake Ranau in Sumatera Selatan.
Nanda et al. (2017) isolated bacteria from Kerinci hot springs with purple
(negative) basil cell forms. On the other hand, Octarya et al. (2011)
managed to isolate bacteria from hot springs Bukik Kili with the genus
Bacillus. Bacteria with basil cell form and gram-positive were also obtained
from the Medang river hot springs by Pitri et al. (2015).
One of the important potentials of thermophilic microorganisms is
their enzymatic activities in high temperature which intensified their
importance in industrial and biotechnological areas. Enzymes obtained from
thermophilic microorganisms were indicated that the strains had developed
special mechanisms genetically and physiologically to utilize available
organic matters. Microorganisms that released thermostable enzymes had
many attractive features such as their ability to minimize the possibility of
microbial contamination in large-scale industrial reactions and operated in
high temperature and for long durations. Based on the results from
hydrolysis enzymes production test, there were no lipase and protease
produced from all isolates, and only a few isolates could produce inulinase.
In this research, we used inulin from dahlia tuber (Dahlia variabilis), that
having secondary metabolites with its potential bioactive compounds as
anti-microbial compounds (Saryono et.al., 2017). Characterization and
identification of extracellular inulinase due by Azhar et.al (2013). The
enzyme producing by bacteria from hot aprings in West Sumatera, with
optimal temperature from 23° to 60°C.
Mehta et al. (2017) approved that lipase could be produced by
fungi. Fungal lipases were extracellular and their production was influenced
by nutritional and physicochemical factors. However, Saryono et.al (2016)
have isolation and characterization inulinase from Aspergillus flavus from
Gmn11.2 Galur Lokal with optimal temperature 50°C, pH 5 at 4 hour
incubation. Mohammad et al. (2017) released that Bacillus strain could
produce lipase. On the other hand, almost isolates showed the ability to
produce cellulase and amylase. Bacillus isolated from Jordanian hot springs
by Mohammad et al. (2017) could produce cellulose.
Conclusion
There were 110 bacterial isolates with the genera of Bacillus,
Thermus, and Pseudomonas. The ability to produce amylase, cellulase,
inulinase and lipase enzymes of bacterial isolates varied at three
temperatures, namely 45, 50, and 55°C. Bacterial isolate was only able to
produce cellulase at all temperatures, while it was unable to produce lipase
at all temperatures. Only a few isolates were capable of producing amylase
and inulinase.
Acknowledgment
This study was funded by Postgraduate Grant Research Year 2017 from The
Ministry of Research, Technology and Higher Education of The Republic of
Indonesia on behalf of Prof. Dr. Saryono.
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