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Article

Isolation and Characterization of Thermophilic Bacteria from Hot Springs in Republic of Korea

1
Department of Bio-Cosmetics, Seowon University, Chung-Ju 28674, Republic of Korea
2
Major in Food Biotechnology, Research Center for Extremophiles and Marine Microbiology, Silla University, Busan 46958, Republic of Korea
3
Department of Food Science and Technology, Pukyong National University, Busan 48513, Republic of Korea
4
Library of Marine Samples, Korea Institute of Ocean Science & Technology (KIOST), Geoje 53201, Republic of Korea
5
National Institute of Biological Resources, Incheon 22689, Republic of Korea
6
Bio-Living Engineering Major, Global Leaders College, Yonsei University, Seoul 03722, Republic of Korea
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Microorganisms 2022, 10(12), 2375; https://doi.org/10.3390/microorganisms10122375
Submission received: 31 October 2022 / Revised: 20 November 2022 / Accepted: 25 November 2022 / Published: 30 November 2022
(This article belongs to the Special Issue Thermophilic Bacteria and Archaea)

Abstract

:
Thermophiles that produce extracellular hydrolases are of great importance due to their applications in various industries. Thermophilic enzymes are of interest for industrial applications due to their compatibility with industrial processes, and the availability of the organisms is essential to develop their full potential. In this study, a culture-dependent approach was used to identify thermophilic bacteria from five hot springs in Republic of Korea. Characterization, taxonomic identification, and extracellular hydrolase (amylase, lipase, and protease) activity of 29 thermophilic bacterial isolates from the Neungam carbonate, Mungang sulfur, Deokgu, Baegam, and Dongnae hot springs were investigated. Identification based on the full-length 16S rRNA gene sequence revealed that strains belonged to the phylum Bacillota and were classified as Aeribacillus, Bacillus, Caldibacillus, Geobacillus, and Thermoactinomyces genera. It was found that 22 isolates could produce at least one extracellular enzyme. Geobacillus, representing 41.4% of the isolates, was the most abundant. The highest amount of proteolytic and lipolytic enzymes was secreted by strains of the genus Geobacillus, whereas Caldibacillus species produced the highest amount of amylolytic enzyme. The Geobacillus species producing hydrolytic extracellular enzymes appeared to be the most promising.

1. Introduction

Extremophiles, including thermophilic, psychrophilic, acidophilic, alkaliphilic, and halophilic microorganisms, have been studied in various fields because of their capacity to survive and function under harsh environments [1]. Among the extremophiles, thermophilic microorganisms have been generally isolated from hot springs and the deep sea of the Earth. Thermophiles are divided into moderate thermophiles (optimum growth temperature 50–60 °C), extreme thermophiles (optimum growth temperature 60–80 °C), and hyperthermophiles (optimum growth temperature 80–110 °C) [2]. They are the reservoirs for production of industrially valuable thermostable enzymes that can be used for biotechnological processes at high temperatures [3,4,5,6].
Thermostable bacterial hydrolytic enzymes have been developed and used extensively in biological processes for production, meaning that thermostability makes them highly demandable for eco-friendly industrial purposes, and extensive manufacturing and usage of thermostable enzymes has resulted from continued industrial demand [7,8,9]. Among the commercially valuable enzymes, α-amylases, lipases, and proteases are the major enzymes produced by thermophiles [7,10]. Their importance in biotechnological applications is significant; for example, proteases and amylases are used together in the food industry, detergent industry, and pharmaceuticals [11]. Due to their increased importance, many scientists have focused on their research to discover new thermophilic microorganisms [12,13,14].
Republic of Korea’s Hot Spring Act defines a hot spring as groundwater with temperature of 25 °C or higher with qualified standards. It is difficult to find volcanic-oriented hot springs as the Korean peninsula is known to be distant from high enthalpy geothermal energy. The area of distribution of the hot springs coincides with areas of granite, and the heat is derived from the process of radioactive decay of the granite. Hot springs with temperatures ranging from 25 °C to 78 °C are found in different regions of Republic of Korea. The low-temperature hot springs demonstrate 85.4%. The physical and chemical characteristics of Republic of Korean hot springs are the low proportion of high temperature, low mineral contents, and alkaline pH [15,16,17]. The physiochemical features of hot spring water facilitate growth of thermophiles. Consequently, research on assessment of the possibility of industrial application by isolation of thermophiles from hot spring water in Republic of Korea is necessary. This study aims to develop a continuous approach for screening, isolating, and characterizing novel thermophilic microorganisms with great biotechnological and environmental potential.

2. Materials and Methods

2.1. Sample Collection

Five hot springs in Republic of Korea were chosen to collect the sample, including the Neungam carbonate, Mungang sulfur, Deokgu, Baegam, and Dongnae Yangtangjang. The samples were collected using a pump in a sterilized sampling bottle and immediately screened for bacterial isolation. Additionally, after screening, samples were stored at 4 °C for up to 5 days.

2.2. Isolation of Thermophilic Bacteria

The hot spring water samples were serially diluted using a sterile 0.85% saline buffer. An aliquot of each suspension was spread on marine broth 2216 (MB; BD Difco) supplemented with 1.5% (w/v) agar (MA) plates and incubated at 60 °C for five days. Morphologically different colonies were selected, and, after several transfers using the same solid medium, pure colonies were obtained. Obtained isolates were suspended in marine broth 2216 supplemented with 10% (v/v) dimethyl sulfoxide (DMSO) and stored at −80 °C for long-time preservation.

2.3. Taxonomic Identification of the Isolates

The isolated strains were identified by 16S rRNA gene sequencing. The 16S rRNA identification was performed by the BIOFACT Co., Ltd. (Daejeon, Republic of Korea) using ABI PRISM 3730XL DNA analyzer (Applied Biosystems, Foster City, CA, USA). Genomic DNA of the strains was isolated using Chelex 100 Boiling Resin (BIO-RAD, Hercules, CA, USA), pro-K, and S-Taq Buffer (BIOFACT, Daejeon, Republic of Korea), followed by PCR amplification of the 16S rRNA gene by Hush Run™ cycler (BIOFACT, Daejeon, Republic of Korea) using universal primers 27F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1492R (5′-GGT TAC CTT GTT ACG ACT T-3′). A 50 µL reaction mixture contained 5 U S-Taq DNA polymerase (BIOFACT, Daejeon, Republic of Korea), 10 mM dNTPs, and 10X S-Taq Reaction Buffer (BIOFACT, Daejeon, Republic of Korea). Identification based on the 16S rRNA sequence of the strains was analyzed using EzBioCloud Database (https://www.ezbiocloud.net/) accessed on 25 March 2022. Multiple sequence alignment was completed by the BioEdit program, and the phylogenetic tree was constructed using the Maximum Likelihood algorithm by the MEGA 6.0 program [18,19].

2.4. Determination of Growth Characteristics

The growth temperature range of the isolates was determined by cultivating at 45, 50, 55, 60, and 65 °C, respectively. The salt tolerance of the isolates was determined using MA medium supplemented with NaCl (w/v) concentration at 3, 6, 9, 12, and 15% incubating the plates at 60 °C. The pH tolerance was determined using MA medium with a pH range of 4.0, 7.0, and 9.0 adjusted with 1N NaOH and 1M HCl. The growth of the isolated bacteria at 60 °C on complex media was tested using nutrient agar (Difco, Sparks, MD, USA), R2A agar (Difco, Sparks, MD, USA), and tryptic soy agar (Difco, Sparks, MD, USA).

2.5. Determination of Hydrolytic Enzyme Activities

The hydrolytic enzyme activity of the isolated strains was tested on a marine agar medium supplemented with specific substrates. After several days of incubation at 60 °C, the enzyme activities were determined by the agar diffusion method. The amount of produced enzymes was determined as enzyme intensity (EI). EI was calculated as follows: (colony diameter + halo zone diameter)/colony diameter [20,21,22]. Each experiment was performed in triplicate.

2.5.1. Amylase Activity

The amylolytic property was identified using the starch hydrolysis method. The strains were inoculated onto marine agar supplemented with 0.2% (w/v) soluble starch (Difco, USA). After incubation for 3 days at 60 °C, agar plates were treated with 1% iodine solution for 2–3 min and presence of transparent zones around the colony indicated amylase activity [23].

2.5.2. Lipase Activity

Lipase activity was determined by the opaque zone around the colony as described by [24]. The strains were inoculated onto marine agar (Difco, Sparks, MD, USA) medium supplemented with 1% (v/v) Tween 80 (Sigma, St. Louis, MO, USA). Tween 80 was added separately after sterilization. After incubation at 60 °C for 3 days, lipase activity was evaluated by an opaque zone around the colony.

2.5.3. Protease Activity

Protease activity was detected by inoculating the strains onto 2% (w/v) skim milk agar (Difco, Sparks, MD, USA) medium. Skim milk was added separately after sterilization. After incubation at 60 °C for 3 days, protease activity was evaluated by clear zone around the colony [25].

2.6. Accession Numbers

The 16S rRNA gene sequences of strains were deposited to the GenBank/EMBL/DDBJ database.

2.7. Deposition of Strains

All strains isolated through this study were deposited in the Microbial Value Enhancement Project, Korea Research Institute of Bioscience and Biotechnology.

3. Results

3.1. Sample Characteristics

The samples from the five hot springs were collected in January 2017. The five hot spring sites were located in different cities in Republic of Korea (Figure 1), with a temperature range between 26 and 60 °C and pH 6.5–9.1 (Table 1).

3.2. Isolation and Growth Characteristics of Thermophilic Bacteria

Within the scope of this study, a total of 29 strains were isolated from hot spring water samples. The screening results showed that three strains were isolated from the Neungam Carbonate, sixteen strains from the Mungang Sulfur, two strains were isolated from the Deokgu, six strains were separated from the Baegam, and two strains from the Dongnae hot springs, respectively. All the strains showed growth on one of the complex media (nutrient agar, R2A, tryptic soy agar), which are widely used for massive cultivation of bacteria in industrial processing, meaning that newly isolated 29 thermophilic bacteria can be cultivated more easily using industrially applied commercial media. Growth of all the strains occurred between 45 °C and 65 °C, indicating that the isolated strains are thermophiles. All the strains showed growth at pH 7.0. On the other hand, any of the strains could grow at acidic (pH 4.0) and alkaline (pH 9.0) conditions. The halophilic nature of the strains showed that all the strains could tolerate 3% (w/v) NaCl concentration and 10 strains could tolerate 6% (w/v) NaCl concentration. Moreover, isolates EF60115, EF60123, and EF60155 could tolerate up to 9% (w/v) NaCl concentration. Any of the strains could tolerate up to 15% (w/v) NaCl condition (Table 2).

3.3. Taxonomic Identification Based on 16S rRNA Gene Sequencing and Phylogenetic Analysis

Taxonomic identification and phylogenetic analysis of the isolates were assessed by 16S rRNA sequencing. The complete 16S rRNA gene sequences of 29 isolates were successfully amplified. A pairwise comparison result of the 16S rRNA gene sequences on the EzBioCloud server showed that there were eight different types of species (Table 3 and Table 4). As shown in Table 3, the strains belong to phylum Bacillota, class Bacilli, and order Bacillales. Further, twenty-eight strains belong to Bacillaceae (96.6%) and one strain belongs to Thermoactinomycetaceae (3.4%). Among the Bacillaceae, four genera were confirmed with five strains of Aeribacillus (17.2%), one strain of Bacillus (3.4%), eleven strains of Caldibacillus (37.9%), and twelve strains of Geobacillus (41.4%). On the other hand, one strain of Thermoactinomyces was identified. Phylogenetic analysis was conducted to evaluate the relationships of the isolated strains with closely related type strains (Figure 2).

3.4. Production of Extracellular Hydrolytic Enzymes

Bacterial isolates were screened for extracellular amylase, protease, and lipase activity. Among the isolates, at least one extracellular enzyme activity was detected in 22 strains (75.9%). Amylase activity was detected in nineteen strains (65.5%), protease activity in ten strains (34.5%), and lipase activity in five strains (17.24%) (Figure 3). The degree of hydrolytic capacity for degrading a certain substrate was evaluated and shown as enzyme intensity (EI) to show which of the isolates produced larger amounts of the enzymes (Table 5). The amylolytic activity was the most common among isolates with the highest amylase activity in strains EF60154 (EI 4.02), EF60094 (EI 3.33), EF60111 (EI 3.16), and EF60034 (EI 3.1) and strains EF60165 (EI 3.67), EF60131 (EI 3.12), EF60136 (EI 3.09), and EF60061 (EI 3.08). The highest protease and lipase activity was detected in strains EF60192 (EI 10.71) and EF60131 (EI 3.46), respectively. Two strains, EF60083 and EF60192, produced all the tested hydrolytic enzymes.

4. Discussion

The region of hot springs that are habitats for thermophiles is limited to a restricted number of sites. In Korea, there are about 400 hot springs, many recognized as having therapeutic and medicinal effects. In these environmental conditions, living organisms have to cope with harsh temperatures and the low availability of nutritional compounds. Biodiversity in these environments is low, but some microorganisms can adapt to these conditions by developing survival strategies.
Bacterial strains isolated in this study are thermophiles that showed growth at relatively high temperatures. One of the important potentials of thermophiles is their productive capacity of thermostable enzymes. Applications of enzymes in industries are numerous; for example, hydrolases (amylases, lipases, and proteases, etc.) account for over 75% of all enzymes produced on a commercial scale. These thermostable hydrolytic enzymes can be extracted from thermophiles because they have many desirable properties, including the capacity to reduce the probability of microbial contamination in large-scale fermentation and the capacity to function for extended periods of time [26,27]. This reason leads to intensive and extended studies to perform fully for investigating such promising microorganisms. The majority (75.8%) of the thermophilic strains produced at least one extracellular hydrolase (Table 4). The amylolytic activity was the most common in isolates with 19 (65.5%) strains. The highest amylase activity was produced by isolate EF60154 from the Baegam hot spring, which was identified as Caldibacillus hisashii. Proteolytic activity was detected in 10 (34.5%) strains, with the highest protease activity in isolates EF60192 and EF60165, which were identified as Geobacillus proteiniphilus. Lipolytic activity was less common among isolates with 5 (17.2%) strains, producing very small amounts. The highest lipase activity was detected in the EF60131 strain, which was identified as Geobacillus lituanicus. Isolates EF60192 and EF60083 that were identified as Geobacillus proteiniphilus and Geobacillus stearothermophilus, respectively, produced all the tested enzymes. Among the isolates, eight strains could produce two extracellular hydrolases. On the other hand, seven strains did not show any enzyme activity. There are numerous biotechnological uses for hydrolases; each one necessitates certain enzyme properties in terms of specificity and thermostability [28]. There may be differences in enzyme production depending on the environmental factors and chemical characteristics of the water samples [29]. Most of these promising thermophilic bacterial isolates belong to the genus Geobacillus. Adaptation of Geobacillus spores to environmental stress and meeting their basic nutritional requirements for growth may be the reason for the occurrence of these species in all five hot springs [30].
The results of this study will achieve great significance in terms of securing the diversity of microbiological resources in Korea, and it is expected that a new strain can be found for production of hydrolytic enzymes in water samples around domestic hot spring regions and basic biological materials for research on bioengineering in food and cosmetics industries.

Author Contributions

Conceptualization, Y.-J.L., S.-B.K. and S.-J.L.; methodology, Y.-J.L. and S.-J.L.; software, D.G.; validation, S.-B.K. and G.N.; formal analysis, H.K.; investigation, Y.-J.L., D.G., D.O., G.E.J. and S.-J.L.; resources, D.G., D.O., G.E.J., H.K. and S.-J.L.; data curation, Y.-J.L., D.G., I.-T.C., Y.-J.J., G.N. and S.-J.L.; writing—original draft preparation, Y.-J.L., D.G. and S.-J.L.; writing—review and editing, Y.-J.L., S.-B.K., G.N., I.-T.C., Y.-J.J. and S.-J.L.; visualization, Y.-J.L. and D.G.; supervision, S.-J.L.; project administration, S.-J.L.; funding acquisition, Y.-J.L., S.-B.K., I.-T.C. and S.-J.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Institute of Biological Resources funded by the Ministry of Environment (NIBR202102109), Basic Research of the NRF (NRF-2020R1F1A1076624, NRF-2021R1F1A1064036), grant from the Ministry of Ocean and Fisheries (PM62830), and grant from the Technology Innovation Program (20015807) funded by the Ministry of Trade, Industry & Energy.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no potential conflicts of interest.

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Figure 1. Map of the hot spring area in Korea where the samples were collected for this study. Neungam and Mungang hot springs are located in Chungju, Deokgu and Baegam hot springs are located in Uljin, and Dongnae (Yangtangjang) hot spring is located in Busan, respectively.
Figure 1. Map of the hot spring area in Korea where the samples were collected for this study. Neungam and Mungang hot springs are located in Chungju, Deokgu and Baegam hot springs are located in Uljin, and Dongnae (Yangtangjang) hot spring is located in Busan, respectively.
Microorganisms 10 02375 g001
Figure 2. Phylogenetic analysis of 29 isolates and their closely related type strains. The phylogenetic tree was constructed based on 16S rRNA gene sequence alignments using the Maximum Likelihood method in MEGA 6.0. Bootstrap values are indicated at the branch points. The scale bar indicates a branch length equivalent to 0.02 changes per nucleotide. Alicyclobacillus acidocaldarius ATCC 27009T and Paenibacillus alginolyticus DSM 5050T served as outgroup.
Figure 2. Phylogenetic analysis of 29 isolates and their closely related type strains. The phylogenetic tree was constructed based on 16S rRNA gene sequence alignments using the Maximum Likelihood method in MEGA 6.0. Bootstrap values are indicated at the branch points. The scale bar indicates a branch length equivalent to 0.02 changes per nucleotide. Alicyclobacillus acidocaldarius ATCC 27009T and Paenibacillus alginolyticus DSM 5050T served as outgroup.
Microorganisms 10 02375 g002
Figure 3. Production of extracellular hydrolase among isolates.
Figure 3. Production of extracellular hydrolase among isolates.
Microorganisms 10 02375 g003
Table 1. Hot spring properties.
Table 1. Hot spring properties.
Hot SpringTemperature (°C)pHChemical CompositionLocation (GPS)
Neungam Carbonate26.2–27.36.5–6.61Carbonate (Na, K, Ca, Mg, Li, Sr, HCO3, CO2, Cl, SO42−, F, dissolved SiO2, Fe, Mn, Zn, Cu, Pb, Al, S)37°05′36.5″ N 127°48′06.6″ E
Mungang Sulfur26.4–28.38.94–9.11Na, Ca, K, SO42−, Cl, H₂S, HCO3, SiO4, Zn, CO3, F, free H₂CO336°53′08.0″ N 127°57′13.9″ E
Deokgu42.38.49K, Na, Ca, Mg, Cl, SO4, CO3, SiO2, F, Li, Sr, Zn, Al, Na-HCO337°04′43.6″ N 129°17′03.4″ E
Baegam45.98.93K, Na, Ca, Mg, SiO2, Li, Sr, Fe, Na-CO336°43′15.0″ N 129°20′22.2″ E
Dongnae59.47.6Na, K, Ca, Mg, Li, Sr, O3, Free CO2, CI, SO4, F, SiO2, Fe, Mn, Zn, Cu, Pb, Al35°13′15.9″ N 129°04′51.5″ E
Table 2. Growth characteristics of aerobically cultured thermophilic bacteria isolated from the diverse hot springs in Republic of Korea.
Table 2. Growth characteristics of aerobically cultured thermophilic bacteria isolated from the diverse hot springs in Republic of Korea.
NoSourceIsolate
Number
Growth Characteristics
MediaNaCl %pHTemperature
NAR2ATSA36912154.07.09.045 °C50 °C55 °C60 °C65 °C
1Neungam Carbonate Hot springEF60032++++++++++
2EF60034+++++++++++
3EF60041+++++++++
4Mungang Sulfur
Hot spring
EF60061++++++++++
5EF60062+++++++++
6EF60065++++++++++
7EF60083++++++++++
8EF60091+++++++++
9EF60094+++++++++++
10EF60095+++++++
11EF60103+++++++++++
12EF60106+++++++++++
13EF60111+++++++++++
14EF60113+++++++++
15EF60115++++++++++++
16EF60123++++++++++++
17EF60131++++++++++
18EF60135++++++++++
19EF60136++++++++++
20Deokgu Hot springEF60141++++++++++
21EF60144+++++++++
22Baegam Hot springEF60151++++++++++
23EF60153+++++++++++
24EF60154+++++++++++
25EF60155++++++++++++
26EF60162+++++++
27EF60165+++++
28Dongnae Hot springEF60192+++++++++
29EF60197++++++++
NA: nutrient agar, MA: marine agar Difco 2216, TSA: tryptic soy agar, +: well–growth, –: no growth.
Table 3. Phylum analysis of aerobically cultured thermophilic bacteria isolated from the diverse hot springs in Republic of Korea.
Table 3. Phylum analysis of aerobically cultured thermophilic bacteria isolated from the diverse hot springs in Republic of Korea.
PhylumClassOrderFamilyGenusClosest SpeciesStrains
BacillotaBacilliBacillalesBacillaceaeAeribacillusAeribacillus pallidus5
BacillusBacillus licheniformis1
CaldibacillusCaldibacillus debilis4
Caldibacillus hisashii6
GeobacillusGeobacillus lituanicus4
Geobacillus proteiniphilus3
Geobacillus stearothermophilus5
ThermoactinomycetaceaeThermoactinomycesThermoactinomyces vulgaris1
Table 4. Taxonomic identification of the isolates based on 16S rRNA gene sequences.
Table 4. Taxonomic identification of the isolates based on 16S rRNA gene sequences.
NoIsolate
Number
Closest StrainClosest Strain NumberSimilarity (%)Deposited NumberGenBank Accession Number
1EF60032Geobacillus stearothermophilusNBRC 1255098.81NMC4-B346ON365874
2EF60034Caldibacillus hisashiiN-1199.79NMC4-B347ON365877
3EF60041Caldibacillus debilisTf99.79NMC4-B348ON365879
4EF60061Geobacillus lituanicusN-399.38NMC4-B352ON365885
5EF60062Caldibacillus hisashiiN-1199.93NMC4-B353ON365925
6EF60065Geobacillus stearothermophilusNBRC 1255099.3NMC4-B355ON365932
7EF60083Geobacillus stearothermophilusNBRC 1255099.49NMC4-B356ON365961
8EF60091Geobacillus lituanicusN-399.72NMC4-B357ON366319
9EF60094Caldibacillus hisashiiN-1199.86NMC4-B359ON366407
10EF60095Caldibacillus debilisTf99.79NMC4-B360ON366406
11EF60103Aeribacillus pallidusKCTC 3564100NMC4-B361ON366400
12EF60106Aeribacillus pallidusKCTC 356499.86NMC4-B362ON430523
13EF60111Caldibacillus hisashiiN-1199.86NMC4-B363ON430527
14EF60113Caldibacillus debilisTf99.79NMC4-B364ON430529
15EF60115Aeribacillus pallidusKCTC 356498.71NMC4-B365MW132409
16EF60123Aeribacillus pallidusKCTC 356499.86NMC4-B366ON430531
17EF60131Geobacillus lituanicusN-399.65NMC4-B367MW131318
18EF60135Bacillus licheniformisATCC 1458099.73NMC4-B370ON430534
19EF60136Geobacillus lituanicusN-399.39NMC4-B371ON430536
20EF60141Caldibacillus debilisTf99.66NMC4-B372MW131319
21EF60144Geobacillus stearothermophilusNBRC 1255099.39NMC4-B373MW131115
22EF60151Geobacillus stearothermophilusNBRC 1255099.02NMC4-B374ON430581
23EF60153Caldibacillus hisashiiN-1199.79NMC4-B375ON430584
24EF60154Caldibacillus hisashiiN-1199.79NMC4-B376ON430575
25EF60155Aeribacillus pallidusKCTC 356499.86NMC4-B377ON430576
26EF60162Geobacillus proteiniphilus101798.8NMC4-B378MW131211
27EF60165Geobacillus proteiniphilus101799.73NMC4-B380ON430578
28EF60192Geobacillus proteiniphilus101798.71NMC4-B381ON430582
29EF60197Thermoactinomyces vulgarisKCTC 907699.66NMC4-B382MW131315
Culture collection numbers: Caldibacillus hisashii N-11T (NBRC 110226); Caldibacillus debilis TfT (DSM 16016); Geobacillus lituanicus N-3T (DSM 15325); Geobacillus proteiniphilus 1017T (KCTC 33986).
Table 5. Amount of extracellular hydrolases produced by thermophilic isolates expressed as EI.
Table 5. Amount of extracellular hydrolases produced by thermophilic isolates expressed as EI.
NoIsolate
Number
Enzyme ActivityEnzyme Intensity (EI)
AmylaseLipaseProteaseAmylaseLipaseProtease
1EF60032+2.33 ± 0.06
2EF60034+3.1 ± 0.15
3EF60041
4EF60061++3.08 ± 0.1 4.65 ± 0.55
5EF60062+2.92 ± 0.17
6EF60065++2.32 ± 0.092.22 ± 0.02
7EF60083+++2.26 ± 0.072.12 ± 0.022.18 ± 0.02
8EF60091+2.92 ± 0.22
9EF60094+3.33 ± 0.17
10EF60095
11EF60103
12EF60106
13EF60111+3.16 ± 0.18
14EF60113+ 2.69 ± 0.38
15EF60115
16EF60123
17EF60131++3.12 ± 0.323.46 ± 0.23
18EF60135++2.15 ± 0.062.25 ± 0.1
19EF60136++3.09 ± 0.07 4.46 ± 0.66
20EF60141+ 2.18 ± 0.06
21EF60144++2.16 ± 0.06 3.65 ± 0.47
22EF60151+2.2 ± 0.08
23EF60153+3.1 ± 0.08
24EF60154+4.02 ± 0.34
25EF60155
26EF60162++2.59 ± 0.34 3.3 ± 0.48
27EF60165++3.67 ± 0.45 7.88 ± 1.07
28EF60192+++2.54 ± 0.152.92 ± 0.1710.71 ± 2.65
29EF60197+ 2.51 ± 0.05
+: enzyme activity, –: no activity.
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Lee, Y.-J.; Ganbat, D.; Oh, D.; Kim, H.; Jeong, G.E.; Cha, I.-T.; Kim, S.-B.; Nam, G.; Jung, Y.-J.; Lee, S.-J. Isolation and Characterization of Thermophilic Bacteria from Hot Springs in Republic of Korea. Microorganisms 2022, 10, 2375. https://doi.org/10.3390/microorganisms10122375

AMA Style

Lee Y-J, Ganbat D, Oh D, Kim H, Jeong GE, Cha I-T, Kim S-B, Nam G, Jung Y-J, Lee S-J. Isolation and Characterization of Thermophilic Bacteria from Hot Springs in Republic of Korea. Microorganisms. 2022; 10(12):2375. https://doi.org/10.3390/microorganisms10122375

Chicago/Turabian Style

Lee, Yong-Jik, Dariimaa Ganbat, DoKyung Oh, HyeWon Kim, Ga Eul Jeong, In-Tae Cha, Seong-Bo Kim, Gaewon Nam, You-Jung Jung, and Sang-Jae Lee. 2022. "Isolation and Characterization of Thermophilic Bacteria from Hot Springs in Republic of Korea" Microorganisms 10, no. 12: 2375. https://doi.org/10.3390/microorganisms10122375

APA Style

Lee, Y.-J., Ganbat, D., Oh, D., Kim, H., Jeong, G. E., Cha, I.-T., Kim, S.-B., Nam, G., Jung, Y.-J., & Lee, S.-J. (2022). Isolation and Characterization of Thermophilic Bacteria from Hot Springs in Republic of Korea. Microorganisms, 10(12), 2375. https://doi.org/10.3390/microorganisms10122375

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