Thermophilic Bacterial Isolates Research Articles

Characterization of thermophilic bacteria from Ie Seum Hot Springs, Aceh Besar, Indonesia as producers of protease enzyme

Abstract. Sabaria E, Yasmin Y, Ismail YS, Bessania MA, Putri I, Fitri L. 2024. Characterization of thermophilic bacteria from Ie Seum Hot Springs, Aceh Besar, Indonesia as producers of protease enzyme. Biodiversitas 25: 1867-1874. Thermophilic bacteria are microorganisms that can survive in high-temperature environments exceeding 75°C. The Ie Seum Hot Springs in Aceh Besar, Indonesia are known to be a place where these thermophilic bacteria inhabit. In such extreme conditions, proteins and enzymes are often denatured, making these bacteria of interest due to their ability to adapt and produce enzymes such as proteases. Proteases are called proteolytic enzymes that can be applied both in the economic and medical fields. This study aimed to isolate and identify thermophilic bacteria species that have the highest potential for protease production in Ie Seum Hot Spring, Aceh Besar by analyzing the 16S rRNA gene and characterizing them using biochemical tests. Based on the result, seven thermophilic bacterial isolates, namely BT1, BT2, BT3, BT4, BT5, BT6, and BT7 were obtained, each showing different colony characteristics. The biochemical tests also revealed differences metabolic activity of each isolate. Of these isolates, BT4 exhibited the highest proteolytic index (4.65). Additionally, the 16S rRNA gene sequence analysis showed that the BT4 strain belongs to the genus Bacillus and has high similarities to Bacillus sp., B. licheniformis and B. sonorensis. These findings suggest that BT4 has significant potential as a source of thermophilic protease enzymes.

Analysis of Amylase Activity in Bacteria Isolated from Hot Spring of Pentadio Resort

The hot spring of Pentadio Resort is one of the geothermal areas likely to be the place of bacteria or microorganisms that can produce amylolytic enzymes. This research aims to analyze bacteria in the Pentadio Resort hot springs that possess the potential to produce amylase enzymes, analyze the characteristics of bacterial isolates that produce amylase enzymes in Pentadio Resort hot springs and determine the types of bacteria that produce amylase enzymes in Pentadio Resort hot springs. The research employs a qualitative and descriptive method. The research involved several methods, including sample collection, media preparation, isolation and selection of thermophilic bacterial isolates, purification of thermophilic bacterial isolates, selection of isolates producing amylase enzymes, identification of selected isolates (motility test, biochemical test, gram staining), and molecular identification. The research yielded seven isolates, and isolate code A6 demonstrated the potential to produce amylase enzymes with a clear zone size of 23,26 mm, bacillus, gram positive, and positive motility. Subsequently, molecular identifidation was carried out in which, based on the results of phylogenetic tree reconstractiopisalate A6 was related to the bacteria Bacillus cereus strain MD 152, which belongs to the Bacillus cereus bacteria

Optimization and Molecular Identification of Protease-Producing Thermophilic Bacterial Isolate TUA-26

Optimization and Molecular Identification of Protease-Producing Thermophilic Bacterial Isolate TUA-26

Enhanced Amylase Activity by Modulating Abiotic Factors and Enzyme Stability in the Thermophilic Bacterial Isolates

Enhanced Amylase Activity by Modulating Abiotic Factors and Enzyme Stability in the Thermophilic Bacterial Isolates

The Potential of Hot Water Mudiak Sapan Thermophilic Bactery Consortium Formulation in Producing Xylanase Enzyme

The xylanase enzyme has high commercial value in the industrial sector, including the food industry, animal feed, bleaching of pulp/pulp, lignocellulosic bioconversion as fuel, and in the food industry, namely the cheese, bread and meat industries, while in the non-food industry this can be used in detergents. Xylanase enzymes can be produced from microorganisms, one of which is thermophilic bacteria. Thermophilic bacteria are known to be able to produce thermostable enzymes and proteins that are heat resistant. Xylanase production in compatible bacterial consortia was higher than monoculture. The purpose of this study was to determine the bicultur consortium that has the potential to produce xylanase enzymes. This research is an experimental research. Enzyme activity testing used the Miller method with DNS (Dinitrosalycilic acid) reagent using a completely randomized design (CRD) with 3 repetitions. Data on the results of xylanase activity were analyzed by ANOVA test and DMRT follow-up test with a 5% significance level.The results of the thermophilic bacterial isolates in the consortium that have the potential to produce xylanase enzymes are MS18 & MSS15, MSS15, MS16 and MS18 & MS16.

Compatibility Test Consorcium of Thermophilic Bacteria Producing Xylanase Enzym from The Hot Water of Mudiak Sapan (MS18, MSS15, MSS11, MS16)

Mudiak Sapan hot spring, South Solok Regency, West Sumatra is one of the hot springs where there are various species of thermophilic bacteria that produce xylanase enzymes. The xylanase enzyme is a group of enzymes that has the ability to hydrolyze xylan (hemicellulose) or polymers into xylose and xylo-oligosaccharides. The purpose of this study was to test the compatibility of bacterial isolates to become a xylanase-producing bacterial consortium. This research is a descriptive research that informs the compatibility of thermophilic bacterial isolates from the Mudiak Sapan hot springs. The method used in this study was disk diffusion, based on the research, it was found that 5 combinations of compatible xylanase enzyme-producing isolates were MS18 x MSS11; MS18 x MSS15; MSS18 x MS16 ; MSS11 x MS16 ; MSS15 x MS16 and 1 combination of incompatible isolates namely MSS11 x MSS15 which had an inhibition zone of 0.645 cm.

Isolation and optimization of thermophilic lipase producing bacteria from soil contaminated with used engines oil

Thirty two of thermophilic bacterial isolates producing lipase were selectively isolated from soil samples contaminated with used engines oil using Tributyrin agar medium. The qualitative assay of lipase activity was done on Tributyrin agar plates by measuring diameter of the clear zone. The isolates 6T, 14T and 29T of the isolated strains exhibited a greater zone of clearance than the others, indicating higher lipase activity. Secondary screening for lipase production was done using liquid basal medium with 1% (w/v) olive oil and pH 7. Results from quantitative assay showed that 6T, 14T and 29T isolates appeared highest ability to produce lipase enzyme 4 U/ml, 10 U/ml and 7 U/mlrespectively. Therefore, the 14T isolate was selected and identified based on their morphological and physiological characteristics which was subsequently identified and designated as Bacillus licheniformis. The effect of incubation time, temperature and pH influencing lipase production from Bacillus licheniformis14T isolate was determine. Results showed that the maximal lipase production was 11 U\ml after 3 day of incubation at medium pH7 and temperature 50 0C.

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

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.

Isolation and Molecular Profiling Of Thermoresistant Bacteria From Chimneys Of Baking Industries Of Madurai, Tamilnadu, India

The present study was chosen to discover the thermophiles isolation from chimneys of baking industries o Madurai, Tamilnadu, India. An attempt has been made to unearth the conserved sequences among them. Phylogenetic neighbour joining tree based on their 16S rRNA showing a relationship with related organisms has been constructed for each isolates keeping a species as an outgroup. In Madurai, baking industries have been found to be one of the hottest area having temperature as high as 120°C. Incubations of the bacterial colonies were done with covered water bath incubator at 70°C for 72-96 hrs in Castenholz TYE broth. The temperature range of 40-100°C, pH range of 4.0-10.0 and incubation period of 24-144 hrs was investigated for the best growth of thermophilic bacteria. Samples were collected in sterilized screw capped vials from different sites of Madurai Baking Industries and brought to the laboratory and kept at 4°C in the refrigerator till further processing. PCR products were sequenced using same primers by a commercial sequencing facility (Xcleris lab). The sequences of these ten bacterial isolates after sequencing were blasted using online NCBI BLAST program. Ten thermotolerant bacteria isolates were isolated and the optimal temperature for growth of the isolates was 70°C and the optimal pH was 7.0. 16S rRNA gene sequences of all these thermophilic bacterial isolates exhibited 98-99% homology with Bacillus sp. The phylogenetic analysis of the 16S rRNA gene sequences also confirmed the relationship of these thermophilic isolates with the genus Bacillus The 16S rRNA gene of all isolates was amplified, sequenced, and insilico analysis of these ten sequences was carried out. Based on BLAST alignment of 16S rRNA gene sequences of these isolates to GenBank sequences, these all isolates belonged to genus Bacillus and showed 98-99% similarity. The present study delves in to isolation, identification, characterisation,and analysis o the genomes of bacterial isolates such as Bacillus sp. VP1, Bacillus sp. VP2, Bacillus sp. VP3,Pseudomonas sp. VP4, Pseudomonas sp VP5,Bacillus licheniformis strain VP7,Bacillus sp. VP8, Bacillus sp. VP9, Bacillus altitudinis sp. VP10, and Bacillus cereus strain VP11.

Molecular Identification, GC-MS Analysis of Bioactive Compounds and Antimicrobial Activity of Thermophilic Bacteria Derived from West Sumatra Hot-Spring Indonesia

Thermophilic bacteria are a source of bioactive compounds that have many benefits for human life. One of them is as a source of antimicrobials. This research aimed to identify and characterize the promising thermophilic bacterial isolates by analyzing bioactive compounds and their potential as antimicrobial agents. Thermophilic bacteria with the code LBKURCC were taken from the collection of the Biochemistry Laboratory of the University of Riau. Forty-four purified strains of thermophilic bacteria were tested for antimicrobial ability. These thermophilic bacteria were taken from hot springs located in the Sumatra provinces of West Sumatra and Riau. Strain LBKURCC218 isolated from Rimbo Panti hot springs in West Sumatra was chosen to further investigate antimicrobials production. Isolates of hot spring bacteria that produced the highest antimicrobial were identified by comparing the similarity of the 16S rRNA gene sequences. BLAST result and phylogenetic tree showed that the selected thermophilic bacterial strain was similar to Bacillus paramicoides with the similarity index of 99.93%. Analysis of bioactive compounds of the ethyl acetate extract of liquid cultures of B. paramycoides LBKURCC218 showed the best producer of antimicrobial compounds compared to other isolates. The most identified compounds from the ethyl acetate extract were Dodecanoic acid, representing 23.62% of the total compounds, followed by 11-Dodecanoic acid at 17.84%. Ethyl acetate extract of B. paramycoides LBKURCC218 has a high inhibition zone against Escherichia coli, Staphylococcus aureus, and Candida albicans.

Effect of Vitamin D3 Supplement on the Semen Quality in Human Patients with Vitamin D Deficiency

Thermophilic bacteria are a source of bioactive compounds that have many benefits for human life. One of them is as a source of antimicrobials. This research aimed to identify and characterize the promising thermophilic bacterial isolates by analyzing bioactive compounds and their potential as antimicrobial agents. Thermophilic bacteria with the code LBKURCC were taken from the collection of the Biochemistry Laboratory of the University of Riau. Forty-four purified strains of thermophilic bacteria were tested for antimicrobial ability. These thermophilic bacteria were taken from hot springs located in the Sumatra provinces of West Sumatra and Riau. Strain LBKURCC218 isolated from Rimbo Panti hot springs in West Sumatra was chosen to further investigate antimicrobials production. Isolates of hot spring bacteria that produced the highest antimicrobial were identified by comparing the similarity of the 16S rRNA gene sequences. BLAST result and phylogenetic tree showed that the selected thermophilic bacterial strain was similar to Bacillus paramicoides with the similarity index of 99.93%. Analysis of bioactive compounds of the ethyl acetate extract of liquid cultures of B. paramycoides LBKURCC218 showed the best producer of antimicrobial compounds compared to other isolates. The most identified compounds from the ethyl acetate extract were Dodecanoic acid, representing 23.62% of the total compounds, followed by 11-Dodecanoic acid at 17.84%. Ethyl acetate extract of B. paramycoides LBKURCC218 has a high inhibition zone against Escherichia coli, Staphylococcus aureus, and Candida albicans.

Isolation and Enzymatic Activity of Thermo-tolerant Bacteria from Waste Dumpsites in Umudikeand Environs

The research studied the isolation of thermo-tolerant bacteria with enzymatic activity from waste dumpsites. 30 soil samples were collected using sterile spatular into sterile universal bottles, labelled and taken to the laboratory analyses. The soil samples were serially diluted and inoculated by spread plate method on different media and incubated at 30oC for 48 hrs. The isolates were observed for colonial morphologies and later sub-cultured to get pure isolates which were stored in agar slants and kept in the refrigerator for further use. The isolates were Gram stained and also subjected to biochemical and sugar fermentation tests. The isolates were then cultured at thermophilic temperature and those with positive results were screened for the production of amylase, protease and lipase activity. The effect of temperature, pH and nitrogen sources on enzyme activity of the thermophilic bacterial isolates was also assessed. Results show that Bacillus subtilis (70.0%), Bacillus licheniformis (43.3%), Bacillus cereus (56.7%), Pseudomonas aeruginosa (56.7%), Streptomyces species (36.7%), Bacillus brevis (43.3%)and Norcadia species (30%) were recovered from the soil samples. All the bacterial isolates tolerated 55oC of temperature while only Bacillus cereus, Bacillus brevis and Norcadia species were isolated at 60oC. All the isolates except Pseudomonas aeruginosa gave amylase activity while all the isolates gave protease activity. All the isolates except Bacillus licheniformis and Nocardia species had lipase activity. Temperature and pH had varied effects on the enzyme activity of the isolates. The potentials of these bacterial isolates as good sources of enzymes at elevated temperatures, varied pH and nitrogenous sources for commercial uses was recognized.

SKRINING BAKTERI TERMOFILIK POTENSIAL AMILOLITIK DARI SUMBER AIR PANAS WAY BELERANG KALIANDA LAMPUNG SELATAN

Thermophilic bacteria that produced amylase and protease have been isolated from Way Belerang hot spring, Kalianda, South Lampung. This research aims to screen and identify thermophilic bacteria that have the potential to produce thermostable amylase and protease enzymes.The research procedures included sampling, isolation of enzyme-producing thermophilic bacteria, a series of phenotypic and biochemical tests, and molecular identification by 16s rRNA. This study used 2 treatments, namely incubation temperature 37 and 50 ºC with 3 repetitions. The results showed that the optimum temperature for growth of thermophilic bacterial isolates and thermophilic bacterial isolates producing amylase enzymes was 50ºC. The bacteria isolate that had the best amylolytic enzyme activity was Isolate A.WB.50.1 with a diameter of the inhibitory zone was 15.44 mm. Isolate A.WB.50.1 has been identified by the species Pseudomonas stutzeri.

Production of Antimicrobial Compounds using Thermophilic Bacteria Species Bacillus subtilis and Bacillus Tequilensis

Infections caused by resistant bacteria are a growing public health problem, consequently, a new source of microorganisms that can be used for antimicrobial production is needed. One of the microorganisms capable of producing antimicrobials is the thermophilic bacteria, namely B. subtilis and B. tequilensis. Due to having hot temperature-resistant enzymes, they are not easily damaged. Therefore, this study aims to produce new antimicrobials from B.subtilis and B.tequilensis. The antimicrobial activity was observed in 5 thermophilic bacterial isolates using the disk diffusion method. The results showed the strongest zone of inhibition (disk diameter = 6mm) or antimicrobial activity against S.aureus which was classified as gram-positive was discovered in B.subtilis UTMP15 (10.27 mm) at the incubation time of 24 hours, and against E.coli classified as gram-negative,it was in B. Subtilis UTMP12 (8.59 mm) at 48 hours. Hence, the isolate is a potential antimicrobial agent.

Comparative growth potential of thermophilic amylolytic Bacillus sp. on unconventional media food wastes and its industrial application.

Amylases take part with vital role in industries such as food, fermentation; starch processing, textile and paper etc. Increasing amylases demand, high nutrient expenditure and environmental pollution have forced to utilize agro-industrial residues as a low-cost feedstock for enzyme production. In present study, three soil samples were collected from agro-industrial waste dumping areas in District Faisalabad. Ten thermophilic bacterial isolates were separated at 55 °C on the basis of colonial morphology, three isolates (F6, F11, F17) showed prominent zone of clearance applying iodine test on starch agar plates. Bacterial isolate F-11 showed highest amylase activity with DNS method and molecularly identified through 16S RNA sequencing as Bacillus sp. with Accession number MH917294. Four unconventional food wastes (banana, lemon, mango and potato) pretreated with 0.8% sulphuric acid concentrations taking 1000 g/L weight released the highest sugars contents and phenolic components. Maximum amylase activity i.e. 29.23 mg/ml was achieved in mango waste at, 40 °C, with pH 6.0 and 0.17% nitrogenous source adding 8% inoculum size (2 days old) using Response Surface Methodology (RSM) for optimization. Crude amylase confirmed its efficiency in starch hydrolysis that suggested it as potential candidate for application in starch industries.

Biological degradation of rice straw with thermophilic lignocellulolytic bacterial isolates and biogas production from total broth by rumen microorganisms

Rice straw, a common agricultural residue, is abundant in Thailand and can be converted to biogas to reduce dependence on fossil resources for energy. Biological degradation of rice straw followed by anaerobic digestion (AD) for biomethane production (BMP) was investigated. Thermophilic lignocellulolytic bacteria for biological degradation were isolated from soil samples collected from Dong Han and Kok Soong National Forest around Roi-Et Rajabhat University, Roi-Et Province, Thailand. Cellulase and ligninolytic enzymes were assayed using standard microbiological methods. Isolate(s) with high cellulolytic and ligninolytic activities were then used to degrade rice straw in basal medium at 50 ± 2 °C for 5, 10, 15, and 20 days. Total broth was anaerobically digested with rumen microorganisms in 1.2-m3 PP digester at 39 ± 0.5 °C for 30 days. A total of 106 thermophilic bacterial isolates were sampled from soil, out of which 96 (90.6%) and 16 (15.1%) isolates tested positive for cellulolytic and ligninolytic activities, respectively. Unlike laccase and manganese peroxidase (MnP), lignin peroxidase (LiP) activity was not detected. Isolate RUFR60 showed both high cellulolytic and ligninolytic activities. Highest biogas and methane (CH4) yields (318.15 and 177.93 mL/gTS, respectively) were recorded from 20 days degraded rice straw which were not significantly (p > 0.05) different from those (315.10 and 171.72 mL/gTS, respectively) recorded from 15 days degraded rice straw. Optimal degradation period of 15 days with thermophilic lignocellulolytic bacteria was efficient and economical for BMP from rice straw.

Adaptive Enrichment of a Thermophilic Bacterial Isolate for Enhanced Enzymatic Activity.

The mimicking of evolution on a laboratory timescale to enhance biocatalyst specificity, substrate utilization activity, and/or product formation, is an effective and well-established approach that does not involve genetic engineering or regulatory details of the microorganism. The present work employed an evolutionary adaptive approach to improve the lignocellulose deconstruction capabilities of the strain by inducing the expression of laccase, a multicopper oxidase, in Geobacillus sp. strain WSUCF1. This bacterium is highly efficient in depolymerizing unprocessed lignocellulose, needing no preprocessing/pretreatment of the biomasses. However, it natively produces low levels of laccase. After 15 rounds of serially adapting this thermophilic strain in the presence of unprocessed corn stover as the selective pressure, we recorded a 20-fold increase in catalytic laccase activity, at 9.23 ± 0.6 U/mL, in an adapted yet stable strain of Geobacillus sp. WSUCF1, compared with the initial laccase production (0.46 ± 0.04 U/mL) obtained with the unadapted strain grown on unprocessed corn stover before optimization. Chemical composition analysis demonstrated that lignin removal by the adapted strain was 22 wt.% compared with 6 wt.% removal by the unadapted strain. These results signify a favorable prospect for fast, cost competitive bulk production of this thermostable enzyme. Also, this work has practical importance, as this fast adaptation of the Geobacillus sp. strain WSUCF1 suggests the possibility of growing industrial quantities of Geobacillus sp. strain WSUCF1 cells as biocatalysts on reasonably inexpensive carbon sources for commercial use. This work is the first application of the adaptive laboratory evolution approach for developing the desired phenotype of enhanced ligninolytic capability in any microbial strain.

Discovery of thermophilic Bacillales using reduced-representation genotyping for identification

BackgroundThis study demonstrates the use of reduced-representation genotyping to provide preliminary identifications for thermophilic bacterial isolates. The approach combines restriction enzyme digestion and PCR with next-generation sequencing to provide thousands of short-read sequences from across the bacterial genomes. Isolates were obtained from compost, hot water systems, and artesian bores of the Great Artesian Basin. Genomic DNA was double-digested with two combinations of restriction enzymes followed by PCR amplification, using a commercial provider of DArTseq™, Diversity Arrays Technology Pty Ltd. (Canberra, Australia). The resulting fragments which formed a reduced-representation of approximately 2.3% of the genome were sequenced. The sequence tags obtained were aligned against all available RefSeq bacterial genome assemblies by BLASTn to identify the nearest reference genome.ResultsBased on the preliminary identifications, a total of 99 bacterial isolates were identified to species level, from which 8 isolates were selected for whole-genome sequencing to assess the identification results. Novel species and strains were discovered within this set of isolates. The preliminary identifications obtained by reduced-representation genotyping, as well as identifications obtained by BLASTn alignment of the 16S rRNA gene sequence, were compared with those derived from the whole-genome sequence data, using the same RefSeq sequence database for the three methods. Identifications obtained with reduced-representation sequencing agreed with the identifications provided by whole-genome sequencing in 100% of cases. The identifications produced by BLASTn alignment of 16S rRNA gene sequence to the same database differed from those provided by whole-genome sequencing in 37.5% of cases, and produced ambiguous identifications in 50% of cases.ConclusionsPreviously, this method has been successfully demonstrated for use in bacterial identification for medical microbiology. This study demonstrates the first successful use of DArTseq™ for preliminary identification of thermophilic bacterial isolates, providing results in complete agreement with those obtained from whole-genome sequencing of the same isolates. The growing database of bacterial genome sequences provides an excellent resource for alignment of reduced-representation sequence data for identification purposes, and as the available sequenced genomes continue to grow, the technique will become more effective.

Optimizing the Reduction of Molybdate by Two Novel Thermophilic Bacilli Isolated from Sinai, Egypt.

The aim of this study was to isolate thermophilic bacteria to be used in in situ bioremediation of molybdate at elevated temperatures. Two molybdate reducing bacteria (Pharon2 and Pharon3) were isolated from the hot Spring at Pharaoh's Bath, Sinai, Egypt. The isolates were identified by 16S rRNA genes sequencing and were submitted to GenBank as Bacillus tequilensis strain Pharon2 (MK078034) and Bacillus sonorensis strain Pharon3 (MK078035). The molybdenum blue production was optimized using multifactorial statistical approaches, Plackett-Burman and central composite designs. According to the results obtained by response optimizer, the maximum molybdenum blue production achieved was 1.04 and 1.12 represented as absorbance at 865nm, with the optimum salt concentration of 1.1 and 2.5%; at pH 7.02 and 7.07; incubation temperature of 46.1 and 52.2 °C; sucrose and glucose as an electron donor for Bacillus tequilensis strain Pharon2 and Bacillus sonorensis strain Pharon3, respectively. In conclusion, the thermophilic bacterial isolates belonging to the genus Bacillus could be used in in situ bioremediation under elevated temperatures. To the best of our knowledge, this is the first record of molybdenum blue production by thermophilic Bacillus tequilensis and Bacillus sonorensis.

Identification of Proteolytic Thermophiles from Moinit Coastal Hot-Spring, North Sulawesi, Indonesia

We have isolated thermophilic bacteria from Moinit coastal hot-spring, North Sulawesi, Indonesia. Of 12 seawater samples collected, we obtained 20 thermophilic bacterial isolates. Sixteen of the isolated thermophiles were found to exhibit protein-degrading ability as indicated by the presence of clearing zones surrounding their colonies on skim milk-containing plates at 65 °C. Five of them with larger proteolytic indexes, designated as 2b, 4b, 9a, 9b, and 12L, were selected for further studies. The ability of these five strains to hydrolyze casein at high temperatures was indicated by relatively high activity of their extracellular proteases at 60 °C. Partial protease-encoding genes were amplified from each isolated strain and sequenced. Further bioinformatics analysis suggests that the extracellular proteases that they produced belong to serine proteases. The integrated morphological, biochemical, and genetic analyses showed the identity of the strains 2b, 4b, and 9a as members of Bacillus, 9b as Pseudomonas, and 12L as Aneurinibacillus. To the best of our knowledge, so far thermophilic Pseudomonas and Aneurinibacillus remain poorly investigated in term of extracellular protease production, suggesting the biotechnological potential of the strains 9b and 12L as the producer of a unique extracellular thermostable protease.