Thermophilic Research Articles

Simultaneous production of d-allulose and d-tagatose from lactose

The high-demand rare sugars d-tagatose and d-allulose may be produced from lactose in a combined process, though this dual conversion has not yet been demonstrated. Four enzymes from thermophilic organisms were cloned and expressed in Escherichia coli to synthesize d-tagatose and d-allulose from lactose: β-galactosidase (Geobacillus stearothermophilus), l-arabinose isomerase (G. stearothermophilus US100), d-glucose isomerase (Weizmannia coagulans) and d-allulose epimerase (Arthrobacter globiformis). A single-pot reaction mixture yielded 9.9 ± 1.5 mg mL−1d-allulose and 10.7 ± 1.1 mg mL−1d-tagatose after 24 h at 70 °C. Enzyme addition order did not have an impact on yields (p = 0.3527). Higher lactose concentrations resulted in higher d-tagatose concentrations (p ≤ 0.001) regardless of when the lactose was added, however, specific yields for d-allulose were higher without adding additional lactose during the reaction (p ≤ 0.001). These experiments demonstrate that a single pot synthesis of d-tagatose and d-allulose is feasible and with more optimization could be used in industrial applications.

Utilization of low-stability variants in protein evolutionary engineering

Evolutionary engineering involves repeated mutations and screening and is widely used to modify protein functions. However, it is important to diversify evolutionary pathways to eliminate the bias and limitations of the variants by using traditionally unselected variants. In this study, we focused on low-stability variants that are commonly excluded from evolutionary processes and tested a method that included an additional restabilization step. The esterase from the thermophilic bacterium Alicyclobacillus acidocaldarius was used as a model protein, and its activity at its optimum temperature of 65 °C was improved by evolutionary experiments using random mutations by error-prone PCR. After restabilization using low-stability variants with low-temperature (37 °C) activity, several re-stabilizing variants were obtained from a large number of variant libraries. Some of the restabilized variants achieved by removing the destabilizing mutations showed higher activity than that of the wild-type protein. This implies that low-stability variants with low-temperature activity can be re-evolved for future use. This method will enable further diversification of evolutionary pathways.

Isolation, identification, and characterization of thermophilic lactic acid bacteria isolated from whey of Kars Kashar cheeses.

Kars Kashar cheese is an artisanal pasta-filata type cheese and geographically marked in Eastern Anatolia of Turkey. The aims of this research were to determine for the first time thermophilic lactic acid bacteria (LAB) of Kars Kashar cheese and characterize the technological properties of obtained isolates. In our research, a number of 15 samples of whey were collected from the different villages in Kars. These samples were incubated at 45 °C and used as the source material for isolating thermophilic LAB. A total of 250 colonies were isolated from thermophilic whey, and 217 of them were determined to be presumptive LAB based on their Gram staining and catalase test. A total of 170 isolates were characterized by their phenotypic properties and identified using the MALDI-TOF mass spectrometry method. Phenotypic identification of isolates displayed that Enterococcus and Lactobacillus were the predominant microbiota. According to MALDI-TOF MS identification, 89 isolates were identified as Enterococcus (52.35%), 57 isolates as Lactobacillus (33.53%), 23 isolates as Streptococcus (13.53%), and one isolate as Lactococcus (0.59%). All thermophilic LAB isolates were successfully identified to the species level and it has been observed that MALDI-TOF MS can be successfully used for the identification of selected LAB. The acidification and proteolytic activities of the isolated thermophilic LAB were examined, and the isolates designated for use as starter cultures were also genotypically defined.

Kinetics and products of Thermotoga maritima β-glucosidase with lactose and cellobiose

Galacto-oligosaccharides (GOS) are prebiotic compounds that are mainly used in infant formula to mimic bifidogenic effects of mother’s milk. They are synthesized by β-galactosidase enzymes in a trans-glycosylation reaction with lactose. Many β-galactosidase enzymes from different sources have been studied, resulting in varying GOS product compositions and yields. The in vivo role of these enzymes is in lactose hydrolysis. Therefore, the best GOS yields were achieved at high lactose concentrations up to 60%wt, which require a relatively high temperature to dissolve. Some thermostable β-glucosidase enzymes from thermophilic bacteria are also capable of using lactose or para nitrophenyl-galactose as a substrate. Here, we describe the use of the β-glucosidase BglA from Thermotoga maritima for synthesis of oligosaccharides derived from lactose and cellobiose and their detailed structural characterization. Also, the BglA enzyme kinetics and yields were determined, showing highest productivity at higher lactose and cellobiose concentrations. The BglA trans-glycosylation/hydrolysis ratio was higher with 57%wt lactose than with a nearly saturated cellobiose (20%wt) solution. The yield of GOS was very high, reaching 72.1%wt GOS from lactose. Structural elucidation of the products showed mainly β(1 → 3) and β(1 → 6) elongating activity, but also some β(1 → 4) elongation was observed. The β-glucosidase BglA from T. maritima was shown to be a very versatile enzyme, producing high yields of oligosaccharides, particularly GOS from lactose.Key points• β-Glucosidase of Thermotoga maritima synthesizes GOS from lactose at very high yield.• Thermotoga maritima β-glucosidase has high activity and high thermostability.• Thermotoga maritima β-glucosidase GOS contains mainly (β1-3) and (β1-6) linkages.Graphical

Alpha Carbonic Anhydrase from Nitratiruptor tergarcus Engineered for Increased Activity and Thermostability.

The development of carbon capture and storage technologies has resulted in a rising interest in the use of carbonic anhydrases (CAs) for CO2 fixation at elevated temperatures. In this study, we chose to rationally engineer the α-CA (NtCA) from the thermophilic bacterium Nitratiruptor tergarcus, which has been previously suggested to be thermostable by in silico studies. Using a combination of analyses with the DEEPDDG software and available structural knowledge, we selected residues in three regions, namely, the catalytic pocket, the dimeric interface and the surface, in order to increase thermostability and CO2 hydration activity. A total of 13 specific mutations, affecting seven amino acids, were assessed. Single, double and quadruple mutants were produced in Escherichia coli and analyzed. The best-performing mutations that led to improvements in both activity and stability were D168K, a surface mutation, and R210L, a mutation in the dimeric interface. Apart from these, most mutants showed improved thermostability, with mutants R210K and N88K_R210L showing substantial improvements in activity, up to 11-fold. Molecular dynamics simulations, focusing particularly on residue fluctuations, conformational changes and hydrogen bond analysis, elucidated the structural changes imposed by the mutations. Successful engineering of NtCA provided valuable lessons for further engineering of α-CAs.

Induction of Apoptosis with Silver Nanoparticles Obtained Using Thermophilic Bacteria.

Yeasts resistant to antifungals have become an increasing risk to human health. One of the best antimicrobial properties is reported to be present in silver nanoparticles (AgNPs); however, little is known about the antimicrobial potential of AgNPs produced using thermophilic bacteria. How AgNPs cause cell death is different depending on the type of the cell, and the mode of death induced is cell-type specific. Apoptosis, one of the types of regulated cell death, can be extremely useful in the fight against infection because surrounding cells that have phagocytic activity can efficiently absorb the apoptotic bodies formed during apoptosis. In the course of this work, for the first time, comprehensive antifungal studies of AgNPs were performed using thermophilic Geobacillus spp. bacteria against Candida guilliermondii, also with the addition of the model yeast Saccharomyces cerevisiae. The determined minimal inhibitory concentrations (MICs) were 10 μg/mL against C. guilliermondii and 50 μg/mL against S. cerevisiae for Geobacillus sp. strain 25 AgNPs, and for Geobacillus sp. 612 the MICs were 5 μg/mL and 25 μg/mL, respectively. It was shown for the first time that the exposure of the yeast cells leads to caspase activation in both S. cerevisiae and C. guilliermondii after exposure to Geobacillus spp. AgNPs. Also, a statistically significant change in the number of cells with permeable membranes was detected. Moreover, it was shown that the antimicrobial effect of the AgNPs is related to ROS generation and lipid peroxidation in C. guilliermondii yeast.

Isolation and Characterization of Thermophilic Bacteria from a Hot Spring in the State of Hidalgo, Mexico, and Geochemical Analysis of the Thermal Water.

Hot springs worldwide can be a source of extremophilic microorganisms of biotechnological interest. In this study, samplings of a hot spring in Hidalgo, Mexico, were conducted to isolate, identify, and characterize morphologically, biochemically, and molecularly those bacterial strains with potential industrial applications. In addition, a physicochemical and geochemical examination of the hot spring was conducted to fully understand the study region and its potential connection to the strains discovered. The hot spring was classified as sulfate-calcic according to the Piper Diagram; the hydrogeochemical analysis showed the possible interactions between minerals and water. Eighteen bacterial strains were isolated with optimal growth temperatures from 50 to 55 °C. All strains are Gram-positive, the majority having a rod shape, and one a round shape, and 17 produce endospores. Hydrolysis tests on cellulose, pectin, and xylan agar plates demonstrated enzymatic activity in some of the strains. Molecular identification through the 16S rDNA gene allowed classification of 17 strains within the Phylum Firmicutes and one within Deinococcus-Thermus. The bacterial strains were associated with the genera Anoxybacillus, Bacillus, Anerunibacillus, Paenibacillus, and Deinococcus, indicating a diversity of bacterial strains with potential industrial applications.

Cloning, expression and purification of cellobiohydrolase gene from Caldicellulosiruptor bescii for efficient saccharification of plant biomass

Anthropogenic activities have led to a drastic shift from natural fuels to alternative renewable energy reserves that demand heat-stable cellulases. Cellobiohydrolase is an indispensable member of cellulases that play a critical role in the degradation of cellulosic biomass. This article details the process of cloning the cellobiohydrolase gene from the thermophilic bacterium Caldicellulosiruptor bescii and expressing it in Escherichia coli (BL21) CondonPlus DE3-(RIPL) using the pET-21a(+) expression vector. Multi-alignments and structural modeling studies reveal that recombinant CbCBH contained a conserved cellulose binding domain III. The enzyme's catalytic site included Asp-372 and Glu-620, which are either involved in substrate or metal binding. The purified CbCBH, with a molecular weight of 91.8 kDa, displayed peak activity against pNPC (167.93 U/mg) at 65°C and pH 6.0. Moreover, it demonstrated remarkable stability across a broad temperature range (60–80°C) for 8 h. Additionally, the Plackett-Burman experimental model was employed to assess the saccharification of pretreated sugarcane bagasse with CbCBH, aiming to evaluate the cultivation conditions. The optimized parameters, including a pH of 6.0, a temperature of 55°C, a 24-hour incubation period, a substrate concentration of 1.5% (w/v), and enzyme activity of 120 U, resulted in an observed saccharification efficiency of 28.45%. This discovery indicates that the recombinant CbCBH holds promising potential for biofuel sector.

A highly efficient method for genomic deletion across diverse lengths in thermophilic Parageobacillus thermoglucosidasius

Parageobacillus thermoglucosidasius is emerging as a highly promising thermophilic organism for metabolic engineering. The utilization of CRISPR-Cas technologies has facilitated programmable genetic manipulation in P. thermoglucosidasius. However, the absence of thermostable NHEJ enzymes limited the capability of the endogenous type I CRISPR-Cas system to generate a variety of extensive genomic deletions. Here, two thermophilic NHEJ enzymes were identified and combined with the endogenous type I CRISPR-Cas system to develop a genetic manipulation tool that can achieve long-range genomic deletion across various lengths. By optimizing this tool—through adjusting the expression level of NHEJ enzymes and leveraging our discovery of a negative correlation between GC content of the guide RNA (gRNA) and deletion efficacy—we streamlined a comprehensive gRNA selection manual for whole-genome editing, achieving a 100 % success rate in randomly selecting gRNAs. Notably, using just one gRNA, we achieved genomic deletions spanning diverse length, exceeding 200 kilobases. This tool will facilitate the genomic manipulation of P. thermoglucosidasius for both fundamental research and applied engineering studies, further unlocking its potential as a thermophilic cell factory.

Comparative analysis of folding and unfolding dynamics and free-energy landscapes in homologous cold shock proteins with variable thermal stabilities

Cold shock proteins from the mesophilic organism (BsCSP), the thermophilic organism (BcCSP), and the hyperthermophilic organism (TmCSP) are homologous proteins with similar native structure but quite different thermal stabilities, which makes them promising candidates for investigating the general and specific aspects the folding and unfolding dynamics of proteins and free-energy landscapes. In this study, we employed magnetic tweezers to explore the force-dependent folding and unfolding rates of BsCSP, BcCSP, and TmCSP at forces from several pN to tens of pN. Our results indicate similar force-dependent folding rates for these three proteins, while the unfolding rate of BsCSP, BcCSP, and TmCSP decreases sequentially, consistent with previous biochemical findings. Comparing with TmCSP, BcCSP shows lower but sufficient stability with folding free energy of 8.0 kBT, and its force-dependent unfolding rates exhibit tremendous nonlinear behavior deviated from Bell's model like TmCSP. BsCSP has very low stability with folding free energy of only 3.9 kBT, and its force-dependent unfolding rates at 3–10 pN exhibit almost linear behavior. Comprehensive free-energy landscapes are constructed, revealing common barriers that governs folding and unfolding dynamics for BcCSP and TmCSP but a wide merged barrier for BsCSP. Our findings provide valuable insights into the folding and unfolding mechanisms of these CSPs, shedding light on the relationship between protein structure, stability, and mechanical properties. Published by the American Physical Society 2024

The Source of Thermophilic Bacteria in Lake Baikal Cold Sediments—Coastal Hydrotherms or Deep Fluids?

The Source of Thermophilic Bacteria in Lake Baikal Cold Sediments—Coastal Hydrotherms or Deep Fluids?

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.

ALACEN: A Holistic Herbaceous Biomass Fractionation Process Attaining a Xylose-Rich Stream for Direct Microbial Conversion to Bioplastics.

Lignocellulose biorefining is a promising technology for the sustainable production of chemicals and biopolymers. Usually, when one component is focused on, the chemical nature and yield of the others are compromised. Thus, one of the bottlenecks in biomass biorefining is harnessing the maximum value from all of the lignocellulosic components. Here, we describe a mild stepwise process in a flow-through setup leading to separate flow-out streams containing cinnamic acid derivatives, glucose, xylose, and lignin as the main components from different herbaceous sources. The proposed process shows that minimal degradation of the individual components and conservation of their natural structure are possible. Under optimized conditions, the following fractions are produced from wheat straw based on their respective contents in the feed by the ALkaline ACid ENzyme process: (i) 78% ferulic acid from a mild ALkali step, (ii) 51% monomeric xylose free of fermentation inhibitors by mild ACidic treatment, (iii) 82% glucose from ENzymatic degradation of cellulose, and (iv) 55% native-like lignin. The benefits of using the flow-through setup are demonstrated. The retention of the lignin aryl ether structure was confirmed by HSQC NMR, and this allowed monomers to form from hydrogenolysis. More importantly, the crude xylose-rich fraction was shown to be suitable for producing polyhydroxybutyrate bioplastics. The direct use of the xylose-rich fraction by means of the thermophilic bacteria Schlegelella thermodepolymerans matched 91% of the PHA produced with commercial pure xylose, achieving 138.6 mgPHA/gxylose. Overall, the ALACEN fractionation method allows for a holistic valorization of the principal components of herbaceous biomasses.

Achieving unprecedented stability in lyophilized recombinase polymerase amplification with thermostable pyruvate kinase from Thermotoga maritima

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41°C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. Considering the onsite use of RPA reagents, lyophilized RPA reagents with long storage stability are highly desired. In this study, as one of the approaches to solve this problem, we attempted to use a thermostable pyruvate kinase (PK). PK gene was isolated from a thermophilic bacterium Thermotoga maritima (Tma-PK). Tma-PK was expressed in Escherichia coli and purified from the cells. Tma-PK exhibited higher thermostability than human PK. The purified Tma-PK preparation was applied to RPA as an ATP-regenerating enzyme. Liquid RPA reagent with Tma-PK exhibited the same performance as that with human PK. Lyophilized RPA reagent with Tma-PK exhibited higher performance than that with human PK. Combined with our previous results of RPA reagents of thermostable Pol from a thermophilic bacterium, Aeribacillus pallidus, the results in this study suggest that thermostable enzymes are preferable to mesophilic ones as a component in lyophilized RPA reagents.

Atrimonas thermophila gen. nov., sp. nov., a novel anaerobic thermophilic bacterium of the phylum Atribacterota isolated from deep subsurface gas field and proposal of Atrimonadaceae fam. nov. within the class Atribacteria in the phylum Atribacterota

A novel anaerobic, thermophilic bacterium of the class Atribacteria, strain M15T, was isolated from a high-temperature gas reservoir, Japan. Cells of strain M15T were gram-negative, short oval-shaped, and lacked flagella. Growth occurred at 45–75 °C (optimum 70–75 °C) and pH 6.5–8.5 (optimum pH 7.5–8.0) and was fast under optimal conditions (doubling time 11.4 h). Yeast extract was required for growth. Fermentative growth with glucose, arabinose, xylose, and cellobiose was observed. The major fermentative end products of glucose were acetate and hydrogen. The major cellular fatty acids were C16:0, iso-C15:0, and C18:0. The genomic G + C content was 46.0 mol%. Fluorescence and electron microscopy observations revealed the intracellular localization of genomic DNA surrounded by a membrane in the cells of strain M15T as reported in a sole validly described species of the class Atribacteria in the phylum Atribacterota, Atribacter laminatus strain RT761T, suggesting that the unique morphological traits are widely shared in this class. Phylogenetic analyses indicated that strain M15T belongs to a distinct family-level lineage in the class Atribacteria and shows low similarities to Atribacter laminatus strain RT761T (16S rRNA gene sequence identity of 90.1 %, average nucleotide identity [ANI] of 66.1 %, average amino acid identity [AAI] of 55.8 %). Phenotypic traits of strain M15T (thermophilic, fast-growing, relatively high G + C content, etc.) were clearly distinct from A. laminatus. Based on these phenotypic and genomic properties, we propose a novel genus and species, Atrimonas thermophila gen. nov., sp. nov. for strain M15T (=JCM39389T, =KCTC25731T) representing a novel family Atrimonadaceae fam., nov. in the class Atribacteria.

Effects of macroalgae and sea urchin grazing pressure on zoantharians growth under laboratory conditions

In the context of ocean warming, thermophilic organisms such as zoantharians are expanding and altering shallow benthic habitats. Here, a four-month laboratory experiment was performed to examine the influence of three types of macroalgae morphotypes common in the Canary Islands (turf algae, Lobophora spp., and crustose coralline algae) on the growth of two zoantharian species, Palythoa caribaeorum and Zoanthus pulchellus. Additionally, the grazing effects of echinoids Diadema africanum and Paracentrotus lividus were assessed as facilitators of substrate colonization by means of controlling macroalgae cover. Colony and algal coverages were measured at the beginning, middle and end of the experiment, and increments were calculated. Results indicated a general decrease in zoantharian colony sizes in contact with different algal types in the absence of sea urchins. However, P. caribaeorum colonies showed significant growth in the presence of D. africanum, highlighting the ecological importance of sea urchins in zoantharian population proliferation and subsequent community modification. This study represents the first investigation into zoantharian-macroalgae interactions under controlled conditions.

Cloning, expression, and characterization of thermostable α-amylase of a thermophilic bacterium isolated from hot-spring of Sikkim and sustainable immobilization on coconut coir

Cloning, expression, and characterization of thermostable α-amylase of a thermophilic bacterium isolated from hot-spring of Sikkim and sustainable immobilization on coconut coir

Detection of risk areas in dairy powder processes: The development of thermophilic spore forming bacteria taking into account their growth limits

Anoxybacillus flavithermus, Geobacillus stearothermophilus and Bacillus licheniformis are the main contaminants found in dairy powders. These spore-forming thermophilic bacteria, rarely detected in raw milk, persist, and grow during the milk powder manufacturing process. Moreover, in the form of spores, these species resist and concentrate in the powders during the processes. The aim of this study was to determine the stages of the dairy powder manufacturing processes that are favorable to the growth of such contaminants. A total of 5 strains were selected for each species as a natural contaminant of dairy pipelines in order to determine the minimum and maximum growth enabling values for temperature, pH, and aw and their optimum growth rates in milk. These growth limits were combined with the environmental conditions of temperature, pH and aw encountered at each step of the manufacture of whole milk, skim milk and milk protein concentrate powders to estimate growth capacities using cardinal models and the Gamma concept. These simulations were used to theoretically calculate the population sizes reached for the different strains studied at each stage in between two successive cleaning in place procedures. This approach highlights the stages at which risk occurs for the development of spore-forming thermophilic bacterial species. During the first stages of production, i.e. pre-treatment, pasteurization, standardization and pre-heating before concentration, physico-chemical conditions encountered are suitable for the development and growth of A. flavithermus, G. stearothermophilus and B. licheniformis. During the pre-heating stage and during the first effects in the evaporators, the temperature conditions appear to be the most favorable for the growth of G. stearothermophilus. The temperatures in the evaporator during the last evaporator effects are favorable for the growth of B. licheniformis. In the evaporation stage, low water activity severely limits the development of A. flavithermus.

Structural basis for the recognition of α-1,6-branched α-glucan by GH13_47 α-amylase from Rhodothermus marinus.

Glycoside hydrolase (GH) family 13 is among the main families of enzymes acting on starch; recently, subfamily 47 of GH13 (GH13_47) has been established. The crystal structure and function of a GH13_47 enzyme from Bacteroides ovatus has only been reported to date. This enzyme has α-amylase activity, while the GH13_47 enzymes comprise approximately 800-900 amino acid residues which are almost double those of typical α-amylases. It is important to know how different the GH13_47 enzymes are from other α-amylases. Rhodothermus marinus JCM9785, a thermophilic bacterium, possesses a gene for the GH13_47 enzyme, which is designated here as RmGH13_47A. Its structure has been predicted to be composed of seven domains: N1, N2, N3, A, B, C, and D. We constructed a plasmid encoding Gly266-Glu886, which contains the N3, A, B, and C domains and expressed the protein in Escherichia coli. The enzyme hydrolyzed starch and pullulan by a neopullulanase-type action. Additionally, the enzyme acted on maltotetraose, and saccharides with α-1,6-glucosidic linkages were observed in the products. Following the replacement of the catalytic residue Asp563 with Ala, the crystal structure of the variant D563A in complex with the enzymatic products from maltotetraose was determined; as a result, electron density for an α-1,6-branched pentasaccharide was observed in the catalytic pocket, and Ile762 and Asp763 interacted with the branched chain of the pentasaccharide. These findings suggest that RmGH13_47A is an α-amylase that prefers α-1,6-branched parts of starch to produce oligosaccharides.

Unveiling Antibacterial Potential and Physiological Characteristics of Thermophilic Bacteria Isolated from a Hot Spring in Iran.

The increasing worldwide demand for antimicrobial agents has significantly contributed to the alarming rise of antimicrobial resistance, posing a grave threat to human life. Consequently, there is a pressing need to explore uncharted environments, seeking out novel antimicrobial compounds that display exceptionally efficient capabilities. Hot springs harbor microorganisms possessing remarkable properties, rendering them an invaluable resource for uncovering groundbreaking antimicrobial compounds. In this study, thermophilic bacteria were isolated from Mahallat Hot Spring, Iran. Out of the 30 isolates examined, 3 strains exhibited the most significant antibacterial activities against Escherichia coli and Staphylococcus aureus. Furthermore, the supernatants of the isolated strains exhibited remarkable antibacterial activity, displaying notable resistance to temperatures as high as 75 °C for 30 min. It was determined that the two strains showed high similarity to the Bacillus genus, while strain Kh3 was classified as Saccharomonospora azurea. All three strains exhibited tolerance to NaCl. Bacillus strains demonstrated optimal growth at pH 5 and 40 °C, whereas S. azurea exhibited optimal growth at pH 9 and 45 °C. Accordingly, hot springs present promising natural reservoirs for the isolation of resilient strains possessing antibacterial properties, which can be utilized in disease treatment or within the food industry.