Microorganisms, based on their temperature requirements, may be classified as psychrophiles (−2 to 20 °C), mesophiles (20–40 °C), and thermophiles and hyperthermophiles (45–113 °C) (Figure 1). While the upper limit for eukaryotic life is ~60 °C, several groups of microorganisms can cope with the stress of high temperature. In general, microorganisms with optimal growth temperatures between 60° and 108 °C are referred to as thermophiles (Tansey and Brock, 1972; Hickey and Singer, 2004). Further, thermophilic microorganisms may be sub-classified as thermophiles: primarily Bacteria that display optimal growth temperature between 60° and 80 °C, and hyperthermophiles: primarily Archaea that grow optimally at 80 °C or above, being unable to grow below 60 °C. Thermophilic microorganisms are generally isolated from several marine and terrestrial geothermally heated habitats (Wang et al., 2015).

Thomas D. Brock, for the first time, reported the existence of microorganisms from the boiling hot springs of Yellowstone National Park (Brock and Freeze, 1969). Since then, a variety of microorganisms adapted to high temperatures have been discovered. These include phototrophs that grow up to 72–73 °C and heterotrophs that grow up to 91 °C (the temperature at which water boils at the elevation of Yellowstone). In 1996, an extraordinary microorganism, Pyrolobus fumarii, from a deep-sea hydrothermal vent was isolated, that was capable of growing up to 113 °C, and was unable to grow below 90 °C (Blöchl et al., 1997). This discovery led to naming a category of thermophiles as hyperthermophiles that grow at 80 °C or above. Since then, over 90 hyperthermophilic species have been discovered, mostly belonging to the domain of Archaea, and some to Bacteria. Throughout evolution, the thermophilic organisms were able to adapt to changing environments, such as, the up-shifts in temperature. The current classification considers thermophiles—all organisms growing above 55 °C, moderate thermophiles—organisms growing above 65 °C, extreme thermophiles—organisms growing above 75 °C, and hyperthermophiles—organisms growing above 90 °C (Imanaka, 2011). Some authors refer hyperthermophiles that include extreme thermophiles as well (Blöchl et al., 1997; Stetter, 2006). Since Brock’s discovery, thermophiles have been discovered from a variety of geothermal areas all over the world, including areas in North America, Iceland, New Zealand, Japan, Italy, India, China, Tibet, Turkey, Tunisia and the Soviet Union.¹

Thermophilic microorganisms are particularly fascinating due to their structural and physiological features allowing them to withstand extremely selective environmental conditions. These properties are often due to specific biomolecules (DNA, lipids, enzymes, osmolites, etc.) that have been studied for years as novel sources for biotechnological and industrial applications (Elleuche et al., 2014). In late 1980s, the first hyperthermophilic enzymes were purified, that proved to be extremely stable at high temperatures (Vieille and Zeikus, 2001; Unsworth et al., 2007). It was also found that these enzymes can be cloned and expressed in mesophilic hosts without losing their active conformation and thermostability. The findings on increasing number of thermostable enzymes from thermophiles opened new possibilities of commercial relevance mainly in starch industry, synthesis of amino acids, petroleum, chemical, pulp and paper industries, and in many other fields (Zeldes et al., 2015). The emerging eco-friendly industrial processes are receiving attention in view of implementing the bio-based technologies and reshaping the biocatalytic functions (Turner et al., 2007; Zimmermann, 2025).

In the present review, we synthesize recent advances in thermophile-derived enzymes (“thermozymes”), highlight the industrial applications supported by their exceptional thermostability, and provide an updated, application-oriented overview of their emerging and established roles in industrial enzyme discovery, along with insights into the conservation of such extreme habitats.

A variety of thermal environments, natural as well as man-made, exist on earth that are populated by the thermophiles. These include volcanic and geothermal areas with temperatures often greater than boiling of water. Themophiles have been studied from hot springs, geysers, volcanoes and deep-sea hydrothermal vents (Figure 2). Thermal environments may originate as a result of solar heating, geothermal activity, intense radiation and combustion processes. Temperatures in solar heated soils and combustion processes can reach up to 60°–70 °C. Thermophiles can also colonize the microenvironments where the temperature is high due to the biological activity, such as compost heaps, saw dust, and the coal refuse piles. Thermophiles are also found in domestic and industrial hot water systems and industrial processes with high temperature. Geothermally heated environments are known to host some of the most remarkable thermophiles and hyperthermophiles (Seckbach, 2013). Although these habitats differ in geological origin, they share physicochemical stresses-elevated temperature, steep thermal gradients, low oxygen availability, and, in many cases, extreme pH or high concentrations of sulfur and metals-that exert strong selective pressure on microbial communities.

Hot springs in volcanic areas may reach high temperatures near boiling point (e.g., above 60 °C). Steam vents (fumaroles) can reach much higher temperatures (up to ~400 °C) due to magmatic heating. In undersea hydrothermal vents (e.g., at seamounts or mid-ocean ridges), fluid temperatures have been measured in the range of 300–400 °C (Barreyre et al., 2025; Schrenk et al., 2013).

Thermophiles have been extensively studied in hot springs and other thermal environments worldwide, encompassing a wide range of temperature conditions. Notable geothermal sites across different continents, including Yellowstone National Park in the USA, Iceland’s geothermal fields, Japan’s solfataric areas, and diverse locations in India such as Bakreshwar (West Bengal), Manikaran (Himachal Pradesh), and Soldhar and Ringigad (Uttarakhand), have yielded significant insights into thermophilic microorganisms (Table 1). These studies highlight the global distribution and diversity of thermophiles thriving in both low- and high-temperature thermal habitats.

A key driver shaping thermophilic microbial communities is the interplay of temperature, pH, redox chemistry, and mineral composition. For example, Sulfur rich environments such as Solfatara fields, are acidic hot springs and boiling mud pots. Such distinct geothermal environments are widespread in volcanic zones and referred to as ‘high temperature fields’ (Schwartz, 1980). Similar volcanic regions along with Yellowstone National Park, possess heating water that happens due to the interaction with magma resulting g in the elevated temperatures/boiling of water (Inskeep et al., 2013). In addition, geysers are the thermal environments that produces jets of steam and hot water above the Earth’s surface. The moment water reaches the surface as steam, it known as fumarole; and becomes “mudpots” when mixed with mud and clay (Inskeep et al., 2013). Most thermophiles isolated from hydrothermal vent systems-where the combination of high temperatures and hydrostatic pressure maintains water in a liquid state are anaerobic microorganisms adapted to these extreme conditions (Reysenbach and Shock, 2002; Schrenk et al., 2013).

Thermophilic microorganisms inhabit various undersea hot springs and hydrothermal vent systems. Black smoker vents emit extremely hot, metal-rich fluids that precipitate metal sulfides upon mixing with cold seawater, forming characteristic chimney structures. The thin walls of these chimneys can reach temperatures of 200–400 °C, creating extreme microsites that host highly specialized archaeal lineages adapted to rapid temperature fluctuations and intense metal stress. Hyperthermophiles also colonize the seawater surrounding active seamounts, where volcanic lava is emitted directly onto the seafloor (Seckbach, 2013). Additionally, shallow marine hydrothermal systems support diverse bacterial and archaeal communities, as demonstrated in studies of vents near Vulcano Island (Antranikian et al., 2017). Together, these environments illustrate how temperature interacts with geochemistry to structure thermophilic ecosystems, selecting for organisms with unique physiological and metabolic traits. Understanding these physicochemical drivers is essential for identifying novel thermophiles and their thermostable enzymes with potential industrial relevance.

Moderate thermophiles consist of diverse taxonomic groups (both prokaryotic and eukaryotic microorganisms) generally have optimum growth between 50 °C and 60 °C temperature ranges (Stetter, 2006; Bender et al., 2022). Phylogenetically, moderate thermophiles often appear closely related to mesophilic organisms, indicating their adaptive capacity to thrive in thermal environments. Recently, thermotolerant bacteria belonging to the genera Bacillus and Paenibacillus have been isolated from hot springs in the temperate regions of the Indian Himalayas, demonstrating growth across a wide temperature range of 20–80 °C with an optimum near 55 °C (Pandey et al., 2015). These bacteria also exhibited tolerance to a broad pH spectrum (4–14) and produced enzymes active in the thermophilic range, underscoring their industrial potential. Notable examples of moderate thermophiles include the cellulolytic bacterium Clostridium thermocellum and the acetogenic bacteria Moorella thermoacetica and Moorella thermoautotrophica (Leschine, 1995; Drake et al., 2008). Aerobic bacteria such as Geobacillus kaustophilus, G. stearothermophilus, G. thermoleovorans, and Bacillus halodurans also fall within this category and have been studied extensively for their thermostable enzymes and biotechnological applications (Nazina et al., 2001; Zeigler, 2014).

Extreme thermophiles, that grow optimally between 60° and 80 °C, are represented in the genera Bacillus, Clostridium, Thermoanaerobacter, Thermus, Fervidobacterium, and Thermotoga. Most of these thermophiles are anaerobic Firmicutes that include cellulolytic (Caldicellulosiruptor saccharolyticus), ethanol producing (Thermoanaerobacterium), and the acetogenic facultative chemolithoautotrophic (Thermoanaerobacterium kivui), and denitrifying (Ammonifex degensii) bacteria. The extreme aerobic thermophiles include Bacillus stearothermophilus (Firmicutes) and within the Gram-negative genus Thermus. The examples of some of the recently reported novel thermophilic species are Sporolituus thermophilus (citrate fermenting anaerobic bacterium), Microaerobacter geothermalis (microaerophilic, nitrate and nitrite reducing bacterium), Nautilia abyssi (the sulfur reducing deep sea bacterium), Anoxybacillus thermarum (the thermal mud-inhabiting), and Caldisericum exile (anaerobic filamentous bacterium of a novel phylum) (Canganella and Wiegel, 2011; Antranikian et al., 2017). Hyperthermophiles represent some of the most ancient and physiologically extreme lineages in both the domains Bacteria and Archaea. Among bacteria, the genera Aquifex and Thermotoga are canonical representatives, both occupying basal phylogenetic positions and exhibiting optimal growth temperatures above 80 °C (Huber and Stetter, 2001). Within Archaea, hyperthermophiles are taxonomically diverse and include members of the genera Pyrodictum, Pyrobaculum, Thermoproteus, Desulfurococcus, Sulfolobus, Methanopyrus, Pyrococcus, Thermococcus, Methanococcus, and Archaeoglobus (Stetter, 1999; Reysenbach and Shock, 2002). These organisms thrive at optimal temperatures ranging from ~80 to 108 °C, inhabiting environments strongly shaped by geothermal activity. Their most common ecological niches include volcanic solfataric fields, terrestrial hot springs, and, most notably, submarine hydrothermal systems (Huber et al., 2000). Hydrothermal vent environments occur at both shallow and abyssal depths and encompass fumaroles, hot springs, mineral-rich sediments, and deep-sea “black smokers,” where vent fluid temperatures can exceed 350–400 °C. While no life forms persist directly in the hottest fluids, hyperthermophiles colonize peripheral mixing zones where steep thermal and chemical gradients allow biological activity (Baross and Hoffman, 1985; Kelley et al., 2001).

The distribution of hyperthermophilic taxa reflects ecological partitioning across hydrothermal gradients. Genera such as Pyrococcus, Pyrodictum, Thermococcus, Methanococcus, Archaeoglobus, and Thermotoga have been recovered from both shallow and deep-sea vent systems, highlighting their physiological versatility (Stetter, 1999; Huber et al., 2000). By contrast, Methanopyrus is often associated with greater depths and higher-pressure regimes (Kurr et al., 1991), whereas Aquifex has been isolated primarily from shallow hydrothermal environments (Deckert et al., 1998). Hyperthermophiles are metabolically diverse, with pathways including sulfate and sulfur reduction, nitrate reduction, and iron reduction, reflecting adaptations to energy sources available in geothermal systems (Reysenbach and Shock, 2002). Their metabolic versatility makes them critical drivers of biogeochemical cycling in extreme environments. The known upper temperature limit for life has been repeatedly revised as new isolates emerge. Pyrolobus fumarii, a vent archaeon, was long regarded as the most thermophilic organism, with an optimum growth temperature of 106 °C and a maximum near 113 °C (Blöchl et al., 1997). More recently, a strain of Methanopyrus kandleri (strain 116), recovered from a deep-sea hydrothermal vent, demonstrated active growth at 122 °C under elevated hydrostatic pressure, setting a new upper boundary for life as currently known (Takai et al., 2008). These findings not only expand our understanding of microbial limits but also inform discussions on the potential for life in extraterrestrial high-temperature environments. Examples of thermophiles belonging to Bacteria and Archaea are given in Table 2.

A range of thermophilic fungi belonging to Zygomycetes (Rhizomucor miehei, R. pusillus), Ascomycetes (Chaetomium thermophile, Thermoascus aurantiacus, Dactylomyces thermophilus, Melanocarpus albomyces, Talaromyces thermophilus, T. emersonii, Thielavia terrestris), Basidiomycetes (Phanerochaete chrysosporium) and Hyphomycetes (Acremonium alabamensis, A. thermophilum, Myceliophthora thermophila, Thermomyces lanuginosus, Scytalidium thermophilum, Malbranchea cinnamomea) have been isolated from composts, soils, nesting materials of birds, wood chips and many other sources (Tansey, 1973; Maheshwari et al., 2000; Salar and Aneja, 2007; Ingersoll, 2023). Some algae such as Synechococcus (the cyanobacterial genus) have been found in the thermophilic mat communities growing at temperatures above 60 °C (Klatt et al., 2011; George et al., 2023). The moderately hot runoff channels and pools below 60 °C are found populated by cyanobacterial mats. In these mats, the diazotrophic cyanobacteria such as Fischerella, Calothrix, and Pleurocapsa grow in nitrogen poor waters at lower temperatures, whereas Synechococcus and Phormidium mats are favored by nitrogen rich waters (Hongmei et al., 2005; George et al., 2023). The protozoa (Cothuria sp. Oxytricha falla, Cercosulcifer hamathensis, Tetrahymena pyriformis, Cyclidium citrullus, Naegleria fowleri) are also reported to grow at high temperatures (Tarkington and Zufall, 2021; Iyevhobu et al., 2025). Some examples of thermophilic microorganisms are presented in Table 3.

Until the 60s, the microorganisms were classified as prokaryotes or eukaryotes. In 1965, it was found that certain molecules could act as evolutionary chronometers. The evolutionary distance between two organisms may be inferred from the differences in the amino acid or nucleotide sequences of homologous macromolecules isolated from both (Woese, 1965). The molecules that were proposed as good evolutionary chronometers are ATPase, the RecA protein, and the rRNAs. These molecules were probably essential even for the most primitive cells (Woese and Fox, 1977). Thus, the changes in the sequence of the genes encoded them allowed the establishment of the evolutionary relationships among different microorganisms. In 1977, Carl Woese proposed 16S rRNA molecule as an optimal prokaryote chronometer that led to the prokaryote phylogenetic tree and the discovery of a new group of microorganisms-the Archaea (Woese and Fox, 1977; Woese et al., 1990). The phylogenetic tree of life, constructed from prokaryote 16S rRNA sequences and eukaryote 18S rRNA sequences, subdivide the living organisms into three Domains namely Bacteria, Archaea and Eukarya (Gribaldo and Brochier-Armanet, 2006; Forterre, 2015). In Bacteria and Archaea, 16S rRNA consists of about 1,500 bases. Hyperthermophiles are found in both the Archaea and Bacteria and occupy the most basal positions of the phylogenetic tree (DiGiacomo et al., 2022). Deeply branching lineages in the phylogenetic tree provide evidence of early divergence events. The separation of Bacteria from the common stem shared by Archaea and Eukarya represents one of the earliest and most basal splits in the tree of life. Short branch lengths indicate relatively low rates of molecular evolution. Unlike the eukaryal domain, the bacterial and archaeal domains contain several lineages that are both basal and slowly evolving. Notably, these lineages are predominantly occupied by hyperthermophiles, which cluster near the root of the universal phylogenetic tree. Among them, anaerobic thermophilic Archaea inhabit some of the most extreme environments and correspond to the deepest, most conserved branches in the tree of life. They are found to often use the substrates that are thought to have been dominant in the primordial terrestrial makeup, indicating that they could have been the first living forms on this planet (DiGiacomo et al., 2022).

In Bacteria, hyperthermophiles are primarily represented by the genera Thermotoga and Aquifex, which occupy some of the most basal lineages within the domain. Cultured Archaea can be classified into two major phyla: Crenarchaeota and Euryarchaeota. The phylum Crenarchaeota, composed entirely of extreme thermophiles and hyperthermophiles, includes genera that are deeply branching and exhibit short branch lengths in rRNA-based phylogenetic trees. Representative hyperthermophilic genera within Crenarchaeota include Sulfolobus, Desulfurococcus, Pyrodictium, Thermofilum, Thermoproteus, and Pyrolobus. Members of Euryarchaeota are more broadly distributed ecologically, yet the most basal lineages within this phylum also correspond to hyperthermophilic genera, such as Methanococcus, Thermococcus, Methanopyrus, and Pyrococcus (de Miguel Bouzas et al., 2006; Stetter, 2006; Canganella and Wiegel, 2011).

Environmental samples can be investigated through culture dependent and culture-independent approach for thermophilic microorganisms. In the culture-dependent methods, the specific microbial colonies can be obtained on solid media following purification through repeated subculturing (Madigan et al., 2019). Pure isolates of bacteria and fungi, typically involves the sequencing of 16S rRNA gene and ITS region, respectively for a preliminary identification (Janda and Abbott, 2007). A polyphasic approach is widely accepted for the specie-level identification which integrates phenotypic traits with genotypic information for more accurate classification (Vandamme et al., 1996). The pure microbial cultures can be preserved in certified microbial culture collections for future ecological and biotechnological studies (Tindall, 2007). When discussing thermophiles, we encounter a range of challenges associated with culturing methods. The solidifying media is unstable at high temperatures that make the process tedious for obtaining pure cultures following the aforementioned method, in addition longer incubation periods are required due to the slow growth of microorganisms (Amann et al., 1995). Above all, the culturing method is highly biased due to the variable requirements of different microbial groups (selective media composition and growth conditions) that provide a less accurate picture of microbial diversity (Staley and Konopka, 1985).

Recent advances have significantly expanded the study of thermophiles. High-pressure microfluidic platforms enable rapid phenotyping of thermophilic archaea under simultaneous high-temperature and high-pressure conditions (e.g., Thermococcus barophilus) (Cario et al., 2022). In parallel, cell-free systems derived from Thermus thermophilus have been adapted for in vitro transcription-translation inside microfluidic droplets, allowing ultrahigh-throughput screening of thermostable enzymes directly (Ribeiro et al., 2024). Additionally, modified iChip cultivation strategies have facilitated the isolation of previously uncultured thermo-tolerant bacteria from hot springs (Zhao et al., 2023). Streamlined discovery pipelines that incorporate modern bioprospecting strategies-such as (ultra)high-throughput screening, microfluidics, and metagenomic mining-have greatly accelerated the identification and functional characterization of thermozymes with industrial potential.

On the other hand, culture-independent methods are advantageous due to molecular techniques such community DNA extraction from environmental samples following PCR amplification of the universal markers. It allows the detection of diverse microbial taxa, purely independent of its culturing requirement (Muyzer et al., 1993). Another method, to digest amplified PCR product with restriction enzymes followed by investigating the banding pattern on electrophoretic gel results in the fingerprinting of the community diversity, referred as amplified ribosomal DNA restriction analysis (ARDRA) (Rameshkumar et al., 2012). The efficiency of such culture independent approaches can be understood by the fact that these methods have found to uncover several taxonomic groups which were previously not detected by any of the culturing methods (Hugenholtz et al., 1998). However, the culture-independent methods are not completely unbiased. Some factors, like DNA extraction efficiency, PCR conditions, variation in rRNA gene copy number etc. strongly influence the outcomes for diversity analysis (Suzuki and Giovannoni, 1996; Polz and Cavanaugh, 1998). Mindful integration of both the approaches is important to achieve the high accuracy is suggested for microbial diversity in such extreme environments (Lagier et al., 2012).

The relative abundance of Archaea and Bacteria in thermal environments was initially studied using culture-based techniques. This led to the assumption that Archaea dominate the thermal environments, probably due to the biases associated to the growth media and culture conditions. With the emergence of application of molecular methods such as slot-blot hybridizations of rRNA utilizing oligonucleotide probes targeting the 16S rRNA of Archaea and Bacteria, it was found that bacteria constituted the major population in thermal environments. Culture independent studies based on the sequencing of 16S ribosomal RNA (rRNA) obtained directly from thermophilic biotopes have revealed the existence of an enormous variety of microorganisms growing at high temperatures that are otherwise not cultivable. Some new methods have been developed for studying the thermophiles such as a novel micromanipulation method that allows the isolation of a single cell from a mixed culture using “optical tweezers.” An isolation strategy that combines in situ 16S rRNA sequence analysis and specific whole-cell hybridization, with the isolation of the identified single cell by “optical tweezers” has also been proposed (de Miguel Bouzas et al., 2006). Ferrer et al. (2016) and Berini et al. (2017) followed metagenomic approach for studying bioprospecting success of microbial enzymes.

Thermophilic microorganisms adapt to the thermal environments in which they have to live and survive by virtue of various mechanisms. The cell membrane of thermophiles is made up of saturated fatty acids that provide a hydrophobic environment for the cell (Koga, 2012). This helps to keep the cell rigid enough to live at elevated temperatures. The Archaea, which compose most of the hyperthermophiles, contain lipids linked with ether on the cell wall. This layer is much more heat resistant than a membrane formed of fatty acids. High temperature increases the fluidity of membranes (Jain et al., 2014). To maintain optimal membrane fluidity the cell must adjust the composition of the membrane including the amount and type of lipids. The typical fatty acid bilayer structure of the bacterial cytoplasmic membrane would become disrupted at extreme temperatures while the archaeal monolayer membrane composed of phytanyl chains connected to glycerol with ether links is much more resistant (Řezanka et al., 2023).

DNA, RNA, and protein hold the machinery of the cell and they get affected by temperature. At high temperatures, denaturation of their native structures takes place. Some mechanisms, related to nucleic acids that protect these biomolecules against high temperature have been studied. Methylation at 2’OH increases RNA stability by protecting it from the hydrolysis of phospho-di-ester bonds. Apart from this, they also increase the stability by the accumulation of positively charged compounds (polyamines) or ions (K⁺) against the negative charge of phosphate group in the back bone that helps in increasing the Tm of the nucleic acids (Hori et al., 2018). Monovalent and divalent salts enhance the stability of nucleic acids as these salts screen the negative charges of the phosphate groups, and protect the DNA from depurination and hydrolysis. The G–C pair of nucleic acids is more thermostable than the A–T or A–U pairs because of the additional hydrogen bond. But elevated G + C ratios are not found among thermophilic prokaryotes due to the stability of the chromosomal DNA, although thermostability is correlated with G + C content of their ribosomal and transfer RNAs (Hickey and Singer, 2004). Thermophiles also tolerate high temperature by using increased interactions that non-thermotolerant organisms use, namely, electrostatic, disulfide bridge and hydrophobic interactions.

Thermophiles contain thermostable proteins that can resist denaturation and proteolysis. The specialized proteins (chaperonins) produced by these organisms help in refolding of proteins to their native form, after their denaturation. Temperature also affects the structure and function of proteins. Proteins found in thermophiles are generally stable due to the occurrence of various structural changes. It includes the packing of the proteins, increase in ion-pair content, formation of higher-order oligomers, increase in hydrogen bonds, changes in the hydrophobicity, reduction in the amount of thermolabile amino acids, increase in proline, etc. These peculiar features of thermophiles that occur at physiological and genetical levels make their proteins different from that of mesophiles (Counts et al., 2017; Go et al., 2024).

To ensure the integrity of genetic information and to carry out metabolic functions, protection of DNA, especially in thermal environments, will be essential. There are several mechanisms used by thermophiles to maintain the stability of their DNA at high temperatures that are likely to work in synergy. The DNA of thermophiles produce a particular type of DNA topoisomerase, called reverse DNA gyrase, that introduces positive super coils in the DNA molecule at the expense of ATP. This confers a greater stability and renders to DNA more resistant to thermal denaturation. This results in raising the melting point of the DNA to at least as high as the organism’s maximum temperature for growth. Another mechanism associated to some thermophilic organisms is the involvement of some histone-like proteins that helps to preserve the duplex structure of DNA. Another mechanism known to protect DNA from denaturation is the presence in the cytoplasm of some hyperthermophilic methanogenes of large amounts of cyclic 2,3-potasium-bi-phospho-glycerate, that avoids chemical damage such as DNA depurinization. Although high temperature results in denaturation and chemical modification in DNA, yet the DNA of hyperthermophiles, such as Pyrococcus furiosus, is known to be more stable in vivo than that of a mesophile such as Escherichia coli (Lehmann et al., 2023).

DNA transfer in Bacteria and Archaea seems to have played a major role, with respect to adaptation of thermophiles to high temperatures. There are three main mechanisms associated with transferring of DNA: natural transformation, conjugation and transduction. The natural transformation refers to the uptake of DNA from the external environment, mostly emerging from lysed cells. Incoming DNA can be degraded and/or can be incorporated into the chromosomal DNA. The recipient cell is usually in charge of DNA uptake. On the other hand, conjugation is a more invasive mechanism where the donor has control over the transferred DNA. It requires direct contact between two cells that may not be closely related. Mostly, small plasmids are transferred in this process. Conjugation is considered to be the main mechanism responsible for horizontal gene transfer (HGT). Transduction involves viruses that function as vehicles enabling DNA exchange between closely related species. Transfer of DNA via gene transfer agents (GTAs) which are virus-like elements encoded by the host genome, membrane vesicles and nanotubes, which are cellular protrusions that can bridge neighboring cells, are some other mechanisms that are also discussed for DNA transfer (Rothschild and Mancinelli, 2001; van Wolferen et al., 2013). DNA exchange has been demonstrated to occur in the archaeon Sulfolobus acidocaldarius by Grogan (1996).

The breakthrough research example was set by the discovery of DNA polymerase from a thermophilic bacterium (Thermus aquaticus) that was isolated from a hot spring in Yellowstone National Park. The enzyme became the driving ingredient in the polymerase chain reaction that made DNA testing possible and now it supports the billion dollars DNA replication industry. Enzymes from thermophilic microorganisms, often referred to as thermozymes, possess important characteristics such as temperature, chemical, and pH stability. Recent developments in research have shown that thermophiles are a good source of novel catalysts. As illustrated in Figure 3, the bioprospecting workflow typically involves steps from sample collection and isolation of thermophiles to enzyme screening, characterization, and subsequent optimization for industrial applications.

Thermostable polymer-degrading enzymes such as amylases, cellulases, chitinases, lipases, proteases pullulanases, and xylanases are being increasingly recognized for their role in food, chemical, pharmaceutical, paper and pulp, textiles, biorefineries, biofuels, and waste-treatment industries. Overexpression of thermozymes in standard E. coli allows the production of much larger quantities of enzymes, which are easy to purify by heat treatment. Most archaeal genes cloned in E. coli have been successfully expressed under the control of strong promoters. Once expressed in mesophilic hosts (prokaryotic/eukaryotic), the thermophilic enzymes are easier to purify by heat treatment (Haki and Rakshit, 2003; Koma et al., 2006; Tong et al., 2021). Furthermore, a recent study has demonstrated the application of thermozymes in industrial settings, such as the use of hyperthermoacidic proteases, amylases, and endoglucanases from thermophilic Archaea for efficient removal of thermophilic biofilms from stainless-steel surfaces, highlighting their practical relevance in food and dairy industry sanitation (Nam and Flint, 2023). Table 4 summarizes examples of thermozymes employed in different industrial applications.

The research areas relevant to thermophilic microorganisms are expanding across diverse fields. Microbiome investigations through culture-independent methods continue to unravel hidden functional capabilities within microbial communities. Although thermophilic fungi-the only eukaryotic lineage capable of thriving at elevated temperatures-have long been proposed as a rich source of thermostable enzymes, their enzyme repertoires remain underrepresented in public databases and in systematic enzyme discovery efforts compared to bacterial and archaeal thermophiles (Maheshwari et al., 2000; Mohammad et al., 2023). Promoting systematic research on thermozymes from thermophilic fungi could uncover novel enzymes with unique properties, offering significant advantages for industrial and pharmaceutical applications. Ongoing improvements in cultivation techniques and molecular biology are essential for advancing biotechnological research. Future investigations could focus on enhancing the thermal stability and catalytic efficiency of thermozymes through protein engineering and bioinformatics, thereby broadening their industrial and pharmaceutical applications. For example, the thermal stability and catalytic efficiency of thermozymes can be enhanced through protein engineering and bioinformatics tools, expanding their applications in industrial processes and pharmaceutical biotransformations. This will facilitate the discovery and deployment of new thermostable enzymes to meet growing demands in industry and clinical sectors.

In addition, adaptation strategies, including mechanisms of DNA transfer under extreme conditions and structural and functional adaptations in these organisms, remain important topics in fundamental research. Finally, the conservation of natural thermal environments requires focused attention from environmental managers. Initiatives such as the protection of the Geyser Basins of Yellowstone National Park under the “Geyser Protection Area” (Barrick, 2010) serve as examples of such efforts. Overall, a roadmap for future research should focus on: (i) integrating advanced omics approaches with improved cultivation strategies, (ii) engineering novel thermozymes for diverse industrial applications, (iii) exploring fundamental adaptive mechanisms in thermophiles, and (iv) conserving thermophilic ecosystems to enable sustainable utilization and study of these microorganisms.