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# "Freezing" Thermophiles: From One Temperature Extreme to Another

Tetyana Milojevic, Margaret Anne Cramm, Casey Hubert, Frances Westall

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Tetyana Milojevic, Margaret Anne Cramm, Casey Hubert, Frances Westall. "Freezing" Thermophiles: From One Temperature Extreme to Another. Microorganisms, 2022, 10 (12), pp.2417. 10.3390/microorganisms10122417. [https://doi.org/10.3390/microorganisms10122417](https://doi.org/10.3390/microorganisms10122417)

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Review

# “Freezing” Thermophiles: From One Temperature Extreme to Another

Tetyana Milojevic <fnref n="1" />,* , Margaret Anne Cramm <fnref n="2" />,† , Casey R. J. Hubert <fnref n="2" /> and Frances Westall <fnref n="3" />

**Abstract:** New detections of thermophiles in psychrobiotic (i.e., bearing cold-tolerant life forms) marine and terrestrial habitats including Arctic marine sediments, Antarctic accretion ice, permafrost, and elsewhere are continually being reported. These microorganisms present great opportunities for microbial ecologists to examine biogeographical processes for spore-formers and non-spore-formers alike, including dispersal histories connecting warm and cold biospheres. In this review, we examine different examples of thermophiles in cryobiotic locations, and highlight exploration of thermophiles at cold temperatures under laboratory conditions. The survival of thermophiles in psychrobiotic environments provokes novel considerations of physiological and molecular mechanisms underlying natural cryopreservation of microorganisms. Cultures of thermophiles maintained at low temperature may serve as a non-sporulating laboratory model for further exploration of metabolic potential of thermophiles at psychrobiotic temperatures, as well as for elucidating molecular mechanisms behind natural preservation and adaptation to psychrobiotic environments. These investigations are highly relevant for the search for life on other cold and icy planets in the Solar System, such as Mars, Europa and Enceladus.

**Keywords:** thermophiles; low temperature; cold environments

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**Citation:** Milojevic, T.; Cramm, M.A.; Hubert, C.R.J.; Westall, F. "Freezing" Thermophiles: From One Temperature Extreme to Another.
*Microorganisms* **2022**, *10*, 2417.
https://doi.org/10.3390/microorganisms10122417

Academic Editors: Alain Dolla and Jean Luc Cayol

Received: 30 September 2022

Accepted: 30 November 2022

Published: 6 December 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

## 1. Introduction

A variety of extremophilic microorganisms have been found in environments characterized by parameters extending beyond the range of their physiological activity [1,2], such as thermophilic bacterial endospores in low temperature marine settings [3]. The occurrence of thermophiles (organisms which grow above 40 °C) in psychrobiotic environments has been described in several independent investigations (Figure 1, Table 1). Thermophiles detected in cold marine and terrestrial habitats span several phyla across the tree of life, with both Bacteria and Archaea (Table 1). Recent 16S rRNA gene sequencing studies have uncovered a number of putative thermophiles in icy [4–7] and permafrost environments [8–11], permanently cold (−2 °C to +4 °C) ocean sediments [12–19], and in cool soils with temperatures never exceeding 25 °C [20–25] (Figure 1).

<footnote n="1">¹ Exobiology Group, CNRS-Centre de Biophysique Moléculaire, University of Orléans, Rue Charles Sadron, CEDEX 2, 45071 Orléans, France</footnote>

<footnote n="2">² Geomicrobiology Group, Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB T2N 1N4, Canada</footnote>

<footnote n="3">³ Exobiology Group, CNRS-Centre de Biophysique Moléculaire, Rue Charles Sadron, CEDEX 2, 45071 Orléans, France</footnote>

<footnote>* Correspondence: tetyana.milojevic@cnrs-orleans.fr; Tel.: +33-2-3825-5548</footnote>

<footnote>† Current address: School of Geography, Queen Mary University of London, Mile End Rd., Bethnal Green, London E1 4NS, UK.</footnote>
<figure><img src="image_6.png" /><figcaption>**Figure 1.** A conceptual framework for comprehensive assessment and understanding of the presence of thermophiles in cold marine and terrestrial habitats.</figcaption></figure>

<table><caption>**Table 1.** "Freezing" thermophiles in cold environments.</caption><thead><tr><th>Microorganisms</th><th>Growth Temperature Ranges</th><th>Psychrobiotic Environments</th><th>Temperature of Psychrobiotic Environments</th><th>References</th></tr></thead><tbody><tr><td colspan="5">"Arctic thermophiles" and other "cold marine sediment thermophiles"</td></tr><tr><td>Thermophilic Desulfotomaculum spp., Caminicella sp., Caloranaerobacter sp.</td><td>45 to 64 °C</td><td>marine surface sediment, Smeerenburgfjorden, Svalbard</td><td>-2 to +4 °C</td><td>[3,14]</td></tr><tr><td>Desulfotomaculum spp.</td><td>50 °C</td><td>marine surface sediment, west Spitsbergen, Svalbard</td><td>-2 to +7 °C</td><td>[19]</td></tr><tr><td>Desulfotomaculum arcticum</td><td>26 to 46.5 °C</td><td>marine sediment, Nordfjorden, Svalbard</td><td>+1.8 °C</td><td>[26]</td></tr><tr><td>Desulfotomaculum geothermicum, D. halophilum, D. thermosapovorans, D. arcticum, D. kuznetsovii, D. alkaliphilum, D. aeronauticum, D. australicum</td><td>46 to 69 °C</td><td>marine surface sediment, Aarhus Bay</td><td>0 to 15 °C</td><td>[13]</td></tr><tr><td>115 thermophilic Firmicutes primarily affiliated to Clostridiales, Bacillales, and Thermoanaerobiales orders</td><td>50 °C</td><td>marine surface sediments from 111 deep water location in Eastern Gulf of Mexico</td><td>not available</td><td>[17]</td></tr></tbody></table>
<table><caption>**Table 1. Cont.**</caption><thead><tr><th>Microorganisms</th><th>Growth Temperature Ranges</th><th>Psychrobiotic Environments</th><th>Temperature of Psychrobiotic Environments</th><th>References</th></tr></thead><tbody><tr><td colspan="5">"Arctic thermophiles" and other "cold marine sediment thermophiles"</td></tr><tr><td>146 thermophilic phylotypes affiliated to the <i>Bacillales</i>, and <i>Clostridiales</i> orders</td><td>50 °C</td><td>marine surface sediment from 81 locations around the world's ocean</td><td>0 to 50 °C</td><td>[16]</td></tr><tr><td><i>Geobacillus stearothermophilus</i></td><td>65 °C</td><td>marine sediment from basins of the continental slope from Ensenada, Mexico to Santa Catalina Island</td><td>~4 °C</td><td>[12]</td></tr><tr><td>Thermophilic bacteria primarily affiliated with <i>Tepidibacter Defluvialea</i>, <i>Bacillus</i>, and <i>Clostridiisalibacter</i> genera</td><td>50 °C</td><td>marine seawater and surface sediment, Aarhus Bay, Denmark</td><td>max. 15 °C</td><td>[27]</td></tr><tr><td><i>Desulfotomaculum kuznetsovii</i></td><td>50 to 70 °C</td><td>marine sediment, Aarhus Bay, Denmark</td><td>max. 15 °C</td><td>[13]</td></tr><tr><td><i>Thermosediminibacter oceani</i>, <i>Thermopsediminibacter litoriperuensis</i></td><td>52 to 76 °C</td><td>deep sea sediments, Peru Margin, eastern equatorial Pacific</td><td>Not available</td><td>[14]</td></tr><tr><td colspan="5">Cold seawater hyperthermophiles</td></tr><tr><td>Thermophilic and hyperthermophilic microorganisms <i>Hydrogenimonaceae</i>, <i>Aquificales</i> families</td><td>45 to 95 °C</td><td>open ocean seawater</td><td>3-30 °C</td><td>[28]</td></tr><tr><td>Hyperthermophiles, various <i>Thermococcus</i>-species</td><td>55 to 98 °C</td><td>low-temperature seawater</td><td>6 °C</td><td>[29]</td></tr><tr><td>Thermophilic and hyperthermophilic microorganisms, the genus <i>Thermococcus</i></td><td>55 to 98 °C</td><td>hydrothermal fluids in the northeastern Pacific Ocean</td><td>3-30 °C</td><td>[30]</td></tr><tr><td>Hyperthermophilic methanogenes <i>Methanocaldococcus</i> spp.</td><td>48 to 94 °C</td><td>low-temperature hydrothermal fluids of Axial Volcano and Endeavour Segment, northeastern Pacific Ocean</td><td>20-34 °C, 6 °C</td><td>[31]</td></tr><tr><td>Hyperthermophilic archaea of the order <i>Thermococcales</i></td><td>55 to 98 °C</td><td>diffuse hydrothermal vents, the northeast Pacific Ocean</td><td>4 °C</td><td>[32]</td></tr><tr><td>Hyperthermophiles <i>Pyrodictium</i>, <i>Pyrococcus</i>, <i>Thermococcus</i>, <i>Archaeoglobus</i> spp.</td><td>60 to 110 °C</td><td>open-sea waters next to Macdonald Seamount</td><td>4 °C</td><td>[33]</td></tr><tr><td colspan="5">Thermophiles in cool soils</td></tr><tr><td>Unclassified thermophilic bacillus, <i>Geobacillus thermoglucosidasius</i></td><td>40 to 80 °C</td><td>cool soil environments in Northern Ireland and Bolivia</td><td>max. 25 °C</td><td>[20]</td></tr><tr><td>53 thermophilic, aerobic, spore-forming and non-spore-forming bacteria; <i>Geobacillus thermoleovorans</i> <i>Geobacillus caldoxylosilyticus</i></td><td>optimum 70 °C</td><td>subsurface soil environment in Northern Ireland</td><td>max. 25 °C</td><td>[24]</td></tr><tr><td>Thermophilic bacteria <i>Geobacillus</i> spp. <i>Thermobacterium</i> spp.</td><td>36 to 65 °C</td><td>Icelandic basaltic and rhyolitic glass and minerals, sub-Arctic environments</td><td>-20.1-44.5 °C</td><td>[34]</td></tr></tbody></table>
<table><caption>**Table 1. Cont.**</caption><thead><tr><th>Microorganisms</th><th>Growth Temperature Ranges</th><th>Psychrobiotic Environments</th><th>Temperature of Psychrobiotic Environments</th><th>References</th></tr></thead><tbody><tr><td colspan="5">Thermophiles in cool soils</td></tr><tr><td>Geobacillus stearothermophilus</td><td>25 to 70 °C</td><td>Icelandic soil</td><td>max. 27 °C</td><td>[35]</td></tr><tr><td>Thermophilic bacteria</td><td>55 °C</td><td>Soil, Point Barrow, Alaska</td><td>max. ~20 °C</td><td>[36]</td></tr><tr><td colspan="5">Ice and permafrost thermophiles</td></tr><tr><td>Facultative chemolithoautotroph, thermophile from hot springs <i>Hydrogenophilus thermoluteolus</i></td><td>50 to 87 °C</td><td>Subglacial Lake Vostok's accretion ice, water</td><td>-2 °C</td><td>[5]</td></tr><tr><td>Thermophiles</td><td>50 to 87 °C</td><td>Subglacial Lake Vostok's accretion ice, water</td><td>-2 °C</td><td>[4,7]</td></tr><tr><td>Thermophilic and thermotolerant species: <i>Thauera hydrothermalis</i>, <i>Desulfurella acetivorans</i>, <i>Hydrogenophilus thermoluteolus</i>, <i>Bacillus</i> sp., <i>Thermobispora bispora</i>, <i>Anoxybacillus flavithermus</i>, <i>Thermosynechococcus</i> sp., <i>Caldimonas</i> sp., <i>Gordonia</i> sp., <i>Geobacillus</i> sp., <i>Amphibacillus</i> sp.</td><td>25 to 87 °C</td><td>Accretion ice originated from the embayment region of Lake Vostok</td><td>below 0 °C</td><td>[37]</td></tr><tr><td>Thermophilic community, Hyperthermophiles</td><td>55 to 95 °C</td><td>Frozen volcanic ash and scoria, the volcanic deposits of Deception Volcano</td><td>-2 °C</td><td>[10]</td></tr><tr><td>Thermophilic members of the genera <i>Geobacillus</i>, <i>Brevibacillus</i>, <i>Anoxybacillus</i>, <i>Thermus</i>, and <i>Bacillales</i></td><td>35 to 79 °C</td><td>Glaciers, Deception Volcano</td><td>0 °C</td><td>[38]</td></tr><tr><td>Thermophilic methanogens, acetogens and sulfate reducers growing on CO<sub>2</sub> and H<sub>2</sub>; extremely thermophilic bacteria of the genus <i>Caldicellulosiruptor</i></td><td>optimum 65 to 75 °C</td><td>Permafrost in the Kamchatka peninsula</td><td>-2 °C</td><td>[10]</td></tr><tr><td>Moderate thermophiles</td><td>-</td><td>Antarctic permafrost</td><td>max. 0 °C</td><td>[8]</td></tr><tr><td>Thermophilic non-spore-forming bacteria of the genus <i>Geobacillus</i></td><td>37 to 78 °C</td><td>Permafrost from volcanic sedimentary rocks of the Deception Island</td><td>-2 °C</td><td>[39]</td></tr><tr><td colspan="5">Laboratory models for the exploration of thermophiles in cold environments</td></tr><tr><td>Extreme thermophile <i>Methanobacterium thermoautotrophicum</i></td><td>78 °C</td><td>Several months of laboratory cultivation with the addition of sterile anaerobic sediments</td><td>15 °C</td><td>[40]</td></tr><tr><td>Geobacillus thermoleovorans strain T80</td><td>50-75 °C</td><td>Long-term (9-month) laboratory cultivation with soil microcosms</td><td>4 °C</td><td>[22]</td></tr><tr><td>Extreme thermoacidophile <i>Metallosphaera sedula</i></td><td>50-80 °C</td><td>2 months of laboratory cultivation in the presence of sterile multiametallic meteorite material (stony chondrite H5 type NWA1172)</td><td>12 °C</td><td>[41]</td></tr></tbody></table>
Thermophilic spores in cold marine sediments have been explored in detail as models for understanding passive dispersal and biogeography. They comprise >10% of the total endospore population in permanently cold sediments from Aarhus Bay [27] and their dormancy enables their rate of dispersal into the cold seabed to be estimated [3]. Spores are specialized cellular structures that protect cells from what would be lethal stresses such as radiation, desiccation, toxins, and extreme heat or cold, and allow the cells to withstand periods of nutrient starvation [42,43]. Spore formation can contribute to the survival of thermophiles in Arctic sediments through dormancy [9,26]. In soils, thermophilic spores are found in abundances of >1.5 × 10⁴ per gram [20,35]. It is intriguing how these thermophilic populations withstand psychro- and mesobiotic environments where temperatures are significantly below their minimum requirement for growth [25,44]. Typically, minimum temperatures reported for enabling growth and metabolic activity of thermophiles are obtained from experimental data using relatively short incubations and artificial culture conditions that differ from environmental conditions that the microorganisms were isolated from. Laboratory growth conditions are not normally designed to detect thermophiles at lower temperatures. The experimental data suggest that the extreme thermoacidophilic archaeon *Metallosphaera sedula* withstands cold stress and can be maintained at low temperature, when cultivated with multimetallic mineral material as the sole energy source [41]. These observations, after their further thorough analysis and validation, may open an avenue to investigate strategies and underlying molecular mechanisms of survival of thermophilic species in psychrobiotic environments (Figure 1).

Furthermore, unraveling the thermal limits of microbial life and the survival limits of thermophiles in low temperature habitats is also decisive for envisaging the microbial survival during transfer through space and between celestial bodies, e.g., in the context of lithopanspermia. Microbial "space travelers" are subject to extreme temperature fluctuation during their interstellar transit [45,46]. Microbial cryopreservation studies are also helpful in terms of defining possible spatial and temporal near-surface microenvironments suitable for microbial life beyond Earth, e.g., on Mars, where microorganisms may be protected not only against harsh radiation, but also from extreme temperature fluctuations (Figure 1). Recently, microniches in the Martian regolith containing chaotropic ions that could favor microbial growth and preservation at low temperature and thus support the potential Martian biosphere [47], have been proposed by Chin et al. [48]. Moreover, comprehensive investigations into the survival limits of thermophiles in low temperature habitats may add new knowledge on ancestral thermophiles with simpler and smaller genomes in cool primordial environments and complement the on-going scientific debates concerning the psychrophilicity or thermophilicity of a last universal common ancestor [49–53].

1.1. "*Arctic Thermophiles*" and *Cold Seawater Hyperthermophiles*

The discovery of thermophiles in any cold environment is always a somewhat peculiar
event, and observations of these misplaced microbes are downplayed in polar habitats.
Thermophilic spore-forming bacteria ("thermospores") in the marine sediments of Sval-
bard, at almost 80° North, have been reported on different occasions [3,19,26]. This is
consistent with a long history of thermophiles detections in sediments with in situ tem-
peratures too cold for their growth [12,13,36,54]. They remain dormant and inactive at the
cold temperatures at which they are found and are detected only after pasteurization and
high-temperature incubation of marine sediment. Arctic thermospores in Svalbard fjord
sediments typically grow at 45-65 °C. Research has focused on their occurrence as spores
and how this dormancy mechanism equips these bacteria for long-term viability in psy-
chrobiotic environments as members of the microbial seed bank and rare biosphere [55,56].

As dormant spores, these thermophiles offer models for studying microbial biogeog-
raphy and dispersal. Psychrobiotic environments maintain the dormant phenotype of
thermophilic spores and in this state, they are not influenced by factors typically shaping
biogeography that rely on microbial activity and growth, such as environmental selection,
drift and mutation [19]. Thus, the dormant phenotype of thermophilic endospores enables
focused investigations of passive microbial dispersal, which is another factor shaping microbial biogeography. For example, rates of dispersal can be quantified by evaluating the number of non-growing spores in a sediment depth profile in relation to the sediment accumulation rate in that location. This is shown through high temperature germination experiments by Hubert et al. [3], where a stable abundance of thermophilic spores (10⁵ g⁻¹ sediment) with depth was combined with the sedimentation rate (determined by ²¹⁰Pb decay to be 0.2 cm y⁻¹ in this Svalbard fjord) pointing to a constant flux of thermophilic spores into the seabed at a deposition rate of 10⁸ m⁻² y⁻¹. Using a similar high temperature incubation approach, de Rezende et al. [15] determined a comparable thermospore deposition rate (10⁷ m⁻² y⁻¹) in Baltic Sea sediment, and furthermore identified some of the same thermophile phylotypes as those being deposited in the Svalbard fjord sediments, despite >3000 km distance between the two locations. At a finer geographic scale, Arctic thermophilic spores enriched from marine sediment from 10 different fjord sites along the west coast of Spitsbergen, Svalbard showed biogeographic patterns suggesting different warm-to-cold dispersal histories and source environments for different taxa [19]. Conversely, return of thermospores from cold to hot environments through sediment burial has been suggested by Heuer et al. [57] and Gittens et al. [58]. These studies highlight the utility of thermophilic spores for exploring the temporal and lateral extent of microbial dispersal.

These observations have led to new hypotheses about dispersal vectors linking warm habitats to the cold ocean, and the fluxes needed to facilitate such dispersal. Several studies have shown that marine microbial diversity and biogeography are influenced by ocean currents [59–62]. These influences determine distributions of marine microorganisms in general, including thermophilic spores, and imply that microbial propagules can be transported long distances via connected water bodies. Thermophilic spores have also been detected in the water column (through incubating filters at high temperature) above the cold marine sediments in which they have been studied [15,27] suggesting passive transport from seawater into the seabed via sedimentation. Particulate transport from the water column into the sediments is often facilitated by what are known as marine flocs or marine snow. This could serve as a vector for transport of microbial propagules. Furthermore, Müller et al. [16] identified an association between the connectivity of a marine sediment sites to the global ocean circulation and the diversity and richness of its thermophilic spore-forming populations. Areas more connected to global ocean currents show a greater diversity of thermophiles, and these connected sites share a greater number of thermospore phylotypes in common.

The dispersal of thermophilic spores into cold sediments (Table 1, Figure 1) raises questions about their origins, i.e., the warm habitat(s) that they must be supplied from. In the examples above, a recurring result of 16S rRNA gene and other marker gene sequence analyses have shown that cold sediment thermophiles are often most closely related to *Clostridia* from warm sub-marine deep biosphere habitats (e.g., petroleum reservoirs and mid ocean ridge systems) [3,14–17,63]. Typically, several taxonomic groups of thermophilic spores are identified in a given location, indicating the possibility that multiple warm source habitats are seeding thermophilic spores into the ocean and eventually into the studied cold ocean sediments [19]. Similarly, thermophiles found in terrestrial cold environments may be dispersed via atmospheric currents, as has been speculated by Ziegler [25]. In this way, studies of thermophiles existing as dormant microbial propagules in permanently cold environments hint at underlying mechanisms of microbial passive dispersal that contribute to marine microbial diversity, and are separate from confounding factors of environmental selection, drift, and mutation. While these dispersal mechanisms may be identified through the study of thermospore biogeography, the mechanisms themselves may influence and contribute to marine microbial diversity beyond the dormant microbiome. Additionally, activity assays that retain taxonomic identity, such as meta-transcriptomics and genomic-based stable isotope probing, should be used to investigate whether low-level psychrotolerant activity by spore-forming thermophiles occurs, and whether the presence and activity of thermophiles shapes the active microbial community in cold marine sedi-
ment. Along with spores of thermophiles detected in psychrobiotic and mesobiotic marine sediments, spreading and long-term survival of non-spore-forming hyperthermophiles at relatively low temperatures (2 to 4 °C) in cold, oxic seawater has also been reported [33,64] (Table 1, Figure 1). Thermophilic and hyperthermophilic microorganisms (e.g., belonging to the genus *Thermococcus*) have been cultured from 3–30 °C hydrothermal fluids in the northeastern Pacific Ocean [28]. Laboratory studies show that hyperthermophiles (various *Thermococcus*-species) can survive at least 9 months in cold surroundings when stored in low-temperature seawater [30]. Hyperthermophilic methanogens have been isolated from low-temperature (20–34 °C and 6 °C) hydrothermal fluids of Axial Volcano and Endeavour Segment in the northeastern Pacific Ocean [29]. Furthermore, hyperthermophilic *Thermococcales* show long-term survival at low temperatures (4 °C), which is important for the wide distribution, dispersal, and dominance of these archaea at deep-sea hydrothermal vents [31]. The presence of hyperthermophilic microorganisms in low-temperature vent fluids emanating from the seafloor suggests that they originate from a subseafloor hydrothermal habitat where temperatures are within their growth range. It has been proposed that hyperthermophiles present in low abundance in cold seawater in a hibernation-like state, are incidentally transferred to the outside of black smokers and actively occupy specific niches in chimney walls, where conditions are optimal for their physiology [32].

The dispersal of hyperthermophilic microorganisms via cold seawater (Table 1, Figure 1) raises questions about how these heat-loving non-sporulating cells can withstand conditions detrimental to their physiological requirements. Adherence to surfaces and motility have been suggested as factors promoting long-term survival of black smoker-associated hyperthermophiles in cold seawater [30,32]. The exact mechanism of microbial colonization of newly formed black smokers still remains to be investigated experimentally. The capabilities for thermo- and chemotaxis enable cells to quickly react to encountering high temperatures, e.g., by means of very fast motility the cells swim in the direction of the warm temperature, moving from very cold to hot environments. After reaching an optimal temperature region they can “explore” the surface via a zigzag-movement and use their motility to establish contact, adhering with their flagella at a suitable place and forming biofilms. Wirth (2017) [32] proposes that additional adhesins may contribute to permanent colonization of black smokers. In this way, studies of these cold seawater hyperthermophiles are revealing possible passive dispersal mechanisms facilitating global connectivity of marine habitats important for microbial distribution.

## 1.2. *Thermophiles in Cool Soils*

The presence of high numbers of thermophilic bacteria in cool soil environments has been shown for subsurface soil samples from different continents [20–25]. Following enrichment at 70 °C and 80 °C, five highly thermophilic aerobic bacteria were isolated from cool soil environments in Northern Ireland and Bolivia [20]. The thermophilic isolates showed high growth rates at 70 °C, some with doubling times shorter than 30 min, and were characterized by unusual cellular morphology, including long flexible cells as well as short rods. Based on 16S rRNA gene sequence analysis, these isolates are closely related to *Geobacillus thermoglucosidasius* and an unclassified member of the thermophilic geobacilli (98%) [20]. The isolation of 53 thermophilic, aerobic, spore-forming and non-spore-forming bacteria was reported for the subsurface soil environment in Northern Ireland [24]. The majority were affiliated with *Geobacillus thermoleovorans* (n = 27) or *Geobacillus caldoxylosilyticus* (n = 18). Several isolates exhibited only 93% similarity with *Geobacillus toebii* strain F70, thus likely representing new species of *Geobacillus*. Interestingly, closely related bacteria are widely distributed in other soil environments, suggesting that—contrary to the thermospores in cold marine sediments—the soil thermophiles might not be introduced from an external source, but are rather autochthonous members of these environments [24]. Alternatively, an estimated thermophile input rate of 1.4 × 10⁵ cells m⁻² yr⁻¹ in Irish soils has been suggested, sustaining a viable population of thermophiles in cold temperate environments. Plate counting suggests that these thermophiles may comprise up to 10% of the
total cultivable aerobic bacteria in cool soils (1.5–8.8 × 10⁴ colony forming units g⁻¹) [22,23]. Dispersal may be facilitated by continuous transfer into soils via cloud droplets, providing a constant supply of thermophiles into these environments [22,23].

The isolation of thermophilic bacteria from cool soils has elicited a research interest concerning the possible metabolic activity by these microorganisms in conditions that differ from their optimum requirement for growth (Figure 1). Survival of a representative cool soil isolate, *Geobacillus thermoleovorans* strain T80, was investigated at low temperatures, revealing an increase in its population during a long-term (9-month) cultivation at 4 °C [22]. This increase was attributed to very slow growth rates accompanied by negligible rates of cell death. Interestingly, cultures of *G. thermoleovorans* maintained at temperatures slightly higher (25 °C) but still below the organism's growth-permissive thermal range, did not show a similar population size increase. The increase of cell density only at 4 °C appears to reflect a preservation mechanism for suppressing cell death. Microcosms spiked with *G. thermoleovorans* also showed that the surviving viable geobacilli could persist at low temperatures after 1 week of cultivation [22].

The hypothesized existence of nanoniches in mesobiotic environments has been proposed by Wiegel et al. [65] based on similar observations of anaerobic alkalithermophiles isolated from mesobiotic environments that grow faster than those isolates from thermobiotic habitats. It was proposed that transient niche establishments in these environments provide short-term limited conditions for alkaliphilic thermophilic bacteria and therefore enable elevated growth rate in order to cope with this limitation [65]. At the molecular level, it has been proposed that microorganisms capable of growth at temperatures considered both thermobiotic and mesobiotic may encode different sets of key enzymes whereby gene expression and protein synthesis of either set is regulated by temperature [40].

Investigating microbiological populations within Icelandic basaltic and rhyolitic glass and minerals (−20.1–44.5 °C), researchers detected putative thermophiles as a minor component of microbial communities of these volcanic rocks [66]. Once again, these microbial species are atypical for a sub-Arctic environment where the temperature does not approach the range required for thermophile activity. *Geobacillus* and *Thermobacterium* sequences have been identified via G2 PhyloChip-assisted microarray analysis of 16S rRNA gene amplicons from the whole rock community genomic DNA at this site [34,67]. Seven thermophiles belonging to the genus *Geobacillus* were isolated from the rock Icelandic community, with several exhibiting a minimum growth temperature of 36 °C [66]. These thermophiles may be dispersed from Icelandic hot springs or from aerosols originating outside Iceland. Year-long measurements of the temperatures in crystalline basalt and obsidian glass at 2 cm depth reveal that the thermal conductivity of the rocks was sufficient to transiently allow for temperatures above 36 °C, up to 44.5 °C. The low albedo of these rocks has been suggested to facilitate their thermal conductivity such that the near-surface environment of these igneous materials can support transient growth of thermophiles in summer months [66]. Icelandic thermophiles are proposed to transiently contribute to local biogeochemical processes, since their microclimatic environment inside the rock pores provides a certain potential for their temporary "re-awakening" during the warm periods of the year. Therefore, similar to the aforementioned *Geobacillus* isolates in cool soils, these thermophiles may be indigenous inhabitants, not necessarily exogenous propagules, and could perform ecologically relevant functions. In this way they may be similar to thermophilic bacteria found in temperate soils that participate in C, N, S, and P cycling [68,69] and treatment of wheat crops with thermophilic *Ureibacillus* which benefits the crops by reducing toxic metals and increasing plant stress tolerance [70].

### 1.3. *Ice and Permafrost Thermophiles*

Accretion and glacial ice of Lake Vostok is another permanently cryobiotic environment where molecular signatures for thermophilic life have been reported (Table 1). Underlying a 4 km thick Antarctic ice sheet, the subglacial Lake Vostok represents an extreme environment with pressure approaching 400 atmospheres, an ultra-low nutrient
supply from the glacier melt, water temperature averaging –2 °C, and a possible excess of oxygen and nitrogen [4,5]. This environment was assumed to be lifeless until DNA-based assays suggested that this is a unique ecosystem supporting a diverse microbiota, but at cell abundances lower than in most environments on Earth [71]. Thermophiles have been detected in samples of Lake Vostok accretion ice, i.e., naturally frozen lake water accreted to the glacier floating above the lake. Microbial life resident in the subglacial Lake Vostok is represented by the facultative chemolithoautotroph *Hydrogenophilus thermoluteo-lus* (*Proteobacteria*, class *Hydrogenophilalia*), a non-spore-forming thermophile that has also been isolated from hot springs. Initially, *H. thermoluteolus* was detected in a sample of a Lake Vostok borehole drilled at a depth of 3607 m [5]. Later, *H. thermoluteolus* has been recognized with high confidence as one 16S rRNA bacterial phylotype with relevance to the lake environment [4]. The authors applied stringent decontamination techniques and contaminant database criteria, performing molecular analyses in two different laboratories. The results of 16S rRNA gene sequencing have been confirmed by targeted analysis of *cbbL/rbcL* (encoding RubisCO in most chemoautotrophs) and *hox/hya/frh/hup* (encoding a NiFe hydrogenase) genes from *H. thermoluteolus* in the same samples [4]. More recent inves-tigations of Lake Vostok ecology based on metagenomic and metatranscriptomic analyses of accretion ice, has uncovered the presence of additional putative thermophiles residing in both types of accretion ice [7]. Naturally, the presence of drilling fluid traces in polar ice cores raises the important question of contamination during sampling, representing a real challenge for associated molecular biological studies. As an approach to address this contamination issue, the analysis of the drilling fluids obtained from a Lake Vostok borehole has been performed [72]. The 16S rRNA gene amplicon sequencing of samples derived from the drilling fluids used during the ice core retrieval process [72] revealed dominated putative hydrocarbon-degrading organisms and less represented populations that cannot metabolize hydrocarbons. Alekhina et al. [72] proposed that all the bacteria present in these drilling fluid samples must be considered as contaminants in the context of Lake Vostok analyses. However, *Hydrogenophilus* spp. were not detected in drilling fluids samples. The very low organic carbon and nutrient contents in accretion ice of Lake Vostok suggests that conditions favor chemolithoautotrophic life. The existence of deep faults at the bottom of the lake has been proposed, where hydrogen would permit chemolithotrophs such as *H. thermoluteolus* to flourish. Indeed, Lake Vostok is a tectonically controlled subglacial lake with recently recorded minor tectonic activity. The hydrothermal plumes caused by episodic seismotectonic activity in deep faults in the bedrock of the lake could release sediments along with microbial cells from faults towards vents in the freezing zone, where the microbes may have been entrapped in the ice in course of accretion [4–6]. The cultivation and recovery of viable *Hydrogenophilus* spp. from the accretion ice would offer direct evidence that the frozen cells in Lake Vostok can be revived.

A number of thermophilic and thermotolerant chemolithoautotrophs that use in-organic sources of energy (e.g., hydrogen, nitrogen, sulfur, or iron) to support their metabolism have recently been detected by metatranscriptomic and metagenomic ap-proach in samples of accretion ice originated from the embayment region of Lake Vostok (3563 + 3585 m) [37]. The large number of unique and total sequences of these microorgan-isms was found to be in line with increases in concentrations of nutrients and microelements (amino acids, organic carbon, Na+, Ca2+, Mg2+, Cl- and SO42-). The authors suggest that the influence of the hydrothermal activity located several kilometers away, or the pres-ence of saltwater in this region (e.g., a brine layer) may contribute to a high number of thermophilic and thermotolerant organisms. Further metaproteomic and metabolomic profiling of these environmental samples might be helpful in order to characterize the activity of thermophiles in such icy environments.

Fascinating hydrothermal springs that occur in the continuous permafrost zone have described to harbour thermophilic microbial communities [73]. Thermophilic species in the permafrost of Kamchatka peninsula and Deception Island have been revealed among microbial communities of frozen sedimentary deposits [8–11] (Table 1, Figure 1). These
studies were primarily conducted by the research group led by the late David Gilichinsky, a
leading permafrost scientist who focused on microbial preservation in ancient ice. Mironov
et al. [10] reported the presence of a thermophilic community in frozen volcanic ash and
scoria. In this study, hyperthermophiles were isolated from a borehole in the volcanic
deposits of Deception Volcano. These cultures were shown to utilize sulfur as an electron
acceptor and grow optimally at 85–95 °C. Mironov et al. [10] described thermophilic
methanogens, acetogens and sulfate reducers growing on CO2 and H2 that were enriched
from permafrost in the Kamchatka peninsula. Optimal growth between 65 and 75 °C
was observed for these cultures, including one that shares 100% rRNA gene sequence
identity to extremely thermophilic bacteria of the genus *Caldicellulosiruptor* [10]. A group
of moderate thermophiles isolated from Antarctic permafrost (six surface and subsurface-
isolated isolates) has been also previously described [8]. Mironov et al. [39] reported on
thermophilic non-spore-forming bacteria of the genus *Geobacillus* in permafrost samples
from volcanic sedimentary rocks of the Deception Island. Naturally, the isolation of
viable thermophiles and hyperthermophiles raised questions about the origin of these
microorganisms and their lifestyle in such environments. It was suggested that some groups
of anaerobic thermophilic bacteria, associated with volcanic activity, might have survived in
permafrost over long periods, becoming embedded and stored in frozen sediments [9]. The
authors considered a volcanic eruption or surrounding strata associated with volcanoes as
possible sources of thermophiles that were isolated from permafrost [9]. Microbial airborne
dispersal is most probably facilitated by sedimentation of ashes, pyroclastic material, and
atmospheric precipitation [9]. Thermophilic microbial communities in permafrost regions
(Table 1) may represent the most ancient storage and persistence of thermophiles preserved
in the frozen state.

Applying cultivation techniques and 16S rRNA gene sequencing, a recent study re-
ported the presence of culturable thermophiles along the extreme temperature gradient
in Deception volcano [38]. Thermophilic members of the genera Geobacillus, Brevibacil-
lus, Anoxybacillus, Thermus, and Bacillales order were recovered from both fumaroles and
glaciers, with environmental temperatures between 80 and 0 °C. The majority of these
described recovered thermophilic isolates in the Deception glaciers (0 °C, Table 1) were
spore-forming bacteria with a good culturability and a pronounced resistance towards
desiccation and UV-C irradiation [38]. The authors proposed the coexistence and a potential
interplay between thermophiles and psychrophiles in polar volcano Deception. The results
of this work indicate that these thermophiles remain viable even when environmental
conditions are outside their growth range [38].

1.4. *Laboratory Models for the Exploration of Thermophiles in Cold Environments*

Our understanding of the genetics and physiology of how thermophiles adapt to the
cold is still in its infancy. Consequently, physiological capacities, metabolic pathways and
molecular mechanisms underlying preservation of thermophiles in cold environments have
not been thoroughly investigated. Molecular approaches targeting global gene expression,
proteomics and metabolomics profiles could deliver an enhanced view of the specific
adaptation of thermophilic life to low temperatures. The major limitation in this field comes
from a restricted cultivation of thermophiles at low temperature regiment underlaboratory
conditions. The rare exception is the *G. thermoleovorans* strain T80, which was cultivated
at 4 °C during a long-term period (9-month) and in soil microcosms at low temperatures
during short-term experiments (1 week) [22].

Wiegel [40] described that extreme thermophile *Methanobacterium thermoautotrophicum*
can grow between 22 and 78 °C and the addition of sterile anaerobic sediments permitted
its incubations at lower temperatures. Moreover, *M. thermoautotrophicum* strain JW 501
was capable of steady methane production at 15 °C over several months, suggesting some
metabolic activity under non-growth permissive temperature [40].

The extreme thermoacidophile *Metallosphaera sedula* is among thermophiles supported
at cold temperatures under the laboratory conditions (Table 1, Figure 2). *M. sedula* is
a facultative chemolithotroph and metal-mobilizing archaeon, which respires iron and reduced inorganic sulfur compounds [74–82]. *M. sedula* is known as an obligate thermophile with a temperature range from 50 to 80 °C permissive for growth and an optimum of 73 °C [83,84]. It is worth noting, that *M. sedula* was first isolated from the sediments of Pisciarelli solfatara (Naples, Italy), where the temperature was much lower, between 25 and 52 °C [83]. Interestingly, none of the energy sources that the organism uses at higher temperatures (pyrite, chalcopyrite, and other inorganic electron donors) are able to support the cultivation of *M. sedula* in this lower temperature range [83,84]. The only exception is the chemolithoautotrophically grown *M. sedula* cultivated at low temperatures (Figure 2) [41] in the presence of preliminary powdered and sterilized multimetallic meteorite material (the stony chondrite H5 type NWA1172 [81,85]) as the sole energy source. Supplementation with NWA 1172 permitted the cultivation of *M. sedula* during two months at the average temperature of 12 °C [41]. The presence of *M. sedula* cells in these experiments was confirmed by multi-labeled fluorescence in situ hybridization (MiL-FISH) with an *M. sedula*-specific ribosomal RNA-targeted probe [81,86], as well as sequencing of the *M. sedula* specific marker gene *msed0966* (Figure 2B) [41]. Interestingly, cold-maintained *M. sedula* cells were characterized by a branched extracellular matrix on the cell surface (Figure 2C,D). This layer of cellular appendages wrapping around the colonies of *M. sedula*, together with a tendency to clump and condense into cellular aggregates, may represent a possible adaptive strategy for withstanding cold conditions [41]. *M. sedula* cells maintained at low temperature thus offer a laboratory model to investigate metabolic potential of non-spore-forming extreme thermophiles in cold environments, microbial strategies, and molecular mechanisms behind natural cryopreservation.

<figure><img src="image_7.png" /><figcaption>**Figure 2.** The cultivation of *M. sedula* cells with the stony meteorite NWA 1172 as the sole energy</figcaption></figure>
source at the cold temperature regime. (A) The cultures of *M. sedula* at low temperature in 1 L glassblower modified Schott-bottle bioreactors. *M. sedula* cultures were incubated at the average temperature of 12 °C in an ice-supplemented environment (modified from Milojevic et al. [41]). (B) The multi-labeled fluorescence in situ hybridization (MiL-FISH) of *M. sedula* cells supplemented with the stony meteorite NWA 1172 as the sole energy source at low temperature. An overlaid epifluorescence image shows overlap of specific probe with DAPI signals (modified from Milojevic et al. [26]). Cultures of *M. sedula* were examined with MiL-FISH [78,83] following 2 months of cultivation at 12 °C. (C) A scanning electron microscopy (SEM) image of *M. sedula* supplemented with the stony meteorite NWA 1172 as the sole energy source at low temperature. (D) A higher magnification SEM image displaying single cells of *M. sedula* covered with extracellular matrix. Cells of *M. sedula* are indicated by the white arrows. Cultures of *M. sedula* were examined with SEM after 2 months of cultivation at the average temperature of 12 °C.

In similar work, Chin et al. [48] also proposed that specific additives such as chaotropic metabolites and generally chaotropic environments can increase tolerance to subzero temperatures, extending growth windows for microbial survival in cold environments. Taken together, several studies indicate that the utilization of specific additives, including energy substrates and/or nutrients, e.g., the addition of sterile anaerobic sediments [40], genuine soil microcosms [22], and metal-rich mineral material [41], can dramatically influence the resistance to cold stress, increasing the preservation of thermophiles in the cold.

## 1.5. Potential for Thermophiles in Extraterrestrial Environments

In terms of astrobiology and the search for extraterrestrial life, the existence of thermophiles in cold environments is of great interest. Mars was habitable in the past, having liquid water, organic molecules and sources of energy (redox gradients, heat, radiation) [87]. Indeed, investigations of the Curiosity rover in Gale Crater revealed that the lake waters had a relatively neutral pH, as documented by the smectite clays, minerals, such as magnetite, Fe-sulphates and -sulphides, compounds indicating variable redox states, and the relatively long-lived presence of water, all suggesting the existence of habitable conditions about 3.6 Ga [47,88]. Moreover, recent investigations have detected organic molecules in situ [89–91]. Mars has also provided the essential palette of biological building blocks: energy sources, Carbon, Hydrogen, Nitrogen, Oxygen, Phosphorous and Sulfur (CHNOPS) elements, as well as important catalytic transition metals associated with life, as we know it. However, while traces of past life may be found at the surface of the ancient terranes of Mars, the objectives of the Mars Science Laboratory and the Curiosity rover [47], the Mars 2020 mission [92], and the ExoMars 2022 mission with its Rosalind Franklin rover [93], extant life, if it still exists, will be in the subsurface. Mars is very cold; its surface may be compared with permafrost and its subsurface aquifers are most likely frozen. However, in the very recent past (~50,000 y) volcanic activity has occurred [94]. Molten lavas rising through the cryosphere and permafrost could create habitable niches with temperatures ranging from thermobiotic to the psychrobiotic, thus permitting cold-preserved cells to revive and metabolise and, possibly, reappear briefly at the surface of the planet with any volcanically-associated hydrothermal fluids.

The icy satellites, Europa and Enceladus, orbiting around Jupiter and Saturn, respectively, are considered to be habitable and potentially inhabited [95–98]. Mobility of the icy crust and plumes of volatiles (water, salts and organic molecules (the latter for Enceladus)) [99–101] ejected from their surfaces testify to the presence of liquid water below the icy shells. In both cases, the presence of a fractionated core and mantle, plus tidal heating due to gravitational pull from the parent planets, suggest the possibility of hydrothermal activity at the rock/water interface at the bottom of the oceans. Missions, such as Europa Clipper and the proposed Europa Lander [102,103], aim to search for signatures of life in the plumes and/or icy crust of Europa. If life exists on either of these two icy bodies, it may
well be some form of thermophilic life form, that could be trapped in the ice (as at Lake Vostok) or expelled in the plumes.

## 1.6. Molecular Targets to Address Mechanisms of Survival of Thermophiles in Psychrobiotic Environments

The metabolic and molecular mechanisms that could allow viability and even minimal metabolic activity of thermophiles in cold environments are largely unknown and unexplored. Thermophilic life forms have been reported in a variety of natural environments with different degree of human influence ranging from such areas as Lake Vostok with no human habitation to soil environments close to settlements with a high index of anthropogenic influence (Table S1). Apparently, anthropogenic activity can be excluded as a factor affecting the distribution of thermophiles in cold settings. At the same time, the analysis of extremophilic properties of the reported thermophiles in Table 1 shows that "freezing" thermophiles are polyextremophilic microorganisms capable of tolerating several environmental extremes (Table 2). Apart from heat resistance, predominant majority of these thermophiles have metal tolerance or even resistance and require no oxygen for their lifestyle. Almost a half of these thermophiles can live on the account of inorganic carbon source (CO₂), frequently in extreme pH conditions and in salt rich environments. Some of them are radiation tolerant species and several can withstand or even flourish in high pressure conditions (Table 2). "Freezing" thermophiles in Table 1 are almost equally represented by both spore-forming and non-spore-forming species. Hence, the physiological and molecular mechanisms of their adaptation to cold environments might go beyond the formation of endospores. Other extremophilic properties summarized in Table 2 (e.g., resistance against metals, pH extremes, radiation, high pressure and salinity) can also contribute to the explanation of the presence of these organisms in cold settings. Further focusing on the mechanisms of the maintenance and persistence of thermophilic microorganisms in psychrobiotic environments may lead to the development of new concepts of microbial physiological plasticity.

<table><caption>**Table 2.** Extremophilic properties of "freezing thermophiles" presented in Table 1.</caption><thead><tr><th>Thermophile</th><th>Heat Resistance</th><th>Metal Tolerance</th><th>Radiation Resistance</th><th>Pressure Resistance</th><th>Desiccation Resistance</th><th>pH Extremes</th><th>Autotrophy</th><th>O₂</th><th>Spore</th><th>Salt Tolerance</th><th>Ref.</th></tr></thead><tbody><tr><td>Desulfotomaculum spp.</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>—</td><td>+</td><td>+</td><td>[104,105]</td></tr><tr><td>Caminicella spp.</td><td>+</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>—</td><td>—</td><td>+</td><td>+</td><td>[106]</td></tr><tr><td>Caloranaerobacter spp.</td><td>+</td><td>+</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>—</td><td>—</td><td>+</td><td>+</td><td>[107]</td></tr><tr><td>Bacillales</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>—</td><td>+/-</td><td>+</td><td>+</td><td>[108,109]</td></tr><tr><td>Thermoanaerobiales</td><td>+</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>+</td><td>—</td><td>—</td><td>+</td><td>N/A</td><td>[110,111]</td></tr><tr><td>Geobacillus spp.</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+/-</td><td>+/-</td><td>+</td><td>—</td><td>[112]</td></tr><tr><td>Tepidibac-ter</td><td>—</td><td>+</td><td>+</td><td>N/A</td><td>+</td><td>N/A</td><td>—</td><td>—</td><td>+</td><td>+</td><td>[113,114]</td></tr><tr><td>Clostridiisalibacter</td><td>—</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>—</td><td>—</td><td>+</td><td>+</td><td>[115]</td></tr><tr><td>Thermosediminibacter</td><td>+</td><td>+</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>—</td><td>—</td><td>—</td><td>—</td><td>[54]</td></tr><tr><td>Thermo-coccus spp.</td><td>+</td><td>+</td><td>+</td><td>+</td><td>N/A</td><td>+</td><td>—</td><td>—</td><td>—</td><td>+</td><td>[116,117]</td></tr><tr><td>Methanocaldococcus spp.</td><td>+</td><td>+</td><td>N/A</td><td>N/A</td><td>N/A</td><td>—</td><td>+</td><td>—</td><td>—</td><td>—</td><td>[118]</td></tr><tr><td>Pyrodictium</td><td>+</td><td>+</td><td>N/A</td><td>+</td><td>N/A</td><td>—</td><td>+</td><td>—</td><td>—</td><td>+</td><td>[119]</td></tr><tr><td>Pyrococcus</td><td>+</td><td>+</td><td>+</td><td>+</td><td>N/A</td><td>—</td><td>—</td><td>—</td><td>—</td><td>N/A</td><td>[120–122]</td></tr><tr><td>Archaea-globus</td><td>+</td><td>+</td><td>+</td><td>+</td><td>+</td><td>N/A</td><td>+/-</td><td>—</td><td>—</td><td>+</td><td>[123]</td></tr><tr><td>Hydrogeno-philus thermo-luteolus</td><td>+</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>—</td><td>+</td><td>+</td><td>—</td><td>N/A</td><td>[124]</td></tr><tr><td>Thauera hydrother-malis</td><td>+</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>+</td><td>—</td><td>—</td><td>—</td><td>N/A</td><td>[125]</td></tr></tbody></table>
<table><caption>**Table 2. Cont.**</caption><thead><tr><th>Thermophile</th><th>Heat Resistance</th><th>Metal Tolerance</th><th>Radiation Resistance</th><th>Pressure Resistance</th><th>Desiccation Resistance</th><th>pH Extremes</th><th>Autotrophy</th><th>O<sub>2</sub></th><th>Spore</th><th>Salt Tolerance</th><th>Ref.</th></tr></thead><tbody><tr><td>Desulfurella acetivorans</td><td>+</td><td>+</td><td>N/A</td><td>N/A</td><td>N/A</td><td>+</td><td>+/-</td><td>-</td><td>-</td><td>N/A</td><td>[126]</td></tr><tr><td>Thermobispora bispora</td><td>+</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>-</td><td>+</td><td>+</td><td>N/A</td><td>[127]</td></tr><tr><td>Anoxybacillus flavi-thermus</td><td>+</td><td>+</td><td>+</td><td>N/A</td><td>N/A</td><td>-</td><td>+</td><td>-</td><td>+</td><td>N/A</td><td>[128]</td></tr><tr><td>Thermosynechococcus spp.</td><td>+</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>N/A</td><td>+</td><td>+</td><td>+</td><td>N/A</td><td>[129]</td></tr><tr><td>Caldimonas spp.</td><td>+</td><td>+</td><td>N/A</td><td>N/A</td><td>N/A</td><td>-</td><td>-</td><td>+/-</td><td>-</td><td>N/A</td><td>[130]</td></tr><tr><td>Caldicellulosiruptor Methanobacterium thermoautotrophicum</td><td>+</td><td>+</td><td>N/A</td><td>N/A</td><td>N/A</td><td>-</td><td>-</td><td>-</td><td>-</td><td>N/A</td><td>[131]</td></tr><tr><td>Metallo-sphaera sedula</td><td>+</td><td>+</td><td>+</td><td>N/A</td><td>N/A</td><td>+</td><td>+</td><td>-</td><td>-</td><td>-</td><td>[132,133]</td></tr><tr><td>Metallo-sphaera sedula</td><td>+</td><td>+</td><td>+</td><td>N/A</td><td>+</td><td>+</td><td>+</td><td>+</td><td>-</td><td>N/A</td><td>[77,80,134]</td></tr></tbody></table>

The molecular strategies that enable these microbes to retain viability beyond their growth range still remain to be substantially discovered. Here, among prospective directions for the future research is a pull of respiratory enzymes such as for instance high affinity NiFe-hydrogenases. This family of microbial enzymes is responsible for scavenging of atmospheric gases and providing a sufficient energy input for long-term persistence in stress-inducing conditions [135]. Among the unusual properties of these enzymes are oxygen insensitivity and a broad thermal stability window (−4 °C to +60 °C) [135]. The insensitivity of these enzymes to environmental stress factors, such as temperature, might also corroborate energy production that takes place even under unfavorable and hostile conditions [135]. Microbial mesophilic isolates from temperate forest and agricultural soils exhibited high-affinity H₂ uptake due to the presence of these enzymes [136] that can be activated in response to energy limitation to persist in the absence of microbial growth [135]. The thermoacidophilic methanotroph *Methylacidiphilum fumariolicum* SolV is capable of gas (e.g., H₂ and CO₂) fixation [137] under moderate temperatures employing both oxygen-sensitive and -insensitive high-affinity NiFe-hydrogenases [138,139]. This area of enzymes of energy metabolism in thermophiles along with investigations of the minimal energetics required for maintenance of thermophiles viability in psychrobiotic environments is of great potential for future research prospects to address knowledge gaps in molecular strategies of “freezing” thermophiles. As for non-spore-forming thermophiles, the focus of future research might concentrate on a mineral protection crust [80–82], which augments the structural stability of prokaryotic cell envelops [140] and can protect their cells from harsh extracellular environment, including stress with cold temperatures [41] and desiccation [134]. Apart from it, natural cryoprotectants (e.g., trehalose, prolyne, glycine betaine, and any other yet to be discovered), which thermophilic spore-forming bacteria and prokaryotic non-spore-formers produce to preserve their membrane integrity during freezing, are among other interesting targets for further research in this area. All these above-mentioned potential molecular strategies of survival are presented in Table S2 as potential targets for the future research on “freezing” thermophiles.

## 2. Materials and Methods

A review of the occurrence of thermophilic microorganisms in psychrobiotic environments worldwide (cold marine sediments, cold seawaters, ice, permafrost, cool soils, etc.), and in laboratory conditions was performed. Articles published, with no restriction on language and with no date restrictions, were included, according to the requirements of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) [141]. PubMed/MEDLINE, Web of Science, Scopus, and Google Scholar databases were used in
all cases. The keywords used were "thermophiles", "cold temperature(s)", "cold temper-
ature(s)", "permafrost" and "ice". In addition, the bibliographies of the included papers
were screened to incorporate additional sources that might have been overlooked during
the primary database search. The results included microbiological studies with review and
research articles, with case reports being included. The articles were chosen using the fol-
lowing inclusion criteria: worldwide studies reporting direct detection of thermophiles in
one of the environments of interest (cold marine sediments, cold seawaters, ice, permafrost,
cool soils, laboratory culture media, etc.) with full text available. The following exclusion
criteria were used: methodological studies aiming only to the development or improvement
of microbial detection methods, studies performed in warm or hot environments, studies
without available full text, and duplicates.

**3. Conclusions**

The presence of thermophilic microbes in psychrobiotic environments (Figure 1, Table 1) has been known for many years [12]. The biogeographic patterns of their dispersal and distribution have been intensively discussed [15,16,20,21,24] and unexpected high abundances and dispersal rates of spore-forming thermophiles into psychrobiotic environments have been reported [3,15]. A general graphical representation of all psychrobiotic environments with detected thermophiles along with some of their physiological strategies for survival there is given in Figure 1. At the same time, the origin, dispersal, supply and preservation mechanisms of non-spore-forming thermophiles native to hyper- and thermobiotic habitats in mesobiotic/psychrobiotic environments remain enigmatic. Our understanding of the molecular machinery and physiology of non-spore-forming thermophiles in response to conditions of psychrobiotic/mesobiotic environments remains limited mainly due to a failure to support cultivation of thermophilic species in artificial culture media at low temperatures. Supplementation with specific additives (e.g., multimetallic mineral materials [26] or soil microcosms [22,68] may offer further possibilities to study the physiological and molecular mechanisms implicated in the survivability of non-spore-forming thermophiles at deep subfreezing temperatures. Importantly, future investigations should deliver a systematic study to determine which constituents of these materials are the crucial ingredients to ensure thermophiles culture preservation at low temperatures.

Novel insights into the physiology of thermophiles in cold environments (Figure 1) can also be gained from the functional studies of thermophiles maintained at cold temperatures by means of targeting specific genes, examining global gene expression using microarrays for mRNA levels and total intracellular and extracellular proteins and metabolites using proteomic and metabolomic profiling (Table S2). Additional focus on membrane lipids, particularly for strains such as *M. sedula* and *G. thermoleovorans*, under different growth temperatures, will also be important. These kinds of insights can be combined with bio-physical and biochemical studies to investigate the structural and functional properties of protein machinery implicated in the preservation of thermophiles at low temperatures (Table S2). Apart from state-of-the-art molecular techniques, it is important to emphasize that future research strategies should be directed towards incubation of thermophiles under simulated or/and in situ environmental conditions in order to minimize any pos-sible discrepancies arising from artificial culture conditions. Such a multi-disciplinary approach can dramatically enhance our understanding of unique ways that certain ther-mophiles evolved adapting to low temperature environments. Furthermore, thermophilic microorganisms are widely known for their biotechnological potential, e.g., in the industrial production of bio-based chemicals, renewable biofuels, nutrition supplements, fertilizers, polymeric substances, biologically active compounds with antiviral, antiseptic, and anti-cancer activities, bioremediation and biomining operations, to name only a few major ones. Operating some of these bioprocesses over a wide range of temperatures might certainly boost bioprocessing by growing or maintaining thermophiles under low temperatures. Low temperature regiment of the selected thermophiles might be an attractive target from
the side of metabolic engineering, yielding the increased survival rates in cold areas. Finally, increased understanding of thermophiles in psychrobiotic environments is essential in aiding the search for past, or extant life, on other planets, such as Mars or some of the icy satellites orbiting Jupiter and Saturn.

**Supplementary Materials:** The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms10122417/s1, Table S1: Some properties of psychrophilic habitats of thermophilic microorganisms; Table S2: Main strategies and molecular targets to address molecular mechanisms of survival of thermophiles in psychrobiotic environment.

**Funding:** This research was funded by European Research Council, grant number ERC-2020-COG-ERC, CONSOLIDATOR GRANT 101001311 and the Austrian Science Foundation (FWF Elise-Richter Research fellowship V333). The APC was funded by MDPI.

**Data Availability Statement:** Data supporting reported results can be found in Supplementary Materials.

**Acknowledgments:** The research leading to these results has received funding from the European Research Council under the European Union's Horizon 2020 Framework Programme/H2020 Excellent Science ERC Grant Agreement n. [101001311] and the Austrian Science Foundation (FWF Elise-Richter Research fellowship V333) to Tetyana Milojevic.

**Conflicts of Interest:** The authors declare no conflict of interest.

## References

1. Savage, K.N.; Krumholz, L.R.; Oren, A.; Elshahed, M.S. Haladaptatus paucihalophilus gen. nov., sp. nov., a halophilic archaeon isolated from a low-salt, sulfide-rich spring. *Int. J. Syst. Evol. Microbiol.* **2007**, *57*, 19–24. [CrossRef] [PubMed]

2. Usami, R.; Echigo, A.; Fukushima, T.; Mizuki, T.; Yoshida, Y.; Kamekura, M. Alkalibacillus silvisoli sp. nov., a alkaliphilic moderate halophile isolate from non-saline forest soil in Japan. *Int. J. Syst. Evol. Microbiol.* **2007**, *57*, 770–774. [CrossRef] [PubMed]

3. Hubert, C.; Loy, A.; Nickel, M.; Arnosti, C.; Baryani, C.; Brüchert, V.; Ferdelman, T.; Finster, K.; Christensen, F.M.; De Rezende, J.R.; et al. A constant flux of diverse thermophilic bacteria into the cold Arctic seabed. *Science* **2009**, *325*, 1541–1544. [CrossRef] [PubMed]

4. Lavire, C.; Normand, P.; Alekhina, I.; Bulat, S.; Prieur, D.; Birrien, J.L.; Fournier, P.; Hänni, C.; Petit, J.R. Presence of Hydrogenophilus thermoluteolus DNA in accretion ice in the subglacial Lake Vostok, Antarctica, assessed using rrs, cbb and hox. *Environ. Microbiol.* **2006**, *8*, 2106–2114. [CrossRef] [PubMed]

5. Bulat, S.A.; Alekhina, I.A.; Blot, M.; Petit, J.-R.; de Angelis, M.; Wagenbach, D.; Lipenkov, V.Y.; Vasilevya, L.P.; Wloch, D.M.; Raynaud, D.; et al. DNA signature of thermophilic bacteria from the aged accretion ice of Lake Vostok, Antarctica: Implications for searching for life in extreme icy environments. *Int. J. Astrobiol.* **2004**, *3*, 1–12. [CrossRef]

6. Bulat, S.A. Microbiology of the subglacial Lake Vostok: First results of borehole-frozen lake water analysis and prospects for searching for lake inhabitants. *Philos. Trans. A Math Phys. Eng. Sci.* **2016**, *374*, 20140292. [CrossRef] [PubMed]

7. Rogers, S.O.; Shtarkman, Y.M.; Koçer, Z.A.; Edgar, R.; Veerapaneni, R.; D'Elia, T. Ecology of subglacial lake Vostok (Antarctica), based on metagenomic/metatranscriptomic analyses of accretion ice. *Biology* **2013**, *2*, 629–650. [CrossRef]

8. Gilichinsky, D.A.; Wilson, G.S.; Friedmann, E.I.; McKay, C.P.; Sletten, R.S.; Rivkina, E.M.; Vishnivetskaya, T.A.; Erokhina, L.G.; Ivanushkina, N.E.; Kochkina, G.A.; et al. Microbial populations in Antarctic permafrost: Biodiversity, state, age, and implication for astrobiology. *Astrobiology* **2007**, *7*, 275–311. [CrossRef]

9. Demidov, N.E.; Gilichinsky, D.A. Terrestrial Permafrost Models and Analogues of Martian Habitats and Inhabitants. In *Permafrost Soils. Soil Biology*, Margesin, R., Ed.; Springer: Berlin, Heidelberg, Germany, 2009; Volume 16, p. 334.

10. Mironov, V.A.; Rivkina, E.M.; Vishnivetskaya, T.A.; Shcherbakova, V.; Gilichinsky, D.A. Thermophilic Life Inside the Permafrost. In *Proceedings of the AbSciCon 2010, Legue City, TX, USA*, 26–29 April 2010.

11. Shcherbakova, V.; Rivkina, E.; Pecheritsyna, S.; Laurinavichius, K.; Suzina, N.; Gilichinky, D. Methanobacterium arcticum sp. nov., a methanogenic archaeon from Holocene Arctic permafrost. *Int. J. Syst. Evol. Microbiol.* **2011**, *61*, 144–147. [CrossRef]

12. Bartholomew, J.W.; Paik, G. Isolation and identification of obligate thermophilic spore forming bacilli from ocean basin cores. *J. Bacteriol.* **1966**, *92*, 635–638. [CrossRef]

13. Isaksen, M.F.; Bak, F.; Jørgensen, B.B. Thermophilic sulfate-reducing bacteria in cold marine sediment. *FEMS Microbiol. Ecol.* **1994**, *14*, 1–8. [CrossRef]

14. Hubert, C.; Arnosti, C.; Brüchert, V.; Loy, A.; Vandieken, V.; Jørgensen, B.B. Thermophilic anaerobes in Arctic marine sediments induced to mineralize complex organic matter at high temperature. *Environ. Microbiol.* **2010**, *12*, 1089–1104. [CrossRef] [PubMed]

15. de Rezende, J.R.; Kjeldsen, K.U.; Hubert, C.R.; Finser, K.; Loy, A.; Jørgensen, B.B. Dispersal of thermophilic Desulfotomaculum endospores into Baltic Sea sediments over thousands of years. *ISME J.* **2013**, *7*, 72–84. [CrossRef] [PubMed]

16. Müller, A.L.; de Rezende, J.R.; Hubert, C.R.; Kjeldsen, K.U.; Lagkouvardos, I.; Berry, D.; Jørgensen, B.B.; Loy, A. Endospores of thermophilic bacteria as tracers of microbial dispersal by ocean currents. *ISME J.* **2014**, *8*, 1153–1165. [CrossRef] [PubMed]
17. Chakraborty, A.; Ellefson, E.; Li, C.; Gittins, D.; Brooks, J.M.; Bernard, B.B.; Hubert, C.R.J. Thermophilic endospores associated with migrated thermogenic hydrocarbons in deep Gulf of Mexico marine sediments. *ISME J.* **2018**, *12*, 1895–1906. [CrossRef]

18. Cramm, M.A.; Chakraborty, A.; Li, C.; Ruff, S.E.; Jørgensen, B.B.; Hubert, C.R. Freezing tolerance of thermophilic bacterial endospores in marine sediments. *Front. Microbiol.* **2019**, *10*, 945. [CrossRef]

19. Hanson, C.A.; Müller, A.L.; Loy, A.; Dona, C.; Appel, R.; Jørgensen, B.B.; Hubert, C.R. Historical factors associated with past environments influence the biogeography of thermophilic endospores in arctic marine sediments. *Front. Microbiol.* **2019**, *10*, 245. [CrossRef]

20. Marchant, R.; Banat, I.M.; Rahman, T.J.; Berzano, M. The frequency and characteristics of highly thermophilic bacteria in cool soil environments. *Environ. Microbiol.* **2002**, *4*, 595–602. [CrossRef]

21. Marchant, R.; Banat, I.M.; Rahman, T.J.; Berzano, M. What are high-temperature bacteria doing in cold environments? *Trends. Microbiol.* **2002**, *10*, 120–121. [CrossRef]

22. Marchant, R.; Franzetti, A.; Pavlostathis, S.G.; Tas, D.O.; Erdbrugger, I.; Unyanar, A.; Mazmanci, M.A.; Banat, I.M. Thermophilic bacteria in cool temperate soils: Are they metabolically active or continually added by global atmospheric transport? *Appl. Microbiol. Biotechnol.* **2008**, *78*, 841–852. [CrossRef]

23. Marchant, R.; Banat, I.M.; Franzetti, A. Thermophilic bacteria in cool soils: Metabolic activity and mechanisms of dispersal. In *Biogeography of Microscopic Organisms. Is Everything Small Everywhere?* Fontaneto, D., Ed.; Cambridge University Press: Cambridge, UK, 2011; Volume 1, pp. 43–57.

24. Rahman, T.J.; Marchant, R.; Banat, I.M. Distribution and molecular investigation of highly thermophilic bacteria associated with cool soil environments. *Biochem. Soc. Trans.* **2004**, *32*, 209–213. [CrossRef] [PubMed]

25. Zeigler, D.R. The Geobacillus paradox: Why is a thermophilic bacterial genus so prevalent on a mesophilic planet? *Microbiology* **2014**, *160*, 1–11. [CrossRef] [PubMed]

26. Vandieken, V.; Knoblauch, C.; Jørgensen, B.B. Desulfotomaculum arcticum sp. nov., a novel spore-forming, moderately thermophilic, sulfate-reducing bacterium isolated from permanently cold fjord sediment of Svalbard. *Int. J. Sys. Evol. Microbiol.* **2006**, *56*, 687–690. [CrossRef] [PubMed]

27. Volpi, M.; Lomstein, B.A.; Sichert, A.; Røy, H.; Jørgensen, B.B.; Kjeldsen, K.U. Identity, abundance, and reactivation kinetics of thermophilic fermentative endospores in cold marine sediment and seawater. *Front. Microbiol.* **2017**, *8*, 131. [CrossRef]

28. Holden, J.F.; Summit, M.; Baross, J.A. Thermophilic and hyperthermophilic microorganisms in 3–30 °C hydrothermal fluids following a deep-sea volcanic eruption. *FEMS Microbiol. Ecol.* **1998**, *25*, 33–41. [CrossRef]

29. Eecke, H.C.V.; Butterfield, D.A.; Huber, J.A.; Lilley, M.D.; Olson, E.J.; Roe, K.K.; Evans, L.J.; Merkel, A.Y.; Cantin, H.V.; Holden, J.F. Hydrogen-limited growth of hyperthermophilic methanogens at deep-sea hydrothermal vents. *Proc. Natl. Acad. Sci. USA* **2012**, *109*, 13674–13679. [CrossRef]

30. Mora, M.; Bellack, A.; Ugele, M.; Hopf, J.; Wirth, R. The temperature gradient forming device: An accessory unit for normal light microscopes to study the biology of hyperthermophilic microorganisms. *Appl. Environ. Microbiol.* **2014**, *80*, 4764–4770. [CrossRef]

31. Huber, J.A.; Butterfield, D.A.; Baross, J.A. Diversity and distribution of subseafloor Thermococcales populations in diffuse hydrothermal vents at an active deep-sea volcano in the northeast Pacific Ocean. *J. Geophys. Res.* **2006**, *111*, G04016. [CrossRef]

32. Wirth, R. Colonization of Black Smokers by Hyperthermophilic Microorganisms. *Trends Microbiol.* **2017**, *2*, 92–99. [CrossRef]

33. Gonnella, G.; Böhnke, S.; Indenbirken, D.; Garbe-Schönberg, D.; Seifert, R.; Mertens, C.; Kurtz, S.; Perner, M. Endemic hydrothermal vent species identified in the open ocean seed bank. *Nat. Microbiol.* **2016**, *1*, 16086. [CrossRef]

34. Kelly, L.C.; Cockell, C.S.; Piceno, Y.M.; Andersen, G.L.; Thorsteinsson, T.; Marteinsson, V. Bacterial diversity of weathered terrestrial Icelandic volcanic glasses. *Microb. Ecol.* **2010**, *60*, 740–752. [CrossRef] [PubMed]

35. Fields, M.L.; Chen Lee, P.P. Bacillus stearothermophilus in soils of Iceland. *Appl. Microbiol.* **1974**, *28*, 638–640. [CrossRef] [PubMed]

36. McBee, R.H.; McBee, V.H. The incidence of thermophilic bacteria in Arctic soils and waters. *J. Bacteriol* **1956**, *71*, 182–185. [CrossRef] [PubMed]

37. Gura, C.; Rogers, S.O. Metatranscriptomic and metagenomic analysis of biological diversity in subglacial Lake Vostok (Antarctica). *Biology* **2020**, *9*, 55. [CrossRef] [PubMed]

38. Bendia, A.G.; Araújo, G.G.; Pulschen, A.A. Surviving in hot and cold: Psychrophiles and thermophiles from Deception Island volcano, Antarctica. *Extremophiles* **2018**, *22*, 917–929. [CrossRef] [PubMed]

39. Mironov, V.A.; Shcherbakova, V.A.; Rivkina, E.M.; Gilichinsky, D.A. Thermophilic bacteria of the genus Geobacillus from permafrost volcanic sedimentary rocks. *Microbiology* **2013**, *82*, 389–392. [CrossRef]

40. Wiegel, J. Temperature spans for growth: Hypothesis and discussion. *FEMS Microbiol. Rev.* **1990**, *75*, 155–170. [CrossRef]

41. Milojevic, T.; Zebec, Z.; Schimak, M.P. Cultivation with powdered meteorite (NWA 1172) as the substrate enhances low-temperature preservation of the extreme thermoacidophile Metallosphaera sedula. *Front. Astron. Space Sci.* **2020**, *7*, 37. [CrossRef]

42. Setlow, P. Mechanisms which contribute to the long-term survival of spores of Bacillus species. *J. Appl. Bacteriol.* **1994**, *76*, 49S–60S. [CrossRef]

43. Nicholson, W.L.; Munakata, N.; Horneck, G.; Melosh, H.J.; Setlow, P. Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. *Microbiol. Mol. Biol. Rev.* **2000**, *64*, 548–572. [CrossRef]

44. Godon, J.J.; Galès, A.; Latrille, E.; Ouichanpagdee, P.; Seyer, J.P. An "overlooked" habitat for thermophilic bacteria: The phyllosphere. *BioDiscovery* **2020**, *23*, e47033. [CrossRef]
45. Kawaguchi, Y.; Yang, Y.; Kawashiri, N.; Shiraishi, K.; Takasu, M.; Narumi, I.; Satoh, K.; Hashimoto, H.; Nakagawa, K.; Tanigawa, Y.; et al. The possible interplanetary transfer of microbes: Assessing the viability of Deinococcus spp. under the ISS Environmental conditions for performing exposure experiments of microbes in the Tanpopo mission. *Orig. Life Evol. Biosph.* **2013**, *43*, 411–428. [CrossRef] [PubMed]

46. Kawaguchi, Y.; Yokobori, S.; Hashimoto, H.; Yano, H.; Tabata, M.; Kawai, H.; Yamagishi, A. Investigation of the Interplanetary Transfer of Microbes in the Tanpopo Mission at the Exposed Facility of the International Space Station. *Astrobiology* **2016**, *16*, 363–376. [CrossRef] [PubMed]

47. Grotzinger, J.P. Habitability, Taphonomy, and the Search for Organic Carbon on Mars. *Science* **2014**, *343*, 386–387. [CrossRef] [PubMed]

48. Chin, J.P.; Megaw, J.; Magill, C.L.; Nowotarski, K.; Williams, J.P.; Bhaganna, P.; Linton, M.; Patterson, M.F.; Underwood, G.J.C.; Mswaka, A.Y.; et al. Solutes determine the temperature windows for microbial survival and growth. *Proc. Natl. Acad. Sci. USA* **2010**, *107*, 7835–7840. [CrossRef]

49. Wächtershäuser, G. Groundworks for an evolutionary biochemistry: The iron-sulphur world. *Prog. Biophys. Mol. Biol.* **1992**, *58*, 85–201. [CrossRef]

50. Bada, J.L.; Bigham, C.; Miller, S.L. Impact melting of frozen oceans on the early Earth: Implications for the origin of life. *Proc. Natl. Acad. Sci. USA* **1994**, *91*, 1248–1250. [CrossRef]

51. Bada, J.L.; Lazcano, A. Origin of life. Some like it hot, but not the first biomolecules. *Science* **2002**, *296*, 1982–1983. [CrossRef]

52. Stetter, K.O. Hyperthermophiles in the history of life. *Philos. Trans. R. Soc. Lond. B Biol. Sci.* **2006**, *361*, 1837–1842. [CrossRef]

53. Akanuma, S.; Nakajima, Y.; Yokobori, S.; Kimura, M.; Mase, T.; Miyazono, K.; Tanokura, M.; Yamagishi, A. Experimental evidence for the thermophilicity of ancestral life. *Proc. Natl. Acad. Sci. USA* **2013**, *110*, 11067–11072. [CrossRef]

54. Lee, Y.; Wagner, I.D.; Brice, M.E.; Kevbrin, V.V.; Mills, G.L.; Romanek, C.S.; Wiegel, J. Thermosediminibacter oceani gen. nov., sp. nov. and Thermosediminibacter litoriperuensis sp. nov., new anaerobic thermophilic bacteria isolated from Peru Margin. *Extremophiles* **2005**, *9*, 375–383. [CrossRef] [PubMed]

55. Lynch, M.D.J.; Neufled, J.D. Ecology and exploration of the rare biosphere. *Nat. Rev. Microbial.* **2015**, *13*, 217–229. [CrossRef] [PubMed]

56. Pedrós-Alió, C. The rare bacterial biosphere. *Ann. Rev. Mar. Sci.* **2012**, *4*, 449–466. [CrossRef] [PubMed]

57. Heuer, V.B.; Inagaki, F.; Morono, Y.; Kubo, Y.; Spivack, A.J.; Viehweger, B.; Treude, T.; Beulig, F.; Schubotz, F.; Tonai, S.; et al. Temperature limits to deep subseafloor life in the Nankai Trough subduction zone. *Science* **2020**, *370*, 1230–1234. [CrossRef]

58. Gittins, D.A.; Desiage, P.-A.; Morrison, N.; Rattray, J.E.; Bhatnagar, S.; Chakraborty, A.; Zorz, J.; Li, C.; Horanszky, O.; Cramm, M.A.; et al. Geological processes mediate a microbial dispersal loop in the deep biosphere. *Sci. Adv.* **2022**, *8*, eabn3485. [CrossRef]

59. Agogué, H.; Lamy, D.; Neal, P.R.; Sogin, M.L.; Herndl, G.J. Water mass-specificity of bacterial communities in the North Atlantic revealed by massively parallel sequencing. *Mol. Ecol.* **2011**, *20*, 258–274. [CrossRef]

60. Hamdan, L.; Coffin, R.; Sikaroodi, M.; Greinert, J.; Treude, T.; Gillevet, P.M. Ocean currents shape the microbiome of Arctic marine sediments. *ISME J.* **2013**, *7*, 685–696. [CrossRef]

61. Wilkins, D.; van Sebille, E.; Rintoul, S.R.; Lauro, F.M.; Cavicchioli, R. Advection shapes Southern Ocean microbial assemblages independent of distance and environment effects. *Nat. Comm.* **2013**, *4*, 2457. [CrossRef]

62. Salazar, G.; Cornejo-Castillo, F.M.; Benítez-Barrios, V.; Fraile-Nuez, E.; Álvarez-Salgdo, A.; Duarte, C.M.; Gasol, J.M.; Acinas, S.G. Global diversity and biogeography of deep-sea pelagic prokaryotes. *ISME J.* **2016**, *10*, 596–608. [CrossRef]

63. Bell, E.; Sherry, A.; Pilloni, G.; Súarez-Súarez, A.; Cramm, M.A.; Cueto, G.; Head, I.M.; Hubert, C.R.J. Sediment cooling triggers germination and sulfate reduction by heat-resistant thermophilic spore-forming bacteria. *Environ. Microbiol.* **2020**, *22*, 456–465. [CrossRef]

64. Huber, R.; Stotters, P.; Cheminee, J.L.; Richnow, H.H.; Stetter, K.O. Hyperthermophilic archaebacteria within the crater and open-sea plume of erupting Macdonald Seamount. *Nature* **1990**, *45*, 179–182. [CrossRef]

65. Wiegel, J. Thermophiles: Anaerobic alkalithermophiles. In *Extremophiles*, 3rd ed.; Bitton, G., Ed.; John Wiley & Sons Inc.: New York, NY, USA, 1998; Volume 3, pp. 3127–3140.

66. Cockell, C.; Cousins, C.; Wilkinson, P.T.; Olsson-Francis, K.; Rozitis, B. Are thermophilic microorganisms active in cold environments? *Int. J. Astrobiol.* **2015**, *14*, 457–463. [CrossRef]

67. Kelly, L.C.; Cockell, C.S.; Herrera-Belaroussi, A.; Piceno, Y.; Andersen, G.L.; DeSantis, T.; Brodie, E.; Thorsteinsson, T.; Marteinsson, V.; Poly, F.; et al. Bacterial diversity of terrestrial crystalline volcanic rocks, Iceland. *Microb. Ecol.* **2011**, *62*, 69–79. [CrossRef] [PubMed]

68. Portillo, M.C.; Santana, M.; Gonzalez, J.M. Presence and potential role of thermophilic bacteria in temperate terrestrial environments. *Naturwissenschaften* **2012**, *99*, 43–53. [CrossRef]

69. Santana, M.M.; Portillo, M.C.; Gonzalez, J.M.; Clara, M.I.E. Characterization of new soil thermophilic bacteria potentially involved in soil fertilization. *J. Plant Nutr. Soil Sci.* **2013**, *176*, 47–56. [CrossRef]

70. Santana, M.M.; Carvalho, L.; Melo, J.; Araújo, M.E.; Cruz, C. Unveiling the hidden interaction between thermophiles and plant crops: Wheat and soil thermophilic bacteria. *J. Plant Interact.* **2020**, *15*, 127–138. [CrossRef]

71. Karl, D.M.; Bird, D.F.; Björkman, K.; Houlihan, T.; Shackelford, R.; Tupas, L. Microorganisms in the accreted ice of Lake Vostok, Antarctica. *Science* **1999**, *286*, 2144–2147. [CrossRef]
72. Alekhina, I.A.; Marie, D.; Petit, J.R.; Lukin, V.V.; Zubkov, V.M.; Bulat, S.A. Molecular analysis of bacterial diversity in kerosene-based drilling fluid from the deep ice borehole at Vostok, East Antarctica. *FEMS Microbiol. Ecol.* **2007**, *59*, 289–299. [CrossRef]

73. Kochetkova, T.V.; Toshchakov, S.V.; Zayulina, K.S.; Elcheninov, A.G.; Zavarzina, D.G.; Lavrushin, V.Y.; Bonch-Osmolovskaya, E.A.; Kublanov, I.V. Hot in Cold: Microbial Life in the Hottest Springs in Permafrost. *Microorganisms* **2020**, *8*, 1308. [CrossRef]

74. Peeples, T.L.; Kelly, R.M. Bioenergetic response of the extreme thermoacidophile Metallosphaera sedula to thermal and nutritional stress. *Appl. Environ. Microbiol.* **1995**, *61*, 2314–2321. [CrossRef]

75. Beblo, K.; Rabbow, E.; Rachel, R.; Huber, H.; Rettberg, P. Tolerance of thermophilic and hyperthermophilic microorganisms to desiccation. *Extremophiles* **2009**, *13*, 521–531. [CrossRef] [PubMed]

76. Beblo, K.; Douki, T.; Schmalz, G.; Rachel, R.; Wirth, R.; Huber, H.; Reitz, G.; Rettberg, P. Survival of thermophilic and hyper-thermophilic microorganisms after exposure to UV-C, ionizing radiation and desiccation. *Arch. Microbiol.* **2011**, *193*, 797–809. [CrossRef]

77. Mukherjee, A.; Wheaton, G.H.; Blum, P.H.; Kelly, M. Uranium extremophily is an adaptive, rather than intrinsic, feature for extremely thermoacidophilic Metallosphaera species. *Proc. Natl. Acad. Sci. USA* **2012**, *109*, 16702–16707. [CrossRef] [PubMed]

78. Maezato, Y.; Johnson, T.; McCarthy, S.; Dana, K.; Blum, P. Metal resistance and lithoautotrophy in the extreme thermoacidophile Metallosphaera sedula. *J. Bacteriol.* **2012**, *194*, 6856–6863. [CrossRef] [PubMed]

79. McCarthy, S.; Ai, C.; Wheaton, G.; Tevatia, R.; Eckrich, V.; Kelly, R.; Blum, P. Role of an archaeal PitA transporter in the copper and arsenic resistance of Metallosphaera sedula, an extreme thermoacidophile. *J. Bacteriol.* **2014**, *196*, 3562–3570. [CrossRef] [PubMed]

80. Blazevic, A.; Albu, M.; Mitsche, S.; Rittmann, S.K.M.; Habler, G.; Milojevic, T. Biotransformation of scheelite CaWO4 by the extreme thermoacidophile Metallosphaera sedula: Tungsten–microbial interface. *Front. Microbiol.* **2019**, *10*, 1492. [CrossRef]

81. Milojevic, T.; Kölbl, D.; Ferrière, L.; Albu, M.; Kish, A.; Flemming, R.L.; Koeberl, C.; Blazevic, A.; Zebec, Z.; Rittmann, S.K.M.; et al. Exploring the microbial biotransformation of extraterrestrial material on nanometer scale. *Sci. Rep.* **2019**, *9*, 18028. [CrossRef]

82. Milojevic, T.; Albu, M.; Kölbl, D.; Kothleitner, G.; Bruner, R.; Morgan, M.L. Chemolithotrophy on the Noachian Martian breccia NWA 7034 via experimental microbial biotransformation. *Commun. Earth Environ.* **2021**, *2*, 39. [CrossRef]

83. Huber, G.; Spinnler, C.; Gambacorta, A.; Stetter, K.O. Metallosphaera sedula gen. and sp. nov. represents a new genus of aerobic, metal-mobilizing, thermoacidophilic archaebacteria. *Syst. Appl. Microbiol.* **1989**, *12*, 38–47. [CrossRef]

84. Auernik, K.S.; Kelly, R.M. Identification of components of electron transport chains in the extremely thermoacidophilic crenarchaeon Metallosphaera sedula through iron and sulfur compound oxidation transcriptomes. *Appl. Environ. Microbiol.* **2008**, *74*, 7723–7732. [CrossRef]

85. Russell, S.; Zipfel, J.; Grossman, J.; Grady, M. The Meteoritical Bulletin No. 86. *Meteorit. Planet Sci. (Suppl.)* **2002**, *37*, 157–184. [CrossRef]

86. Kölbl, D.; Pignitter, M.; Somoza, V.; Schimak, M.P.; Strbak, O.; Blazevic, A.; Milojevic, T. Exploring fingerprints of the extreme thermoacidophile Metallosphaera sedula grown on synthetic Martian regolith materials as the sole energy sources. *Front. Microbiol.* **2017**, *8*, 1918. [CrossRef] [PubMed]

87. Westall, F.; Foucher, F.; Bost, N.; Bertrand, M.; Loizeau, D.; Vago, J.L.; Kminek, G.; Gaboyer, F.; Campbell, K.A.; Bréhéret, J.G.; et al. Biosignatures on Mars: What, Where, and How? Implications for the Search for Martian Life. *Astrobiology* **2015**, *11*, 998–1029. [CrossRef]

88. Losa-Adams, E.; Gil-Lozano, C.; Fairén, A.G.; Bishop, J.L.; Rampe, E.B.; Gago-Duport, L. Long-lating habitable periods in Gale Crater constrained by glauconitic clays. *Nat. Astron.* **2021**, *5*, 936–942. [CrossRef]

89. Freissinet, C.; Glavin, D.P.; Mahaffy, P.R.; Miller, K.E.; Eigenbrode, J.L.; Summons, R.E.; Brunner, A.E.; Buch, A.; Szopa, C.; Archer, P.D., Jr.; et al. Organic molecules in the Sheepbed Mudstone, Gale Crater, Mars. *J. Geophys. Res. Planets* **2015**, *3*, 495–514. [CrossRef] [PubMed]

90. Eigenbrode, J.L.; Summons, R.E.; Steele, A.; Freissinet, C.; Millan, M.; Navarro-Gonazález, R.; Sutter, B.; McAdam, A.C.; Franz, H.B.; Glavin, D.P.; et al. Organic matter preserved in 3-billion-year-old mudstones at Gale crater, Mars. *Science* **2018**, *6393*, 1096–1101. [CrossRef] [PubMed]

91. Szopa, C.; Freissinet, C.; Glavin, D.P.; Millan, M.; Buch, A.; Franz, H.; Summons, R.E.; Sumner, D.Y.; Sutter, B.; Eigenbrode, J., et al. First detections of dichlorobenzene isomers and trichloromethylpropane from organic matter indigenous to Mars mudstone in Gale Crater, Mars: Results from the Sample Analysis at Mars instrument onboard the Curiosity Rover. *Astrobiology* **2020**, *20*, 2. [CrossRef]

92. Farley, K.A.; Williford, K.H.; Stack, K.M.; Bhartia, R.; Chen, A.; de la Torre, M.; Hand, K.; Goreva, Y.; Herd, C.D.K.; Hueso, R.; et al. Mars 2020 mission overview. *Space Sci. Rev.* **2020**, *216*, 1–41. [CrossRef]

93. Vago, J.L.; Westall, F.; Pasteur Instrument Teams LSSWG; Other Contributors; ExoMars Project Team. Habitability on Early Mars and the Search for Biosignatures with the ExoMars Rover. *Astrobiology* **2017**, *17*, 471–510. [CrossRef] [PubMed]

94. Horvath, D.G.; Moitra, P.; Hamilton, C.W.; Craddock, R.A.; Andrews-Hanna, J.C. Evidence for geologically recent explosive volcanism in Elysium Planitia, Mars. *Icarus* **2021**, *365*, 114499. [CrossRef]

95. Kargel, J.S.; Kaye, J.Z.; Head, J.W.; Marion, G.M.; Sassen, R.; Crowley, J.K.; Prieto Ballesteros, O.; Grant, S.A.; Hogenboom, D.L. Europa's Crust and Ocean: Origin, Composition, and the Prospects for Life. *Icarus* **2000**, *148*, 226–265. [CrossRef]

96. Marion, G.M.; Fritsen, C.H.; Eicken, H.; Payne, M.C. The Search for Life on Europa: Limiting Environmental Factors, Potential Habitats, and Earth Analogues. *Astrobiology* **2003**, *3*, 785–811. [CrossRef] [PubMed]
97. Parkinson, C.D.; Barr, A.C.; Liang, M.C.; Yung, Y.; Kirschivnk, J.L. Planetary Conditions for Habitability on Enceladus. *Orig. Life Evol. Biosph.* **2008**, *38*, 355–369. [CrossRef] [PubMed]

98. Greenberg, R. Transport Rates of Radiolytic Substances into Europa's Ocean: Implications for the Potential Origin and Maintenance of Life. *Astrobiology* **2010**, *10*, 275–283. [CrossRef]

99. McCord, T.B.; Hansen, G.B.; Fanale, F.P.; Carlson, R.W.; Matson, D.L.; Johnson, T.V.; Smythe, W.D.; Crowley, J.K.; Martin, P.D.; Ocampo, A.; et al. Salts on Europa's Surface Detected by Galileo's Near Infrared Mapping Spectrometer. *Science* **1998**, *280*, 1242–1245. [CrossRef] [PubMed]

100. Jia, X.; Kivelson, M.G.; Khurana, K.K.; Kurth, W.S. Evidence of a plume on Europa from Galileo magnetic and plasma wave signatures. *Nat. Astron.* **2018**, *2*, 459–464. [CrossRef]

101. Khawaja, N.; Postberg, F.; Hillier, J.; Klenner, F.; Kempf, S.; Nölle, L.; Reviol, R.; Zou, Z.; Srama, R. Low-mass nitrogen-oxygen-bearing, and aromatic compounds in Enceladean ice grains. *Mon. Notices R. Astron. Soc.* **2019**, *489*, 5231–5243. [CrossRef]

102. Phillips, C.B.; Pappalardo, R.T. Europa Clipper Mission Concept: Exploring Jupiter's Ocean Moon. *EOS* **2014**, *95*, 165–167. [CrossRef]

103. Hendrix, A.R.; Hurford, T.A.; Barge, L.M.; Bland, M.T.; Bowman, J.S.; Brinckerhoff, W.; Buratti, B.J.; Cable, M.L.; Castillo-Rogez, J.; Collins, G.C.; et al. The NASA Roadmap to Ocean Worlds. *Astrobiology* **2019**, *19*, 1–27. [CrossRef] [PubMed]

104. O'Sullivan, L.; Roussel, E.; Weightman, A.; Webster, G.; Hubert, C.R.J.; Bell, E.; Head, I.; Sass, H.; Parkes, R.J. Survival of *Desulfotomaculum* spores from estuarine sediments after serial autoclaving and high-temperature exposure. *ISME J.* **2015**, *9*, 922–933. [CrossRef]

105. Kim, J.H.; Stegeman, H.; Farkas, J. Preliminary studies on radiation resistance of thermophilic anaerobic spores and the effect of gamma radiation on their heat resistance. *Int. J. Food Microbiol.* **1987**, *5*, 129–136. [CrossRef]

106. Karine, A.; Pignet, P.; Zbinden, M.; Quillevere, M.; Duchiron, F.; Donval, J.-P.; Lesongeur, F.; Raguenes, G.; Crassous, P.; Querellou, J.; et al. Caminicella sporogenes gen. nov., sp nov., a novel thermophilic spore-forming bacterium isolated from an East-Pacific Rise hydrothermal vent. *Int. J. Syst. Evol. Microbiol.* **2002**, *52*, 1621–1628.

107. Wery, N.; Moricet, J.M.; Cueff, V.; Jean, J.; Pignet, P.; Lesongeur, F.; Cambon-Bonavita, M.A.; Barbier, G. Caloranaerobacter azorensis gen. nov., sp. nov., an anaerobic thermophilic bacterium isolated from a deep-sea hydrothermal vent. *Int. J. Syst. Evol. Microbiol.* **2001**, *51*, 1789–1796. [CrossRef]

108. Yilmaz, E.I. Metal tolerance and biosorption capacity of Bacillus circulans strain EB1. *Res. Microbiol.* **2003**, *154*, 409–415. [CrossRef] [PubMed]

109. Setlow, P. Observations on research with spores of Bacillales and Clostridiales species. *J. Appl. Microbiol.* **2019**, *126*, 348–358. [CrossRef]

110. Kublanov, I.V.; Prokofeva, M.I.; Kostrikina, N.A.; Kolganova, T.V.; Tourova, T.P.; Wiegel, J.; Bonch-Osmolovskaya, E.A. *Thermoanaerobacterium aciditolerans* sp. nov., a moderate thermoacidophile from a Kamchatka hot spring. *Int. J. Syst. Evol. Microbiol.* **2007**, *57*, 260–264.

111. Liu, S.Y.; Rainey, F.A.; Morgan, H.W.; Mayer, F.; Wiegel, J. *Thermoanaerobacterium aotearoense* sp. nov., a slightly acidophilic, anaerobic thermophile isolated from various hot springs in New Zealand, and emendation of the genus *Thermoanaerobacterium*. *Int. J. Syst. Bacteriol.* **1996**, *46*, 388–396. [CrossRef]

112. Watanabe, T.; Furukawa, S.; Hirata, J.; Koyama, T.; Ogihara, H.; Yamasaki, M. Inactivation of Geobacillus stearothermophilus spores by high-pressure carbon dioxide treatment. *Appl. Environ. Microbiol.* **2003**, *69*, 7124–7129. [CrossRef]

113. Azua-Bustos, A.; Fairén, A.G.; Silva, C.G.; Carrizo, D.; Fernández-Martínez, M.A.; Arenas-Fajardo, C.; Fernández-Sampedro, M.; Gil-Lozano, C.; Sánchez-García, L.; Ascaso, C.; et al. Inhabited subsurface wet smectites in the hyperarid core of the Atacama Desert as an analog for the search for life on Mars. *Sci. Rep.* **2020**, *10*, 19183. [CrossRef]

114. Tan, H.Q.; Wu, X.Y.; Zhang, X.Q.; Wu, M.; Zhu, X.F. Tepidibacter mesophilus sp. nov., a mesophilic fermentative anaerobe isolated from soil polluted by crude oil, and emended description of the genus Tepidibacter. *Int. J. Syst. Evol. Microbiol.* **2012**, *62*, 66–70. [CrossRef]

115. Liebgott, P.P.; Joseph, M.; Fardeau, M.L.; Cayol, J.L.; Falsen, E.; Chamkh, F.; Qatibi, A.I.; Labat, M. Clostridiisalibacter paucivorans gen. nov., sp. nov., a novel moderately halophilic bacterium isolated from olive mill wastewater. *Int. J. Syst. Evol. Microbiol.* **2008**, *58*, 61–67. [CrossRef]

116. Jolivet, E.; L'haridon, S.; Corre, E.; Forterre, P.; Prieur, D. *Thermococcus gammatolerans* sp. nov., a hyperthermophilic archaeon from a deep-sea hydrothermal vent that resists ionizing radiation. *Int. J. Syst. Evol. Microbiol.* **2003**, *53*, 847–851. [CrossRef]

117. Keller, M.; Braun, F.J.; Dirmeier, R.; Hafenbradl, D.; Burggraf, S.; Rachel, R.; Stetter, K.O. *Thermococcus alcaliphilus* sp. nov., a new hyperthermophilic archaeum growing on polysulfide at alkaline pH. *Arch. Microbiol.* **1995**, *164*, 390–395. [CrossRef]

118. Orange, F.; Westall, F.; Disnar, J.R.; Prieur, D.; Bienvenu, N.; Défarge, C. Experimental silicification of the extremophilic archaea *Pyrococcus abyssi* and *Methanocaldococcus jannaschii*: Applications in the search for evidence of life in early earth and extraterrestrial rocks. *Geobiology* **2009**, *7*, 403–418. [CrossRef]

119. Parameswaran, A.K.; Schicho, R.N.; Soisson, J.P.; Kelly, R.M. Effect of hydrogen and carbon dioxide partial pressures on growth and sulfide production of the extremely thermophilic archaebacterium Pyrodictium brockii. *Biotechnol. Bioeng.* **1988**, *32*, 438–443. [CrossRef]

120. Fiala, G.; Stetter, K.O. *Pyrococcus furiosus* sp. nov. represents a novel genus of marine heterotrophic archaebacteria growing optimally at 100 °C. *Arch. Microbiol.* **1986**, *145*, 56–61. [CrossRef]
121. Birrien, J.-L.; Zeng, X.; Jebbar, M.; Cambon-Bonavita, M.-A.; Quérellou, J.; Oger, P.; Bienvenu, N.; Xiao, X.; Prieur, D. *Pyrococcus yayanosii* sp. nov., an obligate piezophilic hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent. *Int. J. Syst. Evol. Microbiol.* **2011**, *61*, 2827–2831. [CrossRef] [PubMed]

122. Jolivet, E.; Matsunaga, F.; Ishino, Y.; Forterre, P.; Prieur, D.; Myllykallio, H. Physiological responses of the hyperthermophilic archaeon "Pyrococcus abyssi" to DNA damage caused by ionizing radiation. *J. Bacteriol.* **2003**, *185*, 3958–3961. [CrossRef] [PubMed]

123. Beblo-Vranesevic, K.; Galinski, E.A.; Rachel, R.; Huber, H.; Rettberg, P. Influence of osmotic stress on desiccation and irradiation tolerance of (hyper)-thermophilic microorganisms. *Arch. Microbiol.* **2017**, *199*, 17–28. [CrossRef]

124. Hayashi, N.R.; Ishida, T.; Yokota, A.; Kodama, T.; Igarashi, Y. Hydrogenophilus thermoluteolus gen. nov., sp. nov., a thermophilic, facultatively chemolithoautotrophic, hydrogen-oxidizing bacterium. *Int. J. Syst. Bacteriol.* **1999**, *49*, 783–786. [CrossRef] [PubMed]

125. Yang, L.; Muhadesi, J.B.; Wang, M.M.; Wang, B.J.; Liu, S.J.; Jiang, C.Y. Thauera hydrothermalis sp. nov., a thermophilic bacterium isolated from hot spring. *Int. J. Syst. Evol. Microbiol.* **2018**, *68*, 3163–3168. [CrossRef] [PubMed]

126. Bonch-Osmolovskaya, E.A.; Sokolova, T.G.; Kostrikina, N.A.; Zavarzin, G.A. *Desulfurella acetivorans* gen. nov. and sp. nov.—A new thermophilic sulfur-reducing eubacterium. *Arch. Microbiol.* **1990**, *153*, 151–155. [CrossRef]

127. Liolios, K.; Sikorski, J.; Jando, M.; Lapidus, A.; Copeland, A.; Glavina, T.; Rio, D.; Nolan, M.; Lucas, S.; Tice, H.; et al. Complete genome sequence of Thermobispora bispora type strain (R51). *Stand Genom. Sci.* **2010**, *2*, 318–326. [CrossRef]

128. Schultz, J.; Parise, M.T.D.; Parise, D.; Medeiros, L.G.; Sousa, T.J.; Kato, R.B.; Uetanabaro, A.P.T.; Araújo, F.; Ramos, R.T.J.; de Castro Soares, S.; et al. Unraveling the Genomic Potential of the Thermophilic Bacterium *Anoxybacillus flavithermus* from an Antarctic Geothermal Environment. *Microorganisms* **2022**, *10*, 673. [CrossRef]

129. Nakamura, Y.; Kaneko, T.; Sato, S.; Ikeuchi, M.; Katoh, H.; Sasamoto, S.; Watanabe, A.; Iriguchi, M.; Kawashima, K.; Kimura, T.; et al. Complete genome structure of the thermophilic cyanobacterium Thermosynechococcus elongatus strain BP-1. *DNA Res.* **2002**, *9*, 123–130. [CrossRef]

130. Takeda, M.; Kamagata, Y.; Ghiorse, W.C.; Hanada, S.; Koizumi, J. *Caldimonas manganoxidans* gen. nov., sp. nov., a poly(3-hydroxybutyrate)-degrading, manganese-oxidizing thermophile. *Int. J. Syst. Evol. Microbiol.* **2002**, *52*, 895–900.

131. Rainey, F.A.; Donnison, A.M.; Janssen, P.H.; Saul, D.; Rodrigo, A.; Bergquist, P.L.; Daniel, R.M.; Stackebrandt, E.; Morgan, H.W. Description of *Caldicellulosiruptor saccharolyticus* gen. nov., sp. nov.: An obligately anaerobic, extremely thermophilic, cellulolytic bacterium. *FEMS Microbiol. Lett.* **1994**, *120*, 263–266. [CrossRef]

132. Zeikus, J.G.; Wolfe, R.S. *Methanobacterium thermoautotrophicus* sp. n., an anaerobic, autotrophic, extreme thermophile. *J. Bacteriol.* **1972**, *109*, 707–715. [CrossRef]

133. Lorowitz, W.H.; Nagle, D.P.; Ralph, S. Tanner Anaerobic oxidation of elemental metals coupled to methanogenesis by Methanobacterium thermoautotrophicum. *Environ. Sci. Technol.* **1992**, *26*, 1606–1610. [CrossRef]

134. Kölbl, D.; Blazevic, A.; Albu, M.; Fasching, C.; Milojevic, T. Desiccation of the Extreme Thermoacidophile Metallosphaera sedula Grown on Terrestrial and Extraterrestrial Materials. *Front. Astron. Space Sci.* **2020**, *7*, 41. [CrossRef]

135. Greening, C.; Constant, P.; Hards, K.; Morales, S.E.; Oakeshott, J.G.; Russell, R.J.; Taylor, M.C.; Berney, M.; Conrad, R.; Cook, G.M. Atmospheric hydrogen scavenging: From enzymes to ecosystems. *Appl. Environ. Microbiol.* **2015**, *81*, 1190–1199. [CrossRef] [PubMed]

136. Constant, P.; Chowdhury, S.P.; Pratscher, J.; Conrad, R. Streptomycetes contributing to atmospheric molecular hydrogen soil uptake are widespread and encode a putative high-affinity [NiFe]-hydrogenase. *Environ. Microbiol.* **2010**, *12*, 821–829. [CrossRef] [PubMed]

137. Picone, N.; Mohammadi, S.S.; Waajen, A.C.; van Alen, T.A.; Jetten, M.S.M.; Pol, A.; Op den Camp, H.J.M. More Than a Methanotroph: A Broader Substrate Spectrum for Methylacidiphilum fumariolicum SolV. *Front. Microbiol.* **2020**, *11*, 604485. [CrossRef] [PubMed]

138. Mohammadi, S.S.; Pol, A.; van Alen, T.V.; Jetten, M.; op den Camp, H.J.M. Methylacidiphilum fumariolicum SolV, a thermoacidophilic 'Knallgas' methanotroph with both an oxygen-sensitive and -insensitive hydrogenase. *ISME J.* **2016**, *11*, 4. [CrossRef] [PubMed]

139. Schmitz, R.A.; Pol, A.; Mohammadi, S.S.; Hogendoorn, C.; van Gelder, A.H.; Jetten, M.S.M.; Daumann, L.J.; Op den Camp, H.J.M. The thermoacidophilic methanotroph Methylacidiphilum fumariolicum SolV oxidizes subatmospheric H₂ with a high-affinity, membrane-associated [NiFe] hydrogenase. *ISME J.* **2020**, *4*, 1223–1232. [CrossRef] [PubMed]

140. Kish, A.; Miot, J.; Lombard, C.; Guigner, J.; Bernard, S.; Zirah, S.; Guyot, F. Preservation of archaeal surface layer structure during mineralization. *Sci. Rep.* **2016**, *6*, 26152. [CrossRef]

141. Moher, D.; Liberati, A.; Tetzlaff, J.; Altman, D.G. Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. *BMJ* **2009**, *339*, 332–336. [CrossRef]