Evolution and Physiology of Amphibious Yeasts.

Since the emergence of the first fungi some 700 million years ago, unicellular yeast-like forms have emerged multiple times in independent lineages via convergent evolution. While tens to hundreds of millions of years separate the independent evolution of these unicellular organisms, they share remarkable phenotypic and metabolic similarities, and all have streamlined genomes. Yeasts occur in every aquatic environment yet examined. Many species are aquatic; perhaps most are amphibious. How these species have evolved to thrive in aquatic habitats is fundamental to understanding functions and evolutionary mechanisms in this unique group of fungi. Here we review the state of knowledge of the physiological and ecological diversity of amphibious yeasts and their key evolutionary adaptations enabling survival in aquatic habitats. We emphasize some genera previously thought to be exclusively terrestrial. Finally, we discuss the ability of many yeasts to survive in extreme habitats and how this might lend insight into ecological plasticity, including amphibious lifestyles.

A Na pump with reduced stoichiometry is up-regulated by brine shrimp in extreme salinities.

Brine shrimp (Artemia) are the only animals to thrive at sodium concentrations above 4 M. Salt excretion is powered by the Na+,K+-ATPase (NKA), a heterodimeric (αß) pump that usually exports 3Na+ in exchange for 2 K+ per hydrolyzed ATP. Artemia express several NKA catalytic α-subunit subtypes. High-salinity adaptation increases abundance of α2KK, an isoform that contains two lysines (Lys308 and Lys758 in transmembrane segments TM4 and TM5, respectively) at positions where canonical NKAs have asparagines (Xenopus α1's Asn333 and Asn785). Using de novo transcriptome assembly and qPCR, we found that Artemia express two salinity-independent canonical α subunits (α1NN and α3NN), as well as two ß variants, in addition to the salinity-controlled α2KK. These ß subunits permitted heterologous expression of the α2KK pump and determination of its CryoEM structure in a closed, ion-free conformation, showing Lys758 residing within the ion-binding cavity. We used electrophysiology to characterize the function of α2KK pumps and compared it to that of Xenopus α1 (and its α2KK-mimicking single- and double-lysine substitutions). The double substitution N333K/N785K confers α2KK-like characteristics to Xenopus α1, and mutant cycle analysis reveals energetic coupling between these two residues, illustrating how α2KK's Lys308 helps to maintain high affinity for external K+ when Lys758 occupies an ion-binding site. By measuring uptake under voltage clamp of the K+-congener 86Rb+, we prove that double-lysine-substituted pumps transport 2Na+ and 1 K+ per catalytic cycle. Our results show how the two lysines contribute to generate a pump with reduced stoichiometry allowing Artemia to maintain steeper Na+ gradients in hypersaline environments.

Rubisco kinetic adaptations to extreme environments.

Photosynthetic and chemosynthetic extremophiles have evolved adaptations to thrive in challenging environments by finely adjusting their metabolic pathways through evolutionary processes. A prime adaptation target to allow autotrophy in extreme conditions is the enzyme Rubisco, which plays a central role in the conversion of inorganic to organic carbon. Here, we present an extensive compilation of Rubisco kinetic traits from a wide range of species of bacteria, archaea, algae, and plants, sorted by phylogenetic group, Rubisco type, and extremophile type. Our results show that Rubisco kinetics for the few extremophile organisms reported up to date are placed at the margins of the enzyme's natural variability. Form ID Rubisco from thermoacidophile rhodophytes and form IB Rubisco from halophile terrestrial plants exhibit higher specificity and affinity for CO2 than their non-extremophilic counterparts, as well as higher carboxylation efficiency, whereas form ID Rubisco from psychrophile organisms possess lower affinity for O2. Additionally, form IB Rubisco from thermophile cyanobacteria shows enhanced CO2 specificity when compared to form IB non-extremophilic cyanobacteria. Overall, these findings highlight the unique characteristics of extremophile Rubisco enzymes and provide useful clues to guide next explorations aimed at finding more efficient Rubiscos.

Exploring Andean High-Altitude Lake Extremophiles through Advanced Proteotyping.

Quickly identifying and characterizing isolates from extreme environments is currently challenging while very important to explore the Earth's biodiversity. As these isolates may, in principle, be distantly related to known species, techniques are needed to reliably identify the branch of life to which they belong. Proteotyping these environmental isolates by tandem mass spectrometry offers a rapid and cost-effective option for their identification using their peptide profiles. In this study, we document the first high-throughput proteotyping approach for environmental extremophilic and halophilic isolates. Microorganisms were isolated from samples originating from high-altitude Andean lakes (3700-4300 m a.s.l.) in the Chilean Altiplano, which represent environments on Earth that resemble conditions on other planets. A total of 66 microorganisms were cultivated and identified by proteotyping and 16S rRNA gene amplicon sequencing. Both the approaches revealed the same genus identification for all isolates except for three isolates possibly representing not yet taxonomically characterized organisms based on their peptidomes. Proteotyping was able to indicate the presence of two potentially new genera from the families of Paracoccaceae and Chromatiaceae/Alteromonadaceae, which have been overlooked by 16S rRNA amplicon sequencing approach only. The paper highlights that proteotyping has the potential to discover undescribed microorganisms from extreme environments.

Novel endolithic bacteria of phylum Chloroflexota reveal a myriad of potential survival strategies in the Antarctic desert.

The ice-free McMurdo Dry Valleys of Antarctica are dominated by nutrient-poor mineral soil and rocky outcrops. The principal habitat for microorganisms is within rocks (endolithic). In this environment, microorganisms are provided with protection against sub-zero temperatures, rapid thermal fluctuations, extreme dryness, and ultraviolet and solar radiation. Endolithic communities include lichen, algae, fungi, and a diverse array of bacteria. Chloroflexota is among the most abundant bacterial phyla present in these communities. Among the Chloroflexota are four novel classes of bacteria, here named Candidatus Spiritibacteria class. nov. (=UBA5177), Candidatus Martimicrobia class. nov. (=UBA4733), Candidatus Tarhunnaeia class. nov. (=UBA6077), and Candidatus Uliximicrobia class. nov. (=UBA2235). We retrieved 17 high-quality metagenome-assembled genomes (MAGs) that represent these four classes. Based on genome predictions, all these bacteria are inferred to be aerobic heterotrophs that encode enzymes for the catabolism of diverse sugars. These and other organic substrates are likely derived from lichen, algae, and fungi, as metabolites (including photosynthate), cell wall components, and extracellular matrix components. The majority of MAGs encode the capacity for trace gas oxidation using high-affinity uptake hydrogenases, which could provide energy and metabolic water required for survival and persistence. Furthermore, some MAGs encode the capacity to couple the energy generated from H2 and CO oxidation to support carbon fixation (atmospheric chemosynthesis). All encode mechanisms for the detoxification and efflux of heavy metals. Certain MAGs encode features that indicate possible interactions with other organisms, such as Tc-type toxin complexes, hemolysins, and macroglobulins.IMPORTANCEThe ice-free McMurdo Dry Valleys of Antarctica are the coldest and most hyperarid desert on Earth. It is, therefore, the closest analog to the surface of the planet Mars. Bacteria and other microorganisms survive by inhabiting airspaces within rocks (endolithic). We identify four novel classes of phylum Chloroflexota, and, based on interrogation of 17 metagenome-assembled genomes, we predict specific metabolic and physiological adaptations that facilitate the survival of these bacteria in this harsh environment-including oxidation of trace gases and the utilization of nutrients (including sugars) derived from lichen, algae, and fungi. We propose that such adaptations allow these endolithic bacteria to eke out an existence in this cold and extremely dry habitat.

The biotechnological potential of the Chloroflexota phylum.

In the next decades, the increasing material and energetic demand to support population growth and higher standards of living will amplify the current pressures on ecosystems and will call for greater investments in infrastructures and modern technologies. A valid approach to overcome such future challenges is the employment of sustainable bio-based technologies that explore the metabolic richness of microorganisms. Collectively, the metabolic capabilities of Chloroflexota, spanning aerobic and anaerobic conditions, thermophilic adaptability, anoxygenic photosynthesis, and utilization of toxic compounds as electron acceptors, underscore the phylum's resilience and ecological significance. These diverse metabolic strategies, driven by the interplay between temperature, oxygen availability, and energy metabolism, exemplify the complex adaptations that enabled Chloroflexota to colonize a wide range of ecological niches. In demonstrating the metabolic richness of the Chloroflexota phylum, specific members exemplify the diverse capabilities of these microorganisms: Chloroflexus aurantiacus showcases adaptability through its thermophilic and phototrophic growth, whereas members of the Anaerolineae class are known for their role in the degradation of complex organic compounds, contributing significantly to the carbon cycle in anaerobic environments, highlighting the phylum's potential for biotechnological exploitation in varying environmental conditions. In this context, the metabolic diversity of Chloroflexota must be considered a promising asset for a large range of applications. Currently, this bacterial phylum is organized into eight classes possessing different metabolic strategies to survive and thrive in a wide variety of extreme environments. This review correlates the ecological role of Chloroflexota in such environments with the potential application of their metabolisms in biotechnological approaches.

Extremophiles and their expanding biotechnological applications.

Microbial life is not restricted to any particular setting. Over the past several decades, it has been evident that microbial populations can exist in a wide range of environments, including those with extremes in temperature, pressure, salinity, and pH. Bacteria and Archaea are the two most reported types of microbes that can sustain in extreme environments, such as hot springs, ice caves, acid drainage, and salt marshes. Some can even grow in toxic waste, organic solvents, and heavy metals. These microbes are called extremophiles. There exist certain microorganisms that are found capable of thriving in two or more extreme physiological conditions simultaneously, and are regarded as polyextremophiles. Extremophiles possess several physiological and molecular adaptations including production of extremolytes, ice nucleating proteins, pigments, extremozymes and exopolysaccharides. These metabolites are used in many biotechnological industries for making biofuels, developing new medicines, food additives, cryoprotective agents etc. Further, the study of extremophiles holds great significance in astrobiology. The current review summarizes the diversity of microorganisms inhabiting challenging environments and the biotechnological and therapeutic applications of the active metabolites obtained as a response to stress conditions. Bioprospection of extremophiles provides a progressive direction with significant enhancement in economy. Moreover, the introduction to omics approach including whole genome sequencing, single cell genomics, proteomics, metagenomics etc., has made it possible to find many unique microbial communities that could be otherwise difficult to cultivate using traditional methods. These findings might be capable enough to state that discovery of extremophiles can bring evolution to biotechnology.

Iron bioleaching and polymers accumulation by an extreme acidophilic bacterium.

In many European regions, both local metallic and non-metallic raw materials are poorly exploited due to their low quality and the lack of technologies to increase their economic value. In this context, the development of low cost and eco-friendly approaches, such as bioleaching of metal impurities, is crucial. The acidophilic strain Acidiphilium sp. SJH reduces Fe(III) to Fe(II) by coupling the oxidation of an organic substrate to the reduction of Fe(III) and can therefore be applied in the bioleaching of iron impurities from non-metallic raw materials. In this work, the physiology of Acidiphilium sp. SJH and the reduction of iron impurities from quartz sand and its derivatives have been studied during growth on media supplemented with various carbon sources and under different oxygenation conditions, highlighting that cell physiology and iron reduction are tightly coupled. Although the organism is known to be aerobic, maximum bioleaching performance was obtained by cultures cultivated until the exponential phase of growth under oxygen limitation. Among carbon sources, glucose has been shown to support faster biomass growth, while galactose allowed highest bioleaching. Moreover, Acidiphilium sp. SJH cells can synthesise and accumulate Poly-ß-hydroxybutyrate (PHB) during the process, a polymer with relevant application in biotechnology. In summary, this work gives an insight into the physiology of Acidiphilium sp. SJH, able to use different carbon sources and to synthesise a technologically relevant polymer (PHB), while removing metals from sand without the need to introduce modifications in the process set up.

Diversity and enzymatic, biosurfactant and phytotoxic activities of culturable Ascomycota fungi present in marine sediments obtained near the South Shetland Islands, maritime Antarctica.

We studied the culturable fungal community recovered from deep marine sediments in the maritime Antarctic, and assessed their capabilities to produce exoenzymes, emulsifiers and metabolites with phytotoxic activity. Sixty-eight Ascomycota fungal isolates were recovered and identified. The most abundant taxon recovered was the yeast Meyerozyma guilliermondii, followed by the filamentous fungi Penicillium chrysogenum, P. cf. palitans, Pseudeurotium cf. bakeri, Thelebolus balaustiformis, Antarctomyces psychrotrophicus and Cladosporium sp. Diversity indices displayed low values overall, with the highest values obtained at shallow depth, decreasing to the deepest location sampled. Only M. guilliermondii and P. cf. palitans were detected in the sediments at all depths sampled, and were the most abundant taxa at all sample sites. The most abundant enzymes detected were proteases, followed by invertases, cellulases, lipases, carrageenases, agarases, pectinases and esterases. Four isolates showed good biosurfactant activity, particularly the endemic species A. psychrotrophicus. Twenty-four isolates of P. cf. palitans displayed strong phytotoxic activities against the models Lactuca sativa and Allium schoenoprasum. The cultivable fungi recovered demonstrated good biosynthetic activity in the production of hydrolytic exoenzymes, biosurfactant molecules and metabolites with phytotoxic activity, reinforcing the importance of documenting the taxonomic, ecological and biotechnological properties of fungi present in deep oceanic sediments of the Southern Ocean.

Fungal diversity present in snow sampled in summer in the north-west Antarctic Peninsula and the South Shetland Islands, Maritime Antarctica, assessed using metabarcoding.

We assessed the fungal diversity present in snow sampled during summer in the north-west Antarctic Peninsula and the South Shetland Islands, maritime Antarctica using a metabarcoding approach. A total of 586,693 fungal DNA reads were obtained and assigned to 203 amplicon sequence variants (ASVs). The dominant phylum was Ascomycota, followed by Basidiomycota, Mortierellomycota, Chytridiomycota and Mucoromycota. Penicillium sp., Pseudogymnoascus pannorum, Coniochaeta sp., Aspergillus sp., Antarctomyces sp., Phenoliferia sp., Cryolevonia sp., Camptobasidiaceae sp., Rhodotorula mucilaginosa and Bannozyma yamatoana were assessed as abundant taxa. The snow fungal diversity indices were high but varied across the different locations sampled. Of the fungal ASVs detected, only 28 were present all sampling locations. The 116 fungal genera detected in the snow were dominated by saprotrophic taxa, followed by symbiotrophic and pathotrophic. Our data indicate that, despite the low temperature and oligotrophic conditions, snow can host a richer mycobiome than previously reported through traditional culturing studies. The snow mycobiome includes a complex diversity dominated by cosmopolitan, cold-adapted, psychrophilic and endemic taxa. While saprophytes dominate this community, a range of other functional groups are present.

Enzymes and biosurfactants of industrial interest produced by culturable fungi present in sediments of Boeckella Lake, Hope Bay, north-east Antarctic Peninsula.

This study characterized cultivable fungi present in sediments obtained from Boeckella Lake, Hope Bay, in the north-east of the Antarctic Peninsula, and evaluated their production of enzymes and biosurfactants of potential industrial interest. A total of 116 fungal isolates were obtained, which were classified into 16 genera within the phyla Ascomycota, Basidiomycota and Mortierellomycota, in rank. The most abundant genera of filamentous fungi included Pseudogymnoascus, Pseudeurotium and Antarctomyces; for yeasts, Thelebolales and Naganishia taxa were dominant. Overall, the lake sediments exhibited high fungal diversity and moderate richness and dominance. The enzymes esterase, cellulase and protease were the most abundantly produced by these fungi. Ramgea cf. ozimecii, Holtermanniella wattica, Leucosporidium creatinivorum, Leucosporidium sp., Mrakia blollopis, Naganishia sp. and Phenoliferia sp. displayed enzymatic index > 2. Fourteen isolates of filamentous fungi demonstrated an Emulsification Index 24% (EI24%) ≥ 50%; among them, three isolates of A. psychrotrophicus showed an EI24% > 80%. Boeckella Lake itself is in the process of drying out due to the impact of regional climate change, and may be lost completely in approaching decades, therefore hosts a threatened community of cultivable fungi that produce important biomolecules with potential application in biotechnological processes.

Unraveling the multiplicity of geranylgeranyl reductases in Archaea: potential roles in saturation of terpenoids.

The enzymology of the key steps in the archaeal phospholipid biosynthetic pathway has been elucidated in recent years. In contrast, the complete biosynthetic pathways for proposed membrane regulators consisting of polyterpenes, such as carotenoids, respiratory quinones, and polyprenols remain unknown. Notably, the multiplicity of geranylgeranyl reductases (GGRs) in archaeal genomes has been correlated with the saturation of polyterpenes. Although GGRs, which are responsible for saturation of the isoprene chains of phospholipids, have been identified and studied in detail, there is little information regarding the structure and function of the paralogs. Here, we discuss the diversity of archaeal membrane-associated polyterpenes which is correlated with the genomic loci, structural and sequence-based analyses of GGR paralogs.

Antarctic marine sediment as a source of filamentous fungi-derived antimicrobial and antitumor compounds of pharmaceutical interest.

Antarctica harbors a microbial diversity still poorly explored and of inestimable biotechnological value. Cold-adapted microorganisms can produce a diverse range of metabolites stable at low temperatures, making these compounds industrially interesting for biotechnological use. The present work investigated the biotechnological potential for antimicrobial and antitumor activity of filamentous fungi and bacteria isolated from marine sediment samples collected at Deception Island, Antarctica. A total of 89 microbial isolates were recovered from marine sediments and submitted to an initial screening for L-glutaminase with antitumoral activity and for antimicrobial metabolites. The isolates Pseudogymnoascus sp. FDG01, Pseudogymnoascus sp. FDG02, and Penicillium sp. FAD33 showed potential antiproliferative action against human pancreatic carcinoma cells while showing no toxic effect on non-tumor cells. The microbial extracts from unidentified three bacteria and four filamentous fungi showed antibacterial activity against at least one tested pathogenic bacterial strain. The isolate FDG01 inhibited four bacterial species, while the isolate FDG01 was active against Micrococcus luteus in the minimal inhibitory concentration of 0.015625 µg mL -1. The results pave the way for further optimization of enzyme production and characterization of enzymes and metabolites found and reaffirm Antarctic marine environments as a wealthy source of compounds potentially applicable in the healthcare and pharmaceutical industry.

Extremophiles in a changing world.

Extremophiles and their products have been a major focus of research interest for over 40 years. Through this period, studies of these organisms have contributed hugely to many aspects of the fundamental and applied sciences, and to wider and more philosophical issues such as the origins of life and astrobiology. Our understanding of the cellular adaptations to extreme conditions (such as acid, temperature, pressure and more), of the mechanisms underpinning the stability of macromolecules, and of the subtleties, complexities and limits of fundamental biochemical processes has been informed by research on extremophiles. Extremophiles have also contributed numerous products and processes to the many fields of biotechnology, from diagnostics to bioremediation. Yet, after 40 years of dedicated research, there remains much to be discovered in this field. Fortunately, extremophiles remain an active and vibrant area of research. In the third decade of the twenty-first century, with decreasing global resources and a steadily increasing human population, the world's attention has turned with increasing urgency to issues of sustainability. These global concerns were encapsulated and formalized by the United Nations with the adoption of the 2030 Agenda for Sustainable Development and the presentation of the seventeen Sustainable Development Goals (SDGs) in 2015. In the run-up to 2030, we consider the contributions that extremophiles have made, and will in the future make, to the SDGs.

The potential of plant growth-promoting bacteria isolated from arid heavy metal contaminated environments in alleviating salt and water stresses in alfalfa.

Co-evolution of plant beneficial microbes in contaminated environments enhances plant growth and mitigates abiotic stress. However, few studies on heavy metal (HM) tolerant plant growth-promoting bacteria (PGPB) promoting crop growth in Morocco's farming areas affected by drought and salinity are available. Plant associated bacteria tolerant to HM and able to produce indole acetic acid and siderophores, display ACC-deaminase activity and solubilize phosphate, were isolated from long-term metal exposed environments. Tolerance to HM and biofilms formation in the absence or presence of HM were assessed. A consortium including two Ensifer meliloti strains (RhOL6 and RhOL8), one Pseudomonas sp. strain (DSP17), and one Proteus sp. strain (DSP1), was used to inoculate alfalfa (Medicago sativa) seedlings under various conditions, namely, salt stress (85 mM) and water stress (30% water holding capacity). Shoot and root dry weights of alfalfa were measured 60 days after sowing. In the presence of HM, DSP17 showed the greatest auxin production, whereas RhOL8 had the highest ACC-deaminase activity and DSP17 formed the densest biofilm. Root dry weight increased 138% and 195% in salt and water stressed plants, respectively, regarding non-inoculated controls. Our results confirm the improvement of alfalfa growth and mitigation of salt and drought stress upon inoculation.

Thermochronologic perspectives on the deep-time evolution of the deep biosphere.

The Earth's deep biosphere hosts some of its most ancient chemolithotrophic lineages. The history of habitation in this environment is thus of interest for understanding the origin and evolution of life. The oldest rocks on Earth, formed about 4 billion years ago, are in continental cratons that have experienced complex histories due to burial and exhumation. Isolated fracture-hosted fluids in these cratons may have residence times older than a billion years, but understanding the history of their microbial communities requires assessing the evolution of habitable conditions. Here, we present a thermochronological perspective on the habitability of Precambrian cratons through time. We show that rocks now in the upper few kilometers of cratons have been uninhabitable (>∼122 °C) for most of their lifetime or have experienced high-temperature episodes, such that the longest record of habitability does not stretch much beyond a billion years. In several cratons, habitable conditions date back only 50 to 300 million years, in agreement with dated biosignatures. The thermochronologic approach outlined here provides context for prospecting and interpreting the little-explored geologic record of the deep biosphere of Earth's cratons, when and where microbial communities may have thrived, and candidate areas for the oldest records of chemolithotrophic microbes.

DNA Polymerase I Large Fragment from Deinococcus radiodurans, a Candidate for a Cutting-Edge Room-Temperature LAMP.

In recent years, the loop-mediated isothermal amplification (LAMP) technique, designed for microbial pathogen detection, has acquired fundamental importance in the biomedical field, providing rapid and precise responses. However, it still has some drawbacks, mainly due to the need for a thermostatic block, necessary to reach 63 °C, which is the BstI DNA polymerase working temperature. Here, we report the identification and characterization of the DNA polymerase I Large Fragment from Deinococcus radiodurans (DraLF-PolI) that functions at room temperature and is resistant to various environmental stress conditions. We demonstrated that DraLF-PolI displays efficient catalytic activity over a wide range of temperatures and pH, maintains its activity even after storage under various stress conditions, including desiccation, and retains its strand-displacement activity required for isothermal amplification technology. All of these characteristics make DraLF-PolI an excellent candidate for a cutting-edge room-temperature LAMP that promises to be very useful for the rapid and simple detection of pathogens at the point of care.

Potential applications of extremophilic bacteria in the bioremediation of extreme environments contaminated with heavy metals.

Protecting the environment from harmful pollutants has become increasingly difficult in recent decades. The presence of heavy metal (HM) pollution poses a serious environmental hazard that requires intricate attention on a worldwide scale. Even at low concentrations, HMs have the potential to induce deleterious health effects in both humans and other living organisms. Therefore, various strategies have been proposed to address this issue, with extremophiles being a promising solution. Bacteria that exhibit resistance to metals are preferred for applications involving metal removal due to their capacity for rapid multiplication and growth. Extremophiles are a special group of microorganisms that are capable of surviving under extreme conditions such as extreme temperatures, pH levels, and high salt concentrations where other organisms cannot. Due to their unique enzymes and adaptive capabilities, extremophiles are well suited as catalysts for environmental biotechnology applications, including the bioremediation of HMs through various strategies. The mechanisms of resistance to HMs by extremophilic bacteria encompass: (i) metal exclusion by permeability barrier; (ii) extracellular metal sequestration by protein/chelator binding; (iii) intracellular sequestration of the metal by protein/chelator binding; (iv) enzymatic detoxification of a metal to a less toxic form; (v) active transport of HMs; (vi) passive tolerance; (vii) reduced metal sensitivity of cellular targets to metal ions; and (viii) morphological change of cells. This review provides comprehensive information on extremophilic bacteria and their potential roles for bioremediation, particularly in environments contaminated with HMs, which pose a threat due to their stability and persistence. Genetic engineering of extremophilic bacteria in stressed environments could help in the bioremediation of contaminated sites. Due to their unique characteristics, these organisms and their enzymes are expected to bridge the gap between biological and chemical industrial processes. However, the structure and biochemical properties of extremophilic bacteria, along with any possible long-term effects of their applications, need to be investigated further.

Dramatic loss of microbial viability in bentonite exposed to heat and gamma radiation: implications for deep geological repository.

Bentonite is an integral part of the engineered barrier system (EBS) in deep geological repositories (DGR) for nuclear waste, but its indigenous microorganisms may jeopardize long-term EBS integrity. To predict microbial activity in DGRs, it is essential to understand microbial reactions to the early hot phase of DGR evolution. Two bentonites (BCV and MX-80) with varied bentonite/water ratios and saturation levels (compacted to 1600 kg.m- 3 dry density/powder/suspension), were subjected to heat (90-150 °C) and irradiation (0.4 Gy.h- 1) in the long-term experiments (up to 18 months). Molecular-genetic, microscopic, and cultivation-based techniques assessed microbial survivability. Exposure to 90 °C and 150 °C notably diminished microbial viability, irrespective of bentonite form, with negligible impacts from irradiation or sample type compared to temperature. Bentonite powder samples exhibited microbial recovery after 90 °C heating for up to 6 months but not 12 months in most cases; exposure to 150 °C had an even stronger effect. Further long-term experiments at additional temperatures combined with the mathematical prediction of temperature evolution in DGR are recommended to validate the possible evolution and spatial distribution of microbially depleted zones in bentonite buffer around the waste canisters and refine predictions of microbial effects over time in the DGR.

Extremophiles in Space Exploration.

In the era of deep space exploration, extremophile research represents a key area of research w.r.t space survival. This review thus delves into the intriguing realm of 'Space and Astro Microbiology', providing insights into microbial survival, resilience, and behavioral adaptations in space-like environments. This discussion encompasses the modified behavior of extremophilic microorganisms, influencing virulence, stress resistance, and gene expression. It then shifts to recent studies on the International Space Station and simulated microgravity, revealing microbial responses that impact drug susceptibility, antibiotic resistance, and its commercial implications. The review then transitions into Astro microbiology, exploring the possibilities of interplanetary transit, lithopanspermia, and terraforming. Debates on life's origin and recent Martian meteorite discoveries are noted. We also discuss Proactive Inoculation Protocols for selecting adaptable microorganisms as terraforming pioneers. The discussion concludes with a note on microbes' role as bioengineers in bioregenerative life support systems, in recycling organic waste for sustainable space travel; and in promoting optimal plant growth to prepare Martian and lunar basalt. This piece emphasizes the transformative impact of microbes on the future of space exploration.