搜索结果：Extremophiles : life under extreme conditions

The first forms of life on Earth were microbial, preceding the evolution of multicellular life by more than two billion years. Based on our current understanding of the origin of life, it is likely that the first life forms on any extraterrestrial world would also be microbial. Due to the extreme temperatures, radiation or aridity on most planetary surfaces, such extraterrestrial microbes would most likely dwell in subsurface environments. Earth's subsurface features a wide range of environments, including deep marine sediments, crustal aquifers, rock fracture fluids, hydrocarbon reservoirs, caves and permafrost soils. These environments are known to host an immense diversity of life forms, predominantly microbes that survive or even thrive under extreme conditions and energy scarcity. Life's ability to endure and possibly evolve in Earth's subsurface lends credence to the possible existence of life beyond our planet and provides a blueprint for the extraterrestrial life forms and biosignatures we might expect. The exploration of space via extraterrestrial samples analysed on Earth, in situ extraterrestrial analyses, and remote sensing continue to advance our search for and understanding of potential biosignatures on other planetary bodies. But by investigating Earth's deep, dark and isolated ecosystems, we not only broaden our understanding of life's adaptability but also refine our strategies and technologies for detecting life on other planets and moons. Subsurface exploration is not just a frontier of Earth science-it is a cornerstone of astrobiology and in the pursuit of understanding the multitude of processes that could create and sustain life anywhere. In this opinion article, we discuss the latest highlights in subsurface research and technology, how Earth's subsurface environments serve as models for potential environments on other planetary bodies, why insights into subsurface microbiomes inform the search for life elsewhere, and which technologies and developments will advance the field in the future.

Extremophiles are microorganisms that thrive in environments previously thought to be uninhabitable, including extreme temperature, salinity, pH, pressure, and radiation. These organisms, found in Archaea, Bacteria, and Eukarya, exhibit distinct structural, metabolic, and genetic adaptations, such as enhanced enzyme stability, efficient DNA repair mechanisms, and robust stress-response systems that enable survival under extreme conditions. Understanding these adaptation mechanisms is key to engineering similar traits in mesophilic organisms. This review discusses the diversity of extremophiles and presents phylogenetic and comparative genomic insights which may provide insights into the origins and evolution of early life on Earth We highlight recent advances in CRISPR/Cas-based genome editing, genome-scale metabolic modeling (GEM), and synthetic biology that have expanded the use of extremophiles in sustainable industrial biotechnology. The exceptional stability and catalytic efficiency of extremozymes under harsh conditions underscore their potential in various biotechnological applications. Finally, we discuss the ecological significance of extremophiles in climate change mitigation and outline current challenges and future directions in extremophile research.

Astrobiology assesses the habitability of planetary bodies and the potential for extraterrestrial life. Analog environments on Earth serve as sites for studying extreme environments that resemble extraterrestrial conditions, aiding in validating life-detection methods, mission instrumentation, and biosignature preservation. These environments function as a source of model microorganisms and communities that define the habitability and biochemistry of such extraterrestrial environments. Well-known analog environments include the Atacama Desert (Chile) for space mission validation, the McMurdo Dry Valleys (Antarctica) for Mars analog studies, and Rio Tinto (Spain) for extreme acidic environments. Although significant research has been conducted on these sites, various alternative environments may also offer valuable opportunities for astrobiological studies. Saudi Arabia encompasses a variety of pristine (or with minimal anthropic influence) extreme environments with conditions analogous to extraterrestrial settings (e.g., deserts and salt flats as analogs to Mars, and terrestrial and marine volcanic fields as analogs to icy moons), yet their potential remains largely unexplored. Recent studies have identified a volcanic crater with sodium phosphates and chlorates that mimics Enceladus’s ocean chemistry, and researchers have cultured Halalkalibacterium halodurans strains with adaptations to survive these conditions, offering valuable biological models. Additionally, complex metabolic landscapes with implications for icy moon habitability have been observed in Red Sea systems, which could be employed as valuable natural laboratories in astrobiological research. Furthermore, these findings underscore the potential of the Saudi Arabian extremophilic microbiome for space-related research. This review explores the microbial diversity of extreme environments in Saudi Arabia, emphasizing their potential as new terrestrial analogs to Mars and icy moons and the role of their microbiomes as terrestrial proxies for extraterrestrial life.

Bacterial metal recovery is regarded as an environmentally friendly alternative to chemical hydrometallurgical waste metal recycling. However, conventional studies are generally conducted under neutral pH conditions, even though typical acidic metal leachate is strongly acidic. Previously, we isolated the bacterial strain Priestia sp. Mn7, which can grow under neutral pH and recover metals (Co, Cu, Li, Mn, and Ni) under pH 1.5. This study aimed to clarify this strain's metal tolerance and recovery mechanisms under pH 1.5 conditions. Metal recovery test was conducted using pH 1.5 solution without the five metal species described above. Enhanced expression of genes related to the sugar ATP binding cassette transporter and increased sugar concentration in metal-abundant solution indicated that the strain expels sugar from the cell as a metal stress response under strongly acidic conditions. Dead cells did not recover metals except for Mn. Additionally, the recovered metals were distributed on the cell surface. These results indicated that the strain recovers tested metals via bioprecipitation related to sugar expulsion. To the best of our knowledge, this is the first study to elucidate the metal tolerance and recovery mechanisms of acid-tolerant bacteria under pH 1.5, further contributing to the understanding and utilization of acid-tolerant bacteria.

Microbes from terrestrial extreme environments enable testing of biosignature production in conditions relevant to astrobiological targets. Mars, which was likely more conducive to life during early warmer and wetter epochs, has inspired missions that search for signs of early life in the surficial rock record, including mineral or organic biosignatures. Microbial iron reduction is a common and ancient metabolism that may have also operated on other rocky celestial bodies. To investigate biosignature production during iron reduction, a Shewanella sp. (strain BF02_Schw) isolated from a subglacial discharge known as Blood Falls, Antarctica, was incubated with the electron acceptor ferrihydrite (Fh). Biosignatures associated with Fh reduction were identified using a suite of techniques currently utilized or proposed for Mars missions, including X-ray diffraction and infrared, Mössbauer, and Raman spectroscopy. The biotic origin of features was validated by transcriptional changes observed between treatments with and without Fh and comparison to killed controls. In live treatments, Fh was reduced to magnetite and goethite, both detected in Martian lacustrine basins. Several soluble and volatile metabolites were also detected, including riboflavin and dimethyl sulfide (DMS), which could be astrobiological indicators of active microbial processes. While none of the identified biosignatures individually would serve as definitive proof of life (past or present), detecting concomitant features associated with known terrestrial biotic processes would provide compelling rationale for more targeted life detection missions. Terrestrial extremophiles can support the exploration of astrobiologically relevant microbial processes, validation of life detection instrumentation, and potentially the discovery of new biomarkers.IMPORTANCECulture-based experiments with terrestrial extremophiles can elucidate biosignatures that may be analogous to those produced under extraterrestrial conditions, and thus inform sampling and technology strategies for future missions. Here, we demonstrate the production of several biosignatures under iron-reducing conditions by Shewanella sp. BF02_Schw, originally isolated from an Antarctic analog feature. These biosignatures could be detectable using flight-ready instrumentation. Growth experiments with terrestrial extremophiles can identify biosignatures measurable by current methodologies and inform the development and optimization of techniques for detecting extant or extinct life on other worlds.

NASA cleanrooms, which are critical for assembling space mission components, are maintained under stringent decontamination protocols to minimize biological contamination. These environments are characterized by nutrient-poor and oligotrophic conditions, leading to low microbial loads. Despite extensive cleaning, oligotrophs capable of surviving in such conditions continue to persist, often remaining undetected due to their low abundance, resistance to environmental stresses, and difficulties in biomolecule extraction. Even with shotgun metagenome sequencing technologies, these microbes may go undetected or be underrepresented due to their robust cell walls and the absence of reference genomes in publicly available databases. Over a 6-month study of Mars 2020 mission cleanrooms, 182 bacterial strains belonging to 19 families were identified using a whole-genome sequencing (WGS) approach. Among these, 14 novel Gram-positive species were discovered, including eight spore formers. Though the novel species comprised only 0.001% of the sequencing data, their successful cultivation allowed for functional characterization. Through WGS data mining, genomic traits associated with resilience in extreme conditions were revealed. These species were found to be involved in nitrogen cycling, carbohydrate metabolism, and radiation resistance, traits essential for survival in extreme environments. Furthermore, 12 biosynthetic gene clusters were identified, including those linked to ectoine and [Formula: see text]-poly-L-lysine production, suggesting potential biotechnological applications. These findings highlight the hidden microbial diversity within cleanrooms and emphasize the necessity of advanced detection strategies. A better understanding of these microbes will provide insights into extremophiles with applications in biotechnology, medical research, and life support systems for future space exploration missions.IMPORTANCEDespite strict decontamination protocols, NASA cleanrooms harbor low-biomass microbial communities adapted to nutrient-poor environments. These oligotrophic microbes often go undetected in shotgun metagenomics methods due to their low abundance, resistance to lysis, and lack of reference genomes. Standard shotgun metagenome sequencing methods fail to retrieve them, as dominant microbial DNA overshadows rare species. Over 6 months of monitoring Mars 2020 mission cleanrooms, 182 bacterial strains from 19 families were identified, including 14 novel Gram-positive species, 8 of which were spore formers. Though present at 0.001% abundance in sequencing data, we successfully cultured them, enabling functional characterization. These microbes exhibited roles in nitrogen cycling, carbohydrate metabolism, and radiation resistance, with 12 biosynthetic gene clusters linked to ectoine and [Formula: see text]-poly-L-lysine production. These findings highlight the previously underestimated microbial diversity in cleanrooms and emphasize the need for advanced detection strategies to explore extremophiles with applications in biotechnology and space exploration.

The red microalga Cyanidioschyzon merolae inhabits extreme environments with high temperatures (40-56 °C), high acidity (pH 0.05-4), and high concentrations of heavy metals that are lethal to most forms of life. However, information is scarce on the precise adaptation mechanisms of this extremophile to such hostile conditions. Gaining such knowledge is important for understanding the evolution of microorganisms in the early stages of life on Earth characterized by such extreme environments. Through an analysis of the re-programming of the global transcriptome upon the long-term (up to 15 days) exposure of C. merolae to extremely high concentrations of nickel (1 and 3 mM), the key adaptive metabolic pathways and associated molecular components were identified. Our work shows that the long-term Ni exposure of C. merolae leads to the lagged metabolic switch demonstrated via the transcriptional upregulation of the metabolic pathways critical for cell survival. DNA replication, cell cycle, and protein quality control processes were upregulated, while downregulation occurred with energetically costly processes, including the assembly of the photosynthetic apparatus and lipid biosynthesis. This study paves the way for future multi-omic studies of the molecular mechanisms of abiotic stress adaptation in phototrophs, as well as the future development of rational approaches to the bioremediation of contaminated aquatic environments.

Extremophiles are microorganisms capable of living on Earth in ecological niches characterized by peculiar conditions, including extreme temperatures and/or pH, high salt concentrations, and the presence of heavy metals. The development of unique structural and functional adaptation strategies has stimulated an increasing scientific interest since their discovery. The importance of extremophiles lies in their exploitability in significant bioprocesses with several biotechnological applications and their role as a fundamental source of numerous high-value-added biomolecules. This review aims to examine the diversity and specificities of extremophilic archaea and bacteria, with particular emphasis on their potential applications and development in biotechnology and biomedicine. The use of extremophiles and their extremozymes has allowed applications in several fields, such as bioremediation, sustainable agriculture, the recovery of bioactive molecules for use in bioenergy, biomedicine, and nanoparticle production. The comprehension and exploitation of the complex molecular mechanisms that enable life in extreme environments represent a challenge to mitigate current climate change problems and to invest in sustainable development towards a green transition.

The hadal zone, one of Earth's most extreme ecosystems, harbors diverse and unique microbial communities adapted to its harsh environmental conditions, including high hydrostatic pressure (HHP) and low temperatures. Within these communities, deep-sea fungi play a critical role in geochemical cycling and marine ecosystem functioning; however, research on their cultivable strains and adaptation mechanism remains scarce. In this study, the piezo-tolerant fungus Aspergillus sydowii DM1, isolated from the Mariana Trench sediments (10,898 m), was selected as a representative strain. A comprehensive genome analysis using high-throughput sequencing revealed a genome size of 34.5 Mb, with 12,241 predicted genes. Functional annotations across multiple databases identified a substantial number of pathways associated with environmental adaptations, including extensive carbohydrate, amino acid, sulfur, and nitrogen metabolic pathways. Among them, the HOG (high-osmolarity glycerol) signal pathway, which responds to external stimuli, was indicated to play a crucial role. To study the HOG signal pathways in more detail, we developed a knockout technique for A. sydowii and constructed a hog1 mutant strain (ΔAshog1). The ΔAshog1 strain displayed notable differences in colony phenotype, spore production, secondary metabolites, and oxidative stress tolerance compared to the wild type. Furthermore, the Ashog1 gene was found to regulate reactive oxygen species (ROS) and ATP levels in response to osmotic pressure and HHP, suggesting a role of hog1 in the fungal adaptation to this extreme environment. Our study serves as an ideal candidate for exploring gene functions in extreme microorganisms and carries significant implications for understanding the adaptive mechanisms of hadal microorganisms. Research on the genomes and gene functions of hadal zone fungi is crucial for understanding life's adaptation to extreme environments. However, current studies on constructing genetic operation systems for marine-derived filamentous fungi are scarce, and research on HHP environments in related fields is virtually non-existent. Our study highlights the critical role of the HOG-mediated pathway in regulating stress responses and metabolic processes in extremophiles, a regulatory mechanism that had not been previously investigated under HHP conditions. Notably, the whole-genome annotation of the hadal fungus Aspergillus sydowii DM1 advances our understanding of the life processes of hadal fungi. The development of gene knockout technology, combined with insights into stress adaptation and metabolic regulation in A. sydowii strain DM1, provides a strong foundation for future research and biotechnological applications.

Nature is home to a wide range of species that thrive in extreme conditions. Despite the identification and study of many extremophilic organisms, significant questions remain regarding the limits of life and the potential for enhancing, combining, or transferring extreme characteristics to other organisms. In previous works of our group, several genes retrieved from environmental extremophiles using functional metagenomics were shown to increase the tolerance of the model bacterium Escherichia coli towards different stress conditions. Here, we proposed to evaluate whether the rational combination of those resistance genes isolated from environmental extremophiles and involved in different molecular mechanisms enhanced the cross-protection of E. coli to extreme conditions. Data revealed that the simultaneous introduction in E. coli of environmental extremophilic resistance genes involved in protein degradation, biofilm formation, oxidative stress, and DNA protection resulted in strongly enhanced, non-additive effects, significantly increasing survival rate under perchlorate exposure, UV radiation, and low pH compared to the individual introduction of these genes. Our findings supports that the introduction of multiple resistance genes isolated from environmental extremophiles that belong to diverse biological processes of stress adaptation may be crucial for engineering of multi-resistant species of interest in biomanufacturing and astrobiology.

The study of extremophiles can lead to the discovery of highly tolerant enzymes of value to biotechnology, and extreme environments are typically sampled to facilitate their discovery. Here, we show that extreme acidotolerant filamentous fungi, able to grow at pH&#x2009;<&#x2009;1, can be found when sampling highly diverse environments, from industrial sites with low pH to typical non-acidic plant and soil samples. Over 100 fungal strains were isolated from over 2,000 samples taken from across Vietnam, and many of the strains were able to grow over wide pH ranges, displaying either acidotolerance or clear acidophilicity. ITS sequencing revealed that 63 isolates represent 12 previously undescribed species, with the majority from the Talaromyces or Penicillium genera. We furthermore report the rediscovery of the previously lost, historically significant acidophile Acontium velatum. Screening of selected fungal secretomes for polysaccharide-cleaving activity revealed that many show broad tolerance to harsh conditions (pH, temperature, organic solvents). Our work greatly expands on the diversity of identified extreme acidotolerant and acidophilic filamentous fungi, which can serve as sources of industrially relevant enzymes. For most species identified, acid tolerance or acidophilicity has not previously been reported, and our results showcase that acidophilicity is more widespread than previously appreciated.

Psychrophilic organisms are able to grow at temperatures down to -15 &#xb0;C, while hyperthermophiles can multiply at temperatures up to 122 &#xb0;C. What structural changes in extremophile proteins are needed to maintain stable and biochemically active structures under such conditions? Understanding how such extremophiles accomplish this is relevant for human health, biotechnology, and our search for life elsewhere in the universe. The purpose of the current study is to report and compare the structures of four rubredoxins (Rds), the first ever two experimental psychrophile bacteria structures (from Gram-positive Clostridium psychrophilum and Gram-negative Polaromonas glacialis) and two hyperthermophiles from the Gram-negative Thermotoga maritima bacterium and the archaeon Pyrococcus yayanosii, also a piezophile, as part of a program to understand structural variations that support both stability and function under extreme conditions. These structures were obtained using synchrotron radiation X-ray diffraction at 100 K. All four structures had the expected overall rubredoxin fold. Rubredoxin from the only aerobic psychrophilic bacterium Polaromonas glacialis had larger variations in sequence and structure, whereas the other psychrophilic bacterium showed properties closely related to hyperthermophile rubredoxins. Multi-subunit structures showed similar RMSD variability independent from their thermal adaptation status. We propose including functional information in the analysis since temperature optimization may not be the only determinant for a specific protein adaptation.

Chitin is the second most abundant polysaccharide and can serve as a carbon and nitrogen source for microorganisms in various ecosystems, including hypersaline environments. However, chitin metabolism in extremely halophilic archaea of the class Halobacteria has not been systematically analyzed, and the pathways of N-acetylglucosamine (GlcNAc) utilization in these organisms remain poorly understood. In this study, we performed a large-scale comparative genomic analysis of the class Halobacteria to assess prevalence and organization of genes, presumably involved in chitin utilization. Potential chitinolytic species are unevenly distributed across the class Halobacteria and tend to cluster within only 7 out of its 117 recognized genera. Glycoside hydrolases from GH18 and GH3 families were found as predominant endochitinases and &#x3b2;-hexosaminidases, respectively. Most putative chitinolytic haloarchaea lacked the genes involved in GlcNAc catabolism in bacteria and hyperthermophilic archaeon Thermococcus kodakaraensis. However, many of them harbor enzymes of pathways previously proposed for Natrarchaeobius and nanohaloarchaeon Ca. "Nanohalobium constans". Analysis of gene neighborhoods demonstrated a conserved organization of the gene clusters associated with chitin metabolism in some genomes, resembling the clusters previously described for Natrarchaeobius species. Overall, our results indicate that Halomicrobium, Natrarchaeobius, Natrialba, Saliphagus, Halocatena, Haladaptatus, and Salinarchaeum genera are enriched in species with chitinolytic potential.

Hypersaline environments exhibit extreme physiochemical conditions yet support diverse microbial communities. These communities are not only ecologically important but also possess substantial potential for biotechnological exploitation. In this study, we employed a comparative metagenomic approach to assess microbial diversity using two distinct methodologies: (1) direct DNA extraction from raw sediment, and (2) DNA extraction following halophilic enrichment in selective media. Sediment samples were collected from multiple sites and pooled together within the Rann of Kachchh and close-by saltpans and were analysed using 16S rRNA sequencing coupled with bioinformatics pipelines. The results revealed pronounced differences in microbial community composition between the two approaches. Raw sediment samples exhibited significantly higher alpha diversity, with dominant taxa including Halobacterota, Cyanobacteria, and Desulfobacterota, with a substantial proportion of unclassified genera. In contrast, enriched samples were dominated by fast-growing, culturable genera such as Halobacterium, Alkalibacillus, and Candidatus haloredivivus. Principal Coordinate Analysis (PCoA) of beta diversity demonstrated distinct clustering between raw and enriched communities, even within samples from the same sites, underscoring the selective bias introduced by enrichment procedures. These findings emphasise that the methodological choice strongly influences the observed microbial diversity. The aim of this study was to compare microbial community composition in raw hypersaline sediments and enrichment cultures using metagenomic sequencing, to evaluate how enrichment selectively favours specific halophilic taxa. This comparative approach allows identification of the microbial groups that rapidly proliferate under controlled hypersaline conditions, thereby complementing direct environmental sequencing. By integrating both direct and enrichment-based metagenomic approaches, a more comprehensive understanding of microbial community structure in hypersaline environments can be achieved.

Investigating the survival limits of extremophilic microorganisms exposed to simulated space conditions can shed light on the ability of terrestrial microorganisms to survive and propagate on other planetary bodies. Although microbes can be found in all environmental niches on Earth, this study focuses on psychrophilic and psychrotolerant microorganisms (prokaryotes and eukaryotes) which have been isolated from locations of interest such as icy moon analogue environments (e.g. Canadian high arctic, Antarctica) and cleanrooms, which might be relevant for forward planetary protection. Our research aimed to reproduce conditions for microorganisms on spacecraft travelling to the outer solar system which could contaminate the icy moon's subsurface oceans. The microorganisms were grown under oligotrophic conditions in minimal media supplemented with only a single carbon source and exposing them to extreme conditions, in terms of temperature fluctuations, in terms of freeze and thaw cycles, and radiation, as they occur during the space travel to the outer solar system. Our results in combination with future metagenome data and phenotype prediction tools will allow the identification of planetary protection relevant microorganisms in spacecraft assembly cleanrooms and on spacecraft and support the development of a target-oriented planetary protection constraints for missions to the icy moons. This article is part of the theme issue 'Planetary Protection for sustainable space exploration'.

暂无摘要（点击查看详情）

Extremolytes - unique compatible solutes produced by extremophiles - protect biological structures like membranes, proteins, and DNA under extreme conditions, including extremes of temperature and osmotic stress. These compounds hold significant potential for applications in pharmaceuticals, healthcare, cosmetics, and life sciences. However, despite their considerable potential, only a limited number of extremolytes - most notably ectoine and hydroxyectoine - have achieved commercial relevance, primarily due to the absence of efficient production strategies for the majority of other extremolytes. Cyclic 2,3-diphosphoglycerate (cDPG), a unique metabolite found in certain hyperthermophilic methanogenic Archaea, plays a key role in thermoprotection and is synthesized from 2-phosphoglycerate (2PG) through a two-step enzymatic process involving 2-phosphoglycerate kinase (2PGK) and cyclic-2,3-diphosphoglycerate synthetase (cDPGS). In this study, we present the development of an efficient in vitro enzymatic approach for the production of cDPG directly from 2,3-diphosphoglycerate (2,3DPG), leveraging the activity of the cDPGS from Methanothermus fervidus (MfcDPGS). We optimized the heterologous production of MfcDPGS in Escherichia coli by refining codon usage and expression conditions. The purification process was significantly streamlined through an optimized heat precipitation step, coupled with effective stabilization of MfcDPGS for both usage and storage by incorporating KCl, Mg2+, reducing agents and omission of an affinity tag. The recombinant MfcDPGS showed a Vmax of 38.2&#x202f;U&#x202f;mg-1, with KM values of 1.52&#x202f;mM for 2,3DPG and 0.55&#x202f;mM for ATP. The enzyme efficiently catalyzed the complete conversion of 2,3DPG to cDPG. Remarkably, even at a scale of 100&#x202f;mM, it achieved full conversion of 37.6&#x202f;mg of 2,3DPG to cDPG within 180&#x202f;min, using just 0.5&#x202f;U of recombinant MfcDPGS at 55&#xb0;C. These results highlight that MfcDPGS can be easily produced, rapidly purified, and sufficiently stabilized while delivering excellent conversion efficiency for cDPG synthesis as value added product. Additionally, a kinetic model for MfcDPGS activity was developed, providing a crucial tool to simulate and scale up cDPG production for industrial applications. This streamlined process offers significant advantages for the scalable synthesis of cDPG, paving the way for further biochemical and industrial applications of this extremolyte.

Manganese (Mn) plays a vital role in soil chemistry, plant nutrition, and contaminant cycling, with certain microorganisms capable of transforming soluble Mn(II) into insoluble biogenic manganese oxides. Among these microorganisms, filamentous fungi are especially effective Mn oxidizers, often surpassing bacteria under challenging environmental conditions. The Acremonium-like fungi, historically recognized for their ecological versatility and production of bioactive metabolites, are now known to be highly polyphyletic, with species spanning multiple genera within the family Bionectriaceae. Despite their diversity, the ecological functions of these fungi remain poorly understood, and Mn-oxidizing activity has not previously been reported from this family. In this study, we surveyed saline-alkali soils in Dongying and Binzhou, Shandong Province, China. Seven Acremonium-like fungal isolates from halophytic rhizospheres were recovered from Dongying, but not from Binzhou. Multilocus phylogenetic analyses using ITS (internal transcribed spacer), 28S (28S ribosomal large subunit), tef1-&#x3b1; (elongation factor 1-alpha), and rpb2 (second largest subunit of RNA polymerase II), along with detailed morphological characterization, demonstrated that these isolates represent three previously undescribed species within the genera Acremonium, Protocreopsis, and Verruciconidia. Remarkably, all isolates exhibited Mn(II)-oxidizing activity, representing the first report of this trait in Bionectriaceae. These findings broaden the taxonomic and ecological understanding of Acremonium-like fungi and suggest their potential role in manganese cycling in extreme soils. Their tolerance to environmental stress, combined with Mn-oxidizing capabilities, indicates potential applications in bioremediation, soil health enhancement, and biotechnology. By uncovering novel extremophilic fungi with functional significance, this study provides insights into microbial adaptation in harsh environments and identifies promising resources for the sustainable management of saline-alkali soils.

Enzymes derived from extremophiles, or extremozymes, possess unique properties that enable them to function under extreme environmental conditions. Microbial communities in subterranean ecosystems have evolved specialized metabolic pathways to survive, leading to the discovery of bioactive molecules with diverse biotechnological and industrial applications as well as the development of sustainable methods for habitat restoration. This study aimed to identify cultivable microorganisms producing industrially relevant enzymes, such as laccases, proteases, and urethanases, from extremophiles in the Dinaric Karst subterranean ecosystems, which are known as biodiversity hotspot. A total of 40 samples were collected from six caves and an abandoned railway tunnel, now a key roost for a large Myotis myotis maternity colony. Cave samples were taken from the entrance, twilight, and dark zones, including soil, sediments, moonmilk, mineral deposits, bedrock deposits, insect remains, entomophagous fungi, wall biofilm, and guano from various bat species. Following microbial cultivation, 207 colonies were screened for enzymatic activity using substrate-specific assays. Functional analysis identified one microorganism exhibiting strong laccase activity, seven capable of degrading polyurethane, and numerous protease-producing colonies. Notably, this study constitutes the inaugural report on discovering polyurethane-degrading microorganisms in karst caves. Molecular identification revealed microbial genera, including Bacillus, Pseudomonas, Serratia, Paenibacillus, and Priestia. These findings underscore the biotechnological potential of subterranean extremophiles and highlight the importance of conserving these ecosystems. Further characterization of these enzymes may drive advancements in environmental remediation, waste recycling, and sustainable industrial processes.