Environmental specificity of karst cave habitats evidenced by diverse symbiotic bacteria in Opiliones.

BACKGROUND: Karst caves serve as natural laboratories, providing organisms with extreme and constant conditions that promote isolation, resulting in a genetic relationship and living environment that is significantly different from those outside the cave. However, research on cave creatures, especially Opiliones, remains scarce, with most studies focused on water, soil, and cave sediments. RESULTS: The structure of symbiotic bacteria in different caves were compared, revealing significant differences. Based on the alpha and beta diversity, symbiotic bacteria abundance and diversity in the cave were similar, but the structure of symbiotic bacteria differed inside and outside the cave. Microorganisms in the cave play an important role in material cycling and energy flow, particularly in the nitrogen cycle. Although microbial diversity varies inside and outside the cave, Opiliones in Beijing caves and Hainan Island exhibited a strong similarity, indicating that the two environments share commonalities. CONCLUSIONS: The karst cave environment possesses high microbial diversity and there are noticeable differences among different caves. Different habitats lead to significant differences in the symbiotic bacteria in Opiliones inside and outside the cave, and cave microorganisms have made efforts to adapt to extreme environments. The similarity in symbiotic bacteria community structure suggests a potential similarity in host environments, providing an explanation for the appearance of Sinonychia martensi in caves in the north.

Origin and modern microbial ecology of secondary mineral deposits in Lehman Caves, Great Basin National Park, NV, USA.

Lehman Caves is an extensively decorated high desert cave that represents one of the main tourist attractions in Great Basin National Park, Nevada. Although traditionally considered a water table cave, recent studies identified abundant speleogenetic features consistent with a hypogenic and, potentially, sulfuric acid origin. Here, we characterized white mineral deposits in the Gypsum Annex (GA) passage to determine whether these secondary deposits represent biogenic minerals formed during sulfuric acid corrosion and explored microbial communities associated with these and other mineral deposits throughout the cave. Powder X-ray diffraction (pXRD), scanning electron microscopy with electron dispersive spectroscopy (SEM-EDS), and electron microprobe analyses (EPMA) showed that, while most white mineral deposits from the GA contain gypsum, they also contain abundant calcite, silica, and other phases. Gypsum and carbonate-associated sulfate isotopic values of these deposits are variable, with δ34SV-CDT between +9.7‰ and +26.1‰, and do not reflect depleted values typically associated with replacement gypsum formed during sulfuric acid speleogenesis. Petrographic observations show that the sulfates likely co-precipitated with carbonate and SiO2 phases. Taken together, these data suggest that the deposits resulted from later-stage meteoric events and not during an initial episode of sulfuric acid speleogenesis. Most sedimentary and mineral deposits in Lehman Caves have very low microbial biomass, with the exception of select areas along the main tour route that have been impacted by tourist traffic. High-throughput 16S rRNA gene amplicon sequencing showed that microbial communities in GA sediments are distinct from those in other parts of the cave. The microbial communities that inhabit these oligotrophic secondary mineral deposits include OTUs related to known ammonia-oxidizing Nitrosococcales and Thaumarchaeota, as well as common soil taxa such as Acidobacteriota and Proteobacteria. This study reveals microbial and mineralogical diversity in a previously understudied cave and expands our understanding of the geomicrobiology of desert hypogene cave systems.

Microbiological exploration of the Cueva del Viento lava tube system in Tenerife, Canary Islands.

Cueva del Viento, located in the Canary Islands, Spain, is the Earth's sixth-longest lava tube, spanning 18,500 m, and was formed approximately 27,000 years ago. This complex volcanic cave system is characterized by a unique geomorphology, featuring an intricate network of galleries. Despite its geological significance, the geomicrobiology of Cueva del Viento remains largely unexplored. This study employed a combination of culture-dependent techniques and metabarcoding data analysis to gain a comprehensive understanding of the cave's microbial diversity. The 16S rRNA gene metabarcoding approach revealed that the coloured microbial mats (yellow, red and white) coating the cave walls are dominated by the phyla Actinomycetota, Pseudomonadota and Acidobacteriota. Of particular interest is the high relative abundance of the genus Crossiella, which is involved in urease-mediated biomineralization processes, along with the presence of genera associated with nitrogen cycling, such as Nitrospira. Culture-dependent techniques provided insights into the morphological characteristics of the isolated species and their potential metabolic activities, particularly for the strains Streptomyces spp., Paenarthrobacter sp. and Pseudomonas spp. Our findings underscore the potential of Cueva del Viento as an ideal environment for studying microbial diversity and for the isolation and characterization of novel bacterial species of biotechnological interest.

A Second Fungal Outbreak in Castañar Cave, Spain, Discloses the Fragility of Subsurface Ecosystems.

Castañar is a cave with strict visitor control measures since it was open to public visits in 2003. However, in recent years, the cave suffered two fungal outbreaks, the first in 2008 and controlled by cleaning the contaminated sediments and subsequent closure of the cave until 2014. The cave was reopened but limited to a maximum of 450 visitors/year. Despite these restrictions on visit, the cave experienced a second outbreak in 2021, originating from the installation of a steel grating walkway, aiming at protecting the ground sediments from the visitors' footsteps. Here, we conducted an analysis using Next-Generation Sequencing and culture-dependent techniques to investigate the fungal communities related to the second outbreak and compare with those present before the cave suffered the outbreak. The results show that the most abundant fungi involved in the 2021 outbreak were already detected in 2020, and even in 2008 and 2009, although the main species that originating both outbreaks were different, likely due to the different carbon sources introduced into the cave.

Microclimate, airborne particles, and microbiological monitoring protocol for conservation of rock-art caves: The case of the world-heritage site La Garma cave (Spain).

Cave heritage is often threatened by tourism or even scientific activities, which can lead to irreversible deterioration. We present a preventive conservation monitoring protocol to protect caves with rock art, focusing on La Garma Cave (Spain), a World Heritage Site with valuable archaeological materials and Palaeolithic paintings. This study assessed the suitability of the cave for tourist use through continuous microclimate and airborne particles monitoring, biofilm analysis, aerobiological monitoring and experimental visits. Our findings indicate several factors that make it inadvisable to adapt the cave for tourist use. Human presence and transit within the cave cause cumulative effects on the temperature of environmentally very stable and fragile sectors and significant resuspension of particles from the cave sediments. These environmental perturbations represent severe impacts as they affect the natural aerodynamic control of airborne particles and determine bacterial dispersal throughout the cave. This monitoring protocol provides part of the evidence to design strategies for sustainable cave management.

Habitat-related variability in the morphological and taxonomic diversity of microbial communities in two Hungarian epigenic karst caves.

The physical and chemical characteristics of the bedrock, along with the geological and hydrological conditions of karst caves may influence the taxonomic and functional diversity of prokaryotes. Most studies so far have focused on microbial communities of caves including only a few samples and have ignored the chemical heterogeneity of different habitat types such as sampling sites, dripping water, carbonate precipitates, cave walls, cave sediment and surface soils connected to the caves. The aim of the present study was to compare the morphology, the composition and physiology of the microbiota in caves with similar environmental parameters (temperature, host rock, elemental and mineral composition of speleothems) but located in different epigenic karst systems. Csodabogyós Cave and Baradla Cave (Hungary) were selected for the analysis of bacterial and archaeal communities using electron microscopy, amplicon sequencing, X-ray diffraction, and mass spectroscopic techniques. The microbial communities belonged to the phyla Pseudomonadota, Acidobacteriota, Nitrospirota and Nitrososphaerota, and they showed site-specific variation in composition and diversity. The results indicate that morphological and physiological adaptations provide survival for microorganisms according to the environment. In epigenic karst caves, prokaryotes are prone to increase their adsorption surface, cooperate in biofilms, and implement chemolithoautotrophic growth with different electron-donors and acceptors available in the microhabitats.

Metagenome Analysis of Speleothem Microbiome from Subterranean Cave Reveals Insight into Community Structure, Metabolic Potential, and BGCs Diversity.

The Borra caves, the second largest subterranean karst cave ecosystem in the Indian sub-continent, are located at the Ananthagiri hills of Araku Valley in the Alluri district of Andhra Pradesh, India. The present investigation applied a shotgun metagenomic approach to gain insights into the microbial community structure, metabolic potential, and biosynthetic gene cluster (BGC) diversity of the microbes colonizing the surface of the speleothems from the aphotic zone of Borra caves. The taxonomic analysis of the metagenome data illustrated that the speleothem-colonizing core microbial community was dominated mainly by Alpha-, Beta-, and Gamma-Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. The key energy metabolic pathways analysis provides strong evidence of chemolithoautotrophic and chemoheterotrophic modes of nutrition in the speleothem-colonizing microbial community. Metagenome data suggests that sulfur reducers and sulfur-disproportionating microbes might play a vital role in energy generation in this ecosystem. Our metagenome data also suggest that the dissimilatory nitrifiers and nitrifying denitrifiers might play an essential role in conserving nitrogen pools in the ecosystem. Furthermore, metagenome-wide BGCs mining retrieved 451 putative BGCs; NRPS was the most abundant (24%). Phylogenetic analysis of the C domain of NRPS showed that sequences were distributed across all six function categories of the known C domain, including several novel subclades. For example, a novel subclade had been recovered within the LCL domain clade as a sister subclade of immunosuppressant cyclosporin encoding C domain sequences. Our result suggested that subterranean cave microbiomes might be a potential reservoir of novel microbial metabolites.

Environmental Drivers of the Moonmilk Microbiome Diversity in Some Temperate and Tropical Caves.

Moonmilk is a cave deposit that was used for medical and cosmetic purposes and has lately raised interest for its antimicrobial potential. We studied five moonmilk samples from four caves with different microclimatic conditions, two temperate in north-western and northern Romania (Ferice, Fața Apei, and Izvorul Taușoarelor caves) and one tropical in Minas Gerais, Brazil (Nestor Cave). The physicochemical and mineralogical analyses confirmed the presence of calcite and dolomite as the main phase in the moonmilk. A 16S rRNA gene-based metabarcoding approach showed the most abundant bacteria phyla Proteobacteria, GAL15, Actinobacteriota, and Acidobacteriota. The investigated caves differed in the dominant orders of bacteria, with the highest distance between the Romanian and Nestor Cave samples. Climate and, implicitly, the soil microbiome can be responsible for some differences we found between all the samples. However, other factors can be involved in shaping the moonmilk microbiome, as differences were found between samples in the same cave (Ferice). In our five moonmilk samples, 1 phylum, 70 orders (~ 36%), and 252 genera (~ 47%) were unclassified, which hints at the great potential of cave microorganisms for future uses.

New insights into the structure, microbial diversity and ecology of yellow biofilms in a Paleolithic rock art cave (Pindal Cave, Asturias, Spain).

In the absence of sunlight, caves harbor a great diversity of microbial colonies to extensive biofilms with different sizes and colors visible to the naked eye. One of the most widespread and visible types of biofilm are those with yellow hues that can constitute a serious problem for the conservation of cultural heritage in many caves, such as Pindal Cave (Asturias, Spain). This cave, declared a World Heritage Site by UNESCO for its Paleolithic parietal art, shows a high degree of development of yellow biofilms that represents a real threat to the conservation of painted and engraved figures. This study aims to: 1) identify the microbial structures and the most characteristic taxa composing the yellow biofilms, 2) seek the linked microbiome reservoir primarily contributing to their growth; 3) seed light on the driving vectors that contribute to their formation and determine the subsequent proliferation and spatial distribution. To achieve this goal, we used amplicon-based massive sequencing, in combination with other techniques such as microscopy, in situ hybridization and environmental monitoring, to compare the microbial communities of yellow biofilms with those of drip waters, cave sediments and exterior soil. The results revealed microbial structures related to the phylum Actinomycetota and the most characteristic bacteria in yellow biofilms, represented by the genera wb1-P19, Crossiella, Nitrospira, and Arenimonas. Our findings suggest that sediments serve as potential reservoirs and colonization sites for these bacteria that can develop into biofilms under favorable environmental and substrate conditions, with a particular affinity for speleothems and rugged-surfaced rocks found in condensation-prone areas. This study presents an exhaustive study of microbial communities of yellow biofilms in a cave, which could be used as a procedure for the identification of similar biofilms in other caves and to design effective conservation strategies in caves with valuable cultural heritage.

Methanotrophs dominate methanogens and act as a methane sink in a subterranean karst cave.

Karst caves are potential sinks of atmospheric methane due to microbial consumption. However, knowledge gaps on methanogens (methane producing microorganisms) and their interaction with methane-oxidizing bacteria (MOB) hinder our further understanding about methane dynamics in karst caves. Here we reported methanogenic community composition and their interaction with MOBs in the Heshang Cave to comprehensively understand methane cycling in subsurface biosphere. MOBs in karst cave were dominated by high-affinity MOB, upland soil cluster (USC), with USCγ pmoA gene abundance within the range of 1.34 × 104 to 1.8 × 107 copies·g-1 DW. In contrast, methanogens were dominated by Methanoregula and cluster ZC-I. The mcrA numbers were 7.21 × 103 to 8.31 × 104 copies·g-1 DW, 1-3 orders of magnitude lower than those of MOB. The inter-domain network analysis indicated that MOBs and methanogens cooperated more in the interior of the cave. Despite of the higher number of methanogenic nodes in the network, MOB dominated the keystone taxa, suggesting a leading functional role of MOB. MOB in caves showed a comparable with or higher potential methane oxidizing rate (PMOR, 0.63 ng CH4·g-1 DW·h-1 in sediment versus 11.02 ng CH4·g-1 DW·h-1 in weathered rock) than those in soils, whereas methane produced by methanogens was undetected. Collectively, high absolute abundances of MOB, high PMORs, the dominance of methanotrophic keystone taxa in the inter-domain network confirmed the superiority of MOBs over methanogens in the oligotrophic karst cave, mounting new evidence on caves as an important methane sink in terms of the interaction between methanogens and MOBs.

Speleomycology of Air in Stopica Cave (Serbia).

Fungi can colonize organic matter present in subterranean sites and have a significant role as dwellers in different microniches of cave habitats. In order to analyze the content of airborne fungal propagules in different parts of "Stopica Cave," a touristic site in Serbia, air sampling was carried out in three seasons during 2020, prior to and during the onset of COVID-19 pandemic. Culturable mycobiota was identified using both microscopic techniques and ITS region/BenA gene barcoding, while multivariate analyses were employed to establish the link between fungal taxa and different environmental factors. The maximal measured fungal propagule concentrations were recorded during spring sampling which were based on fungal propagule concentration categories; the cave environment matches the category V. A total of 29 fungal isolates were identified, while Aspergillus, Cladosporium, Fusarium, Lecanicillium, Mucor, and Penicillium were the most diverse genera. According to the trophic mode, most of the isolated fungal species were pathotrophs (75.86%), but when regarding ecological guilds, the most dominant were undefined saprobes and animal pathogens (41.38% for each). Show caves are especially vulnerable to human impacts, and the fungal propagules' concentration within the caves could be good indices for the level of ecological disturbance.

Tourism affects microbial assemblages in show caves.

Anthropogenic disturbance on natural ecosystems is growing in frequency and magnitude affecting all ecosystems components. Understanding the response of different types of biocoenosis to human disturbance is urgently needed and it can be achieved by adopting a metacommunity framework. With the aid of advanced molecular techniques, we investigated sediment communities of Fungi, Bacteria and Archaea in four Italian show caves, aiming to disentangle the effects induced by tourism on their diversity and to highlight changes in the driving forces that shape their community composition. We modelled diversity measures against proxies of tourism pressure. With this approach we demonstrate that the cave tourism has a direct effect on the community of Bacteria and an indirect influence on Fungi and Archaea. By analysing the main driving forces influencing the community composition of the three microbial groups, we highlighted that stochastic factors override dispersal-related processes and environmental selection in show caves compared to undisturbed areas. Thanks to this approach, we provide new perspectives on the dynamics of microbial communities under human disturbance suggesting that a proper understanding of the underlying selective mechanisms requires a comprehensive and multi-taxonomic approach.

First Insights into the Bacterial Diversity of Mount Etna Volcanic Caves.

While microbial communities in limestone caves across the world are relatively understood, knowledge of the microbial composition in lava tubes is lagging behind. These caves are found in volcanic regions worldwide and are typically lined with multicolored microbial mats on their walls and ceilings. The Mount Etna (Sicily, S-Italy) represents one of the most active volcanos in the world. Due to its outstanding biodiversity and geological features, it was declared Natural Heritage of Humanity by the UNESCO in 2013. Despite the presence of more than 200 basaltic lava tubes, the microbial diversity of these hypogean systems has never been investigated so far. Here, we investigated bacterial communities in four lava tubes of Mount Etna volcano. Field emission scanning electron microscopy (FESEM) was carried out for the morphological characterization and detection of microbial features. We documented an abundant presence of microbial cells with different morphotypes including rod-shaped, filamentous, and coccoidal cells with surface appendages, resembling actinobacteria reported in other lava tubes across the world. Based on 16S rRNA gene analysis, the colored microbial mats collected were mostly composed of bacteria belonging to the phyla Actinomycetota, Pseudomonadota, Acidobacteriota, Chloroflexota, and Cyanobacteria. At the genus level, the analysis revealed a dominance of the genus Crossiella, which is actively involved in biomineralization processes, followed by Pseudomonas, Bacillus, Chujaibacter, and Sphingomonas. The presence of these taxa is associated with the carbon, nitrogen, and ammonia cycles, and some are possibly related to the anthropic disturbance of these caves. This study provides the first insight into the microbial diversity of the Etna volcano lava tubes, and expands on previous research on microbiology of volcanic caves across the world.

Microbial diversity and proxy species for human impact in Italian karst caves.

To date, the highly adapted cave microbial communities are challenged by the expanding anthropization of these subterranean habitats. Although recent advances in characterizing show-caves microbiome composition and functionality, the anthropic effect on promoting the establishment, or reducing the presence of specific microbial guilds has never been studied in detail. This work aims to investigate the whole microbiome (Fungi, Algae, Bacteria and Archaea) of four Italian show-caves, displaying different environmental and geo-morphological conditions and one recently discovered natural cave to highlight potential human-induced microbial traits alterations. Results indicate how show-caves share common microbial traits in contrast to the natural one; the first are characterized by microorganisms related to outdoor environment and/or capable of exploiting extra inputs of organic matter eventually supplied by tourist flows (i.e. Chaetomium and Phoma for fungi and Pseudomonas for bacteria). Yet, variation in microalgae assemblage composition was reported in show-caves, probably related to the effect of the artificial lighting. This study provides insights into the potential microbiome cave contamination by human-related bacteria (e.g. Lactobacillus and Staphylococcus) and commensal/opportunistic human associated fungi (e.g. Candida) and dermatophytes. This work is critical to untangle caves microbiome towards management and conservation of these fragile ecosystems.

Major impacts of caving activities on cave microbial diversity: case study of Morca Cave, Turkey.

Some of microorganisms identified in cave ecosystems have been reported to play a permanent and strategic role for maintaining life of these environments. Human entrance into caves can induce some changes of cave physicochemical parameters which ultimately impacts the living organisms. In these facts, for the first time, Morca Cave was explored in a purpose to evaluate the impacts that can be caused by the human activities on microbial diversity in a limited period of time. Morca is a karts cave located in the Taseli Plateau in the Middle Taurus mountains in Turkey. The first entrance into this cave was in 2018, and expeditors reached to - 500 m. During the second expedition in 2019, a camp was established at the - 1040-m depth during 4 days. In order to evaluate the human impacts in a new explored cave, this camping depth is chosen to be our studied area because it was its first entrance. Before the installation and at the end of the camp, sediments and surface samples were taken from different points of the camp area and around. Sequencing of 16 s rRNA of each sample to isolate DNA by using the next-generation sequencing (NGS) method was performed. The profile of the microbial diversity before the camping revealed that the class Thermoplasmata was dominated the archaea group and Gammaproteobacteria and Alphaproteobacteria were the most dominant bacterial classes. After the camp, most studied sites were noted with a decrease of microbial diversity especially the previous cited classes strains. Bacteria belonging to Bacilli class have increased after the camp. Increase of bacteria that are belonging to Bacteroidia has also observed in the most active areas. This present study highlight how cave microbial diversity can respond to the human activities within a short period inside a closed cave. Furthermore, it may constitute a solid basis and support on the improvement of techniques for cave management and expedition planning for the conservation of cave nature.

Microscale dynamics of dark zone alterations in anthropized karstic cave shows abrupt microbial community switch.

Strong anthropization of karstic caves may result in formation of various wall alterations including dark zones, whose microbial community differs from that of non-altered surfaces nearby. Dark zones grow quickly and without gradual visual changes, leading to the hypothesis of a simple process rather than complex microbial successions, but this is counter-intuitive as underground microbial changes are typically slow and dark zones are microbiologically very distinct from unmarked surfaces. We tested this hypothesis in Paleolithic Lascaux Cave, across two years of microscale sampling. Indeed, Illumina MiSeq metabarcoding evidenced only three community stages for bacteria, fungi and all microeukaryotes together (i.e. unmarked surfaces, newly-formed dark zones and intermediate/old dark zones) and just two stages for archaea (unmarked surfaces vs dark zones), indicating abrupt community changes. The onset of dark zone formation coincided with the development of Ochroconis fungi, Bacteroidota and the bacterial genera Labrys, Nonomuraea and Sphingomonas, in parallel to Pseudomonas counter-selection. Modeling of community assembly processes highlighted that the dynamics of rare taxa in unmarked surfaces adjacent to dark zones and in newly-formed dark zones were governed in part by deterministic processes. This suggests that cooperative relationships between these taxa might be important to promote dark zone formation. Taken together, these findings indicate an abrupt community switch as these new alterations form on Lascaux cave walls.

Comparison of Actinobacteria communities from human-impacted and pristine karst caves.

Actinobacteria are important cave inhabitants, but knowledge of how anthropization and anthropization-related visual marks affect this community on cave walls is lacking. We compared Actinobacteria communities among four French limestone caves (Mouflon, Reille, Rouffignac, and Lascaux) ranging from pristine to anthropized, and within Lascaux Cave between marked (wall visual marks) and unmarked areas in different rooms (Sas-1, Passage, Apse, and Diaclase). In addition to the 16S rRNA gene marker, 441 bp fragments of the hsp65 gene were used and an hsp65-related taxonomic database was constructed for the identification of Actinobacteria to the species level by Illumina-MiSeq analysis. The hsp65 marker revealed higher resolution for species and higher richness (99% operational taxonomic units cutoff) versus the 16S rRNA gene; however, more taxa were identified at higher taxonomic ranks. Actinobacteria communities varied between Mouflon and Reille caves (both pristine), and Rouffignac and Lascaux (both anthropized). Rouffignac displayed high diversity of Nocardia, suggesting human inputs, and Lascaux exhibited high Mycobacterium relative abundance, whereas Gaiellales were typical in pristine caves and the Diaclase (least affected area of Lascaux Cave). Within Lascaux, Pseudonocardiaceae dominated on unmarked walls and Streptomycetaceae (especially Streptomyces mirabilis) on marked walls, indicating a possible role in mark formation. A new taxonomic database  was developed. Although not all Actinobacteria species were represented, the use of the hsp65 marker enabled species-level variations of the Actinobacteria community to be documented based on the extent of anthropogenic pressure. This approach proved effective when comparing different limestone caves or specific conditions within one cave.

Community assembly, potential functions and interactions between fungi and microalgae associated with biodeterioration of sandstone at the Beishiku Temple in Northwest China.

Fungi, cyanobacteria and algae are specific microbial groups associated with the deterioration and safety of stone monuments. In this study, high-throughput sequencing analysis was used to investigate the diversity, distributions, ecological functions, and interaction patterns of both the fungal and microalgal (including cyanobacteria and algae) communities on sandstone in the Beishiku Temple, located on the ancient Silk Road. The results showed that the core phyla of fungi were affiliated with unclassified Lecanoromycetes, Engyodontium, Knufia, Epicoccum, Endocarpon, and Cladosporium of Ascomycota whereas the phyla of microalgae were dominated by prokaryotic Cyanobacteria and eukaryotic Chlorophyta. The environmental factors of temperature, relative humidity, and light intensity were monitored simultaneously. The structure of the microbial communities was much more strongly shaped by soluble Cl-, Na+, NO3- ions than by the light intensity, moisture content or temperature, especially for the weathered sandstone located outside the caves. The co-occurrence network analysis suggested that a more stable community structure was evident outside the caves than inside. The stronger positive connections and coexistence patterns that were detected indicate a strong adaptability of fungi and microalgae to the distinct oligotrophic microhabitats on sandstone. The metacommunity co-occurrence network exhibited the ecological predominance of fungi, and most of the functional fungi in the biofilms outside the caves belonged to the Lichenized group, based on the FUNGuild prediction. These findings highlight the ecology and functions of stone-inhabiting microorganisms to further advance the current understanding and knowledge of sandstone biodeterioration for protection and management.

Metagenome-Assembled Genomes from Monte Cristo Cave (Diamantina, Brazil) Reveal Prokaryotic Lineages As Functional Models for Life on Mars.

Microbial communities have been explored in various terrestrial subsurface ecosystems, showing metabolic potentials that could generate noteworthy morphological and molecular biosignatures. Recent advancements in bioinformatic tools have allowed for descriptions of novel and yet-to-be cultivated microbial lineages in different ecosystems due to the genome reconstruction approach from metagenomic data. Using shotgun metagenomic data, we obtained metagenome-assembled genomes related to cultivated and yet-to-be cultivated prokaryotic lineages from a silica and iron-rich cave (Monte Cristo) in Minas Gerais State, Brazil. The Monte Cristo Cave has been shown to possess a high diversity of genes involved with different biogeochemical cycles, including reductive and oxidative pathways related to carbon, sulfur, nitrogen, and iron. Three genomes were selected for pangenomic analysis, assigned as Truepera sp., Ca. Methylomirabilis sp., and Ca. Koribacter sp. based on their lifestyles (radiation resistance, anaerobic methane oxidation, and potential iron oxidation). These bacteria exhibit genes involved with multiple DNA repair strategies, starvation, and stress response. Because these groups have few reference genomes deposited in databases, our study adds important genomic information about these lineages. The combination of techniques applied in this study allowed us to unveil the potential relationships between microbial genomes and their ecological processes with the cave mineralogy and highlight the lineages involved with anaerobic methane oxidation, iron oxidation, and radiation resistance as functional models for the search for extant life-forms outside our planet in silica- and iron-rich environments and potentially on Mars.

Prauserella cavernicola sp. nov., isolated from cave rock.

A novel actinomycete, designated strain ASG 168T, was isolated from cave rock collected from Stegodon Sea Cave in Thailand. Long chains of non-motile spores that were oval or spherical in shape with a smooth surface developed on aerial mycelia. Substrate mycelia fragmented into irregular rod-shaped elements. A polyphasic taxonomic study showed that strain ASG 168T had typical characteristics of members of the genus Prauserella. 16S rRNA gene sequence analysis indicated that strain ASG 168T shared 97.5 % similarity with Prauserella marina MS498T and 96.7 % with Prauserella coralliicola SCSIO 11529T. Average nucleotide identity values with P. coralliicola SCSIO 11529T and P. marina MS498T were 82.98 and 76.08 %, respectively. The cell-wall peptidoglycan contained meso-diaminopimelic acid. The whole-cell sugars contained ribose, arabinose and galactose. The predominant menaquinone was MK-9(H4). The predominant fatty acids were iso-C16 : 0, C16 : 0 and summed feature 3 (C16 : 1 ω7c/C16 : 1 ω6c). The phospholipid profile consisted of phosphatidylethanolamine, phosphatidylmethylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, phosphatidylinositol mannosides and two unknown phospholipids. The G+C content of the genomic DNA was 70.6 mol%. Differentiation of strain ASG 168T from closely related species was evident from digital DNA-DNA hybridization values of 29.2 and 21.3 % with P. coralliicola and P. marina, respectively. Based on comparative analysis of phenotypic, chemotaxonomic and genotypic data, the novel actinomycete strain ASG 168T (=TBRC 13679T=NBRC 114887T) is proposed to be the type strain of a novel species, Prauserella cavernicola sp. nov.