Cave Thiovulum (Candidatus Thiovulum stygium) differs metabolically and genomically from marine species.

Thiovulum spp. (Campylobacterota) are large sulfur bacteria that form veil-like structures in aquatic environments. The sulfidic Movile Cave (Romania), sealed from the atmosphere for ~5 million years, has several aqueous chambers, some with low atmospheric O2 (~7%). The cave's surface-water microbial community is dominated by bacteria we identified as Thiovulum. We show that this strain, and others from subsurface environments, are phylogenetically distinct from marine Thiovulum. We assembled a closed genome of the Movile strain and confirmed its metabolism using RNAseq. We compared the genome of this strain and one we assembled from public data from the sulfidic Frasassi caves to four marine genomes, including Candidatus Thiovulum karukerense and Ca. T. imperiosus, whose genomes we sequenced. Despite great spatial and temporal separation, the genomes of the Movile and Frasassi Thiovulum were highly similar, differing greatly from the very diverse marine strains. We concluded that cave Thiovulum represent a new species, named here Candidatus Thiovulum stygium. Based on their genomes, cave Thiovulum can switch between aerobic and anaerobic sulfide oxidation using O2 and NO3- as electron acceptors, the latter likely via dissimilatory nitrate reduction to ammonia. Thus, Thiovulum is likely important to both S and N cycles in sulfidic caves. Electron microscopy analysis suggests that at least some of the short peritrichous structures typical of Thiovulum are type IV pili, for which genes were found in all strains. These pili may play a role in veil formation, by connecting adjacent cells, and in the motility of these exceptionally fast swimmers.

Unearthing Neanderthal population history using nuclear and mitochondrial DNA from cave sediments.

Bones and teeth are important sources of Pleistocene hominin DNA, but are rarely recovered at archaeological sites. Mitochondrial DNA (mtDNA) has been retrieved from cave sediments but provides limited value for studying population relationships. We therefore developed methods for the enrichment and analysis of nuclear DNA from sediments and applied them to cave deposits in western Europe and southern Siberia dated to between 200,000 and 50,000 years ago. We detected a population replacement in northern Spain about 100,000 years ago, which was accompanied by a turnover of mtDNA. We also identified two radiation events in Neanderthal history during the early part of the Late Pleistocene. Our work lays the ground for studying the population history of ancient hominins from trace amounts of nuclear DNA in sediments.

Ancient DNA and multimethod dating confirm the late arrival of anatomically modern humans in southern China.

The expansion of anatomically modern humans (AMHs) from Africa around 65,000 to 45,000 y ago (ca. 65 to 45 ka) led to the establishment of present-day non-African populations. Some paleoanthropologists have argued that fossil discoveries from Huanglong, Zhiren, Luna, and Fuyan caves in southern China indicate one or more prior dispersals, perhaps as early as ca. 120 ka. We investigated the age of the human remains from three of these localities and two additional early AMH sites (Yangjiapo and Sanyou caves, Hubei) by combining ancient DNA (aDNA) analysis with a multimethod geological dating strategy. Although U-Th dating of capping flowstones suggested they lie within the range ca. 168 to 70 ka, analyses of aDNA and direct AMS 14C dating on human teeth from Fuyan and Yangjiapo caves showed they derive from the Holocene. OSL dating of sediments and AMS 14C analysis of mammal teeth and charcoal also demonstrated major discrepancies from the flowstone ages; the difference between them being an order of magnitude or more at most of these localities. Our work highlights the surprisingly complex depositional history recorded at these subtropical caves which involved one or more episodes of erosion and redeposition or intrusion as recently as the late Holocene. In light of our findings, the first appearance datum for AMHs in southern China should probably lie within the timeframe set by molecular data of ca. 50 to 45 ka.

What's the temperature in tropical caves?

Hourly temperature was measured for approximately one year at 17 stations in three caves in Quintana Roo, Mexico. Thirteen of these stations were in the extensive twilight zones of all three caves. All seventeen stations showed seasonality in temperature with a 3°C drop during the Nortes season. Two of the caves, Muévelo Sabrosito and Muévelo Rico, showed greater variability during the winter months while in Río Secreto (Tuch) variability was greatest during the rainy season. Río Secreto is less open to the surface than the other two. All sites also showed a daily temperature cycle, although it was very faint in some Río Secreto (Tuch) sites. While temperature variability is diminished relative to surface variation, its temporal pattern is worthy of further study.

Streptomyces from traditional medicine: sources of new innovations in antibiotic discovery.

Given the increased reporting of multi-resistant bacteria and the shortage of newly approved medicines, researchers have been looking towards extreme and unusual environments as a new source of antibiotics. Streptomyces currently provides many of the world's clinical antibiotics, so it comes as no surprise that these bacteria have recently been isolated from traditional medicine. Given the wide array of traditional medicines, it is hoped that these discoveries can provide the much sought after core structure diversity that will be required of a new generation of antibiotics. This review discusses the contribution of Streptomyces to antibiotics and the potential of newly discovered species in traditional medicine. We also explore how knowledge of traditional medicines can aid current initiatives in sourcing new and chemically diverse antibiotics.

Nutrient availability in tropical caves influences the dynamics of microbial biomass.

Few studies have evaluated the trophic level in tropical caves, and none related the microbial biomass dynamics in the immobilization of carbon and nitrogen. Here, four tropical caves of Terra Ronca State Park, Brazil, were studied: Angélica, São Bernardo, Terra Ronca I, and Terra Ronca II caves. Physical, chemical, and microbiological parameters (microbial biomass and respiration) were estimated in the dry and wet seasons. São Bernardo, Terra Ronca I, and Terra Ronca II caves presented higher nitrogen and microbial biomass nitrogen (MBN) values in the wet season than in the dry season. On the other hand, the Angélica cave showed larger amounts of nitrogen and lower MBN values in the dry season. These results indicate that caves can be adjusted in two ecological theories known as "stoichiometric decomposition" and "microbial nitrogen mining"-to the effects of nutrient availability on organic matter decomposition. The caves studied showed different environmental dynamics in relation to organic matter decomposition, which allows them to be considered unique and possess specific characteristics. Microbial biomass dynamics can be an important parameter to evaluate the availability of nutrients and ecological dynamics of the trophic network in subterranean environments.

Shelter in Smolen III - A unique example of stratified Holocene clastic cave sediments in Central Europe, a lithostratigraphic stratotype and a record of regional paleoecology.

A cave site Shelter in Smolen III (southern Poland) contains an approximately 2-m-thick stratified sequence of Upper Pleistocene and Holocene clastic sediments, unique for Central Europe. The sequence contents abundant fossil fauna, including mollusk, rodent and bat remains. The cave sites with long profiles of subfossil fauna present a great value for reconstructions of regional terrestrial paleoenvironment. We explore the stratigraphy of this site through analyses of the lithology and geochemistry of sediments, radiocarbon dating of faunal and human remains and charcoals, and archaeological study, as well as the paleoecology derived from the taxonomic composition of fossil faunal assemblages. Our data show that the entire period of the Holocene is recorded in the rockshelter, which makes that site an exceptional and highly valuable case. We present paleoenvironmental reconstructions of regional importance, and we propose to regard Shelter in Smolen III as a regional stratigraphic stratotype of Holocene clastic cave sediments.

Radon (222Rn) concentrations in the touristic Jumandy cave in the Amazon region of Ecuador.

This work consists of the detection and quantification of the concentration levels of radioactive gas radon-222 (222Rn) of natural origin, as well as the determination of the critical points and the estimation of the effective dose absorbed by the tourists and guides inside the Jumandy cavern in Napo, Ecuador. According to the feasibility map of uranium of Ecuador, the study area is located in one of the top-priority areas for obtaining uranium, suggesting possible radioactivity in this unstudied region. The measurements were carried out from July to October of 2017, in three different monitoring points inside the cavern. The average radon concentrations measured in the cavern exceeded the maximum recommended environmental level by a factor of 28, and the effective dose absorbed by the guides exceeded the recommended maximum by a f actor of 10. Meteorological parameters such as temperature and relative humidity have an impact on the 222Rn concentrations in different parts of the cave.

Profiling the Bacterial Diversity in a Typical Karst Tiankeng of China.

While karst tiankengs have a higher capacity to act as safe havens for biodiversity in changing climates, little is known about their soil microorganisms. To fill this gap, we investigate the distribution and driving factors of the bacterial community in karst tiankeng systems. There is a significant difference in the soil characteristics between the inside and the outside of a karst tiankeng. At the karst tiankeng considered in this study, the bacterial composition, in terms of the operational taxonomic unit (OTU), was found to be significantly different in different soil samples, taken from diverse sampling sites within the collapsed doline or the external area, and showed a high habitat heterogeneity. The dominant phylum abundances vary with the sampling sites and have their own indicator taxa from phylum to genus. Unlike the primary controlling factors of plant diversity, the microclimate (soil moisture and temperature), soil pH, and slope dominated the distribution of the bacterial community in karst tiankeng systems. Our results firstly showed the distribution characteristics of bacterial communities and then revealed the importance of microhabitats in predicting the microbial distribution in karst tiankeng systems.

Improved Polycyclic Aromatic Hydrocarbon and n-Alkane Determination in Speleothems through Cleanroom Sample Processing.

Interest in paleoenvironmental reconstructions from biomarkers in speleothems is increasing, thanks in part to the capacity of speleothems to grow continuously and to resist postdepositional alteration. In particular, the possibility exists to link high-resolution and accurately dated fire and vegetation records with isotopic data of climatic and paleoenvironmental interactions at the local and regional scale. However, the scarcity of existing methods for the quantification of organic molecules in stalagmites, together with the issues of sample availability, contamination, and low concentrations, complicate this approach. In this work, we developed a novel method for the simultaneous determination of 18 polycyclic aromatic hydrocarbons (PAHs) and 26 n-alkanes (C10-C35) and then tested it on "clean" calcite and aragonite stalagmite samples from cave KNI-51 in the Australian tropics. The method involves subsampling by using a hand-held drill, complete dissolution of the matrix in hydrochloric acid, then liquid-liquid extraction, and GC-MS analysis. Sample preparation was carried out in a 10 000 class clean room built entirely in stainless steel to avoid contamination. Detection limits were 0.3-9 ng for PAHs and 6-44 ng for n-alkanes. Measurable concentrations of fire-derived PAH compounds, namely, phenanthrene, pyrene, benzo( e)pyrene, and indeno(123- cd)pyrene, were detected in only one sample, which dates to the year ∼2004 CE, when a fire burned vegetation over the cave; n-alkanes were detected in all samples in the range C23-C35, with no odd-even preference.

Microbial eukaryotes in the suboxic chemosynthetic ecosystem of Movile Cave, Romania.

Movile Cave is a small system of partially inundated galleries in limestone settings close to the Black Sea in Southeast Romania. Isolated from the surface for 6 million years, its sulfidic, methane and ammonia-rich waters harbour unique chemosynthetic prokaryotic communities that include sulphur and ammonium-metabolizing chemolithotrophs, methanogens, methanotrophs and methylotrophs. The cave also harbours cave-dwelling invertebrates and fungi, but the diversity of other microbial eukaryotes remained completely unknown. Here, we apply an 18S rRNA gene-based metabarcoding approach to study the composition of protist communities in floating microbial mats and plankton from a well-preserved oxygen-depleted cave chamber. Our results reveal a wide protist diversity with, as dominant groups, ciliates (Alveolata), Stramenopiles, especially bicosoecids, and jakobids (Excavata). Ciliate sequences dominated both, microbial mats and plankton, followed by either Stramenopiles or excavates. Stramenopiles were more prominent in microbial mats, whereas jakobids dominated the plankton fraction of the oxygen-depleted water column. Mats cultured in the laboratory were enriched in Cercozoa. Consistent with local low oxygen levels, Movile Cave protists are most likely anaerobic or microaerophilic. Several newly detected OTU clades were very divergent from cultured species or environmental sequences in databases and represent phylogenetic novelty, notably within jakobids. Movile Cave protists likely cover a variety of ecological roles in this ecosystem including predation, parasitism, saprotrophy and possibly diverse prokaryote-protist syntrophies.

Ornithinimicrobium cavernae sp. nov., an actinobacterium isolated from a karst cave.

A novel actinobacterium, designated strain CFH 30183T, was isolated from a soil sample collected from a karst cave in Luoyang, Henan Province. The taxonomic position of the strain was investigated using a polyphasic approach. Cells of strain CFH 30183T were observed to be Gram-stain positive, motile, asporogenous and coccoid to rod shaped. The strain was found to be aerobic and oxidase positive. On the basis of 16S rRNA gene sequence analysis, strain CFH 30183T was found to be closely related to Ornithinimicrobium murale 01-Gi-040T (97.8% sequence identity). The ANIb/ANIm values between strain CFH 30183T and O. murale DSM 22056T were found to be 80.3%/85.9%. Strain CFH 30183T was found to grow optimally at 28-32 °C, at pH 8.0-9.0 and in the presence of up to 7% NaCl (w/v). Whole cell hydrolysates of strain CFH 30183T contained L-ornithine as the diagnostic diamino acid, and arabinose, glucose, mannose and rhamnose as whole cell sugars. The respiratory quinone was determined to be MK-8(H4), while the major fatty acids were found to consist of iso-C15:0 and iso-C16:0. The polar lipids profile was found to include diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, an unidentified phospholipid, an unidentified phosphoglycolipid and four unidentified lipids. The DNA G + C content of strain CFH 30183T was calculated to be 70.9%. Based on the phenotypic, genotypic and phylogenetic data obtained, strain CFH 30183T is considered to represent a novel species of the genus Ornithinimicrobium, for which the name Ornithinimicrobium cavernae sp. nov. is proposed. The type strain is CFH 30183T (= KCTC 49018T = CGMCC 1.16393T).

Radiolysis via radioactivity is not responsible for rapid methane oxidation in subterranean air.

Atmospheric methane is rapidly lost when it enters humid subterranean critical and vadose zones (e.g., air in soils and caves). Because methane is a source of carbon and energy, it can be consumed by methanotrophic methane-oxidizing bacteria. As an additional subterranean sink, it has been hypothesized that methane is oxidized by natural radioactivity-induced radiolysis that produces energetic ions and radicals, which then trigger abiotic oxidation and consumption of methane within a few hours. Using controlled laboratory experiments, we tested whether radiolysis could rapidly oxidize methane in sealed air with different relative humidities while being exposed to elevated levels of radiation (more than 535 kBq m-3) from radon isotopes 222Rn and 220Rn (i.e., thoron). We found no evidence that radiolysis contributed to methane oxidation. In contrast, we observed the rapid loss of methane when moist soil was added to the same apparatus in the absence of elevated radon abundance. Together, our findings are consistent with the view that methane oxidizing bacteria are responsible for the widespread observations of methane depletion in subterranean environments. Further studies are needed on the ability of microbes to consume trace amounts of methane in poorly ventilated caves, even though the trophic and energetic benefits become marginal at very low partial pressures of methane.

Microbial megacities fueled by methane oxidation in a mineral spring cave.

Massive biofilms have been discovered in the cave of an iodine-rich former medicinal spring in southern Germany. The biofilms completely cover the walls and ceilings of the cave, giving rise to speculations about their metabolism. Here we report on first insights into the structure and function of the biofilm microbiota, combining geochemical, imaging and molecular analytics. Stable isotope analysis indicated that thermogenic methane emerging into the cave served as an important driver of biofilm formation. The undisturbed cavern atmosphere contained up to 3000 p.p.m. methane and was microoxic. A high abundance and diversity of aerobic methanotrophs primarily within the Methylococcales (Gammaproteobacteria) and methylotrophic Methylophilaceae (Betaproteobacteria) were found in the biofilms, along with a surprising diversity of associated heterotrophic bacteria. The highest methane oxidation potentials were measured for submerged biofilms on the cavern wall. Highly organized globular structures of the biofilm matrix were revealed by fluorescent lectin staining. We propose that the extracellular matrix served not only as an electron sink for nutrient-limited biofilm methylotrophs but potentially also as a diffusive barrier against volatilized iodine species. Possible links between carbon and iodine cycling in this peculiar habitat are discussed.

Cultivable microscopic fungi from an underground chemosynthesis-based ecosystem: a preliminary study.

Movile Cave, a unique groundwater ecosystem in southern Romania, was discovered in 1986. This chemoautotrophic cave contains an abundant and diverse fauna with terrestrial and aquatic invertebrate communities, including 33 endemic species. Since its discovery, studies have focused mainly on cave chemoautotrophic bacteria, while the microfungal community has been largely neglected. In this study, we determined the microfungal species living on various substrates in Movile Cave and compared this spectrum with the mycobiota detected outside the cave (outside air-borne and soil-borne microfungi). To investigate all of the niches, we collected samples for two consecutive years from the dry part of the cave (cave air and sediment, corroded limestone walls, isopod feces, and isopod and spider cadavers) and from the post-siphon part of the cave, i.e., Airbell II (sediment and floating microbial mat). A total of 123 microfungal species were identified from among several hundred isolates. Of these, 96 species were only detected in the cave environment and not outside of the cave, while 90 species were from the dry part of the cave and 28 were from Airbell II. The most diverse genera were Penicillium (at least 18 species) and Aspergillus (14 species), followed by Cladosporium (9 species). Surprisingly, high CFU counts of air-borne microfungi were found inside the cave; they were even higher than outside the cave during the first year of investigation.

Transport-Induced Spatial Patterns of Sulfur Isotopes (δ34S) as Biosignatures.

Cave minerals deposited in the presence of microbes may host geochemical biosignatures that can be utilized to detect subsurface life on Earth, Mars, or other habitable worlds. The sulfur isotopic composition of gypsum (CaSO4·2H2O) formed in the presence of sulfur-oxidizing microbes in the Frasassi cave system, Italy, was evaluated as a biosignature. Sulfur isotopic compositions (δ34SV-CDT) of gypsum sampled from cave rooms with sulfidic air varied from -11 to -24‰, with minor deposits of elemental sulfur having δ34S values between -17 and -19‰. Over centimeter-length scales, the δ34S values of gypsum varied by up to 8.5‰. Complementary laboratory experiments showed negligible fractionation during the oxidation of elemental sulfur to sulfate by Acidithiobacillus thiooxidans isolated from the caves. Additionally, gypsum precipitated in the presence and absence of microbes at acidic pH characteristic of the sulfidic cave walls has δ34S values that are on average 1‰ higher than sulfate. We therefore interpret the 8.5‰ variation in cave gypsum δ34S (toward more negative values) to reflect the isotopic effect of microbial sulfide oxidation directly to sulfate or via elemental sulfur intermediate. This range is similar to that expected by abiotic sulfide oxidation with oxygen, thus complicating the use of sulfur isotopes as a biosignature at centimeter-length scales. However, at the cave room (meter-length) scale, reactive transport modeling suggests that the overall ∼13‰ variability in gypsum δ34S reflects isotopic distillation of circulating H2S gas due to microbial sulfide oxidation occurring along the cave wall-atmosphere interface. Systematic variations of gypsum δ34S along gas flow paths can thus be interpreted as biogenic given that slow, abiotic oxidation cannot produce the same spatial patterns over similar length scales. The expression and preservation potential of this biosignature is dependent on gas flow parameters and diagenetic processes that modify gypsum δ34S values over geological timescales. Key Words: Gypsum-Sulfur isotopes-Biosignature-Sulfide oxidation-Cave. Astrobiology 18, 59-72.

Sources of sulphate minerals in limestone cave-a possible evidence of anthropogenic activity: a case study in Crna Jama Cave (Slovenia).

In the caves, the formation of cave minerals is a consequence of a variety of chemical reactions, some of them also due to human activity. There are many caves in Slovenia, but sulphate minerals are not very often reported and analysed. In this study, the presence of sulphate minerals is detected by SEM/EDS analysis of speleothems from Crna Jama, a cave near Kocevje (southern Slovenia). The cave is characterised by its dark, almost black colour on cave walls, floor and speleothems. Anthropogenic influence in the cave is still visible, including the remains of a fireplace, some inscriptions on the walls and wooden containers. The analyses of some of the black-coated speleothems reveal the presence of calcium sulphate, confirmed by XRD as gypsum. Gypsum crystals are around 50 µm in size, and they occur in thin crusts. Additionally, some rare authigenic baryte crystals a few micrometres in size are detected. The sulphates δ34S value in gypsum found on dark coloured speleothems is + 10.4‰ Vienna Canyon Diablo Troilite (VCDT), while the sulphate δ34S of the bedrock is + 8.6‰ VCDT. The more likely source of sulphate ions is thus biomass burning rather than bedrock. Also, bedrock and biomass ash are a very probable source of calcium and barium. The highly probable pyrogenous origin of sulphates draws attention to human impact on cave mineralogy.

Nitrates and phosphates in cave waters of Kraków-Czestochowa Upland, southern Poland.

The paper presents the varied presence of nitrates and phosphates in water from caves located in Czestochowa and Kraków, in urban, strongly anthropogenic conditions, representing the vadose zone of the fissure-karstic-porous massif of Upper Jurassic limestones. Hydrochemical research was carried out by the authors in the Cave on the Stone in Czestochowa in 2012-2015, in caves of the Zakrzówek horst from 1996 to 2002, and in the Dragon's Cave by the research team of J. Motyka in 1995-1998. A number of NO3 and PO4 measurements were performed in waters sampled at these research sites: 20 measurements each of NO3 and PO4 at the Cave on the Stone, 228 of NO3 and 422 of PO4 at Zakrzówek, and 19 each of NO3 and PO4 at the Dragon's Cave. To assess the quality aspect of N and P compounds in waters from the Cave on the Stone, the results of geochemical modelling were processed using PHREEQC software. In cave waters, the oxidised form of nitrogen NO3- predominates; in surface waters in the vicinity, unoxidised forms prevail: NH4+, NH3, and NH4SO4-. Among phosphorus speciations, dissolved forms are dominant: HPO42-, H2PO4-, and the insoluble form CaHPO4; in surface waters, these forms are practically absent. Transformations of water chemistry in 'urban' caves, often centuries old, manifest themselves in, inter alia, the occurrence of multi-ionic waters, including seasonal variations and extremely diversified concentrations, with very high concentrations in subpopulations of NO3 (0.2-485 mg dm-3) and P (0.02-6.87 mg dm-3). The common presence of NO3 in waters of the phreatic zone of the Czestochowa Upland, an area developed in an agricultural direction, is documented by, inter alia, the exploitation of intakes supplying the city of Czestochowa (10-57 mg dm-3, 2011) and crenological studies from 2008 to 2015 (NO3, 2-58 mg dm-3), at simultaneously low phosphate concentrations (PO4, 0.02-0.24 mg dm-3).

Limnodrilus sulphurensis n. sp., from a sulfur cave in Colorado, USA, with notes on the morphologically similar L. profundicola (Clitellata, Naididae, Tubificinae).

A new species of the tubificine genus Limnodrilus is described and COI barcoded from Sulphur Cave and associated springs in Colorado, USA. The habitats are characterized by high sulfide concentrations. The new species, L. sulphurensis, is distinguished from all congeners by the elongate, nearly parallel teeth of chaetae in its anterior segments. It has a penis sheath resembling that of L. profundicola; consequently, museum specimens and new collections are examined here to resolve some of the taxonomic confusion surrounding that widespread, but uncommon species.