Frasassi Cave Research Articles

Groundwater dilution and flow conditions in sulfuric acid caves: the case study of Frasassi (Italy)

This study analyzes the dilution process of the sulfidic groundwater in the Frasassi caves due to the recharge of O2-rich freshwater and its influence on the sulfuric acid speleogenesis and morphogenesis. The drainage pattern and the seasonal changes of the chemo-physical characteristics of the groundwater through a year-long monitoring and the measurements of the groundwater levels are presented. The inflow of water infiltrating from the karst surface influenced the sulfidic groundwater parameters, reflecting the seasonal meteoric cycle. On the contrary, the Sentino River, which represents the local base level, directly influenced the groundwater only in the most external part of the cave. The water level measurements evidenced a low hydraulic gradient (~3‰), due to the high karstification, and also some differences in the permeability depending on the drainage direction. Most cave pools are isolated on the surface and connected to each other through a network of submerged passages. In the present conditions, a surface layer of bicarbonate water forms above the sulfidic water in a large part of the cave, where it impedes subaerial corrosion by released acidic gases. Conversely, the distribution of residual gypsum deposits and corrosional wall features in the upper old cave levels demonstrate that large interfaces between sulfidic water and cave atmosphere existed during some periods of the cave history. Here, the release of acidic gases (CO2 and H2S) and the production of H2SO4 from H2S oxidation caused the widespread subaerial corrosion which significantly contributed to the morphogenesis. The comparison between these residual morphologies and the active processes shows that morphogenesis in the cave has evolved through time, influenced by hydrodynamic conditions, in turn depending on the general morphological and hydrogeological setting of the whole karst area.

Sulfur disproportionating microbial communities in a dynamic, microoxic-sulfidic karst system.

Biogeochemical sulfur cycling in sulfidic karst systems is largely driven by abiotic and biological sulfide oxidation, but the fate of elemental sulfur (S0 ) that accumulates in these systems is not well understood. The Frasassi Cave system (Italy) is intersected by a sulfidic aquifer that mixes with small quantities of oxygen-rich meteoric water, creating Proterozoic-like conditions and supporting a prolific ecosystem driven by sulfur-based chemolithoautotrophy. To better understand the cycling of S0 in this environment, we examined the geochemistry and microbiology of sediments underlying widespread sulfide-oxidizing mats dominated by Beggiatoa. Sediment populations were dominated by uncultivated relatives of sulfur cycling chemolithoautotrophs related to Sulfurovum, Halothiobacillus, Thiofaba, Thiovirga, Thiobacillus, and Desulfocapsa, as well as diverse uncultivated anaerobic heterotrophs affiliated with Bacteroidota, Anaerolineaceae, Lentimicrobiaceae, and Prolixibacteraceae. Desulfocapsa and Sulfurovum populations accounted for 12%-26% of sediment 16S rRNA amplicon sequences and were closely related to isolates which carry out autotrophic S0 disproportionation in pure culture. Gibbs energy (∆Gr ) calculations revealed that S0 disproportionation under in situ conditions is energy yielding. Microsensor profiles through the mat-sediment interface showed that Beggiatoa mats consume dissolved sulfide and oxygen, but a net increase in acidity was only observed in the sediments below. Together, these findings suggest that disproportionation is an important sink for S0 generated by microbial sulfide oxidation in this oxygen-limited system and may contribute to the weathering of carbonate rocks and sediments in sulfur-rich environments.

Thiovibrio frasassiensis gen. nov., sp. nov., an autotrophic, elemental sulphur disproportionating bacterium isolated from sulphidic karst sediment, and proposal of Thiovibrionaceae fam. nov.

A novel, autotrophic, mesophilic bacterium, strain RS19-109T, was isolated from sulphidic stream sediments in the Frasassi Caves, Italy. The cells of this strain grew chemolithoautotrophically under anaerobic conditions while disproportionating elemental sulphur (S0) and thiosulphate, but not sulphite with bicarbonate/CO2 as a carbon source. Autotrophic growth was also observed with molecular hydrogen as an electron donor, and S0, sulphate, thiosulphate, nitrate and ferric iron as electron acceptors. Oxygen was not used as an electron acceptor and sulphide was not used as an electron donor. Weak growth was observed with sulphate as an electron acceptor and organic carbon as an electron donor and carbon source. The strain also showed weak growth by fermentation of tryptone. It grew at pH 5.5–7.5 (optimum, pH 7.0), 4–35 °C (optimum, 30 °C) and between 0–1.7 % NaCl. Strain RS19-109T was found to be phylogenetically distinct based on 16S rRNA gene sequence similarity (89.2 %) to its closest relative, Desulfurivibrio alkaliphilus AHT2T. The draft genome sequence for strain RS19-109T had average nucleotide identity, average amino acid identity and in silico DNA–DNA hybridization values of 72.2, 63.0 and 18.3 %, respectively, compared with the genome sequence of D. alkaliphilus AHT2T. On the basis of its physiological and genomic properties, strain RS19-109T is proposed as the type strain of a novel species of a novel genus, Thiovibrio frasassiensis gen. nov., sp. nov. A novel family, Thiovibrionaceae fam. nov., is proposed to accommodate Thiovibrio within the order Desulfobulbales . Strain RS19-109T has been deposited at the DSMZ-German Collection of Microorganisms and Cell Cultures (=DSM 115074T) and the American Type Culture Collection (=ATCC TSD-325T).

Frasassi Caves and Surroundings: A Special Vehicle for the Geoeducation and Dissemination of the Geological Heritage in Italy

One of the most fascinating aspects of the work of geologist lies in knowing how to read the physical landscape as an expression of the geological and geomorphological phenomena that shaped the Earth’s relief over time. The necessity to disseminate these vast areas of knowledges and skills starts from here, with the aim to enhance the concept of geodiversity and to raise awareness for its protection and promotion. This work aims to share some examples of projects realized following the subscription of agreement between different public authorities in the Apennine territory of Marche Region in Italy, such as the Geology Section of the University of Camerino (MC), “Consorzio Frasassi” (which manages the underground karst complex of Frasassi caves) and municipality of Genga (AN), where Frasassi caves are located. Thanks to this partnership, our research group realized didactic geological notebooks for school groups visiting the caves and interactive museum laboratories in 3D, showing the geological evolution of the area. This sharing of knowledge will contribute to educate communities about the importance of the geological heritage. At the same time, this project can serve as motivation to establishing the same type of collaborations in those territories where similar projects can be replicated.

The fascinating biology of stinky caves

The discovery of Movile Cave in 1986, similarly to the discovery of the deep-sea vents 9 years earlier, showed that, when redox interfaces are present in subsurface ecosystems, diverse and abundant biological communities can thrive based primarily on autochthonous food produced in situ by chemosynthesis. Additional caves, partially flooded by sulfidic water have been discovered, i.e. the Frasassi caves in Italy, Ein-Nur Cave and Ayyalon Cave in Israel, Melissotrypa Cave in Greece, Tashan Cave in Iran, caves in the Sharo-Argun Valley in the Caucasus Mountains, etc. All of these are inhabited by numerous endemic cave-adapted organisms present in dense populations sustained by a rich food base generated within the subterranean environment by microorganisms that use atmospheric oxygen to oxidize the reduced chemical compounds present in the water (H2S, NH4+, CH4). Stable isotope data has shown that the biological communities found in sulfidic caves are not dependent on carbon fixation in green plants that use light energy in the process of photosynthesis. Special adaptations have been identified in some of these inhabitants that allow them to cope with the adverse environmental conditions such as toxicity of H2S, hypoxia, and extreme pH values. Symbiotic associations between crustaceans, protozoa, and sulfide oxidizing bacteria were discovered in several sulfidic caves. New species of microorganisms have also been described from these unusual subsurface environments. Unusual microbiomes have recently been discovered in dry caves that contain chemoclines between heavy volcanic gasses dominated by CO2, but also containing H2S and CH4, and atmospheric air that floats on top of the volcanic gas emissions. Unique sulfur mineralization, microbial biofilms containing new species of bacteria that thrive at extremely pH values, symbiotic relationships between bacteria and fungi have been discovered in Sulfur Cave in Romania, Bosoletto Cave in Italy, and Sousaki caves in Greece. The extreme life conditions encountered in these caves are of special interest to astrobiologists using these caves as a model for better understanding conditions that life may be present in on other planets and celestial bodies in our Solar System and beyond.

Organic Stabilization of Extracellular Elemental Sulfur in a Sulfurovum-Rich Biofilm: A New Role for Extracellular Polymeric Substances?

This work shines light on the role of extracellular polymeric substance (EPS) in the formation and preservation of elemental sulfur biominerals produced by sulfur-oxidizing bacteria. We characterized elemental sulfur particles produced within a Sulfurovum-rich biofilm in the Frasassi Cave System (Italy). The particles adopt spherical and bipyramidal morphologies, and display both stable (α-S8) and metastable (β-S8) crystal structures. Elemental sulfur is embedded within a dense matrix of EPS, and the particles are surrounded by organic envelopes rich in amide and carboxylic groups. Organic encapsulation and the presence of metastable crystal structures are consistent with elemental sulfur organomineralization, i.e., the formation and stabilization of elemental sulfur in the presence of organics, a mechanism that has previously been observed in laboratory studies. This research provides new evidence for the important role of microbial EPS in mineral formation in the environment. We hypothesize that the extracellular organics are used by sulfur-oxidizing bacteria for the stabilization of elemental sulfur minerals outside of the cell wall as a store of chemical energy. The stabilization of energy sources (in the form of a solid electron acceptor) in biofilms is a potential new role for microbial EPS that requires further investigation.

Porrhomma frasassianum spec. nov. from a sulfidic cave, Italy (Araneae: Linyphiidae)

Porrhomma frasassianumspec. nov., a blind troglobitic spider from the sulfidic Grotta del Fiume – Grotta Grande del Vento cave system (Italy), is described and illustrated. The relationship between this new species and other European species of Porrhomma is discussed, its distribution in the Frasassi cave system is analysed and the spider species cohabiting with the newly described species are listed.

Barite replacement boxwork in the Frasassi caves (Italy)

The Frasassi caves, located in the Sentino River Gorge in Ancona Province (Marche, Italy), contains boxwork in a small zone in the inner part of the cave system where it is closely associated with subaqueous corrosion produced by sulfuric acid speleogenesis. The boxwork consists of barite that replaces calcite spar-filled veins and limestone porosity and projects from the corroded cave walls. The replacement involved only the calcite that protrudes from the rock surfaces, indicating that the process took place in the cave environment, and therefore the boxwork is not simply a product of differential corrosion. I hypothesize that the boxwork formed in nearly stagnant bodies of sulfidic water as a consequence of lateral recharge of O2-rich bicarbonate water moving upward along the walls because of its lower density. The acidity produced by the oxidation of H2S enhanced limestone corrosion and incision of rills and channels, while also releasing sulfate ions that induced the precipitation of barite. This sulfuric acid process was sufficiently slow to dissolve the micritic bedrock and replace spar with barite, preserving the shape of the network of fissures and pores formerly filled by calcite.

Non contact shape and dimension measurements by LIDAR techniques of one of the biggest Italian caverns

Many measurements techniques have been proposed and used for the “digitalization of things”: laser scanner, high resolution camera, depth cam, structured light 3D scanner, hyper-spectral sensor, multi-spectral cam, photo modelling, etc. In this work LIDAR measurement techniques has been applied for the digitalization of a cave room of about 180 x 120 m and a height recently discovered up to 250 m, named Abisso Ancona in the Frasassi caves, in the municipality of Genga, Ancona province, Italy.

Climate variability on the Adriatic seaboard during the last glacial inception and MIS 5c from Frasassi Cave stalagmite record

A stalagmite (FR16) from Frasassi Cave, located near the Adriatic coast of the Italian peninsula, offers a 16 kyr petrographic and stable isotope record spanning from 112.8 ± 1.5 ka to 96.6 ± 1.0 ka, corresponding to the interval from marine isotope stage (MIS) 5c to MIS 5d. The physical characteristics of FR16 calcite, allowed for a thorough understanding of the processes controlling isotopic changes, because fabrics as well are controlled by the environmental parameters within the cave that reflect external climate forcing. The FR16 δ13C profile matches fabric changes where columnar fabric usually corresponds to more negative values and micrite/microsparite layers to less negative δ13C values, and with the North Greenland Ice-core Project (NGRIP) δ18O record with more negative δ13C values during interstadials GI24 and GI23. This suggests a hydrological sensitivity for the two proxies, and their variability can be linked to recharge changes. On the other hand, the response to Greenland events appears subdued in the δ18O record and the trend mimics instead the Northern Hemisphere (NH) summer insolation with more negative δ18O values corresponding to the maximum insolation centred at ca. 105 ka. This behaviour can be explained by the higher sensitivity of δ18O to rainfall seasonality, moisture source and trajectories, whose combined effect attenuated the stadials/intersadials signal in the stalagmite.

Large-scale microalgal biomass production for hydrothermal liquefaction – petroleum refinery approach

Sulfide oxidation forms a critical step in the global sulfur cycle, although this process is notoriously difficult to constrain due to the multiple pathways and highly reactive intermediates involved. Multiple sulfur isotopes (δ34S and Δ33S) can provide a powerful tool for unravelling sulfur cycling processes in modern (and ancient) environments, although they have had limited application to systems with well-resolved oxidative S cycling. In this study, we report the major (δ34S) and minor (Δ33S) isotope values of sulfur compounds in streams and sediments from the sulfidic Frasassi cave system, Marche Region, Italy. These microaerophilic cave streams host prominent white biofilms dominated by chemolithotrophic organisms that oxidize sulfide to S0, allowing us to estimate S isotope fractionations associated with in situ sulfide oxidation and to evaluate any resulting isotope biosignatures. Our results demonstrate that chemolithotrophic sulfide oxidation produces 34S enrichments in the S0 products that are larger than those previously measured in laboratory experiments, with 34εS0–H2S of up to 8‰ calculated. These small reverse isotope effects are similar to those produced during phototrophic sulfide oxidation (⩽7‰), but distinct from the small normal isotope effects previously calculated for abiotic oxidation of sulfide with O2 (∼−5‰). An inverse correlation between the magnitude of 34εS0–H2S effects and sulfide availability, along with substantial differences in Δ33S, both support complex sulfide oxidation pathways and intracellular recycling of S intermediates by organisms inhabiting the biofilms. At the ecosystem level, we calculate fractionations of less than 40‰ between sulfide and sulfate in the water column and in the sediments. These fractionations are smaller than those typically calculated for systems dominated by sulfate reduction (>50‰), and contrast with the commonly held assumption that oxidative recycling of sulfide generally increases overall fractionations. The relatively small fractionations appear to be related to the sequestration of S0 in the biofilms (either intra- or extra-cellularly), which removes this intermediate substrate from fractionation by further disproportionation or oxidation reactions. In addition, the net 33λH2S–SO4 values calculated in this system are larger than data published for systems dominated by reductive sulfur cycling, partially due to the isotopic imprint of chemolithotrophic sulfide oxidation on the aqueous sulfide pool. These distinct isotopic relationships are retained in the sedimentary sulfur pool, suggesting that trends in 34S and 33S values could provide an isotopic fingerprint of such chemolithotrophic ecosystems in modern and ancient environments.

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.

Sulphur-oxidising bacteria isolated from deep caves improve the removal of arsenic from contaminated harbour sediments

ABSTRACTAcidophilic S-oxidising bacteria isolated from sulphur-rich deep caves (Frasassi, Italy), characterised by relatively low temperature, were tested for their ability to mobilise (semi-)metals from contaminated sediments. Sediment samples from two commercial Italian seaports were used to set up bioleaching experiments. The effect of different growth substrates was also investigated. Our experiments revealed that S-oxidising bacteria isolated from Frasassi caves have a high potential to remove As from contaminated marine sediments, as never reported before. Although As solubilisation efficiency was quite low (i.e. about 30%), only a small amount of As was associated with non-residual fractions of the sediment. On the contrary, the solubilisation efficiencies of Zn and Ni (20% and 10%, respectively) were lower than those previously obtained by the use of other acidophilic bacteria and mainly influenced by the experimental conditions rather than by the presence of the S-oxidising bacteria. Results presented here open new perspectives in bioleaching applications for the remediation of contaminated sediments. Indeed, microbial strains adapted to relatively low-temperature environments could improve sediment bioleaching at temperature regimes where mesophilic and thermophilic strains are not favoured. Such strains could be exploited for developing selective bioremediation procedures for sediments contaminated with As, to be applied in multistep biotreatment processes.

Groundwater biodiversity in a chemoautotrophic cave ecosystem: how geochemistry regulates microcrustacean community structure

The Frasassi cave system in central Italy hosts one of the few known examples of a groundwater metazoan community that is supported by sulfur-based lithoautotrophic microbes. Despite the challenging conditions represented by high concentrations of H2S and low concentrations of O2, this cave system is home to many invertebrate species. Here, we analyzed the copepods inhabiting sulfidic lakes and non-sulfidic dripping pools in order to investigate how environmental conditions in sulfidic waters regulate the spatial distribution of the cave microcrustacean community over time. We also sampled copepod assemblages of sulfidic lakes under conditions of both high and low H2S concentration. Cluster analysis and canonical correspondence analysis separated the copepod assemblages inhabiting dripping pools from those of sulfidic lakes. H2S concentration, pH and O2 concentration were identified as the main factors regulating community structure. These results indicate that the distribution of groundwater copepods within the cave system is ecologically and spatially structured. Sulfidic lakes showed lower Simpson dominance, higher Shannon diversity and higher Pielou equitability at higher H2S concentrations. The complex community structure of the copepods of the Frasassi cave system suggests that a chemosynthetically produced food source facilitated the colonization of stygobionts in sulfidic groundwater due to their tolerance to the environmental conditions.

Draft Genome Sequence of Lampenflora Chlorobium limicola Strain Frasassi in a Sulfidic Cave System.

The draft genome sequence of Chlorobium limicola strain Frasassi was assembled from metagenomic sequencing of a green mat in an artificially lighted aquarium inside the Frasassi caves in Italy. The genome is 2.08 Mbp in size and contains the necessary genes for anoxygenic photosynthesis and CO2 fixation.

Sulfur isotope values in the sulfidic Frasassi cave system, central Italy: A case study of a chemolithotrophic S-based ecosystem

Sulfide oxidation forms a critical step in the global sulfur cycle, although this process is notoriously difficult to constrain due to the multiple pathways and highly reactive intermediates involved. Multiple sulfur isotopes (δ34S and Δ33S) can provide a powerful tool for unravelling sulfur cycling processes in modern (and ancient) environments, although they have had limited application to systems with well-resolved oxidative S cycling. In this study, we report the major (δ34S) and minor (Δ33S) isotope values of sulfur compounds in streams and sediments from the sulfidic Frasassi cave system, Marche Region, Italy. These microaerophilic cave streams host prominent white biofilms dominated by chemolithotrophic organisms that oxidize sulfide to S0, allowing us to estimate S isotope fractionations associated with in situ sulfide oxidation and to evaluate any resulting isotope biosignatures. Our results demonstrate that chemolithotrophic sulfide oxidation produces 34S enrichments in the S0 products that are larger than those previously measured in laboratory experiments, with 34εS0–H2S of up to 8‰ calculated. These small reverse isotope effects are similar to those produced during phototrophic sulfide oxidation (⩽7‰), but distinct from the small normal isotope effects previously calculated for abiotic oxidation of sulfide with O2 (∼−5‰). An inverse correlation between the magnitude of 34εS0–H2S effects and sulfide availability, along with substantial differences in Δ33S, both support complex sulfide oxidation pathways and intracellular recycling of S intermediates by organisms inhabiting the biofilms. At the ecosystem level, we calculate fractionations of less than 40‰ between sulfide and sulfate in the water column and in the sediments. These fractionations are smaller than those typically calculated for systems dominated by sulfate reduction (>50‰), and contrast with the commonly held assumption that oxidative recycling of sulfide generally increases overall fractionations. The relatively small fractionations appear to be related to the sequestration of S0 in the biofilms (either intra- or extra-cellularly), which removes this intermediate substrate from fractionation by further disproportionation or oxidation reactions. In addition, the net 33λH2S–SO4 values calculated in this system are larger than data published for systems dominated by reductive sulfur cycling, partially due to the isotopic imprint of chemolithotrophic sulfide oxidation on the aqueous sulfide pool. These distinct isotopic relationships are retained in the sedimentary sulfur pool, suggesting that trends in 34S and 33S values could provide an isotopic fingerprint of such chemolithotrophic ecosystems in modern and ancient environments.

Fate of sulfide in the Frasassi cave system and implications for sulfuric acid speleogenesis

The oxidation of hydrogen sulfide (H2S) has led to the formation of some of the world's largest caves through a process known as sulfuric acid speleogenesis (SAS). Here we present a multi-year study of the large, sulfidic, and actively-forming Frasassi cave system, Italy. We show that despite the presence of abundant sulfide-oxidizing biofilms in Frasassi streams, H2S(g) degassing to the cave atmosphere was the major sink for dissolved sulfide. Degassing rates ranged from 0.9 to 80μmolm−2s−1, whereas microbial oxidation rates were between 0.15 and 2.0μmolm−2s−1. Furthermore, microsensor measurements showed that sulfuric acid is not a major end product of microbial sulfide oxidation in the streams. Our results suggest that subaerial SAS will be important for karstification, and more important than subaqueous SAS, wherever ground waters with high sulfide concentrations emerge as flowing streams in contact with cave air.

Niphargus–Thiothrix associations may be widespread in sulphidic groundwater ecosystems: evidence from southeastern Romania

Niphargus is a speciose amphipod genus found in groundwater habitats across Europe. Three Niphargus species living in the sulphidic Frasassi caves in Italy harbour sulphur-oxidizing Thiothrix bacterial ectosymbionts. These three species are distantly related, implying that the ability to form ectosymbioses with Thiothrix may be common among Niphargus. Therefore, Niphargus–Thiothrix associations may also be found in sulphidic aquifers other than Frasassi. In this study, we examined this possibility by analysing niphargids of the genera Niphargus and Pontoniphargus collected from the partly sulphidic aquifers of the Southern Dobrogea region of Romania, which are accessible through springs, wells and Movile Cave. Molecular and morphological analyses revealed seven niphargid species in this region. Five of these species occurred occasionally or exclusively in sulphidic locations, whereas the remaining two were restricted to nonsulphidic areas. Thiothrix were detected by PCR on all seven Dobrogean niphargid species and observed using microscopy to be predominantly attached to their hosts' appendages. 16S rRNA gene sequences of the Thiothrix epibionts fell into two main clades, one of which (herein named T4) occurred solely on niphargids collected in sulphidic locations. The other Thiothrix clade was present on niphargids from both sulphidic and nonsulphidic areas and indistinguishable from the T3 ectosymbiont clade previously identified on Frasassi-dwelling Niphargus. Although niphargids from Frasassi and Southern Dobrogea are not closely related, the patterns of their association with Thiothrix are remarkably alike. The finding of similar Niphargus–Thiothrix associations in aquifers located 1200 km apart suggests that they may be widespread in European groundwater ecosystems.

Nitrogen fixation in distinct microbial niches within a chemoautotrophy-driven cave ecosystem

Microbial sulfur and carbon cycles in ecosystems driven by chemoautotrophy—present at deep-sea hydrothermal vents, cold seeps and sulfidic caves—have been studied to some extent, yet little is known about nitrogen fixation in these systems. Using a comprehensive approach comprising of 15N2 isotope labeling, acetylene reduction assay and nitrogenase gene expression analyses, we investigated nitrogen fixation in the sulfide-rich, chemoautotrophy-based Frasassi cave ecosystem (Italy). Nitrogen fixation was examined in three different microbial niches within the cave waters: (1) symbiotic bacterial community of Niphargus amphipods, (2) Beggiatoa-dominated biofilms, which occur at the sulfide–oxygen interface, and (3) sulfidic sediment. We found evidence for nitrogen fixation in all the three niches, and the nitrogenase gene (homologs of nifH) expression data clearly show niche differentiation of diazotrophic Proteobacteria within the water streams. The nifH transcript originated from the symbiotic community of Niphargus amphipods might belong to the Thiothrix ectosymbionts. Two abundantly expressed nifH genes in the Beggiatoa-dominated biofilms are closely related to those from Beggiatoa- and Desulfovibrio-related bacteria. These two diazotrophs were consistently found in Beggiatoa-dominated biofilms collected at various time points, thus illustrating species-specific associations of the diazotrophs in biofilm formation, and micron-scale niche partitioning of sulfur-oxidizing and sulfate-reducing bacteria driven by steep redox gradients within the biofilm. Finally, putative heterotrophs (Geobacter, Azoarcus and Desulfovibrio related) were the active diazotrophs in the sulfidic sediment. Our study is the first to shed light on nitrogen fixation in permanently dark caves and suggests that diazotrophy may be widespread in chemosynthetic communities.

Exploring the sulfide tolerance of ectosymbioticNiphargusamphipods from the Frasassi caves, central Italy

Two species of the crustacean amphipod genus Niphargus inhabit the sulfidic groundwaters of the Frasassi caves in central Italy, and both harbor filamentous, sulfide-oxidizing Thiothrix ectosymbionts. As sulfide is toxic to most aerobic organisms, it appeared possible that the ectosymbionts could help their Niphargus hosts with detoxification processes. In this study, mortality due to sulfide was compared between Niphargus individuals with ectosymbionts and individuals whose ectosymbionts had been killed by antibiotic treatment. Both Frasassi-dwelling Niphargus species revealed exceptionally high tolerances to sulfide compared to other amphipod species studied so far. Niphargus individuals without viable ectosymbionts tolerated sulfide levels exceeding those occurring in Frasassi cave waters. Thus, the amphipods may employ Thiothrix-independent mechanisms for sulfide resistance.