Background: Movile Cave (SE Romania) is a chemoautotrophically-based ecosystem fed by hydrogen sulfide-rich groundwater serving as a primary energy source analogous to the deep-sea hydrothermal ecosystems. Our current understanding of Movile Cave microbiology has been confined to the sulfidic water and its proximity, as most studies focused on the water-floating microbial mat and planktonic accumulations likely acting as the primary production powerhouse of this unique subterranean ecosystem. By employing comprehensive genomic-resolved metagenomics, we questioned the spatial variation, chemoautotrophic abilities, ecological interactions and trophic roles of Movile Cave’s microbiome thriving beyond the sulfidic-rich water.Results: A customized bioinformatics pipeline led to the recovery of 106 high-quality metagenome-assembled genomes from 7 cave sediment metagenomes. Assemblies’ taxonomy spanned 19 bacterial and three archaeal phyla withAcidobacteriota,Chloroflexota,Proteobacteria,Planctomycetota,Ca.Patescibacteria,Thermoproteota,Methylomirabilota,andCa.Zixibacteria as prevalent phyla. Functional gene analyses predicted the presence of CO₂fixation, methanotrophy, sulfur and ammonia oxidation in the explored sediments. Species Metabolic Coupling Analysis of metagenome-scale metabolic models revealed the highest competition-cooperation interactions in the sediments collected away from the water. Simulated metabolic interactions indicated autotrophs and methanotrophs as major donors of metabolites in the sediment communities. Cross-feeding dependencies were assumed only towards 'currency' molecules and inorganic compounds (O₂, PO₄³⁻, H⁺, Fe²⁺, Cu²⁺) in the water proximity sediment, whereas hydrogen sulfide and methanol were assumedly traded exclusively among distant gallery communities.Conclusions: These findings suggest that the primary production potential of Movile Cave expands way beyond its hydrothermal waters, enhancing our understanding of the functioning and ecological interactions within chemolithoautotrophically-based subterranean ecosystems.

Introduction: Karst caves are characterized by relatively constant temperature, lack of light, high humidity, and low nutrients availability. The diversity and functionality of the microorganisms dwelling in caves micro-habitats are yet underexplored. Therefore, in-depth investigations of these ecosystems aid in enlarging our understanding of the microbial interactions and microbially driven biogeochemical cycles. Here, we aimed at evaluating the diversity, abundance, distribution, and organic substrate preferences of microbial communities from Peștera cu Apă din Valea Leșului (Leșu Cave) located in the Apuseni Mountains (North-Western Romania).Materials and Methods: To achieve this goal, we employed 16S rRNA gene amplicon sequencing and community-level physiological profiling (CLPP) paralleled by the assessment of environmental parameters of cave sediments and water.Results and Discussion: Pseudomonadota (synonym Proteobacteria) was the most prevalent phylum detected across all samples whereas the abundance detected at order level varied among sites and between water and sediment samples. Despite the general similarity at the phylum-level in Leșu Cave across the sampled area, the results obtained in this study suggest that specific sites drive bacterial community at the order-level, perhaps sustaining the enrichment of unique bacterial populations due to microenvironmental conditions. For most of the dominant orders the distribution pattern showed a positive correlation with C-sources such as putrescine, γ-amino butyric acid, and D-malic acid, while particular cases were positively correlated with polymers (Tween 40, Tween 80 and α-cyclodextrin), carbohydrates (α-D-lactose, i-erythritol, D-mannitol) and most of the carboxylic and ketonic acids. Physicochemical analysis reveals that sediments are geochemically distinct, with increased concentration of Ca, Fe, Al, Mg, Na and K, whereas water showed low nitrate concentration. Our PCA indicated the clustering of different dominant orders with Mg, As, P, Fe, and Cr. This information serves as a starting point for further studies in elucidating the links between the taxonomic and functional diversity of subterranean microbial communities.

Background: Movile Cave (Dobrogea, SE Romania) hosts a subterranean chemoautotrophically-based ecosystem supported by a sulfidic thermal aquifer analogous to the deep-sea hydrothermal ecosystems. Our current understanding of Movile Cave microbiology has been confined to the thermal water proximity (no more than 2 m distant), with most studies focusing on the water-floating mat, which likely acts as the primary production powerhouse in this sulfidic ecosystem. To gain more insightful information on the functioning of the sulfidic Movile Cave ecosystem, we employed a metagenomics-resolved approach to reveal the microbiome diversity, metabolic potential, and interactions and infer its roles within the food webs in the sediments beyond the sulfidic thermal waters. Results: A customized bioinformatics pipeline led to the recovery of 106 high-quality metagenome-assembled genomes from 7 cave sediment metagenomes. Assemblies’ taxonomy spanned 19 bacterial and three archaeal phyla with Acidobacteriota, Chloroflexota, Proteobacteria, Planctomycetota, Ca . Patescibacteria, Thermoproteota, Methylomirabilota , and Ca . Zixibacteria as prevalent phyla. Functional gene analyses allowed prediction of CO ₂ fixation, methanotrophy, sulfur and ammonia oxidation as possibly occurring in the explored sediments. Species Metabolic Coupling Analysis of metagenome-scale metabolic models revealed the highest competition-cooperation interactions in the sediments collected at the farthest distance from the sulfidic water. As a result of simulated metabolic interactions, autotrophs and methanotrophs were hypothesized as major donors of exchanged metabolites in the sediment communities. Cross-feeding dependencies were assumed only towards ‘currency’ molecules and inorganic compounds (O ₂ , PO ₄ ^3- , H ⁺ , Fe ²⁺ , Cu ²⁺ ) in the sediment nearby sulfidic water, whereas hydrogen sulfide and methanol are predictably traded exclusively among communities dwelling in the distant gallery. Conclusions: These findings suggest that the primary production potential of the Movile Cave expands way beyond its hydrothermal waters, enhancing our understanding of ecological interactions inside chemolithoautotrophically based subterranean ecosystems and their functioning.

Cave ecosystems comprise specialized microbial communities that play essential roles in biogeochemical cycles; yet their metabolic capabilities and ecological functions are not yet fully understood. As conventional cultivation techniques offer limited insights into the metabolic capabilities, methods based on direct functionality screening could provide more in-depth knowledge of cave microbial activity. In this study, we utilized the Community-Level Physiological Profiling (CLPP) based on Biolog® EcoPlate™ approach to assessing carbon substrate utilization by microbial communities associated with pool water, sediment, sediment (limon), and moonmilk from five caves in Romania. Principal Component Analysis (PCA) and Generalized Additive Models (GAMs) statistics were employed to infer the patterns of C-substrate metabolization and their environmental drivers. Environmental variables such as sodium (Na) and electrical conductivity (EC) significantly impacted C-utilization capabilities as indicated by both PCA and GAM. The latter analysis elucidated non-linear relationships between variables, such as EC, Na, and Mg, and microbial metabolic diversity indices. However, distinct C utilization patterns were detected among sampled sites and chemical types. Unlike moonmilk samples whose associated microbial communities appeared as exhibiting low C-substrate utilization, the highest activity was shown in cave pool water samples with the associated microbial communities extensively consuming D-galacturonic acid and Tween 80. Conversely, substrates like L-threonine and α-ketobutyric acid showed limited utilization across all cave samples. Average Well Color Development (AWCD) and Shannon diversity indices indicated that microbial communities associated to samples from Cloșani and Muierilor caves demonstrated the highest metabolic diversity. Our findings suggested that metabolic profiling using Biolog®EcoPlates™ method combined with multivariate statistical methods might prove as suitable approach to effectively screen for cave microbial functionality and the probable environmental drivers. Besides, this work distinguishes from similar studies by relying on GAM analysis to predict the environmental factors governing the microbially-mediated organic carbon degradation in subterranean ecosystems.

Cave ecosystems comprise specialized microbial communities that play essential roles in biogeochemical cycles; yet their metabolic capabilities and ecological functions are not fully understood. As conventional cultivation techniques provide limited insights into the metabolic capabilities, methods based on direct functionality screening may improve our knowledge of cave microbial activity. In this study, the Community-Level Physiological Profiling (CLPP) based on Biolog® EcoPlate™ approach was applied to assess carbon (C) substrate utilization by microbial communities associated with 36 environamental samples collected from five karstic caves (Cloșani, Ferice, Leșu, Muierilor, and Topolnița) in Romania. Principal Component Analysis (PCA) and Generalized Additive Models (GAMs) statistics were employed to infer the patterns of C substrate metabolization and their environmental drivers. Environmental variables such as sodium (Na) and electrical conductivity (EC) significantly impacted C substrate utilization capabilities as indicated by both PCA and GAM. The latter analysis elucidated non-linear relationships between variables, such as EC, Na, and Mg, and microbial metabolic diversity indices. However, distinct C substrate utilization patterns were detected among sampled sites and chemical types. Unlike moonmilk samples where associated microbial communities appeared as exhibiting low C substrate utilization, the highest activity was shown in cave pool water samples with the associated microbial communities extensively consuming D-galacturonic acid and Tween 80. Conversely, substrates like L-threonine and α-ketobutyric acid showed limited utilization across all cave samples. Average Well Color Development (AWCD) and Shannon diversity indices indicated that microbial communities associated to samples from Cloșani and Muierilor caves demonstrated the highest metabolic diversity. Our findings suggested that metabolic profiling using Biolog® EcoPlate™ method combined with multivariate statistical methods might prove a suitable analysis approach to effectively screen for cave microbial functionality and the probable environmental drivers. Besides, this work distinguishes from similar studies by relying on GAM analysis to predict the environmental factors governing the microbially-mediated organic carbon degradation in subterranean ecosystems. Article history: Received 28 February 2025; Revised 30 April 2025; Accepted 18 May 2025; Available online 25 June 2025