Untargeted molecular analyses of complex mixtures are relevant for many fields of research, including geochemistry, pharmacology, and medicine. Ultrahigh-resolution mass spectrometry is one of the most powerful tools in this context. The availability of open scripts and online tools for specific data processing steps such as noise removal or molecular formula assignment is growing, but an integrative tool where all crucial steps are reproducibly evaluated and documented is lacking. We developed a novel, server-based tool (ICBM-OCEAN, Institute for Chemistry and Biology of the Marine Environment, Oldenburg−complex molecular mixtures, evaluation & analysis) that integrates published and novel approaches for standardized processing of ultrahigh-resolution mass spectrometry data of complex molecular mixtures. Different from published approaches, we offer diagnostic and validation tools for all relevant steps. Among other features, we included objective and reproducible reduction of noise and systematic errors, spectra recalibration and alignment, and identification of likeliest molecular formulas. With 15 chemical elements, the tool offers high flexibility in formula attribution. Alignment of mass spectra among different samples prior to molecular formula assignment improves mass error and facilitates molecular formula confirmation with the help of isotopologues. The online tool and the detailed instruction manual are freely accessible at www.icbm.de/icbm-ocean.

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Hydrothermal plumes transport reduced chemical species and metals into the open ocean. Despite their considerable spatial scale and impact on biogeochemical cycles, niche differentiation of abundant microbial clades is poorly understood. Here, we analyzed the microbial ecology of two bathy- (Brothers volcano; BrV-cone and northwest caldera; NWC) and a mesopelagic (Macauley volcano; McV) plumes on the Kermadec intra-oceanic arc in the South Pacific Ocean. The microbial community structure, determined by a combination of 16S rRNA gene, fluorescence in situ hybridization and metagenome analysis, was similar to the communities observed in other sulfur-rich plumes. This includes a dominance of the vent characteristic SUP05 clade (up to 22% in McV and 51% in BrV). In each of the three plumes analyzed, the community was dominated by a different yet uncultivated chemoautotrophic SUP05 species, here, provisionally named, Candidatus Thioglobus vadi (McV), Candidatus Thioglobus vulcanius (BrV-cone) and Candidatus Thioglobus plumae (BrV-NWC). Statistical analyses, genomic potential and mRNA expression profiles suggested a SUP05 niche partitioning based on sulfide and iron concentration as well as water depth. A fourth SUP05 species was present at low frequency throughout investigated plume samples and may be capable of heterotrophic or mixotrophic growth. Taken together, we propose that small variations in environmental parameters and depth drive SUP05 niche partitioning in hydrothermal plumes.