Description: Most of the microbes assumed to colonize the seafloor and its subsurface resist cultivation. Thus, their catalytic capabilities and contribution to element cycling remain enigmatic. In marine sediments, it is estimated that microbial dark matter accounts for up to 91%. Although sequence-based approaches allow culture-independent insights into taxonomic diversity, and metabolic potential, they rely on database comparisons. Therefore, sequence data can only reflect what is already known. Indeed, for more than one-third of the genes found in genomes from uncultured bacteria, no predicted function can be assigned. Besides the lack of homologous genes, a further challenge when investigating metatranscriptomes is the community bias caused by sample processing delays. Hence, the need for in situ fixation of RNA, particularly for samples from greater water depths and longer sample transport times, is a prerequisite to obtain unbiased metatranscriptomic data reflecting gene expression profiles in its original state. Although activity-based searches for novel enzymes by means of metagenomic fosmid libraries allow to link previously not allocated genes to a function, they are hampered by expression limitations in the surrogate host. In situ monitoring of chemical compounds can provide rates, but do not allow assignment of responsible organisms and may also overlook possible intermediates and reactions. Here we present a holistic approach that combines in situ incubation and rate measurements at the seafloor of Boknis Eck (BE, Eckernfoerde Bay, south-western Baltic Sea) with activity-based metagenomic screening and metatranscriptomics. At BE, the seasonal stratification of the water column causes pronounced hypoxia, sporadically even anoxia. Once a month we deploy a mini chamber lander system for short-term in situ incubations. Embedded sensor technology allows monitoring of chemical dynamics (e.g., oxygen, sulphide) and sampling under pre-defined chemical conditions (e.g. if oxygen drops during incubation under a specific threshold value). Metagenomic and metatranscriptomic data will be used to determine the abundance of identified novel enzymes with specific functions (CO2 reduction, H2 conversion, etc.) that have been detected by means of activity-based searches. This comparison can give an estimate of how important the novel enzyme and its function might actually be for the investigated microbial community and thus for ecosystem functioning. Our results suggest that such combined approaches have the potential to open a window into the metabolic network of uncultured microbes and their catalytic ability in the biogeochemical cycling of key elements.

Description: Iron is an essential micronutrient often limiting the growth of marine microorganisms in wide areas of the world’s oceans. In high concentrations, iron, by contrast, is potentially toxic and usually leads to irreversible cell encrustation followed by cell death. To counteract both, microorganisms have evolved the strategy of producing organic iron-binding molecules, so called iron-ligands, enabling them to improve the bioavailability and uptake of iron as well as to mitigate its potentially toxic effects. Hydrothermal vents are among the major sources of iron in the oceans. These dynamic habitats host a variety of metabolically highly specialized and versatile microbes that not only have to cope with partially high iron concentrations but may also be able to mediate the availability of inorganic hydrothermal iron by actively producing iron-ligands. However, hardly any information exists to-date describing the impact of increasing iron concentrations on hydrothermal plume microbial communities and their potential to form iron-ligands. We therefore set up microcosm experiments with hydrothermal plume material in artificial seawater along an iron gradient ranging from 0 to 10 mM. We found that the microbial community at low iron concentrations (0.1 to 100 μM) differs significantly from that found in the original non-treated plume sample, allowing a certain group of Epsilonproteobacteria to become dominant (up to 93% of the overall community). The microbial community detected at 10 mM is by contrast more similar to that found in the original plume sample and consists mainly of one gammaproteobacterial group (up to 97% of the overall community). We further analyzed these results in the context of ligand concentrations and structural diversity and found indications for microbially mediated iron-ligand formation. This is the first holistic experimental approach linking studies of hydrothermal vent microbial community composition with the geochemistry involved in organic iron-ligand formation.

Description: Molecular hydrogen (H2) can serve as an energy source for microorganisms in marine seafloor habitats. Microbial hydrogen oxidation plays a pivotal role in primary biomass production but also fosters fermentation by maintaining low hydrogen concentrations in different seafloor habitats, e.g. in deep-sea hydrothermal vent systems and anoxic sediments. The interconversion of H2 to protons and electrons is catalyzed by hydrogenase enzymes, which so far have all been described as metalloenzymes requiring a complex maturation apparatus. Here we will present data from recent hydrogen amended sediment incubation experiments where we monitored hydrogen consumption alongside with shifts in the respective microbial communities based on RNA analyses. Hydrogen consumption was observed in all incubations, yet differences occurred in the specific rates and in the communities apparently responsible for the utilization of the hydrogen. Since the majority of the microbes are currently not culturable, we recently established a function-based screen to recover hydrogenases from metagenomic fosmid libraries. We will introduce some novel hydrogenases recovered by applying this screen to fosmid libraries of seafloor habitats.

Description: In seafloor habitats molecular hydrogen (H2) can play a pivotal role in the metabolisms of a multitude of microorganisms. Microbial hydrogen oxidation fuels primary biomass production but also fosters fermentation by maintaining low hydrogen concentrations in different seafloor habitats, e.g. in deep-sea hydrothermal vent systems and anoxic sediments. The hydrogen-based energy conservation starts with the enzymatic interconversion of H2 to protons and electrons, catalyzed by hydrogenases. These key enzymes so far have all been described as metalloenzymes requiring a complex maturation apparatus. Here we will present data from two different approaches to uncover the microbial hydrogen oxidation potential in seafloor habitats. The first approach comprises hydrogen amended short-term sediment incubation experiments where we monitored hydrogen consumption alongside with shifts in the respective bacterial and archaeal communities based on RNA analyses. We observed hydrogen consumption in all of our incubations, yet differences occurred in the specific rates and in the communities apparently responsible for hydrogen utilization. Given the unculturable microbial majority and the need for culture-independent techniques, we recently established a function-based screen to recover hydrogenases from metagenomic fosmid libraries. We will introduce some novel hydrogenases recovered by applying this screen to fosmid libraries of seafloor habitats.

Description: Anthropogenic activities are modifying the oceanic environment rapidly and are causing ocean warming and deoxygenation, affecting biodiversity, productivity, and biogeochemical cycling. In coastal sediments, anaerobic organic matter degradation essentially fuels the production of hydrogen sulfide and methane. The release of these compounds from sediments is detrimental for the (local) environment and entails socio-economic consequences. Therefore, it is vital to understand which microbes catalyze the re-oxidation of these compounds under environmental dynamics, thereby mitigating their release to the water column. Here we use the seasonally dynamic Boknis Eck study site (SW Baltic Sea), where bottom waters annually fall hypoxic or anoxic after the summer months, to extrapolate how the microbial community and its activity reflects rising temperatures and deoxygenation. During October 2018, hallmarked by warmer bottom water and following a hypoxic event, modeled sulfide and methane production and consumption rates are higher than in March at lower temperatures and under fully oxic bottom water conditions. The microbial populations catalyzing sulfide and methane metabolisms are found in shallower sediment zones in October 2018 than in March 2019. DNA-and RNA profiling of sediments indicate a shift from primarily organotrophic to (autotrophic) sulfide oxidizing Bacteria, respectively. Previous studies using data collected over decades demonstrate rising temperatures, decreasing eutrophication, lower primary production and thus less fresh organic matter transported to the Boknis Eck sediments. Elevated temperatures are known to stimulate methanogenesis, anaerobic oxidation of methane, sulfate reduction and essentially microbial sulfide consumption, likely explaining the shift to a phylogenetically more diverse sulfide oxidizing community based on RNA.

Description: Iron (Fe) is an essential trace element for life. In the ocean, Fe can be exceptionally scarce and thus biolimiting or extremely enriched causing microbial stress. The ability of hydrothermal plume microbes to counteract unfavorable Fe-concentrations up to 10 mM is investigated through experiments. While Campylobacterota (Sulfurimonas) are prominent in a diverse community at low to intermediate Fe-concentrations, the highest 10 mM Fe-level is phylogenetically less diverse and dominated by the SUP05 clade (Gammaproteobacteria), a species known to be genetically well equipped to strive in high-Fe environments. In all incubations, Fe-binding ligands were produced in excess of the corresponding Fe-concentration level, possibly facilitating biological Fe-uptake in low-Fe incubations and detoxification in high-Fe incubations. The diversity of Fe-containing formulae among dissolved organics (SPE-DOM) decreased with increasing Fe-concentration, which may reflect toxic conditions of the high-Fe treatments. A DOM-derived degradation index (IDEG) points to a degradation magnitude (microbial activity) that decreases with Fe and/or selective Fe-DOM coagulation. Our results show that some hydrothermal microbes (especially Gammaproteobacteria) have the capacity to thrive even at unfavorably high Fe-concentrations. These ligand-producing microbes could hence play a key role in keeping Fe in solution, particularly in environments, where Fe precipitation dominates and toxic conditions prevail.

Description: During research cruise AL595 (31.5. - 20.6.2023) onboard research vessel ALKOR, investigations were carried out at the Grimsey Hydrothermal Field offshore Northern Iceland as part of the Helmholtz InnoPool project “High CO2 – metabolic responses and bioeconomic opportunities”. For the first time, the Hover-AUVs Anton and Luise were successfully operated at water depths of up to 400m, which is close to the maximum operational depth of 500m specified for these Girona 500 AUVs. AUV Anton was used to measure high resolution multibeam data with a horizontal resolution of approximately 40cm cov- ering a total area of ca. 1.4km2. AUV Luise acquired five photo-mosaics with sub-centimeter resolution covering a total area of ca. 5.000m2. In addition, both AUVs carried CTD probes, which will allow to investigate the local distribution of hydrothermal activity. Both high-resolution bathymetry and pho- togrammetry data yield new insights into the morphology and overall structure of the vent site and its surrounding, which will be valuable for the interpretation of geophysical data previously acquired in the working area. Sampling with a multicorer (three successful deployments), a 300cm long gravity corer (five successful deployments), a BIGO lander (two successful deployments) and casts with the CTD- rosette (seven deployments) generated fluid, pore-fluid and sediment samples to be analyzed by the working groups Geomicrobiology and Biogeochemistry, Marine Natural Products and Marine Geochem- istry at GEOMAR and at Matís (Iceland, Natural Products only). Lab work to be carried out in the home labs will yield insights into the physiological adaptation of microbial communities and individual microbes to very high CO2 concentrations and will explore microbial utilization of CO2 for establishing CO2-based bioeconomic value chains.

Description: In order to expand the knowledge of microbial ecosystems from deep-sea hydrothermal vent systems located on the Central and South-East Indian Ridge, we sampled hydrothermal fluids, massive sulfides, ambient water and sediments of six distinct vent fields. Most of these vent sites were only recently discovered in the course of the German exploration program for massive sulfide deposits and no previous studies of the respective microbial communities exist. Apart from typically vent-associated chemosynthetic members of the orders Campylobacterales , Mariprofundales , and Thiomicrospirales , high numbers of uncultured and unspecified Bacteria were identified via 16S rRNA gene analyses in hydrothermal fluid and massive sulfide samples. The sampled sediments however, were characterized by an overall lack of chemosynthetic Bacteria and the presence of high proportions of low abundant bacterial groups. The archaeal communities were generally less diverse and mostly dominated by members of Nitrosopumilales and Woesearchaeales , partly exhibiting high proportions of unassigned Archaea. Correlations with environmental parameters were primarily observed for sediment communities and for microbial species (associated with the nitrogen cycle) in samples from a recently identified vent field, which was geochemically distinct from all other sampled sites. Enrichment cultures of diffuse fluids demonstrated a great potential for hydrogen oxidation coupled to the reduction of various electron-acceptors with high abundances of Hydrogenovibrio and Sulfurimonas species. Overall, given the large number of currently uncultured and unspecified microorganisms identified in the vent communities, their respective metabolic traits, ecosystem functions and mediated biogeochemical processes have still to be resolved for estimating consequences of potential environmental disturbances by future mining activities.