Dissimilatory sulfur metabolism coupled to anaerobic oxidation of methane

The seafloor and its microbial inhabitants play an important role in the biogeochemical cycling of elements. These environments are generally anoxic but contain high concentrations of sulfate penetrating from the overlying seawater. The main carbon mineralization processes such as the anaerobic oxidation of methane (AOM; Eq. 1) are therefore generally coupled to sulfate reduction. CH4 SO42 → HCO3 HS H2O (Eq. 1) AOM plays a crucial role in both carbon and sulfur cycling. It oxidizes the majority of the methane a potent greenhouse gas diffusing from the seafloor and prevents its escape to the atmosphere. Methane oxidation also returns the carbon trapped in the form of recalcitrant methane back to the carbon cycle as carbon dioxide. The AOM-coupled sulfate reduction consumes a large portion of the downwards sulfate flux and forms sulfide, which diffuses upwards towards the seafloor where it supports free-living sulfide- and sulfur-oxidizers but also gutless worms, clams and mussels that rely for their nutrition on the thiotrophic symbionts. Despite the pronounced effect of AOM on the sediment geochemistry little is known about its biology. The organisms responsible for AOM a consortium of methanotrophic archaea and Deltaproteobacteria have been identified in situ but their slow metabolism complicates growing them in pure cultures and renders the physiological investigations challenging. So far, AOM research has predominantly focused on the C1 metabolism of the methanotrophic archaea. The investigations presented in this thesis address the dissimilatory sulfur metabolism of the organisms involved in AOM and the mechanisms of its coupling to methane oxidation. Chapters 2 and 3 describe the purification and characterization of the three known enzymes involved in dissimilatory sulfate reduction (SR enzymes; ATP sulfurylase, APS reductase, sulfite reductase). The enzymes were purified from a naturally enriched microbial mat using liquid chromatography. The identity of the SR enzymes was confirmed by N-terminal amino acid sequencing and their activity in total cell extracts as well as in individual chromatography fractions was quantified by corresponding enzyme essays. Our aim was to assign these enzymes to a particular organism in the mat sample. For this purpose, polyclonal antibodies against the purified ATP sulfurylase and sulfite reductase were used APS reductase could not be sufficiently purified for antibody generation in situ in the original environmental sample as well as in our other enrichment cultures. This combination of environmental proteomics and immunolocalization allowed us to unambiguously assign the isolated SR enzymes exclusively to the bacterial partner. The archaea did not express detectable amounts of the identified SR enzymes themselves and therefore likely depend on their bacterial partners to perform the sulfate reduction. These results are presented as manuscripts in revision (Manuscript 1) and in preparation (Manuscript 2). The following Chapter 4 introduces experiments that were performed in order to elucidate sulfur transfer and speciation in AOM consortia. We used stable and radioactive sulfur isotopes to follow sulfur exchange between the medium and biomass and on a single cell level among individual cells. Based on our results and thermodynamic consideration we propose a model, in which DSS bacteria reduce sulfate to a zerovalent sulfur compound (probably polysulfide) that might be utilized by ANME as an electron acceptor for methane oxidation. Thus, unexpectedly, ANME participate in the dissimilatory sulfur metabolism coupled to AOM. Our combined data suggest that ANME obtain this compound from the associated bacteria. Such sulfur shuttling between two organisms not only represents a unique mechanism for a syntrophic relationship but also has significant implications for our understanding of sulfur transformations in the AOM zones in marine sediments. These results are presented as a manuscript in preparation (Manuscript 3)

2. Wochenbericht FS Poseidon Cruise 539 [POS539]

11.11.2019 – 17.11.2019, Varna (BG) - Varna (BG), (06.11.2019 - 22.11.2019

2. Wochenbericht FS Poseidon Cruise 539 [POS539]

11.11.2019 – 17.11.2019, Varna (BG) - Varna (BG), (06.11.2019 - 22.11.2019

3. Wochenbericht FS Poseidon Cruise 539 [POS539]

18.11.2019 – 22.11.2019, Varna (BG) - Varna (BG), (06.11.2019 - 22.11.2019

Anaerobic metabolism of Foraminifera thriving below the seafloor

Foraminifera are single-celled eukaryotes (protists) of large ecological importance, as well as environmental and paleoenvironmental indicators and biostratigraphic tools. In addition, they are capable of surviving in anoxic marine environments where they represent a major component of the benthic community. However, the cellular adaptations of Foraminifera to the anoxic environment remain poorly constrained. We sampled an oxic-anoxic transition zone in marine sediments from the Namibian shelf, where the genera Bolivina and Stainforthia dominated the Foraminifera community, and use metatranscriptomics to characterize Foraminifera metabolism across the different geochemical conditions. Relative Foraminifera gene expression in anoxic sediment increased an order of magnitude, which was confirmed in a 10-day incubation experiment where the development of anoxia coincided with a 20-40-fold increase in the relative abundance of Foraminifera protein encoding transcripts, attributed primarily to those involved in protein synthesis, intracellular protein trafficking, and modification of the cytoskeleton. This indicated that many Foraminifera were not only surviving but thriving, under the anoxic conditions. The anaerobic energy metabolism of these active Foraminifera was characterized by fermentation of sugars and amino acids, fumarate reduction, and potentially dissimilatory nitrate reduction. Moreover, the gene expression data indicate that under anoxia Foraminifera use the phosphogen creatine phosphate as an ATP store, allowing reserves of high-energy phosphate pool to be maintained for sudden demands of increased energy during anaerobic metabolism. This was co-expressed alongside genes involved in phagocytosis and clathrin-mediated endocytosis (CME). Foraminifera may use CME to utilize dissolved organic matter as a carbon and energy source, in addition to ingestion of prey cells via phagocytosis. These anaerobic metabolic mechanisms help to explain the ecological success of Foraminifera documented in the fossil record since the Cambrian period more than 500 million years ago

Diverse methylotrophic methanogenic archaea cause high methane emissions from seagrass meadows

© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Schorn, S., Ahmerkamp, S., Bullock, E., Weber, M., Lott, C., Liebeke, M., Lavik, G., Kuypers, M. M. M., Graf, J. S., & Milucka, J. Diverse methylotrophic methanogenic archaea cause high methane emissions from seagrass meadows. Proceedings of the National Academy of Sciences of the United States of America, 119(9), (2022): e2106628119, https://doi.org/10.1073/pnas.2106628119.Marine coastlines colonized by seagrasses are a net source of methane to the atmosphere. However, methane emissions from these environments are still poorly constrained, and the underlying processes and responsible microorganisms remain largely unknown. Here, we investigated methane turnover in seagrass meadows of Posidonia oceanica in the Mediterranean Sea. The underlying sediments exhibited median net fluxes of methane into the water column of ca. 106 µmol CH4 ⋅ m−2 ⋅ d−1. Our data show that this methane production was sustained by methylated compounds produced by the plant, rather than by fermentation of buried organic carbon. Interestingly, methane production was maintained long after the living plant died off, likely due to the persistence of methylated compounds, such as choline, betaines, and dimethylsulfoniopropionate, in detached plant leaves and rhizomes. We recovered multiple mcrA gene sequences, encoding for methyl-coenzyme M reductase (Mcr), the key methanogenic enzyme, from the seagrass sediments. Most retrieved mcrA gene sequences were affiliated with a clade of divergent Mcr and belonged to the uncultured Candidatus Helarchaeota of the Asgard superphylum, suggesting a possible involvement of these divergent Mcr in methane metabolism. Taken together, our findings identify the mechanisms controlling methane emissions from these important blue carbon ecosystems.This project was funded by theMax Planck Society

Report and preliminary results of R/V POSEIDON cruise POS539, Varna (Bulgaria) - Varna (Bulgaria) November 6 - November 21, 2019

The R/V POSEIDON cruise POS539 took place in the northwestern basin of the Black Sea (42°30’N to 44°N and 29°E to 31°E). The overarching aim of the campaign was to obtain sediment and water samples, including suspended particle material, from the various redox zones of the Black Sea. The campaign lasted between November 6th and November 21st 2019 and the collected samples were taken in order to investigate the activity and physiology of microorganisms involved in the conversion of nitrogen compounds and degradation of organic carbon under various oxygen conditions. The main topics of the cruise were: (a) to quantify the contribution of archaeal nitrifiers to the nitrogen and carbon cycles, b) to measure the production and consumption of the powerful greenhouse gases CH4 and N2O, c) to record palaeoenvironmental changes in high resolution, and d) to describe the complexity and identity of biopolymers. For this, water and sediment samples were retrieved from 10 discrete shelf and slope stations. First, ‘deep water’ transect was conducted, which included three stations with water depths over 2000 m. The second perpendicular transect encompassed stations that gradually transitioned from the deep parts of the slope towards the shelf (ca. 80 m depth). Additionally, two stations were setup north and south of the shelf transect, respectively, for paleoceanographic studies. Throughout the cruise the weather conditions were overwhelmingly good, only towards the end of the campaign gusty winds of 7 Bft were recorded. The recorded oceanographic conditions were in agreement with the expected water properties at all stations. Station activities were completed on November 20th at 14:00 local board time. On November 21st at 10:30 local time, R/V POSEIDON reached the port of Varna, Bulgaria, thus concluding the POS539 expedition. Analyses and results from the samples and experiments will provide a basis for our understanding of the microbial control on the carbon and nitrogen cycle of the Black Sea.13032

Dark aerobic sulfide oxidation by anoxygenic phototrophs in anoxic waters

Anoxygenic phototrophic sulfide oxidation by green and purple sulfur bacteria (PSB) plays a key role in sulfide removal from anoxic shallow sediments and stratified waters. Although some PSB can also oxidize sulfide with nitrate and oxygen, little is known about the prevalence of this chemolithotrophic lifestyle in the environment. In this study, we investigated the role of these phototrophs in light‐independent sulfide removal in the chemocline of Lake Cadagno. Our temporally resolved, high‐resolution chemical profiles indicated that dark sulfide oxidation was coupled to high oxygen consumption rates of ~9 μM O2·h−1. Single‐cell analyses of lake water incubated with 13CO2 in the dark revealed that Chromatium okenii was to a large extent responsible for aerobic sulfide oxidation and it accounted for up to 40% of total dark carbon fixation. The genome of Chr. okenii reconstructed from the Lake Cadagno metagenome confirms its capacity for microaerophilic growth and provides further insights into its metabolic capabilities. Moreover, our genomic and single‐cell data indicated that other PSB grow microaerobically in these apparently anoxic waters. Altogether, our observations suggest that aerobic respiration may not only play an underappreciated role in anoxic environments but also that organisms typically considered strict anaerobes may be involved

Ideas and Perspectives: A Strategic Assessment of Methane and Nitrous Oxide Measurements In the Marine Environment

In the current era of rapid climate change, accurate characterization of climate-relevant gas dynamics-namely production, consumption, and net emissions-is required for all biomes, especially those ecosystems most susceptible to the impact of change. Marine environments include regions that act as net sources or sinks for numerous climateactive trace gases including methane (CH4) and nitrous oxide (N2O). The temporal and spatial distributions of CH4 and N2O are controlled by the interaction of complex biogeochemical and physical processes. To evaluate and quantify how these mechanisms affect marine CH4 and N2O cycling requires a combination of traditional scientific disciplines including oceanography, microbiology, and numerical modeling. Fundamental to these efforts is ensuring that the datasets produced by independent scientists are comparable and interoperable. Equally critical is transparent communication within the research community about the technical improvements required to increase our collective understanding of marine CH4 and N2O. A workshop sponsored by Ocean Carbon and Biogeochemistry (OCB) was organized to enhance dialogue and collaborations pertaining to marine CH4 and N2O. Here, we summarize the outcomes from the workshop to describe the challenges and opportunities for near-future CH4 and N2O research in the marine environment

Dissimilatorischer Schwefelmetabolismus gekoppelt an anaerobe Oxidation von Methan

The seafloor and its microbial inhabitants play an important role in the biogeochemical cycling of elements. These environments are generally anoxic but contain high concentrations of sulfate penetrating from the overlying seawater. The main carbon mineralization processes such as the anaerobic oxidation of methane (AOM; Eq. 1) are therefore generally coupled to sulfate reduction. CH4 SO42 → HCO3 HS H2O (Eq. 1) AOM plays a crucial role in both carbon and sulfur cycling. It oxidizes the majority of the methane a potent greenhouse gas diffusing from the seafloor and prevents its escape to the atmosphere. Methane oxidation also returns the carbon trapped in the form of recalcitrant methane back to the carbon cycle as carbon dioxide. The AOM-coupled sulfate reduction consumes a large portion of the downwards sulfate flux and forms sulfide, which diffuses upwards towards the seafloor where it supports free-living sulfide- and sulfur-oxidizers but also gutless worms, clams and mussels that rely for their nutrition on the thiotrophic symbionts. Despite the pronounced effect of AOM on the sediment geochemistry little is known about its biology. The organisms responsible for AOM a consortium of methanotrophic archaea and Deltaproteobacteria have been identified in situ but their slow metabolism complicates growing them in pure cultures and renders the physiological investigations challenging. So far, AOM research has predominantly focused on the C1 metabolism of the methanotrophic archaea. The investigations presented in this thesis address the dissimilatory sulfur metabolism of the organisms involved in AOM and the mechanisms of its coupling to methane oxidation. Chapters 2 and 3 describe the purification and characterization of the three known enzymes involved in dissimilatory sulfate reduction (SR enzymes; ATP sulfurylase, APS reductase, sulfite reductase). The enzymes were purified from a naturally enriched microbial mat using liquid chromatography. The identity of the SR enzymes was confirmed by N-terminal amino acid sequencing and their activity in total cell extracts as well as in individual chromatography fractions was quantified by corresponding enzyme essays. Our aim was to assign these enzymes to a particular organism in the mat sample. For this purpose, polyclonal antibodies against the purified ATP sulfurylase and sulfite reductase were used APS reductase could not be sufficiently purified for antibody generation in situ in the original environmental sample as well as in our other enrichment cultures. This combination of environmental proteomics and immunolocalization allowed us to unambiguously assign the isolated SR enzymes exclusively to the bacterial partner. The archaea did not express detectable amounts of the identified SR enzymes themselves and therefore likely depend on their bacterial partners to perform the sulfate reduction. These results are presented as manuscripts in revision (Manuscript 1) and in preparation (Manuscript 2). The following Chapter 4 introduces experiments that were performed in order to elucidate sulfur transfer and speciation in AOM consortia. We used stable and radioactive sulfur isotopes to follow sulfur exchange between the medium and biomass and on a single cell level among individual cells. Based on our results and thermodynamic consideration we propose a model, in which DSS bacteria reduce sulfate to a zerovalent sulfur compound (probably polysulfide) that might be utilized by ANME as an electron acceptor for methane oxidation. Thus, unexpectedly, ANME participate in the dissimilatory sulfur metabolism coupled to AOM. Our combined data suggest that ANME obtain this compound from the associated bacteria. Such sulfur shuttling between two organisms not only represents a unique mechanism for a syntrophic relationship but also has significant implications for our understanding of sulfur transformations in the AOM zones in marine sediments. These results are presented as a manuscript in preparation (Manuscript 3)