Final Report Summary - DARCLIFE (Deep subsurface Archaea: carbon cycle, life strategies, and role in sedimentary ecosystems)
Archaea are ubiquitous inhabitants of marine sediments, particularly of the marine deep sedimentary biosphere. They mediate important processes controlling greenhouse gases and nutrients. To date, no representatives have been cultured from these habitats. The DARCLIFE project was devoted to these poorly understood benthic archaea and aimed to elucidate their role in sedimentary ecosystems and carbon cycling and to examine their strategies for coping with extreme energy limitation. DARCLIFE used an integrated, interdisciplinary research approach in which the analysis of intact archaeal membrane lipids was a central focus. These lipids encode information related to both the taxonomy of their microbial producers and their adaptation to their habitat; in addition they serve as proxies for live cells in natural environments. DARCLIFE substantially advanced analytical protocols for the detection of intact membrane lipids that led to the discovery of several novel lipid classes in natural environments. We probed contrasting sedimentary settings as natural laboratories to examine archaeal lipids and genes in the context of their geochemical environment. Two research expeditions were tailored to the objectives of DARCLIFE, proposed and carried out in the Western Mediterranean and Eastern Mediterranean Sea, the Marmara Sea and the Black Sea (M84/1 of RV Meteor and POS450 of RV Poseidon, funded by the Deutsche Forschungsgemeinschaft); their stringent sampling strategy was key to seamlessly combining the lipid-based characterization of benthic archaea with DNA-based phylogenetic classification and an in-depth geochemical examination of their habitat. We also repeatedly conducted field work at an estuarine site in North Carolina, USA, which is known to harbor uniquely high concentrations of the Miscellaneous Crenarchaeotal Group (MCG), a globally distributed group of archaea inhabiting Earth’s subsurface. Additional samples were collected during cruises of opportunity or were selected from our archives of global subseafloor samples. In a number of archaeal cultures, we addressed how growth conditions impact their cellular lipidome in order to strengthen our interpretation of environmental distributions.
We developed and applied a new generation of analytical protocols for microbial lipid analysis in complex sample matrices, improved the robustness of their quantification, developed a systematic framework aiding their mass spectrometric identification in complex mixtures, and achieved at least two orders of magnitude higher sensitivity. Instead of a few tens of polar lipids, we can now routinely monitor up to one thousand compounds in sediments and environmental samples and thus obtain a richer, more differentiated view of microbial communities. This advancement of analytical methods for lipid detection and characterization resulted in the discovery of multiple series of novel archaeal lipids that had not been described in culture before but seem widespread in the marine subseafloor. Combination with stable isotope assays has provided constraints on lipid turnover and the metabolic strategies of benthic archaea. For example, we found that the typical glycerol backbone of some tetraether lipids was substituted with a butanetriol or pentanetriol moiety, which are unconventional lipid building blocks and thus fundamentally violate what were thought to be conserved pathways of lipid biosynthesis. These novel lipids appear characteristic of benthic archaea and have been tentatively linked to members of the MCG. Distributions of these compounds in the marine subseafloor suggest that the extension of the glycerol backbone by one or two C atoms results in improved stability of intact polar lipids and may thus lower expenditures for repair of the cell membrane, a strategy that may be crucial for microbes persisting for millennia under severe energy limitation. The isotopic compositions of butanetriol derivatives are consistent with CO2 serving as the C substrate and this notion was supported by our efforts to reconstruct MCG genomes, contradicting other lines of evidence that suggest MCG actively degrade sedimentary organic matter. Similarly, at hydrocarbon seep settings in the Gulf of Mexico and in the Indian Ocean, large ranges of isotopic compositions of archaeal lipid classes testified to the diversity of carbon substrates and carbon metabolisms utilized by the archaeal seep communities. In particular, another group of novel lipids with strong biomarker potential for benthic archaea are unsaturated glycerol-based isoprenoidal tetraethers, in which the biphytane moieties carry multiple double bonds instead of or in addition to cycloalkylations. These molecules were likely previously overlooked with conventional analytical protocols; we have subsequently found these unsaturated forms to be widespread in benthic environments and have tentatively linked them to heterotrophic euryarchaeota affiliated with the Marine Benthic Group D. Parallel with the advancement of analytical protocols, our development of a novel stable isotope probing technique based on dual labeling with deuterated water and 13C bicarbonate has helped to unveil the modes of carbon flow in benthic archaeal communities. Specifically, we could show that moderately thermophilic anaerobic methane oxidizing archaea inhabiting hydrothermally heated sediments in the Guaymas Basin seem to derive their carbon for biosynthesis from the inorganic carbon pool rather than from methane, as previously assumed. Together with the evidence for inorganic carbon being utilized by members of the MCG mentioned above, this finding suggests that the capability to augment heterotrophic pathways with CO2 assimilation may be common among benthic archaea.
We also critically evaluated archaeal polar lipids as life markers in subseafloor sediments by experimentally determining their degradation kinetics and modeling the fate of these lipids under subseafloor conditions. Our results suggest that only about one tenth of the polar lipid pool is associated with live cells in deeply buried sediments; the remainder represents an accumulated fossil lipid pool. Consequently, our previous lipid-derived estimates of archaeal biomass in the subseafloor published in 2008 were too high; our new data based on a census of microbial lipids conducted in the DARCLIFE project supports this assessment and suggests more balanced, variable contributions of Archaea and Bacteria to subseafloor biomass. Modeling efforts suggest that the turnover of archaeal communities in the deep subseafloor is in the range of a few ten thousand years. The corresponding slow growth was observable also in our efforts to enrich selected benthic archaea and explains their recalcitrance to isolation attempts. In order to expand our understanding of archaeal adaptation to energy stress and the associated effects on the lipidome, we grew cultures of relevant model species at the Archaea Center in Regensburg and at MARUM. The results obtained with Nitrosopumilus maritimus and Methanothermobacter thermautotrophicus support our hypothesis that the strong predominance of archaeal glycolipids over phospholipids generally observed in the subseafloor results from an adaptive response to severe energy stress; phospholipids on the other hand appear to be generally more prominent in actively growing populations, which also explains their accumulation at geochemical interfaces as found during our environmental surveys.
Eight PhD students and 12 postdoctoral scientists were involved in the DARCLIFE project at different levels of intensity, the research has so far resulted in 36 publications, with two thirds being core papers, and the other third stimulated through group-internal and external collaborations enabled by the project. Three central PhD projects are about to be finalized and the senior team members are currently concluding the final statistical analyses and modeling. Consequently, a substantial number of manuscripts is under preparation on topics such as the multivariate relationships between the ~100,000 microbial species, up to ~1,000 microbial lipids, and a rich geochemical dataset including mineralogy, redox elements, carbon isotopes, and thousands of molecular formulas for dissolved organic compounds per sample. Important byproducts of the DARCLIFE project include a new molecular imaging approach that shows great potential for the sub-mm-scale interrogation of lipid records in sediment and geobiological materials and a rich dataset of dissolved organic matter compositions that provides an unprecedented view into the sources and diagenetic processes influencing this pool in marine pore waters. Collectively, the discoveries and scientific progress achieved during the DARCLIFE project were critically dependent on the concerted effort and momentum developed by a larger team that could only be assembled in the framework of an ERC Advanced grant project.
We developed and applied a new generation of analytical protocols for microbial lipid analysis in complex sample matrices, improved the robustness of their quantification, developed a systematic framework aiding their mass spectrometric identification in complex mixtures, and achieved at least two orders of magnitude higher sensitivity. Instead of a few tens of polar lipids, we can now routinely monitor up to one thousand compounds in sediments and environmental samples and thus obtain a richer, more differentiated view of microbial communities. This advancement of analytical methods for lipid detection and characterization resulted in the discovery of multiple series of novel archaeal lipids that had not been described in culture before but seem widespread in the marine subseafloor. Combination with stable isotope assays has provided constraints on lipid turnover and the metabolic strategies of benthic archaea. For example, we found that the typical glycerol backbone of some tetraether lipids was substituted with a butanetriol or pentanetriol moiety, which are unconventional lipid building blocks and thus fundamentally violate what were thought to be conserved pathways of lipid biosynthesis. These novel lipids appear characteristic of benthic archaea and have been tentatively linked to members of the MCG. Distributions of these compounds in the marine subseafloor suggest that the extension of the glycerol backbone by one or two C atoms results in improved stability of intact polar lipids and may thus lower expenditures for repair of the cell membrane, a strategy that may be crucial for microbes persisting for millennia under severe energy limitation. The isotopic compositions of butanetriol derivatives are consistent with CO2 serving as the C substrate and this notion was supported by our efforts to reconstruct MCG genomes, contradicting other lines of evidence that suggest MCG actively degrade sedimentary organic matter. Similarly, at hydrocarbon seep settings in the Gulf of Mexico and in the Indian Ocean, large ranges of isotopic compositions of archaeal lipid classes testified to the diversity of carbon substrates and carbon metabolisms utilized by the archaeal seep communities. In particular, another group of novel lipids with strong biomarker potential for benthic archaea are unsaturated glycerol-based isoprenoidal tetraethers, in which the biphytane moieties carry multiple double bonds instead of or in addition to cycloalkylations. These molecules were likely previously overlooked with conventional analytical protocols; we have subsequently found these unsaturated forms to be widespread in benthic environments and have tentatively linked them to heterotrophic euryarchaeota affiliated with the Marine Benthic Group D. Parallel with the advancement of analytical protocols, our development of a novel stable isotope probing technique based on dual labeling with deuterated water and 13C bicarbonate has helped to unveil the modes of carbon flow in benthic archaeal communities. Specifically, we could show that moderately thermophilic anaerobic methane oxidizing archaea inhabiting hydrothermally heated sediments in the Guaymas Basin seem to derive their carbon for biosynthesis from the inorganic carbon pool rather than from methane, as previously assumed. Together with the evidence for inorganic carbon being utilized by members of the MCG mentioned above, this finding suggests that the capability to augment heterotrophic pathways with CO2 assimilation may be common among benthic archaea.
We also critically evaluated archaeal polar lipids as life markers in subseafloor sediments by experimentally determining their degradation kinetics and modeling the fate of these lipids under subseafloor conditions. Our results suggest that only about one tenth of the polar lipid pool is associated with live cells in deeply buried sediments; the remainder represents an accumulated fossil lipid pool. Consequently, our previous lipid-derived estimates of archaeal biomass in the subseafloor published in 2008 were too high; our new data based on a census of microbial lipids conducted in the DARCLIFE project supports this assessment and suggests more balanced, variable contributions of Archaea and Bacteria to subseafloor biomass. Modeling efforts suggest that the turnover of archaeal communities in the deep subseafloor is in the range of a few ten thousand years. The corresponding slow growth was observable also in our efforts to enrich selected benthic archaea and explains their recalcitrance to isolation attempts. In order to expand our understanding of archaeal adaptation to energy stress and the associated effects on the lipidome, we grew cultures of relevant model species at the Archaea Center in Regensburg and at MARUM. The results obtained with Nitrosopumilus maritimus and Methanothermobacter thermautotrophicus support our hypothesis that the strong predominance of archaeal glycolipids over phospholipids generally observed in the subseafloor results from an adaptive response to severe energy stress; phospholipids on the other hand appear to be generally more prominent in actively growing populations, which also explains their accumulation at geochemical interfaces as found during our environmental surveys.
Eight PhD students and 12 postdoctoral scientists were involved in the DARCLIFE project at different levels of intensity, the research has so far resulted in 36 publications, with two thirds being core papers, and the other third stimulated through group-internal and external collaborations enabled by the project. Three central PhD projects are about to be finalized and the senior team members are currently concluding the final statistical analyses and modeling. Consequently, a substantial number of manuscripts is under preparation on topics such as the multivariate relationships between the ~100,000 microbial species, up to ~1,000 microbial lipids, and a rich geochemical dataset including mineralogy, redox elements, carbon isotopes, and thousands of molecular formulas for dissolved organic compounds per sample. Important byproducts of the DARCLIFE project include a new molecular imaging approach that shows great potential for the sub-mm-scale interrogation of lipid records in sediment and geobiological materials and a rich dataset of dissolved organic matter compositions that provides an unprecedented view into the sources and diagenetic processes influencing this pool in marine pore waters. Collectively, the discoveries and scientific progress achieved during the DARCLIFE project were critically dependent on the concerted effort and momentum developed by a larger team that could only be assembled in the framework of an ERC Advanced grant project.