Search

The identification of reliable biomarkers, such as amino acids, is key for the search of extraterrestrial life. A large number of microorganisms metabolize, synthesize, take up and excrete amino acids as part of the amino acid metabolism during aerobic and/or anaerobic respiration or in fermentation. In this work, we investigated whether the anaerobic microbial metabolism of amino acids could leave a secondary biosignature indicating biological activity in the environment around the cells. The observed fingerprints would reflect the physiological capabilities of the specific microbial community under investigation. The metabolic processing of an amino acid mixture by two distinct anaerobic microbial communities collected from Islinger Mühlbach (ISM) and Sippenauer Moor (SM), Germany was examined. The amino acid mixture contained L-alanine, β-alanine, L-aspartic acid, DL-proline, L-leucine, L-valine, glycine, L-phenylalanine and L-isoleucine. In parallel, an amino acid spiked medium without microorganisms was used as a control to determine abiotic changes over time. Liquid chromatography mass spectrometry (LC-MS) was used to track amino acid changes over time. When comparing to the control samples that did not show significant changes of amino acids concentrations over time, we found that glycine was almost completely depleted from both microbial samples to less than 3% after the first two weeks- This results indicates a preferential use of this simple amino acid by these microbial communities. Although glycine degradation can be caused by abiotic processes, these results show that its preferential depletion in an environment would be consistent with the presence of life. We found changes in most other amino acids that varied between amino acids and communities, suggesting complex dynamics with no clear universal pattern that might be used as a signature of life. However, marked increases in amino acids, caused by cellular synthesis and release into the extracellular environment (e.g., alanine), were observed and could be considered a signature of metabolic activity. We conclude, that substantial anomalous enhancements of some amino acids against the expected abiotic background concentration may be an agnostic signature of the presence of biological processes.

During the project “Mars Analogues for Space Exploration” (MASE) several microbial isolates were obtained from extreme terrestrial environments. Some of these strains were subjected to Mars relevant environmental stress factors in the laboratory under controlled conditions; e.g. radiation, low water activity, high salt concentrations, or oxidizing compounds. All sampling, isolation, and cultivation steps, as well as the stress tests were performed under anoxic conditions. So far, five only distantly related microorganisms are under detailed investigation: Buttiauxella sp. MASE-IM-9, Clostridium sp. MASE-IM-4, Halanaerobium sp. MASE-BB-1, Trichococcus sp. MASE-IM-5, and Yersinia intermedia MASE-LG-1. It was shown that tolerance to desiccation and to ionizing radiation, applied separately, was not correlated as it is known for example for deinococcal representatives. If desiccation and exposure to radiation were applied together typical additive effects as well as an enhanced radiation tolerance could be observed. The survival after addition of oxidizing compounds (hydrogen peroxide; perchlorates) for dedicated time points (15 minutes; 24 hours) was very divers. The comparison of the five microorganisms revealed that Trichococcus sp. MASE-IM-5 was the most sensitive strain and survived only 20 mM hydrogen peroxide for 15 minutes and 24 hours, respectively. The most tolerant organism was Buttiauxella sp. MASE-IM-9 which was able to survive 230 mM hydrogen peroxide for both tested time points. In between, the other Bacteria were arranged with different tolerances against hydrogen peroxide (Buttiauxella sp. MASE-IM-9 > Yersinia intermedia MASE-LG-1 > Halanaerobium sp. BB-1 > Clostridium sp. MASE-IM-4 > Trichococcus sp. MASE-IM-5). Comparable species specific results were obtained after the addition of different perchlorates. In general, the strategy of the MASE project has proven to be useful to gain new model organisms. The isolated and characterized MASE strains have so far unknown high tolerances against cell damaging treatments and may serve as model organisms for future space exposure experiments.

The search for traces of life is one of the principal aims of Mars exploration. Within the MASE project (Mars Analogues for Space Exploration) we work to improve approaches and methods for biomarker detection and extraction from Mars analogue sites. One promising strategy to study the preservation of biosignatures in Mars conditions consists of the combined study of biomarkers detection and monitoring physicochemical parameters in mineralized samples. We have observed that it there is a correlation between biomarker detection and changes in oxi-reduction potential (ORP) during mineralization process. In addition, these methods and the study of samples from MASE sites have enabled us to develop an antibody microarray for competitive sandwich immunoassays as a potential tool for the detection of biomarkers in salty and anaerobic conditions.

International audience

The Mars Analogues for Space Exploration (MASE) project is bringing new insight about Mars potential habitability but also some new knowledge about Earth organisms and the functioning of extreme terrestrial ecosystems. The overall aim of the MASE project is to study a variety of Mars-like environments in order to further our understanding of Martian habitability, as well as our ability to detect organisms that might be present on Mars. For communication with the scientific community, we are benefiting from Open Access scientific publications and international conferences. Mars exploration missions are massive undertakings that are managed and implemented by space agencies/industry sector in a collaborative manner. Activities performed and results obtained from MASE have a direct impact on a search for life mission design and planning. Thus, MASE is in regular contact with the European Space Agency and activities to engage with other agencies will be soon taken into action. Providing a link between life on the Earth and life on Mars, has the potential to raise sig- nificant interest from the general public. For the project to reach this audience, we targeted journalists from science popular magazines and also organized a set of press conferences. This approach dramatically increased the visibility of the MASE project on outreach publications and media platforms. Alternative communication channels as press releases, newsletters and blog entries have also been regularly used to communicate with broader audiences with a positive outcome. Moreover, MASE scientist have been quite involved in outreach events to promote astrobiological research at local level. More recently, the MASE project have cre- ated a booklet title “A guide to Martian landscapes on Earth” to promote Mars analogues research. This hard-copy printed product resulted very successful to attract the attention of both specialized and general public audiences in conferences and science outreach events. A variety of internet based products are also used on regular basis to convey the main outcomes of the MASE project: MASE website www.mase.esf.org Twitter @MarsAnalogues Facebook MASE @MarsAna- logues These social media platforms have been proved to be the fastest and more effective way to internationally communicate MASE outcomes. Through Facebook, we have mostly engaged with general audience interested on space and astrobiology research topics, while Twitter audience is narrower and directly linked with the astrobiology scientific community.

During the development of the Mars Analogues for Space Exploration project (MASE) several microbial isolates of the Tessaracoccus genus, a denitrifying actynomicetes, were obtained from enrichment cultures of samples from cores drilled at different depths in the Iberian Pyrite Belt. Some of these strains, isolates SSRT5 and SSRT8, were subjected to Mars environmental stress tests under controlled laboratory conditions; e.g. high salt concentra- tions, temperature, oxidizing compounds, radiation and low water activity. All sampling, isolation, and cultivation steps, as well as the stress tests were performed under strict anoxic conditions. The differences found between these isolates are remarkable. Isolate SSRT8 was able to grow efficiently at low temperature (4 oC). It was able to grow in the presence of 300 mM Ca, Mg and Na perchlorates and to resist up to 1M concentration of these salts. The sensitivity of isolate SSRT5 was dependent on the cation of the perchlorate. Isolate SSRT8 was more resistant to extended exposures to low temperatures (-80 oC) and exposure to 500Gy X-Ray radiation. Both isolates are extremely sensitive to freeze and thaw cycles, desiccation and starvation. Genomic comparison of both isolates is currently underway to detect the een two isolates from the same ecosystem.

Introduction: The Mars Analogues for Space Ex-ploration (MASE) project is brining new insight about Mars potential habitability but also some new knowledge about Earth organisms and the functioning of extreme terrestrial ecosystems. The overall aim of the MASE project is to study a variety of Mars-like environments in order to further our understanding of Martian habitability, as well as our ability to detect organisms that might be present on Mars. This collabo-rative, 4 research project supported by the European Commission’s Seventh Framework Programme has been running since January 2014 and a variety of dis-semination and communication activities has been per-formed since then to increase visibility of Mars ana-logues research.

Assessing the habitability of Mars and detecting life, if it ever existed there, depends on knowledge of whether the combined environmental stresses experienced on Mars are compatible with life as we know it and whether a record of that life could ever be detected. So far, only few investigations were performed to understand the combined effect of different environmental stresses on survival and growth of anaerobic and extremophilic organisms. In the space experiment MEXEM (formerly known as MASE-in-SPACE) the hypothesis will be tested that selected terrestrial organisms, enrichment cultures and original samples from extreme Mars-analogue environments on Earth are able to withstand the Martian environ- mental stress factors due to their highly effective cellular and molecular adaptation and repair mechanisms. In addition, artificially fossilized and aged isolates from Mars-analogue environments on Earth will be examined and assessed with respect to their suitability for biosigna- ture identification. MEXEM samples will be (i) oxygen-depleted natural sediment samples, (ii) natural sediments spiked with selected, defined strains representative for the respective analogue site, (iii) individual (facultative) anaerobic / micro-aerophilic species including ciliates and viruses, (iv) defined mixtures of these biological entities, (v) isolated strains from samples collected inside the ISS and (vi) artificially fossilized isolates from the natural environments. Most of these samples and isolates were obtained from Mars-analogue envi- ronments on Earth in the frame of the EC funded project MASE (Grant Agreement 607297) and from the space experiment EXTREMOPHILES (PI C. Moissl-Eichinger). MEXEM will be flown outside on the ISS in the new exobiology facility ESA is building now. It offers the possibility to simulate of the martian environment, in particular the martian UV cl e, which cannot be done in the lab, but also martian atmosphere and pressure in LEO.

During the project MASE (Mars Analogues for Space Exploration) several microbial isolates were obtained and some of these strains were subjected to Mars relevant environmental stress factors in the laboratory under controlled conditions; e.g. radiation, low water activity, high salt concentrations, oxidizing compounds. All sampling, isolation, and cultivation steps, as well as the stress tests were performed under anoxic conditions. So far, five only distantly related microorganisms are under detailed investigation: \textit{Buttiauxella} sp. MASE-IM-7, \textit{Clostridium} sp. MASE-IM-4, \textit{Halanaerobium} sp. MASE-BB-1, \textit{Trichococcus} sp. MASE-IM-5, and \textit{Yersinia} sp. MASE-LG-1. It was shown that tolerance to desiccation and to ionizing radiation, applied separately was not correlated. If desiccation and exposure to radiation was applied together typical additive effects could be observed in each species. The survival after addition of oxidizing compounds (hydrogen peroxide; perchlorates) for a dedicated time (15 minutes; 24 hours) was very divers. If the five microorganisms were compared with each other, \textit{Trichococcus} sp. MASE-IM-5 was the most sensitive strain and survived only 10 mM hydrogen peroxide for 15 minutes and 24 hours, respectively. The most tolerant organism was \textit{Halanaerobium} sp. MASE-BB-1 which was able to survive 100 mM hydrogen peroxide for both tested time points. In between, the other Bacteria were arranged with different tolerances against hydrogen peroxide. Comparable, species specific results were obtained after the addition of different perchlorates. In general, the strategy of the MASE project has proven to be useful to gain new model microorganisms. The isolated and characterized MASE strains have so far unknown high tolerances against cell damaging treatments and may serve as model organisms for future space exposure experiments.

Osmotic stress is one of the major limitations for cell growth. Microorganisms can evolve adaptations to abiotic stresses like high salt concentrations in the environment. Sensor and signal transduction networks provide information to the cell about the osmolarity of its surroundings leading to an immediate metabolic response to counteract the osmotic stress. Some of these adaptations can be structural, some are metabolic. Our current knowledge of microbial responses to osmotic challenges is based on studies of representative bacteria, archaea, and eukaryotic microbes that is mainly focusing on the effect of NaCl. There is still a lack of understanding whether and how different salts, for example NaCl versus MgSO4 alter the response of a microorganism to salt induced stress conditions. We chose Yersinia intermedia MASE-LG1 a strain isolated from an Icelandic lake as test organism. It is known for its abilities to adapt to a wide variety of habitats of rapidly changing environmental conditions. In order to identify which roles the different salts play in the global metabolic response, Y. intermedia was exposed sustained salt stress induced by either MgSO4 or NaCl. After metabolite extraction, metabolic profiles from three replicate cultures of Y. intermedia MASE-LG-1 grown under 3 different conditions (e.g. control salt stressed in MgSO4, and salt stressed in NaCl) were obtained. Generally, changes in numerous metabolites mainly in the amino acid metabolisms were observed in stressed samples compared to the control. To a lesser extent the carbohydrate metabolism was also affected. Looking at the effect of the different kations, the results clearly indicated significant differences in response to salt stress induced by the magnesium salt compared to sodium chloride. The results suggest that the amino acid synthesis, reflecting the general activity of translation operations, dominates the reaction to osmotic stress. These adaptations might provide necessary energy and building blocks to fuel processes conveying salt tolerance like the biosynthesis of compatible solutes. In addition we were able to identify metabolites which are linked to osmoprotective activity. The outcome of this study will have impact on our understanding of how microorganisms adapt to hostile environmental conditions.

Introduction: Assessing the biological origin of microstructures present in rocky materials is very chal-lenging mainly due to the degradation of microbial remains during diagenesis and to mineralogical processes leading to abiotic artifacts. To better understand the processes leading to microfossil formation and preservation, artificial mineralisation of microorganisms was first undertaken with the silicification of Cyanobacteria [1], followed by the mineralisation of eukaryotic and bacterial models [2] or of natural communities [3] and has since been notably extended to hyperthermophilic Archaea [4].

The concept of present and/or past extraterrestrial life is thrilling and tackled intensively throughout the last decades, yet remains notional. Some regions of extraterrestrial bodies (e.g. the Mars) are in general considered as habitable; however, are ruled by extreme physical and chemical variables, which constrain the possibility of life. Similar settings (at least to a certain extent) exist on Earth and function as analogue model sites in many studies to elucidate basic information on the limits of life. One crucial feature, which distinguishes Earth from extraterrestrial bodies, is the absence of oxygen in the atmosphere. Terrestrial Mars analogue, anoxic settings are hardly described, in par-ticular with respect on the hosted microbial communities. The MASE (Mars Analogues for Space Exploration) project tackled to understand specifically anaerobic life thriving in a number of various Mars analogue settings. Within the frame of this project, a diverse set of extreme and anoxic Mars analogue environments were sampled and microbiologically investigated by combining cultivation based and cultivation-independent analyses. This study included (i) a widescale cultivation approach targeting the anaerobic microbial fraction and (ii) an amplicon sequencing approach focusing on the viable Archaeome and Bacteriome.

Life is a physico-chemical process by which tell-tale signals or traces are left on the environment. These signals are indicators of life and are known as biomarkers. Besides, the traces of some kinds of microorganisms can be well preserved, provided that they are rapidly mineralized and that the sediments in which they occur are rapidly cemented [1]. The search for these traces of life is one of the main objectives of Mars exploration [1] and to improve and optimize the search and detection of them forms part of MASE project targets. In MASE project (Mars Analogues for Space Exploration) we work to improve approaches and methods for biomarker detection in samples with low biomass from Mars analogue sites. A developed antibody multiarray competitive immunoassay (MACIA) for the simultaneous detection of compounds of a wide range of molecular sizes or whole spores and cells [2] [3] has revealed as suitable option to achieve this purpose.

The MASE (Mars Analogues for Space Exploration) [1] project is a four-year collaborative research project supported by the European Commission Seventh Framework Contract. The aim of the project is to understand how combined environmental stresses influence the habitability of a number of Mars analogue environments in Europe and beyond. Field sites sampled for MASE include deep subsurface salts at Boulby Mine in the UK, sulfidic springs In Germany and an acidic cold lake in lceland. Samples and isolates have also been provided to the project from acidic deep subsurface environments at the Rio Tinto in Spain as well as permafrost sites in Russian Siberia and northern Canada. Crucial to assessing the habitability of any environmental system to be used as an astrobiological analogue • whether for life in general or, as in this case, for anaerobic microorganisms • is a detailed understanding of their geological and physiochemical context [2, 3]. One of the key outcomes of the MASE project is a comparison and synthesis of just such a set of context data from a varied set of analogue sites, the core of which is presented here, and complemented by an analysis of field samples to detect and quantify amino acids, other organics as well as biologically relevant molecules. We show that anaerobic environments provide some of the best fidelity environments for examining the potential habitability of environments on Mars, which are also anaerobic, but that even these analogue environments show the signatures of Earth's very different geological history, such as high carbon abundance in some environments (from 0.1% in lcelandic lake sediments to 22. 7% in deep .permafrost environments).

Astrobiology seeks to understand the limits of life and to determine the physiology of organisms in order to be able to better assess the potential habitability of other worlds and improve our ability to assay them for the presence of life. To successfully achieve this we require representative microorganisms from environments on Earth that in physical and/or chemical conditions approximate to extraterrestrial environments. The most challenging of these environments with respect to the sample collection and follow on isolation and cultivation of microorganisms are anaerobic environments. Here we describe a systematic approach to this challenge and aim to provide a guideline for future fieldwork and sampling campaigns. We selected a number of anaerobic environments based on characteristics that make them analogous to past and present locations on Mars (Icelandic lakes, sulfidic springs, deep hypersaline environments, acidic iron-rich environments, and permafrost). We implemented a culturing approach to enrich organisms from these environments under anaerobic conditions using a defined medium that would allow for all organisms to be grown under identical culturing conditions m future physiological comparisons. We then isolated anaerobic microorganisms, carried out a study of their basic physiology and deposited these organisms in the DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) culture collection to make them available to astrobiologists and microbiologists. This project represents the first attempt to implement a coordinated effort from the selection of extraterrestrial analog sites through to the isolation and the characterisation of organisms and their deposition within a culture collection.

The search for life beyond Earth is challenging and requires, as a prerequisite, intensive research on microbial life in similar, extreme environments on Earth. Mars analogue sites are characterised by e.g. anoxic conditions, organic compound limitation, low temperatures, high salinity or presence of oxidising compounds, and consequently represent the chemical and physical borders of life as we know it. The analysis of microorganisms withstanding such conditions is embedded in the European Commission-funded MASE (Mars Analogues for Space Exploration; (http://mase.esf.org/) project. Combining a broad spectrum of interdisciplinary expertise, the European project members aim at a better understanding of habitability, microbial lifestyles and biomarker preservation in Mars analogues. For the first time, the selected sites (e.g. salt mine, sulfidic springs) have undergone a profound analysis of their microbial communities on various levels, including vast cultivation of anaerobic microorganisms and molecular screening. In this work, we applied propidium monoazide in order to distinguish between cells with intact membrane (considered as viable) and dead cells on molecular stage, followed by DNA extraction, and amplicon-sequencing of the archaeal and bacterial 16S rRNA genes. The geochemistry of the sites was comprehensively investigated (i.e. elemental analysis, amino acid chirality, minerology), to determine triggers for microbial community composition. We aim to set up a model of potential metabolism reactions based on the different setting conditions and compare it with microbiome data. Consequently, we will obtain insights into the prerequisites of possible extra-terrestrial life forms and into their lifestyles, which may enable them to thrive under most extreme conditions.

During the project MASE (Mars Analogues for Space Exploration) several isolates were obtained and these strains were subjected to Mars relevant environmental stress factors in the laboratory under controlled conditions; e.g. radiation, low water activity, high salt concentrations, oxidizing compounds. First screening steps were always the microorganisms’ tolerance to survive times of desiccation and exposure to ionizing radiation. Both treatments were performed under anoxic conditions.

Assessing the habitability of Mars and detecting life, if it was ever there, depends on knowledge of whether the combined environmental stresses experienced on Mars are compatible with life and whether a record of that life could ever be detected. Many combinations of Mars relevant stress factors, such as high radiation dose rates and high UV uences combined with high salt concentrations, and low water activity, have not been investigated. In particular, the response of anaerobic organisms to Mars-like stress factors and combinations thereof are not known. In the EC project MASE (Mars Analogues for Space Exploration) we address these limitations by characterising different Mars analogue environments on Earth, isolating microorganisms from these sites and exposing them to Mars relevant stress factors alone and in combination. We want to find out, if these bacteria respond in an additive or synergistic way and if they would be able to survive on Mars. So far, eight only distantly related microorganisms are under detailed investigation, e.g Yersinia sp., Halanaerobium sp., Acidiphilum sp. Desulfovibrio sp.. Unexpectedly, a Yersinia strain turned out to be quite resistant, especially against desicca- tion and oxidising compounds, whereas a Desulfovibrio sp. strain exhibit a relatively high radiation resistance. The future experiments aim at the identification of the underlying cellu- lar and molecular mechanisms and the comparison to other new isolates from Mars analogue environments on Earth in the MASE project.

Introduction: The Life Detection System (LDS) is designed as a two modules system for microbial life detection under growing conditions. The microbes growth is followed by redox, pH and conductivity parameters but others parameters can be monitored as well if needed. The experiments presented in this paper follow the physicochemical parameter in a growth culture under fossilization/mineralization-induced process with the objectives of biomarkers detection. The study of biomarkers detection [1] and fossilization process is crucial from an astrobiological point of view for the search for life on Mars as it has been reported that life can survive on Mars surface conditions under protected microniches [2]. At the same time, and using LDS system, we can follow the modification of some parameters on the media that could drive the process.

MASE (Mars Analogues for Space Exploration) is a four year collaborative research project supported by an EC FP7 contract. Its aim is to understand how combined environmental stresses affect the habitability of a number of Mars analogue environments on Earth, specifically for anaerobic organisms. Crucial to assessing the habitability of any environmental system is a detailed understanding of the geological, physiochemical and biological context in which the environment is set. One of the key outcomes of MASE is a comparison and synthesis of just such a collection of context data from a varied set of Mars analogue sites.

© 2015 Kopf et al. Ocean Sampling Day was initiated by the EU-funded Micro B3 (Marine Microbial Biodiversity, Bioinformatics, Biotechnology) project to obtain a snapshot of the marine microbial biodiversity and function of the world's oceans. It is a simultaneous global mega-sequencing campaign aiming to generate the largest standardized microbial data set in a single day. This will be achievable only through the coordinated efforts of an Ocean Sampling Day Consortium, supportive partnerships and networks between sites. This commentary outlines the establishment, function and aims of the Consortium and describes our vision for a sustainable study of marine microbial communities and their embedded functional traits.

Ocean Sampling Day was initiated by the EU-funded Micro B3 (Marine Microbial Biodiversity, Bioinformatics, Biotechnology) project to obtain a snapshot of the marine microbial biodiversity and function of the world’s oceans. It is a simultaneous global mega-sequencing campaign aiming to generate the largest standardized microbial data set in a single day. This will be achievable only through the coordinated efforts of an Ocean Sampling Day Consortium, supportive partnerships and networks between sites. This commentary outlines the establishment, function and aims of the Consortium and describes our vision for a sustainable study of marine microbial communities and their embedded functional traits.

DNA microarrays are currently in use almost exclusively as research tools for gene expression analysis and their application for the identification of organisms is still in its infancy, only documented by a few studies on mammals, bacteria, and viruses. The "Fish & Chips" project aims to demonstrate that DNA chips can be a new innovative tool for the identification of marine animals and phytoplankton. To achieve this goal, fishes, invertebrates, and phytoplankton were sampled in European seas and taxonomically classified. Fragments of their mitochondrial 16S, cyt b, and COI genes and from the nuclear 18S genes were sequenced and served as molecular markers to enable probe design for the microarrays. An on-line data base containing the sequences and all relevant information of the samples has been implemented. A first prototype of a "Fish Chip",is based on more than 400 sequences from the 16S rRNA gene belonging to 46 species. A second microarray prototype serves to identify flatfishes from the North Sea based on COI-and 16S sequences from 70 individuals of 17 fish species. A "Phytoplankton Chip" is now available with probes for all microalgal classes and many toxic species, as well as a "Invertebrate Chip". The results show that this approach is feasible.

High-temperature (250°C) hydrothermal vents and massive anhydrite deposits have been found in a shallow water, sediment-filled graben near 66°36′N in the Tjornes Fracture Zone north of Iceland. The site is located about 30 km offshore, near the small island of Grimsey. The main vent field occurs at a depth of 400 m and consists of about 20 large-diameter (up to 10 m) mounds and 1–3 m chimneys and spires of anhydrite and talc. A north–south alignment of the mounds over a 1-km strike length of the valley floor suggests that their distribution is controlled by a buried fault. Widespread shimmering water and extensive white patches of anhydrite in the sediment between the mounds indicates that the entire 1-km2 area occupied by the vents is thermally active. A 2-man research submersible JAGO was used to map the area and to sample vent waters, gases, and chimneys. Actively boiling hydrothermal vents occur on most of the mounds, and extensive two-phase venting indicates that the field is underlain by a large boiling zone (200×300 m). The presence of boiling fluids in shallow aquifers beneath the deposits was confirmed by sediment coring. The highest-temperature pore fluids were encountered in talc- and anhydrite-rich sedimentary layers that occur up to 7 m below the mounds. Baked muds underlie the talc and anhydrite layers, and pyrite is common in stockwork-like fractures and veins in the hydrothermally altered sediments. However, massive sulfides (pyrite–marcasite crusts) were found in only one relict mound. Subseafloor boiling has likely affected the metal-carrying capacity of the hydrothermal fluids, and deposition of sulfides may be occurring at greater depth. Although the mounds and chimneys at Grimsey resemble other deposits at sedimented ridges (e.g. Middle Valley, Escanaba Trough, Guaymas Basin), the shallow water setting and extensive boiling of the hydrothermal fluids represent a distinctive new type of seafloor hydrothermal system.