Community Research and Development Information Service - CORDIS

FP7

MASE Report Summary

Project ID: 607297
Funded under: FP7-SPACE
Country: France

Final Report Summary - MASE (Mars Analogues for Space Exploration)

Executive Summary:
Was Mars habitable? If it was, what sorts of life could have thrived there and would we ever be able to detect signatures of its presence? The EU MASE (Mars Analogues for Space Exploration) project was established to advance our understanding of the past habitability of Mars and the signatures of life.

The project took a multiple pronged approach to the challenge. Samples were collected from diverse ‘analogue’ field sites across Europe that have similarities to Mars, for example, acidic environments in the Rio Tinto in Spain, cold volcanic environments in Iceland, sulfur-rich springs in Germany and other locations. These samples were used to isolate anaerobic microorganisms. We focused on anaerobic microbes because the vast majority of work has been done with aerobic microbes. However, the atmosphere of Mars is low in oxygen and therefore the most interesting organisms to study with respect to the planet’s habitability are those that grow without oxygen.

The organisms we isolated have been deposited in DSMZ, the German Culture Collection and are now available to the international community. A total of 33 species and 42 enrichment cultures out of over 1500 enrichment cultures obtained in project were studied. Extensive work was undertaken to better understand the response of some of these organisms to simulated Mars conditions s, such as salt, pH, temperature, oxidants and radiation, some of the of extreme conditions on Mars. A detailed investigation of the microbes under individual and combined Mars extreme conditions was carried out. This work significantly advanced our understanding of how anaerobic microbes respond to and grow under Martian conditions and whether some extremes are additive in their effects on life or whether they act against each other. In a nutshell, this work allowed us to better understand the limits to life on Mars. In addition, state-of-the-art studies were carried out using ‘-omics’ technologies to study how microbes respond to salt and radiation stress and the molecular basis of their response.

Our project was also interested in understanding how anaerobic microbes fossilize under Martian conditions. The fossilization of one of our model organisms, Yersinia sp. MASE-LG1, was accomplished with and without radiation stress. Experiments were carried out on ageing of the organisms and the simulation of thermal aging prior to fossilization. During the project, the first fossilization of a community was also undertaken. During the project, early Earth material was investigated to understand the potential of ancient environments on Earth as analogues for the organisms in the fossilised samples. This work has significantly pushed forwards our ability to know how to detect life on Mars and its remnants.

The samples, isolates and fossilised samples were also used to advance life detection. The Signs of Life Immunoassay Detection (SOLID) instrument uses antibodies to detect biomolecules. During the project, the SOLID instrument was used to analyse samples from the field sites. This work allowed us to follow the loss of biosignatures during fossilization and is an important step in using the SOLID instrument in planetary and field samples. The project also developed the DTIVA instrument to follow the changes in metabolic and redox properties of cells in anaerobic conditions. The DTIVA instrument is designed to look for metabolic traces of life. This work constitutes an important development in bringing the DTIVA closer to real deployment in field and planetary situations.

Throughout the project, strong interactions between MASE partners were developed to maximise the outputs from the project. At least ten papers have been published or are nearing completion. The project has ensured that all of its outputs are Open Access. They have been published in journals such as PLoS ONE, Scientific Reports, Astrobiology and others.

The impact of the MASE project has been widespread. The project has made new microorganisms available to the community that can be used for future astrobiology experiments. New insights have been gained into how anaerobic microbes cope with extremes. As well as helping us assess the habitability of Mars, it is noteworthy that some of these microbes such as Yersinia species, have medical interest, allowing our work to achieve translation to new fields. Our work has been used to develop life detection technologies. Throughout the project, the work was disseminated to a wide public audience.
Project Context and Objectives:
The surface of Mars is known to have hosted bodies of liquid water in its past. Since about 3.9 billion years ago the planet has progressively transitioned to a desiccated state on its surface. Today, the lack of liquid water and the high flux of both ultraviolet and ionising radiation renders much of the surface of Mars uninhabitable. However, a crucial question still remains: was Mars habitable in the past and could it even be habitable today beneath its surface? This question strikes at the heart of astrobiology since Mars may hold clues to the origin and evolution of life on our own planet. Even if Mars is eventually found to be lifeless, we are faced with questions about what features of the planet rendered it devoid of life – a lack of an origin of life or something missing for life?

To better understand the habitability of Mars we need to study how organisms that most closely match current and Mars environmental conditions behave under Martian conditions and what their preservation potential is. We also need to link these scientific investigations into developing appropriate life detection technologies that would give us the best chance of detecting that life, if it ever existed.

The EU MASE (Mars Analogues for Space Exploration) project was designed to address these challenges. The project had four broad areas of investigation: 1) to enrich and isolate novel microorganisms from environments similar to environments on Mars, 2) to investigate their response to simulated martian stresses, 3) to study their potential for preservation, 4) to consider and test life detection technologies that could be used to investigate these organisms. These investigations were ordered under six main objectives and eight work packages.

In particular, MASE focused on the study of anaerobic microorganisms (i.e. organisms that grow without oxygen). The vast majority of studies of microorganisms in Mars-like conditions have focused on aerobic microorganisms. However, the surface of Mars today has very low oxygen (0.13%) and the subsurface, like the Earth’s subsurface, is expected to be oxygen-free. Therefore, the most likely organisms to be able to survive and grow on Mars are anaerobes. MASE was focused on carrying out the first systematic study of microbial enrichment and isolation under anaerobic conditions with the objective of comparing organisms between different Mars-like stress conditions. The study of Mars over the last two decades has shown that the planet is not homogeneous. Instead, it has hosted a wide variety of conditions that vary between low and high salt, low and high pH, presence and absence of oxidants. Recognising this variation, the MASE project enriched and isolated organisms from environments that broadly represent the variety of Mars environments, including acidic, neutral pH, salty and freshwater volcanic environments.

Although demonstrating growth and survival under Mars-like conditions is one aspect of the search for life on Mars, another crucial aspect is whether organisms can be preserved. That an environment can sustain life does not imply that the same conditions are conducive to its preservation. To investigate whether microorganisms are likely to be preserved on Mars we need to extend studies of the preservation of anaerobic microorganisms into environments with carbonates, silica, salt conditions and other chemical and physical conditions relevant to the Martian surface. The MASE project accomplished these objectives by using the enriched and isolated anaerobic organisms to study preservation potential.

The context of all of this work is therefore to being a greater congruence of planetary sciences and microbiology, to align the new knowledge of past and present conditions on Mars with new knowledge of the limits and capabilities of anaerobic microorganisms in order to refine our understanding of the habitability of Mars, the potential of the planet to preserve life and enhance our ability to find it.

Objective 1: Collect new samples from analogue sites that have been identified by the project team in order to isolate novel organisms to study effects of analogue conditions.
Objective 1 was focused on the collection of samples from analogue sites that would provide the material for undertaking enrichments and obtaining isolates of anaerobic microorganisms. This objective specifically relates to our objective of using terrestrial space-analogue environments to better understand the limits of life and habitability of extraterrestrial environments. During the project we successfully obtained samples from the originally proposed environments: Boulby Mine (UK), sulfidic springs (Germany), volcanic lakes (Iceland) and Rio Tinto (Spain).

We also successfully gained access to permafrost samples obtained from the Canadian (Herschel Island) and Siberian Arctic (Lena River Delta) to allow us to attempt enrichments from icy anaerobic environments. The team also took part in the AMADEE field campaign which was focused on the study of glacial life in Austria. The latter field site was a target of opportunity and provided the chance to augment our attempts to isolate and obtain samples from cold anaerobic glacial-like environments. Samples from all of these field sites were distributed from WP4 to other work packages to carry out the studies described in this report.

As well as the collection of samples, we also needed to know the environmental context of the sample collection. During the project a full geochemical and amino acid analysis was carried out on the samples and from these data habitability tables were obtained summarising the plausible energy and nutrient sources available for life. These have been used to link the geochemical and microbiology data from the sites and gather the required information for assessing anaerobic sites as Mars analogues.

This objective was primarily undertaken in the framework of WP4 in collaboration with WP5 and 6.

Objective 2: Isolate and characterise anaerobic microorganisms from selected analogue environments that closely match environmental conditions that might have been habitable on early Mars and identify model organisms.
During the course of the project, using samples obtained in Objective 1, we produced over 1500 enrichments across institutions. These enrichments involved the use of Mars-relevant electron donors and acceptors such as PAHs, amino acids etc. From our total enrichments, 33 species and 42 enrichment cultures for further study were obtained. From these sets of enrichments seven model organisms were made available to other work packages e.g. WP6, 7 and 8 in order to allow the workflow as described in the proposal. We also deposited twelve of these organisms in the DSMZ culture collection and these organisms are now available to the wider community. These were Aeromonas sp. (provided by Graz), Acidiphilium sp. (provided by INTA-CAB), SipMet I (provided by Graz), Clostridium sp. (provided by Graz), Trichococcus sp. (provided by Graz), Yersinia sp. (provided by UEDI), and Halanerobium sp. (provided by Graz) at DLR. We also wanted to know how these isolates sat within the wider environmental context of anaerobic enrichments. During the project, we extracted DNA from the environmental samples and phylogenetic analysis of the anaerobic organisms was undertaken using a protocol that allows us to investigate the active members of the community. Thus, as well as defining specific organisms, we have accomplished the first study of the extreme anaerobic microbiome of Mars analogue environments.

Part of the MASE project was to investigate how organisms can adapt to Mars-like stresses. In support of this objective we undertook a whole genome analysis DNA of Halanaerobium sp. and Yersinia sp. MASE-LG1. These genomes have been deposited in public databases and will allow any research group to investigate the genetic basis of extreme adaptations in these organisms.
For all of our strains, work was undertaken to characterise them under basic Mars stresses, such as salt, pH, temperature, oxidants and radiation. This work has allowed for direct intercomparison between the organisms since they have all been isolated using the same basal ‘MASE medium’.

This objective was primarily undertaken in the framework of WP5 and 6.

Objective 3: Study the responses of organisms to realistic combined environmental stresses that might have been experienced in habitable environments on Mars.
During the project, we successfully carried out stress tests on the model organisms selected for MASE (as above). Consistent with the stress plan developed in the early part of the project, we undertook both individual and combined stresses on organisms. Individual stresses were desiccation, temperature, salts and oxidising compounds, ionizing radiation, nutrient limitation and alkali/acid. The combined stresses focused on desiccation and radiation. These stress tests were carried out in detail on Yersinia sp., leading to a separate publication on the response of this model organism to Martian stresses.

We were also able to carry out comprehensive combined stress tests on six model organisms to allow for a comparison of how anaerobic organisms cope with both desiccation and ionising radiation exposure. Other stress tests that we were able to investigate included the effects of nutrient stress on desiccation and irradiation. Given the weak effects of nutrient stress on cell survival it was decided to concentrate on combinations of desiccation and ionizing radiation effects during the project. These studies are the first comprehensive study of the responses of anaerobic organisms to Mars-like stresses.

MASE was also focused on the study of microbial growth and the physiological and metabolic responses of organisms to Mars-like stresses. During the project, we carried out a comprehensive study of the metabolomics responses of Yersinia sp. to the effects of NaCl, MgSO4 and ionising radiation separately and in combination. The rationale for this work was to better understand the metabolic basis of the observed effects of Mars-like stresses on an anaerobic microorganism using two salts prevalent on Mars and their effects in combination with another important Mars stress (radiation). This work constituted the first detailed study of the metabolic responses of an anaerobic microorganism to Mars-like conditions.

This objective was primarily undertaken in the framework of WP5 and 6.

Objective 4: Carry out the artificial fossilisation and aging of stressed cells to study the potential for their preservation in the rock record.
During the project, the enrichments and isolated obtained under Objectives 1 and 2 were successfully used to carry out fossilization experiments. The fossilization of Yersinia sp. MASE-LG1 was accomplished with and without radiation stress. Experiments were also carried out on ageing of the organisms and the simulation of thermal aging prior to fossilization. During the project, the first fossilization of a community was also undertaken.

During the project, archaea material was also investigated to understand the potential of ancient environments on Earth as analogues for the organisms in the fossilised samples. In this approach material from the archean is used to identify biosignatures and morphological remains of life that in themselves are a focus of research output, but can also be used as a comparison with artificial aged and fossilised samples. This work was published during the project.

During MASE, considerable progress was made in developing and implementing analytical methods for studying fossilised and aged samples. Included within this suite of methods were Raman, GCMS and electron microscopy (SEM and TEM). These methods were used to both analyse archean material and also to examine the extent of detectable biosignatures in the artificially fossilised materials.

In addition to the direct study of fossilization, work was also conducted to study how the fossilized cell biosignatures and metabolism changed during the process of preservation to better elaborate the biological processes occurring. This was done by studying the detectability of antibodies to cells and their metabolic activity using instruments developed in WP8.

This objective was primarily undertaken in the framework of WP7 in collaboration with WP5, 6 and 8.

Objective 5: Test technology for life detection approaches on other planetary surfaces.
In this project several technologies were selected for testing life detection. The Signs of Life Immunoassay Detection (SOLID) instrument uses antibodies to detect biomolecules. During the project the SOLID instrument was used to analyse samples from the field sites, particularly Boulby and the sulfidic springs and in collaboration with WP7, it was used to examine the effects of fossilization on biosignature detection using SOLID. This work has allowed us to follow the loss of biosignatures during fossilization and is an important step in using the SOLID instrument in planetary and field samples. The work with the environmental samples also led to development of a new microarray using markers obtained from MASE sites. This work will allow the SOLID instrument to be used to specifically study samples of anaerobic biosignatures.

In collaboration with WP7, MASE also developed the DTIVA instrument to follow the changes in metabolic and redox properties of cells in anaerobic conditions. The DTIVA instrument is designed to look for metabolic traces of life, yet has not been used on natural samples and particularly samples undergoing fossilisation. This work constitutes an important development in bringing the DTIVA closer to real deployment in field and planetary situations.

In addition to SOLID and DTIVA, we have also tested a new method for the study of carbon and other elements in samples from analog environments using laser-induced breakdown spectroscopy. This method allows for the highly accurate and rapid determination of elements within samples. In MASE, we applied the method to the study of our permafrost samples subjected to artificial heating to simulate preservation on Mars and to study whether biosignatures and carbon signatures of life could be detected using the new instrument.

This objective was primarily undertaken in the framework of WP8 in collaboration with WP5, 6 and 7.

Objective 6: Synthesise this knowledge into a new synthetic understanding of the potential for life on Mars and the use of Mars analogue research to advance space exploration
Objective 6 was focused on bringing together the advances and outputs in Objectives 1 to 5 into a coherent whole. We achieved this objective by two primary routes. First, during MASE, strong interactions between work packages were developed that allowed for synthesis. Papers written by the MASE team incorporate synthetic cross-links between projects. Examples include the publication of the anaerobic microbiome of analogue sites (WP5 and 7), the publication looking at fossilization of cells using SOLID (WP7 and 8), the publication examining effects of stress on the metabolome of an anaerobic microorganisms (WP5 and 6). Second, we coordinated the project in such a way as to ensure that organisms selected for study were common to all work packages and agreed by WP leaders to ensure that comparable results could be achieved across the study of stress, preservation and life detection.

Throughout the MASE project, we have recognised that the habitability and the preservation potential of organisms are two different problems. By isolating organisms under Mars relevant conditions, subjecting them to Mars-relevant stresses and then investigating their preservation potential we have, for the first time, integrated the study of anaerobic microbiology to the potential for preservation and thus the detectability of life, which advances space exploration.

MASE has also achieved synthesis by investigating a very wide diversity of stresses and combined stresses relevant to Mars. We now know that Mars has diverse chemical and physical extremes and that no one extreme characterises the whole planet. In this project, we linked anaerobic microbiology to study of stresses that cut across a diversity of potential Martian environments, past and present, this significantly advancing our understanding of what the limits might be to life on Mars and how different stresses might change the potential for life. One significant approach has been to use the same enrichment methods and media for all organisms allowing for a direct comparison of physiological and metabolic responses of diverse organisms to Martian conditions.

This objective was achieved by a synthesis and publication of data across all work packages.

Project Results:
Work Package 4 – Methodology for Sample management and experimental process
1.Brief summary with WP description, partners involved and main achievements
WP description
The overall objective of this WP is to support the campaigns at the selected terrestrial analogue sites to study their geological context, and to acquire pre-screened samples using in-situ portable instruments. Samples from the selected field sites will be analysed in the laboratory to further investigate the mineralogy, organic matter content, and biota using extraction and analytical methods. This approach allows us to obtain new science results through a combination of field research, coordinated multi-instrument data and ground sample analysis. It provides geological and biological context and refines search methodologies and strategies including operational concepts in conditions similar to those expected on distant planets.

Partners involved
Work package leader: UNIVERSITEIT LEIDEN
Partners involved: DLR, UAM, MATIS OHF, UEDIN, INTA, CNRS, NERC and MUG

Main achievements
We characterized new anoxic field sites in UK, Iceland, and Germany, and compared these with isolates and samples provided from permafrost in Canada and Russia alongside isolates from Spain. The aim of this study was both, to investigate these extreme environments on Earth and to characterize their potential as future analogue regions for life detection on Mars. The dataset used for this habitability assessment is comprised of both in-situ measurements and post-sampling laboratory investigations. The different analysis techniques included inductively coupled plasma atomic emission spectroscopy, elemental analyses, total organic carbon analysis, water activity measurement and sequencing. From the multi-site comparison it is evident that anoxic analogue sites can be used to understand the physical and chemical limits to anaerobic life and their relevance to Mars. An important factor which influences habitability is the salinity. We found that in many of these sites there were diverse cations and anions that might not only provide electron donors and acceptors for growth, but also nutrients required for life. Overall, our study showed that anaerobic microbial communities from Mars-analogue settings on Earth possess an impressive machinery to withstand physical and chemical pressures. During the work package implementation successful links were made with most other work packages that have led to publishable results. The WP progressed according to the overall objectives and tasks set out in the original proposal.

2. Summary of activities and progress since the start of the project
Task 4.1 – Field Trips Preparation Documentation
This report package includes a reconnaissance of analogue sites accessed during MASE field campaigns, compiling relevant context information for each site: the Boulby Potash Mine (UK), Grænavatn Lake (Iceland), and Sulfidic Springs, Regensburg (Germany). Parameters including location, infrastructure and logistics, broad site description and geological setting, sampling site description, known microbiology, relevance to Mars, environmental conditions and any available geochemical information has been compiled. Statements on field and lab instrument readiness were also included in this work package. The task was successfully completed within the original timeframe and the results were presented to the EU in D4.1.

Task 4.2 - Boulby Mine Campaign Documentation and Habitability Assessment
Boulby is a potash mine in the north-east of the UK that contains sequences of Permian halite and sulfate deposits and partly anoxic conditions. The Boulby Mine provides an analogue for evaporite deposits on Mars which have been detected in numerous locations. All relevant information about the Boulby Mine field campaign and the sample context was compiled, including an assessment of the habitability. The task was successfully completed within the original timeframe and the results were presented to the EU in D4.2.

Task 4.3 - Iceland Site Campaign Documentation and Habitability Assessment
Grænavatn is an Icelandic “green lake”. As eruption material has been found to overlay glacial moraine, the Grænavatn “maar” likely dates from the early postglacial period. The near-lakeshore environment at Grænavatn is characterised by periodic desiccation and an acidic pH, together with volcanic rock-water interactions. These combined characteristics are similar to Mars conditions. All relevant information about the Iceland field campaign and sample context was compiled, including an assessment of the habitability. The task was successfully completed within the original timeframe and the results were presented to the EU in D4.3.

Task 4.4 - Sippenauer Moor Campaign Documentation and Habitability Assessment
The Sippenauer Moor and Islinger Mühlbach areas are characterised by a main sulfidic spring, emanating into a streamlet where whitish mats of sulfide-oxidizing bacteria cover the submerged surfaces. These aquifers are very similar to sulfidic cave springs that are rich in sulfide, ammonia and sulfate, but poor in dissolved organic carbon, suggesting that the major microbial community of the biotopes are chemolithoautothrophs. All relevant information about the Sippenauer Moor field campaign and the sample context was compiled, including an assessment of the habitability. The task was successfully completed within the original timeframe and the results were presented to the EU in D4.4.

Task 4.5 Documentation on-site context for samples/isolates made available for the MASE project
This deliverable provides environment and context information for samples not collected through MASE field campaigns but made available by partner organisations. It covers four sample strains and three enrichment cultures from boreholes drilled at Rio Tinto (Spain) and 12 permafrost samples from Herschel Island (Canada) and Yedoma (Russia). Exploration by the NASA rover Opportunity has revealed sulfate- and hematite-rich sedimentary rocks exposed in craters and other surface features of Meridiani Planum on Mars. Modern, Holocene and Plio- Pleistocene deposits of the Río Tinto, southwestern Spain, provide at least a partial environmental analogue to Meridiani Planum rocks. Mars is also known to host permafrost under its surface, particularly at high latitudes. If this permafrost has melted in its past, then liquid water-rich subsurface permafrost environments are plausible analogue environments for habitable locations on Mars.
Permafrost is known to host psychrophilic and psychrotolerant organisms with the ability to metabolize in low nutrient environment, survive accumulated damage from inactivity for long periods of time, and tolerate high salt where they have been in contact with cryopegs (regions of salt water inclusions in permafrost). Originally permafrost samples should have been provided from the Arctic, Antarctic and Kamchatka. Corrective actions have been taken to obtain alternative permafrost samples for analyses. Permafrost samples were provided late and this deliverable was therefore submitted in 2015.

Task 4.6 – Permafrost 1 and 2 samples laboratory analysis report
Permafrost samples were collected from two sites: Herschel Island, Canada (Permafrost 1) and the Yedoma in Russian Siberia (Permafrost 2). Herschel is a 15 by 8 km island located in the Beufort Sea off the north coast of Canada’s Yukon territory. The island was formed by glacier thrusting approximately 30 kyr ago. Permafrost on Herschel is composed of perennially frozen ice-rich sediments. The Yedoma is a million km2 region of Pleistocene-age permafrost in the north of Russian Siberia and is characterised by its organic-rich nature (about 2% carbon by mass) with an ice content in the range of 50–90%. The report presents the results of the laboratory analysis performed on the Permafrost 1 and 2 samples. The carbon and nitrogen content of the solid samples was analysed with high temperature combustion elemental analyser. Originally permafrost samples should have been provided from the Arctic, Antarctic and Kamchatka. Corrective actions have been taken to obtain alternative permafrost samples for analyses. The task was successfully completed and the results were presented to the EU in D4.6.

Task 4.7 – Multi-site comparison and synthesis
For the MASE project several analogue environments in Europe were selected according to the following criteria: 1) each site should correspond to a particular putative environment representative of past or present-day Mars; 2) each environment should be limited in nutrients; 3) each site should be anoxic in nature. In this multi-site comparison we provide a synthesis of datasets in order to draw conclusions on the characteristics of the MASE field sites: Boulby Mine (UK), Grænavatn (Iceland), the Sippenauer Moor/Islinger Mühlbach (Germany), samples from Permafrost regions in Canada and Russia, and samples from Rio Tinto (Spain). These sites, when taken in combination, are representative analogues of a range of conditions on Mars, throughout the planet’s history. The dataset used for this habitability assessment is comprised of both in-situ measurements and post-sampling laboratory investigations. Field samples were kept at 4 °C when distributed to partner institutions. While the degree of water activity was relatively constant for most sites, in the range of 0.93–0.96, the measurements for the Boulby sites were in the range of 0.74. A result to be expected when taking into account the site’s salinity. In-situ measurements at the field sites visited by MASE scientists indicated a neutral pH value except for the acidic environment of Grænavatn. The percentage of organic and inorganic carbon and nitrogen for sediment samples was measured and showed that the values of organic carbon is 12–25 times higher than inorganic carbon in all samples, with values from the Yedoma permafrost samples dominating in total organic carbon. However, many of these sites bear the imprint of 2.5 Ga of oxygenic photosynthesis with abundances of organic carbon in excess of expected abundances in Mars environments.

3. Summary of deliverables and milestones
All deliverables and milestones were completed according to the work plan.
D4.1 Field Trips Preparation Documentation [Month 6]
D4.2 Boulby Mine Campaign Documentation and Habitability Assessment [Month 17]
D4.3 Iceland Site Campaign Documentation and Habitability Assessment [Month 19]
D4.4 Sippenauer Moor Campaign Documentation and Habitability Assessment [Month 21]
D4.5 Documentation on-site context for samples/isolates made available for the MASE project [Month 18]
D4.6 Permafrost 1 and 2 samples laboratory analysis report [Month 18]
D4.7 Multi-site comparison and synthesis [Month 46]

MS15 Successful Field Campaign - Boulby Mine, UK [Month 6]
MS16 Successful Field Campaign – Iceland [Month 8]
MS17 Successful Field Campaign - Sippenauer Moor, Germany [Month 10]

4. Significant results
The WP4 of the MASE project provided crucial support to the field campaigns at selected terrestrial analogue sites Boulby Potash Mine (UK), Grænavatn Lake (Iceland), Sulfidic Springs, Regensburg (Germany), as well as permafrost (Canada and Russia) and Rio Tinto isolates (Spain), to study their geological context and/or assist in the sampling (see D4.1-4.6). In the course of the MASE project, environmental parameter and the microbial community of samples from these terrestrial nutrient-poor, anoxic early and present Mars analogue sites, ranging from hypersaline environments, acidic aquatic settings, cold sulfidic springs, to permafrost were analysed (see D4.7). Our understanding of the habitability of Mars is hampered by a lack of knowledge of the stress responses of anaerobic organisms. In this project representative (facultative) anaerobic microorganisms were isolated from the terrestrial MASE field sites and further exposed to stresses typical for the Martian environment. The aim was to gain fundamental insights into the limits of anaerobic microbial life on Earth and to use these data to assess the habitability of Mars. The facultative anaerobic Yersinia strain MASE-LG-1 DSM 102845 was obtained from the Icelandic lake Grænavatn. MASE data showed that anaerobic organisms have the capacity to survive and grow in physical and chemical stresses, imposed individually or in combination that are associated with Martian environments.

Sixty-nine microbial enrichments and 31 isolates were obtained from the MASE sites. In total, 1034 enrichment attempts were performed with varying supplements (95% targeting heterotrophs and 5% targeting autotrophs). In sum 69 enrichments were successfully obtained. Growth was detected in 6.9% of all enrichment attempts targeting heterotrophs and in 1.9% of all enrichments targeting autotrophs. In further purification steps, 31 pure isolates were obtained. Thirty isolates belonged to the domain of the Bacteria (mostly Proteobacteria), and 1 isolate was affiliated to the domain of Archaea (Methanomethylovorans sp. MASE-SM-1). All of them grew under anoxic conditions and required organic medium supplements. The MASE project provides the first comprehensive microbial catalogue and a number of potential model microorganisms of anoxic environments that exhibit a wide range of physical and chemical, astrobiology-relevant extremes, including high salinity, extreme pH and temperature, as well as nutrient depletion.

In summary, WP4 achieved all of its objectives, tasks and milestones ranging from the selection of anoxic analogue field sites, the collection of anoxic samples for the characterization of the whole anaerobic community by sequencing, the enrichment and isolation of anaerobic organisms, their deposition within a culture collection for long-term storage as a pre-requisite for studies in physiology, biochemistry and other investigations.

5. Main difficulties encountered since the start of the project
Deviation from the content of the Research plan
During the first reporting period we had a deviation in the plan when the MASE Team was unable to acquire permafrost samples as originally planned. However, this was corrected in the second reporting period and the samples were acquired from Canada and Russia. Deliverable 4.5 and 4.6 had a short delay at submission.

6. Success indicators and criteria
WP 4 – Methodology for sample management and experimental process. There was one success criterion associated with this work package:
1) The collection of ten samples from each of the field samples to be used in WP5.
This success criterion was achieved (see Tasks 4.2, 4.3, 4.4 and 4.6).


Work Package 5 – Sample strains collection and documentation
1.Brief summary with WP description, partners involved and main achievements
WP description
The overall objective of this WP is to select the organisms to be used to study the effects of extra-terrestrial stress and that will be used in fossilisation and study the survival of biomarkers. This aim will be achieved by first identifying existing model anaerobic organisms from the selected field sites and in culture collections to be used in stress and fossilisation tests. We will enrich new chemoorganotrophic and chemolithotrophic organisms from anaerobic samples collected from the analogue environments -as well as from the permafrost samples provided- and then isolate organisms from these enrichments to be used in the stress and fossilisation tests (in addition to those provided from Rio Tinto). We will then prepare these cultures for the stress and fossilisation tests to be used in follow-on WPs. In addition to collection and isolation of organisms we will also carry out genetic identification of the strains and characterisation of basic physiology. Following stress tests this WP will also carry out analysis of the organisms and their response to stress.

Partners involved
WP leader: UEDIN
Partners involved: DLR, UAM, MATIS OHF, UNIVERSITEIT LEIDEN, INTA, CNRS, NERC and MUG

Main achievements
The WP progressed according to the overall objectives and tasks set out in the original proposal. Over 1,500 enrichments led to the successful acquisition of all model organisms required for the project and all subsequent tasks were successfully achieved using samples acquired with WP4. Objectives and tasks were completed in experiments on basic physiology, metabolic activity and interactions of model microbes, including a pioneering study of the changes in metabolic responses of organisms to extreme conditions (salt and radiation) on Mars (particularly in collaboration with WP6). During the WP implementation successful links were made with other partners that have led to publishable results (WP4, 6, 7 and 8). The central objective of this WP – to deliver organisms for subsequent stress and fossilisation tests was successfully achieved as is apparent from the follow-on success of WP6 and 7. Some small technical deviations in the protocols for implementation were made during the project including late acquisition of permafrost samples, but these alterations were accommodated within WP4 and permafrost samples were successfully acquired and analyzed accordingly. There have been no major disruptions to WP5 implementation according to the protocols and tasks agreed with the EU within the framework of the MASE project.

2. Summary of activities and progress since the start of the project
Task 5.1 Definition of the pre- and post-test analysis protocols
The task was successfully completed within the original timeframe and the results of the task were presented to the EU in D5.1 (Pre- and post-stress analysis protocol).

Task 5.2 Identify and select existing model organisms
The task was successfully completed within the original timeframe and the results of the task were presented to the EU in D5.2 (A collection of existing model organisms from analogue environments). More specifically, the early enrichment of a Yersinia species allowed us to begin stress tests early in the WP timeline prior to selecting other organisms (Task 5.3).

Task 5.3 Enrich and culture available and new model organisms and microbial communities
The task was successfully completed within the original timeframe and the results of the task were presented to the EU in D5.3 (A collection of new samples and isolates for enrichment from analogue sites).

In total over 1.500 inoculations were set up using samples acquired in WP4 using different supplements to enrich for microbial specialists and select for specific metabolisms ranging from PAHs, amino acids, C1 compounds, iron reducers, halophiles and sulphate reducers. Temperature was varied in order to obtain psychrophiles. This task was successfully performed with partners at GRAZ, UEDI, UAM, INTA-CAB and UEDI and lead to the isolation of 33 species and 42 enrichment cultures. Selected isolates (7 model organisms in total) were made available to other WPs e.g. WP6, WP7 and WP8 in order to allow the workflow as described in the proposal.
Within this task there was a delay in obtaining samples from permafrost. However, this problem was solved during the second reporting period and enrichments were successfully established.

Task 5.4 Selection of model organisms
Protocols were successfully identified, documented and agreed early in the project duration (and subsequently used to deliver tasks in WP6 – see WP6).
We successfully fulfilled D5.4, which provided a list of isolates that all members agreed on to be further analyzed in WP 6, 7, and 8. D5.4 also contained first experiments to obtain information on their basic physiological limits. The selection was made according to D5.2 listing the requirements for WP6 experiments. The enrichments for further fossilization were selected based on the criteria defined by WP8 and required additional analysis apart from cell counting as a high phylogenetic diversity was favoured. Denaturing gradient gel electrophoresis (DGGE) was used to characterise these enrichments needed for WP7.
Additionally, the depositing of the newly obtained isolated at the DSMZ was achieved. A total of 12 isolates were deposited with DSMZ and we may continue to deposit isolates beyond the life time of MASE.
All methods used to implement the enrichment and isolation of organisms were published in D5.5 and also in a paper published in the International Journal of Astrobiology.

Task 5.5 Carry out basic physiological tests
Basic physiological tests were carried out according to the protocols stated in D5.4 for the isolates including Yersinia intermedia MASE-LG1, Halanaerobium sp. and Trichocococcus sp. The results obtained from Yersinia intermedia MASE-LG1 strain were included in the publication in collaboration with WP6 concerning stress tests (see Significant Results). Further basic physiology tests with the selected model organisms was undertaken.

Task 5.6 Pre and post-stress analysis
This task was completed with the following strains: Yersinia intermedia MASE-LG1 (provided by Graz), Aeromonas sp. (provided by Graz), Acidiphilium sp. (provided by INTA-CAB), Buttiauxella (provided by Edinburgh), Clostridium sp. (provided by Graz), Trichococcus sp. (provided by Graz) and Halanaerobium sp. (provided by Graz) at DLR (see WP6 summary). Pre- and post-stress analyses have been undertaken with these model organisms. These tests were carried out according to what was agreed in task 5.1 (D5.1).

Consistent with the original proposal the following analysis was undertaken: (i) the determination of growth and reproduction by mainly MPN assay, (ii) the determination of the integrity of cellular components using fluorescence in situ hybridisation for RNA integrity and PMA treatment. (iii) the determination of metabolic products. Additionally API tests were used to characterise the strains. Metabolomics experiments on Yersinia intermedia MASE-LG1. (stressed and unstressed conditions) were successfully undertaken and these data have been accepted for publication, (iv) protein expression via proteomics (see associated investigator section). This experiment was done in collaboration and identical conditions were applied as for metabolomics. This work is still underway and will continue beyond the lifetime of the project, (v) gene expression via transcriptomics. This approach was not undertaken because of time constrains in the project. Instead, we directed our focus at metabolomics which was undertaken as described in Significant Results. (vi) genome sequencing for two to three isolates. In order to enhance the proteome and transcriptome interpretations fully annotated genomes were obtained from three isolates, Halanaerobium sp., Buttiauxella sp. and Yersinia intermedia MASE-LG1. The outcome is explained in more detailed in D5.6.

A total of 25 original samples were subjected to Illumina MISeq amplicon 16S rRNA sequencing for molecular microbial community assessment. This work was accomplished in collaboration with GRAZ (WP6). For amplicon generation, primer sets targeting general Bacteria and in addition specific for Archaea were used. The analysis was performed using R (version 3.2.2) and the packages DADA2. For diversity analysis, classification was carried out using the SILVA database. For functional gene prediction, the dataset was reclassified using the Greengenes database in order to implement them into the workflow of the functional gene prediction tool PICRUSt.

3. Summary of deliverables and milestones
All deliverables and milestones were completed according to the work plan.
D5.1 Pre- and post-stress analysis protocol [Month 3]
D5.2 A collection of existing model organisms from analogue environments [Month 5]
D5.3 A collection of new samples and isolates for enrichment from analogue sites [Month 10]
D5.4 A collection of organisms for stress tests and information on their basic physiological limits [Month 20]
D5.5 Publication of isolation methods and techniques [Month 36]
D5.6 Post-stress test analysis information [Month 46]

MS18 Successful collection of samples from analogue sites [Month 10]
MS19 Successful enrichment of new organisms from analogue samples [Month 12]
MS20 Collection and archiving of isolated organisms from analogue enrichments [Month 20]
MS21 Selection of new model organisms [Month 20]
MS22 Agreement on test plan [Month 3]
MS25 Agreement on fossilisation protocol [Month 6]

4. Significant results
Enrichments and novel organisms
The WP succeeded in isolating the organisms needed for the project. Out of ~1,500 enrichments, most significant was the remarkable number of isolates (a total of 33) and enrichments (summary available in D7.3) obtained and provided by UAM, INTA-CAB, GRAZ, UEDI and MATIS indicating a successful collaboration and task management which provided the baseline for an effective implementation into other WPs e.g. 6, 7, and 8 and led to success in WPs 5, 6, 7 and 8. A diverse set of different model organisms were successfully selected in agreement with the consortium. A subset of those were used for fossilization experiments in WP8. Twelve of these isolates were successfully submitted to DSMZ and are now available to the wider community (DSM 102845, Yersinia aldovae LG-1; DSM 102846, Yersinia enterocolitica ssp. pal. MASE -SM-6; DSM 102847, Citrobacter gillenii MASE-SM-7; DSM 102848, Yersinia enterocolitica ssp. pal. MASE-SM-9; DSM 102849, Yersinia enterocolitica ssp. pal. MASE-SM-10; DSM 1022991, Yersinia massiliensis MASE -SM-8; DSM 103793, Obesumbacterium - like bacterium MASE-JM3 (probably Hafnia alvei); DSM 103794, Obesumbacterium - like bacterium MASE-IM3; DSM 105071, Buttiauxella sp. MASE IM-9; DSM 105632, Trichococcus sp.; DSM 105631, Clostridium sp.; DSM 105537, Halanaerobium sp.). All of the methods developed in MASE and used for the isolation of organisms are detailed in the MASE publication, Cockell et al., 2017).

The novel isolates that we have obtained will be much more useful to the community if their tolerances of Martian stresses can ultimately be linked to their genetic pathways. To advance this work, we carried out whole genome sequencing of Halanaerobium sp., Buttiauxella sp. and Yersinia sp. MASE-LG1. These data have now been uploaded to public databases and can be used by the wider community.

Basic physiological tests and post-stress analysis
Basic physiological tests were successfully completed. The most significant results were obtained from Yersinia intermedia MASE-LG1 and have been written up and accepted for publication in PLoS as the first comprehensive study of an anaerobic microorganism under Mars-simulated stresses in conjunction with WP6. The results of these tests provided the baseline data for determining the experimental conditions for the metabolomics experiments as microbial cells need to remain viable and active growth is required in order to obtain the required cell mass.

In addition to basic characterisation, WP5 also had the responsibility to undertake post-stress analysis. When the MASE project was conceived it was decided to use ‘omics’ technologies to investigate the response of organisms under simulated Martian stresses. We chose to apply metabolomics. Metabolomics experiments were successfully carried out after a test run with high salt samples to confirm the reliability of the results at UEDI. This is also the first metabolomics study that investigated metabolic differences between two salts prevalent on Mars – magnesium sulfate and sodium chloride salt stressed cells. Furthermore, little is known about how radiation affects the metabolites and which metabolites are affected by reduction or accumulation, so we also studied ionizing radiation. The known osmoprotectants were identified as well as other metabolites, providing the first comprehensive overview of the metabolic responses of an anaerobic organism to Mars-relevant stresses. The data is to be published in Frontiers in Microbiology.

The isolates in the context of the anaerobic microbiome
Part of the WP5 objective was to understand how the isolates fitted into the overall anaerobic microbiome. When we chose the sampling sites, we were uncertain whether we would be able to extract enough DNA for further downstream analysis as the environmental conditions were extreme. In some samples salt concentration and pH can lead to difficulties in the DNA isolation process. We encountered several difficulties but we were able to optimize the protocol accordingly so that we were able to obtain results from 16S rRNA sequencing of PMA and non PMA treated samples. Only one sample did not work (Boulby mine) due to limitations in the PMA treatment method with respect to high salt concentrations. This is a significant success. The results are currently in submission from UGRAZ as a lead author.

In addition, functional genes were successfully predicted in order to get greater insights in Mars-relevant pathways present: e.g. methane metabolism, nitrogen metabolism etc. and comparison between PMA treated (viable) fraction and non PMA treated samples can be made.

In summary, WP5 achieved all of its objectives, tasks and milestones with significant outputs that span a range of achievements including: new isolates deposited in an international culture collection, novel genome sequences for the wider community, new insights into basic physiological capabilities of organisms under Mars-relevant stresses, new insights into the anaerobic microbiome of extreme environments, the use of omics technologies to provide the first global insights into the metabolic responses of anaerobic microorganisms to Mars-like environments, new peer-reviewed scientific work making these advances available to the wider community.

5. Main difficulties encountered since the start of the project
5.1 Deviation from the content of the Research plan
During the first reporting period we had a deviation in the plan when we are were unable to acquire permafrost samples. However, this was corrected in the second reporting period and the samples were acquired and used for subsequent culturing.
The other deviation we had was that we were unable to carry out transcriptomics on the samples. This method has a high time effort and cost associated with it. When faced with a choice of ‘omics’ methods, we decided to use metabolomics. The reason for this is that the method is more generic and gives a better idea of the overall global metabolic response of an organism to stress that does not depend on detailed genetic knowledge which we did not have for our novel organisms. Our decision allowed us to focus on the metabolome which has led to a significant publication.

5.2 Deviation from objectives
There were no deviations from the project objectives.

5.3 Deviation from schedule
See 3.1. Otherwise there were no deviations from the project objectives.

6. Success indicators and criteria
WP 5 - Investigation of survivability and adaptability of model organisms to Mars relevant environmental conditions. There were three success criteria associated with this WP:
1) The successful cultivation of 15 enrichment cultures for WP6 (3 per analogue site) and one new model organism from each site.
This success criteria was achieved (see Task 5.1 and 5.2).
2) The identification and provision of three available model organisms to WP6
This success criteria was achieved (see Task 5.3 and 5.4).
3) The testing of at least three parameters (pH, temperature and salinity) on the selected enrichments and model organisms
This success criteria was achieved (see Task 5.5).


Work Package 6 - Investigation of survivability and adaptability of model organisms to Mars relevant environmental conditions
1. Brief summary with WP description, partners involved and main achievements
WP description
To determine the limits of life on Earth and to assess the potential habitability of past and present Mars, selected model organisms, enrichment cultures, and communities as well as fossilized microorganisms from Earth analogue environments will be exposed to different physical and chemical stressors with relevance to Mars. These organism samples from the analogue sites will be identified, characterized and cultured prepared in WP5 and communities obtained from the analogue sites. The physiological potential of the organisms to resist stressors will be tested under standardized conditions in the planetary and space simulation facilities of DLR. The stressors will be applied first alone and then, according to the outcome of the first tests, in further series of experiments in combination.
With this approach the potential synergistic effects of environmental stressors will be analyzed in detail. To ensure that the project begins early, already available isolates from the targeted Earth analogue environments will be tested as pure cultures.
The following environmental parameters are to be tested: while the priority will be on ionizing radiations in form of X-rays, and desiccation, the following environmental parameters are also to be tested: low pressure, simulated Mars atmosphere, varying temperatures (–25°C and +80 °C), salts including oxidizing chemical compounds (perchlorates, hydrogen peroxide), and high / low pH.

Partners involved
WP leader: DLR
Partners involved: UEDIN, UAM, MATIS OHF, UNIVERSITEIT LEIDEN, INTA, CNRS, MUG

Main achievements
The WP progressed according to the overall objectives and tasks set out in the original proposal. Thereby, WP6 was concerned to develop a comprehensive test plan for stress tests, following by an optimization of the sample preparation procedure and the stress tests protocol, to design, manufacture and test an exposure and transport box, and to perform all stress tests with available model organisms.

2. Summary of activities and progress since the start of the project
Task 6.1 Test plan for stress tests
The task was successfully completed within the proposed timeframe and the results of the task were presented to the EU in D6.1 (Test plan for stress tests).

Task 6.2 Optimization of sample preparation
The task was successfully completed within the proposed timeframe and the results of the task were presented to the EU in D6.2 (Test plan for stress tests). The early enrichment of Yersinia intermedia MASE-LG-1 allowed us to begin stress tests early in the WP timeline (Task 6.4).

Task 6.3 Transfer device for anaerobic samples
The task was successfully completed within the proposed timeframe and the results of the task were presented to the EU in D6.3 (Transfer device for anaerobic samples). The description of the transport and exposure box has been included in the publication Beblo-Vranesevic et al. 2017 (PLOSOne 12(10):e0185178).

Task 6.4 Stress test report
The stress tests have been applied successfully, the results were documented and presented to the EU in D6.4 (Stress test report).
The following organisms were used for the stress tests: Acidiphilium sp. PM (DSM 24941), Aeromonas sp. MASE-IM-1, Buttiauxella sp. MASE-IM-9 (DSM 105071), Clostridium sp. MASE-IM-4 (DSM 105631), Halanaerobium sp. MASE-BB-1 (DSM 105537), Methanomethylovorans sp. MASE-SM-1, Methanomethylovorans sp. MASE-SM-1, Tessaracoccus profundi SSRT8, Tessaracoccus lapidicaptus SSRT5, Trichococcus sp. MASE-IM-5 (DSM 105632), Yersinia intermedia MASE-LG-1 (DSM 102845).

Most of the results achieved have been published in two papers and a third publication including the results of the combined stress tests is in preparation.

3. Summary of deliverables and milestones
All deliverables and milestones were completed according to the work plan.
D6.1 Test plan for stress tests [Month 2]
D6.2 Optimization of sample preparation [Month 9]
D6.3 Transfer device for anaerobic samples [Month 9]
D6.4 Stress test report [Month 46]

MS22 Agreement on test plan [Month 3]
MS23 Protocol for sample preparation and analysis [Month 9]
MS24 Completion of transfer device [Month 9]

4. Significant results
A diverse set of model organisms were selected in agreement with the consortium. With these model organisms the stress tests started, but only organisms that showed survival after desiccation and a stable growth were used for further tests.

The most results were obtained from Yersinia intermedia MASE-LG-1 since it was the first isolated strain with a stable and reproducible growth. According to the defined core stresses within T6.1 / T6.3 the following single and combined stress tests were performed with Y. intermedia MASE-LG-1.

The results of these tests provided the baseline data for comparison with the data achieved for the other model organisms.

As a first step the tolerance of the model organism to desiccation and ionizing radiation as single stresses was tested.
The six vegetative strains Acidiphilium sp. PM, Buttiauxella sp. MASE-IM-9, Clostridium sp. MASE-IM-4, Halanaerobium sp. MASE-BB-1, Trichococcus sp. MASE-IM-5, and Y. intermedia MASE-LG-1 showed different survival after desiccation and after exposure to radiation. It shall be noted, that no sporulation was observed microscopically in the cultures of Clostridium sp. MASE-IM-4.
The combination of desiccation and ionizing radiation led to additive effects in all model organisms. The survival was lowered compared to the survival after ionizing radiation alone. Nevertheless, there were some specificities: first, dried Y. intermedia MASE-LG-1 cells seemed to be more tolerant to ionizing radiation than cells which were irradiated in liquid. Second Clostridium sp. MASE-IM-4 and Trichococcus sp. MASE-IM-5 were able to tolerant higher doses of ionizing radiation in a dried form compared to the survival after irradiation in liquid.
In nearly all cases, the application of Martian atmosphere during storage led to an additional lowering of the survival of about one to two orders of magnitude.
We also tested perchlorate salts that are found on Mars. Perchlorates had a significant negative influence on the desiccation tolerance of all model organisms. In the presence of perchlorates, the survival was reduced up to six orders of magnitude more than what was observed after desiccation for 24 h without perchlorates. In the most cases, the reduction in the survival after desiccation was the same for both concentrations (0.5%-1.0%) for each perchlorate.

5. Main difficulties encountered since the start of the project
5.1. Deviation from the content of the Research plan
There were no deviations from the research plan.

5.2. Deviation from objectives
There were no deviations from the project objectives.

5.3. Deviation from schedule
There were no deviations from the project objectives.

6. Success indicators and criteria
WP 6 - Investigation of survivability and adaptability of model organisms to Mars relevant environmental conditions. There was one success criteria associated with this WP:

1) The completion of the stress tests for all of the enrichments and isolates identified in the success criteria for WP5. This success criterion was achieved (see Task 6.4).


Work Package 7 – Fossilisation of organisms and their biosignatures
1. Brief summary with WP description, partners involved and main achievements
WP description
WP7 follows on from WP6 and uses the stressed organisms to investigate their fossilisation potential and to study the way in which stress alters their ability to be fossilised using mineral solutions, such as silica, gypsum and others. MASE strategy is to examine the fossilisation potential of similar organisms exposed to a range of stresses so that comparisons can be made and the effects of growth under different conditions on fossilisation potential determined. Although the project could attempt many types of fossilisation media, the strategy is to use protocols that are likely to represent the chemical conditions for the formation of mineral solutions on Mars. Artificial aging (metamorphism under martian conditions) was used to study how subsequent exposure to pressure and temperature representative of burial of the fossilised organisms under 5 km of Martian sediments and volcanics affects the preservation of biosignatures. These biosignatures of the artificially prepared fossils have been compared to the fossil traces of chemotrophic microorganisms in ancient rocks from the Barberton, South Africa and Pilbara, Australia to gain new insights on how organisms have been fossilised in the natural environment and how they might be fossilised on Mars.

Partners involved
The MASE team for the collection, characterisation, culturing and stressing of the chosen microbial species and communities;
CNRS-CBM-ORLEANS Fréderic Foucher for Raman
CNRS-ISTO-ORLEANS Pascale Gautret (SEM, PIXE), Claude Le Milbeau (GCMS, PYR-GCMS), Rémy Guegan (IR), Rémy Champallier (autoclaves)
AIFIRA-CENBG-Gradignan Stéphanie Sorieul (PIXE)
UNIV. BOLOGNA Barbara Cavalazzi (ancient biosignature characterisation)
Royal Observatory, Brussels, Lena Noak, Elodie Gloessner, Ozgur Karatakin (martian metamorphism models)

Main achievements
1. Successful fossilisation, for up to six months, of Yersinia in silica and gypsum to simulate mineralisation processes on Earth and Early Mars with characterisation of morphological and organochemical biosignatures during the early-late diagenetic phase showing preservation of morphology but changes to the composition of the biomolecules with time. Results published in Gaboyer et al. 2017, Scientific Reports (list below).
2. Successful fossilisation of the Glacier community in silica and a mixture of salts (gypsum and epsomite) to simulate mineralisation processes specifically on Mars in an evaporitic environment. Publication in preparation (Gaboyer et al. list below).
3. Initiation of fossilisation of Halanaerobium sp. MASE-BB-1 and Buxttiauxella sp. MASE-IM-9.
4. Artificial aging under simulated Mars metamorphic conditions of fossilised MASE microorganisms. Further analysis of biosignatures in progress.
5. Confirmation of differing degrees of fossilisation depending on the mineralising medium, with silica demonstrated to be an excellent agent of preservation.
6. Demonstration of the utility of using a number of different techniques (SEM/TEM microscopy, IR and RAMAN spectroscopy, Rock Eval, and GCMS) to document biosignatures in fossilised material.
7. Confirmation that the generic test for biosignatures SOLID (partner CAB) is unable to detect any biosignature in fossilised material.
8. Fundamental characterisation of Mars-analogue volcanic sediments from the Palaeoarchaean of South Africa and their biosignatures. Field study, optical microscopy, Raman spectroscopy and SEM analyses of natural samples have yielded a comprehensive understanding of the geological and mineralogical associations of these carbonaceous biosignatures. Their association with volcanic substrates is clear, as are their growth models i.e. growing around loci of volcanic particles. A publication is in review (Hickman-Lewis et al., Precambrian Research).
9. Successful quantification of the trace element composition of Early Archaean microfossils as a novel biosignature. Publication in preparation.


2. Summary of activities and progress since the start of the project
Task 7.1 Definition of the protocol for fossilisation concerned identifying the optimum protocol for fossilisation. Fossilisation solutions of silica, calcium carbonate, and gypsum were tested. After initial unsuccessful fossilisation tests using CaCO3, further experiments concerned only silica and gypsum. These fossilisation protocols protocols are described in D7.1 and in Gaboyer et al., (2017).

Task 7.2 Selection and fossilisation of controls and of microorganisms exposed to stress tests
(Batch 1). The strain Yersinia was first fossilised in its unstressed form and then, when the cultures stressed under Mars analogue conditions (desiccation and irradiation) were available, in the stressed form. Our results showed silica fossilised cells more rapidly than gypsum. There was no difference in the reaction of stressed or unstressed cells to fossilisation, except that a biomolecule indicative of stress was detected in the stressed cells.
The strains selected for the first fossilisation tests are described in D7.2 and in Gaboyer et al., (2017).

A second batch (Batch 2) for fossilisation consisted of unstressed communities collected from the different sites including As and the Glacier community from the Austrian Alps. These communities were fossilised in silica and an evaporite mixture, with silica producing the most rapidly and successfully fossilised cells. The results are described in D7.3 and a publication is in preparation.

A third batch (Batch 3) consisted of two strains of organisms that were enriched and exposed to the stresses that simulate environmental conditions on Mars (WP5 and WP6). These organisms included Halanaerobium sp. MASE-BB-1 and Buxttiauxella sp. MASE-IM-9. They were fossilised in conditions imitating a hydrothermal environment, i.e. exposed to volcanic particles at 80°C and then fossilised in silica. This task was not able to be completed because the postdoc contract ran out (September 2017) but the samples will be analysed in the future. This task is described in D7.3.

Task 7.3 Artificial aging of the fossilised microorganisms and controls concerns the artificially fossilised model organisms, unstressed communities, and stressed communities. We used a model of metamorphic conditions on Mars (produced by the Royal Observatory of Brussels) to experimentally age. The experiment has been completed but the samples not yet analysed because of expiry of the postdoc contract (September 2017). This task is described in D7.7.

Task 7.4 Analysis of fossilised and aged cells and control communities and detection of their biosignatures. We used a range of techniques to document the changes produced in the fossilised cells, specifically morphological and organochemical. It was clear that, after 1 month of fossilisation, there was not enough carbon to undertake the organochemical analyses. Future experiments of this kind need to ensure adequate initial biomass.
Thus, because of the lack of carbon, it was not possible to undertake the other foreseen analyses (isotopes, elemental composition.
D7.4-7.6 describe the results of the fossilisation experiments of the three batches of microorganisms.

Task 7.5 Report on results of fossilisation and comparison with biosignatures in very ancient rocks. In fact this task was continued throughout the project in parallel with the experimental fossilisation and aging, specifically because of the time consuming nature of the ancient biosignature characterisation. For this, samples of Early Archaean age rocks from Barberton in South Africa and the Pilbara in Australia already available in the collection of F. Westall and collected in 2014 in the context of the MASE project were used. The Early Archaean rocks contain fossilised (silicified) remains of chemotrophic microorganisms that represent excellent analogues for the kind of chemotrophs that could have lived (or still live, in the subsurface) on Mars.
The biosignatures of the Early Archaean chemotrophic colonies were characterised morphologically (optical, SEM and TEM microscopy, by Raman spectroscopy, by TofSIMS for the molecular composition, by SIMS for C isotopes, and by PIXE for elemental composition. Note that the amount of carbon available for analysis was greater than in the artificially fossilised samples but still insufficient for certain analyses, such as in situ isotopes.
A comparison between the naturally fossilised, Early Archaean biosignatures with the artificially-fossilised cultures and communities was more limited than anticipated because of the lack of sufficient carbon in the latter for organo-geochemical analyses and because of the unexpected length of time needed for the experiments (the postdoc contract ran out before the experiments could be terminated. Nevertheless, considering the available information, the artificial fossilisation experiments provide useful information pertaining to early-late diagenetic alteration during fossilisation, phases that the ancient microfossil communities had undergone as well.
These results are described in D7.8 and in Hickman-Lewis et al. (in review) and (in prep.).

3. Summary of deliverables and milestones
All deliverables and milestones were completed according to the work plan.
D7.1 Report on protocols and approaches to be used for fossilisation and list of organisms to be fossilised [Month 3]
This deliverable is a report on protocols to be used for fossilisation based of the results of previous fossilisation experiments with amendments. It had originally been decided to fossilise the model organisms using silica and calcium carbonate but the latter mineral did not produce satisfactory results and, thus silica and gypsum were used, the latter also a mineral of relevance for Mars.
In the course of the experiments, further changes were made to the fossilisation protocol to answer additional questions about fossilisation of Mars. The communities were fossilised in silica and an evaporitic mixture (gypsum and epsomite) to simulate evaporitic conditions. Halanaerobium sp. MASE-BB-1 and Buxttiauxella sp. MASE-IM-9 were exposed at high temperatures (80°C) and a basaltic environment before silicification to simulate hydrothermal environments.

D7.2 List the model organisms that have been selected for the first fossilisation Batch [Month 3]
This deliverable describes the model organism selected for the first fossilisation experiment (Batch 1), i.e. Yersinia.

D7.3 Lists of communities that have been collected for fossilisation in Batch 2 [Month 13]
This deliverable describes the List of communities collected for fossilisation in Batch 2. These include the As community and the Glacier community from the Austrian Alps.

D7.4 Artificially fossilised and aged samples from Batch 1 - Descriptive Report [Month 12]
This deliverable describes the results of the fossilisation of samples from Batch 1, i.e. Yersinia. In this experiment, silica and gypsum were used to fossilise stressed and unstressed cells. There was not difference in the reaction to fossilisation of the stressed and unstressed cells. Cells fossilised with silica were more rapidly encapsulated in the mineral than those fossilised in gypsum. In both cases, the minerals formed deposits around the cells and, in the case of gypsum, the mineral deposited within the cell. A small proportion of viable cells still survived to the end of the experiment, more in the unstressed cells compared to the stressed cells.
Analysis of the organic cell components showed some change in the ratio of different components with time, linked most likely to lysis and degradation of the fossilised cells, but after 1 month of fossilisation there was not sufficient carbon to undertake the analyses.

Since all the aging experiments were conducted together, towards the end of the MASE project, the results are described in Deliverable 7.7.

D7.5 Artificially fossilised and aged samples from Batch 2 - Descriptive Report [Month 23]
This deliverable describes the artificially fossilised and aged samples from Batch 2, the communities of organisms. The results are very similar to results for fossilised Yersinia, silica more rapidly encapsulating the cells than the evaporite mixture. Desiccation produced more significant morphological changes than previously noted.
This deliverable also reports the protocol for the artificial aging experiment based on calculations made by colleagues at the Royal Observatory of Brussels relating to metamorphic conditions on Mars.

D7.6 Artificially fossilised and aged samples from Batch 3 - Descriptive Report [Month 44]
This deliverable describes the artificially samples fossilised from Batch 3, Halanaerobium sp. MASE-BB-1 and Buxttiauxella sp. MASE-IM-9. Owing to delays in the previous experiments, the fossilisation of these samples has only recently commenced. These samples were first exposed to hydrothermal-like conditions (80°C and grains of basalt) and then immersed in a solution of silica. No further studies were possible because of the ending of the postdoc contract (September 2017).

D7.7 Report on results from the analyses performed on fossilised and aged samples [Month 46]
This report results from the analyses performed on fossilised and aged samples. In this report the salient points of the previous fossilisation results are brought together, presenting a summary of the results of fossilisation and aging experiments, with characterisation of the morphological and organo-chemical biosignatures where possible. These experiments were undertaken to simulate the potential fossilisation of Mars-analogue microorganisms and their “aging” (or metamorphism) under one set of potential Mars metamorphic conditions.
The three batches of Mars analogue microorganisms included: Yersinia intermedia MASE-LG-1 strain, a community of microorganisms isolated from the Austrian Alps (Glacier community), and Halanaerobium sp. MASE-BB-1 and Buxttiauxella sp. MASE-IM-9. The microorganisms were fossilised for up to 6 months in mineralising solutions that included silica (very common in hydrothermal environments), calcium carbonate (common on the post Archaean Earth), and calcium sulphate (gypsum, a common secondary deposit on post Noachian Mars). The resulting biosignatures of the fossilised microorganisms were analysed by electron microscopy for their morphological characteristics, IR and RAMAN spectroscopy, Rock Eval and GCMS for the mineralogical and organic compositions.

D7.8 Report on comparison with Early Archaean biosignatures [Month 46]
This is a report on the characteristics of the biosignatures of Early Archaean chemotrophic microorganisms and their comparison with the biosignatures of the artificially fossilised cells. The ancient biosignatures characteristics include cellular and colony morphology, organochemical, geochemical and mineralogical signatures. The artificially fossilised cells represent an early-late diagenetic stage of fossilisation compared to the ancient biosignatures, but demonstrate the feasibility of preserving cellular features and carbon in the geological record.

MS25 Agreement on fossilisation protocol [Month 6]
The initial protocol for fossilisation was established later because of the late recruitment of the postdoc for the project.
MS26 Fossilised and aged samples [Month 38]
The samples processed through articificial fossilisation and ageing were made available in a stepwise manner throughout the project, as soon as they were produced: first Yersinia (2015), then the communities (2016), and now the two enrichments, Halanaerobium sp. MASE-BB-1 and Buxttiauxella sp. MASE-IM-9 (2017).

4. Significant results
Successful fossilisation, for up to six months, of a variety of Mars analogue anaerobic microorganisms including Yersinia intermedia MASE-LG-1 strain, a community of microorganisms isolated from the Austrian Alps (Glacier community), and Halanaerobium sp. MASE-BB-1 and Buxttiauxella sp. MASE-IM-9 in solutions of Mars relevant minerals (silica, gypsum and epsomite) to simulate mineralisation processes on Early Mars. Differing degrees of fossilisation were shown, with silica demonstrated to be an excellent agent of preservation. Publication Gaboyer et al., 2017.

Morphological and geochemical analysis of artificially fossilised model organisms. Analyses were conducted using optical microscopy, confocal laser scanning Raman spectroscopy, scanning electron microscopy, transmission electron microscopy, Rock-Eval, GCMS and particle induced X-ray emission spectroscopy showed differential preservation of cells depending on fossilising mineral and changes to the molecular compositions due to early diagenetic processes.

Mars-analogue volcanic sediments from the Palaeoarchaean of South Africa and their biosignatures have been thoroughly characterised. Field study, optical microscopy, Raman spectroscopy and SEM analyses of natural samples have yielded a comprehensive understanding of the geological and mineralogical associations of these carbonaceous biosignatures. Their association with volcanic substrates is clear, as are their growth models i.e. growing around loci of volcanic particles.

High-resolution geochemical analyses of Palaeoarchaean biosignatures by SEM and PIXE has revealed significant results. Our highest-resolution analyses show that a number of transition metals with known roles in biology seem to show enrichment within certain morphologies of carbonaceous material. Particular enrichments exist in carbonaceous clots. Bio-essential and bio-functional elements (C, N, P, S, K, V, Fe, Co, Ni, Zn and As) concentrated by carbonaceous material in 3.33 Ga cherts support that such morphologies originate from decaying biomass

5. Main difficulties encountered since the start of the project
- Late start of postdoc (September 2014)
- Lack of sufficient starting biomass for the fossilisation experiments to undertake all the biosignature analyses originally planned
- The time-consuming nature of the experiments meant that the tasks were not completed by the end of the project (but they will be carried on in the future)

6. Success indicators and criteria
WP 7 - Fossilisation of organisms and their biosignatures. There were three success criteria associated with this WP:
1) The successful fossilisation and characterisation of two identified model organisms.
One model organism was fossilised, Yersinia (see Task 7.1)
2) The successful fossilisation and characterisation of three new stressed model organisms.
The stressed organism fossilised was Yersinia (See Task 7.2 and 7.3)
3) The successful fossilisation and characterisation of five new stressed enrichments.
Two stressed enrichments were fossilised, Halanaerobium sp. MASE-BB-1 and Buxttiauxella sp. MASE IM-9 (See Task 7.3 and 7.4)


Work Package 8 – Biomarkers and life detection strategies
1. Brief summary with WP description, partners involved and main achievements
WP description
The main objective of this WP is the development of protocols and technology for automated identification of life and/or signs of life. These new protocols and technologies will be tested and implemented in MASE Earth Analogues field sites and on artificial fossils produced in WP7.

Partners involved
WP leader: INTA
Partners involved: UEDIN, DLR, CNRS, UAM

Main achievements
WP8 objective was focused on the deployment of technologies in the field sites for the detection of biomarkers and the assessment of habitability. The WP was centred on three methods for the detection of extant life: i) the detection of ATP, ii) optimisation methods for the extraction of nucleic acids from the very diverse chemical conditions expected in the analogue sites and iii) immunoassay life detection methods by hybridization with antibodies. These methods make assumptions about Martian biochemistry, if it exists, but provide a way to assay for molecules that represent simple molecules (ATP) to complex long-chain molecules (DNA) and therefore may be regarded as model methods for the detection of a diversity of other molecules that could exist. The purpose of this work was to improve approaches and methods for biomarker extraction in samples with low biomass that are analogous to Mars.
Two crucial steps addressed in this WP were miniaturisation and automation. In the time frame of this project we didn´t expect to undertake these optimisations for all technologies that might be deployed on Mars. Our study was focused on antibody detection of biomarkers such as ATP and other cell constituents and molecular biology methods for life detection, including the exploration of novel technologies. These technologies were tested in the field sites we identified in MASE.
As a summary, on WP8 we attempted to investigate and document: 1) how the technologies perform in the environmental conditions of each of our field sites with respect to the physical and chemical conditions to be found in each field site; 2) how the technologies could be improved for robustness in the field sites and with special reference to expected conditions on Mars. This part of the assessment was focused on conditions in the sites relevant to Mars (e.g. acidity in the Rio Tinto, high salt concentrations in the Boulby Mine, low temperatures in Iceland) and how the instruments perform under these conditions, 3) whether the instruments could be left unattended (automation) and what steps would be necessary to advance the instruments so that they could operate in an autonomous mode in the selected environments and ultimately on Mars. A series of milestone and deliverables within this WP were focused on making these assessments individually in each field site.

2. Summary of activities and progress since the start of the project
Task 8.1 Identify and select techniques for identification of life
Task 8.2 Integration of protocols as tools to be tested on field campaigns
Task 8.3 Improvement and miniaturisation of protocols and techniques
Task 8.4 Carry out test on field campaigns

3. Summary of deliverables and milestones
All deliverables and milestones were completed according to the work plan.
D8.1 List of Life detection protocols tested in laboratory [Month 10]
D8.2 Report on the protocols to be used in analogue sites [Month 18]
D8.3 Report on MASE campaigns [Month 30]
D8.4 Report on validation of protocols and techniques to be implemented in analogues sites [Month 36]

MS25 Agreement on fossilisation protocol [Month 6]
MS27 WP8 campaign - Boulby Mine [Month 34]
MS28 WP8 Test Campaign - Sippenauer Moor [Month 36]

4. Significant results
The most significant results got by WP8 are the implementation of the analytical techniques on the two field campaigns held in the MASE Project framework. Anaerobic source in Sipennauer Moor area near Regensburg (Germany) and Boulby Mine (United Kingdom) were the two analogues visited for science work on the MASE context.
The main objective and motivation for these two campaigns was the verification in the implement of improvements proposed in original proposal and tested along the progress of the project. Both sites visited were previously demonstrated as a good interest sites for MASE project since they have Mars analogue conditions. At the same time both sites are completely different in physico-chemical parameters and environmental characteristics what means that both of them are complementary for testing the protocols and tools under different Martian conditions. Sipennauer Moor is liquid anaerobic sulfur driven environment in contrast to Boulby mine, which is salty ecosystem. Salt samples are really difficult to manage for some of the techniques used on the analysis, allowing us to test the protocols and devices under real stressful conditions. Results obtained in the first campaign at Regensburg sample site (before the implementation of improvements mentioned) will be compared to those obtained from this recent campaign. At the same time, and as it was not possible to test the technique directly in the first Boulby campaign, it will be used in situ this time with some improvements already applied on the techniques. Besides, improvements in the processing of salty samples will be included. The comparison of the results before and after the implementation of the improvements allowed us to present conclusions for adapting the techniques to real stressful environments.

Sipennauer Moor anaerobic source field campaign (Regensburg):
This campaign took place from July 13 to 14th 2016 and involved 4 scientists from MASE project: Petra Schwendner, Christine Moissl-Eichinger, Laura García-Descalzo and Felipe Gómez and two collaborators from CAB (Miriam García-Villadangos and Juan Manuel Manchado)

Sampling
Samples were taken under sterile and anaerobic conditions and they were processed within the next hour by SOLID at the Regensburg University facilities due to the weather conditions.

Methodology of the automated technique
Briefly, after loading up to 0.5 g of soil or ground sample into SOLID extraction cell (EC), the instrument was set to run autonomously. SOLID pumps 2.5 mL of extraction-incubation buffer (NaCl 0,3M, Tris-HCl 0,4M pH=8, 0.1 % Tween 20) into the extraction cell, and a piston closes hermetically the EC, which is sealed by a Teflon membrane in the opposite side. The sonicator horn is tight contact to the Teflon membrane, allowing the transmission of the vibrations to the inside of the chamber. After 10 minutes of sonication, a motor valve is opened while the piston pushes downward the liquid extract to the 10µm filter. The filtrate is injected into the SAU, flooding one of the flow cells and contacting with one of the LDChip antibody microarrays. An internal recirculation circuit allows the sample to be re-circulated for up to 1 h in order to enhance the reaction kinetics between target analytes and capturing antibodies on the microarray. Then, the sample is discarded into a waste reservoir, and the microarray cell is washed out with the incubation buffer to remove the non-specifically bound compounds. Then a mixture of fluorescently labeled antibodies, preloaded in a different reservoir, is added to the chamber and incubated for 1 h. A last wash out removes the excess of fluorescent antibodies and leaves the microarray ready for fluorescent detection. A red laser beam excites the bound fluorescent antibodies and the fluorescent signal is captured by a highly sensitive CCD detector and stored as an image file. Fluorescent spots are quantified with GenePix Pro software (Molecular Devices, CA, USA). A blank assay (only buffer instead of sample) is run in one of the flow cells and revealed with the same fluorescent antibodies. The blank image is used as a negative control to subtract spot intensities in the rest of the tested samples. Images, quantification and analysis of the data were performed as described (Parro et al., 2011).

Parro, V., de Diego-Castilla, G., Moreno-Paz, M., Blanco, Y., Cruz-Gil, P., Rodríguez-Manfredi, J.A., Fernández-Remolar, D., Gómez, F., Gómez, M.J., Rivas, L.A., Demergasso, C., Echeverría, A., Urtuvia, V.N., Ruiz-Bermejo, M., García-Villadangos, M., Postigo, M., Sánchez-Román, M., Chong-Díaz, G. and Gómez-Elvira, J. (2011) A microbial oasis in the hypersaline Atacama subsurface discovered by a life detector chip: implications for the search for life on Mars. Astrobiology 11(10): 969-96.

Results
Both, liquid and solid samples were taken from an anaerobic spring in Sippenauer Moore. Liquid samples were tested both directly and concentrated by centrifugation. A replicate of each sample was done.

The subsequent treatment of numeric data obtained from the fluorescence intensity signals will give us more specific conclusions but the comparison of the images of these signals between first assays in Regensburg at December 2014 and same site in July 2016 reveals a clear increase in cleaner assay and the good implementation of the improvements mentioned in previous deliverables.
We expect that further analysis of the data generated will support this statement.

Modifications applied to optimization of results on MASE samples:
- Reduction in labeled Ab concentration 1/3000 – high crosslinking in MASE samples.
- BSA in sample from the origin of protocol (avoiding unspecific links) similar effect as concentration-reduction.
- Modification on the comparison rates (2.5 higher than the control)
- Water samples – increasing concentration till 40 fold.
- Reducing in number of antibodies of chips: change from Chip451 to Chip168.
- Increasing in wash steps between incubation of the sample and incubation with labelled antibodies as well as after this last step.
By including the mentioned readjustments of the technique tested since previous Regensburg campaign and subsequent improvements reached along the progress of the project we can say those have been proved/ verified as successful in view of results obtained.

Boulby Mine field campaign (United Kingdom):
Sampling
The field campaign to Boulby mine was focussed on the testing of technology (several techniques and instruments) for the study of ancient evaporite deposits.
This campaign took place from July 18th to 20th 2016 and involved 18 scientists.
The objective of the campaign was to collect samples cleanly for further investigation whilst at the same time testing technology that could be used to study salt deposits on Mars. The data will be used to write a paper on biosignatures in ancient salt deposits, implications for Mars missions and lessons learned for instrument use in the mining environment.
Small drill holes were done on the polygonal features area for sampling at the surface and subsurface of the features and edges. Samples of salts from the interior and edges of polygons were provided for analysis in the laboratory. We expect to validate improvements mentioned before in the processing and analysis of these salty samples.

Analysis of samples
The overall objective is to determine whether the edges of polygons preferentially contain biosignatures using SOLID instrument (for SOLID methodology see 1. Regensburg field campaign) with some modifications (including the improvements of the methodology for salty samples, see below), or whether they preserve a signature of environmental conditions during the Permian ~250 Myr ago. Preliminary findings showed that biomarkers specific to life could be detected and that salt from the roadways, which was used as a positive control, had a high microbial bioload. The team were able to use their experience in the lab to learn how to optimise the instrument for use in deep subsurface environments and developed the protocols for examining high salt samples. This work will continue at the Spanish Centre for Astrobiology.
As indicated before, slightly modified protocols have been tested for improving results in the signal interference of salt. The best one observed to validate results obtained in field was:
-Enlarging amount of extraction buffer from 1ml to 1,5 ml
- Adding a sonication cycle and increasing the time of each cycle as well (4 sonication cycles instead of 3 and of 60 seconds each one instead of 30).
- Leave the sonicated samples to deposit sediments and filter as much liquid as possible through a 5 microns nylon filter.
It has also been observed as an enhancement to include an extra and longer step of washing after and before the labelled antibodies incubation time.

Results
Two sets of samples were taken from polygons in the two different days of sampling (19th-20th). First data obtained from immunoassays performance in the mine and in the lab.

Several techniques and tools by a number of teams were tested during the campaign and further analyses will be carried out at different laboratories. The involved techniques were: X-ray diffraction, X-ray fluorescence, DNA and aminoacid techniques to detect biosignatures including hopenoids and other cell components, laser ablation instrument to detailed elemental composition analysis, inductively coupled plasma mass spectrometry and spectroscopy of the samples.

All the results produced from these techniques will be written and published in a scientific journal.

DTIVA significant results:
The parameters of conductivity, pH and redox potential have been monitored in MASE cultures of Yersinia sp. using the DTIVA microsensors. The results show a regular modification of the daily cycle in several of the parameters. The adequate controls have been carried out to rule out that this modification could be due to the growth and fossilization of the bacteria and there is no external variable that could influence.

Conclusions
The main conclusions of the field-testing campaigns in a very graphic description are the following:
- The reported capacity to identify specific biomarkers on the sample
- The possibility to study the biomarkers evolution and organics preservation with time and under different stressed conditions, which is being used on fossilization experiments on laboratory batch studies
- The real necessity of a MASE special microarray to be developed for particular epitopes and specific bacterial proteins identification. This microarray will allow us to the identification of representative microorganisms in extreme environments. This MASE microarray is practically developed and it could be used for further fossilization studies in the future
- The technical difficulties on some samples. During the first Regensburg campaign it was pointed out the high fluorescence background on some microarrays and the impossibility of final identification depending on the sample. The reduction on the fluorescence with specific improvements on the protocols for MASE studies was effective. During the second Regensburg campaign the whole microarrays set were optima for total samples identification.
- The technical difficulties on salt samples: The first Boulby samples studied on laboratory were difficult to process for immune assays using SOLID. Similarly to the previous point, several improvements in the protocols were needed. During the second and final Boulby campaign those salty samples were not a problem for biomarkers identification
- Those previous reported improvements on the techniques allow MASE team for further fossilization studies. The comparison of the identified biomarkers before and after fossilization reports organics preservation and evolution of organic matter under harsh conditions.

5. Main difficulties encountered since the start of the project
5.1. Deviation from the content of the Research plan
There were no deviations from the research plan.

5.2. Deviation from objectives
There were no deviations from the project objectives.

5.3. Deviation from schedule
There were no deviations from the project objectives.

6. Success indicators and criteria
WP 8 - Biomarkers and Life detection strategies. There was one success criteria associated with this WP:
1) The implementation of five techniques for the identification of life on: the original
samples, isolates, enrichments and fossilised samples.
Life is a physico-chemical process that use energy gradient for auto-organisation. In this process, on Earth, life leaves tell-tale signals in the substrate that use as energy input and in the atmosphere. The main problem in detecting biomarkers is that the expected signal would probably be weak. Then, we need much specialised techniques to detect unambiguously the presence of biomarkers that would confirm the presence of life. Some work has been developed in the field in order to adapt some protocols for future space missions. The field work was located at the Rio Tinto mouth area in Huelva (Spain). The necessity of developing new microbial life detection devices for astrobiological purposes was shown during those campaigns. The techniques tested at the laboratory for final selection of the definitive techniques to be used on the MASE field sites were:

Raman spectroscopy
ATP as a biomarker of viable microorganisms
DTIVA: Automated tools for microbial life detection
Antibody microarray immunoassays

The techniques identified and selected for further studies on the field sites were:
Antibody microarray immunoassays
DTIVA: Automated tools for microbial life detection

Potential Impact:
The MASE project, in addition to science return, was also focused on producing impact and ensuring potential exploitation. The project was strongly motivated by the writing of peer-reviewed publications, which it has achieved. However, apart from this academic impact, MASE also focused on a number of avenues for ensuring its impact in the wider community and in industry, the latter particularly focused on technology development for future space missions and the development of new knowledge for informing space missions. There were several avenues by which we ensured that this objective was met:

Objective 1. Two of the project work packages were focused on the production of enrichments of anaerobic microbes and then the isolation of microbes needed for the MASE studies. We were keen to ensure that these microbes were available both during and after the MASE project to the international community. One way to do this is to make them available through microbial culture collections. We chose to make our organisms available through DSMZ. The DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) is one of the largest biological resource centers worldwide. Its collections currently comprise more than 50,000 items, including about 27,000 different bacterial and 4,000 fungal strains, 800 human and animal cell lines, 700 plant cell lines, 1,400 plant viruses and antisera, and 13,000 different types of bacterial genomic DNA. All biological materials accepted in the DSMZ collection are subject to extensive quality control and physiological and molecular characterization by our central services. In addition, DSMZ provides an extensive documentation and detailed diagnostic information on the biological materials. The unprecedented diversity and quality management of its bioresources render the DSMZ an internationally known supplier for science, diagnostic laboratories, national reference centers, as well as industrial partners.

To achieve this objective we established and early collaboration with Pakall Rudiger at DSMZ. After basic physiological characterisation of the strains by the MASE partners, the isolates were sent to DSMZ for further characterisation. After this process they have been deposited in DSMZ with DSMX numbers as follows: DSM 102845 Yersinia aldovae LG-1; DSM 102846 Yersinia enterocolitica ssp. pal. MASE -SM-6; DSM 102847 Citrobacter gillenii MASE-SM-7; DSM 102848 Yersinia enterocolitica ssp. pal. MASE-SM-9; DSM 102849 Yersinia enterocolitica ssp. pal. MASE-SM-10; DSM 1022991 Yersinia massiliensis MASE -SM-8; DSM 103793 Obesumbacterium - like bacterium MASE-JM3 (probably Hafnia alvei); DSM 103794 Obesumbacterium - like bacterium MASE-IM3; DSM 105071 Butiauxella sp. MASE IM-9; DSM 105632 Trichococcus sp.; DSM 105631 Clostridium sp.; DSM 105537 Halanaerobium sp.

This is a non-exhaustive list, as there remain some isolates to be sent to DSMZ. Other scientists around the world can now obtain these isolates from DSMZ. One major impact of this approach is that other scientists can make detailed physiological comparisons between these organisms since they have all been isolated using the same MASE medium. For example, in our own studies, six of the isolates were investigated for their radiation and desiccation tolerance. This was made possible because all the isolates had been obtained and grown in the same media. Other scientists will be able to carry out similar astrobiological comparisons using isolates obtained in MASE medium.

Objective 2. One important exploitation objective of MASE is to use the data to improve our ability to know whether biosignatures of life would be preserved on Mars and in what state we might find them. Work package 7 in the MASE project had a very strong element of exploitation. The experiments were focused on investigating the fossilisation of microorganisms in different solution such as silica solutions and sulfate solutions analogous to the types of solutions that might be found on Mars. The results of these experiments are scientific observations, but they also feed directly into space mission exploitation, such as the ExoMars mission. For example, what are the best places to search for life? Given a certain type of rock, such as silica or sulfate, would we expect biosignatures to be preserved? These considerations directly affect a number of mission and practical concerns which include: 1) where should be land to have the best chances of testing for the presence of life?, 2) if certain samples are better for preserving biosignatures, where would we be most likely to find them?, 3) what sorts of instruments do we need to develop to look for biosignatures in different rock types?, 4) what are the mechanisms that determine how biosignatures are preserved in different environments?
These results from MASE will be published in the peer-reviewed literature, but they will also be read and used by those individuals involved in landing site selection and the development of new instruments for future missions.

Objective 3. From the beginning the MASE project was focused on using the field sites and samples we obtained for testing life detection equipment. This was a key goal of the exploitation part of the project. To accomplish this, a dedicated work package (No 8) was implemented to test life detection technologies.
To achieve this, two of the field campaigns during the project were used to test technologies: the anaerobic springs in Sipennauer Moor area near Regensburg (Germany) and Boulby Mine (United Kingdom). The main objective and motivation for these two campaigns was the verification of improvements in the instruments. Both sites are completely different in physico-chemical parameters and environmental characteristics which means that both of them are complementary for testing the protocols and tools under different conditions. Sipennauer Moor is liquid anaerobic sulfur driven environment in contrast to Boulby mine, which is a salty ecosystem. Salt samples are difficult to manage for some of the techniques used in the analysis, allowing us to test the protocols and devices under real conditions. The comparison of the results before and after the implementation of the improvements allowed us to iterate improvements in the equipment.

One of the instruments tested is an immunoassay instrument called SOLID (Sign of Life Immunoassay Detection), which uses antibodies raised against specific molecules from microorganisms to test for life. The instrument was tested in the field campaigns and samples from MASE were used to raise new antibodies for future iterations of the chip.

In addition to the development of immunoassay methods, the project also achieved exploitation of the results in the development of the DTIVA instrument. This is an instrument that tries to detect life by looking at changes in the chemistry of solutions in which life might exist. The parameters of conductivity, pH and redox potential were investigated in MASE cultures of Yersinia sp. using the DTIVA microsensors. The results show a regular modification of the daily cycle in several of the parameters. The adequate controls were carried out to investigate whether observed changes were real or could be attributed to the organisms.

During MASE, we also brought other equipment on line including: X-ray diffraction, X-ray fluorescence, DNA and amino acid techniques to detect biosignatures including hopenoids and other cell components, laser ablation instrument to detailed elemental composition analysis, inductively coupled plasma mass spectrometry and spectroscopy of the samples.

The significance of these outcomes is that anaerobic cultures of microbes have been used to advance our ability to develop technology and instrumentation for future astrobiology missions and life detection.

Objective 4. The MASE project also achieved wider societal impact. The question of whether Mars ever supported life is one of enormous public interest as well as scientific interest. Our project, by investigating the response of microbes to Mars conditions, their preservation potential and developing new instrumentation gathered a great deal of knowledge to inform this search. One spin-out from this work was the societal impact, primarily through our dissemination activities which are described in detail in this section of the report. The dissemination activities not only inform people about the MASE project, but they have also provided a means to education the public about the work carried out in MASE and its implications for the search for life beyond Earth. Often the public has a very positive and sometimes over-positive view of the possibilities for life. However, there are a number of factors that make it difficult to search for life particularly on Mars. First, many environments may not be habitable at all, so they may not contain life. To really determine whether Mars ever supported life we need a more realistic insight into what sorts of organisms might live there. MASE significantly improved our insight into these potential stresses that might prevent life from thriving. Second, even if life did exist on Mars, it is not certain that biosignatures of it remain. This would depend on the rock type and the environments in which it thrived. By looking at preservation in different types of environment, MASE improved our ability to predict where we might look for life.
In summary, MASE was able to provide to the public a better and more realistic insight into the potential for life on Mars and the complications and challenges in finding its remains. This is a significant societal impact since it provides the backdrop for improving our ability to explain Europe’s ambitions in space and can help the public understand why the search for life on Mars is not a trivial undertaking and will take some time to develop.

The overall aim of the Mars Analogues for Space Exploration (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 collaborative, four years research project supported by the European Commission’s FP7 Programme, was kicked-off in January 2014 and a variety of dissemination and communication activities were performed during its life cycle. All MASE communication and dissemination aspects were managed by the European Science Foundation in order to promote the project on the international scientific landscape as to participate to the general public awareness on Mars analogues research and astrobiology. A total of 69 dissemination activities were carried out during the MASE project including a project website, social media platforms, newsletters, an educational booklet, press conferences, attendance to scientific conferences and outreach events, a project workshop and research publications. The main outcomes related to communication and dissemination activities were:
- Definition of the project logo, visual identity and communication tools
The project logo was discussed with all members of the scientific committee and ideas were presented to a graphic designer who suggested several options. Committee members agreed on a logo that showed between brackets the Earth with the planet Mars in the background (See attachment). The acronym of the project is written in the same colour as the planet Mars and the full name of the project is written below the acronym in light grey.

- Development and implementation of a project website
The MASE project website is accessible on http://mase.esf.org/ or http://mase-eu.org since 18 March 2014. It presents the project, its objective and general principles as well as its partners. The website also provide a ‘news’ section with the last updates of the project. The MASE website will remain active until 2020 in order to make publicly available all MASE related publications. Since the beginning of the project, the MASE website has received almost 9200 visits which approximately represents 6.30 visits day/on average, mainly from internet users from United States, Russia, United Kingdom and France.

- Setting up of social media tools
The MASE project @MarsAnalogues was active on two social media platforms: Twitter and Facebook. By the end of the project, the MASE “community page” was followed by a total 304 users, gathered a total of 311 likes, and 17074 people checked out our Facebook updates. The MASE Twitter account generated a total of 251 tweets and it was followed by a total of 196 individuals. Both platforms were regularly updated and Twitter was mainly used during MASE sample campaigns and attendance to conferences.

- Setting up of a professional social media platform
Research Gate is a social networking site for scientists and researchers to share papers, ask and answer questions, and find collaborators. An account to feature the MASE project was created in July 2016, and since then regular updates related to conference papers and research publications were published. By December 2017, the account accumulated a total of 93 followers and research items were read a total of 1379 times. The MASE project on Research Gate will remain active and publications outcome will be accessible through it.

- MASE newsletters
Between 2016-2017 a total of 3 newsletters were produced and disseminated through the MASE website, Facebook, Twitter and European Astrobiology Network Association mailing list. These newsletters provided regular updates on the progress of the MASE project including conferences attended, scientific publications, outreach activities, events of general interest for the astrobiology community, short interviews with ours young investigators and MASE communication channels. A final MASE newsletter is expected to be published in Spring 2018. All MASE newsletters are available in the section News of the MASE website http://mase.esf.org/news.html

- Mars on Earth: Seeking Martian landscapes close to home
The Science Communication and Society MSC Programme at Leiden University in collaboration with the MASE consortium elaborated an educational booklet about how astrobiology research on Earth can help us to search for Martian life. MASE postdoctoral researcher Euan Monaghan and Assistant Professor in Astronomy & Society at the Leiden Observatory Pedro Russo, supervised the work of a 3 master students towards designing it. Hardcopies of the MASE outreach booklet “Mars on Earth: Seeking Martian landscapes close to home” were widely distributed at the scientific institutions of all MASE partners, a variety of scientific events (e.g. EANA and AbSciCon) and publicised online through the MASE website, Facebook and Twitter. The MASE outreach booklet is available in the section News of the MASE website http://mase.esf.org/news/article/mars-on-earth-seeking-martian-landscapes-close-to-home-1169.html

- Setting up of media relation and press conference
An important aspect of the MASE communication strategy was the setting up of a network of science journalists and communication officers. This network was composed of 13 communication professionals and it was activated during major milestones of the project. During the MASE project a total of 3 press conferences were organized. On 4 April 2014, in conjunction with the first sampling campaign in Boulby mine (UK), ESF organised a press conference targeted to its network of science journalists and communication officers. This press event, attended by four journalists, involved a presentation of the MASE project as well as a visit of the Boulby salt mine and the Boulby International Subsurface Astrobiology Laboratory (BISAL). The press conference was preceded by the dissemination of a press release via various media information channels. The press release was echoed on numerous scientific research-related websites and the one of the journalist attending wrote a full article in the online version of the Daily Mail. On 25 November 2015, a second press conference was held at the German Aerospace Center in Köln. The main goal of this press conference was to inform the participants on the importance of ground research facilities to support space exploration in general and astrobiology. Dr. Charles Cockell gave a presentation about Mars analogues environments found on Earth, and this was followed by a second presentation by Dr. Elke Rabbow about planetary simulation facilities. These presentations were followed by questions from the audience and a guided visit to the DLR facilities. Six different media and one freelance journalist registered to attend the press conference. Through the press release and the press conference, MASE received strong media coverage on radio, TV, online platforms and a newspapers. 16 articles were published in German newspapers including three in publications of national relevance altogether. The last MASE press conference took place on 29th March 2017 at the Centre de Biophysique Moleculaire (CBM, CNRS) in Orléans. A total of 10 journalists where gathered to learn more about the MASE project and in particular, the research carried out by the Exobiology team lead by Dr. Frances Westall. The Press Conference took place on in the morning of 29th April 2017 and was broadcasted in the local TV channel of Orléans in the evening and on the MASE Twitter account. Journalists who attended the event had the opportunity to interview MASE scientists Dr. Frances Westall and Dr. Frederic Gaboyer and visit the “lithotheque- rock collection”, the International Space Analogue Rockstore (ISAR) and the analytical platform of Raman spectroscopy and Atomic Force Microscopy. Journalists representing press (Le Figaro, La Recherche, L’Hebdo Orléans, Usbek & Rica, La croix, AFP, Science et Avenir and Futura Sciences), radio (France Bleue Orléans) and TV (France 3 Centre Val de Loire) attended the press conference.

- Presentation of the project to scientific events
Since the beginning of the project in 2014, the MASE team attended regularly scientific conferences across Europe and USA. As result, the MASE research has been presented in the format of oral presentations/posters in a total of 23 international scientific events (See List of Dissemination Activities).

- Project workshop
The project workshop was implemented as a session of the European Astrobiology Network Association (EANA) in August 2017 in Aarhus (Denmark). Initially the plan was to hold a MASE only workshop, but with the EANA conference occurring towards the end of the project it seemed beneficial to hold the workshop as part of EANA so that the wider community could take part in the discussion and see the results of the work accomplished in MASE. In this way, full community discussion and participation in the MASE results could be undertaken. To achieve our goal, a special session of MASE talks was presented on 15 August during EANA. The talks were planned to cover a range of the work packages to give the conference delegates an understanding of the scope of research carried out under MASE and to provide the basis for discussion afterwards. The MASE special session coincided with the EANA poster session in the evening at which it was possible for researchers interested to discuss the work and liaise with scientists involved in MASE. We also ensured that MASE had a number of posters that provided the focal point for continued discussion of the MASE project and the results presented in the afternoon special session. We found this format to be successful since many of the participants at EANA would not normally have attended a specific workshop for anaerobic microbiology. By conducting the workshop in this way, we were able to reach out to the wider astrobiology community and encourage discussion about future astrobiology experiments.

- International relations and the role of Associated Investigators
Creating and maintaining relations and collaborations outside the remit of the current partnership was an important element of the MASE project. To this end the MASE project developed the concept of MASE Associated Investigators (AIs). MASE AI were individual investigators (possibly representing a team/group) from Europe or beyond whose research activities and interest were relevant and potentially complementary to the MASE research plan or MASE resulting initiatives. By the end of the MASE project in December 2017, a total of eight AIs joined the project and their research topics can be consulted in the MASE website.

- MASE Scientific publications
To date the MASE project has produced a total of 8 publications which are publicly available in the MASE website and the MASE Research Gate account. One publication is under review and ten other publications are under preparation (up to February 2018). Research collaborations within the MASE team have also resulted in 6 related publications (See List of Dissemination Activities).

List of Websites:
Website: mase.esf.org

Prof. Charles Cockell: Scientific Coordinator, University of Edinburgh - c.s.cockell@ed.ac.uk
Dr. Petra Rettberg: The German Aerospace Center - petra.rettberg@dlr.de
Prof. Ricardo Amils: Universidad Autonoma de Madrid - ramils@cbm.uam.es
Dr. Viggo thor Marteinsson: MATIS ltd. - viggo@matis.is
Prof. Pascale Ehrenfreund: Leiden Institute of Chemistry - p.ehrenfreund@chem.leidenuniv.nl
Dr. Felipez Gomez Gomez: Instituto Nacional de Tecnica Aeroespacial/Centro de Astrobiologia - gomezgf@cab.inta-csic.es
Dr. Christine Moissl-Eichinger: Medical University of Graz - christine.moissl-eichinger@medunigraz.at
Dr. Frances Westall: Centre National de la Recherche Scientifique - frances.westall@cnrs.fr
Mr. Nicolas Walter: Administrative Coordinator, European Science Foundation - nwalter@esf.org
Dr. Patricia Cabezas: Administrative Coordinator, European Science Foundation - pcabezas@esf.org

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