Selection of Anabaena sp. PCC 7938 as a Cyanobacterium Model for Biological ISRU on Mars.

Crewed missions to Mars are expected to take place in the coming decades. After short-term stays, a permanent presence will be desirable to enable a wealth of scientific discoveries. This will require providing crews with life-support consumables in amounts that are too large to be imported from Earth. Part of these consumables could be produced on site with bioprocesses, but the feedstock should not have to be imported. A solution under consideration lies in using diazotrophic, rock-weathering cyanobacteria as primary producers: fed with materials naturally available on site, they would provide the nutrients required by other organisms. This concept has recently gained momentum but progress is slowed by a lack of consistency across contributing teams, and notably of a shared model organism. With the hope to address this issue, we present the work performed to select our current model. We started with preselected strains from the Nostocaceae family. After sequencing the genome of Anabaena sp. PCC 7938-the only one not yet available-we compared the strains' genomic data to determine their relatedness and provide insights into their physiology. We then assessed and compared relevant features: chiefly, their abilities to utilize nutrients from Martian regolith, their resistance to perchlorates (toxic compounds present in the regolith), and their suitability as feedstock for secondary producers (here a heterotrophic bacterium and a higher plant). This led to the selection of Anabaena sp. PCC 7938, which we propose as a model cyanobacterium for the development of bioprocesses based on Mars's natural resources. IMPORTANCE The sustainability of crewed missions to Mars could be increased by biotechnologies which are connected to resources available on site via primary producers: diazotrophic, rock-leaching cyanobacteria. Indeed, this could greatly reduce the mass of payloads to be imported from Earth. The concept is gaining momentum but progress is hindered by a lack of consistency across research teams. We consequently describe the selection process that led to the choice of our model strain, demonstrate its relevance to the field, and propose it as a shared model organism. We expect this contribution to support the development of cyanobacterium-based biotechnologies on Mars.

Avoidance of protein oxidation correlates with the desiccation and radiation resistance of hot and cold desert strains of the cyanobacterium Chroococcidiopsis.

To investigate the relationship between desiccation and the extent of protein oxidation in desert strains of Chroococcidiopsis a selection of 10 isolates from hot and cold deserts and the terrestrial cyanobacterium Chroococcidiopsis thermalis sp. PCC 7203 were exposed to desiccation (air-drying) and analyzed for survival. Strain CCMEE 029 from the Negev desert and the aquatic cyanobacterium Synechocystis sp. PCC 6803 were further investigated for protein oxidation after desiccation (drying over silica gel), treatment with H2O2 up to 1 M and exposure to γ-rays up to 25 kGy. Then a selection of desert strains of Chroococcidiopsis with different survival rates after prolonged desiccation, as well as Synechocystis sp. PCC 6803 and Chroococcidiopsis thermalis sp. PCC 7203, were analyzed for protein oxidation after treatment with 10 and 100 mM of H2O2. Results suggest that in the investigated strains a tight correlation occurs between desiccation and radiation tolerance and avoidance of protein oxidation.

Preservation of Biomarkers from Cyanobacteria Mixed with Mars-Like Regolith Under Simulated Martian Atmosphere and UV Flux.

The space mission EXPOSE-R2 launched on the 24th of July 2014 to the International Space Station is carrying the BIOMEX (BIOlogy and Mars EXperiment) experiment aimed at investigating the endurance of extremophiles and stability of biomolecules under space and Mars-like conditions. In order to prepare the analyses of the returned samples, ground-based simulations were carried out in Planetary and Space Simulation facilities. During the ground-based simulations, Chroococcidiopsis cells mixed with two Martian mineral analogues (phyllosilicatic and sulfatic Mars regolith simulants) were exposed to a Martian simulated atmosphere combined or not with UV irradiation corresponding to the dose received during a 1-year-exposure in low Earth orbit (or half a Martian year on Mars). Cell survival and preservation of potential biomarkers such as photosynthetic and photoprotective pigments or DNA were assessed by colony forming ability assays, confocal laser scanning microscopy, Raman spectroscopy and PCR-based assays. DNA and photoprotective pigments (carotenoids) were detectable after simulations of the space mission (570 MJ/m(2) of UV 200-400 nm irradiation and Martian simulated atmosphere), even though signals were attenuated by the treatment. The fluorescence signal from photosynthetic pigments was differently preserved after UV irradiation, depending on the thickness of the samples. UV irradiation caused a high background fluorescence of the Martian mineral analogues, as revealed by Raman spectroscopy. Further investigation will be needed to ensure unambiguous identification and operations of future Mars missions. However, a 3-month exposure to a Martian simulated atmosphere showed no significant damaging effect on the tested cyanobacterial biosignatures, pointing out the relevance of the latter for future investigations after the EXPOSE-R2 mission. Data gathered during the ground-based simulations will contribute to interpret results from space experiments and guide our search for life on Mars.

Detection of macromolecules in desert cyanobacteria mixed with a lunar mineral analogue after space simulations.

In the context of future exposure missions in Low Earth Orbit and possibly on the Moon, two desert strains of the cyanobacterium Chroococcidiopsis, strains CCMEE 029 and 057, mixed or not with a lunar mineral analogue, were exposed to fractionated fluencies of UVC and polychromatic UV (200-400 nm) and to space vacuum. These experiments were carried out within the framework of the BIOMEX (BIOlogy and Mars EXperiment) project, which aims at broadening our knowledge of mineral-microorganism interaction and the stability/degradation of their macromolecules when exposed to space and simulated Martian conditions. The presence of mineral analogues provided a protective effect, preserving survivability and integrity of DNA and photosynthetic pigments, as revealed by testing colony-forming abilities, performing PCR-based assays and using confocal laser scanning microscopy. In particular, DNA and pigments were still detectable after 500 kJ/m(2) of polychromatic UV and space vacuum (10(-4) Pa), corresponding to conditions expected during one-year exposure in Low Earth Orbit on board the EXPOSE-R2 platform in the presence of 0.1 % Neutral Density (ND) filter. After exposure to high UV fluencies (800 MJ/m(2)) in the presence of minerals, however, altered fluorescence emission spectrum of the photosynthetic pigments were detected, whereas DNA was still amplified by PCR. The present paper considers the implications of such findings for the detection of biosignatures in extraterrestrial conditions and for putative future lunar missions.

Dependence of cyanobacterium growth and Mars-specific photobioreactor mass on total pressure, pN2 and pCO2.

In situ resource utilization systems based on cyanobacteria could support the sustainability of crewed missions to Mars. However, their resource-efficiency will depend on the extent to which gases from the Martian atmosphere must be processed to support cyanobacterial growth. The main purpose of the present work is to help assess this extent. We therefore start with investigating the impact of changes in atmospheric conditions on the photoautotrophic, diazotrophic growth of the cyanobacterium Anabaena sp. PCC 7938. We show that lowering atmospheric pressure from 1 bar down to 80 hPa, without changing the partial pressures of metabolizable gases, does not reduce growth rates. We also provide equations, analogous to Monod's, that describe the dependence of growth rates on the partial pressures of CO2 and N2. We then outline the relationships between atmospheric pressure and composition, the minimal mass of a photobioreactor's outer walls (which is dependent on the inner-outer pressure difference), and growth rates. Relying on these relationships, we demonstrate that the structural mass of a photobioreactor can be decreased - without affecting cyanobacterial productivity - by reducing the inner gas pressure. We argue, however, that this reduction would be small next to the equivalent system mass of the cultivation system. A greater impact on resource-efficiency could come from the selection of atmospheric conditions which minimize gas processing requirements while adequately supporting cyanobacterial growth. The data and equations we provide can help identify these conditions.

Cultivation and nutritional characteristics of Chlorella vulgaris cultivated using Martian regolith and synthetic urine.

Long-term spatial missions will require sustainable methods for biomass production using locally available resources. This study investigates the feasibility of cultivating Chlorella vulgaris, a high value microalgal specie, using a leachate of Martian regolith and synthetic human urine as nutrient sources. The microalga was grown in a standard medium (BBM) mixed with 0, 20, 40, 60, or 100 % Martian medium (MM). MM did not significantly affect final biomass concentrations. Total carbohydrate and protein contents decreased with increasing MM fractions between 0 % and 60 %, but biomass in the 100% MM showed the highest levels of carbohydrates and proteins (25.2 ± 0.9 % and 37.1 ± 1.4 % of the dry weight, respectively, against 19.0 ± 1.7 % and 32.0 ± 2.7 % in the absence of MM). In all MM-containing media, the fraction of the biomass represented by total lipids was lower (by 3.2 to 4.5%) when compared to BBM. Conversely, total carotenoids increased, with the highest value (97.3 ± 1.5 mg/100 g) measured with 20% MM. In a three-dimensional principal component analysis of triacylglycerols, samples clustered according to growth media; a strong impact of growth media on triacylglycerol profiles was observed. Overall, our findings suggest that microalgal biomass produced using regolith and urine can be used as a valuable component of astronauts' diet during missions to Mars.

The MaMBA facility as a testbed for bioregenerative life support systems.

The Moon and Mars Base Analog (MaMBA) is a concept for an extraterrestrial habitat developed at the Center of Applied Space Technology and Microgravity (ZARM) in Bremen, Germany. The long-term goal of the associated project is to create a technologically functioning prototype for a base on the Moon and on Mars. One key aspect of developing such a prototype base is the integration of a bioregenerative life support system (BLSS) and its testing under realistic conditions. A long-duration mission to Mars, in particular, will require BLSS with a reliability that can hardly be reached without extensive testing, starting well in advance of the mission. Standards exist for comparing the capabilities of various BLSS, which strongly focus on technological aspects. These, we argue, should be complemented with the use of facilities that enable investigations and optimization of BLSS prototypes with regard to their requirements on logistics, training, recovery from failure and contamination, and other constraints imposed when humans are in the loop. Such facilities, however, are lacking. The purpose of this paper is to present the MaMBA facility and its potential usages that may help close this gap. We describe how a BLSS (or parts of a BLSS) can be integrated into the current existing mock-up at the ZARM for relatively low-cost investigations of human factors affecting the BLSS. The MaMBA facility is available through collaborations as a test platform for characterizing, benchmarking, and testing BLSS under nominal and off-nominal conditions.

An airlock concept to reduce contamination risks during the human exploration of Mars.

Protecting the Martian environment from contamination with terrestrial microbes is generally seen as essential to the scientific exploration of Mars, especially when it comes to the search for indigenous life. However, while companies and space agencies aim at getting to Mars within ambitious timelines, the state-of-the-art planetary protection measures are only applicable to uncrewed spacecraft. With this paper, we attempt to reconcile these two conflicting goals: the human exploration of Mars and its protection from biological contamination. In our view, the one nominal mission activity that is most prone to introducing terrestrial microbes into the Martian environment is when humans leave their habitat to explore the Martian surface, if one were to use state-of-the-art airlocks. We therefore propose to adapt airlocks specifically to the goals of planetary protection. We suggest a concrete concept for such an adapted airlock, believing that only practical and implementable solutions will be followed by human explorers in the long run.

Plant and microbial science and technology as cornerstones to Bioregenerative Life Support Systems in space.

Long-term human space exploration missions require environmental control and closed Life Support Systems (LSS) capable of producing and recycling resources, thus fulfilling all the essential metabolic needs for human survival in harsh space environments, both during travel and on orbital/planetary stations. This will become increasingly necessary as missions reach farther away from Earth, thereby limiting the technical and economic feasibility of resupplying resources from Earth. Further incorporation of biological elements into state-of-the-art (mostly abiotic) LSS, leading to bioregenerative LSS (BLSS), is needed for additional resource recovery, food production, and waste treatment solutions, and to enable more self-sustainable missions to the Moon and Mars. There is a whole suite of functions crucial to sustain human presence in Low Earth Orbit (LEO) and successful settlement on Moon or Mars such as environmental control, air regeneration, waste management, water supply, food production, cabin/habitat pressurization, radiation protection, energy supply, and means for transportation, communication, and recreation. In this paper, we focus on air, water and food production, and waste management, and address some aspects of radiation protection and recreation. We briefly discuss existing knowledge, highlight open gaps, and propose possible future experiments in the short-, medium-, and long-term to achieve the targets of crewed space exploration also leading to possible benefits on Earth.

On the growth dynamics of the cyanobacterium Anabaena sp. PCC 7938 in Martian regolith.

The sustainability of crewed infrastructures on Mars will depend on their abilities to produce consumables on site. These abilities may be supported by diazotrophic, rock-leaching cyanobacteria: from resources naturally available on Mars, they could feed downstream biological processes and lead to the production of oxygen, food, fuels, structural materials, pharmaceuticals and more. The relevance of such a system will be dictated largely by the efficiency of regolith utilization by cyanobacteria. We therefore describe the growth dynamics of Anabaena sp. PCC 7938 as a function of MGS-1 concentration (a simulant of a widespread type of Martian regolith), of perchlorate concentration, and of their combination. To help devise improvement strategies and predict dynamics in regolith of differing composition, we identify the limiting element in MGS-1 - phosphorus - and its concentration-dependent effect on growth. Finally, we show that, while maintaining cyanobacteria and regolith in a single compartment can make the design of cultivation processes challenging, preventing direct physical contact between cells and grains may reduce growth. Overall, we hope for the knowledge gained here to support both the design of cultivation hardware and the modeling of cyanobacterium growth within.

Biosignature stability in space enables their use for life detection on Mars.

Two rover missions to Mars aim to detect biomolecules as a sign of extinct or extant life with, among other instruments, Raman spectrometers. However, there are many unknowns about the stability of Raman-detectable biomolecules in the martian environment, clouding the interpretation of the results. To quantify Raman-detectable biomolecule stability, we exposed seven biomolecules for 469 days to a simulated martian environment outside the International Space Station. Ultraviolet radiation (UVR) strongly changed the Raman spectra signals, but only minor change was observed when samples were shielded from UVR. These findings provide support for Mars mission operations searching for biosignatures in the subsurface. This experiment demonstrates the detectability of biomolecules by Raman spectroscopy in Mars regolith analogs after space exposure and lays the groundwork for a consolidated space-proven database of spectroscopy biosignatures in targeted environments.

A Low-Pressure, N2/CO2 Atmosphere Is Suitable for Cyanobacterium-Based Life-Support Systems on Mars.

The leading space agencies aim for crewed missions to Mars in the coming decades. Among the associated challenges is the need to provide astronauts with life-support consumables and, for a Mars exploration program to be sustainable, most of those consumables should be generated on site. Research is being done to achieve this using cyanobacteria: fed from Mars's regolith and atmosphere, they would serve as a basis for biological life-support systems that rely on local materials. Efficiency will largely depend on cyanobacteria's behavior under artificial atmospheres: a compromise is needed between conditions that would be desirable from a purely engineering and logistical standpoint (by being close to conditions found on the Martian surface) and conditions that optimize cyanobacterial productivity. To help identify this compromise, we developed a low-pressure photobioreactor, dubbed Atmos, that can provide tightly regulated atmospheric conditions to nine cultivation chambers. We used it to study the effects of a 96% N2, 4% CO2 gas mixture at a total pressure of 100 hPa on Anabaena sp. PCC 7938. We showed that those atmospheric conditions (referred to as MDA-1) can support the vigorous autotrophic, diazotrophic growth of cyanobacteria. We found that MDA-1 did not prevent Anabaena sp. from using an analog of Martian regolith (MGS-1) as a nutrient source. Finally, we demonstrated that cyanobacterial biomass grown under MDA-1 could be used for feeding secondary consumers (here, the heterotrophic bacterium E. coli W). Taken as a whole, our results suggest that a mixture of gases extracted from the Martian atmosphere, brought to approximately one tenth of Earth's pressure at sea level, would be suitable for photobioreactor modules of cyanobacterium-based life-support systems. This finding could greatly enhance the viability of such systems on Mars.

Microbiome dynamics during the HI-SEAS IV mission, and implications for future crewed missions beyond Earth.

BACKGROUND: Human health is closely interconnected with its microbiome. Resilient microbiomes in, on, and around the human body will be key for safe and successful long-term space travel. However, longitudinal dynamics of microbiomes inside confined built environments are still poorly understood. Herein, we used the Hawaii Space Exploration Analog and Simulation IV (HI-SEAS IV) mission, a 1 year-long isolation study, to investigate microbial transfer between crew and habitat, in order to understand adverse developments which may occur in a future outpost on the Moon or Mars. RESULTS: Longitudinal 16S rRNA gene profiles, as well as quantitative observations, revealed significant differences in microbial diversity, abundance, and composition between samples of the built environment and its crew. The microbiome composition and diversity associated with abiotic surfaces was found to be rather stable, whereas the microbial skin profiles of individual crew members were highly dynamic, resulting in an increased microbiome diversity at the end of the isolation period. The skin microbiome dynamics were especially pronounced by a regular transfer of the indicator species Methanobrevibacter between crew members within the first 200 days. Quantitative information was used to track the propagation of antimicrobial resistance in the habitat. Together with functional and phenotypic predictions, quantitative and qualitative data supported the observation of a delayed longitudinal microbial homogenization between crew and habitat surfaces which was mainly caused by a malfunctioning sanitary facility. CONCLUSIONS: This study highlights main routes of microbial transfer, interaction of the crew, and origins of microbial dynamics in an isolated environment. We identify key targets of microbial monitoring, and emphasize the need for defined baselines of microbiome diversity and abundance on surfaces and crew skin. Targeted manipulation to counteract adverse developments of the microbiome could be a highly important strategy to ensure safety during future space endeavors. Video abstract.

A Desert Cyanobacterium under Simulated Mars-like Conditions in Low Earth Orbit: Implications for the Habitability of Mars.

In the ESA space experiment BIOMEX (BIOlogy and Mars EXperiment), dried Chroococcidiopsis cells were exposed to Mars-like conditions during the EXPOSE-R2 mission on the International Space Station. The samples were exposed to UV radiation for 469 days and to a Mars-like atmosphere for 722 days, approaching the conditions that could be faced on the surface of Mars. Once back on Earth, cell survival was tested by growth-dependent assays, while confocal laser scanning microscopy and PCR-based assay were used to analyze the accumulated damage in photosynthetic pigments (chlorophyll a and phycobiliproteins) and genomic DNA, respectively. Survival occurred only for dried cells (4-5 cell layers thick) mixed with the martian soil simulants P-MRS (phyllosilicatic martian regolith simulant) and S-MRS (sulfatic martian regolith simulant), and viability was only maintained for a few hours after space exposure to a total UV (wavelength from 200 to 400 nm) radiation dose of 492 MJ/m2 (attenuated by 0.1% neutral density filters) and 0.5 Gy of ionizing radiation. These results have implications for the hypothesis that, during Mars's climatic history, desiccation- and radiation-tolerant life-forms could have survived in habitable niches and protected niches while transported.

Dried Biofilms of Desert Strains of Chroococcidiopsis Survived Prolonged Exposure to Space and Mars-like Conditions in Low Earth Orbit.

Dried biofilms and dried multilayered planktonic counterparts obtained from three desert strains of Chroococcidiopsis were exposed to low Earth conditions by using the EXPOSE-R2 facility outside the International Space Station. During the space mission, samples in Tray 1 (space vacuum and solar radiation, from λ ≈ 110 nm) and Tray 2 (Mars-like UV flux, λ > 200 nm and Mars-like atmosphere) received total UV (200-400 nm) fluences of about 4.58 × 102 kJ/m2 and 4.92 × 102 kJ/m2, respectively, and 0.5 Gy of cosmic ionizing radiation. Postflight analyses were performed on 2.5-year-old samples due to the space mission duration, from launch to sample return to the lab. The occurrence of survivors was determined by evaluating cell division upon rehydration and damage to the genome and photosynthetic apparatus by polymerase chain reaction-stop assays and confocal laser scanning microscopy. Biofilms recovered better than their planktonic counterparts, accumulating less damage not only when exposed to UV radiation under space and Mars-like conditions but also when exposed in dark conditions to low Earth conditions and laboratory control conditions. This suggests that, despite the shielding provided by top-cell layers being sufficient for a certain degree of survival of the multilayered planktonic samples, the enhanced survival of biofilms was due to the presence of abundant extracellular polymeric substances and to additional features acquired upon drying.

Evaluation of the Resistance of Chroococcidiopsis spp. to Sparsely and Densely Ionizing Irradiation.

Studying the resistance of cyanobacteria to ionizing radiation provides relevant information regarding astrobiology-related topics including the search for life on Mars, lithopanspermia, and biological life-support systems. Here, we report on the resistance of desert cyanobacteria of the genus Chroococcidiopsis, which were exposed (as part of the STARLIFE series of experiments) in both hydrated and dried states to ionizing radiation with different linear energy transfer values (0.2 to 200 keV/µm). Irradiation with up to 1 kGy of He or Si ions, 2 kGy of Fe ions, 5 kGy of X-rays, or 11.59 kGy of γ rays (60Co) did not eradicate Chroococcidiopsis populations, nor did it induce detectable damage to DNA or plasma membranes. The relevance of these results for astrobiology is briefly discussed. Key Words: Ionizing radiation-Linear energy transfer-Lithopanspermia-Cyanobacterial radioresistance-Chroococcidiopsis-Mars. Astrobiology 17, 118-125.

A first step toward liposome-mediated intracellular bacteriophage therapy.

OBJECTIVES: The emergence of antibiotic-resistant bacteria presents a severe challenge to medicine and public health. While bacteriophage therapy is a promising alternative to traditional antibiotics, the general inability of bacteriophages to penetrate eukaryotic cells limits their use against resistant bacteria, causing intracellular diseases like tuberculosis. Bacterial vectors show some promise in carrying therapeutic bacteriophages into cells, but also bring a number of risks like an overload of bacterial antigens or the acquisition of virulence genes from the pathogen. METHODS: As a first step in the development of a non-bacterial vector for bacteriophage delivery into pathogen-infected cells, we attempted to encapsulate bacteriophages into liposomes. RESULTS: Here we report effective encapsulation of the model bacteriophage λeyfp and the mycobacteriophage TM4 into giant liposomes. Furthermore, we show that liposome-associated bacteriophages are taken up into eukaryotic cells more efficiently than free bacteriophages. CONCLUSION: These are important milestones in the development of an intracellular bacteriophage therapy that might be useful in the fight against multi-drug-resistant intracellular pathogens like Mycobacterium tuberculosis.