Activity of methanogenic archaea under simulated Mars analog conditions

Assumed comparable environmental conditions of early Mars and early Earth in 3.7 Ga ago – at a time when first fossil records of life on Earth could be found – suggest the possibility of life emerging on both planets in parallel. As conditions changed, the hypothetical life on Mars either became extinct or was able to adapt and might still exist in biological niches. The controversial discussed detection of methane on Mars led to the assumption, that it must have a recent origin – either abiotic through active volcanism or chemical processes, or through biogenic production. Spatial and seasonal variations in the detected methane concentrations and correlations between the presence of water vapor and geological features such as subsurface hydrogen, which are occurring together with locally increased detected concentrations of methane, gave fuel to the hypothesis of a possible biological source of the methane on Mars. Therefore the phylogenetically old methanogenic archaea, which have evolved under early Earth conditions, are often used as model-organisms in astrobiological studies to investigate the potential of life to exist in possible extraterrestrial habitats on our neighboring planet. In this thesis methanogenic archaea originating from two extreme environments on Earth were investigated to test their ability to be active under simulated Mars analog conditions. These extreme environments – the Siberian permafrost-affected soil and the chemoautotrophically based terrestrial ecosystem of Movile cave, Romania – are regarded as analogs for possible Martian (subsurface) habitats. Two novel species of methanogenic archaea isolated from these environments were described within the frame of this thesis. It could be shown that concentrations up to 1 wt% of Mars regolith analogs added to the growth media had a positive influence on the methane production rates of the tested methanogenic archaea, whereas higher concentrations resulted in decreasing rates. Nevertheless it was possible for the organisms to metabolize when incubated on water-saturated soil matrixes made of Mars regolith analogs without any additional nutrients. Long-term desiccation resistance of more than 400 days was proven with reincubation and indirect counting of viable cells through a combined treatment with propidium monoazide (to inactivate DNA of destroyed cells) and quantitative PCR. Phyllosilicate rich regolith analogs seem to be the best soil mixtures for the tested methanogenic archaea to be active under Mars analog conditions. Furthermore, in a simulation chamber experiment the activity of the permafrost methanogen strain Methanosarcina soligelidi SMA-21 under Mars subsurface analog conditions could be proven. Through real-time wavelength modulation spectroscopy measurements the increase in the methane concentration at temperatures down to -5 °C could be detected. The results presented in this thesis contribute to the understanding of the activity potential of methanogenic archaea under Mars analog conditions and therefore provide insights to the possible habitability of present-day Mars (near) subsurface environments. Thus, it contributes also to the data interpretation of future life detection missions on that planet. For example the ExoMars mission of the European Space Agency (ESA) and Roscosmos which is planned to be launched in 2018 and is aiming to drill in the Martian subsurface

Bacterial Growth in Chloride and Perchlorate Brines: Halotolerances and Salt Stress Responses of Planococcus halocryophilus

Extraterrestrial environments encompass physicochemical conditions and habitats that are unknown on Earth, such as perchlorate-rich brines that can be at least temporarily stable on the martian surface. To better understand the potential for life in these cold briny environments, we determined the maximum salt concentrations suitable for growth (MSCg) of six different chloride and perchlorate salts at 25°C and 4°C for the extremotolerant cold- and salt-adapted bacterial strain Planococcus halocryophilus. Growth was measured through colony-forming unit (CFU) counts, while cellular and colonial phenotypic stress responses were observed through visible light, fluorescence, and scanning electron microscopy. Our data show the following: (1) The tolerance to high salt concentrations can be increased through a stepwise inoculation toward higher concentrations. (2) Ion-specific factors are more relevant for the growth limitation of P. halocryophilus in saline solutions than single physicochemical parameters like ionic strength or water activity. (3) P. halocryophilus shows the highest microbial sodium perchlorate tolerance described so far. However, (4) MSCg values are higher for all chlorides compared to perchlorates. (5) The MSCg for calcium chloride was increased by lowering the temperature from 25°C to 4°C, while sodium- and magnesium-containing salts can be tolerated at 25°C to higher concentrations than at 4°C. (6) Depending on salt type and concentration, P. halocryophilus cells show distinct phenotypic stress responses such as novel types of colony morphology on agar plates and biofilm-like cell clustering, encrustation, and development of intercellular nanofilaments. This study, taken in context with previous work on the survival of extremophiles in Mars-like environments, suggests that high-concentrated perchlorate brines on Mars might not be habitable to any present organism on Earth, but extremophilic microorganisms might be able to evolve thriving in such environments.EC/FP7/339231/EU/Habitability of Martian Environments: Exploring the Physiological and Environmental Limits of Life/HOM

Determination of Geochemical Bio-Signatures in Mars-Like Basaltic Environments

Bio-signatures play a central role in determining whether life existed on early Mars. Using a terrestrial basalt as a compositional analog for the martian surface, we applied a combination of experimental microbiology and thermochemical modeling techniques to identify potential geochemical bio-signatures for life on early Mars. Laboratory experiments were used to determine the short-term effects of biota on the dissolution of terrestrial basalt, and the formation of secondary alteration minerals. The chemoorganoheterotrophic bacterium, Burkholderia sp. strain B_33, was grown in a minimal growth medium with and without terrestrial basalt as the sole nutrient source. No growth was detected in the absence of the basalt. In the presence of basalt, during exponential growth, the pH decreased rapidly from pH 7.0 to 3.6 and then gradually increased to a steady-state of equilibrium of between 6.8 and 7.1. Microbial growth coincided with an increase in key elements in the growth medium (Si, K, Ca, Mg, and Fe). Experimental results were compared with theoretical thermochemical modeling to predict growth of secondary alteration minerals, which can be used as bio-signatures, over a geological timescale. We thermochemically modeled the dissolution of the basalt (in the absence of biota) in very dilute brine at 25°C, 1 bar; the pH was buffered by the mineral dissolution and precipitation reactions. Preliminary results suggested that at the water to rock ratio of 1 × 107, zeolite, hematite, chlorite, kaolinite, and apatite formed abiotically. The biotic weathering processes were modeled by varying the pH conditions within the model to adjust for biologic influence. The results suggested that, for a basaltic system, the microbially-mediated dissolution of basalt would result in “simpler” secondary alteration, consisting of Fe-hydroxide and kaolinite, under conditions where the abiotic system would also form chlorite. The results from this study demonstrate that, by using laboratory-based experiments and thermochemical modeling, it is possible to identify secondary alteration minerals that could potentially be used to distinguish between abiotic and biotic weathering processes on early Mars. This work will contribute to the interpretation of data from past, present, and future life detection missions to Mars

Transitory Microbial Habitat in the Hyperarid Atacama Desert

Traces of life are nearly ubiquitous on Earth. However, a central unresolved question is whether these traces always indicate an active microbial community or whether, in extreme environments, such as hyperarid deserts, they instead reflect just dormant or dead cells. Although microbial biomass and diversity decrease with increasing aridity in the Atacama Desert, we provide multiple lines of evidence for the presence of an at times metabolically active, microbial community in one of the driest places on Earth. We base this observation on four major lines of evidence: a physico-chemical characterization of the soil habitability after an exceptional rain event, identified biomolecules indicative of potentially active cells [e.g., presence of ATP, phospholipid fatty acids (PLFAs), metabolites, and enzymatic activity], measurements of in situ replication rates of genomes of uncultivated bacteria reconstructed from selected samples, and microbial community patterns specific to soil parameters and depths. We infer that the microbial populations have undergone selection and adaptation in response to their specific soil microenvironment and in particular to the degree of aridity. Collectively, our results highlight that even the hyperarid Atacama Desert can provide a habitable environment for microorganisms that allows them to become metabolically active following an episodic increase in moisture and that once it decreases, so does the activity of the microbiota. These results have implications for the prospect of life on other planets such as Mars, which has transitioned from an earlier wetter environment to today's extreme hyperaridity. [Abstract copyright: Copyright © 2018 the Author(s). Published by PNAS.

New simulants for martian regolith: Controlling iron variability

Existing martian simulants are predominantly based on the chemistry of the average ‘global’ martian regolith as defined by data on chemical and mineralogical variability detected by orbiting spacecraft, surface rovers and landers. We have therefore developed new martian simulants based on the known composition of regolith from four different martian surface environments: an early basaltic terrain, a sulfur-rich regolith, a haematite-rich regolith and a contemporary Mars regolith. Simulants have been developed so that the Fe2+/Fe3+ ratios can be adjusted, if necessary, leading to the development of four standard simulants and four Fe-modified simulants. Characterisation of the simulants confirm that all but two (both sulfur-rich) are within 5 wt% of the martian chemistries that they were based on and, unlike previous simulants, they have Fe2+/Fe3+ ratios comparable to those found on Mars. Here we outline the design, production and characterisation of these new martian regolith simulants. These are to be used initially in experiments to study the potential habitability of martian environments in which Fe may be a key energy source

Microbial Hotspots in Lithic Macrohabitats Inferred from DNA Fractionation and Metagenomics in the Atacama Desert

The existence of microbial activity hotspots in temperate regions of Earth is driven by soil heterogeneities, especially the temporal and spatial availability of nutrients. Here we investigate whether microbial activity hotspots also exist in lithic microhabitats in one of the most arid regions of the world, the Atacama Desert in Chile. While previous studies evaluated the total DNA fraction to elucidate the microbial communities, we here for the first time use a DNA separation approach on lithic microhabitats, together with metagenomics and other analysis methods (i.e., ATP, PLFA, and metabolite analysis) to specifically gain insights on the living and potentially active microbial community. Our results show that hypolith colonized rocks are microbial hotspots in the desert environment. In contrast, our data do not support such a conclusion for gypsum crust and salt rockenvironments, because only limited microbial activity could be observed. The hypolith community is dominated by phototrophs, mostly Cyanobacteria and Chloroflexi, at both study sites. The gypsum crusts are dominated by methylotrophs and heterotrophic phototrophs, mostly Chloroflexi, and the salt rocks (halite nodules) by phototrophic and halotolerant endoliths, mostly Cyanobacteria and Archaea. The major environmental constraints in the organic-poor arid and hyperarid Atacama Desert are water availability and UV irradiation, allowing phototrophs and other extremophiles to play a key role in desert ecology

Machine Learning Algorithms Applied to Identify Microbial Species by Their Motility

(1) Background: Future missions to potentially habitable places in the Solar System require biochemistry-independent methods for detecting potential alien life forms. The technology was not advanced enough for onboard machine analysis of microscopic observations to be performed in past missions, but recent increases in computational power make the use of automated in-situ analyses feasible. (2) Methods: Here, we present a semi-automated experimental setup, capable of distinguishing the movement of abiotic particles due to Brownian motion from the motility behavior of the bacteria Pseudoalteromonas haloplanktis, Planococcus halocryophilus, Bacillus subtilis, and Escherichia coli. Supervised machine learning algorithms were also used to specifically identify these species based on their characteristic motility behavior. (3) Results: While we were able to distinguish microbial motility from the abiotic movements due to Brownian motion with an accuracy exceeding 99%, the accuracy of the automated identification rates for the selected species does not exceed 82%. (4) Conclusions: Motility is an excellent biosignature, which can be used as a tool for upcoming life-detection missions. This study serves as the basis for the further development of a microscopic life recognition system for upcoming missions to Mars or the ocean worlds of the outer Solar System

Machine Learning Algorithms Applied to Identify Microbial Species by Their Motility

(1) Background: Future missions to potentially habitable places in the Solar System require biochemistry-independent methods for detecting potential alien life forms. The technology was not advanced enough for onboard machine analysis of microscopic observations to be performed in past missions, but recent increases in computational power make the use of automated in-situ analyses feasible. (2) Methods: Here, we present a semi-automated experimental setup, capable of distinguishing the movement of abiotic particles due to Brownian motion from the motility behavior of the bacteria Pseudoalteromonas haloplanktis, Planococcus halocryophilus, Bacillus subtilis, and Escherichia coli. Supervised machine learning algorithms were also used to specifically identify these species based on their characteristic motility behavior. (3) Results: While we were able to distinguish microbial motility from the abiotic movements due to Brownian motion with an accuracy exceeding 99%, the accuracy of the automated identification rates for the selected species does not exceed 82%. (4) Conclusions: Motility is an excellent biosignature, which can be used as a tool for upcoming life-detection missions. This study serves as the basis for the further development of a microscopic life recognition system for upcoming missions to Mars or the ocean worlds of the outer Solar System.EC/H2020/957272/EU/PortPath: A Portable Device for Detecting Pathogens/PortPathTU Berlin, Open-Access-Mittel – 202