THE BOSS EXPERIMENT OF THE EXPOSE-R2 MISSION: BIOFILM VERSUS PLANKTONIC CELLS

In the BOSS experiment (biofilm organisms surfing space), which was performed in the context of the successfully finalized EXPOSE-R2 mission, an international consortium of scientists investigated the ability of a variety of organisms to survive in space and on Mars as a function of their life style. The question in focus is whether there are different strategies for individually living microorganisms (planktonic state) compared to a microbial consortium of the same cells (biofilm state) to cope with the unique mixture of extreme stress factors including desiccation, gamma-, ionizing- and UV radiation in this environment. Biofilms, in which the cells are encased in a self-produced matrix of excreted extracellular polymeric substances, are one of the oldest clear signs of life on Earth. Since they can become fossilized they might also be detected as the first life forms on other planets and moons of the solar system and are therefore ideal candidates for astrobiological investigations. As an example for the organisms that attended the EXPOSER2 mission the results of the ight and mission ground reference analysis of Deinococcus geothermalis are presented. Deinococcus geothermalis is a non-spore-forming, gram-positive, orange-pigmented representative of the Deinococcus family which is unparalleled in its poly-extreme resistances to a variety of environmental stress factors on Earth. The results demonstrate that Deinococcus geothermalis remains viable in the desiccated state over almost 2 years, whereas culturability was preserved in biofilm cells at a significantly higher level than in planktonic cells. Furthermore, cells of both sample types were able to survive simulated space and Martian conditions and showed high resistance towards extra-terrestrial UV radiation. Additionally results of cultivation-independent investigations of pigment stability, membrane integrity, enzyme activity, ATP content and DNA integrity will be discussed.To conclude, biofilms exhibit an enhanced rate of survival compared to their planktonic counterparts when exposed to space and Martian conditions. This seems to indicate an advantage of living as a biofilm when facing the poly-extreme conditions of space or Mars. The findings will contribute to the understanding of the opportunities and limitations of life under the extreme environmental conditions of space or other planets as function of the state of life and aims to contribute to the understanding of the adaptation mechanisms that allow microorganisms to survive in extreme environments, possibly including space and the surface of Mars

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

Unterschiede in der Toleranz von Biofilmen und planktonischen Zellen von Deinococcus geothermalis gegenüber Austrocknung sowie simulierten Weltraum- und Marsbedingungen

The formation of biofilms is one of the most successful survival strategies of bacteria on Earth. Aggregated and embedded in a matrix of extracellular polymeric substances (EPS), biofilm cells are often more tolerant to environmental stress than single, planktonic cells of the same species. If microorganisms were to travel through space or to reside on a Mars-like planet, their survival in these hostile environments might be enhanced if they are organised in biofilms. The aim of this study was to test this hypothesis. Deinococcus geothermalis DSM 11300 was chosen as a model organism due to its high intrinsic tolerance to both desiccation and radiation. Biofilms generated at the solid-air inter-face of membranes placed on R2A agar medium and, for comparison, membrane-deposited planktonic cells grown in R2A broth were air-dried overnight and exposed to various stressors relevant for space and Mars environments, including prolonged desiccation, vacuum, simulated Martian atmosphere, low and high temperatures, and UV radiation. The EPS of D. geothermalis were isolated using a cation-exchange resin extraction method. They contained significant amounts of proteins and polysaccharides, both of which might promote the retention of water under dehydrating conditions, as well as relatively low amounts of extracellular DNA (eDNA), which could have a structural role within the EPS matrix. In biofilms, the EPS were of distinct spatial arrangement. Environmental scanning electron microscopy provided evidence for an EPS layer covering the uppermost layer of cells. Three different polysaccharide fractions – presumably galactosides – of different spatial distribution and arrangement were identified within the matrix using fluorescently-labelled lectins. When tryptic soy agar (TSA) was used as a nutrient source instead of R2A, biofilms of different morphology and EPS composition were formed. High peptone concentrations in the TSA medium (20 g l 1) caused the cells to form highly cohesive aggregates. Dispersal of these cell aggregates could be achieved by treatment with proteinase K, suggesting the involvement of proteins, possibly in the form of adhesins or type IV pili, in the cell-to-cell attachment of the organism. Following exposure to stress conditions, the viability of the organisms was assessed and compared to non-exposed cells. Since many bacteria are able to enter a viable but non-culturable (VBNC) state as a response to stress, cultivation-independent viability markers (membrane integrity, ATP levels, presence of 16S rRNA) were analysed in addition to the determination of colony counts. During prolonged desiccation, biofilms sustained viability significantly longer than planktonic cells: Compared to non-desiccated samples, a desiccation period of 56-61 days reduced the culturability of biofilms and planktonic cells to 5.6% and 0.8%, respectively. Membrane integrity was maintained to a high degree in biofilms, whereas more than 60% of planktonic cells showed signs of membrane damage following desiccation. Whilst biofilm cells sustained their initial ATP levels, planktonic cells experienced a 1-log reduction in ATP upon dehydration. When desiccated biofilms and planktonic cells of D. geothermalis were exposed to vac-uum, artificial Martian atmosphere, repeated thaw-freeze cycles, or extreme temperatures ( 25 °C; +60 °C), their viability in terms of culturability and membrane integrity remained unchanged compared to dry but non-exposed controls. UV irradiation – either monochro-matic (254 nm; ≥ 1 kJ m 2) or polychromatic (200-400 nm; > 5.5 MJ m-2 for planktonic cells and > 270 MJ m-2 for biofilms) – significantly reduced the culturability of D. geothermalis in both its biofilm and planktonic form. Survival seemed to be insignificantly enhanced when the cells were irradiated in artificial Martian atmosphere instead of space-like vacuum. Under both desiccation and UV irradiation, biofilms exhibited a decline in culturable cells whilst total cell counts and cultivation-independent viability parameters remained rela-tively stable. This suggests that a part of the population became VBNC as a response to these stressors. Induction of the VBNC state might confer an increased tolerance towards stress to D. geothermalis. In conclusion, a significant fraction of the population of D. geothermalis sustained viability under all stress conditions tested, with biofilm cells often being more stress-tolerant than planktonic cells. It seems that the increased stress tolerance of biofilms is a result of the induction of a VBNC state and the protective effect of the EPS matrix. Judging from the re-sults obtained in this study, D. geothermalis might survive in space or on Mars for a limited period of time, especially if shielded against the harmful extraterrestrial UV radiation.Die Bildung von Biofilmen ist eine der erfolgreichsten Überlebensstrategien von Bakterien. In Biofilmen sind die Mikroorganismen aggregiert und in eine Matrix aus extrazellulären polymeren Substanzen (EPS) eingebettet, welche den Zellen eine erhöhte Toleranz gegenüber verschiedenen Umweltstressoren verleiht. Diese Schutzfunktion des Biofilms könnte es Bakterien ermöglichen, in so unwirtlichen Habitaten wie dem Weltraum oder dem Mars zu überleben. Das Ziel dieser Studie war es, diese Hypothese zu untersuchen. Als Modellorganismus wurde Deinococcus geothermalis DSM 11300 wegen seiner au-ßerordentlichen Resistenz gegenüber Austrocknung und Strahlungen ausgewählt. Biofilme von D. geothermalis wurden auf der Oberfläche von auf Nähragar (R2A) platzierten Membranen angezüchtet. Zu Vergleichszwecken wurden in Nährbouillon (R2B) kultivierte planktonische Zellen ebenfalls auf Membranen aufgetragen. Die membranassoziierten Zellen wurden jeweils über Nacht an der Luft getrocknet, und verschiedenen Weltraum- und Mars-relevanten Stressoren (Austrocknung, Vakuum, künstliche Marsatmosphäre, hohe und tiefe Temperaturextreme, UV-Strahlung) ausgesetzt. Die EPS von D. geothermalis wurden mit Hilfe eines Kationen-Austauschharzes iso-liert. In den EPS dominierten Proteine und Polysaccharide, welche hygroskopisch sein und damit zur Rückhaltung von Wasser im austrocknenden Biofilm beitragen können. Außerdem wurde extrazelluläre DNA (eDNA), die eine strukturelle Rolle in der EPS-Matrix haben könnte, nachgewiesen. Rasterelektronenmikroskopische Untersuchungen von Biofilmen zeigten, dass die oberste Zellschicht von einer EPS-Schicht überlagert ist, die zur Wasserrückhaltung beitragen könnte. Mit Hilfe von fluoreszenzmarkierten Lektinen wurden drei galaktosid¬haltige Polysaccharid-Fraktionen von unterschiedlicher Struktur und Verteilung in der EPS-Matrix identifiziert. Wurde Caso-Agar anstelle von R2A als Nährmedium eingesetzt, so bildete D. geo-thermalis Biofilme von unterschiedlicher Morphologie und EPS-Zusammensetzung. Außer-dem formten die Zellen hier bedingt durch den hohen Pepton-Anteil im Medium (20 g l-1) stark kohäsive Aggregate. Diese Aggregate konnten durch das Enzym Proteinase K dispergiert werden, was darauf hindeutet, dass Proteine an der interzellulären Anheftung von D. geothermalis beteiligt sind. Nach der Exposition gegenüber verschiedenen Stressoren wurde die Vitalität der Zel-len analysiert und mit der Vitalität ungestresster Zellen verglichen. Da viele Bakterien in der Lage sind, als Stressantwort in einen Zustand überzugehen, in dem sie zwar vital, jedoch nicht mehr kultivierbar sind („viable but non-culturable“, VBNC), wurden neben der Bestimmung der Koloniezahl auch kultivierungsunabhängige Vitalitätsmarker (Membranintegrität, ATP, 16S rRNA) herangezogen. Unter Austrocknungsbedingungen zeigten Biofilme eine deutlich höhere Vitalität als planktonische Zellen: Verglichen mit ungestressten Zellen verringerte sich die Kultivierbarkeit von D. geothermalis während einer Austrocknungsperiode von 56-61 Tagen auf 5.6% für Biofilme und 0.8% für planktonische Zellen. Der Großteil der Biofilm-Zellen war in der Lage, die Integrität ihrer Zellmembran aufrecht zu erhalten, wohingegen mehr als 60% der planktonischen Zellen Membranschäden aufwiesen. Während Biofilme außerdem recht stabile ATP-Konzentrationen aufwiesen, sorgte die Austrocknung bei planktonischen Zellen für eine Verringerung der ATP-Konzentration um eine Größenordnung. Die Exposition von Biofilmen und planktonischen Zellen gegenüber Vakuum, künstlicher Marsatmosphäre, wiederholten Gefrierzyklen oder extremen Temperaturen ( 25 °C; +60 °C) hatte keinen messbaren Einfluss auf ihre Kultivierbarkeit und Membranintegrität. Mono- oder polychromatische UV-Bestrahlung (254 nm, ≥ 1 kJ m 2; 200-400 nm, > 5.5 MJ m-2 für planktonische Zellen und > 270 MJ m-2 für Biofilme) sorgte hingegen für eine signifikante Abnahme der Kultivierbarkeit von sowohl planktonischen, als auch Biofilm-Zellen. Unter Austrocknung und UV-Bestrahlung zeigten Biofilm-Zellen eine signifikante Abnahme der Kultivierbarkeit, wohingegen kultivierungsunabhängige Vitalitätsmarker deutlich weniger beeinträchtigt wurden. Dies könnte darauf hindeuten, dass ein Teil der Biofilm-Population als Antwort auf diese beiden Stressoren in den VBNC-Zustand übergegangen ist. Die Induktion des VBNC-Zustands könnte für D. geothermalis mit einer erhöhten Stresstoleranz einhergehen. Zusammenfassend lässt sich sagen, dass ein deutlicher Teil der Population von D. geothermalis während der Exposition gegenüber verschiedenen Stressoren vital blieb. Dabei waren Biofilme oft toleranter als planktonische Zellen. Es scheint als sei die erhöhte Stresstoleranz von Biofilm-Zellen auf die Induktion des VBNC-Zustands, sowie auf die schützende Funktion der EPS-Matrix zurückzuführen. In Anbetracht der hier erlangten Ergebnisse erscheint es möglich, dass D. geothermalis die im Weltraum und auf dem Mars vorherrschenden Bedingungen für eine bestimmte Zeit überleben könnte, insbesondere wenn die Zellen gegen die schädliche extraterrestrische Strahlung geschützt sind

Survival of Deinococcus geothermalis in Biofilms under Desiccation and Simulated Space and Martian Conditions

Biofilm formation represents a successful survival strategy for bacteria. In biofilms, cells are embedded in a matrix of extracellular polymeric substances (EPS). As they are often more stress-tolerant than single cells, biofilm cells might survive the conditions present in space and on Mars. To investigate this topic, the bacterium Deinococcus geothermalis was chosen as a model organism due to its tolerance toward desiccation and radiation. Biofilms cultivated on membranes and, for comparison, planktonically grown cells deposited on membranes were air-dried and exposed to individual stressors that included prolonged desiccation, extreme temperatures, vacuum, simulated martian atmosphere, and UV irradiation, and they were exposed to combinations of stressors that simulate space (desiccation + vacuum + UV) or martian (desiccation + Mars atmosphere + UV) conditions. The effect of sulfatic Mars regolith simulant on cell viability during stress was investigated separately. The EPS produced by the biofilm cells contained mainly polysaccharides and proteins. To detect viable but nonculturable (VBNC) cells, cultivation-independent viability indicators (membrane integrity, ATP, 16S rRNA) were determined in addition to colony counts. Desiccation for 2 months resulted in a decrease of culturability with minor changes of membrane integrity in biofilm cells and major loss of membrane integrity in planktonic bacteria. Temperatures between -25°C and +60°C, vacuum, and Mars atmosphere affected neither culturability nor membrane integrity in both phenotypes. Monochromatic (254 nm; ‡1 kJ m⁻²) and polychromatic (200–400 nm; >5.5 MJ m⁻² for planktonic cells and >270 MJ m⁻² for biofilms) UV irradiation significantly reduced the culturability of D. geothermalis but did not affect cultivation-independent viability markers, indicating the induction of a VBNC state in UV-irradiated cells. In conclusion, a substantial proportion of the D. geothermalis population remained viable under all stress conditions tested, and in most cases the biofilm form proved advantageous for surviving space and Mars-like conditions

Tolerances of Deinococcus geothermalis Biofilms and Planktonic Cells Exposed to Space and Simulated Martian Conditions in Low Earth Orbit for Almost Two Years

Fossilized biofilms represent one of the oldest known confirmations of life on the Earth. The success of microbes in biofilms results from properties that are inherent in the biofilm, including enhanced interaction, protection, and biodiversity. Given the diversity of microbes that live in biofilms in harsh environments on the Earth, it is logical to hypothesize that, if microbes inhabit other bodies in the Universe, there are also biofilms on those bodies. The Biofilm Organisms Surfing Space experiment was conducted as part of the EXPOSE-R2 mission on the International Space Station. The experiment was an international collaboration designed to perform a comparative study regarding the survival of biofilms versus planktonic cells of various microorganisms, exposed to space and Mars-like conditions. The objective was to determine whether there are lifestyledependent differences to cope with the unique mixture of stress factors, including desiccation, temperature oscillations, vacuum, or a Mars-like gas atmosphere and pressure in combination with extraterrestrial or Marslike ultraviolet (UV) radiation residing during the long-term space mission. In this study, the outcome of the flight and mission ground reference analysis of Deinococcus geothermalis is presented. Cultural tests demonstrated that D. geothermalis remained viable in the desiccated state, being able to survive space and Mars-like conditions and tolerating high extraterrestrial UV radiation for more than 2 years. Culturability decreased, but was better preserved, in the biofilm consortium than in planktonic cells. These results are correlated to differences in genomic integrity after exposure, as visualized by random amplified polymorphic DNA–polymerase chain reaction. Interestingly, cultivation-independent viability markers such as membrane integrity, ATP content, and intracellular esterase activity remained nearly unaffected, indicating that subpopulations of the cells had survived in a viable but nonculturable state. These findings support the hypothesis of long-term survival of microorganisms under the harsh environmental conditions in space and on Mars to a higher degree if exposed as biofilm