Aggregated Cell Masses Provide Protection against Space Extremes and a Microhabitat for Hitchhiking Co-Inhabitants Jennifer Wadsworth, 1 Petra Rettberg, 2 and Charles S. Cockell 1 Abstract The European Space Agency’s EXPOSE facility, located on the outside of the International Space Station, was used to investigate the survival of cell aggregates of a cyanobacterium, Gloeocapsa sp., in space and simulated martian conditions for 531 days in low Earth orbit as part of the ‘‘Biofilm Organisms Surfing Space’’ (BOSS) experiments. Postflight analysis showed that the cell aggregates of the organism conferred protection against space conditions compared to planktonic cells. These cell aggregates, which consisted of groups of metabolically inactive cells that do not form structured layered biofilms, demonstrated that disordered ‘‘primitive’’ aggregates of sheathed cells can provide protection against environmental stress such as UV radiation. Furthermore, the experiment demonstrated that the cyanobacterial cell aggregates provided a microhabitat for a smaller bacterial co-cultured species that also survived in space. This observation shows that viable cells can ‘‘hitchhike’’ through space within the confines of larger protecting cells or cell aggregates, with implications for planetary protection, human health, and other space microbiology applications. Key Words: Space radiation—Biofilms—Radiation protection—Cyanobacteria— Mars—EXPOSE. Astrobiology 19, 995–1007. 1. Introduction I n their natural habitats, many known microorgan- isms form biofilms as opposed to existing in a free- swimming, planktonic state. While a biofilm is not required for bacterial viability, it has developed as an evolutionary solution to enhance survival. The International Union of Pure and Applied Chemistry’s definition of a biofilm is an ‘‘Ag- gregate of microorganisms in which cells that are frequently embedded within a self-produced matrix of extracellular polymeric substances (EPSs) adhere to each other and/or to a surface’’ (Vert et al., 2012). Along with benefits such as metabolism coordination (Camilli and Bassler, 2006), bio- films have been shown to be crucial in providing a barrier between the bacterial population and unfavorable environ- mental conditions (Edwards et al., 2005). These include toxic substances (Flemming, 1993) as well as damaging environ- mental factors, for example high-UV environments on Earth such as in the Atacama Desert and Antarctica (Los Rı ´os et al., 2003; Wierzchos et al., 2006; Flemming et al., 2016). The low Earth orbit (LEO) environment consists of multiple such extreme conditions that pose many challenges to life, including high-UV and ionizing radiation exposure. It is thought that the protective characteristics of biofilms allow them to confer protection to microbes in these harsh, non-terrestrial conditions (Billi et al., 2000; Cockell et al., 2005; Baque ´ et al., 2013b). Low Earth orbit extends from 160 to 2000 km above the surface of Earth and is still within Earth’s upper atmosphere and magnetosphere. While the magnetosphere is able to de- flect some of the high-energy radiation found in space, the LEO radiation environment is still more hazardous than that of Earth’s surface, especially due to galactic cosmic rays (GCR) and shortwave UV radiation (Horneck et al., 1996; Benton and Benton, 2001). Temperatures can fluctuate be- tween -120°C in environments shaded by parts of the spacecraft and +120°C in full sunlight (de Groh and Finck- enor, 2016), while acceleration due to gravity is 1 · 10 -6 g (Downey, 2016). The atmospheric pressure of the upper at- mosphere above 100 km drops to 3.2 · 10 -2 Pa (Squire et al., 1997), essentially a vacuum. These LEO conditions that are practically and economi- cally problematic to reproduce on Earth have been exploited outside the International Space Station (ISS) to test the re- sponse and degradation of various organisms and substances including prokaryotes, eukaryotes, and chemical compounds (Horneck et al., 1984; Sancho et al., 2007; Cottin et al., 2008, 2017). Since its installation on the ISS in 2008, the 1 UK Centre for Astrobiology, School of Physics and Astronomy, University of Edinburgh, Edinburgh, UK. 2 Institute of Aerospace Medicine, DLR, Cologne, Germany. ASTROBIOLOGY Volume 19, Number 8, 2019 ª Mary Ann Liebert, Inc. DOI: 10.1089/ast.2018.1924 995 Downloaded by 84.80.16.35 from www.liebertpub.com at 08/08/19. For personal use only.
Transcript
Aggregated Cell Masses Provide Protection against SpaceExtremes and a Microhabitat for Hitchhiking Co-Inhabitants
Jennifer Wadsworth,1 Petra Rettberg,2 and Charles S. Cockell1
Abstract
The European Space Agency’s EXPOSE facility, located on the outside of the International Space Station, wasused to investigate the survival of cell aggregates of a cyanobacterium, Gloeocapsa sp., in space and simulatedmartian conditions for 531 days in low Earth orbit as part of the ‘‘Biofilm Organisms Surfing Space’’ (BOSS)experiments.
Postflight analysis showed that the cell aggregates of the organism conferred protection against space conditionscompared to planktonic cells. These cell aggregates, which consisted of groups of metabolically inactive cells thatdo not form structured layered biofilms, demonstrated that disordered ‘‘primitive’’ aggregates of sheathed cells canprovide protection against environmental stress such as UV radiation. Furthermore, the experiment demonstratedthat the cyanobacterial cell aggregates provided a microhabitat for a smaller bacterial co-cultured species that alsosurvived in space. This observation shows that viable cells can ‘‘hitchhike’’ through space within the confines oflarger protecting cells or cell aggregates, with implications for planetary protection, human health, and other spacemicrobiology applications. Key Words: Space radiation—Biofilms—Radiation protection—Cyanobacteria—Mars—EXPOSE. Astrobiology 19, 995–1007.
1. Introduction
In their natural habitats, many known microorgan-isms form biofilms as opposed to existing in a free-
swimming, planktonic state. While a biofilm is not requiredfor bacterial viability, it has developed as an evolutionarysolution to enhance survival. The International Union of Pureand Applied Chemistry’s definition of a biofilm is an ‘‘Ag-gregate of microorganisms in which cells that are frequentlyembedded within a self-produced matrix of extracellularpolymeric substances (EPSs) adhere to each other and/or to asurface’’ (Vert et al., 2012). Along with benefits such asmetabolism coordination (Camilli and Bassler, 2006), bio-films have been shown to be crucial in providing a barrierbetween the bacterial population and unfavorable environ-mental conditions (Edwards et al., 2005). These include toxicsubstances (Flemming, 1993) as well as damaging environ-mental factors, for example high-UV environments on Earthsuch as in the Atacama Desert and Antarctica (Los Rıos et al.,2003; Wierzchos et al., 2006; Flemming et al., 2016).
The low Earth orbit (LEO) environment consists ofmultiple such extreme conditions that pose many challengesto life, including high-UV and ionizing radiation exposure.It is thought that the protective characteristics of biofilms
allow them to confer protection to microbes in these harsh,non-terrestrial conditions (Billi et al., 2000; Cockell et al.,2005; Baque et al., 2013b).
Low Earth orbit extends from 160 to 2000 km above thesurface of Earth and is still within Earth’s upper atmosphereand magnetosphere. While the magnetosphere is able to de-flect some of the high-energy radiation found in space, theLEO radiation environment is still more hazardous than thatof Earth’s surface, especially due to galactic cosmic rays(GCR) and shortwave UV radiation (Horneck et al., 1996;Benton and Benton, 2001). Temperatures can fluctuate be-tween -120�C in environments shaded by parts of thespacecraft and +120�C in full sunlight (de Groh and Finck-enor, 2016), while acceleration due to gravity is 1 · 10-6 g(Downey, 2016). The atmospheric pressure of the upper at-mosphere above 100 km drops to 3.2 · 10-2 Pa (Squire et al.,1997), essentially a vacuum.
These LEO conditions that are practically and economi-cally problematic to reproduce on Earth have been exploitedoutside the International Space Station (ISS) to test the re-sponse and degradation of various organisms and substancesincluding prokaryotes, eukaryotes, and chemical compounds(Horneck et al., 1984; Sancho et al., 2007; Cottin et al.,2008, 2017). Since its installation on the ISS in 2008, the
1UK Centre for Astrobiology, School of Physics and Astronomy, University of Edinburgh, Edinburgh, UK.2Institute of Aerospace Medicine, DLR, Cologne, Germany.
ASTROBIOLOGYVolume 19, Number 8, 2019ª Mary Ann Liebert, Inc.DOI: 10.1089/ast.2018.1924
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European Space Agency’s EXPOSE facility has hosted threelong-term missions with the goal of providing a platform forEuropean scientists to expose samples in situ to conditionsthat are often outside the scope of laboratory simulations.The platform was first located on the European ColumbusModule (EXPOSE-E), while the subsequent missions(EXPOSE-R and EXPOSE-R2) were attached to the RussianZvezda module (Rabbow et al., 2017).
By using ESA’s EXPOSE-R2 facility, the ‘‘Biofilm Or-ganisms Surfing Space’’ (BOSS; principal investigator:Petra Rettberg) experimental setup allowed an internationalteam to subject a variety of samples to the LEO environ-ment. The Edinburgh experiment that was integrated intothe BOSS setup was used to expose cyanobacterial samplesto LEO conditions for a total of 531 days. The selectedmodel organism, Gloeocapsa, does not form well-definedlayered biofilms, but rather an amorphous cellular mass(ACM), and was used to investigate the protection affordedby a simple, biofilm-like cellular mass. The LEO environ-ment was used to create a set of ‘‘space’’ and ‘‘simulatedmartian’’ conditions (see Table 1) on the EXPOSE platformin which to test the protection potential of ACMs. In addi-tion to Gloeocapsa’s own viability, its ability to harborsecondary, co-cultured species was investigated.
In this work, we describe the postflight analyses of theprimary model organism and the ability of a ‘‘hitchhiking’’secondary organism to survive prolonged exposure to thehostile conditions of LEO. These findings have implicationsnot only for planetary protection but also for understandingthe possibilities of microbial—and pathogen—survival onspace stations.
2. Materials and Methods
2.1. Model organism selection and preparation
The model organism used in this experiment was thecyanobacterial species Gloeocapsa sp. The bacterium issurrounded by a concentric gelatinous sheath that can alsoenvelop multicellular groups of bacteria, especially recentlydivided pairs, thereby forming an ACM (Whitton and Potts,2007) (Fig. 1).
The gelatinous sheath of Gloeocapsa sp. has previouslybeen shown to provide effective protection against UV ra-diation (Garcia-Pichel et al., 1993).
The cyanobacterium Chroococcidiopsis, related toGloeocapsa sp., similarly possesses a gelatinous sheath andhas been shown to have a heightened tolerance for radiationexposure (Billi et al., 2000; Cockell et al., 2005) and has
Table 1. BOSS Condition Parameters
Parameter Space conditions Simulated martian conditions
Light exposure No long-pass cutoff filters, full solar spectrum(>120 nm)
>200 nm cutoff filters
Light intensity MgF2 neutral density filters, 0.1% of incident light Quartz neutral density filters, 0.1% of incident lightPressure 1.33 · 10-3 to 1.33 · 10-4 Pa (near-vacuum) 1000 Pa (artificial martian gas mixture)
‘‘Space’’ and ‘‘simulated martian’’ condition parameters of the BOSS EXPOSE-R2 mission. Cutoff filters provided samples with asimulated martian wavelength range. Incident light was limited to 0.1% of its original intensity by neutral density filters in all top-layersamples to avoid over-exposure; bottom-layer samples were exposed to all parameters but shielded from light. None of the samples wereshielded from ionizing particle radiation sources.
FIG. 1. Model organism Gloeocapsa. Model organism Gloeocapsa in liquid media. Each bacterium is *6mm in size butcan form larger aggregates many hundred micrometers in diameter. (a) A single, larger aggregate; scale bar = 200mm.Individual Gloeocapsa cells (source: http://fmp.conncoll.edu) shown at higher resolution; white arrow indicates newlydivided cells sharing the same gelatinous sheath (b).
been used in BOSS experiments by other investigator teams(Baque et al., 2013a). Although Gloeocapsa sp. cell ag-gregates lack the more defined structure of biofilms, non-planktonic culture samples will be defined as ‘‘biofilms’’ inthis work.
The Gloeocapsa sp. strain was originally found in anenvironmental sample taken from the upper greensand layerof the limestone cliffs in Devon, UK, by Olsson-Franciset al. and was isolated by exposure of the environmentalsample to the LEO environment for 10 days (Olsson-Franciset al., 2010). It was further used in the previous EXPOSE-Espace experiment ADAPT (principal investigator: PetraRettberg), where it survived 548 days exposed to LEO ifshielded from UV exposure (Cockell et al., 2011). Due to itsability to survive longer-term LEO exposure, it was chosenas a model organism for the BOSS experiment.
Despite steps taken to obtain a monoculture of the cya-nobacteria, a co-cultured, nonpathogenic bacterial specieswas present in all BOSS cyanobacterial cultures. The co-cultured species in EXPOSE-R2 was also isolated from thesame cell culture as Gloeocapsa sp. during the EXPOSE-Emission and was identified as an a-proteobacteria isolaterelated to Geminicoccus roseus (Foesel et al., 2007). It isdescribed as a diplococcoid bacterium containing caroten-oids and low amounts of bacteriochlorophyll a, giving itscolonies a pink coloration.
‘‘Biofilm’’ samples were fabricated by gently homoge-nizing, in a hand-operated glass homogenizer, cells from a3-month-old culture of Gloeocapsa until a solution of uni-form green cells was obtained. Cells were checked bybright-field microscopy to ensure minimum disruption to thecell membranes had occurred but to achieve as near aspossible a suspension of single cells. It should be noted thatcell aggregates always remained, and it was not possible toachieve a completely homogeneous single cell suspensionwithout severe disruption to many cells. Then 250 mL ofcells was pipetted as uniformly as possible onto porous,sintered, silica discs with 10 mm diameter, 3 mm thickness,and a pore size of 100–160mm (Scientific Glass, UK). Oncethe cells were absorbed into the disc, the discs were trans-ferred into plastic 6-well plates, and BG-11 medium wasadded until the discs were covered in liquid and the platescovered to avoid evaporation. The cells were left for 3months at ambient laboratory conditions (21�C and light ofapproximately 50mmol/m2/s) to allow for the formation ofcell aggregates within the glass discs. Cells were observedto form well-defined cell aggregates or clumps within therock pore spaces. After this period, the discs were re-moved and allowed to dry in laboratory conditions for2 days. ‘‘Planktonic’’ samples were obtained by pipetting250 mL of homogenized cells, as described above, asuniformly as possible onto glass discs. The glass discswere then dried similarly to the ‘‘biofilm’’ samples.Identical control cyanobacterial samples for the BOSSexperiment were prepared as described above. Both sam-ples and controls were sent to DLR (Deutsches Zentrumfur Luft- und Raumfahrt e.V.), the German Space Agency,for integration into the EXPOSE-R2 hardware and themission ground reference (MGR) experiment, respec-tively. Due to concerns over the quality of the groundreferences, this paper will solely focus on the ISS sampledata to avoid inaccurate conclusions.
2.2. EXPOSE-R2 platform
The EXPOSE-R2 facility is box-shaped with the dimen-sions 480 · 390 mm area, 140 mm height, and an approxi-mate mass of 44 kg. It is equipped with UV radiation sensors(OEC GmbH, Germany), a radiometer (Dexter 6M ThinFilm Based Thermopile Detector), and temperature sensors.Each sample compartment containing the Edinburgh sam-ples for the BOSS experiments has two layers for samples(tray 1 and 2, compartment 3; Fig. 2), with the upper samplecarrier containing samples that are subjected to the fullrange of environmental factors including UV radiation andthe lower sample carrier containing samples that are ex-posed to the same factors with the exception of UV radia-tion. In addition to the UV-exposed top layer and darkcontrol bottom layer, the samples were exposed to ‘‘space’’and ‘‘simulated martian’’ conditions to see the degree ofACM protections under these different environments; seeTable 1 for an overview of the condition parameters. Thefilter frame windows on top of each sample tray allow forwavelength exposure of top-layer UV-exposed samples tobe controlled, that is, cutoff filters for the simulated martianflux (Rabbow et al., 2017).
Samples were exposed to their respective conditions for531 days. This does not include the first 62 days of themission, in which samples were protected from solar irra-diation during a period of outgassing. Previous flights hadexperienced discoloration of the sample windows, thoughtto be caused by radiation reacting with the pressurizedcontents prior to outgassing, resulting in the precautionarymeasures taken on this flight. Additionally, approximately 6
FIG. 2. Positioning of samples in BOSS setup in theEXPOSE-R2 platform. The upper sample carrier holdssamples exposed to the full range of environmental factorsincluding UV radiation (top layer). The lower sample carriercontains samples that are exposed to the same factors withthe exception of UV (bottom layer); the position of theEdinburgh experiments is marked with the red square.Figure taken from Rabbow et al. (2017). (Credit: ReneDemets, ESA; available via license CCBY4.0)
AGGREGATED CELLS PROVIDE PROTECTION AGAINST SPACE 997
weeks passed between the landing of the samples and arrivalat the lab for postflight analyses.
The calculated mean fluence (means of the correspondingindividually determined fields of view of the samples percompartment) of the biologically active wavelength range of200–400 nm was 458 – 32 MJ/m2 for tray 1 and 492 – 66MJ/m2 for tray 2. Postflight data confirmed the temperaturerange to have been between -20.9�C and 57.98�C. The at-mospheric pressure outside the Zvezda module was not di-rectly measured by equipment on board the EXPOSE-R2platform but was confirmed by Russian agency RSC Energiato vary between 1.33 · 10-3 and 1.33 · 10-4 Pa. For a moredetailed description of the EXPOSE-R2 setup, please con-sult the work of Rabbow et al. (2017).
There are three main ionizing radiation sources contrib-uting to the radiation environment outside the ISS: the Innerand Outer Radiation Belt (South Atlantic Anomaly protonsand electrons, respectively), GCR, and solar particle events.Maximum doses from these sources were determined to be844, 82, and 2960mGy/d, respectively, and the daily-averageddose rate calculated to be 71.6mGy/d (Dachev et al., 2017).Ionizing radiation caused by a solar energetic particle eventon June 22, 2015, was measured by the R3DR2 instrument onboard EXPOSE-R2. The increased resulting dose from theevent was determined to be >5000 mGy/h (Dachev et al.,2016).
The BOSS samples were stored on board the ISS beforethe return to Earth for 136 days. After arriving, they weretransported to DLR, Cologne, Germany, where the sampleswere confirmed to have not exceeded their shock and tem-perature limits and were sterilely prepared and sent to therespective investigators.
Table 2 gives an overview of the experimental samplelabels and respective conditions.
Samples 1-3-t-11 to 2-3-b-12 contained only biofilms (inblack); samples 1-3-t-15 to 2-3-b-16 contained planktoniccells (in blue). Henceforth, ‘‘top-layer’’ will denote samplesexposed to the incident solar radiation in the upper sample
carrier, and ‘‘bottom-layer’’ will denote the covered samplesfrom the lower sample carrier.
2.3. Postflight analyses
The following postflight analysis techniques were per-formed on the samples: liquid culturing, agar plate culturing,bright-field and fluorescent microscopy, transmission elec-tron microscopy, scanning electron microscopy, and Ramanspectroscopy.
Upon arrival at the Edinburgh lab, the sintered discsamples including duplicates were split into approximately1/3 sections. One section of each condition was used for
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liquid cultivation and one for agar plate cultivation, whilethe extra sections were kept at -80�C for the further assays.
2.3.1. Culturing. In order to establish whether cells wereable to be revived, samples were cultured in liquid mediaand on agar plates. This was conducted as a plus/minusassay to determine whether the cells were culturable, that is,able to replicate. As Gloeocapsa sp. cells form aggregates,single colony counting was found to be imprecise and notrepresentative of actual cell numbers. For liquid cultivation,one-third of one of the duplicates was placed in BG-11medium (Sigma-Aldrich, 73816) and aerobically cultured atroom temperature (21�C) for 3 months under ambient labo-ratory illumination (intensity = 50mmol/m2/s). For agar platecultivation, one-third of one of the duplicate disc sections wasgently ground up with mortar and pestle in sterile water andthen spread onto BG-11 agar plates. Plates were incubated 3months as above. Both positive and negative liquid and platecontrols for the medium were established, using Chroo-coccidiopsis sp. 029 (Cockell et al., 2005) as positive controlsto demonstrate healthy cyanobacterial growth in the chosenmedium and medium sterility.
2.3.2. Staining. Live/Dead stain (SYTO 9 and propi-dium iodide). In order to determine the overall viability ofsamples in addition to culturing, the LIVE/DEAD BacLightBacterial Viability kit was used (Thermo Fisher Scientific,catalog number L7012), which indicates cell viability as afunction of membrane integrity. Five microliters of SYTO 9dye (1.67 mM) and propidium iodide (PI, 1.67 mM) werecombined. One microliter of this solution was added to H2O tomake a 1:10 working stock solution. Five microliters of theworking stock solution was added per 200mL of sample. Afterincubation for 20 min at room temperature and protected fromlight, 10mL per sample was trapped on a microscope slide andviewed with a fluorescent microscope (Leica DM4000 B).SYTO 9 has an excitation wavelength of 485 nm and anemission wavelength of 498 nm (source: LIVE/DEAD Bac-Light Bacterial Viability kit online protocol).
Fluorescein diacetate. To further establish the extent ofdamage to cells, their enzymatic activity was measured byusing fluorescein diacetate (FDA) supplied by ThermoFisher Scientific (catalog number: F1303). Active enzymes(esterase) hydrolyze the dye, which is indicated by a changein fluorescence. Fifty milligrams of FDA was added to10 mL of acetone, of which 1 mL was added per 200 mL ofsample and incubated for 20 min at room temperature. Tenmicroliters per sample was trapped on a microscope slideand viewed by fluorescence microscopy (see above) at anexcitation wavelength of 485 nm.
DiBAC4(3). An additional assay to establish cell damageis the measurement of membrane potential, which can bedone by using DiBAC4(3) (bis-(1,3-dibutylbarbituric acid)trimethine oxonol). If a cell is depolarized, the dye can enterand bind to proteins, which is indicated by a change influorescence. DiBAC4(3) was supplied by Thermo FisherScientific (catalog number: B438). One microliter of thesolution was added per 100mL of sample and incubated atroom temperature for 20 min. The dye has an excitation max-imum of 490 nm and emission maxima of 516 nm (source:Thermo Fisher Scientific online protocol).
2.3.3. Transmission electron microscopy prepara-tion. Transmission electron microscopy (TEM) sampleswere centrifuged at 10,000g/min for 5 min with two washingsteps with phosphate-buffered saline. The supernatant wasdiscarded after each wash, and the sample was fixed afterthe second wash with 3% glutaraldehyde in 0.1 M sodiumcacodylate buffer (pH 7.3). After 2 h of fixation, sampleswere washed three times in 0.1 M sodium cacodylate for10 min each. Specimens were then post-fixed in 1% osmiumtetroxide in 0.1 M sodium cacodylate for 45 min, then wa-shed three times for 10 min of 0.1 M sodium cacodylatebuffer. These samples were then dehydrated in 50%, 70%,90%, and 3 · 100% ethanol for 15 min each, then in two10 min changes in propylene oxide. Samples were thenembedded in TAAB 812 resin. One micrometer–thick sec-tions were cut on a Leica Ultracut ultramicrotome, stainedwith Toluidine Blue, and viewed in a light microscope toselect suitable areas for investigation. Ultra-thin sections,60 nm thick, were cut from selected areas, stained in uranylacetate and lead citrate. A Philips/FEI CM120 Biotwin TEMwas used to image samples.
2.3.4. Scanning electron microscopypreparation. Scanningelectron microscopy (SEM) sample preparation is identical tothe TEM sample preparation up until, and including, dehy-dration using 50%, 70%, 90%, and 3 · 100% ethanol.
Each dehydration was followed by critical point dryingwith liquid carbon dioxide. After mounting on aluminumstubs with carbon tabs attached, the specimens were sputter-coated with 20 nm gold palladium. A Hitachi 4700 II coldfield-emission SEM was used to image samples with aworking distance of 14.8 mm and 5 kV laser.
2.3.5. Raman spectroscopy. Raman signatures weremeasured with a Renishaw inVia confocal Raman microscope:laser wavelength = 514 nm at 1% laser power (0.15 mW), ex-posure time per second = 1. Spectra were analyzed with theWiRE Raman analysis software version 4.4.
3. Results
3.1. Culturing
As shown in Table 3, after the first 3 months of culturing,only one of the biofilm samples (1-3-b-11) that was exposedto the space conditions (i.e., vacuum and full solar spec-trum) was culturable. While all the duplicate bottom layersof biofilm samples under simulated martian conditions werereculturable in both liquid and on plates, only one of the top-layer biofilms under these conditions was able to be recultured.
Cultured planktonic cells (shown in blue) presented thefewest viable samples. None of the top-layer or bottom-layer samples that were exposed to space conditions retainedviability. Only two planktonic samples (one top-layer andone bottom-layer) exposed to the less harsh simulatedmartian conditions showed growth.
The 9-month growth data shown in Table 3 indicates thatin both the liquid and agar cultures top-layer and bottom-layer biofilm samples that had shown growth after 3 monthsappeared to have lost viability (i.e., no visible cell colonies).All biofilm samples that had been exposed to space condi-tions and cultured in liquid media had died after 9 months ofculturing, while bottom-layer biofilm samples exposed to
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simulated martian conditions survived. Fewer planktonicsamples were still viable than at the 3-month time point,with only two bottom-layer samples from the simulatedmartian environment that still showed growth with no ob-servable growth in any of the other conditions.
In conclusion, the growth assay demonstrated that thebiofilms showed the most viable samples under bottom-layer simulated martian conditions, with no UV-radiation-exposed samples surviving the exposure to space conditions.Moreover, the planktonic top- and bottom-layer cells underspace conditions produced no reculturable samples.
The only planktonic samples that were still viable after 9months were those that had been under the simulated mar-tian conditions and protected from UV, of which 50% re-mained culturable.
3.2. Fluorescence microscopy
In order to assess the nature of damage to cells under thevarious conditions, multiple fluorescent stains were appliedand analyzed under the microscope. Bright-field images ofsamples show the clumped colonies of cells and the dis-tinctive green coloring of the cyanobacteria as seen inFig. 1a. However, the retention of pigmentation is not in-dicative of undamaged cells. Internal damage may still havebeen sustained, which can be visualized by the use of stains.
3.2.1. Live/Dead. The SYTO 9 PI live/dead stain is atwo-component stain, which indicates cell viability as afunction of membrane integrity. SYTO 9 is green fluoresc-ing and permeates all cell membranes and stains nucleicacids. PI, on the other hand, cannot permeate cell mem-branes except if they are ruptured and stains the nucleicacids red. Below are selected representative live/dead im-ages from various conditions.
Figure 3a shows a top-layer biofilm sample exposed to thesimulated martian conditions displaying enhanced mem-brane damage shown by a dull green fluorescence, whichcorresponds to the low number of reculturable samples after3 months. Similar to the growth observed under the spaceand simulated martian conditions, the top-layer and bottom-layer samples exposed to space conditions (not shown) sus-tained more damage than those from the simulated martianenvironment. There were, however, small areas of greenfluorescence that may indicate intact cells. The planktoniccells exposed to the space and simulated martian environ-ments (top-layer space sample in Fig. 3b) also showed ex-tensive damage shown by red/dull green cells. However,despite the culture data showing reduced viability ofplanktonic samples, in comparison to the biofilm samples,the live/dead stains reveal sections of live, bright greenfluorescing cells, especially in samples from the simulatedmartian conditions (data not shown).
3.2.2. Fluorescein diacetate. Fluorescein diacetate (FDA)is itself nonfluorescent. However, when accumulated and hy-drolyzed by live cells it becomes fluorescent; dead cells cannothydrolyze FDA and do not fluoresce. This stain can therebyindicate whether a cell is enzymatically active (green) or not.This gives extra information regarding cell damage, as thelive/dead stain on its own cannot show us whether a ‘‘live’’cell is metabolically active.
Both biofilm top-layer samples subjected to space andsimulated martian conditions showed no signs of enzymaticactivity (simulated martian conditions shown in Fig. 3c).However, similar to results with the live/dead stain in Fig. 3a,3b, the top-layer planktonic samples exposed to space andsimulated martian conditions showed no signs of enzymaticactivity (not shown). Bottom-layer cells exposed to the sim-ulated martian environment (Fig. 3d) showed greater activityalong the periphery of the cell, indicating protection offeredby the surrounding cells that showed no enzymatic activity.
3.2.3. DiBAC4(3) and autofluorescence. Bis-(1,3-dibutyl-barbituric acid) trimethine oxonol, or DiBAC4(3) for short, is apotential-sensitive molecule that can enter depolarized cellsand bind to proteins or the membrane, staining them green.Thus, green staining indicates the loss of potential. The Di-BAC4(3) images are shown in conjunction with the naturalautofluorescence of the cyanobacteria, which appears red inhealthy cells.
One key observation was the loss of membrane potentialindicated by green fluorescence (Fig. 3e, red arrow) ac-companying a lack of red autofluorescence in the samples(Fig. 3f), suggesting the loss of membrane potential couldbe coupled with further cell damage as indicated by the lossof autofluorescence. Figure 3e also demonstrates the local-ized retention of membrane potential (absence of fluores-cence, blue arrow) in the same cell aggregate, which wasaccompanied by the preserved autofluorescence in the sameregion in Fig. 3f.
Top-layer biofilms under space conditions showed completeloss of autofluorescence in certain parts of the culture, accom-panied by green fluorescence indicating loss of membrane po-tential (Fig. 3e, 3f). However, samples from the simulatedmartian environment were still able to retain their auto-fluorescence and not suffer substantial membrane potential loss(not shown). Top- and bottom-layer planktonic samples showedno signs of fluorescence, indicating an intact membrane po-tential and retention of autofluorescence after exposure to bothspace and simulated martian conditions (not shown).
3.3. Transmission electron microscopy
Transmission electron microscopy allows for high mag-nification and resolution of ultra-thin fixed sections of cellsand their interior. TEM was performed on select samples tofurther visualize cell integrity.
The top-layer biofilms showed increased cell lysis andlack of a cell sheath (white arrow, Fig. 4a). Similarly, theplanktonic cells exposed to both space and simulated martianenvironments (not shown) show the dark sheath detachedfrom the cell and cell lysis. In addition, there was evidence ofa smaller bacterial species (dark spots in Fig. 4a, blue arrows)that was sometimes found within the lysed cyanobacterialcell. The additional bacteria will be further discussed in thesection ‘‘Bacterial co-culture’’ (Section 3.6).
3.4. Scanning electron microscopy
In addition to TEM, SEM was performed, which enableshigh-resolution 3D images of surface topography. SEM wasperformed to visualize general structural damage to the cellsand in the hope of providing evidence of the smaller bacteriaseen in the TEM photo, in Fig. 4a. SEM evidence of the
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FIG. 3. Fluorescence microscopy assays showing damage to cell components. Live/Dead stain showing intact membraneintegrity in green of top-layer biofilm sample from simulated martian conditions (2-3-t-12) (a) and top-layer planktonicsample from space conditions (1-3-t-15) (b). FDA stain showing metabolic activity (in green) of top-layer biofilm samplefrom simulated martian conditions (2-3-t-12) (c) and bottom-layer planktonic sample from simulated martian conditions (2-3-b-15) (d). DiBAC4(3) stain of top-layer biofilm sample from space conditions (1-3-t-11) showing lack of membranepotential in green (red arrow) and localized unstained retained potential (blue arrow) (e) and natural autofluorescence in redin the same sample (f).
AGGREGATED CELLS PROVIDE PROTECTION AGAINST SPACE 1001
additional bacterium is discussed in the section ‘‘Bacterial co-culture’’ (Section 3.6). Figure 4b of the bottom-layer biofilmsample (simulated martian conditions) clearly shows signifi-cant rupturing of the cyanobacterial membrane and addition-ally shows an example of a puncturing of the cell membranewith the cell maintaining most of its structural integrity (ar-rows). The photo also shows the variability of cyanobacterialsurface texture of either smooth or ‘‘framboidal.’’
3.5. Raman spectroscopy
Raman spectroscopy was used to assess whether, and towhat extent, the cyanobacteria’s carotenoids had been
damaged. This not only provides additional information onthe scope of structural damage to the cell but also givesinsight into the role of biofilms in the preservation of de-tectable biosignatures.
Characteristic Raman scattering allows for the identifica-tion of chemical compounds and crystal structure. Ramananalysis of biological samples can result in very noisy signals.However, carotenoids lend themselves well to this analysistechnique, as they have a very distinct signal pattern con-sisting of three main peaks at *1000, 1150, and 1500 cm-1
(corresponding to a C–CH, C–C, and C = C chemical bond,respectively) when excited at 514 nm (‘‘control’’ sample,Fig. 5) and are considered key biomarkers for pigmented life-forms. Peak heights indicate substance concentration (in thiscase carotenoid) and were used to demonstrate the state ofpreservation or damage to the cell. Peak heights are shown inTable 4.
When comparing the top-layer samples from space andmartian conditions (1-3-t-11 and 2-3-t-11, respectively), themartian condition sample showed a higher intensity andclearer Raman signal. When the intensities are overlaid(Fig. 5), we get a clearer picture of signal clarity as well aspeak height. Radiation is deleterious to biomarkers, as itdegrades organic matter. It is therefore not surprising thatthe samples exposed with cutoff filters were less degradedthan those exposed to unfiltered radiation in space. A healthycontrol sample (green, Fig. 5) was added for an additio-nal comparison between top-layer and dark sample signalretention.
The top-layer planktonic cell samples (1-3-t-15 and 2-3-t-15, blue in Table 4) showed the sample exposed to the spaceconditions (1-3-t-15) having higher peaks than the top-layersample (2-3-t-15) from the simulated martian conditions—areversal of the trend demonstrated in the biofilm samples.Additionally, when compared to the biofilm samples (blackin Table 4) exposed to space conditions, the peak heights arecomparable but generally higher than both biofilm carot-enoid peaks. Yet, when compared to biofilm peaks undersimulated martian conditions, the planktonic cells’ carotenoidpeaks are consistently lower than those of the biofilms.Moreover, in the context of all other conditions (Fig. 5), theplanktonic samples (1-3-t-15 and 2-3-t-15, in blue) show thedeterioration of signal clarity in spite of relatively tall peakheight of 1-3-t-15, as seen in Table 4.
3.6. Bacterial co-culture
Evidence of an additional bacterial species was observedin Fig. 4, most likely to be the co-cultured, pigmented a-proteobacterial species. The secondary species was presentin the Gloeocapsa sp. environmental samples and was usedto test the ability of the cyanobacteria to harbor an addi-tional species in hostile conditions.
Pink-colored colonies thought to be the proteobacteriawere observed on the agar plate cultures of the EXPOSE-R2cyanobacteria after 3 months of culturing. However, the co-culture was unable to be isolated under lab conditions in avariety of media and temperatures. Figure 6a shows live(green) cyanobacteria cells but also much smaller live cellswithin and around the cyanobacterial structures and a ‘‘jet’’of these smaller bacteria erupting from the larger cyano-bacteria aggregate after slight pressure was applied to the
FIG. 4. TEM and SEM images showing damage and co-cultured bacteria. Ultra-thin section of top-layer biofilmTEM image showing loss of gelatinous sheath (white arrow,a) and evidence of smaller co-cultured bacteria (blue ar-rows, a) in top-layer biofilm sample from space conditions(1-3-t-11). SEM image showing surface topography of thesample and cell rupturing (white arrows, b).
sample under the microscope slide. At higher magnification,these smaller cells were clearly distinct from the cyano-bacteria aggregates, as they showed no sign of sheath oraggregates that even single cyanobacteria demonstrate(Whitton and Potts, 2007). They were also detectable in theTEM pictures in Fig. 6c, 6d. Coccoidal shapes correspondingto the smaller bacteria size are also visible in the SEM imagein Fig. 6b. In the TEM pictures, multiple smaller bacteria
were often observed to be located within the ruptured largercyanobacteria cells.
4. Discussion
Space conditions and other extreme environments, suchas the surface of Mars, can be simulated to a certain extenton the surface of Earth. However, short of sending samplesto other planetary bodies, sending samples into LEO pro-vides a practical way of obtaining accurate information onbacterial viability after exposure to similarly extreme con-ditions. LEO enables exposure to environmental factorsgenerally outside the scope of simulation such as GCR andshortwave UV radiation (<200 nm). ESA’s EXPOSE plat-form provides an opportunity for scientists to subject a va-riety of taxa and molecules to these extreme environments,with objectives varying from endurance testing of materialsto probing the preservation of biomarkers.
In this study, we exposed a cyanobacterium, Gloeocapsa,to LEO conditions for 1.5 years in the framework of theBOSS experiment with a two-fold goal: first, of determiningthe ability of simple cell aggregates to protect cyanobacterialcultures from space and a simulated martian environmentcompared to planktonic cells; and second, to investigate abilityof the cyanobacterial species to provide a secondary bacterialspecies with a microhabitat in these harsh conditions.
FIG. 5. Overlay of Raman spectra. Overlay of biofilm (black, gray), planktonic (blue), and control (green) sample Ramanspectra with relative intensity shown on the y axis (arbitrary units). Sample conditions: top-layer biofilm under spaceconditions (1-3-t-11); top-layer biofilm under simulated martian conditions (2-3-t-11); top-layer planktonic cells underspace conditions (1-3-t-15); top-layer planktonic cells under simulated martian conditions (2-3-t-15); bottom-layer biofilmunder space conditions (1-3-b-11); bottom-layer biofilm under simulated martian conditions (2-3-b-12); bottom-layerplanktonic cells under space conditions (1-3-b-15); bottom-layer planktonic cells under simulated martian conditions (2-3-b-16).
Peaks are at *1000, 1150, and 1500 cm-1 from the various experi-mental conditions. Black indicates biofilm samples used in BOSS; blueindicates BOSS planktonic samples. As shown in Table 2, ‘‘11’’ and‘‘12’’ samples and ‘‘15’’ and ‘‘16’’ samples are duplicates, respectively.
AGGREGATED CELLS PROVIDE PROTECTION AGAINST SPACE 1003
Gloeocapsa sp. cells form loosely structured ACMs inlieu of a well-structured biofilm. Although it is unclearwhether gelatinous amorphous structures could be consid-ered a primitive ‘‘proto’’-biofilm, the hypothesis of theEdinburgh BOSS experiment is that less-ordered cell ag-gregates also share protective characteristics of more-ordered biofilms and protect organisms against deleteriousenvironmental conditions.
Results show that, while Gloeocapsa sp. biofilm samplesstill sustained cellular damage and were mostly reculturableonly when protected from UV exposure, biofilm Ramansignal retention was much greater than that of planktoniccells.
The protection by biofilms is further supported by growthresults from the planktonic samples. Planktonic cells show
the lowest viability, with only two samples in total beingculturable and both under the less harsh, simulated martianconditions. This is shown in Fig. 6c, 6d, where planktoniccells show the loss of the gelatinous sheath and rupturedcells to a greater extent than in biofilm samples.
Interestingly, the planktonic stain results show more in-tact cells, despite the low viability of the growth assays.This may suggest that the culture is in a ‘‘viable but notculturable’’ state, similarly shown in Deinococcus geother-malis by Frosler et al. (2017), where the model bacteriawere exposed to simulated space and martian conditions.Live/dead stains show small patches of live cells, especiallyin the sample from simulated martian conditions, which isconsistent with FDA results showing small areas of enzy-matic activity. The planktonic DiBAC4(3)-stained samples
FIG. 6. Evidence of co-cultured bacteria. Live/dead stains show smaller green fluorescent cells (a) in sample 1-3-b-16(bottom-layer planktonic cells under space conditions). SEM shows potential bacteria cells on the surface of cyanobacteriacells (white arrows, b). TEM pictures of samples show smaller bacteria inside lysed cells of cyanobacteria in sample 2-3-t-15 (top-layer planktonic cells under simulated martian conditions) and sample 1-3-b-12 (bottom-layer biofilm sample underspace conditions) (c, d).
show the highest intact membrane potential coupled withpreserved autofluorescence out of all the conditions.
In spite of the apparent preservation of cell integrity in thefluorescent microscopy pictures, the planktonic Ramanspectra show the most deteriorated carotenoid signal out ofall sample conditions. The planktonic spectra of the space-exposed samples are comparable in peak height to bothbiofilm samples, despite the lack of culturable cells from thesamples exposed to the space environment, which was ini-tially surprising. However, when planktonic spectra fromthe simulated martian conditions are compared to thespectra of biofilms exposed to simulated martian conditions,there is a hierarchy with biofilm samples having the clearestpeak signatures, followed by planktonic samples. Moreover,when all spectra are overlaid and compared by signal clarity,the spectra of the planktonic cells were diminished incomparison to other conditions. The clearest signal is seenin the biofilm sample exposed to simulated martian condi-tions, which is the least harsh and is consistent with otherresults for this condition. These data not only support thehypothesis of the ability of disordered cell aggregates toprotect cellular structures, such as pigments, in living cells,but show that these aggregates also contribute to the pres-ervation of biosignatures after cell death.
Some of the results we observed with planktonic samplesmay be caused by the incomplete separation of cells fromtheir aggregated form during the preflight preparation.Gloeocapsa sp. cells do not naturally appear in planktonicform; rather, they form aggregates often directly after mi-tosis (Whitton and Potts, 2007). Therefore, assuming thepreflight separation was successful, any consequent growthmay have resulted in the reformation of aggregates. Ad-ditionally, cells may have re-aggregated due to the desic-cation process and clustered within the disc pores. Thiswould explain the damage still caused by the absence of anintact biofilm, yet the retention of smaller aggregates wouldallow for a subset of cells to be cultured. Additionally, thesmaller clumps of cells would be able to fit into smallerpores on the sintered discs before being desiccated. Thiswould result in the ‘‘planktonic’’ cells being protected bythe small aggregates as well as the discs themselves. Al-though it is shown to not be substantial enough protection toallow for a high number of culturable samples, it wouldexplain the partially preserved membrane integrity and en-zymatic activity. Evidence of these smaller aggregates per-sisting in planktonic cell samples can be seen in thefluorescent stain microscopic pictures (Fig. 3).
As stated, the Gloeocapsa sp. species is of particular in-terest with regard to its biofilm, as it produces an ACM thatlacks a layered biofilm structure. There have been previousstudies showing the protective capacity of biofilms (Flem-ming, 1993; Los Rıos et al., 2003; Wierzchos et al., 2006).However, the Edinburgh BOSS results demonstrate that theGloeocapsa sp. cell aggregates are also able to confer pro-tection. These results show that spatially random cell divisioncan give rise to cell aggregates that can provide protectionagainst extreme radiation conditions in analogy to morestructured layered biofilms. Biofilms have previously beenshown to provide protection against harsh conditions, whichsuggests that under high-UV radiation fluxes, such as mayhave been present on early Earth, simple, more primitive cellaggregation can also provide significant protection.
While the focus of BOSS was protection from a biogenicsubstance for one species, the secondary hypothesis testedwith these experiments was that of interspecies protection.
Despite the samples being prepared as a cyanobacterialmonoculture, the postflight growth assays showed that a co-cultured, pink proteobacteria had survived LEO exposure,which was most likely to be the bacterial species cohabit-ing with the cyanobacteria when it was first isolated inEXPOSE-E (Olsson-Francis et al., 2010). Although it was notobservable on all agar plates (i.e., duplicates), it was observedfor each condition. This is surprising in the case of theplanktonic samples, as it would be expected that they pro-vide little additional protection when compared to biofilmprotective properties (Baque et al., 2013b). However, asdiscussed in the previous section, it is unlikely that thesamples were truly planktonic, but instead remained insmaller aggregates.
More compelling evidence of the cyanobacteria providinga microhabitat for the secondary bacteria was seen underlive/dead stain. TEM images shown in Figs. 4a and 6c, 6dshed more detailed light on the exact location of the co-culture. The proteobacteria are often observed to be withinruptured cyanobacteria cells. This suggests that the lysedcyanobacteria may have provided enhanced protection to theco-cultured bacteria, though the relationship between thetwo cultures is as yet uncharacterized. Although we cannotquantify whether the cyanobacteria extend the lifetime ofthe co-cultured species or prevent it from being killedcompletely, the observation at a minimum demonstrates thatlarge cells, whether they retain viability or not, can harborhitchhiking smaller species of microorganisms that are ca-pable of surviving under prolonged space conditions. Thishas significant implications for planetary protection. If anorganism is determined to not be able to survive space orspecific planetary conditions, and thereby is not deemed athreat to planetary protection protocols, it may still be har-boring one or multiple additional species that were protectedfrom extreme conditions by the known organism. Ad-ditionally, the experiments show that even if steps are takento prepare monocultures, especially of environmental sam-ples, this does not exclude the possibility that co-culturedmicrobes may still be present, such as within larger bacterialagglomerates. Most bacterial cultures naturally exist in amultispecies environment as opposed to monocultures oftengrown in the labs, which may account for the vast number of‘‘unculturable’’ bacterial and archaean species to date(Stewart, 2012). As the likelihood of co-culture species’presence is high, planetary protection protocols may have tobe adjusted to take more realistic, multispecies contamina-tion and respective protection into consideration. Thesefindings are also significant regarding understanding andcontrolling the microbiome of the ISS. It is already estab-lished that biofilm cultures contribute to the deterioration ofmaterials on board (Gu et al., 1998). However, our workshows that they may also harbor secondary species, whichcould potentially be pathogens—even if the biofilm-formingcells themselves are no longer viable—which is of greatconcern to astronaut health (Novikova et al., 2006).
Finally, the results of the BOSS and co-culture experi-ments provide information on the survival of microbial-associated biosignatures that might be sought in martianplanetary exploration missions. The biofilm data show that
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under simulated martian conditions for 1.5 years carotenoidpigment is still detectable. Appropriate instrumentation de-sign and testing to establish upper bounds of detectabilitycan influence future Mars lander missions and can inform ondata from ESA’s ExoMars rover or NASA’s 2020 Marsrover that are equipped with instruments such as Ramanspectroscopy.
5. Conclusion
To test the shielding properties of biofilms, cyanobacteriawere exposed to the conditions of LEO and a martian en-vironment simulated in space for 1.5 years. Upon return toEarth, biofilm samples and planktonic samples that had beenexposed to simulated martian conditions were able to berecultured. In spite of the ability of biofilm samples to berecultured and to maintain a Raman signal for carotenoids,fluorescence microscopy revealed the presence of damagesustained by various cell components. While fluorescencemicroscopy of the planktonic cells in the BOSS experimentshowed less damage than the biofilms, culturing and Ramananalysis revealed the more extensive damage to the plank-tonic cells. These results demonstrate the increased protec-tion biofilms can provide to shield from extreme radiationconditions such as those found on Mars. In addition tofactors that enable the survival of single species exposed tomultiple extreme conditions, the bacterial cultures them-selves were shown to allow for smaller co-cultivated speciesto survive within the cell aggregate space. Together, theseresults illustrate the need for planetary protection studiesand space station microbiome studies and protocols to takemultispecies aggregates into consideration.
Acknowledgments
This work was supported by a UK Space Agency AuroraStudentship Grant (STFC ST/M003612/1) to J.W. C.S.C.was supported by the Science and Technology facilitiesCouncil: ST/M001261/1. The support of the Wellcome TrustMulti User Equipment Grant (WT104915MA) is acknowl-edged for use of the TEM.
Author Disclosure Statement
No competing financial interests exist.
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Address correspondence to:Jennifer Wadsworth
University of EdinburghJames Clerk Maxwell Building
1. Cottin Hervé, Rettberg Petra. 2019. EXPOSE-R2 on the International Space Station (2014–2016): Results from the PSS andBOSS Astrobiology Experiments. Astrobiology 19:8, 975-978. [Abstract] [Full Text] [PDF] [PDF Plus]
