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Home , Biosignature, Enceladus Life Finder

Communication Towards Determining Retention in Icy World Plumes

Kathryn Bywaters 1,*, Carol R. Stoker 2,*, Nelio Batista Do Nascimento Jr. 2 and 2, Lawrence Lemke † 1 SETI Institute, Moffett Field, CA 94043, USA 2 NASA Ames Research Center, Space Science Division, Moffett Field, CA 94035, USA; nelio.b.nascimentojr@.gov (N.B.D.N.J.); [email protected] (L.L.) * Correspondence: [email protected] (K.B.); [email protected] (C.R.S.); Tel.: +1-650-604-2295 (K.B.); +1-650-604-6490 (C.R.S.) Retired. †  Received: 21 March 2020; Accepted: 14 April 2020; Published: 16 April 2020 

Abstract: With the discovery of the persistent jets of water being ejected to space from , an understanding of the effect of the space environment on potential and in them is necessary for planning life detection missions. We experimentally determine the survivability of microbial cells in liquid medium when ejected into vacuum. Epifluorescence microscopy, using a stain, and SEM imaging were used to interrogate the cellular integrity of E. coli after ejected through a pressurized nozzle into a vacuum chamber. The experimental samples showed a 94% decrease in visible intact E. coli cells but showed a fluorescence residue in the shape of the sublimated droplets that indicated the presence of . The differences in the experimental conditions versus those expected on Enceladus should not change the analog value because the process a sample would undergo when ejected into space was representative. E. coli was selected for testing although other types could vary physiologically which would affect their response to a vacuum environment. More testing is needed to determine the dynamic range in concentration of cells expected to survive the plume environment. However, these results suggest that lipids may be directly detectable evidence of life in icy world plumes.

Keywords: icy world; plume; life detection; microbes; lipids; ; Enceladus;

1. Introduction In the search for life outside of Earth it is necessary to quantify the potential abundances of biosignatures, including whole single-celled organisms, in target environments. Sample collection techniques aside, which can also affect the integrity of the biosignatures being collected, the physical processes of the target environment can have an effect on the retention of biosignatures. Knowledge of the types and expected quantities of these biosignatures will enable detection strategies and sensitivity limits to be set for proposed life detection instrumentation. To this end, an understanding of the effect of the physical environment on organisms and biosignatures is essential. One planetary environment that is of growing interest for life detection missions are icy moons, particularly Enceladus and Europa. Enceladus has a sub-ice-shell global ocean [1] of salty water [2]. It is estimated that the ocean is 26–31 km thick, beneath a 21–26 km thick icy crust [1]. The discovery, by the Cassini orbiter mission, of plumes of water carrying fine icy particles venting from four long fractures in the south polar region of Enceladus [3–6], along with evidence indicating ongoing hydrothermal activity [7], has focused attention on sampling the potentially habitable environment of the subsurface liquid water on that body [8–11]. The persistent jets of water being ejected provide a source of potentially life-bearing material that is emitted to space.

Life 2020, 10, 40; doi:10.3390/life10040040 www.mdpi.com/journal/life Life 2020, 10, 40 2 of 12 Life 2020, 10, x 2 of 12

TheThe habitability habitability of of the the Enceladus Enceladus subsurfacesubsurface oc oceanean has has been been inferred inferred from from the the Cassini Cassini orbiter orbiter whichwhich sampled sampled plume-derived plume-derived material.material. Cassini detected detected frozen frozen droplets droplets of of water water [4] [ 4with] with salinity salinity comparablecomparable to to Earth’s Earth’s ocean ocean suggesting suggesting the the water water is inis in contact contact with with a rocky a rocky core core [12 [12].]. Both Both H2 Hand2 and CO 2 haveCO been2 have detected been detected in the plume,in the plume, as have as organic have orga carbonnic molecules [13,14 ].[13,14]. Nitrogen was also was present also aspresent NH3 which as NH is3 readilywhich is used readily by microbes.used by microbes. The major The elements major elements needed needed for life for (C, life H, (C, N, H, O, N, P, S)O, haveP, beenS) have detected been or detected are expected or are to expected be present to [be15 ].pres McKayent [15]. et al. McKay [15] conducted et al. [15] anconducted extensive an consideration extensive ofconsideration the habitability of ofthe the habitability Enceladus of subsurface the Enceladus ocean, subsurface including ocean, a breakdown including of a thebreakdown requirements of the for life,requirements limits of life andfor life, how limits the Enceladus of life and ocean ho compares.w the Enceladus Based on ocean these considerations,compares. Based it seemson these likely thatconsiderations, the subsurface it seems ocean likely of Enceladus that the subsurface would be habitableocean of Enceladus to terrestrial would microbes be habitable if introduced to terrestrial there, microbes if introduced there, although whether the conditions allowed for the origin of life on although whether the conditions allowed for the origin of life on Enceladus is an open question [16]. Enceladus is an open question [16]. The potential habitability of icy moons has led to studies addressing possible microbial survival The potential habitability of icy moons has led to studies addressing possible microbial survival and growth [17–22], as well as estimates of concentrations [23–25] in these environments. and growth [17–22], as well as estimates of biomass concentrations [23–25] in these environments. The results show that, under certain conditions (degree of convection, mantle rheology, etc.), there The results show that, under certain conditions (degree of convection, mantle rheology, etc.), there wouldwould be enoughbe enough chemical chemical energy energy to sustain to sustain an ecosystem an ecosystem of chemoautotrophic of chemoautotrophic organisms. organisms. The EuropaThe LanderEuropa Study Lander 2016 Study Report 2016 [23 Report], using [23], Earth using analogue Earth analogue environments environments (i.e., subglacial (i.e., subglacial lakes), placed lakes), the upperplaced range the ofupper cell densityrange of at cell approximately density at approxim 100 cellsately per mL 100 and cells set per that mL value and asset the that limit value of detectionas the forlimit life detectionof detection instruments. for life detection instruments. NeveuNeveu et et al. al. [26 [26]] presented presented NASA’s NASA’s Life Life Detection Detection Ladder, Ladder, which which outlined outlined the methodsthe methods for extantfor lifeextant detection life beyonddetection Earth. beyond One Earth. of the One given of criteriathe given for acriteria convincing for a life-detectionconvincing life-detection measurement wasmeasurement that the measurement was that the context measurement must be sucontextfficiently mu “survivable”.st be sufficiently The term“survivable”. survivable The was term used tosurvivable address the was issue used that to physical,address the chemical, issue that and phys geologicalical, chemical, conditions and geological in the environment conditions in that the the sampleenvironment encounters that had the notsample destroyed encounters the targeted had not signs destroyed of life. Herethe targeted we investigated signs of thelife. survivability Here we ofinvestigated microbial cells the insurvivability simulated of icy microbial world plumes cells in simulated as they are icy introduced world plumes into as the they highly are introduced desiccating conditionsinto the highly of vacuum. desiccating Thiswork conditions begins of to vacuum. experimentally This work deduce begins the to survivabilityexperimentally and deduce thereby the the potentialsurvivability abundances and thereby of organisms the potential/ abundances that of could organisms/biomarkers be expected in these that environments. could be expected in Ifthese there environments. is life in the oceans of icy moons that is being injected into space by the plumes (Figure1), it becomesIf there imperative is life in the to oceans quantitatively of icy moons understand that is being the einjectedffects of into this space process by the on plumes the integrity (Figure of cells1), andit becomes potential imperative biosignatures. to quantitatively Here we understand investigate the what effects would of this happen process toon a the specific integrity kind of of cells and potential biosignatures. Here we investigate what would happen to a specific kind of terrestrial microbes inhabiting a liquid water environment if they were injected into vacuum. We terrestrial microbes inhabiting a liquid water environment if they were injected into vacuum. We measure the physical response of injected by plumes into the highly desiccating measure the physical response of microorganisms injected by plumes into the highly desiccating conditions of vacuum. It has been established that and even organisms can survive conditions of vacuum. It has been established that biomolecules and even organisms can survive space vacuum [27,28] even though they experience dehydration and cellular damage [29,30]. Simple space vacuum [27,28] even though they experience dehydration and cellular damage [29,30]. Simple andand complex complex organic organic molecules molecules havehave alsoalso beenbeen shown to to be be ubiquitous ubiquitous in in space space [31,32]. [31,32 ].However, However, thethe retention retention rate rate of ofintact intact cellscells and degree degree of of cellular cellular structural structural damage damage has hasyet to yet be to described be described or orquantified. quantified.

FigureFigure 1. 1.Schematic Schematic depictingdepicting hydrothermalhydrothermal heat heat transfer transfer from from an an icy icy moon moon core core to toliquid liquid water. water. CracksCracks in in an an icy icy crust crust overlying overlying the the liquid liquid allows allows plumes plumes that that erupt erupt into into space space potentially potentially carrying carrying cells andcells/or biomarkers.and/or biomarkers.

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2. Materials and Methods Escherichia coli (E. coli) was selected as the for testing. There are a wide range of model organisms from different target environments that could have been tested, such as a methanogen as this could harness the energy flux present on Enceladus [14], Nautilia profundicola, an anaerobic nitrate-reducing bacterium from an active seafloor vent, Colwellia psychrerythraea, a heterotrophic marine bacterium ubiquitous in cold marine ecosystems, or Bacillus pumilus a spore-forming bacterium that have shown especially high resistance to ultraviolet (UV) radiation. However, for the scope of this work we selected E. coli as a point of reference by which to begin measuring the effects of the plume environment on cell retention due to the vast body of work that has already been conducted on this organism. E. coli strain OP50 was maintained by weekly transfers to LB Broth (LB Broth: (1) 0.17 M NaCl (Fisher; S271), (2) 5 g yeast extract (Alfa Aesar; H26769), and (3) 10 g tryptone (Fisher; BP1421) added to 0.8 L of DiH2O. The pH was adjusted to 7.5 with NaOH, filled to 1 L, and then autoclaved). Cells were grown to late log phase and harvested by centrifugation at 6000 rpm for 10 min. Cells were washed to remove the LB broth (due to high intrinsic fluorescence) for all experiments. The cell samples were centrifuged for 10 min in 1.5 mL tubes. Being careful not to disturb the cell pellet, the supernatant was then removed and cells were resuspended in PBS by quickly vortexing (PBS: (1) 0.14 M NaCl (Fisher; S271), (2) 0.003 M KCl (Sigma; P-9333), (3) 0.01 M Na2HPO4 (Fisher; BP332), and (4) 0.002 KH2PO4 (Sigma; P5379) added to 0.8 L of DiH2O. The pH was adjusted to 8.3, filled to 1 L and then autoclaved). This was repeated for a total of three washes. Growth media and PBS buffer were made using ultra high purity water. A pH range of 7.5–8.3 was used: (1) to ensure E. coli cells were not disrupted and (2) because it is reasonable for seawater on Earth. A wide range of pH values have been proposed for Enceladus, potentially > 10 [33] and as low as 2.6 for Europa [34]. However, this paper only provides a first quantitative data point and more experimentation is needed. Primuline was selected as the fluorescence stain due to the fact that it is a non-destructive lipid stain. The case has been made that lipids are the universal biomarkers of [35] and therefore lipids are likely candidate measurement targets for a life detection mission. The use of a lipid stain also allowed for the visualization of the cellular membranes and helped determine if they had remained intact (this does not differentiate between live/dead cells). Primuline (MP Biomedicals 195454) was added to cells resuspended in the PBS buffer, to achieve cell concentration of ~105 cells/mL, at a concentration of 100 µM. A cell concentration of ~105 cells/mL was chosen to ensure that a sufficient number of cells would be captured in the sample collectors. Cell/dye combination was prepared in 50 mL Falcon tubes, quickly vortexed for mixing and then incubated for 10 min at room temperature in the dark. After the incubation period, stained cells were then washed again. Experiments were performed in a vacuum chamber of 463 mm diameter (473 L) (Figure2). Experiments were performed at starting pressures between 8.00 and 8.67 Pa. The chamber was depressurized using a Sargent Welsh Direct Torr 8851 roughing vacuum pump. Sample collectors (SEM capsules and microscope slides) were mounted on a custom-made acrylic plate that was fixed to one of the flanges in the chamber (Figure3). Life 2020, 10, 40 4 of 12 LifeLife 2020 2020, 10, 10, x, x 4 4of of 12 12

(a(a) ) (b(b) )

FigureFigure 2. 2. ( a()a )Experiments Experiments were were were performed performed in in in a a avacuum vacuum chamber chamber with with with a a a463 463 mm mm diameter diameter (473 (473 L), L), evacuatedevacuated with with a aroughing roughing pump. pump.pump. ( b (b) )Image Image of of injection injection system. system. (1) (1) Injection Injection Deck, Deck, (2) (2) Gas Gas Bottle, Bottle, (3) (3) SolenoidSolenoid vale, vale, (4) (4) Directional Directional valve, valve, (5) (5) Piston, Piston, (6) (6) In In Internalternalternal nozzle nozzle feed, feed, (7) (7) Action Action Trigger, Trigger, (8) (8) Solution Solution Reservoir,Reservoir, and andand (9) (9)(9) External ExternalExternal test testtest nozzle. nozzle.nozzle.

(a(a) ) (b(b) )

FigureFigure 3. 3. ( a(()aa )Simplified) Simplified Simplified illustration illustration illustration of of ofthe the the chamber chamber chamber geomet geomet geometry.ry.ry. The The The parts parts parts are are (1) are (1) the (1)the outer the outer outer volume volume volume of of the the of chamber,thechamber, chamber, (2) (2) nozzle (2)nozzle nozzle and and andtubing, tubing, tubing, (3) (3) (3)spray spray spray pattern, pattern, pattern, (4) (4) (4)flange, flange, flange, (5) (5) (5) arm, arm, arm, (6) (6) (6) plate plate plate holding holding sample samplesample collectors,collectors, and andand (7) (7)(7) SEM SEMSEM collection collection collection cups. cups. ( b(b) ) Sample Sample collectorscollectors collectors (photo(photo (photo insert insert insert upper upper upper left) left) left) were were were mounted mounted mounted on onaon custom-madea acustom-made custom-made acrylic acrylic acrylic plate plate plate (red (red (red arrows arrows arrows indicate indicate indicate where where where they they they were were were mounted) mounted) mounted) that that that was was was fixed fixed fixed to an to to arman an armmountedarm mounted mounted on one on on ofone one the of flanges.of the the flanges. flanges. Photo Photo insertPhoto atinsert insert bottom at at bottom showsbottom theshows shows arm theand the arm platearm and insideand plate plate the inside chamber.inside the the chamber.chamber. Injection of E. coli was performed using a high-pressure nozzle system. A double-acting pneumatic pistonInjectionInjection/cylinder ofof (Bimba, E.E. colicoli PN waswas 0074-DXP) performedperformed was using actuatedusing aa high-pressureviahigh-pressure a short pulse nozzle ofnozzle high-pressure system.system. A COA double-actingdouble-acting2 gas supplied pneumaticatpneumatic 5.8 MPa piston/cylinder topiston/cylinder force E. coli containing (Bimba, (Bimba, PN fluidPN 0074-DXP) 0074-DXP) samples through was was actuated actuated an injection via via a nozzle.a short short pulse Highpulse injectionof of high-pressure high-pressure pressures COCO2 gas2 gas supplied supplied at at 5.8 5.8 MPa MPa to to force force E. E. coli coli containing containing fluid fluid samples samples thro throughugh an an injection injection nozzle. nozzle. High High injectioninjection pressures pressures were were needed needed to to ensure ensure the the smallest smallest mean mean droplet droplet size size which which was was 35 35 μ μmm for for the the

Life 2020, 10, 40 5 of 12 were needed to ensure the smallest mean droplet size which was 35 µm for the ceramic nozzle per the manufacture’s specification. Two different hollow-cone type nozzles were experimented with: a ceramic misting nozzle (Aeromist Inc. Arizona 1 ” NPT Ceramic Nozzle SKU: 52252 rated 5.1 L/h) 4 ≈ or a stainless-steel nozzle (Aeromist high pressure anti drip 0.012X 10/24 SKU: 51433 rated 6.0 L/h). ≈ Both nozzles had orifices of 0.3175 mm in diameter. The nozzle in use was located on axis at the apex of the vacuum chamber, pointing downward toward the collector plate. The piston/cylinder assembly was configured to act as a metering pump. To charge the cylinder, a ball valve was manually opened, and 5 mL of test fluid was manually injected into the base port of the cylinder from a sterile syringe, causing the piston to displace to its limit in one direction. During injection into the vacuum chamber, the ball valve was closed and CO2 pressure was directed through the nose cylinder port to the opposing face of the piston by an electrically operated on/off solenoid valve (Peter Paul Electronics model 19052), causing the piston to displace to its limit in the other direction, thereby forcing the metered fluid through the nozzle. Pressure in the chamber was monitored with a convection gauge sensor tube (ConvecTech Part CVT-275-101) and controller (Terranova 906A) convection gauge. A humidity sensor (Adafruit BME280 Temperature Humidity Pressure sensor) was mounted under the sample holder. Prior to injection of fluids containing , the internal chamber was cleaned with ethanol and the entire steel chamber was heated to sterilize by wrapping it with heat tape and covering with a Rockwool insulating blanket. The chamber temperature was measured at 5 different locations with an average temperature of 149 ◦C and maintained at this temperature for 12 h. The sample holders were cleaned using ethanol directly before insertion into the chamber. For sample collection, QuantomiX WETSEM® (QX-102) capsules were selected for use because they do not require any treatment prior to viewing. 3 WETSEM® capsules were mounted along with 3 microscope slides on the sample collection plate (Figure2). SEM capsules and microscope slides were mounted so that typical areas of the chamber were sampled to account for potential variability in sample distribution. Injections of fluid into the chamber consisted of: (1) a negative control; no E. coli (under vacuum), (2) a positive control; E. coli under standard atmospheric pressure and (3) the experimental sample; E. coli under vacuum. The collection area of the SEM capsules was 28.27 mm2 and 645.16 mm2 for the microscope slides. Chamber pressure prior to injection was between 8.00–8.67 Pa. This pressure was the practical minimum achievable with the available apparatus but was found to be sufficient to produce rapid or flash freezing of the injected drops. Upon fluid injections the injected material froze rapidly, then the pressure rose in the chamber because evaporation of the water drops produced water vapor that quickly filled the small chamber volume to a typical value of ~800 Pa (Figure4). After fluid was injected into the chamber, the vacuum pump was restarted, and pumping continued until drops had fully sublimated whence the samples were removed from the chamber. For comparison with the vacuum sublimated ice drops, the water drops from positive controls, after being removed from the chamber, were allowed to dry before imaging. There were experimental differences that affect the fidelity of this experiment to plumes. Table1 compares the experimental parameters for these to those measured or expected at Enceladus. Epifluorescence microscopy was conducted on the microscope slides and imaged using a Leica DMi8 S Platform with a 63X objective (Leica HCX PL APO) and a filter set with 327–402 nm excitation passband, 409 DM, and 417–477 nm emission passband. The WETSEM® capsules were imaged using a Hitachi S-4800 tabletop SEM, resolution 2 nm at 1 kV. Three samples were collected on microscope slides for each of the negative control, positive control and experimental sample. Cell counts were performed via epifluorescence microscopy on each microscope slide per sample, counting areas were 0.15 mm2. Using ImageJ each cell was selected, and area traced by hand to delineate between nearly intact and damaged/destroyed cells. Any selected cell with a surface area of less than 0.3 µm2 was not counted. LifeLife2020 2020, 10, 10, 40, x 66 ofof 12 12

Figure 4. Pressure (Pa) in the chamber before and after injection; time of injection T = 0. Primary data Figure 4. Pressure (Pa) in the chamber before and after injection; time of injection T = 0. Primary data is available in Excel file S1. is available in Excel file S1. Table 1. Comparisons of experimental parameters to those measured or expected at Enceladus. Table 1. Comparisons of experimental parameters to those measured or expected at Enceladus. Parameter Measured or Expected at Enceladus Experimental Parameter Measured or Expected at Enceladus Experimental Pressure 1.3 10 9 Pa [33] 8.67 Pa Pressure 1.3× ×− 10−9 Pa [33] 8.67 Pa Temperature 273 K [36] 296 K Temperature 273 K [36] 296 K pH 9–11 [33] 7.5–8.3 pH 9–11 [33] 7.5–8.3 Injection velocity 100 m/s 20 m/s DropletInjection size velocity 1–10 µ 100m [m/s37] 20 m/s 35 µm CellsDroplet size 100 cells/ 1–10mL (for μm Europa) [37] 35~10 μm5 cells/mL OrganismCells 100 Methanogen cells/mL (for * Europa) ~105 cells/mLEscherichia coli Organism Methanogen * Escherichia coli * Due to energy availability. * Due to energy availability.

3. ResultsEpifluorescence microscopy was conducted on the microscope slides and imaged using a Leica DMi8There S Platform was a diwithfference a 63X inobjective the performance (Leica HCX between PL APO) the and two a filter different set with nozzle 327–402 types nm used. excitation Cell countspassband, of positive 409 DM, controls and 417–477 (E. coli nmunder emission standard passband. atmospheric The WETSEM pressure)® capsules injected were into theimaged chamber using usinga Hitachi the ceramic S-4800 vaporizertabletop SEM, nozzle resolution and stainless-steel 2 nm at 1 kV. nozzle Three were samples compared were and collected showed on a microscope significant decreaseslides for in each the cell of retentionthe negative with control, the stainless-steel positive control nozzle. and Only experimental approximately sample. 2% of Cell all visible counts intact were E.performed coli cells were via epifluorescence preserved using microscopy the stainless-steel on each nozzlemicroscope (Figure slide S1). per All sample, experimental counting parameters areas were were0.15 consistentmm2. Using between ImageJ each the usecell ofwas the selected, ceramic and vaporizer area traced nozzle by hand and stainless-steelto delineate between nozzle nearly with theintact only and variable damaged/destroyed being the nozzle cells. type. Any The selected ceramic cell with nozzle a surface showed area no of degradation less than 0.3 in μE.m2 coli wascell not countscounted. by comparison. Therefore, the experiments used only the ceramic nozzle and those results are reported here. 3. ResultsPressure in the chamber was monitored and recorded (Figure4). At the start the chamber containedThere room was air.a difference The vacuum in the pump performance took ~2 hbetween to achieve the ourtwo target different value nozzle (8.67 types Pa). Whenused. theCell samplecounts was of positive injected controls pressure ( reachedE. coli under 797.27 standard Pa before atmospheric decreasing again. pressure) During injected and immediately into the chamber after injectionusing the into ceramic the chamber, vaporizer it was repeatedlynozzle and witnessed stainless-steel that only nozzle under were the experimental compared and conditions showed the a dropletssignificant which decrease were visiblein the cell with retention the naked with eye the rapidly stainless-steel froze: small nozzle. drops Only bounced approximately off the collection 2% of all platevisible and intact larger E. dropscoli cells quickly were frozepreserved on it. using the stainless-steel nozzle (Figure S1). All experimental parametersThe SEM were images consistent of positive between controls the use (under of the ceramic atmospheric vaporizer pressure) nozzle using and stainless-steel the ceramic nozzle nozzle showedwith theE. only coli cellsvariable intact being (Figure the 5nozzlea). The type. extent The of ceramic the damage, nozzle if showed any, to theno degradation integrity of thein E. cell coli wallscell counts could notby comparison. be determined, Therefore, however, the the experiment overall appearances used only of the the ceramic cells was nozzle that theyand those were intact.results Contraryare reported to the here. results seen in the positive controls, the SEM images of E. coli injected into the chamber underPressure vacuum in (8.67 the Pa) chamber showed was a substantial monitored decreaseand recorded in visible (Figure intact 4). cells. At the However, start the a residuechamber appearedcontained which room outlined air. The thevacuum droplets pump (Figure took5b–d). ~2 h Theto achieve water inour the target droplets value had (8.67 sublimated Pa). When (due the tosample being underwas injected vacuum), pressure leaving reached behind 797.27 what Pa was before believed decreasing to be cellular again. During remnants—The and immediately residue observedafter injection in the SEMinto images.the chamber, it was repeatedly witnessed that only under the experimental conditions the droplets which were visible with the naked eye rapidly froze: small drops bounced off the collection plate and larger drops quickly froze on it.

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The SEM images of positive controls (under atmospheric pressure) using the ceramic nozzle showed E. coli cells intact (Figure 5a). The extent of the damage, if any, to the integrity of the cell walls could not be determined, however, the overall appearance of the cells was that they were intact. Contrary to the results seen in the positive controls, the SEM images of E. coli injected into the chamber under vacuum (8.67 Pa) showed a substantial decrease in visible intact cells. However, a residue appeared which outlined the droplets (Figure 5b–d). The water in the droplets had sublimatedLife 2020, 10, 40 (due to being under vacuum), leaving behind what was believed to be cellular7 of 12 remnants—The residue observed in the SEM images.

(a) (b)

(c) (d)

FigureFigure 5. 5. SEMSEM images images of of (a (a) )a a positive positive control; control; E.E. coli coli underunder atmospheric atmospheric pressure, pressure, ( (bb)) an an overview overview of of thethe WETSEM ®®viewing viewing window window for for the the experimental experimental samples; samples;E. coli E.after coli beingafter injectedbeing injected into vacuum, into vacuum,(c) the residue (c) the fromresidue a droplet from a indroplet the experimental in the experimental sample, sample, and (d) and a close (d) a up close of the up residueof the residue from a fromdroplet a droplet in the experimentalin the experimental sample. sample.

ToTo confirm confirm if if the the residue residue in in the the SEM SEM images images was was in in fact fact cellular cellular biomarkers, biomarkers, E.E. coli coli waswas stained stained withwith PrimulinePrimuline priorprior to to injection injection into into the chamberthe chamber and then and imaged then withimaged an epifluorescencewith an epifluorescence microscope. microscope.The fluorescence The images of E. coli imagesstained of withE. coli Primuline stained with were Primuline consistent were with theconsistent SEM images with (Figurethe SEM6). imagesThe positive (Figure controls 6). Theshowed positiveE. controls coli cells showed intact withE. coli little cells to intact no stain with fluorescence little to no stain outside fluorescence of the cell outsidestructures of the (little cell background structures fluorescence).(little background The experimentalfluorescence). samples The expe showedrimental a fluorescence samples showed residue a fluorescencein the form ofresidue the sublimated in the form droplets of the sublimated indicating thedroplets presence indicating of lipids, the with presence only fewof lipids, intact with cells onlyobserved. few intact Cell countscells observed. performed Cell showed counts that performed there was showed a 94% reduction, that there withwas ana 94% uncertainty reduction, of 6%,with in anvisible uncertainty intact E. of coli6%,cells in visible when intact subjected E. coli to cells injection when subjected into the vacuum to injection chamber into the under vacuum experimental chamber underconditions experimental (Figure7 ).conditions (Figure 7).

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(a) (b) (a) (b) Figure 6. Fluorescence images of E. coli stained with Primuline; (a) a positive control; E. coli under Figure 6.6. Fluorescence images of E. coli stained with Primuline; ( a) a positive control; E. coli under atmospheric pressure; (b) the experimental samples; E. coli after being injected into vacuum. Scale bar atmospheric pressure; ( (bb)) the the experimental experimental samples; samples; E.E. coli coli afterafter being being injected injected into into vacuum. vacuum. Scale Scale bar = 10 μm. The other images for the positive control and experimental samples are available in Figure bar= 10= μ10m.µ Them. Theother other images images for the for positive the positive control control and experimental and experimental samples samples are available are available in Figure in S2. FigureS2. S2.

FigureFigure 7.7.Cell Cell counts counts for E.for coli E.under coli atmosphericunder atmospheric pressure pressure (positive control)(positive and control) under theand experimental under the Figure 7. Cell counts for E. coli under atmospheric pressure (positive control) and under the conditions,experimentaln = conditions,3. n = 3. experimental conditions, n = 3. 4. Discussion 4. Discussion 4. DiscussionAny putative life possible in the interior ocean of an icy moon is adapted to a liquid water Any putative life possible in the interior ocean of an icy moon is adapted to a liquid water environment.Any putative It is important life possible to understand in the interior how ocea the processn of an oficy being moon ejected is adapted into the to vacuum a liquid of water space environment. It is important to understand how the process of being ejected into the vacuum of space wouldenvironment. impact It such is important life in order to understand to design missions how the process and choose of being the bestejected instruments into the vacuum for detecting of space it. would impact such life in order to design missions and choose the best instruments for detecting it. Ourwould work impact has focusedsuch life on in E.order Coli ,to a specificdesign missio aqueousns and organism, choose because the best it instruments has been well for studied. detecting This it. Our work has focused on E. Coli, a specific aqueous organism, because it has been well studied. This workOur work has allowed has focused us to on gain E. insightColi, a specific into the aqueous impact on organism, cell membranes because of it beinghas been rapidly well frozen.studied. Much This work has allowed us to gain into the impact on cell membranes of being rapidly frozen. Much morework extensivehas allowed experiments us to gain couldinsight and into should the impact be done on cell to explore membranes alternative of being types rapidly of organisms frozen. Much such more extensive experiments could and should be done to explore alternative types of organisms such asmore methanogens, extensive experiments and to investigate could and a wider should range be done of environmental to explore alternative conditions types such of organisms such may as methanogens, and to investigate a wider range of environmental conditions such organisms may experienceas methanogens, in their and native to investigate habitat. Still, a wider this preliminaryrange of environmental work helps toconditions bridge the such current organisms knowledge may experience in their native habitat. Still, this preliminary work helps to bridge the current knowledge gapexperience between in thetheir modeled native habitat. abundances Still, this of cells preliminary in the liquid work water helps environment to bridge the of current icy world knowledge plumes gap between the modeled abundances of cells in the liquid water environment of icy world plumes andgap experimentallybetween the modeled derived abundances values of what of cells to expect in the atliquid collection. water environment of icy world plumes and experimentally derived values of what to expect at collection. and experimentallyIt is important toderived understand values how of what the experimental to expect at collection. conditions differ from those of the Enceladus It is important to understand how the experimental conditions differ from those of the Enceladus plumesIt is to important ensure that to understand the results arehow relevant. the experimental The chamber conditions pressure differ prior from to injectionthose of the (8.67 Enceladus Pa) was plumes to ensure that the results are relevant. The chamber pressure prior to injection (8.67 Pa) was notplumes chosen to ensure to match that conditions the results in are the relevant. Europa plume The chamber but rather pressure was the prior lowest to pressureinjection that(8.67 we Pa) could was not chosen to match conditions in the Europa plume but rather was the lowest pressure that we could achievenot chosen using to match the available conditions apparatus. in the Europa Additionally, plume but after rather injection was the thelowest water pressure drops that immediately we could achieve using the available apparatus. Additionally, after injection the water drops immediately beganachieve to using evaporate the available into the smallappara volumetus. Additionally, chamber causing after theinject pressureion the inwater the chamberdrops immediately to increase began to evaporate into the small volume chamber causing the pressure in the chamber to increase tobegan 799.94 to Paevaporate of water into vapor. the small The liquid volume water chamber evaporated causing when the pressure injected becausein the chamber the pressure to increase in the to 799.94 Pa of water vapor. The liquid water evaporated when injected because the pressure in the chamberto 799.94 wasPa of below water the vapor. triple The point liquid pressure water ofevap water,orated so waterwhen boiledinjected until because its temperature the pressure reached in the chamber was below the triple point pressure of water, so water boiled until its temperature reached 0chamber◦C. The was evaporation below the is triple a phase point change pressure that of requires water, so heat water (the boiled latent until heat its of temperature vaporization) reached which 0 °C. The evaporation is a phase change that requires heat (the latent heat of vaporization) which was was0 °C. pulled The evaporation from the drops is a phase quickly, change bringing that requires their temperature heat (the latent to the heat freezing of vaporization) point. Removing which was the pulled from the drops quickly, bringing their temperature to the freezing point. Removing the water pulled from the drops quickly, bringing their temperature to the freezing point. Removing the water vapor from the chamber using the vacuum pump took considerable time (Figure 4). In the case of vapor from the chamber using the vacuum pump took considerable time (Figure 4). In the case of

Life 2020, 10, 40 9 of 12 water vapor from the chamber using the vacuum pump took considerable time (Figure4). In the case of Enceladus plumes, the water vapor evaporating from plume drops would be expanding into the unlimited volume of space. While there would be a short period during eruption when the drops would experience higher gas pressure from the water vapor, it would drop very quickly to essentially zero. Compared to an icy world plume of water drops, the high-water vapor pressure in the chamber after injection may have slowed the rate of evaporation of the drops, and therefore slowed the rate of freezing. Nevertheless, large drops were observed to freeze within 2 s and to remain frozen after the pressure increased and until the experiment was stopped. Our apparatus was not cooled so the internal components such as the sample collectors were at room temperature when the fluid injection occurred. Since these components had much greater thermal mass than the ice particles, the ice was continuously heated by the sample capture components causing them to sublime fairly quickly, in effect freeze drying the material in the ice particles. So, in the final state before the chamber was opened, this material, including cells, was both in vacuum and dry. In the Enceladus plume, the ice particles once ejected equilibrate to their equilibrium temperature at the orbit of Saturn and so sublimate very slowly. While the environmental conditions produced in the lab experiment are different in detail than what occurs when water drops erupt from Enceladus, our results are still a valuable analog providing insight into the fate of any microbes carried by the plume drops. The process that water drops undergo when ejected into space was simulated as the drops quickly froze due to the heat loss during vaporization. This rapid freezing is likely responsible for the destruction of cells causing cell lysis. Rapid freezing will certainly occur if liquid water is erupted into vacuum such as occurs in the Enceladus plume. However, this difference should not change the analog value of the experiment because the process a sample would undergo when ejected into space was largely duplicated with flash freezing followed by slow sublimation. From the results presented, it is possible to estimate the potential cell concentrations that might be observed in icy world plume ejecta. There are now several lines of evidence suggesting the existence of plumes at Europa’s surface [38–40]; taking this into account and considering the upper range of cell density of approximately 100 cells per mL derived in the Study 2016 Report [23] combined with our result of a 94% reduction in visible intact E. coli cells, this would mean that the amount of detectable intact cells would be 6 cells per mL. However, the assumption here is that cells on Europa would respond in a similar way as E. coli, which may not be the case. For Earth organisms, we know that there is a wide range in composition and lysability of cell walls and membranes which would affect their response to a vacuum environment. More testing, with a multitude of different organisms, is needed to determine the dynamic range in concentration of cells expected to survive the plume environment. Intact cells might have a low retention rate in plume ejecta; however, our results show that ejection of E. coli from a liquid medium into vacuum causes cell lysis, leaving behind a residue of lipids and potentially other biomarkers such as proteinogenic amino acids. From the lipid residue observed via epifluorescence microscopy, it is reasonable to assume the lipid structure itself is still intact along with other cellular non-polar molecules, at least for a large portion of the sample, even if the cell membrane is not. Presuming intact lipids, the needed concentration to meet the values proposed for the lower limit of detection in the Europa Lander Study 2016 Report [23] would still be a viable option as a for a life detection mission. This result suggests that directly detectable evidence of life in plumes erupted from the ocean of icy moons may be lipids. However, further characterization of the remaining material after exposure to vacuum is warranted to understand if the phospholipid molecules are still intact and, if not, are hydrocarbon tails of these lipids (i.e., fatty acids/isoprenoids) distinguishable from abiogenic sources of these molecules. High resolution microscopy and gas chromatography- (GC-MS) analysis would enable the sample to be interrogated for cellular damage and identification of lipid molecules, respectively. Three key technologies needed for the unambiguous detection of life have been identified; organic Life 2020, 10, 40 10 of 12 analyzer (GC-MS), vibrational spectrometer, and a microscope [23]. Employing these technologies for characterization of the sample could potentially aid in the design of future missions. Our initial testing discovered that cells were destroyed in passage through the stainless-steel nozzle but not through the ceramic nozzle. The stainless-steel nozzle was originally selected because it yielded a mean droplet size ( 10 micrometer) near the target size range of Enceladus’ particles, when ≈ operated at its rated pressure. Destruction of cells in the stainless-steel nozzle was unexpected and showed that the positive control experiments were needed to characterize the behavior of the system. Both nozzle assemblies have spring loaded balls sitting in shaped valve seats that serve as check valves to allow flow in the forward direction only. When no line pressure is applied to the inlet, the seated ball also prevents drips. When sufficient line pressure is applied, the pressure unseats the ball and allows fluid to flow to the discharge orifice. The nominal unseat pressure for the stainless-steel nozzle is 1380 kPa, while the unseat pressure for the ceramic nozzle is 240 kPa. This implies that the ≈ ≈ clearance between the ball and the valve seat is smaller on the stainless valve than on the ceramic valve when fluid is flowing and may have forced the cells through an opening smaller than the cell diameter or generated an internal pressure outside the range E. coli could tolerate. We note this for completeness in case our experimental method is replicated by others. Because the ceramic nozzle did not destroy the cells in passage through the orifice, the ceramic nozzle was used in all data gathering runs even though it produced a somewhat larger mean droplet size ( 35 micrometer). ≈ The Enceladus plumes have particles sizes in the range 1–10 microns [37], the lower end of this range being comparable to the size of an individual microbe. A conceptual study by Porco et al. [37] discusses processes involving the formation and rising of bubbles in the Enceladus ocean thought to create the plume particles. The rising of bubbles to the surface creates drops of different sizes. These bubbles are expected to concentrate the organics and microbial numbers compared to the bulk fluid, with the largest drops having the highest concentrations. These largest are called jet drops; they form as a rebound process after gas filled bubbles burst and they can be as large or larger than the drops in our experiments. The process that we used to produce drops (misting nozzle) is fundamentally different than the process producing them in the plumes. However, this should not materially impact the rapid freezing process that we believe causes cell lysis. The size distribution observed in the Enceladus plumes also does not represent the range of sizes produced by the bursting bubbles, as the largest particles would fall rapidly back to the surface, and the remaining ice particles would decrease in size as they sublimate.

5. Conclusions This work has started to fill in the uncertainties about the survivability of biosignatures in icy world plumes. Flash freezing of the cells, caused by the cooling effect of sublimation in vacuum caused lysing of the cell and thereby the destruction of the cell as a whole. The 94% reduction in visible intact E. coli cells suggested that conditions in the environment that the sample encounters has destroyed the cells to some degree. However, to fully understand how ejection of cells living in a liquid environment into vacuum impacts the potential samples for life detection analyses, it would be necessary to investigate the response of multiple different types of microorganisms to the physical processes undergone in the plume environment. Even in the absence of intact cells, other viable biosignatures could still persist in the plume ejecta, particularly in the form of lipids.

Supplementary Materials: The following are available online at http://www.mdpi.com/2075-1729/10/4/40/s1, Figure S1. Fluorescence images of E. coli stained with Primuline. Figure S2. Cell counts for E. coli under atmospheric pressure (positive control) when the ceramic nozzle was used versus the stainless-steel nozzle, n = 3. Excel file S1: Pressure versus time for experimental trial. Author Contributions: Conceptualization, K.B. and C.R.S.; methodology, K.B., N.B.D.N.J., L.L., and C.R.S.; validation, K.B., N.B.D.N.J., L.L., and C.R.S.; formal analysis, K.B. and C.R.S.; investigation, K.B., N.B.D.N.J., L.L., and C.R.S.; resources, K.B., N.B.D.N.J., and C.R.S.; data curation, K.B., N.B.D.N.J., and C.R.S.; writing—original draft preparation, K.B. and C.R.S.; writing—review and editing, K.B., N.B.D.N.J., L.L., and C.R.S.; visualization, Life 2020, 10, 40 11 of 12

K.B., N.B.D.N.J., and C.R.S.; supervision, C.R.S.; project administration, K.B. and C.R.S.; funding acquisition, C.R.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by a NASA Ames Research Innovation Award. Conflicts of Interest: The authors declare no conflict of interest.

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