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Home , Astrobiology, Extraterrestrial life, Hydrate

ASTROBIOLOGY Volume 3, Number 3, 2003 © Mary Ann Liebert, Inc.

Methodologies and Techniques for Detecting Extraterrestrial (Microbial)

Near-Infrared Detection of Potential Evidence for Microscopic on

J. BRAD DALTON, 1 RAKESH MOGUL, 2 HIROMI K. KAGAWA, 1 SUZANNE L. CHAN, 1 and COREY S. JAMIESON 3

ABSTRACT

The possibility of an within the icy shell of ’s Europa has established that world as a primary candidate in the search for within our . This paper evaluates the potential to detect evidence for microbial life by comparing labora- tory studies of terrestrial with measurements from the Galileo Near Infrared Imaging Spectrometer (NIMS). If the interior of Europa at one harbored life, some evi- dence may remain in the surface materials. Examination of laboratory spectra of terrestrial measured at cryogenic temperatures reveals distorted, asymmetric near- infrared absorption features due to of hydration. The band centers, widths, and shapes of these features closely match those observed in the Europa spectra. These features are strongest in reddish-brown, disrupted terrains such as linea and chaos regions. Narrow spec- tral features due to amide bonds in the microbe provide a means of constraining the abundances of such materials using the NIMS data. The NIMS data of disrupted terrains ex- hibit distorted, asymmetric near-infrared absorption features consistent with the presence of water ice, octahydrate, hydrated salts, and possibly as much as 0.2 mg cm 23 of carbonaceous material that could be of biological origin. However, inherent noise in the ob- servations and limitations of spectral sampling must be taken into account when discussing these findings. Key Words: Extraterrestrial life—Europa— Infrared —Extremo- philes. 3, 505–529.

INTRODUCTION al., 1979; Pappalardo et al., 1999; Kivelson et al., 2000; Stevenson, 2000), but also a significant com- UROPAHASBEENIDENTIFIED as a potential host plement of the biogenic elements (Pierazzo and Efor extraterrestrial life within our Solar Sys- Chyba, 2002). It has been postulated that this tem. Recent work has indicated the likelihood of ocean could have at one time harbored prebiotic not only a possible subsurface ocean (Cassen et compounds or even extraterrestrial life (Reynolds

1SETI Institute, Mountain View, California. 2Wilkes Honor College, Florida Atlantic University, Jupiter, Florida. 3University of Hawaii, Manoa, Hawaii.

505 506 DALTON ET AL. et al., 1983; Sagan et al., 1993; Fanale et al., 1998; diagnostic infrared absorption features have Chyba and Phillips, 2001; Rothschild and Man- proven useful for constraining the abundances of cinelli, 2001; Zolotov and Shock, 2003). several compounds (Dalton, 2000). Because the putative ocean remains inaccessi- Water ice and frost have well-characterized, ble beneath Europa’s icy crust, infrared remote symmetric near-infrared absorption features at sensing of the surface materials has been used to 1.5 and 2.0 mm (Ockman, 1957; Herzberg, 1991; infer details about the interior composition. If in Gaffey et al., 1993). These are evident in the fact the surface composition is indicative of inte- Galileo Near-Infrared Imaging Spectrometer rior composition, studies of surface materials (NIMS) Europa spectrum of icy terrain in Fig. 1 may provide insight into biological and prebiotic (bottom curve). Other regions of the surface dis- processes (Squyres et al., 1983; McCord et al., play distorted, asymmetric features at these po- 1999a; Kargel et al., 2000). The diagnostic spectral sitions, as indicated by the top curve. The pres- characteristics of specific biogenic materials may ence of these distorted absorption features in provide unique (Schulze-Makuch telescopic and spacecraft observations of Europa et al., 2002), which can be tested for in the search (Clark and McCord, 1980; Dalton, 2000) forms the for extraterrestrial life. Infrared detection of evi- basis of the argument for hydrated salts on the dence for life requires that (1) material of biolog- surface (McCord et al., 1998, 1999a). These fea- ical origin be emplaced at the surface; (2) identi- tures are clearly due to water of hydration (Paul- fiable infrared absorption features diagnostic of ing, 1935), though the host is less cer- this material survive exposure to the hostile tain (Gaffey et al., 1993; Carlson et al., 1999b; environment; and (3) spectra of sufficient spectral resolution and signal-to-noise ratio be acquired at spatial scales capable of isolating regions con- 0.8 taining the material of interest. This paper applies these criteria to the examination of recent space- 0.7 craft and laboratory data concerning the spectra 0.6 of Europa and those of terrestrial microorgan- e isms. c n 0.5 a

The surface of Europa is composed mostly of t c Dark terrain e water ice (Pilcher et al., 1972; Clark and McCord, l

f 0.4 1980). More recent ground-based and spacecraft e R

observations have indicated the presence of other d 0.3 e l materials as well. Cometary and meteoric infall a c are expected to provide several elemental and S 0.2 molecular species over the age of the Solar Sys- tem, including biogenic elements (Zahnle et al., 0.1 Icy 1998; Pierazzo and Chyba, 2002). Energetic parti- cle bombardment associated with Jupiter’s mag- 0 0.8 1.2 1.6 2 2.4 netosphere not only delivers elements such as H, S, and O but also drives complex that Wavelength ( mm) is expected to produce a number of additional compounds (Delitsky and Lane, 1997, 1998; FIG. 1.Galileo NIMS spectra of both icy and dark sur- face units on Europa. Lower curve is a 26-NIMSEL av- Cooper et al., 2001; Carlson et al., 2002). Observed erage from an icy region, illustrating the broad 2.0- mm species include O 2, SO2, CO2, and H2O2 (Lane et absorption feature indicating pure H 2O ice. Upper curve al., 1981; Noll et al., 1995; Carlson et al., 1999a,b). is an average of 18 NIMSELS corresponding to dark, red- Current models suggest the presence of sulfuric dish-brown terrain. This curve exhibits the distorted and asymmetric 2.0- mm absorption bands typical of bound acid octahydrate and other compounds water such as water of hydration. Both averages were ex- (Carlson et al., 1999b), simple organics such as tracted from contiguous portions of the G1ENNHILAT01 (Delitsky and Lane, 1998; Chyba, observation (Dalton, 2000). Overlapping data segments 2000), and hydrated sulfate and carbonate salts at every 12th wavelength position are instrumental arti- facts created by stepping the grating to acquire readings (Kargel, 1991, 1998; McCord et al., 1998, 1999a,b, at each detector; the resulting segments are compiled to 2002; Kargel et al., 2000). While no single mater- create each spectrum (Carlson et al., 1992) with redun- ial completely explains the Galileo observations, dancy supplied by the overlapping wavelengths. DETECTION OF MICROBES ON EUROPA 507

McCord et al., 1999b, 2002; Dalton, 2003). Most and Clark, 1998; Marion and Farren, 1999; Zolo- hydrated materials exhibit features at these posi- tov and Shock, 2001), recent laboratory and the- tions, and the hydrated sulfate salts are only one oretical work indicates that some compounds class of with these characteristics (Hunt may be stable on geologic timescales (McCord et et al., 1971; Crowley, 1991; Herzberg, 1991; Dal- al., 2001; Carlson et al., 2002; Orlando, 2002; Pier- ton, 2000). In fact, the hydrate features in the spec- azzo and Chyba, 2002; Mogul et al., 2003). Because tra of the sulfate salts are not intrinsic to the sul- reaction rates depend strongly upon temperature, fates, but rather to the bound water itself and experiments must be performed at the low tem- associated bond shifts (Hunt et peratures relevant to Europa’s surface (80–130 K). al., 1971; Gaffey et al., 1993; Dalton and Clark, Several of the hydrated salts have been postu- 1998; Chaban et al., 2002). Comparison of Galileo lated to exhibit resistance to degradation over the NIMS spectra with Solid State Imager (SSI) data lifetime of Europa’s surface (McCord et al., 2001), indicates that the hydrate absorption features are though Carlson et al. (2002) predict destruction of most pronounced in reddish-brown disrupted many sulfate compounds in less than 4,000 years. terrain units such as linea and chaos regions, and It has also been recently shown that survival rates in certain impact craters (Granahan et al., 1998; for microorganisms and their component materi- McCord et al., 1998; Fanale et al., 1999, 2000), yet als exposed to energetic bombardment virtually nonexistent in the bright plains units. are dependent not only on energy but also upon This has been interpreted as evidence of a link to composition of bombarding ions (Mogul et al., subsurface processes and interior composition 2003). Extrapolation from room temperature ex- (Kargel, 1991; Kargel et al., 2000; Spaun and Head, periments or experiments using and en- 2001; Zolotov and Shock, 2001; McKinnon and ergies other than those present at Europa may not Shock, 2002), though some role is probably be appropriate. As there is presently much dis- played by exogenic processes such as ion im- agreement in the literature regarding destruction plantation and radiolysis (Carlson et al., 1999b, rates and survival lifetimes for complex com- 2002; Cooper et al., 2001; Paranicas et al., 2001). pounds in Europa’s environment, fur- The exact mechanisms of disruption and em- ther work will be needed to constrain the rates of placement of these materials are the subject of destruction for all candidate materials. considerable debate (Squyres et al., 1983; Spaun Because the infrared spectral features of many et al., 1998; Greenberg et al., 1999; Head and Pap- compounds, including water ice and water of hy- palardo, 1999; Prockter and Pappalardo, 2000; dration, are strongly temperature-dependent, in- Figueredo et al., 2002; Prockter et al., 2002). The terpretation of icy satellite spectra must rely on question of whether these surface units could be laboratory measurements performed at or near indicative of oceanic composition remains open. the surface temperatures of these worlds (Fink If there is no connection, then investigation of as- and Sill, 1982; Gaffey et al., 1993; Grundy and trobiological potential at Europa will require ac- Schmitt, 1998; Roush, 2001). Of the Europa sur- cess to the subsurface. For the purposes of this face candidate materials that have been charac- analysis, we shall assume that such a link exists. terized at low temperatures thus far, the hydrated In that case, if the ocean at one time contained sulfur compounds provide the best available some form of organisms, then it may be inferred spectral matches (Carlson et al., 1999b; McCord et that these would be emplaced at the surface as al., 2001) to the hydrate features observed in the well. Europa spectra. Yet many materials have not Once emplaced at the surface, any material been considered simply because they have not yet would be subjected to the hostile space environ- been measured (Dalton, 2002b; Jamieson and Dal- ment. Energetic charged particle bombardment, ton, 2002). The most interesting candidates from ultraviolet photolysis, dehydration, and chemical an astrobiological perspective are those that are breakdown by radical species would all act to de- related to the occurrence of life. Meteoritic and compose exposed materials (Cooper et al., 2001; cometary delivery of over geologic time is Hudson and Moore, 2001; Paranicas et al., 2001; predicted to eventually result in both inorganic Carlson et al., 2002). To be detected, biological or (i.e., sodium carbonates, , carbon prebiotic material would have to persist to the monoxide) and organic (i.e., amino acids, form- present. While the long-term viability of several aldehyde) carbon compounds through a com- candidate materials has been questioned (Dalton bination of endogenic and exogenic processes 508 DALTON ET AL.

(Kvenvolden et al., 1970; Kargel, 1991; Delitsky MATERIALS AND METHODS and Lane, 1997, 1998; McCord et al., 1999a; Chyba and Phillips, 2001; Kempe and Kazmierczak, This investigation was composed of three com- 2002). salts and sulfuric ponents: (1) measurement and characterization of acid are both commonly found in terrestrial hot infrared spectra of microbes and at springs environments, which host extremophilic varying temperatures (measurements at 80–120 K microorganisms (Rothschild and Mancinelli, 2001, were achieved using the specialized cryogenic and references therein). If the surface deposits on vacuum equipment described below); (2) assess- Europa contain these compounds, and these com- ment of the survival of the microbes after expo- pounds are derived from an interior ocean, it may sure to the low pressures and temperatures; and be that the deposits contain other materials from (3) comparison of the spectra with those of Eu- the interior. If the interior once hosted life, some ropa as measured by the Galileo NIMS instru- remnant of this life may be entrained in the sur- ment. face deposits. If, in fact, the surface deposits did contain remnants of biological organisms, what Infrared characterization of microbes would they look like today? Microorganisms possess unique infrared spec- and biomolecules tra because of their water of hydration and bio- Sample selection . Several model microorganisms molecular components. Their spectra contain and biomolecules were chosen for spectroscopic minor contributions from the absorption of func- analysis. The microorganisms selected for this in- tional groups (such as amides, carboxylic acids, vestigation were , Sul- phosphates, and alcohols) found in proteins, folobus shibatae , and Escherichia coli . D. radiodurans , nucleic acids, and ; and ma- was chosen because of its ability to survive in jor absorption contributions arising from the wa- low-pressure and high-radiation environments, ters that hydrogen bond to and hydrate the bio- such as those found in interplanetary space or at . We have measured the infrared the surface of planetary bodies. D. radiodurans can spectra of four different biomolecules at standard survive desiccation and ionizing radiation of 60 temperature and pressure, and three terrestrial Grays/h for 30 h (1,800 Grays; 1 Gray 5 1 J/kg) microorganisms at temperatures relevant to the with no loss of viability (Mattimore and Battista, surface of Europa. In this paper we compare these 1996; Lange et al., 1998; Fredrickson et al., 2000; spectra with those of disrupted terrains on Eu- Venkateswaran et al., 2000). S. shibatae was cho- ropa as measured by the Galileo NIMS instru- sen as an analog to Europan microfauna because ment. of its sulfur-based and the expected Interpretation of these spacecraft observations high sulfur abundance at Europa (Kargel, 1991, is critically dependent upon the spectral resolu- 1998; Fanale et al., 1999; Carlson et al., 1999b, tion of the observations, especially when exam- 2002). S. shibatae thrives and can be cultured in ining subtle spectral changes. Because the mate- acidic environments (Table 1) that contain high rials of interest are found in discrete surface units concentrations of sulfur and sulfur compounds, of disrupted terrain, mixing of signal from adja- including magnesium sulfate salts and sulfuric cent icy terrain can overwhelm the spectral in- acid (Grogan, 1989; Rothschild and Mancinelli, fluence of the target material. Thus, high spatial 2001). Taken together, these attributes render the resolution is just as important as high spectral res- microorganisms as ideal candidates for spectro- olution. In addition, the signal-to-noise ratio de- scopic study and comparison with Europa. As a termines the confidence levels that can be as- control, E. coli was also studied in order to draw cribed to any spectral identification. We have distinctions between the survival and spectral evaluated the spectral correlations between labo- properties unique to extremophiles. ratory and observational spectra in light of these Spectra of the individual biomolecules that sig- three criteria. The results of our investigation pro- nificantly contribute to the near-infrared absorp- vide valuable insights into the search for life on tion of the microbes were measured at room tem- icy satellites, and suggest methods whereby the perature in order to provide insights into the near-infrared biosignatures of life as we know it spectral characteristics of the microbes. Bovine may be used to constrain the astrobiological po- serum albumin, calf thymus DNA, dextrin, and tential of these worlds. L-a-phosphatidylcholine dipalmitoyl were cho- DETECTION OF MICROBES ON EUROPA 509

TABLE 1. GROWTH MEDIUM FOR LABORATORY gation. One system, the Outer Solar System En- CULTURES OF S. SHIBATAE vironment Chamber (OSSEC), resides at the U.S. Medium composition Amount Geological Survey in Denver, CO; the other is the NASA Ames Cryogenic Reflectance Environment Sulfolobus medium (per 1 l) Facility (NACREF) in Mountain View, CA. Though Yeast extract 2 g they differ in terms of cooling capability and Sucrose 2 g 100 3 buffer 10 ml methods, they are similar in other respects. Both 200 3 buffer 5 ml use sapphire (Al 2O3) viewports to illuminate and 1,000 3 buffer 1 ml to measure the sample; both use stainless steel H2SO4 0.2 ml sample cups to minimize spectral contributions Deionized H 2O Remainder 100 3 buffer (per 1 l) from the equipment. Both use diode tem- (NH4)2SO4 130 g perature probes for process monitoring, and both MgSO4 ? 7H2O 25 g can be sealed and operated either in vacuum FeCl3 ? 6H2O 2 g mode or with a dry for frag- 50% H2SO4 1.5 ml 200 3 buffer (per 1 l) ile samples. Both are initially evacuated using ro- 50% H2SO4 5 ml tary and sorption pumps, but the OSSEC utilizes KH2PO4 56 g an ion pump to achieve high vacuum, while the 1% MnCl2 36 ml NACREF is outfitted with a turbomolecular pump. 1% ZnCl 4.4 ml 2 The OSSEC is cooled by means of a coldfinger 1% CuCl2 1 ml 1% Na2MoO4 0.06 ml attached to the sample holder, which extends into 1,000 3 buffer (per 200 ml) a liquid nitrogen bath directly below the sealed CaCl2 ? 2 H2O 14 g sample chamber. The sample temperature is con- The three buffer solutions are combined in the pre- trolled by the addition or removal of liquid ni- scribed ratios with sucrose, yeast extract, and sulfuric trogen. In practice, the OSSEC reliably achieves acid; then deionized water is added to make up 11 of temperatures as low as 80 K. The NACREF fea- solution having a pH of ~2.3. Note the inclusion of and magnesium sulfates, and sulfuric acid, tures a closed-loop helium cryostat and com- compounds predicted to occur on Europa. pressor to cool the samples. The temperature diode readings are read by a programmable tem- perature controller, which adjusts the tempera- sen to act as model , , carbo- ture by means of a resistive heater embedded in hydrate, and samples, respectively. the sample holder. While temperature readings for both systems are good to within 1 K, the Spectrometer system . All laboratory spectra were NACREF provides reliable and convenient tem- acquired using an Analytical Spectral Devices perature control, and can reach temperatures as (ASD) portable spectrometer. This device cov- low as 20 K. ers the visible and near-infrared spectral range The incidence and emission angles for both sys- from 0.35 to 2.5 mm. The visible and near-infrared tems are constrained by the viewport positions. portion of the spectral range (up to 1 mm) utilizes The OSSEC chamber is fixed at 5°for both inci- a silicon detector with a spectral sampling of 1.4 dence and emission, resulting in a phase angle of nm and a resolution of 6 nm. The remainder of the 10°. The NACREF can be set for either normal in- spectral range is covered by a pair of indium anti- cidence and emission, or normal incidence and monide (InSb) detectors with spectral sampling of 15°emission and phase. The latter was used for 2 nm and resolution of 11 nm (Goetz et al., 1998). the measurements in this paper. The OSSEC is il- The ASD spectrometer was programmed to aver- luminated by a 100 W tungsten halogen lamp age 100 measurements, each having a 1–s integra- with a Samlex regulated DC power supply. The tion time, together for each spectrum; after check- NACREF uses a regulated Dolan–Jenner fiber op- ing for consistency, five such spectra acquired at tic power supply and a custom tungsten-halogen each temperature were averaged to produce the re- lamp. To prevent undesirable spectral effects of sults presented here. This corresponds to an inte- dichroic reflector coatings commonly used in the gration time of 500 s per observation. manufacture of such lamps, the reflector hous- ings in our lamps have been coated with a 2.75- Cryogenic environment chambers . Two different mm aluminum film. Light is collected at the sap- cryogenic systems were utilized in this investi- phire viewports for both chambers by a low-OH 510 DALTON ET AL. silica fiber optic, which is standard to the ASD tally dehydrated during this procedure. As a re- spectrometer. sult, the infrared absorption features arising from water of hydration in the D. radiodurans were sig- Sample preparation . Powdered samples of the nificantly reduced. The measurement was later four representative biomolecules were acquired repeated using the NACREF and a new sample from reagent-grade stock and weighed under am- of D. radiodurans . The same procedures were fol- bient laboratory conditions. Approximately 2 cm 3 lowed, except that spectra were acquired every of each sample was placed in a steel sample cup 10 K, and after spectra were acquired at 80 K the and measured using the ASD spectrometer and sample was cooled to 20 K. Improved purging of fiber-optic illuminator at room temperature with the NACREF chamber with nitrogen and the Spectralon® (Labsphere, Inc.) as the reference more rapid cooling of the sample prevented frost standard. migration and eliminated the need to warm the Cultures of D. radiodurans (ATCC #51178), S. sample after vacuum was established. Upon re- shibatae (ATCC #13939), and E. coli strain W1485 moval from the chamber, the sample was rapidly were grown at NASA . D. frozen in liquid nitrogen and placed in the 280°C radiodurans was cultured in a TGY liquid medium freezer to await survivability assessment. (5 g of Tryptone, 3 g of yeast extract, and 1 g of per 1 l) and incubated at 30°C to mid-log Low temperature survivability phase. S. shibatae was aerobically grown in the medium described in Table 1 at 76°C up to late After exposure to temperatures and pressures log phase for about 4 days. E. coli was aerobically corresponding to the polar latitudes of Europa, grown at 37°C in LB medium (1% Bacto-tryptone, all three samples were cultured to evaluate sur- 0.5% Bacto-yeast extract, and 1% sodium chlo- vival rates. Frozen pellets of D. radiodurans ride) for 18 h. Samples of each were centrifuged and E. coli were resuspended in phosphate- to produce ,1 ml of densely packed cells and buffered solution, and the cell numbers were di- frozen in a 280°C freezer. rectly counted under a light microscope using a Microbial samples were transported on dry ice hemocytometer. D. radiodurans cells were plated to the OSSEC facility in Denver. Samples were on TGY agar plates (TGY medium with 15 g of slowly thawed in a water ice bath and placed in agar/l) and incubated at 30°C for 3 days. E. coli the OSSEC under a dry nitrogen atmosphere. The was spread on MacConkey’s medium plates and chamber was purged with nitrogen, then sealed incubated at 37°C for 18 h. Colonies appearing af- and rapidly cooled to 250 K, and then evacuated ter incubation were counted to calculate survival to 1024 Torr. Spectra of each sample were ac- rates. Approximately 0.1 g of frozen S. shibatae quired with the ASD at 20 K intervals from 200 cells were inoculated in 100 ml of medium and to 100 K. Spectra of the S. shibatae growth medium aerobically incubated at 76°C for 3 days. After the (Table 1) were also measured for comparison. The cell number was counted, the culture was diluted growth medium was virtually indistinguishable at 1:100, 1:50, and 1:10 with fresh medium and in- from pure water ice, and therefore not a contrib- cubated for 2 days. Only the 1:10 dilution showed utor to the asymmetric absorption features con- growth; the cell number was counted to calculate sidered below. After each series of spectral mea- the survival rate. Contamination issues were re- surements, the chamber was allowed to warm to solved separately for each strain: The S. shibatae 250 K, then pressurized to 1 atm with nitrogen. culture was grown in its acidic medium at high Still frozen, the samples were removed from the temperature; D. radiodurans was identified by its chamber, transferred to sealed vials, and placed specific reddish-pink colonies; and E. coli was se- on dry ice for the return to NASA Ames. lectively grown on MacConkey’s plates, which Complications were encountered during spec- contain bile salts to inhibit the growth of most en- tral measurements because of migration of water vironmental contaminants (Grogan, 1989). from between the cells to the tops of the samples. Survival rates were calculated separately for The formation of water frost introduced spectral the D. radiodurans measured at the NACREF. Re- features that obscured the spectral signal of the sults from the first set of experiments indicated samples. Warming the sample up to 230–250 K that most of the cells from all three samples had under vacuum efficiently removed this water; been killed in the thawing and refreezing phases, however, the D. radiodurans sample was acciden- not in the subjection to Europa-like conditions. DETECTION OF MICROBES ON EUROPA 511

The second D. radiodurans experiment did not in- spectral units. This will be considered in the Dis- volve long-distance transport or repeated freez- cussion. Offsets between NIMS detectors were re- ing and thawing cycles. The results of this second moved by the same linear multiplicative scaling experiment indicated much higher survival rates. applied to the ASD spectra (Dalton, 2000). To facilitate comparison, laboratory spectra were convolved to the lower spectral resolution Processing of laboratory and spacecraft data and bandpass of the Galileo NIMS instrument All room temperature and NACREF spectra using the Gaussian convolution routine in the were acquired using Spectralon as the reference SpecPr package. Absorption band centers and material, while all measurements with the OSSEC depths were calculated using standard contin- used Halon® (Allied Chemical) as the reference; uum removal methods (Clark and Roush, 1984) the influences of minor spectral variations in the to eliminate interference from other absorption reference materials were corrected using National features in each spectrum, and to remove depen- Bureau of Standards measurements for Spec- dence on the wavelength positions of the differ- tralon and Halon. Because of the long integration ent spectrometer channels. and high signal inherent in the laboratory measurements, the error bars of the resultant RESULTS spectra are too small to be clearly visible in the figures, and are therefore omitted for clarity. Cryogenic spectra of microbe samples All laboratory spectra were processed using the SpecPr program (Clark, 1993) and da Vinci The reasoning underlying this study of the spectral math engine. Vertical offsets at 1.0 and cryogenic spectra of microorganisms involved 1.82 mm due to differences in gain states of the the water of hydration bands in the Europa spec- ASD spectrometer detector electronics were re- tra. While these spectral features are due to wa- moved by linear multiplicative scaling. This pro- ter, this water must be in a bound state, which cedure preserves relative band shapes and depths constrains the permitted vibrational transitions at the expense of absolute reflectance levels. and gives rise to the distorted and asymmetric Infrared spectra of Europa were obtained by features. The difference between spectra of water the Galileo NIMS instrument from 0.7 to 5.2 mm ice and hydrated material is clearly demonstrated at a spectral resolution of 12.5–25 nm and sam- in Fig. 1, where the unmistakable water ice fea- pling interval of 12 nm (Carlson et al., 1992). The tures in the icy terrain are markedly different in NIMS observations (denoted G1ENNHILAT and the spectra from the disrupted terrain. The fact E11ENCYCLOD) used in this paper are available that these disrupted terrains are generally red- to the public and have not had any additional cor- dish-brown is also of note. While this reddish rections or recalibrations beyond the standard tone can be produced by and iron oxides, procedures applied by the Galileo NIMS Team. there are strong absorption features near 0.8–1 These data were downloaded with 228 wave- mm in the spectrum of iron, which are not seen length channels. The G1ENHILAT observation on Europa. Complex sulfur polymers could give was acquired with nominal spatial resolution of rise to similar coloration (Carlson et al., 1999b, 77 km/pixel, while E11ENCYCLOD was at 8.4 2002), but the precise of these polymers km/pixel. Twenty-six spectra of icy terrain and has not yet been determined. Hydrated salts can- 18 spectra of dark terrain in the G1ENNHILAT not explain the coloration, because they are white. data were averaged together using SpecPr to im- An alternative explanation is the pigmentation prove signal-to-noise characteristics. Averages of that is found in many microorganisms. Microor- 21 and 72 spectra from dark terrain, and 160 spec- ganisms also contain significant amounts of wa- tra from icy terrain, in the E11ENCYCLOD ob- ter, much of it as water of hydration. Sulfur- servation were created to compare the spectral metabolizing microbes such as S. shibatae contain features in the two terrain types. It should be sulfur compounds as well, including sulfates. Al- noted that the 72- and 160-spectra averages were though the spectra of microbial samples mea- extracted from a version of the cube that had not sured at room temperature contain water and wa- been reprojected to remove spatial distortions: ter of hydration absorption features, these are at This reprojection procedure may reduce spectral the wrong wavelength positions because they contrast due to spatial averaging of different arise from liquid water. However, at low tem- 512 DALTON ET AL. peratures these shift to longer wavelengths. Again, the question becomes, if microorganisms were emplaced at the surface of Europa, what . .. . would they look like? O The spectra of S. shibatae , D. radiodurans , and E. coli at 120 K are displayed in Fig. 2. The strongest C absorption features are those due to water of hy- C .. dration near 1.0, 1.25, 1.5, and 2.0 mm. These ex- C N hibit the characteristic asymmetry seen in the spectra of dark terrain on Europa. Shortward of 0.8 mm, the spectra of all three drop off toward H the visible, which is to be expected because of their reddish-brown coloration. Finer structure is also apparent in these spectra, which are shown FIG. 3.Portion of a peptide chain. This schematic di- here at the full resolution of the ASD instrument. agram illustates the molecular bonds that give rise to the Not all of this fine structure would be apparent 2.05- and 2.17- mm absorption features in the microbe at NIMS resolution. A wide absorption is dis- spectra. cernible at 2.3 mm that is caused by the C-H stretching mode common to spectra of many or- ganic compounds (Colthup et al., 1990). The most important differences from the hydrate features a C-N-H bending mode; the 2.17- mm feature is a in the Europa spectra are the narrow features at combination of the N-H fundamental with a C-N 2.05 and 2.17 mm. These arise from amide bonds stretching mode (Gaffey et al., 1993). within the peptide linkages of the cellular pro- The spectral characteristics of E. coli are not teins (Fig. 3): The 2.05- mm amide feature is a com- markedly different from those of the extremo- bination of the fundamental N-H vibration with philes. Arising as they do from the same sorts of bonds in the same sorts of compounds, this is not surprising. This suggests that carbon-based mi- crobes will look quite similar in the near-infrared 1 despite major changes in functionality and har- diness. While relative strengths and depths of individual absorption features may vary, the 0.8 features themselves remain the same. It may be

inferred that extraterrestrial microorganisms e

c Escherichia coli could bear strong resemblance to the ones pre- n

a 0.6 t sented here. c e l f e

R Infrared behavior of cell constituents 0.4 d Sulfolobus shibatae e l A better understanding of the sources of these a

c infrared features can be gained from examining S 0.2 spectra of individual cellular components. Figure 4 contains representative spectra of the four spec- Deinococcus radiodurans trally dominant materials other than water itself: 0 protein, nucleic acids, carbohydrates, and lipids. 0.8 1.2 1.6 2 2.4 These were all measured at room temperature with the ASD spectrometer. All four materials are Wavelength ( mm) influenced by water, particularly just short of 2.0 FIG. 2.Near-infrared reflectance spectra of three mi- mm but also near 1.5 mm. The 1.25- and 1.0- mm crobial samples at 120 K. All three exhibit water absorp- water absorptions are weaker and are not easily tion features near 1.0, 1.25, 1.5, and 2.0 mm, with the lat- distinguished in all of the spectra. Some of this ter two having the distorted and asymmetric nature characteristic of hydrates. Additional features at 2.05 and water is simply adsorbed on the powdered sam- 2.17 mm are due to amide bonds in the cellular structures. ples. The 1.5- mm water complex in the protein Spectra have been offset vertically for clarity. and lipid spectra comprises at least three narrow DETECTION OF MICROBES ON EUROPA 513

1.2 absorption at 2.3 mm is quite strong in the lipid spectrum and can also be identified in the pro- Lipid 1 tein and spectra, as expected.

DNA Spectral comparison of Europa e

c 0.8 candidate materials n a t

c The spectra of the primary Europa surface ma- e l f 0.6 terial candidates are shown in Fig. 5 along with e Protein R

C-H representative spectra of S. shibatae . All of these d e

l spectra were measured at 120 K and convolved 0.4 a

c to the Galileo NIMS wavelengths and resolution S corresponding to the G1ENNHILAT observation 0.2 Carbohydrate (bottom curve). Much of the structure evident in the laboratory spectra is subdued at this resolu- tion. The spectrum of sulfuric acid octahydrate is 0 0.8 1.2 1.6 2 2.4 taken from Carlson et al. (1999b). The spectra of bloedite [Na 2Mg(SO4)2?4H2O] and hexahydrite Wavelength ( mm) (MgSO4?6H2O) were taken from Dalton (2000). FIG. 4. Room temperature spectra of the main Based on room temperature measurements, Mc- infrared-active cellular components, vertically offset for Cord et al. (1998) suggested that bloedite and viewing. Dotted lines mark the positions of the 2.05- and hexahydrite may together make up as much as 2.17-mm amide absorption features. These are most preva- 65% of the surface composition in the disrupted lent in the protein, but are also visible in the DNA and lipid spectra. The C-H stretch absorption feature near 2.3 terrains. Carlson et al. (1999b) proposed instead mm common to many organic molecules is apparent in that sulfuric acid octahydrate is the dominant sur- the lipid, protein, and carbohydrate spectra. Water ab- face component. All four materials exhibit dis- sorption features are evident in all four spectra. torted and asymmetric absorption features due to water of hydration near 1.0, 1.25, 1.5, and 2.0 mm. combination bands (Pauling, 1935), which tend to In the Europa spectrum, the band centers are interact more closely in pure water (and ice) to located at 1.01, 1.23, 1.48, and 1.95 mm (dotted create the broad 1.5- mm feature seen in the icy lines). In hexahydrite, all except the 2.0- mm fea- Europa spectrum of Fig. 1. The diagnostic C-H ture fall shortward of these positions. Bloedite

H2SO4 • 8H2O Sulfolobus FIG. 5.Spectra of the S. shibatae Ar- Shibatae chaea, sulfuric acid octahydrate, and the e c hydrated sulfate salts bloedite and n Bloedite a hexahydrite, compared with the dark t c e

Europa terrain spectrum from Fig. 1. l f

Dotted lines denote positions of band e R centers from the Europa spectrum. Oc- d e tahydrate spectrum is from Carlson et al. l Hexahydrite (1999b) and was measured at 140 K. a c

Bloedite and hexahydrite spectra (mea- S sured at 120 K) are from Dalton (2000). The Galileo NIMSspectrum of Europa Europa is the same as in Fig. 1. All spectra are convolved to the NIMS wavelength set with overlapping wavelength positions deleted for clarity.

0.8 1.2 1.6 2 2.4 Wavelength ( mm) 514 DALTON ET AL.

provides a better match for the first three bands, a b c but the 2.0- mm feature is centered at a slightly Sulfolobus longer wavelength. Sulfuric acid octahydrate Shibatae matches well at 1.25 and 1.5 mm (no data were available below 1.0 mm), but the 2.0- mm absorp- H2SO4 • 8H2O tion occurs at a longer wavelength, as in the salts. Some of these wavelength discrepancies and e changes in band shape may be attributable to ra- c 1.5 Bloedite n a diation damage (Nash and Fanale, 1977), reduc- t c e ing the importance of exact matches. Still, it is in- l f Hexahydrite e teresting to note that the first three absorption r

d

band centers of S. shibatae fall slightly longward e t of the Europa positions, but with smaller dis- c e r placements than are observed in the hydrated r o c salts. The 2.0- mm band center in the S. shibatae - Europa m 1 spectrum matches the Europa position exactly. u u n

Each measured spectrum also displays features i t not observed in the Europa spectrum. Hexahy- n o drite and bloedite both exhibit additional bands C within the 1.5- mm complex, some of which are due to separation of combination bands at low temperatures. Hexahydrite has a weak band at 2.2 mm, while bloedite has a stronger one at 2.1 mm. Sulfuric acid octahydrate does not exhibit 0.5 any additional strong absorption features, though it and the salts all have a cation-OH stretching 1.8 1.9 22.1 2.2 2.3 feature at 1.35 mm, which is discernible at NIMS Wavelength ( mm) resolution (Dalton and Clark, 1999). Three fea- tures not apparent in the Europa spectrum of Fig. FIG. 6.Continuum-corrected spectra of the 2.0- mm ab- 5 are displayed by all three microbial samples, as sorption feature for the materials and wavelengths of Fig. 5. Dotted lines denote center positions of (from left represented by S. shibatae : the amide absorption to right): ( a) 2.0-mm water of hydration feature in Europa features at 2.05 and 2.17 mm, and the C-H stretch spectrum; and ( b) 2.05- and ( c) 2.17-mm amide features in at 2.3 mm. S. shibatae spectrum (arrows). Error bars denote standard Because the relative depths, widths, shapes, deviation of the mean for the original 18–spectrum aver- age (Dalton, 2000). and positions of the 1.0-, 1.25-, and 1.5- mm fea- tures in the Europa spectrum can all be repro- duced individually using only water ice of vary- ing grain sizes (McCord et al., 1999a; Dalton, agnostic characteristics. The band center in the 2000), the 2.0- mm absorption feature was used as Europa spectrum at 1.95 mm (dotted line, a) is a diagnostic signal. To highlight this information, most closely matched by the S. shibatae spectrum, the standard continuum removal method of while the three hydrate spectra are slightly longer Clark and Roush (1984) was applied to each of in wavelength, indicating a lower vibrational fre- the laboratory spectra and to the observed spec- quency than that observed at Europa. The addi- trum from Fig. 5. This reduces interference from tional absorption features at 2.1 mm in bloedite continuum absorption and transitions outside the and 2.2 mm in hexahydrite are not evident in the wavelength range of interest. The resulting con- Europa spectrum, though a decrease at 2.2 mm in tinuum-removed spectra are plotted in Fig. 6, the Europa spectrum may be significant. How- with vertical offsets applied for visibility. The cor- ever, this slight decrease is only two channels respondence between the band shapes, widths, wide, and may therefore be due to noise. The depths, and centers in the laboratory and obser- hexahydrite feature is much wider: six channels vational spectra can be ascertained more readily at the resolution of the NIMS observation. this way. Viewed in isolation, the 2.0- mm ab- More telling perhaps would be evidence of the sorption band complex reveals a number of di- amide features at 2.05 and 2.17 mm (dotted lines, DETECTION OF MICROBES ON EUROPA 515 b and c). The feature at 2.05 mm appears to cor- Search for infrared biosignatures on Europa respond to a similar feature in the Europa spec- trum. Though close to the noise level of the ob- To date, examination of the Galileo NIMS and servations, this 3% band depth feature (see Table SSI data has produced few examples of this 2) is 80 nm (six channels) wide. A slight inflec- (Dalton, 2002a). The modest spectral tion at 2.17 mm is barely discernible in the NIMS and spatial resolution of the NIMS data makes it observation. This absorption feature has a band difficult to locate spectra derived primarily from depth of 5% and a width of 120 nm (nine chan- strongly hydrated material and exhibiting suffi- nels), which should make it easier to detect. Un- cient signal to identify these subtle absorptions. fortunately, the short wavelength edge of this fea- Because the wavelengths are not identical in all ture overlaps with a gap between detectors in the NIMS observations, the NIMS channels are not NIMS instrument. At such positions (note the always ideally situated for identifying narrow closely spaced data points near 1.84 and 2.12 mm) features. the absolute reflectance cannot be determined One observation that may represent potential precisely because the NIMS detectors switch from evidence for biosignatures is the E11ENCY- one grating step to the next, introducing a gain CLOD01 image cube described earlier. A false- shift that is difficult to accurately compensate color image in Fig. 7 depicts the NIMS observa- (Carlson et al., 1992; McCord et al., 1999b). This tion overlaid on an SSI mosaic. The SSI image renders any identification suspect. A more robust has a spatial resolution ranging from 300 to 220 identification could be performed with higher m/pixel. The much larger NIMS pixels (8.4 km spectral resolution and signal levels. Despite nominal) have not been smoothed or averaged in these uncertainties, the wavelength positions of any way. While the instantaneous spatial profile the apparent features, and of the 2.0- mm feature is not square, the effective field of view is (R. Carl- itself, align remarkably well with those of the S. son, personal communication, 2003), so these raw shibatae measured at 120 K. Within the limits of pixels give a reasonable indication of the location noise and spectral resolution discussed below, on the surface being sampled. Each pixel repre- the shape of the 2.0- mm band complex in Fig. 6 sents a single spectrum (“NIMSEL”) correspond- comes close to what one might expect to see if mi- ing to that location. In the false-color image, blue croorganisms were in fact trapped near the sur- indicates icy spectral behavior in the 2.0- mm re- face in the disrupted, reddish terrain of Europa. gion, while red corresponds to the highly asym-

TABLE 2. ABSORPTION BAND CENTER POSITIONS, DEPTHS, AND WIDTHSFORTHE THREE MICROBES MEASURED AT 120 K AND FOR THE TWO EXTRACTED SPECTRA FROM DARK TERRAIN IN NIMS OBSERVATION E11ENCYCLOD01

Absorption feature center position, depth, and width

2.0-mm H2O 2.05-mm amide 2.17-mm amide D. radiodurans 1.9468 2.0525 2.1718 Center (mm) 0.2602 0.0284 0.0515 Depth 0.1593 0.0310 0.0531 Width (mm) S. shibatae 1.9577 2.0603 2.1694 Center (mm) 0.3853 0.0688 0.0258 Depth 0.2082 0.0455 0.0463 Width (mm) E. coli 1.9556 2.0535 2.1744 Center (mm) 0.1241 0.0141 0.0176 Depth 0.1734 0.0343 0.0495 Width (mm) 72 NIMSEL 1.9582 2.0459 2.1814 Center (mm) 0.4983 0.0264 0.0238 Depth 0.1954 0.0201 0.0130 Width (mm) 21 NIMSEL 1.9591 2.0431 2.1821 Center (mm) 0.5259 0.0633 0.0212 Depth 0.2055 0.0218 0.0131 Width (mm)

All values were calculated using the methods of Clark and Roush (1984). Band centers are for continuum-corrected spectra. The band depth is defined as the ratio of the reflectance at the band center position divided by the reflectance of the fitted continuum at the same wavelength. Widths are FWHM by the same method. 516 DALTON ET AL. metric absorption features associated with hy- desired spectral features were present, noise in drated material. This hydrated material is other parts of the spectrum made interpretation strongly correlated with the linea and some of the difficult. However, examining other parts of the dark spots in the image. Few areas are composed cube revealed similar features in the other linea. of pure water ice. Spectra were extracted from The southeastern linea is covered by more various regions within the image cube and ex- NIMSELS and, in particular, is more than one amined for evidence of 2.05- mm features. Where NIMSEL wide along much of its length. This al- possible, spectra from contiguous regions were lows a larger number of samples of non–water ice averaged together to improve signal-to-noise ra- terrain. As demonstrated in McCord et al. (1999b), tios. This was complicated by the high degree of the capabilities of the NIMS instrument, com- spatial heterogeneity at small scales on Europa: bined with the spatial heterogeneity and high ra- Even at 220-m resolution, features can be dis- diation environment at Europa, necessitate spec- cerned in the SSI image that are smaller or nar- tral averaging of carefully inspected NIMSELS in rower than the SSI pixels, so finding homoge- order to produce spectra that accurately reflect neous areas the size of the NIMSELS proved the surface constituents of interest. Following challenging. For example, the central stripes of their method, 72 NIMSELS (indicated in red, Fig. linea tend to be composed of icier material than 7) were selected and averaged in Fig. 8. Some the margins, so linea spectra typically have an icy NIMSELS contained noise spikes within the component to them. In the end, many of the spec- wavelength range of interest and were discarded tral averages were partly contaminated by water (black pixels, Fig. 7). In order to compare the spec- ice due to subpixel-scale mixing. tra of the heavily hydrated linea material with the One area of 21 contiguous NIMSELS (green, more icy background plains, a separate average Fig. 7) proved interesting. The averaged spectrum was made using 160 NIMSELS (blue pixels) lo- is shown in Fig. 8 (green curve). This spectrum is cated between the major linea. While some dis- dominated by the asymmetric 2.0- mm hydrate rupted areas and narrow linea traverse these feature. An apparent feature at 2.05 mm takes up plains, NIMSELS showing strong features of hy- several channels, and there is a slight indentation dration were omitted along with those having at 2.17 mm as well. Because the reprojection strong noise spikes (black pixels). process involves some spectral averaging, it may No offset, smoothing, or scaling has been ap- reduce both spectral contrast and variations in plied to the spectral averages in Fig. 8. Overlap- signal, resulting in weaker features but smaller ping wavelengths at the spectrometer grating noise levels. It may also introduce spurious fea- steps have been retained. All error bars indicate tures that typically arise from a single-channel 1-s levels of standard deviation of the mean. The noise spike being averaged into neighboring different amplitudes of error estimates for the 21- spectra. Spectral contrast may be reduced when versus the 72- and 160-NIMSEL averages reflect a spectrum having a specific absorption feature additional processing of the data in the reprojec- is averaged with a spectrum in which that feature tion method. The 2.05- mm absorption feature is absent. An average of 21 spectra is less likely is somewhat weaker in the 72-NIMSEL average to have spurious features, but given the noise lev- than in the 21-NIMSEL average (2.5% of re- els of the NIMS spectra, such an average is still flectance as opposed to 3%, Table 2), and does not suspect. appear to be quite as broad. Of course, this is a The unprojected image cube is shown at the difference of only two channels, but when deal- top of Fig. 7. Spacecraft motion and viewing ing with this few channels it affects the conclu- geometries produce considerable spatial “smear” sions that can be drawn. Similarly, the 2.17- mm in the observation, but the spectra are more rep- absorption feature, if present, can only occupy a resentative of surface material than those from few channels. The 72- and 21-NIMSEL averages the reprojected cube. Major surface features in- both have an apparent feature at this location, ex- cluding the intersecting linea can still be distin- cept that one channel is high ( ,2.19 mm) com- guished in the unprojected cube. Because there is pared with its neighbors. This would seem to ar- not a one-to-one correspondence between pixels gue against an identification based on this in the projected and unprojected cubes, no set of feature. If this single channel were high because NIMSELS in the unprojected cube was able to re- of random noise, then neighboring channels produce the spectrum just described. Where the should not be affected. On the other hand, the ap- DETECTION OF MICROBES ON EUROPA 517

NIMS E11ENCYCLOD

20 N

10 N

210 W

FIG. 7.Galileo NIMS E11ENCYCLOD01 observation. Top: Unprojected NIMS cube, with colored pixels indi- cating NIMSELS extracted for spectral averages in Fig. 8. 0.2 Blue, 160-NIMSEL average of icy plains units; red, 72 NIMS E11 ENCYCLOD01 Observation NIMSELS of dark linea material; black, NIMSELS having large noise spikes in the 2.0- mm range, which have been dropped from the average. Bottom: NIMS E11ENCY- CLOD observation in Mercator projection, overlaid on the 0.15 72 NIMSEL AVERAGE Galileo SSI image to illustrate locations of dark, disrupted surface material. Twenty-one NIMSELS extracted from dark linea terrain in the projected image have been aver- e aged in Fig. 8. c n

a 0.1

t 21 NIMSEL c e l f

FIG. 8.Averaged spectra from observation E11ENCY- e

CLOD01 showing potential evidence of biological ma- R 160 NIMSEL terial in disrupted terrain. A feature corresponding to the 0.05 ICY REGION 2.05-mm amide absorption is apparent in the two spectra from the dark linea in Fig. 7 (red and green curves). The blue curve, an average of 160 spectra from the area be- tween the linea, has a more symmetric 2.0- mm absorption band and no evidence of features at 2.05 and 2.17 mm. A 0 few channels are low at 2.17 mm in the dark terrain curves, 1.8 1.9 2 2.1 2.2 2.3 but one channel is high (at 2.19 mm) making confident as- signment of this feature difficult. Wavelength ( mm) 518 DALTON ET AL. pearance of the same spectral behavior over sev- cells (2.2 3 10210 and 1.1 3 1029 living cells/to- eral channels in both averages might indicate a tal cells). The hardiest of the three was D. radio- systematic effect, possibly due to instrument durans, with a survival of 5 3 1022 living cells/ characteristics. Yet both averages are from dif- total cells. E. coli, although not considered an ferent locations in the observation; and the 160- , had a survival rate of 3.4 3 1028 NIMSEL average shows no such feature. In fact, living cells/total cells, one order of magnitude the 160-NIMSEL average does not exhibit evi- higher than that of S. shibatae . Control samples of dence of either feature, which suggests that these D. radiodurans and E. coli, which had been iden- features may be linked to geographic units after tically stored but not spectrally analyzed, were all. Although it was collected over primarily icy also measured for survivability. Respective sur- terrain, the 160-NIMSEL average does exhibit vival rates for these samples were 5 3 1022 and some asymmetry in the 2.0- mm absorption fea- 4.7 3 1028 living cells/total cells. This indicated ture, and therefore some hydrated material is pre- that cell death occurred primarily because of the sent. However, the 2.05- and 2.17- mm absorptions freezing and thawing that occurred during sam- are not apparent in the 160-NIMSEL average, con- ple preparation and not because of the extremely sistent with a predominantly icy surface. Exami- cold temperatures of the experiment. nation of spectra throughout the E11ENCYC- Survival of D. radiodurans analyzed by the LOD01, E6ENSUCOMP02, and E4ENSUCOMP03 NACREF was also measured at temperatures rel- NIMS observations did not identify evidence of evant not only to Europa (120 K) but also inter- these features, although regions of highly hy- stellar space (20 K). Two aliquots were removed drated material were studied. This indicates that after exposure to the extreme conditions, cul- the 2.05- and 2.17- mm absorptions are not found tured, and measured for survivability. An aver- in all of the dark, disrupted, hydrated terrain. age survival of 71% was achieved after exposure This is not surprising, since the material giving to temperatures and pressures similar to those en- rise to this spectral signature may not be homo- countered in interstellar space. Because D. radio- geneously distributed. Just as the dark material durans is known for its robust nature, this may appears darker and more concentrated in some not be surprising. The two major points to be areas, there may be processes acting to concen- made here are (1) that survival rates were non- trate other constituents within the dark terrains. negligible for even the extreme cold of interstel- lar space and (2) that even E. coli, which is not Survival of organisms subjected to particularly well adapted to such harsh condi- extreme cold tions, exhibited some resistance to destruction by repeated thawing and freezing to extreme tem- The ability of microorganisms to survive ex- peratures. These results have important implica- posure to extreme conditions has implications not tions for the survival of microorganisms at Eu- just for Europa but for the search for life in the ropa and in space. itself, particularly as it relates to the hy- pothesis of . Survival and even meta- bolic activity have been quantified at subzero DISCUSSION temperatures, and certain cells have been found to survive temperatures approaching absolute The application of cryogenic spectra to the zero (Mazur, 1980; Rivkina et al., 2000). Indeed, analysis of remotely sensed spectra of icy satel- storage of frozen and freeze-dried microorgan- lites provides unique insights into planetary isms is a standard practice in re- processes. For this study, the comparison of search (Hay, 1978; Freshney, 2000). Galileo NIMS spectra with the spectra of ex- Culturing of our samples after exposure to Eu- tremophilic microorganisms measured in the lab- ropan temperatures during cryogenic spectro- oratory has implications for the interpretation of scopic measurements revealed viable microor- the Galileo data. Several effects must be taken ganisms in each sample, as determined by the into account when interpreting these results, in- limiting dilution method described in the previ- cluding the intense radiation environment and its ous section. The S. shibatae proved to be the most effects on the survival of not just microorganisms, fragile, with a survival of 1 cell out of a culture but of any organic . Additional work will containing between 4.6 3 109 and 9.2 3 108 total be needed to resolve some of these issues. DETECTION OF MICROBES ON EUROPA 519

Comparison of Galileo and laboratory spectra Examination of the continuum-removed spec- tra in Figs. 9 and 10 clarifies the types of spectral The low-temperature spectra of microbial sam- behavior that would characterize the biosigna- ples in Figs. 2 and 5 display all of the pertinent tures of entrained microorganisms at the surface features of hydrates. The primary water features of an icy satellite. The asymmetric 2.0- mm ab- near 1.0, 1.25, 1.5, and 2.0 mm are extremely sim- sorption feature dominates the spectra of all three ilar to those seen in the NIMS Europa spectra. This microorganisms, and the two spectra of reddish, in itself is not sufficient to claim an identification, disrupted terrain on Europa. There is also some since other materials may also display similar asymmetry in the 160-NIMSEL average of spec- spectral effects. The hydrated materials in Fig. 5 tra from the “icy” plains in the NIMS observa- also have hydrate features at these positions. The tion, which indicates the presence of some hy- overall slopes of the spectra tend to match fairly drated material in spectra making up the average well, although the slope for the sulfuric acid oc- (Fig. 7). The 2.05- and 2.17- mm amide features in tahydrate is somewhat too steep. The band center the microbe spectra (Fig. 9) vary in their relative positions for the microbe specimens as repre- intensities. Again, this can be because of scatter- sented by S. shibatae in Fig. 5 are also much bet- ing from water crystals, changes in ice grain sizes, ter matches to the Europa spectrum. This is sig- or differences in cell composition and/or con- nificant because the wavenumber of an absorption stituent concentrations. Each of these is difficult is directly proportional to the energy difference to control. Note that while the 2.17- mm feature is between molecular states, and the of much weaker than the 2.05- mm feature in the S. these states is proportional to the vibrational fre- shibatae spectrum, the opposite is the case in the quency of the molecule. If the vibrational fre- D. radiodurans spectrum. Both bands appear to quency does not match the observational evi- have similar strengths in the E. coli spectrum. dence, then the molecular bond lengths, bond While the depth of the 2.17- mm feature ranges strengths, and component cannot be iden- from 1.7% to 5.2% of the continuum level within tical. It may be possible to reproduce some of these the 2.0-mm complex, the 2.05- mm feature ranges observed band shifts in the laboratory through charged particle irradiation effects, which alter the chemical and physical properties of the candidate materials. While the relative depths of many 1 bands are closer to the Europa proportions in the e microbe spectrum, this is not as important as band c n a center position because relative depths can be t

c 0.9 e strongly influenced by particle grain size effects, l f scattering from grain boundaries, and mixture e R

proportions. These effects can be difficult to du- d 0.8 e plicate in the laboratory. Much more telling is the t c e reduction in reflected intensity shortward of 1 mm r r in the Europa and S. shibatae spectra. This drop off o c 0.7 -

is not present in the hydrated salts (Hunt et al., m u

1971; Crowley, 1991), but is evident in the spectra u n i of all three samples (Fig. 2). One t 0.6

n E. coli additional consideration is the visible reflectance: o D. radiodurans D. radiodurans , E. coli, and S. shibatae were selected C S. shibatae for this experiment partly because of their red- 0.5 dish-brown coloration, which is similar to that 1.8 1.9 22.1 2.2 2.3 seen on Europa. All of the postulated hydrated Wavelength ( mm) salts and acids are white. Even so, this does not eliminate them from consideration, as it may yet FIG. 9.Continuum-corrected spectra of E. coli, D. ra- be possible to reproduce the observed coloration diodurans, and S. shibatae measured at 120 K. The 2.0- through irradiation of hydrated sulfur com- mm water of hydration band center lies closer to 1.95 mm. The amide absorption features at 2.05 and 2.17 mm vary pounds. Further work will be necessary to inves- in strength among the three samples. No offset has been tigate this possibility. applied after the continuum removal. 520 DALTON ET AL.

NIMS E11 ENCYCLOD01 mm. The apparent depth of this feature is ,2%, 1.2 though slightly higher (2.4%) in the 72-NIMSEL

e spectrum. If the 2.19- mm channel is anomalously c n

a 1 high, this depth could be much greater, but such t

c estimates are highly speculative. e l f The high radiation environment at Europa e 0.8 R

makes interpretation of narrow spectral features d

e particularly challenging. A single radiation hit on t c

e 0.6 a detector may manifest itself as a single channel r r

o spike, often as far as five times the calculated vari- c - ance of the neighboring channels, but frequently

m 0.4

u much smaller and occasionally well within the u

n variance. For this reason, spectral averaging of i t

n 0.2 72 NIMSEL neighboring pixels has been used (McCord et al., o

C 21 NIMSEL 1998, 1999b; Dalton, 2000) to boost signal levels. 160 NIMSEL As described earlier, this can be troublesome for 0 1.8 1.9 22.1 2.2 2.3 two reasons. First, neighboring pixels may not be representative of the material in the original pixel; Wavelength ( mm) the high degree of spatial heterogeneity at small scales on Europa becomes critical. Second, spec- FIG. 10. Continuum-corrected spectra from NIMS E11ENCYCLOD01. Each has been vertically offset for vi- tral averaging can alter feature strengths. A noise sual clarity. The absorption features at 2.0, 2.05, and 2.17 spike may propagate into spectral averages and mm correlate well with those in the microbe spectra create apparent features that are not there, or, al- shown in Fig. 9. ternatively, a feature may be averaged away by the influence of pixels where it is either not pre- sent or is obscured by random noise. between 1.4% and 6.9%. Both are weakest in the Although some pixels with obvious noise E. coli spectrum. The relative depths of these spikes outside the 3 s level were omitted from the bands with respect to each other can thus vary spectral averages (black pixels in Fig. 7), some se- significantly in terrestrial microorganisms and lection effects may remain. The spectral average can therefore be expected to do so in extraterres- from the projected E11 NIMS cube may suffer trial microorganisms as well. More work should from errors introduced in the projection process. be done to quantify these variations and their Artifacts of the instrument and instrument cali- causes. At present, it would appear that the oc- bration may obscure the spectral signal. The large currence of these bands in concert with the 2.0- number of pixels in the unprojected averages (72 mm hydrate feature could be construed as a pos- and 160) combined with the presence of the ap- sible biosignature. parent features in both 72- and 21-NIMSEL aver- The shapes of the continuum-corrected NIMS ages would argue against purely random noise. spectra in Fig. 10 are suggestive of a 2.05- mm fea- Likewise, the similar shapes of the apparent 2.17- ture and possibly a 2.17- mm feature. As these fea- mm feature in both averages yet its absence from tures are superposed upon the 2.0- mm band com- the 160-NIMSEL “icy” average would seem to ar- plex, the continuum-removed spectra clarify their gue against a systematic error. relative contribution. There is clearly no evidence Given the low apparent strength of these fea- for either feature in the icy terrain spectrum, even tures and the high noise levels, we cannot confi- though it shows the influence of hydrates. The dently interpret them as evidence of life on Eu- band depth of the 2.05- mm feature in the 21-NIM- ropa. In particular, the fact that these features are SEL average is 6.3%, while only 2.6% in the 72- so narrow, coupled with their placement within NIMSEL average (Table 2). The weaker signal in the much stronger hydrate absorption feature, the 72-NIMSEL average could be due to compo- makes their detection and verification challeng- nent spectra that do not exhibit the feature (i.e., ing at the sampling and bandpass of the NIMS hydrated material that does not contain amides). Europa observations. The 2.05- mm amide feature The evidence for a 2.17- mm feature is even has a full width at half-maximum (FWHM) of be- weaker, particularly with the high channel at 2.19 tween 30 and 45 nm in the microbial samples, DETECTION OF MICROBES ON EUROPA 521 while the 2.17- mm feature has a FWHM from 46 temperature plasmas have demonstrated the abil- to 53 nm. To reliably detect these features even ity of plasmas to completely degrade D. under low noise conditions would require sam- radiodurans and biomolecules into CO 2, N2, and pling of at least 10 nm, which would place only H2O (Bol’shakov et al., 2003; Mogul et al., 2003). five channels across their FWHM. Given the high However, these experiments also showed that D. radiation environment at Europa, 2–5 nm sam- radiodurans was not significantly degraded by a pling is needed to significantly increase the po- plasma of a atmospheric gas analog. tential for robust discrimination of these biosig- These experiments highlight the importance of against the background continuum. plasma composition on the reaction rates of mi- crobial degradation. The surface gas composition and ion densities at Europa would suggest that Radiation survival of biological material the rate of destruction is not extremely fast. Fur- The radiation environment at Europa also thermore, since much of the destruction at the poses specific challenges not just to the detection surface of Europa is due to radiation-induced of life, but to the survival of the very compounds chemistry and not collisional in nature, the re- that make up most biological matter. Bombard- duced reaction rates prevailing at cryogenic tem- ment of the surface by energetic particles en- peratures cannot be neglected. trained within the Jovian drives In the absence of detailed studies concerning a number of physical and chemical processes that radiolysis of biological material at cryogenic tem- are detrimental to the persistence of complex peratures, some preliminary calculations may be molecules. Ironically, the radiolysis driven by made based on other available information. Mc- particle bombardment has also been invoked as Cord et al. (2001) have suggested that the resis- a method to produce compounds that could be tance of various compounds to destruction by used as energy sources by potential Europan life radiolysis may be estimated based upon the forms (Chyba and Phillips, 2001; Borucki et al., strengths of the various molecular bonds within 2002). The heavy-ion and proton bombardment the compounds. They reason that because the hy- flux is calculated to be between 10 8 and 1010 par- drogen bonds associated with the of hy- ticles/(cm2 s) (Ip et al., 1998; Cooper et al., 2001). dration in many of the hydrated salts are of equal The bulk of the energetic deposition occurs in the or greater strength than those in water ice (Eisen- form of electrons with energies in the 2–700 keV berg and Kauzman, 1969; Novak, 1974), the hy- range with a mean energy near 350 keV (Cooper drated salts should be at least as stable as water et al., 2001). These electrons interact with the sur- ice at the surface of Europa. To support this ar- face water ice to produce H and OH radicals, as gument they also point out that the energies of well as highly reactive species including O, O 2, the water infrared absorption features are simi- and HO2 (Hochanadel, 1960; Johnson and Quick- lar. Following this line of reasoning, one may con- enden, 1997; Carlson et al., 2002). Other elements sider the strengths of the amide bonds that give delivered via the magnetospheric particle flux in- rise to the 2.05- and 2.17- mm absorption features. clude presumably Iogenic sulfur, sodium, potas- First, for comparison, the 2.0- mm water feature it- sium, and chlorine (Carlson et al., 1999b, 2002; self is a combination of the n2 H-O-H symmetric Cooper et al., 2001; Paranicas et al., 2001). Com- bending and the n3 H-O-H asymmetric stretching plex interactions among the incident particles, modes. These involve the O-H bonds, which have their secondary radiolysis products, and the sur- bond energies of ,494 kJ/mol (Table 3). The 2.05- face produce a number of derivative products mm amide feature is also a combination band, (Moore, 1984; Delitsky and Lane, 1997, 1998; Carl- comprising the nN-H fundamental in concert with son et al., 1999a,b, 2002). a C-N-H bending mode (see Fig. 3), while the and/or microorganisms ex- 2.17-mm amide feature combines the nN-H funda- posed to this particle flux are expected to be bro- mental with a C-N stretching mode (Colthup et ken down over geologic timescales. Earlier it was al., 1990; Gaffey et al., 1993). The bonds within the mentioned that D. radiodurans can survive expo- peptide linkage of Fig. 3 and their energies are sures of up to 1,800 Grays; this corresponds to given in Table 3. Examination of Table 3 illus- less than a 3-day exposure at 1 mm depth on Eu- trates that the bonds responsible for the amide ropa’s surface based on dose rates from Parani- features are all at least two orders of magnitude cas et al. (2002). Recent experiments using low- stronger than the hydrogen bonds of water ice 522 DALTON ET AL.

TABLE 3. BOND ENERGIES FOR MOLECULES CONSIDEREDAS EUROPA SURFACE CANDIDATESIN THIS PAPER

Molecule Bond Bond energy (kJ/mol)

H2O H O 493.7 6 0.8 Ices, hydrates Hydrogen ,20 Acid salts [i.e., MH(RCOO)] Hydrogen ,25–35 Peptides, proteins, amino acids C O 525.9 6 0.4 C C 498 6 21 C H 335 6 4 C N 278.2 N H 372 6 8

Sources:Cottrell (1958), Pauling (1960), March (1968), Eisenberg and Kauzman (1969), Darwent (1970), Novak (1974), and Joesten and Schaad (1974). and hydrates, and closer in strength to the cova- products are dependent on temperature, radia- lent O-H bonds of the water molecule. This sug- tion dose, O 2 concentration, and the availability gests the possibility that, at low temperature, the of radical scavengers. Several of these variables amide bonds may be resistant to destruction by are not yet constrained well enough to calculate radiation-induced chemistry, enough so to sur- amide lifetimes at Europa based on existing lab- vive to the current epoch, and possibly even more oratory studies. Qualitatively, however, it is ap- so than the hydrated salts as discussed in McCord parent from chemical models and laboratory et al. (2001). studies that the lifetime of the amide bond may The suggestion that radiation resistance of a be much higher than those of the C-H and hy- compound can be estimated from molecular bond dration bonds, particularly at low temperatures strengths rests upon a number of assumptions, (Matsumoto et al., 1970). The approximate life- and does not take into account the fact that radi- times and G values need to be measured under olytic reactions involve electronic excitations of conditions that duplicate the temperature, den- many electron-volts, which can induce dissocia- sity, and radiation dose at Europa, and will be the tion from excited states. A deeper understanding subject of future studies. The outcome of such of stability issues can be gained from examina- studies may address a fascinating consideration: tion of the underlying mechanisms. The radi- If the detritus of radiation-processed microbes re- olytic destruction of most solutes in dilute aque- tains both hydrate and amide features, then this ous solutions occurs primarily through the could be a powerful to be utilized in diffusion and attack of hydroxyl radicals and sol- the search for life on Europa and other icy satel- vated electrons, which are produced by the radi- lites. If, however, the amide bonds are easily de- olysis of water. This is due to the fact that the stroyed by radiation, then the resulting detritus (water) is in a much higher concentra- may provide an even better spectral match to the tion than the solute of interest (Garrison, 1968; Galileo observations than any other candidate Kminek and Bada, 2001). The cold temperatures material considered thus far. and non-liquid state of the Europan ice crust Whatever their ultimate nature, radiolysis would limit the diffusion of chemically reactive products and implanted species are gradually in- species produced through radiolysis and there- corporated into the surface. Cratering rates and fore increase the apparent lifetime of the biomarker. estimates of impact gardening suggest that the Radiolysis studies on (HCONH 2) have uppermost 1.3 m of the surface can be overturned demonstrated that G values (yield per 100 eV of and well mixed on a geological resurfacing deposited energy) for all products are ,1, and timescale of approximately 10 Ma (Zahnle et al., that the amide bond is actually retained in some 1998; Chyba, 2000; Cooper et al., 2001). Biogenic of these radiolysis products (Matsumoto et al., elements delivered by impacts may have 1970). Investigations of peptides and proteins been mixed all the way down into the ocean over show that radiolysis of peptides also results in re- the age of the Solar System, providing enough tention of the amide bond, along with formation raw material to produce microorganism densities 3 3 of NH3, CO2, H2O2, keto acids, and aldehydes as high as 5 3 10 cells/cm (Whitman et al., 1998; (Garrison, 1987). The respective G values for these Pierazzo and Chyba, 2002). This calculation as- DETECTION OF MICROBES ON EUROPA 523 sumes a global average, and neglects mechanisms (Hunt et al., 1973), which could explain the spec- for concentrating either raw materials or living tral behavior in the NIMS observations shown in systems. Convective overturn, circulation pat- Figs. 6, 8, and 10. The apparent structure at 2.18 terns, diapirism, rifting, and lithospheric brine and 2.21 mm in the NIMS spectra could be due to mobilization caused by subsurface heating are all a number of materials that could obscure poten- mechanisms that may contribute to the concen- tial evidence of amide absorptions in this range. tration of biogenic elements or even biological Alunite [KAl 3(SO4)2(OH)6], for example, has a material both within the icy crust and in the in- combination feature at 2.17 mm caused by the fun- terior ocean (Spaun et al., 1998; Greenberg, 1999; damental O-H stretch together with an Al-O-H Head and Pappalardo, 1999; McKinnon, 1999; bend (Hunt et al., 1971). Several alteration miner- McKinnon and Shock, 2002; Prockter et al., 2002). als formed in the presence of sulfur compounds It is likely that some process would be necessary under terrestrial conditions are known to have to concentrate life-sustaining materials in order absorption features between 2.16 and 2.23 to support life within the ocean; determining the mm arising from this combination. Similarly, nature and viability of such mechanisms is be- Mg-O-H-bearing minerals sometimes exhibit fea- yond the scope of this paper. tures at 2.1, 2.2, and/or 2.3 mm (Hunt and Ash- There may be other substances present in the ley, 1979; Gaffey et al., 1993). It is possible that surface that could account for the observed spec- some other oxide or hydroxides on Europa could tral signature. As mentioned earlier, there are a give rise to the 2.05- and 2.17- mm features. Dam- plethora of hydrated compounds whose spectra age from irradiation may further complicate the have not been measured in the laboratory for picture, inducing wavelength shifts in absorption comparison with the icy satellites. This is a con- positions. Whatever alternative compounds may sequence of the relative infancy of the field of come under consideration, it must be remem- planetary remote sensing, and of the difficulty bered that to explain the observations, the surface inherent in reproducing the relevant temperature materials must exhibit both amide features as and pressure conditions. Given the radiation en- well as water of hydration. vironment at Europa it is reasonable to ask what sorts of compounds might be expected at the sur- Implications of survival face, as a result of chemistry involving either ex- ogenically derived or endogenic species. Irradia- The survival of cultures exposed to low tem- tion of sulfur compounds may play a role in peratures, and the survival of biological material producing some of the observed spectral shifts, exposed to particle bombardment, both have far- and polymeric sulfur rings and chains (S 8, Sx) reaching significance. Microbiologists may not be have been suggested as possible coloring agents surprised to read of the results of our survival ex- (Nash and Fanale, 1977; Carlson et al., 1999b, periments, but the context is slightly different 2002). The 2.05- and 2.17- mm amide bonds do not here. The results described above clearly demon- of necessity imply viable microorganisms at the strate that terrestrial and are ca- surface: These signals may also arise from for- pable of surviving exposure to the conditions of mamide or simple dipeptides. Pierazzo and temperature and pressure prevalent at the sur- Chyba (1999, 2002) have considered the potential face of Europa and in the near-surface environ- for amino acids to be delivered to Europa by ment, with no special preparation . They do not need , and concluded that impact survival of to be particularly hardy organisms, or even ex- such complex molecules is likely to be negligible. tremophiles, to do this. Furthermore, our results The apparent correlation, however, of the amide demonstrate that at least some terrestrial mi- absorption features with disrupted terrain is of croorganisms can survive the cold and vacuum interest since it indicates that while radiation pro- of deep interstellar space. Thus, the only remain- cessing may play a role, there is certainly endo- ing obstacle to panspermia is radiation. Microbes genic material associated with the observed sur- ensconced within crevices and fractures deep in- face features. There is also the possibility of a side silicate or icy celestial objects will be well or similar substance that could explain protected against ultraviolet and charged particle the observed spectral features. Some OH-bearing irradiation; only high-energy particles will pose minerals such as phyllosilicates and various hy- a significant threat. droxides exhibit spectral absorptions near 2.2 mm Figure 11 illustrates the near-infrared spectra 524 DALTON ET AL.

Deinococcus radiodurans at low temperature 0.7

200K

0.6 )

t 120K e s f f o

s 0.5 20K u l FIG. 11.Spectra of D. radiodurans at p ( 200, 120, and 20 K. Little variation is seen e

c with temperature; what is seen here may n 0.4 a

t be experimental artifacts due to small c

e changes in lighting and viewing geometry. l f e

R 0.3

0.2

0.8 1.2 1.6 2 2.4 Wavelength ( mm)

of D. radiodurans at 200, 120, and 20 K. As with tential for accidental contamination of a possi- other heavily hydrated species such as epsomite bly pristine world with terrestrial microbes. (cf. Dalton and Clark, 1998; McCord et al., 1998, While it is highly unlikely that extant - 2002; Dalton, 2000), there is little change in the isms might be found at the surface of Europa, two water absorption features below 200 K. This fact other possibilities remain. One is that the radio- may simplify the search for life and other organic lytically processed detritus of endogenic mi- material beyond the Solar System (Des Marais et croorganisms emplaced at the surface may still al., 2002; Pendleton and Allamandola, 2002) be- be available for study. As shown above, such de- cause only liquid and frozen organisms may need tritus may contain hydrated compounds, amino to be considered. The search for evidence of life acids, amides, and other products of radiation- beyond the Solar System will not be limited to driven organic chemistry. The reasonable as- at -like temperatures, and so these sumption that Europan organisms might at least measurements will prove useful. Further work partially base their upon abundant will need to be done to ensure that other mi- sulfur compounds suggests in turn that radiolyt- croorganisms and materials do not exhibit spec- ically processed sulfur compounds derived from tral changes at low temperatures. living material could exist at the surface. The survival of even the relatively fragile S. The other remaining possibility is that viable shibatae sample has another implication. As organisms could persist trapped in the ice. Based pointed out earlier, the Sulfolobus growth on the resurfacing scenario presented above, if medium is made up largely of sulfur com- the upper 1.3 m has been sufficiently processed pounds already considered to be likely compo- to render it sterile, then an astrobiological ex- nents of the Europan surface and ocean (Table plorer would need to excavate to at least 2 m, or 1) (McCord et al., 1998, 2002; Carlson et al., 1999b, possibly 5 m depth, in order to find organisms 2002; Kargel et al., 2000; Dalton, 2000). Even if that, while subject to extreme cold, have not been life is not found at Europa, it is possible that ter- exposed to radiation in the geologically recent restrial organisms could be used to seed life past. Furthermore, given the ongoing removal of there. The ethical considerations of such activity surface material by sputtering and micromete- are profound, and naturally beyond the scope of orite impact processes, relatively fresh material this investigation. However, the results pre- may be exposed over geologic time. Lag deposits sented here demonstrate that such concepts left behind as more volatile molecules migrate to must be considered carefully, in light of the po- colder surface areas could be expected to accu- DETECTION OF MICROBES ON EUROPA 525 mulate in the darker (and consequently warmer) diation environment, then they will provide a disrupted regions. Near-surface material that has powerful biosignature that can be exploited in the not been subjected to the full surface exposure evaluation of astrobiological potential at Europa. flux may retain greater complements of biologi- On the other hand, if the amide bonds are de- cally derived material. stroyed, the spectrum of the resulting material Noting the band depth of the 2.05- mm absorp- should give a compelling spectral match to the tion feature in the cryogenic S. shibatae samples observations of reddish, disrupted terrains. This to have been around 6.88% of continuum re- has profound implications. flectance (Table 2), and the absorption band The nature of other compounds produced by depth in the 72-NIMSEL observation to be such radiolysis is also of importance. Analysis of ,2.64%, a simple linear scaling may be used to this material could provide insights into the struc- calculate a rough upper limit on surface abun- ture of whatever the actual surface material is on dances. Based on the determination that our cen- Europa, based on spectral similarities. The fam- trifuged samples had cell densities of around 5 3 ily of hydrates and hydrated salts in particular 108 cells/cm3, the cell density of entrained mi- remains largely unexamined under controlled croorganisms at Europa would be about 2 3 108 laboratory conditions that simulate the Europan cells/cm3, or 200 million cells/ml. This corre- environment. It may be possible for not only in- sponds to about 0.2 mg/cm 3 of carbonaceous ma- organic species but also biomolecules or com- terial. Based on our survival rates, we calculate pounds of a prebiotic nature to form hydrates that within 5 m of the surface, in dark, disrupted even when frozen, perhaps under the influence terrain, there could conceivably be as many as 4 3 of ionizing radiation. A great deal of work still 1022 viable cells/cm 3, if the cells were as fragile needs to be done to assess the potential of vari- as the S. shibatae . If, on the other hand, the em- ous candidate materials to explain the observa- placed cells were as hardy as the D. radiodurans , tions. it may be possible to find as many as 1.4 3 108 A comprehensive search of the Galileo NIMS viable cells/cm 3 within 2–5 m of the surface in observations may yet turn up more evidence of dark deposits. If microbes were emplaced at con- narrow absorption features that have hitherto centrations of 10 6 cells/cm3, which is typical for gone unnoticed. Analysis of the Galileo mea- terrestrial saline , there could still be from 1 surements has been hampered by the high radi- cell per 4.6 l of ice to 50,000 cells/cm 3 still viable. ation noise present in the Europa flyby data and Even if microbes were emplaced at a concentra- the associated difficulties in interpreting the tion of 5 3 103 cells/cm3 (Pierazzo and Chyba, significance of small features, compounded by 2002), this could still provide as many as 3.5 3 the lack of relevant laboratory measurements. 103 viable cells/cm 3 if they are as tough as D. ra- Progress on both fronts could yield new insights. diodurans. If, however, they are fragile, this could Finally, future work should include plans for leave as few as 1 viable cell in 10 6 cm3, which a return to Europa, possibly with new spec- could be difficult to search for. It is important to trometers having capabilities that can address bear in mind though that all it takes is one living the questions raised by the NIMS data. An or- cell. Though these calculated values are of ne- biting imaging spectrometer having 2- to 5-nm cessity highly speculative, they provide a valu- sampling and bandpass, with a spatial resolution able starting place for discussion of strategies for up to 100 m, should be capable of acquiring spec- the search for life on Europa. tra of dark, homogeneous surface units with suf- ficient signal to unequivocally identify narrow spectral features such as those common to bio- Future work logical materials. A landed mission, capable of The results presented here suggest several ar- penetrating between 2 to 5 m (if not all the way eas in which further work is needed. The survival to the interior ocean) could collect samples and of both biota and biotic materials subjected to par- perform in situ analyses, which would defini- ticle fluxes at energies, temperatures, and pres- tively answer many questions about the surface sures relevant to Europa will be needed to an- composition. While not a certainty, there is also swer a number of questions. The survival of the the possibility that it may be able to return evi- amide bonds is of particular interest: Should the dence for biological activity in the subsurface of amide bonds survive exposure to the Europan ra- this enigmatic world. 526 DALTON ET AL.

CONCLUSION Kokaly of the U.S. Geological Survey for the Cyani- dium spectrum, which inspired much of this The comparison between observations of Eu- project. We acknowledge helpful reviews by ropa from the Galileo NIMS instrument and lab- Robert Carlson, and by an anonymous reviewer. oratory measurements of microbe samples under This investigation was partly supported by the Na- temperature and pressure conditions relevant to tional Research Council Fellowship program. No the Europan surface provides unique constraints macroscopic organisms were physically harmed on possible evidence for extraterrestrial life. In- during the preparation of this manuscript. frared absorption features due to water of hy- dration and amide bonds within cellular struc- tures may be useful as biosignatures in searches ABBREVIATIONS for past or present biota and biotic materials. Ex- amination of the Galileo NIMS observations sug- ASD, Analytical Spectral Devices; FWHM, full gested tantalizing glimpses of this biosignature; width at half-maximum; NACREF, NASA Ames however, within the constraints of spectral reso- Cryogenic Reflectance Environment Facility; lution and signal-to-noise, these NIMS observa- NIMS, Near Infrared Imaging Spectrometer; tions cannot be considered definitive evidence for OSSEC, Outer Solar System Environment Cham- either extant or extinct life on Europa. However, ber; SSI, Solid State Imager. the possibility of such evidence cannot be ruled out on the basis of available information, either. The data do suggest that recoverable samples of REFERENCES biological material and even viable organisms, if present in these locations, could persist within a Bol’shakov, A.A., Cruden, B.A., Mogul, R., Rao, M.V.V.S., few meters of the surface. Additional laboratory Sharma, S.P., Khare, B.N., and Meyyappan, M. (2003) work at relevant Europan temperature and pres- Radio-frequency oxygen plasma as a sterilization source. AIAA J. (in press). sure conditions, particularly on exposure of bio- Borucki, J.G., Khare, B., and Cruikshank, D.P. (2002) A logical material to particle bombardment and new energy source for organic synthesis in Europa’s spectral analysis of other candidate surface ma- surface ice. J. Geophy s. Res . 107(E11), 5114, doi terials, will be needed to address these questions. 10.1029/2002JE001841. Further analysis of the available NIMS data may Carlson, R.W., Weissman, P.R., Smythe, W.D., Mahoney, yield additional insights. An orbiter with an ad- J.C., and the NIMS and Engineering Teams vanced imaging spectrometer should be able to (1992) Near-Infrared Mapping Spectrometer experi- resolve the narrow spectral features diagnostic of ment on Galileo. Space Sci. Rev . 60, 457–502. Carlson, R.W., Anderson, M.S., Johnson, R.E., Smythe, biological materials, while a landed mission ca- W.D., Hendrix, A.R., Barth, C.A., Soderblom, L.A., pable of excavating several meters may be able to Hansen, G.B., McCord, T.B., Dalton, J.B., Clark, R.N., sample material that has not been substantially Shirley, J.H., Ocampo, A.C., and Matson, D.L. (1999a) altered by radiation, and thus derive new knowl- on the surface of Europa. Science edge of endogenic processes and their relation- 283, 2062–2064. ship to possible biological activity. Future space- Carlson, R.W., Johnson, R.E., and Anderson, M.S. (1999b) craft mission designs should take these results Sulfuric acid on Europa and the radiolytic sulfur cycle. Science 286, 5437–5440. into consideration in order to optimize their ca- Carlson, R.W., Anderson, M.S., Johnson, R.E., Schulman, pability to assess the astrobiological potential of M.B., and Yavrouian, A.H. (2002) Sulfuric acid pro- this icy world. duction on Europa: the radiolysis of sulfur in water ice. 157, 456–463. Cassen, P., Reynolds, R.T., and Peale, S.J. (1979) Is there ACKNOWLEDGMENTS liquid water on Europa? Geophys. Res. Lett . 6, 731–734. Chaban, G.M., Huo, W.M., and Lee, T.J. (2002) Theoreti- Sincere thanks to Roger Clark and Ted Roush cal study of infrared and Raman spectra of hydrated magnesium sulfate salts. J. Chem. Phys . 117, 2532–2537. for access to the cryogenic and spectrometer sys- Chyba, C.F. (2000) Energy for microbial life on Europa. tems, and to Jack Lissauer and Jonathan Trent for Nature 403, 381–382. helpful discussions. Also we are grateful to Wol- Chyba, C.F. and Phillips, C. (2001) Possible fram Zillig of the Max Planck Institute of Bio- and the search for Life on Europa. Proc. Natl. Acad. Sci . chemistry for the S. shibatae culture, and Raymond USA 98, 801–804. DETECTION OF MICROBES ON EUROPA 527

Clark, R.N. (1993) SPECtrum Processing Routines User’s Sharma, S.K., Horton, K., Granahan, J.C., and the Manual Version 3 (program SPECPR). U.S. Geological Galileo NIMS Team (1998) Laboratory simulation of the Survey Open File Report 93– 595. Available at: chemical of Europa’s aqueous phase [ab- http://speclab.cr.usgs.gov/ stract]. Proc. Lunar . Sci . XXIX, 1248. Clark, R.N. and McCord, T B. (1980) The Galilean satel- Fanale, F.P., Granahan, J.C., McCord, T.B., Hansen, G., lites: new near-infrared spectral reflectance measure- Hibbitts, C.A., Carlson, R., Matson, D., Ocampo, A., ments (0.65–2.5 mm) and a 0.325–5 mm summary. Icarus Kamp, L., Smythe, W., Leader, F., Mehlman, R., Greeley, 41, 323. R., Sullivan, R., Geissler, P., Barth, C., Hendrix, A., Clark, Clark, R.N. and Roush, T.L. (1984) Reflectance spec- B., Helfenstein, P., Veverka, J., Belton, M. J. S., Becker, K., troscopy: quantitative analysis techniques for remote Becker, T., and the Galileo NIMS, SSI, UVS Instrument sensing applications. J. Geophys. Res . 89, 6329–6340. Teams (1999) Galileo’s multi-instrument spectral view of Colthup, N.B., Daly, L.H., and Wiberley, S.E. (1990) In- Europa’s surface composition. Icarus 139, 179–188. troduction to Infrared and Raman Spectroscopy , 3rd edit., Fanale, F.P., Granahan, J.C., Greeley, R., Pappalardo, R., Academic Press, San Diego. Head, J., Shirley, J., Carlson, R., Hendrix, A., Moore, J., Cooper, J.F., Johnson, R.E., Mauk, B.H., Garrett, H.B., and McCord, T.B., and Belton, M. (2000) Tyre and Pwyll: Gehrels, N. (2001) Energetic ion and electron irradia- Galileo orbital remote sensing of mineralogy versus tion of the icy Galilean satellites. Icarus 149, 133–159. morphology at two selected sites on Europa. J. Geophys. Cottrell, T.L. (1958) The Strengths of Chemical Bonds , But- Res. 105, 22647–22657. terworths, London. Figueredo, P.H., Chuang, F.C., Rathbun, J., Kirk, R.L., and Crowley, J.K. (1991) Visible and near-infrared (0.4–2.5 Greeley, R. (2002) and origin of Europa’s “Mit- mm) reflectance spectra of playa evaporite minerals. J. ten” feature (Murias Chaos). J. Geophys. Res . 107, doi Geophys Res . 96, 16231–16240. 10.1029/2001JE001591. Dalton, J.B. (2000) Constraints on the surface composition Fink, U. and Sill, G. (1982) The infrared spectral proper- of Jupiter’s moon Europa based on laboratory and ties of frozen volatiles. In Comets, edited by L. Wilken- spacecraft data [Ph.D. dissertation], University of Col- ing, Press, Tucson, pp. 164–202. orado, Boulder. Fredrickson, J.K., Kostandarithes, H.W., Li, S.W., Plymale, Dalton, J.B. (2002a) Detectability of potentially entrained A.E., and Daly, M.J. (2000) Reduction of Fe(III), Cr(VI), microorganisms at the surface of Europa [abstract]. U(VI), and Tc(VII) by Deinococcus radiodurans R1. Appl. Proc. Lunar Planet. Sci. Conf . XXXIII, 1555. Environ. Microbiol . 66, 2006–2011. Dalton, J.B. (2002b) Cryogenic reflectance spectroscopy in Freshney, R.I. (2000) Culture of Cells: A Manual of support of planetary missions. In Proceedings of the Basic Technique , Wiley, New York. NASA Laboratory Workshop , edited by F. Gaffey, S.J., McFadden, L.A., Nash, D., and Pieters, C.M. Salama, NASA CP-2002–211863, NASA, Moffett Field, (1993) Ultraviolet, visible, and near-infrared reflectance CA, pp. 241–244. spectroscopy: laboratory spectra of geologic materials. Dalton, J.B. (2003) Spectral behavior of hydrated sulfate In Remote Geochemical Analysis , edited by C.M. Pieters salts: implications for Europa mission spectrometer de- and P.A.J. Englert, Cambridge University Press, Cam- sign. Astrobiology (in press). bridge, pp. 43–77. Dalton, J.B. and Clark, R.N. (1998) Laboratory spectra of Garrison, W.M. (1968) Radiation chemistry of organo- Europa candidate materials at cryogenic temperatures nitrogen compounds. Curr. Top. Radiat. Res . 4, 45–90. [abstract]. Bull. Am. Astron. Soc . 30, 1081. Garrison, W.M. (1987) Reaction mechanisms in the radi- Dalton, J.B. and Clark, R.N. (1999) Observational constraints olysis of peptides, polypeptides, and proteins. Chem. on Europa’s surface composition from Galileo NIMS data Rev. 87, 381–398. [abstract]. Proc. Lunar Planet. Sci. Conf . XXX, 2064. Goetz, A.F., Heidebrecht, K.B., Kindel, B., and Boardman, Darwent, B. deB. (1970) Bond Dissociation Energies in J.W. (1998) Using ground spectral irradiance for model Simple Molecules , National Standard Reference Data correction of AVIRIS data. In Summaries of the Seventh System Publication 31, U.S. National Bureau of Stan- Annual JPL Airborne Workshop , JPL Publi- dards,Washington, DC. cation No. 97–21, Jet Propulsion Laboratory, Pasadena, Delitsky, M.L. and Lane, A.L. (1997) Chemical schemes CA, pp. 159–168. for surface modification of icy satellites: a road map. J. Granahan, J.C., Fanale, F.P., Carlson, R., Kamp, L., Mat- Geophys. Res . 102, 16385. son, D., Ocampo, A., Smythe, W., Greeley, R., Geissler, Delitsky, M.L. and Lane, A.L. (1998) Ice chemistry on the P., Moore, J. M., Belton, M., and the Galileo SSI and Galilean satellites. J. Geophys. Res . 103, 31391–31403. NIMS Teams (1998) Galileo remote sensing composi- Des Marais, D.J., Harwit, M.O., Jucks, K.W., Kasting, J.F., tional studies of the Tyre Macula region of Europa [ab- Lin, D.N.C., Lunine, J.I., Schneider, J., Seager, S., Traub, stract]. Proc. Lunar Planet. Sci . XXIX, 1291. W.A., and Woolf, N.J. (2002) Remote sensing of plane- Greenberg, R., Hoppa, G.V., Tufts, B.R., Geissler, P., Ri- tary properties and biosignatures on extrasolar terres- ley, J., and Kandel, S. (1999) Chaos on Europa. Icarus trial planets. Astrobiology 2, 153–181. 141, 263–286. Eisenberg, D. and Kauzman, W. (1969) The Structure and Grogan, D.W. (1989) Phenotypic characterization of the Properties of Water , Oxford University Press, London. archaebacterial genus Sulfolobus: comparison of five Fanale, F.P., Li, Y.H., Decarlo, E., Domergue-Schmidt, N., wild-type strains. J. Bacteriol . 171, 6710–6719. 528 DALTON ET AL.

Grundy, W.M. and Schmitt, B. (1998) The temperature- C. (1970) Evidence for extraterrestrial amino acids and dependent near-infrared absorption spectrum of hexag- in the Murchison . Nature 228, onal H2O ice. J. Geophys. Res . 103, 25809–25822. 923–926. Hay, R.J. (1978) Preservation of cell culture stocks in liq- Lane, A.L., Nelson, R.M., and Matson, D.L. (1981) Evi- uid nitrogen. TCA Manual 4, 787–790. dence for sulphur implantation in Europa’s UV ab- Head, J.W. and Pappalardo, R.T. (1999) Brine mobiliza- sorption. Nature 292, 38–39. tion during lithospheric heating on Europa: implica- Lange, C.C., Wackett, L.P., Minton, K.W., and Daly, M.J. tions for formation of chaos terrain, lenticula texture, (1998) Engineering a recombinant Deinococcus radiodu- and color variations. J. Geophys. Res . 104, 27143–27156. rans for organopollutant degradation in radioactive Herzberg, G. (1991) Molecular Spectra and Molecular Struc- mixed waste environments. Nat. Biotechnol . 16, 929–933. ture II, Krieger, Malabar, FL. March, J. (1968) Advanced Organic Chemistry: Reactions, Hobbs, P.V. (1974) Ice Physics , Oxford University Press, Mechanisms and Structure , McGraw-Hill, New York. London. Marion, G.M. and Farren, R.E. (1999) Mineral solubili- Hochanadel, C.J. (1960) Radiation chemistry of water. In ties in the Na-K-Mg-Ca-Cl-SO 4-H2O system: a re-eval- Comparative Effects of Radiation , edited by M. Burton, J.S. uation of the sulfate chemistry in the Spencer-Møller- Kirby-Smith, and J.L. Magee, Wiley, New York, pp. Weare model. Geochim. Cosmochim. Acta 63, 1305– 151–189. 1318. Hudson, R.L. and Moore, M.H. (2001) Radiation chemi- Mattimore, V. and Battista, J.R. (1996) Radioresistance of cal alterations in solar system ices: an overview. J. Geo- Deinococcus radiodurans : functions necessary to survive phys. Res. 106, 33275–33284. ionizing radiation are also necessary to survive pro- Hunt, G.R. and Ashley, R.P. (1979) Spectra of altered rocks longed desiccation. J. Bacteriol . 178, 633–637. in the visible and near infrared. Econ. Geol. 74, Matsumoto, A., Hayashi, N., and Lichtin, N.N. (1970) 60Co 1613–1629. gamma-radiolysis of liquid formamide: gaseous and Hunt, G.R., Salisbury, J.W., and Lenhoff, C. (1971) Visible high-boiling stable products. Radiat. Res . 41, 299–311. and near-infrared spectra of minerals and rocks: IV. Sul- Mazur, P. (1980) Limits to life at low temperatures and at phides and sulphates. Mod. Geol. 3, 1–14. reduced water contents and water activities. Origins Life Hunt, G.R., Salisbury, J.W., and Lenhoff, C.J. (1973) Visi- 10, 137–159. ble and near-infrared spectroscopy of minerals and McCord, T.B., Hansen, G.B., Fanale, F.P., Carlson, R.W., rocks: VI. Additional silicates. Mod. Geol. 4, 85–106. Matson, D.L., Johnson, T.V., Smythe, W.D., Crowley, Ip, W.-H., Williams, D.J., McEntire, R.W., and Mauk, B.H. J.K., Martin, P.D., Ocampo, A., Hibbitts, C.A., Grana- (1998) Ion sputtering and surface erosion at Europa. han, J.C., and the NIMS Team (1998) Salts on Europa Geophys. Res. Lett . 25, 829–832. detected by Galileo’s Near Infrared Mapping Spec- Jamieson, C.S. and Dalton, J.B. (2002) A summary of lab- trometer. Science 280, 1242–1245. oratory spectra for ices of planetary interest. Bull. Am. McCord, T.B., Hansen, G.B., Matson, D.L., Johnson, T.V., Astron. Soc . 34, #37.07. Crowley, J.K., Fanale, F.P., Carlson, R.W., Smythe, Joesten, M.D. and Schaad, L.J. (1974) Hydrogen Bonding , W.D., Martin, P.D., Hibbitts, C.A., Granahan, J.C., Marcel Dekker, New York. Ocampo, A., and the NIMS team (1999a) Hydrated salt Johnson, R.E. and Quickenden, T.I. (1997) Photolysis and minerals on Europa’s surface from the Galileo near- radiolysis of water ice on outer solar system bodies. J. infrared mapping spectrometer (NIMS) investigation. J. Geophys. Res . 102, 10985–10996. Geophys. Res . 104, 11824–11852. Kargel, J.S. (1998) The salt of Europa. Science 280, McCord, T.B., Hansen, GB., Shirley, J.H., and Carlson, 1211–1212. R.W. (1999b) Discussion of the 1.04- mm water ice ab- Kargel, J.S. (1991) Brine volcanism and the interior struc- sorption in the Europa NIMS spectra and a new NIMS ture of and icy satellites. Icarus 94, 368–390. calibration. J. Geophys. Res . 104, 27157–27162. Kargel, J.S., Kaye, J.Z., Head, J.W., III, Marion, G.M., McCord, T.B., Orlando, T.M., Teeter, G., Hansen, G.B., Sassen, R., Crowley, J.K., Prieto-Ballesteros, O., Grant, Sieger, M.T., Petrik, N.G., and van Keulen, L. (2001) S.A., and Hogenboom, D.L. (2000) Europa’s crust and Thermal and radiation stability of the hydrated salt ocean: origin, composition, and the prospects for life. minerals epsomite, mirabilite, and natron under Eu- Icarus 148, 226–265. ropa environmental conditions. J. Geophys. Res . 106(E2), Kempe, S. and Kazmierczak, J. (2002) and early 3311–3320. life on Earth and Europa: favored by an alkaline ocean? McCord, T.B., Teeter, G., Hansen, G.B., Sieger, M.T., and Astrobiology 2, 123–130. Orlando, T.M. (2002) Brines exposed to Europa sur- Kivelson, M.G., Khurana, K.K., Russell, C.T., Volwerk, M., face conditions. J. Geophys. Res . 107, doi 10.1029/ Walker, R.J., and Zimmer, C. (2000) Galileo magne- 2000JE001453. tometer measurements: a stronger case for a subsurface McKinnon, W.B. (1999) Convective instability in Europa’s ocean at Europa. Science 289, 1380–1343. floating ice shell. Geophys. Res. Lett . 26, 951–954. Kminek, G. and Bada, J.L. (2001) The effect of sterilizing McKinnon, W.B. and Shock, E.L. (2002) Ocean karma: gamma radiation on amino acids [abstract]. Proc. Lunar what goes around comes around (or does it?) [abstract]. Planet. Sci . XXXII, 1918. Proc. Lunar Planet. Sci. Conf . XXXIII, 2181. Kvenvolden, K.A., Lawless, J.G., Pering, K., Peterson, E., Mogul, R., Bol’shakov, A.A., Chan, S.L., Stevens, R.M., Flores, J., Ponnamperuma, C., Kaplan, I.R., and Moore, Khare, B.N., Meyyappan, M., and Trent, J.D. (2003) Im- DETECTION OF MICROBES ON EUROPA 529

pact of low temperature plasmas on Deinococcus radio- lution: a mid-ocean ridge-type rift mechanism. J. Geo- durans and biomolecules. Biotechnol. Prog . 19, 776–783, phys. Res. 107, doi 10.1029/2000JE001458. doi 10.1021/bp025665e. Prockter, L.M. and Pappalardo, R.T. (2000) Folds on Eu- Moore, M.H. (1984) Studies of proton-irradiated SO 2 ropa: implications for crustal cycling and accommoda- at low temperatures: implications for . Icarus 59, tion of extension. Science 289, 941–943. 114–128. Reynolds, R.T., Squyres, S.W., Colburn, D.S., and McKay, Nash, D.B. and Fanale, F.P. (1977) Io’s surface composi- C.P. (1983) On the habitability of Europa. Icarus 56, tion based on reflectance spectra of sulfur/salt mixtures 246–254. and proton-irradiation experiments. Icarus 31, 40–80. Rivkina, E.M., Friedmann, E.I., McKay, C.P., and Gilichin- Noll, K.S., Weaver, H.A., and Gonnella, A.M. (1995) sky, D.A. (2000) Metabolic activity of permafrost bac- The albedo spectrum of Europa from 2200 to 3300 teria below the freezing point. Appl. Environ. Microbiol . Angstroms. J. Geophys. Res . 100, 19057–19059. 66, 3230–3233. Novak, A. (1974) Hydrogen bonding in solids. Correla- Rothschild, L.J. and Mancinelli, R.L. (2001) Life in extreme tion of spectroscopic and crystallographic data. Struct. environments. Nature 409, 1092–1101. Bonding 18, 177–212. Roush, T.L. (2001) Physical state of ices in the outer solar Ockman, N. (1957) The infrared-spectra and Raman- system. J. Geophys. Res . 106, 33315–33323. spectra of single crystals of ordinary ice [Ph.D. disser- Sagan, C., Thompson, W.R., Carlson, R., Gurnett, D., and tation], University of Michigan, Ann Arbor. Hord, C. (1993) A search for life from the Galileo space- Orlando, T.M. (2002) Electron- and vacuum ultraviolet craft. Nature 365, 715. photon-induced weathering of outer solar system sur- Schulze-Makuch, D., Irwin, L.N., and Guan, H. (2002) faces [abstract P52C-07]. In EOS Transactions of the Search parameters for the remote detection of extrater- American Geophysical Union, 2002 Fall Meeting Suppl. 83, restrial life. Planet. Space Sci . 50, 675–683. P52C-07. Spaun, N.A. and Head, J.W. (2001) A model of Europa’s Pappalardo, R.T., Belton, M.J.S., Breneman, H.H., Carr, crustal structure: recent Galileo results and implications M.H., Chapman, C.R., Collins, G.C., Denk, T., Fagents, for an ocean. J. Geophys. Res . 106, 7567–7576. S., Geissler, P.E., Giese, B., Greeley, R., Greenberg, R., Spaun, N.A., Head, J.W., Collins, G.C., Prockter, L.M., and Head, J.W., Helfenstein, P., Hoppa, G., Kadel, S.D., Pappalardo, R.T. (1998) Conamara Chaos region, Eu- Klaasen, K.P., Klemaszewski, J.E., Magee, K., McEwen, ropa: reconstruction of mobile ice polygons. Geophys. A.S., Moore, J.M., Moore, W.B., Neukum, G., Phillips, Res. Lett. 25, 4277–4280. C.B., Prockter, L.M., Schubert, G., Senske, D.A., Sulli- Squyres, S.W., Reynolds, R.T., and Cassen, P.M. (1983) van, R.J., Tufts, B.R., Turtle, E.P., Wagner, R., and Liquid water and active resurfacing on Europa. Nature Williams, K.K. (1999) Does Europa have a subsurface 301, 225–226. ocean? Evaluation of the geological evidence . J. Geo- Stevenson, D. (2000) Europa’s ocean—the case strength- phys. Res. 104, 24015–24056. ens. Science 289, 1305–1307. Paranicas, C., Carlson, R.W., and Johnson, R.E. (2001) Whitman, W.B., Coleman, D.C., and Wiebe, W J. (1998) Electron bombardment of Europa. Geophys. Res. Lett . 28, : the unseen majority. Proc. Natl. Acad. Sci . 673–676. USA 95, 6578–6583. Paranicas, C., Mauk, B.H., Ratliff, J.M., Cohen, C., and Venkateswaran, A., McFarlan, S.C., Ghosal, D., Minton, Johnson, R.E. (2002) The ion environment near Europa K.W., Vasilenko, A., Makarova, K., Wackett, L.P., and and its role in surface energetics. Geophys. Res. Lett . 29, Daly, M.J. (2000) Physiologic determinants of radiation 18-1–18-4, doi: 10.1029/2001GL014127. resistance in Deinococcus radiodurans . Appl. Environ. Mi- Pauling, L. (1935) The structure and entropy of ice and crobiol. 66, 2620–2626. other crystals with some randomness of atomic Zahnle, K., Levison, H., and Dones, L. (1998) Cratering arrangement. J. Am. Chem. Soc . 57, 2680–2684. rates on the Galilean satellites. Icarus 136, 202–222. Pauling, L. (1960) The Nature of the Chemical Bond , 3rd edit., Zolotov, M.Y. and Shock, E.L. (2001) The composition and Cornell University Press, Ithaca, NY. stability of salts on the surface of Europa and their Pendleton, Y.J. and Allamandola, L.J. (2002) The organic oceanic origin. J. Geophys. Res . 106, 32815–31827. refractory material in the diffuse : Zolotov, M.Y. and Shock, E.L. (2003) Energy for biologic mid-infrared spectroscopic constraints. Astrophys. J . sulfate reduction in a hydrothermally formed ocean 138, 75–98. on Europa. J. Geophys. Res . 108(E4), 5022, doi 10.1029/ Pierazzo, E. and Chyba, C.F. (1999) survival 2002JE001966. in large cometary impacts. Meteorit. Planet. Sci . 34, 909–918. Pierazzo, E. and Chyba, C.F. (2002) Cometary delivery of Address reprint requests to: biogenic elements to Europa. Icarus 157, 120–127. Dr. J. Brad Dalton Pilcher, C.B., Ridgway, S.T., and McCord, T.B. (1972) SETI Institute, MS 245-3 Galilean satellites: identification of water frost. Science NASA Ames Research Center 178, 1087–1089. Prockter, L.M., Head, J.W., Pappalardo, R.T., Sullivan, Moffett Field, CA 94035-1000 R.J., Clifton, A.E., Giese, B., Wagner, R., and Neukum, G. (2002) Morphology of Europan bands at high reso- E-mail: [email protected]..gov