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Abstract

Europa and Enceladus are key targets to search for evidence of life in our solar system. However, the surface and shallow subsurface of both airless icy moons are constantly bombarded by ionizing radiation that could degrade chemical biosignatures. Therefore, sampling of icy surfaces in future life detection missions to Europa and Enceladus requires a clear understanding of the necessary ice depth where unaltered organic biomolecules might be present. We conducted radiolysis experiments by exposing individual amino acids in ices and amino acids from dead microorganisms in ices to gamma radiation to simulate conditions on these icy worlds. In the pure amino acid samples, glycine did not show a detectable decrease in abundance, whereas the abundance of isovaline decreased by 40% after 4 MGy of exposure. Amino acids in dead Escherichia coli (E. coli) organic matter exhibited a gradual decline in abundances with the increase of exposure dosage, although at much slower rates than individual amino acids. The majority of amino acids in dead A. woodii samples demonstrated a step function decline as opposed to a gradual decline. After the initial drop in abundance with 1 MGy of exposure, those amino acids did not display further decreases in abundance after exposure up to 4 MGy. New radiolysis constants for isolated amino acids and amino acids in dead E. coli material for Europa/Enceladus-like conditions have been derived. Slow rates of amino acid destruction in biological samples under Europa and Enceladus-like surface conditions bolster the case for future life detection measurements by Europa and Enceladus lander missions. Based on our measurements, the “safe” sampling depth on Europa is ∼20 cm at high latitudes of the trailing hemisphere in the area of little impact gardening. Subsurface sampling is not required for the detection of amino acids on Enceladus—these molecules will survive radiolysis at any location on the Enceladus surface. If the stability of amino acids observed in A. woodii organic materials is confirmed in other microorganisms, then the survival of amino acids from a potential biosphere in Europa ice would be significantly increased.

Key Points

Gamma irradiation of pure amino acids, amino acids in ices, and amino acids in biological materials was conducted at liquid nitrogen temperatures to simulate radiolytic degradation of biomolecules in Europa and Enceladus near-surface ices. We observed that amino acids present in biological materials degrade at slower rates relative to free amino acids dispersed in ice.

1. Introduction

Europa and Enceladus have long been considered promising locations for the search for life beyond Earth. Both icy satellites have massive liquid oceans under their ice shells (Chyba and Phillips, 2002; Hand et al., 2009; Porco et al., 2006). Cometary interplanetary dust particles (IDPs) could have delivered the necessary carbon and other chemical ingredients for life as we know it (Pierazzo and Chyba, 2002) to both icy moons. Based on Cassini measurements, Postberg et al. (2023) found phosphorous, the least abundant bioessential element, in the ejected ice grains from Enceladus. Therefore, it is plausible to assume that potential biospheres in those ocean worlds would also be based on complex organic molecules (e.g., amino acids, carboxylic [fatty] acids, and nucleobases). If present, such molecules could be incorporated in the outer ice shell by various mechanisms (Chyba and Phillips, 2002; Barbaro et al., 2017) and be detectable in future lander missions. However, the icy surfaces of both airless icy bodies are exposed to ionizing radiation even though the nature of such radiation is different.

On Europa, ionizing radiation is dominated mostly by energetic electrons and to a lesser extent protons with energies of 10 KeV–100 MeV (Paranicas et al. 2009, see their Figs. 3, 4; Nordheim et al., 2018, see their Fig. 2) from Jupiter’s radiation belts. The bulk of galactic cosmic rays (GCRs) flux is effectively deflected by Jupiter’s magnetosphere, and only protons with energies >13.1 GeV and alpha particles with energies >6.1 GeV (Nordheim et al., 2019, see their Fig. 1, 2) can reach Europa’s surface.

Saturn’s magnetosphere is much weaker than Jupiter’s. Thus, on Enceladus, GCRs are only partially deflected by Saturn’s magnetosphere, and protons with energies >3 GeV can reach Enceladus’s surface (Kotova et al., 2019, see their Fig. 7). GCR’s alpha particles should be deflected even less effectively. Assuming the standard GCR particle flux distribution (Adriani et al., 2011; Maiorov et al, 2011 see their Fig. 1) and the proton access energies in the Saturnian system at the Enceladus’ orbit (Kotova et al., 2019, see their Fig. 7a), the total fraction of GCR protons that reach the surface of Enceladus should be ∼8.5% of the total incident GCR proton flux. In contrast, the flux of the electrons at the Enceladus surface from Saturn’s radiation belt is much weaker than the flux of energetic electrons at Europa’s surface. Specifically, Enceladus’ electron flux with energies ∼1MeV is ∼100 times less than Europa’s corresponding flux, while Enceladus’ electron flux of 6 MeV electrons is 1000 times less than that of Europa (Paranicas et al., 2012, see their Fig. 2; Paranicas et al., 2009 see their Fig. 3).

Any type of ionizing radiation particles (electrons, protons, alpha particles, or their secondaries) can break any molecules while penetrating through the target rock or ice. Therefore, any potential organic molecular biomarker could be destroyed or transformed beyond recognition by radiolysis. To avoid the effects of ionizing radiation, two strategies have been considered in future life search missions, that is, sampling of plumes and drilling into the ice surface (MacKenzie et al., 2021; Hand et al., 2022).

The first strategy allows sampling of unirradiated material with potential organic biomarkers from subsurface oceans without drilling. However, such a strategy cannot be easily implemented on Europa missions because Europa’s plumes, if present at all, would be highly localized and sporadic in time (Paganini et al., 2019). Plumes on Enceladus are more promising for sampling because Enceladus ejects water continuously (Villanueva et al., 2023) above the South Pole. Yet, it is not clear whether ejected material on Enceladus comes from the subsurface ocean or from the partial melting within the ice shell (Meyer et al., 2022). If the latter is true, then such material might not be representative of the Enceladus ocean. There is also an overall challenge on acquiring enough organic material during plumes’ flyby.

The second strategy, drilling, requires knowledge of ice depths where organic biomarkers can survive radiation exposure for long periods of time. The rate of radiation accumulation in the icy subsurface of Europa and Enceladus has been modeled with well-developed codes like GEANT (Paranicas et al., 2009, Nordheim et al., 2018, Teodoro et al., 2017). However, to estimate the degradation rate of organic molecules and ultimately the drilling depth, it is necessary to determine the radiolysis constants for organic biomolecules first. To date, there have been several studies of biomolecules’ radiolytic stability under Europa/Enceladus-like temperature conditions, which have focuses on glycine (Gerakines and Hudson, 2013), thymine (Materese et al., 2020), and uracil (Gerakines et al., 2022). All of those studies considered the radiolysis of pure isolated organic molecules dissolved in ice at various concentrations.

If life is present on either Europa or Enceladus, then we would expect that biomolecules would be incorporated in ice not only as individual free molecules but also as parts of cellular structures. The rate of radiolysis of an organic molecule as a part of a dead cell in ice can be different from the radiolysis of the same free organic molecule dissolved in the ice matrix, where “free” represents the amino acids that are not bound to the surface or bound together as peptides or larger molecules. For example, it is possible that biomolecules in dead cells can be partially protected from radiolytically generated oxidants by cellular membranes and, hence, degrade slower. Slower radiolysis rates would mean shallower drilling requirements for future life-searching missions.

In the present study, we conducted radiolysis experiments by exposing pure amino acids in ices and amino acids derived from dead microorganisms Escherichia coli (E. coli) and Acetobacterium woodii (A. woodii) in ices to gamma radiation as an analog for ionizing radiation exposure on Europa and Enceladus. The choice of microorganisms was done to generalize our findings of amino acids survival in biological materials. E. coli is a Gram-negative, facultative anaerobic prokaryote, while A. woodii is a Gram-positive, homoacetogenic bacterium. For samples with pure amino acids in ices, we have chosen glycine and isovaline. Isovaline is a common non-protein amino acid found in some carbonaceous meteorites and is therefore a good analog for amino acids of exogenous origin on the surface of Enceladus/Europa. Glycine is the simplest amino acid and present in both meteorites and biological organic matter. We also irradiated fused silica samples mixed with amino acids (glycine and isovaline) as an analog of organic molecules associated with some silicate mineral matrix deposited on the surface of Europa and Enceladus via IDPs. We determined new radiolysis constants of amino acids and estimated their survival fractions at various depths on Europa and Enceladus.

2. Methods

2.1. Sample preparation procedure

Stock solutions of individual amino acids and mixtures (1 × 10⁻³ M) were prepared by dissolving solid amino acid standards (97–99% purity from Sigma-Aldrich) in Millipore Integral 10 (18.2 MΩ, < 3 ppb total organic carbon) ultrapure water. The FS-120 fused silica was manufactured by H.P. Technical ceramics (Sheffield, UK). Solid FS-120 was crushed using a porcelain mortar and pestle. The crushed FS-120 was passed through a 150 µm sieve and then baked at 500°C in air overnight to remove organic contaminants. All samples were prepared in 13 mm diameter borosilicate glass test tubes that were wrapped in aluminum foil and then heated in a furnace at 500°C in air overnight. The samples were prepared in an ISO 5 HEPA laminar flow bench with a procedural blank prepared in parallel with each sample type.

The E. coli was cultivated in 1 L of LB media divided into four 1L-size glass Erlenmeyer flasks. Cells were grown to late-log phase at 37°C and then harvested by centrifugation at 8,000 RPM for 10–15 min to make a wet cell pellet. The pellet was washed three times with filtered distilled water. The cell pellet was then autoclaved and freeze-dried to produce a powder of dead cellular material for preparation of the various experimental conditions. The A. woodii was cultivated at 33°C in 1 L of DSMZ medium #135 divided into 4 1L-sized pyrex glass bottles adapted to seal with 20 mm blue butyl vial stoppers. Following the same procedure as that for E. coli, at late-log phase, the A. woodii cells were harvested by combining the growth together and then producing a single large wet cell pellet through centrifugation at 8000 RPM for 10–15 min. As with E. coli, the pellet was washed three times, autoclaved, and then freeze-dried to produce a dead cell powder for preparation of the various experimental conditions.

Samples were prepared at room temperature and then sealed under vacuum. The sample sets were prepared as shown in Table 1 and described as follows:

Table 1.
Experimental Masses, Volumes, and Irradiation Levels Used for This Study

Sample description Irradiation level

0 MGy 1 MGy 2 MGy 3 MGy 4 MGy

Amino acid plus fused silica

Glycine + Fused silica (5017.7 mg of FS + 1 mL of 1 mm glycine) 807.5 mg 882.5 mg 852.5 mg 840.7 mg 847.3 mg

Isovaline + Fused silica (5145.2 mg of FS +1 mL of 1 mM Ival) 862.7 mg 882.2 mg 863.8 mg 860.3 mg 866.7 mg

Amino acid plus ice

Glycine + Ice (1 mM solution) 200 µL 200 µL 200 µL 200 µL 200 µL

Isovaline + Ice (1 mM solution) 200 µL 200 µL 200 µL 200 µL 200 µL

Bio samples + Ice

E. coli + Ice (59.3 mg of E. coli in 10 mL of water) 500 µL 500 µL 500 µL 500 µL 500 µL

A. woodii + Ice (12.0 mg of A. woodii in 10 mL of water) 500 µL 500 µL 500 µL 500 µL 500 µL

Sample description	Irradiation level
Amino acid plus fused silica
Glycine + Fused silica (5017.7 mg of FS + 1 mL of 1 mm glycine)	807.5 mg	882.5 mg	852.5 mg	840.7 mg	847.3 mg
Isovaline + Fused silica (5145.2 mg of FS +1 mL of 1 mM Ival)	862.7 mg	882.2 mg	863.8 mg	860.3 mg	866.7 mg
Amino acid plus ice
Glycine + Ice (1 mM solution)	200 µL	200 µL	200 µL	200 µL	200 µL
Isovaline + Ice (1 mM solution)	200 µL	200 µL	200 µL	200 µL	200 µL
Bio samples + Ice
E. coli + Ice (59.3 mg of E. coli in 10 mL of water)	500 µL	500 µL	500 µL	500 µL	500 µL
A. woodii + Ice (12.0 mg of A. woodii in 10 mL of water)	500 µL	500 µL	500 µL	500 µL	500 µL

Amino acid and ice solutions were prepared by dissolving solid amino acid standards (97–99% purity from Sigma-Aldrich) in ultrapure water.

The glycine and fused silica samples were prepared by weighing out 5017.7 mg of FS-120 fused silica in a 20 mm test tube and adding 1 mL of 1 mM glycine. The entire sample was then dried under vacuum and vortexed to ensure that the glycine and FS-120 were adequately mixed.

The FS-120 and glycine mixture were then divided into five test tubes with approximately 850 mg of the mixture per test tube.

The l-isovaline and fused silica samples were prepared by weighing out 5145.2 mg of FS-120 fused silica in a 20 mm test tube and adding 1 mL of 1 mM l-isovaline. The entire sample was dried under vacuum and vortexed to ensure that the isovaline and FS-120 were adequately mixed. The FS-120 and l-isovaline mixture was then divided into five with approximately 850 mg of the mixture per test tube.

The E. coli powder plus water samples were prepared by weighing out 59.3 mg of the dry E. coli powder and adding 10 mL of ultrapure water in a 10 mL centrifuge tube and vortexing; 500 µL of this suspension was then transferred to each test tube for irradiation.

The A. woodii plus water samples were prepared by weighing out 12.0 mg of dry A. woodii powder into 10 mL of ultrapure water in a 15 mL centrifuge tube and vortexing. This suspension was then divided into five ampoules with 500 µL per test tube.

Procedural blanks were prepared for each sample set and analyzed in parallel and were not exposed to gamma radiation.

Each loaded test tube was converted into an ampoule by forming a neck with an oxy-propane torch. The ampoule was cooled and then connected via a stainless steel ½ inch Ultra-Torr fitting to a vacuum glass line with a liquid nitrogen (LN₂) trap and a turbo drag pump. To reduce adsorbed water and oxygen in the sample tubes, each tube was evacuated until the pressure inside the tube reached ∼20 mTorr and was then flame sealed in vacuo using the torch. For the tubes that contained liquid water, the tubes were first frozen in LN₂ before pumping and subjected to three freeze–pump–thaw cycles to remove dissolved air from the samples before flame sealing.

2.2. Irradiation

The flame-sealed sample tubes were irradiated with gamma rays (∼1 MeV) from a GammaCell 220 ⁶⁰Co radiation unit at a rate of 1.8 Gray (Gy)/s at the Radiation Science & Engineering Center (RSEC) facility at Pennsylvania State University. Gamma irradiation of samples was performed under LN₂ conditions (∼77 K). Samples were placed in a special sample holder and dewar (see Supplementary Figure S1, Supplementary Figure S2). Procedural controls for each sample set were prepared and analyzed in parallel and were not exposed to gamma radiation (0 MGy). Controls were kept at RSEC under LN₂ conditions, while other samples were irradiated.

Supplementary Figures S1 and Supplementary Figures S2 show the sample holder and the placement of the flame-sealed tubes with sample mixtures in a specially designed insulated cylindrical dewar. The sealed sample tubes were irradiated in a vertical position (Supplementary Figure S2). To maintain cold temperature, LN₂ was continuously replenished in the dewar throughout irradiation. Two thermocouples were placed inside the dewar at the top of the sample tubes. As soon as the temperatures readings increased above 77 K, LN₂ was replenished.

The duration of the irradiation to reach of the necessary dosages was adjusted by RSEC to take into account the partial shielding of samples by the dewar itself and by LN₂. Samples were exposed to total accumulated gamma doses of 1.0, 2.0, 3.0, and 4.0 MGy. The 4 MGy dosage corresponds to ∼13 million years of exposure at 10 cm depths on Europa at high latitudes of the trailing hemisphere (an area with the lowest exposure rate on Europa, Nordheim et al., 2018) and 25 million years of exposure in the top 1 m on Enceladus (Teodoro et al., 2017, their Fig. 1). After the desired total dosage was achieved, samples were removed from the irradiation chamber and placed in the external LN₂ dewar.

After all of the samples were irradiated to the specified dosages, they were placed in a stainless steel sample holder for transportation under dry ice (Supplementary Figure S3) to NASA Goddard Space Flight Center (GSFC). Caution in handling the samples upon removal from the LN₂ dewar after irradiation was required. See Supplementary Table S4 for a list of all samples and those that were lost during the workup process. The later three E. coli radiated samples (2, 3, and 4 MGy) all had issues with the glass ampoules becoming brittle or even exploding during or after irradiation. At GSFC, the samples were stored in a −80°C freezer until the tubes were opened for analysis. No attempt was made to analyze the volatile composition of the sample tube headspace after irradiation.

2.3. Sample extraction and analytical procedures

2.3.1. Sample extraction

After irradiation, each ampoule was tested for leaks by placing the sealed ampoule in a 20 mm test tube and submerging it in ultrapure water to find gas leaks. The irradiated samples along with their nonirradiated controls were then scored with a diamond-tipped glass scorer and opened for subsequent analysis. A portion of each sample was removed for analysis. All remaining portions of the samples were taken out of the original irradiated ampoule and stored in glass vials in a −80°C freezer for future analyses. See Supplementary Table S4 for the amount of each irradiated sample that was hot water extracted and subsequently analyzed for this study.

The amino acid plus fused silica samples were extracted in 1 mL of water at 100°C for 24 h. After extraction, the samples were centrifuged at 3000 rpm for 5 min, and 600 µL of the supernatant was transferred into a clean 10 mm tube and dried under vacuum. The samples were then brought up in 100 µL of water, and 10 µL was taken and diluted in another 100 µL of water to ensure that the analytes of interest were within the optimal instrumental measurement range.

The amino acid plus ice samples were flame-sealed separately in a glass ampoule with 1 mL of ultrapure water and extracted at 100°C for 24 h. After extraction, the supernatant was placed in a 10 mm test tube and dried under vacuum. After reconstitution, the samples were diluted three times to ensure that the analyte peaks of interest were within the optimal instrumental measurement range.

The E. coli plus ice and A. woodii plus ice samples were analyzed in triplicate. Owing to the biological nature of these samples, we expected to see more variability between the different aliquots (See Supplementary Table S4). Three aliquots of 100 µL of each sample were flame-sealed separately in a glass ampoule with 1 mL of ultrapure water and extracted at 100°C for 24 h. After extraction, the samples were centrifuged at 3000 rpm for 5 min and split into a hydrolyzed portion and an unhydrolyzed portion. The hydrolyzed portion of the water supernatants was subjected to a 6M HCl vapor hydrolysis procedure at 150°C for 3 h to determine total hydrolyzable amino acid content. The HCl acid-hydrolyzed, hot-water extracts were then desalted by using cation-exchange resin (AG50W-X8, 100–200 mesh, hydrogen form, BIO-RAD), and the amino acids were recovered by elution with 2 M NH₄OH (prepared from ultrapure water and NH₃(g) (AirProducts) in vacuo). The samples were dried under vacuum before being reconstituted, and a portion was taken for derivatization.

Based on our previous analyses of amino acid standards taken through the entire extraction, acid hydrolysis, and desalting procedure, we found no evidence of significant decomposition, racemization, thermal degradation, or carbon isotopic fractionation of the amino acids (Glavin et al., 2010).

2.3.2. Derivatization and analysis

Samples were derivatized with Waters AccQ·Tag reagents according to the manufacturer’s protocol (Boogers, 2008). Briefly, 10 μL of either a standard or a sample extract mix solution was mixed with 70 μL of AccQ·Tag Ultra borate buffer, and 20 μL of AccQ·Tag reagent previously dissolved in 1.0 mL of AccQ·Tag Ultra reagent diluent was added. The reaction was allowed to proceed for 10 min at 55°C. The ultra-high performance liquid chromatography (UHPLC) solvents were prepared according to the Waters manufacturer’s protocol.

The samples were analyzed via the commercial Waters AccQ·Tag protocol on a Waters Acquity UHPLC coupled to a Xevo TQ-S Micro triple quadrupole mass spectrometer. Separation of amino acids by UHPLC was accomplished by injecting 1 μL of the AccQ·Tag derivatized sample onto an Acquity AccQ·Tag Ultra C18, 150 × 2.1 mm column (1.7 μm particle size) maintained at 55°C. Chromatographic separation was achieved by using 100 μL AccQ·Tag concentrate A diluted in 900 μL of ultrapure water as eluent A and Waters AccQ·Tag B as eluent B. Analytes were eluted by using a flow rate of 700 μL/min and the following gradient: 0.00 min (0.1% B), 0.54 min (0.1% B), 5.74 min (10.0% B), 7.74 min (21.2% B), 8.04 min (59.6% B), 8.64 min (59.6% B), 8.73 min (0.1% B), and 10.00 min (0.1% B). The Waters Acquity UHPLC was equipped with a fluorescence detector set to λ_excitation = 266 nm and λ_emission = 473 nm.

The Xevo TQ-S micro triple quadrupole mass spectrometer was equipped with an electrospray ionization source (positive ion mode) by using multiple reaction monitoring (MRM) mode for detection and quantitation of the analytes. The Xevo TQS Micro capillary voltage was set to 1.0 keV, the sampling cone was set to 40°C, the source temperature was set to 150°C, the cone gas flow was set to 50 L/h, the desolvation temperature was set to 500°C, and the desolvation gas flow was set to 1000 L/h. The MRM parameters used for the liquid chromatography triple quadrupole mass spectrometry peak quantifications of the AccQ·Tag derivatives of the 20 standard protein amino acids are in the supporting information Supplementary Table S1.

Selected ion traces were quantified. A linear least-square model was fit to each amino acid in the standard calibration set, and these calibration curves were used to quantify the analytes in the samples. A procedural blank (empty sealed glass tube), which was carried through the same analytical procedures as the samples, was used to subtract procedural and laboratory amino acid background from the samples, which were all analyzed in triplicate. The amino acid concentrations in the procedural blank varied from compound to compound and ranged from 0.001 to 0.1 µM for glycine, serine, and l-alanine. Most of the analyzed samples contained amino acid concentrations in the mM range (>1000× above background). For some of the samples that degraded when irradiated, the detected amino acid levels dropped to near background levels, which increased the quantitation error.

3. Results

3.1. Individual amino acids in ices

We observed radiolytic degradation of the individual amino acids (glycine and isovaline) in icy mixtures (Fig. 1). For both glycine and isovaline, we did not observe measurable degradation in the 1 MGy samples. There was an almost 20% increase in the glycine signal for the 2 MGy sample, which could be attributed to either contamination in that sample or free radicals that led to a different pH or greater abundance of ammonia that could have altered the extraction efficiency and subsequent derivatization efficiency. Please note that only one sample was extracted, but three separate measurements were conducted to obtain error bars for the amino acids in ice measurements (See Supplementary Table S4). After a 4 MGy exposure, the glycine abundance did not change relative to the unirradiated control, whereas isovaline exhibited a gradual decline, with ∼40% of the initial abundances disappearing after 4 MGy. It is convenient to report radiolysis experiment results by using the standard exponential equation: $N / N_{0} = e^{- K_{rad} D}$ where N is the amino acid abundance in an irradiated sample, N₀ is the amino acid abundance in a control (unirradiated) sample, k is the radiolysis constant in MGy⁻¹, and D is the radiation dose in MGy. We plot N/N ₀—the remaining fraction of a specific amino acid in a sample after exposure to a dosage D (e.g., Fig. 1). The radiolysis constants for amino acids were obtained from the slope of a semi-log plot of N/N ₀ versus D using LINEST function in Microsoft Excel 365. We determine the following radiolysis constant for individual isovaline dissolved in ice: k _isovaline (ice in LN₂) = 0.13 ± 0.01 MGy⁻¹.

FIG. 1.

Normalized abundances hot water extracts of individual amino acids + H₂O ice irradiated up to 4 MGy. All samples were irradiated at 77 K (LN₂). The abundances measured after irradiation have been normalized against the control (unirradiated) samples. Uncertainties (δx) were determined as the standard error (δx = σ_x · (n) ^–½), whereby the uncertainties were based on the standard deviation (σ_x) of the average value of triplicate measurements (n = 3). The amount of irradiation for each sample are defined as follows: for 1 MGy, for 2 MGy, for 3 MGy, and for 4 MGy.

3.2. Individual amino acids in fused silica

We observed radiolytic degradation of the same individual amino acids (glycine and isovaline) mixed with fused silica (Fig. 2) at 77K. It was found that both glycine and isovaline degrade in silica mixtures at faster rates then in pure ice samples. Using the approach described above in Section 3.1, it was determined that the radiolysis constants for amino acids in silicates at 77K are: k_glycine (SiO₂ in LN₂) = 0.36 ± 0.04 MGy⁻¹, k_isovaline (SiO₂ in LN₂) = 0.45 ± 0.01 MGy⁻¹.

FIG. 2.

Normalized abundances of glycine plus fused silica and isovaline plus fused silica irradiated up to 4 MGy. All samples were kept at 77K (LN₂). The abundances measured after irradiation have been normalized against the control (unirradiated) samples. Uncertainties (δx) were determined as the standard error (δx = σ_x · (n) ^–½), whereby the uncertainties were based on the standard deviation (σ_x) of the average value of triplicate measurements (n = 3). The amount of irradiation for each sample are defined as follows: for 1 MGy, for 2 MGy, for 3 MGy, and for 4 MGy.

3.3. Radiolytic degradation of E. coli in ice

Figure 3 shows the surviving fraction of amino acids from dead E. coli cellular material dissolved in ice. Unfortunately, sample tubes of this set exposed to 2 MGy and 4 MGy exploded (see Section 2.2). Therefore, for this set, we plot only the data from 1 and 3 MGy samples. All amino acids exhibit a decrease with the increase of radiation dosage. However, with the exception of arginine (Arg) and histidine (His), at least ∼85% of all other amino acids survived 3 MGy of exposure. The radiolysis constant of valine k_valine (E. coli-ice in LN₂) = 0.027 ± 0.02 MGy⁻¹ is much smaller then k_isovaline (ice in LN₂) = 0.13 ± 0.01 MGy⁻¹. Note that isovaline is not common in the terrestrial biosphere, and thus, we compare the degradation of isovaline to the degradation of valine in biological material (since valine and isovaline are similar by mass and composition). The full list of radiolysis constants from this study is provided in Supplementary Table S3, and absolute abundance of amino acids is given in Supplementary Table S2.

FIG. 3.

Amino acid yields from three separate hydrolyzed hot water extracts of E. coli + ice. All samples were kept at 77 K (LN₂) during transport and irradiation before extraction. This figure shows the amino acids grouped by functional group (see Fig. SF4) and then arranged in ascending order according to molecular weight. Uncertainties (δx) were determined as the standard error (δx = σ_x · (n) ^–½), whereby the uncertainties were based on the standard deviation (σ_x) of the average value of triplicate measurements (n = 3). The amount of irradiation for each sample are defined as follows: for 1 MGy, for 3 MGy.

3.4. Radiolytic degradation of A. woodii in ice

Figure 4 shows the survived fraction of amino acids from A. woodii cellular material dissolved in ice. In this set of samples, we also lost a 3 MGy sample because of explosion. However, amino acids in the remaining 1, 2, and 4 MGy irradiated samples show a striking difference in radiolysis pattern compared with the E. coli samples (Fig. 3) and individual amino acids samples (Fig. 1). First, all amino acids in A. woodii ice samples showed a significant drop in abundance after just 1 MGy of exposure (10–35% decline). Second, several amino acids (proline, serine, and valine) demonstrated a gradual decrease in abundance at higher dosages as well with the radiolysis constants: k_proline (A. woodii-ice in LN₂) = 0.115 ± 0.004 MGy⁻¹; k_serine (A. woodii-ice in LN₂) = 0.13 ± 0.01 MGy⁻¹; k_valine (A. woodii-ice in LN₂) = 0.12 ± 0.01 MGy⁻¹. Those radiolysis constants are in good agreement with the radiolysis constant for individual isovaline k_isovaline (ice in LN₂) = 0.13 ± 0.01 MGy⁻¹ (Fig. 1). However, glycine, alanine, leucine, isoleucine, phenylalanine, and aspartic acid did not show any loss between 1 MGy, 2 MGy, and 4 MGy samples. Instead of the expected exponential decline in amino acid abundance with an increase in dosage, we observed a step-function behavior where, after an initial drop, amino acid abundance remained constant at higher dosages. Such a step-function behavior has not been observed in any previously published studies on amino acid radiolysis (Kminek and Bada, 2006; Gerakines and Hudson, 2013; Pavlov et al., 2022).

FIG. 4.

Amino acid yields from hydrolyzed hot water extracts. All samples were kept at 77 K (LN₂) during transport and irradiation before extraction. This figure shows the amino acids grouped by functional group (see Supplementary Figure S4) and then arranged in ascending order according to molecular weight. Uncertainties (δx) were determined as the standard error (δx = σ_x · (n) ^–½), whereby the uncertainties were based on the standard deviation (σ_x) of the average value of triplicate measurements (n = 3). The amount of irradiation for each sample are defined as follows: for 1 MGy, for 2 MGy, and for 4 MGy.

The data for A. woodii show the following trends: (1) the positive amino acids (histidine, arginine, and lysine) have more degradation for the small molecular weight amino acids, (2) the diacids amino acids (aspartic acid and glutamic acid) have more degradation for the large molecular weight amino acids, (3) the polar amino acids (serine and threonine) have similar degradation for both amino acids, regardless of molecular weight, (4) the aliphatic amino acids (glycine, alanine, valine, leucine, and isoleucine) have more degradation the higher the molecular weight, and (5) the aromatic amino acids (proline and phenylalanine) have similar degradation for both amino acids, regardless of molecular weight.

4. Discussion

4.1. Comparison to previous organic radiolysis studies and future work

We used gamma ray irradiation as a proxy for the ionizing radiation on Europa and Enceladus. However, as stated in the introduction, Europa’s surface radiation is dominated by the energetic electrons and to a lesser extent protons of Jupiter’s radiation belt, and Enceladus’ top ice layer is irradiated mainly by GCRs (protons and alpha particles). Gamma photons and energetic electrons cause the degradation of organic molecules similarly by cleaving electrons from the organic molecules, which then degrade during recombination. Blanco et al. (2018) conducted a comparative study of organic degradation by gamma vs. electrons in air, and they concluded that the dose, and not the type, of radiation was the main factor for the radiation damage to molecules. Gamma radiation has also been used as a proxy for GCR irradiation (Bonner et al., 1985; Kminek and Bada, 2006, Quinn et al., 2013, Pavlov et al., 2022) in planetary simulation studies because of low cost and convenience in setup. Protons have a significantly different linear energy transfer behavior than that of electrons and gamma-ray photons. Thus, it is possible that protons can destroy organic molecules with a different efficiency than gamma and electrons. However, no comparative study between gamma and proton radiolytic degradation of organic molecules has been conducted so far and should be addressed in future work.

When comparing radiolysis constants derived in our study (Supplementary Table S3) against previously published studies, it is important to recognize differences in the experimental setup and analysis techniques first. For example, there were several studies on the radiolytic degradation of amino acids under gamma radiation for planetary applications (Kminek and Bada, 2006; Pavlov et al., 2022). Although those studies used analysis techniques similar to ours (UHPLC), they were conducted under higher temperatures, and amino acids were not mixed with ice; thus, these studies were not directly applicable to Europa and Enceladus.

Another set of studies conducted irradiation experiments relevant to the survival of complex organic molecules in ices and at Europa/Enceladus-like temperatures (Gerakines and Hudson, 2013; Materese et al., 2020; Gerakines et al., 2022). However, those studies used 0.8 MeV protons for irradiation instead of gamma, had several times higher concentrations of organic molecules in ices, and used FT-IR technique for analysis.

In our study, free glycine in ice samples did not display a decrease in abundance with irradiation up to 4 MGy (Fig. 1). We did observe a similar lack of degradation in pure dry glycine powder under Mars-like temperatures in our previous study (Pavlov et al., 2022). Interestingly, Gerakines and Hudson et al. (2015) observed degradation of glycine in H₂O:glycine mix of 300:1 at 100 K with the radiolysis constant of 0.05 MGy⁻¹ (their Fig. 5). In our study, samples had H₂O:glycine ratio of 55560:1. Based on Gerakines and Hudson et al. (2013), radiolysis of glycine occurs more effectively in more diluted glycine mixtures (e.g., see their Table 3). Therefore, in our very diluted samples, we should have expected higher radiolysis rates not less. We suggest that proton radiation (used in Gerakines’ studies) is more destructive for glycine in ice mixtures than gamma radiation (used in our study).

As reported in Figure 1, we observed degradation of isovaline in ice with the radiolysis constant of 0.13 ± 0.01 MGy⁻¹. Such a constant is similar to the radiolysis constant for pure isovaline (0.099) reported by Pavlov et al. (2022) (see their Fig. 8). The higher radiolysis rate of isovaline compared with glycine observed by Pavlov et al. (2022) and noted in the current study could be because of isovaline’s larger molecular weight (Kminek and Bada, 2006). Radiolysis of isovaline was not studied previously at Europa/Enceladus-like conditions. However, Materese et al. (2020) studied the radiolysis of thymine (126.1 g/mol, C₅H₆N₂O₂), which is comparable in molecular mass and atomic composition to isovaline (117.15 g/mol, C₅H₁₁NO₂). Materese et al. (2020) reported radiolysis constants of 0.18 ± 0.03 MGy⁻¹ for thymine, which is comparable to what we found for isovaline.

Both glycine and isovaline in fused silica samples exhibit much faster radiolytic degradation rates than individual amino acids in ices or amino acids in E. coli-ice samples (Fig. 2; k_glycine (SiO₂ in LN₂) = 0.36 MGy⁻¹, k_isovaline (SiO₂ in LN₂) = 0.45 MGy⁻¹). Similarly, increased amino acid radiolysis was detected by Pavlov et al. (2022) when fused silica samples spiked with amino acids were gamma irradiated at Mars-like temperatures (218—223 K). However, radiolysis at Mars-like temperatures was even faster — k_isovaline(SiO₂ at 218–223 K) = 2.08 ± 0.14 MGy⁻¹. The difference in radiolysis constants is not surprising, since LN₂ temperatures result in decreased mobility of the oxidative radicals generated during gamma irradiation of SiO₂ (e.g., O). A decrease in radicals’ mobility would result in a less effective destruction of amino acids by radicals. Our results indicate that the rates of potential organic biomolecules’ degradation in silica-rich regions on both Europa and Enceladus are higher than in pure ice, and thus, future mission designers for Europa and Enceladus encounters should be cautious in sampling silica-rich locations on both icy moons.

4.2. Slow degradation in E. coli and step-function decrease in A. woodii amino acids

The E. coli + ice data show an increase of amino acid degradation within some functional amino acid groups with an increase in irradiation amount. This increase in degradation was observed for the similar structure positive amino acids with arginine (174.2 g/mol) having more degradation than the smaller molecular weight lysine (146.190 g/mol). As shown in Supplementary Table S4, the 2 MGy and 4 MGy samples did not survive the radiation process, which made this a smaller dataset than others. We are still able, however, to see degradation in most functional groups with increased irradiation amounts.

In general, degradation of the majority of amino acids in E. coli ice samples is significantly slower than the degradation of amino acids in pure dry amino acid powders (Kminek and Bada, 2006). For example, in dry powders, the amino acids radiolysis constants were reported as k_glycine (dry) = 0.067; k_alanine (dry) = 0.11; k_aspartic (dry) = 0.16; k_glutamic (dry) = 0.17 (Kminek and Bada, 2006). In contrast, the radiolysis constants of the same amino acids in E. coli material in our experiments were determined to be k_glycine (E. coli-ice in LN₂) = 0.033; k_alanine (E. coli-ice in LN₂) = 0.011; k_aspartic (E. coli-ice in LN₂) = 0.061; k_glutamic (E. coli-ice in LN₂) = 0.053. The radiolysis constants for the amino acids in E. coli samples are 2–10 times smaller than the radiolysis constants for pure amino acids powders.

However, the most surprising result came from the A. woodii samples. The abundance of a few amino acids (e.g., proline, serine, and valine) gradually decrease with the increase of exposure at significantly higher rates than the corresponding amino acids in E. coli samples (Supplementary Table S3). However, the majority of amino acids in A. woodii exhibited a step-function decline in abundance (Fig. 4), a significant drop in 1 MGy sample with respect to the unirradiated control, followed by essentially no change between the 1, 2, and 4 MGy samples.

One possible explanation for such behavior might be because of the presence of cellular material in biosamples. As mentioned in Section 2.1, in earlier radiation experiments, both E. coli and A. woodii were washed with DI water, autoclaved, freeze-dried (see section 2.1), and then uniformly distributed in water before freezing. However, despite all these preparation steps, amino acids in the biosamples will be at least partially encapsulated by the membrane and cell wall material of the dead cells. It is plausible that the membrane and cell wall material can preclude or slow down degradation of amino acids by blocking radiolytically produced oxidants in ice. That would explain the relatively slow overall rates of degradation in E. coli.

In addition, the presence of microbial antioxidant enzymes in E. coli (Staerck et al., 2017) may have effectively shielded cellular amino acids present from reactions with reactive oxygen species slowing their transformation. In response to hydrogen peroxide, E. coli have oxidative stress responses that involve the upregulation of ∼35 enzymes (Farr and Kogoma, 1991; Greenberg and Demple, 1989), as well as the upregulation of 40 other enzymes in response to superoxide (Greenberg and Demple, 1989; Walkup and Kogoma, 1989; Greenberg et al., 1990). These oxidative stress response systems are also upregulated and appear to offer protection during osmotic stress (e.g., Smirnova et al., 2000) and cold stress (Smirnova et al., 2001). Given the presence of dozens of E. coli enzymes related to oxidative stress, it seems plausible that some of these antioxidants could have quenched reactive oxygen species present in the ice or as the ice was melted, which may have resulted in protection of the amino acids present.

The step-function behavior of amino acids abundance in A. woodii might also be related to encapsulation. Unlike E. coli, A. woodii is a gram-positive bacteria with an S-layer outside of a thick peptidoglycan cell wall (Mayer et al., 1977) (Pavkov-Keller et al., 2011). S-layer is composed of protein (and peptidoglycan also has peptides). Together, this system of encapsulation can contain a significant fraction of the cells amino acids. Therefore, the initial drop in the amino acid abundance can be because of the destruction of amino acids in the S-layer and thick peptidoglycan cell wall. Once those amino acids are destroyed, the remaining amino acids could be protected by the encapsulation materials from oxidants and thus limit degradation and some amino acids can reform.

As mentioned earlier, even though abundance of the majority of the A. woodii amino acids displays a step-function behavior, a few amino acids (e.g., proline, serine, and valine) continue to degrade at higher dosages (Fig. 4 and Supplementary Fig. S5). Bacterial cell walls have abundant aspartic acid and glutamic acid (Howe et al., 1965) relative to typical proteins (Moura et al., 2013), while there is usually less serine (Howe et al., 1965). In our results, aspartic acid has one of the most pronounced step drops in abundance after 1 MGy, and in contrast, serine appears to show a gradual decline out to 4 MGy, which is consistent with it not being a major portion of the cell wall. Other amino acids are not easily explained with a simple model based on the cell wall. For example, biologically valine and leucine have similar functions and distributions in cell proteins, but leucine has a pronounced step drop in our results, and valine shows a graduate decline.

Future radiation experiments will be required to establish whether membranes from gram-positive microorganisms can result in effective protection of biomolecules from radiolytic products. If true, then the possibility of organic biosignature survival would increase significantly even in places with extremely high ionizing radiation rates (e.g., Europa).

Besides the step-function behavior of the A. woodii amino acid abundance, there were two other abundance trends in irradiated samples. The molecular weight and functional group played important roles in the amount of degradation with increasing radiation. This was especially evident with the large amount of degradation observed for the larger aliphatic amino acids and the small amount of degradation observed for the small aliphatic amino acids. Specifically, the one- and two-carbon aliphatic amino acids glycine (75 g/mol) and alanine (89 g/mol) had low degradation rates (84–88% survival), while the five-carbon and six-carbon aliphatic amino acids, valine (117 g/mol), leucine (131 g/mol), and isoleucine (131 g/mol), had higher degradation rates (64–68% survival). This correlation of increased degradation with molecular weight is consistent with the Kminek and Bada (2006) study. This correlation is also observed for the basic amino acids with similar structures as seen with arginine (174 g/mol) having had more degradation than lysine (146 g/mol). The opposite trend is observed with the amino diacids, with aspartic acid (133 g/mol) showing more degradation with increased radiation than glutamic acid (147 g/mol).

4.3. Implications to sampling on europa and enceladus

The overarching goal of our radiolysis studies was to determine the ice depths on Europa and Enceladus where organic biomolecules (specifically amino acids) are likely to be preserved despite continuous radiolytic degradation. As pointed out by Pavlov et al. (2022), it is important to distinguish the timescale for the degradation of biomolecules and the timescale for the complete destruction of organic matter. For example, Nordheim et al. (2018) (their Fig. 3) calculated the age to accumulate 603 MGy dosage as a function of depth in ice. They point out that 603 MGy is the dosage to break every molecular bond in the target material multiple times. Although such dosage is certainly a sufficient condition to wipe out any complex organic matter, it is not a “necessary” condition to destroy potential organic biomolecules beyond recognition. For example, if a potentially biological amino acid was radiolytically converted to some other organic molecules’ class (e.g., amines), then it would be hardly possible to establish the origin of the radiolysis product. Thus, for the life search planetary missions, the depth of drilling should be dictated not by the survival of organic matter in general, but by the timescale for the preservation of the original organic biomolecules (e.g., amino acids). Nordheim et al., (2018) calculated the destruction of amino acids in the surface ice on Europa (their Fig. 5), and yet the constraint for the depth of drilling (10–20 cm) in their study was based on estimates of 603 MGy dosage accumulation at a particular depth and location.

In our radiation experiments under Europa- and Enceladus-like conditions, all samples and all amino acids, with the exception of pure glycine in ice, displayed a noticeable decrease in abundance after 3–4 MGy of gamma radiation exposure. To determine the ice depths on Europa and Enceladus where a substantial fraction of specific organic biomolecules can survive, radiation accumulation depth profiles and radiolysis constants are needed. For Europa, we used radiation accumulation profiles from the work of Nordheim et al. (2018) (their Supplementary Fig. S1). On Europa, the radiation depth profile is highly dependent on location. The lowest dose-depth profiles and, therefore, the best location for organic survival occur at mid- to high-latitude locations on the trailing hemisphere (>45° N/S, 90°E). In that area, the rate of dose accumulation is ∼0.32 Gy/year at 10 cm ice depth and ∼0.063 Gy/year at 20 cm ice depth.

Using radiolysis constants from our experiments (Supplementary Table S3), we calculated the timescales to accumulate enough dosage to decrease the original abundance of amino acids by 90% (D₁₀) (Table 2). Given the age estimates of Europa’s ice surface at 30–90 Myr (Zahnle et al., 2008), our experimental study results suggest that drilling down to 10 cm on Europa might not be enough to obtain intact amino acids. However, drilling to 20 cm at mid- to high-latitude locations on the trailing hemisphere would be sufficient to sample a large fraction of the unaltered amino acids. At other locations (e.g., leading hemisphere and trailing hemisphere apex), amino acids would not survive even at 1 m depth.

Table 2.
Timescales to Accumulate D₁₀ Dosages (N/N₀ = 0.1) at 10 and 20 cm at Mid- to High-Latitude Locations on the Trailing Hemisphere of Europa

Depth Alanine (E. coli) + Ice Arginine (E. coli) + Ice Serine (E. coli) + Ice Histdine (E. coli) + Ice Isovaline (Individual) + Ice

10 cm 664 Myr 96.2 Myr 56.2 Myr 52.9 Myr 56.2 Myr

20 cm 3322 Myr 481 Myr 281 Myr 265 Myr 281 Myr

Depth	Alanine (E. coli) + Ice	Arginine (E. coli) + Ice	Serine (E. coli) + Ice	Histdine (E. coli) + Ice	Isovaline (Individual) + Ice
10 cm	664 Myr	96.2 Myr	56.2 Myr	52.9 Myr	56.2 Myr
20 cm	3322 Myr	481 Myr	281 Myr	265 Myr	281 Myr

On Enceladus, the main contributors to the radiation accumulation dosage below 1 mm ice depths are GCRs even though they are partially deflected by the Saturnian magnetosphere (see Introduction, Kotova et al., 2019). GCRs with energies >3 GeV (mostly protons and alpha particles) have a high penetration ability into the icy surface. Thus, just as is the case for Mars (Dartnell et al., 2007; Pavlov et al., 2012), there is little depth gradient of energy deposition in the top meter of Enceladus ice. Using radiation accumulation rates of ∼0.16 Gy/year from the work of Teodoro et al., (2017), we calculated timescales to accumulate D₁₀ dosages for the Enceladus surface (Table 3). Given the age of the Enceladus surface, 1–100 Myr (Kargel and Pozio, 1996; Kirchoff et al., 2018), our experiments show that future lander missions at Enceladus may not have to drill the surface at all—at any location on Enceladus, most of the amino acids will survive radiolytic degradation below just 1 mm of ice depths.

Table 3.

Timescales to Accumulate D₁₀ Dosages (10% Survival) at 1 mm Ice Depth of Enceladus

Depth	Alanine (E. coli) + Ice	Arginine (E. coli) + Ice	Serine (E. coli) + Ice	Histidine (E. coli) + Ice	Isovaline (Individual) + Ice
Surface	1308 Myr	189 Myr	111 Myr	104 Myr	110.7 Myr

In the top 1 mm of ice, organic molecules can be altered through UV photooxidation and radiolysis induced by electrons and protons from the Saturnian radiation belts (Nordheim et al., 2017). For example, Hendrix and House (2023) estimated the penetration depth for UV to be around 100 µm and suggested that organics buried beyond this depth would then be protected from UV transformations. Such burial and protection (Hendrix and House, 2023) would be most effective in the southern latitudes where ice particle deposition is fastest (Porco et al., 2006; Kempf et al., 2010; Southworth et al., 2019). In the Saturnian system, charged particle irradiation from the Saturnian radiation belts is less significant than UV for surface organic transformations, and these trapped particles impact Enceladus primarily on the leading and trailing hemisphere in temperate latitudes (Nordheim et al., 2017; Nordheim et al., 2018). Together, these studies suggest that sampling in the southern latitudes of Enceladus is optimal for organics found in the plume. In other locations, there may be subsurface organics from the emplacement of an ice diapir from below. The sampling of organics from below the penetration depth of UV and charge particle irradiation can be done anywhere such diapirs exist such as in the northern hemisphere (Filacchione et al., 2022). Such subsurface organics would be only subject to GCRs, which we find to be relatively small over the lifetime of the oldest Enceladus surface ice.

Note that using GCRs’ radiation accumulation rates from the work of Teodoro et al. (2017) is probably an overestimate for Enceladus. Few details of their calculation were given, but the absolute values of the reported ionization rates (their Fig. 1) are very similar to the calculated ionization rates in Martian permafrost from the work of Dartnell et al. (2007). That leads to the conclusion that the GCR fluxes at the surface of Mars and Enceladus were also assumed similar. However, as stated in the introduction, only protons with energies >3 GeV will reach Enceladus’s surface (Kotova et al., 2019). That would mean that the total cumulative flux of GCRs at the Enceladus surface should be only ∼8.5% of the incident GCR flux at Saturn’s orbit. On the contrary, the GCRs’ flux at the surface of Mars is reduced by the Martian atmosphere. Still, we should expect significantly lower ionization rates in Enceladus’ subsurface than that of Mars. If the ionization rates on Enceladus are indeed significantly lower than were predicted by Teodoro et al., (2017), then the ages to reach D₁₀ dosages (Table 3) in the Enceladus subsurface would be longer as well. That means our conclusion that amino acids would have an excellent chance to survive against radiolysis at any location in the Enceladus ice would be only stronger.

Note that, for estimates in Tables 2 and 3, we used radiolysis constants from E. coli samples and individual isovaline samples. Owing to the step-function dependence of the amino acids in A. woodii, we could not derive radiolysis constants for these samples. Further confirmation of the step-function behavior is needed. If confirmed, then amino acids at least in some terrestrial microorganisms can survive under Europa-like conditions indefinitely.

In addition, the estimates in Tables 2 and 3 do not take into account the impact of gardening on Europa and Enceladus surfaces (Costello et al., 2021). Although impact gardening can expose fresh material, it also buries irradiated material into the subsurface. Given the high efficiency of biomolecules’ radiolysis on Europa, it is critical to avoid heavily gardened surfaces in future sampling.

5. Conclusions

Gamma irradiation of pure amino acids, free amino acids in ices, and amino acids in biological materials was conducted at LN₂ temperatures to simulate radiolytic degradation of biomolecules in Europa and Enceladus ices. Our results suggest that a significant degradation will occur after exposure to just several MGy dosages. Amino acids in dead E. coli organic matter exhibit a gradual decline in abundance with the increase of exposure dosage, although at much slower rates than individual free amino acids. The majority of amino acids in dead A. woodii samples demonstrate a step function decline and not a gradual decline. After the initial drop in abundance after 1 MGy of exposure, those amino acids did not display a further decrease after exposure up to 4 MGy. New radiolysis constants for isolated amino acids and amino acids in biological material for Europa/Enceladus-like conditions have been derived. Using new radiolysis constants, the “safe” drilling depth to look for unaltered amino acids on Europa is 20 cm at high latitudes of the trailing hemisphere. Drilling is not required on Enceladus; amino acids will survive radiolysis below just 1 mm of ice at any location on the Enceladus surface

Footnotes

Acknowledgement

The authors would like to thank Mrs. Candace Davison, Assistant Director for Education and Outreach at RSEC The Pennsylvania State University for helping to set up gamma radiation experiment under liquid nitrogen temperatures.

Authors Disclosure Statement

There are no conflicts of interest.

Funding Information

This study was based upon work supported by NASA under award number 80GSFC21M0002 and NASA’s Planetary Science Division Internal Scientist Funding Program through the Fundamental Laboratory Research (FLaRe) work package at Goddard Space Flight Center. Also, this research was supported by NASA Astrobiology NfoLD award #80NSSC18K1140.

Supplementary Material

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

Supplementary Figure S4

Supplementary Table S1

Supplementary Table S2

Supplementary Table S3

Supplementary Table S4

Abbreviations Used

Associate Editor: Lewis Dartnell

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Supplementary Material

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Sample description	Irradiation level
Sample description	0 MGy	1 MGy	2 MGy	3 MGy	4 MGy
Amino acid plus fused silica
Glycine + Fused silica (5017.7 mg of FS + 1 mL of 1 mm glycine)	807.5 mg	882.5 mg	852.5 mg	840.7 mg	847.3 mg
Isovaline + Fused silica (5145.2 mg of FS +1 mL of 1 mM Ival)	862.7 mg	882.2 mg	863.8 mg	860.3 mg	866.7 mg
Amino acid plus ice
Glycine + Ice (1 mM solution)	200 µL	200 µL	200 µL	200 µL	200 µL
Isovaline + Ice (1 mM solution)	200 µL	200 µL	200 µL	200 µL	200 µL
Bio samples + Ice
E. coli + Ice (59.3 mg of E. coli in 10 mL of water)	500 µL	500 µL	500 µL	500 µL	500 µL
A. woodii + Ice (12.0 mg of A. woodii in 10 mL of water)	500 µL	500 µL	500 µL	500 µL	500 µL

Radiolytic Effects on Biological and Abiotic Amino Acids in Shallow Subsurface Ices on Europa and Enceladus

Abstract

Key Points

1. Introduction

2. Methods

2.1. Sample preparation procedure

2.3. Sample extraction and analytical procedures

2.3.1. Sample extraction

2.3.2. Derivatization and analysis

3. Results

3.1. Individual amino acids in ices

4.1. Comparison to previous organic radiolysis studies and future work

4.2. Slow degradation in E. coli and step-function decrease in A. woodii amino acids

4.3. Implications to sampling on europa and enceladus

Footnotes

Acknowledgement

Authors Disclosure Statement

Funding Information

Supplementary Material

Abbreviations Used

References

Supplementary Material