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The detection of organic molecules associated with life on Mars is one of the main goals of future life-searching missions such as the ESA-Roscosmos ExoMars and NASA 2020 mission. In this work we studied the preservation of 25 amino acids that were spiked onto the Mars-relevant minerals augite, enstatite, goethite, gypsum, hematite, jarosite, labradorite, montmorillonite, nontronite, olivine and saponite, and on basaltic lava under simulated Mars conditions. Simulations were performed using the Open University Mars Chamber, which mimicked the main aspects of the Martian environment, such as temperature, UV radiation and atmospheric pressure. Quantification and enantiomeric separation of the amino acids were performed using gas-chromatography-mass spectrometry (GC-MS). Results show that no amino acids could be detected on the mineral samples spiked with 1 μM amino acid solution (0.1 μmol of amino acid per gram of mineral) subjected to simulation, possibly due to complete degradation of the amino acids and/or low extractability of the amino acids from the minerals. For higher amino acid concentrations, nontronite had the highest preservation rate in the experiments in which 50 μM spiking solution was used (5 μmol/g), while jarosite and gypsum had a higher preservation rate in the experiments in which 25 and 10 μM spiking solutions were used (2.5 and 1 μmol/g), respectively. Overall, the 3 smectite minerals (montmorillonite, saponite, nontronite) and the two sulfates (gypsum, jarosite) preserved the highest amino acid proportions. Our data suggest that clay minerals preserve amino acids due to their high surface areas and small pore sizes, whereas sulfates protect amino acids likely due to their opacity to UV radiation or by partial dissolution and crystallization and trapping of the amino acids. Minerals containing ferrous iron (such as augite, enstatite and basaltic lava) preserved the lowest amount of amino acids, which is explained by iron (II) catalysed reactions with reactive oxygen species generated under Mars-like conditions. Olivine (forsterite) preserved more amino acids than the other non-clay silicates due to low or absent ferrous iron. Our results show that D- and L-amino acids are degraded at equal rates, and that there is a certain correlation between preservation/degradation of amino acids and their molecular structure: alkyl substitution in the α-carbon seem to contribute towards amino acid stability under UV radiation. These results contribute towards a better selection of sampling sites for the search of biomarkers on future life detection missions on the surface of Mars.
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Icarus 277 (2016) 342–353
Contents lists available at ScienceDirect
Icarus
journal homepage: www.elsevier.com/locate/icarus
Influence of mineralogy on the preservation of amino acids under
simulated Mars conditions
Renato dos Santos
a
, Manish Patel
b , c
, Javier Cuadros
d
, Zita Martins
a ,
a
Department of Earth Science and Engineering, Imperial College London, South Kensington Campus, London SW7 2AZ, UK
b
Department of Physical Sciences, The Open University, Milton Keynes MK7 6AA, UK
c
Space Science and Technology Division, Rutherford Appleton Laboratory, Harwell, Oxfordshire, UK
d
Department of Earth Sciences, The Natural History Museum, London SW7 5BD, UK
a r t i c l e i n f o
Article history:
Received 14 December 2015
Revised 18 May 2016
Accepted 19 May 2016
Available online 27 May 2016
Keywo rds:
Mars
Mineralogy
Solar radiation
a b s t r a c t
The detection of organic molecules associated with life on Mars is one of the main goals of future life-
searching missions such as the ESA-Roscosmos ExoMars and NASA 2020 mission. In this work we studied
the preservation of 25 amino acids that were spiked onto the Mars-relevant minerals augite, enstatite,
goethite, gypsum, hematite, jarosite, labradorite, montmorillonite, nontronite, olivine and saponite, and
on basaltic lava under simulated Mars conditions. Simulations were performed using the Open Univer-
sity Mars Chamber, which mimicked the main aspects of the martian environment, such as temperature,
UV radiation and atmospheric pressure. Quantification and enantiomeric separation of the amino acids
were performed using gas-chromatography-mass spectrometry (GC–MS). Results show that no amino
acids could be detected on the mineral samples spiked with 1 μM amino acid solution (0.1 μmol of
amino acid per gram of mineral) subjected to simulation, possibly due to complete degradation of the
amino acids and/or low extractability of the amino acids from the minerals. For higher amino acid con-
centrations, nontronite had the highest preservation rate in the experiments in which 50 μM spiking
solution was used (5 μmol/g), while jarosite and gypsum had a higher preservation rate in the exper-
iments in which 25 and 10 μM spiking solutions were used (2.5 and 1 μmol/g), respectively. Overall,
the 3 smectite minerals (montmorillonite, saponite, nontronite) and the two sulfates (gypsum, jarosite)
preserved the highest amino acid proportions. Our data suggest that clay minerals preserve amino acids
due to their high surface areas and small pore sizes, whereas sulfates protect amino acids likely due to
their opacity to UV radiation or by partial dissolution and crystallization and trapping of the amino acids.
Minerals containing ferrous iron (such as augite, enstatite and basaltic lava) preserved the lowest amount
of amino acids, which is explained by iron (II) catalyzed reactions with reactive ox ygen species gener-
ated under Mars-like conditions. Olivine (forsterite) preserved more amino acids than the other non-clay
silicates due to low or absent ferrous iron. Our results show that D- and L-amino acids are degraded
at equal rates, and that there is a certain correlation between preservation/degradation of amino acids
and their molecular structure: alkyl substitution in the α-carbon seem to contribute towards amino acid
stability under UV radiation. These results contribute towards a better selection of sampling sites for the
search of biomarkers on future life detection missions on the surface of Mars.
©2016 The Authors. Published by Elsevier Inc.
This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ).
1. Introduction
The detection of organic molecules associated with extra-
terrestrial life has been primarily focused on Mars due to its
proximity to Earth, evidences of a congenial past environment and
potential to support microbial life ( Westall et al., 2013 ). Increasing
evidence from NASA’s Opportunity and Curiosity rovers obtained at
different locations indicates that the Red Planet could have indeed
Corresponding author.
E-mail address: z.martins@imperial.ac.uk (Z. Martins).
supported life at the surface in the past (
Arvidson et al., 2014;
Grotzinger et al., 2014 ). Furthermore, the detection of silica-rich
deposits by the Spirit rover in the Gusev crater is also an indi-
cation of an environment able to support life ( Des Marais 2010;
Ruff et al., 2011; Squyres et al., 2008 ). It is also plausible that
life developed underground and biomarkers reached the surface
( Michalski et al., 2013 ). Despite this, the environmental conditions
that prevail now on Mars’ surface are not congenial to life or to the
preservation of biomarkers. Two of the factors contributing to the
harsh current martian environmental conditions are the thin at-
mosphere and the absence of a significant magnetosphere ( Fairén
http://dx.doi.org/10.1016/j.icarus.2016.05.029
0019-1035/© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ).
R. dos Santos et al. / Icarus 277 (2016) 342–353 343
Fig. 1. Powder X-Ray diffraction patterns of hematite (Fe
2
O
3
). The figures indicate the d-spacing of the several peaks in angstroms. The intensity increase at 10 °2
θis
produced by the X-ray fluorescence of Fe.
et al., 2010 ), resulting in the inability to attenuate the intensity of
the multiple forms of solar radiation that reach the planet, such
as UV radiation, galactic cosmic rays and solar energetic particles
( Cockell et al., 20 0 0; Hassler et al., 2014 ). As a result, the martian
regolith is exposed to intense levels of radiation, contributing to
the reactivity of the soil which may destroy potential martian life
and degrade organic molecules ( Dartnell et al., 2007; Quinn et
al., 2013 ). UV radiation leads to the formation of radical species
(e.g. reactive oxygen species such as superoxide and hydroxyl
radicals) by photochemical processes, which cause degradation of
any potential organic compounds present on Mars ( Benner et al.,
20 0 0; Georgiou et al., 2007, Georgiou et al., 2015; Yen et al., 20 0 0 ).
Amino acids, which are the building blocks of proteins and consid-
ered important target molecules in future life-searching missions
( Parnell et al., 2007 ), are known to be subjected to degradation
by UV radiation ( Garry et al., 2006; Noblet et al., 2012 ). A 1.5-year
exposure of glycine and serine to Mars-like surface UV radiation
conditions in low-Earth orbit resulted in complete degradation of
these organic molecules ( Noblet et al., 2012 ).
In order to maximize the chances of finding biomarkers on
Mars, we must determine the most suitable conditions to preserve
them. Preservation of organic molecules on Mars is thought to be
favored in subsurface environments, and also through associations
with specific minerals that may confer protection from the harsh
surface conditions ( Kminek and Bada, 2006; Summons et al., 2011 ,
and references therein; Poch et al., 2015 ). Despite the unfavorable
conditions that are found at the surface, indigenous chlorinated hy-
drocarbons were recently detected on Mars by the Sample Anal-
ysis at Mars (SAM) instrument on-board Curiosity ( Freissinet et
al., 2015 ). The successful detection of organic molecules on sam-
ples from Mars’ surface exposed to ionizing radiation and oxidative
conditions suggests that: 1) the preservation of organic molecules
may not be limited to subsurface environments, and 2) organic
biomarkers may be found on the surface if associated with specific
minerals.
In this paper we examine the preservation under simulated
Mars-like conditions of amino acids that were spiked onto 11
minerals and onto basaltic lava, which are all present on the mar-
tian surface ( Ehlmann and Edwards, 2014 ). The simulations were
performed using a custom-built Mars environmental simulation
chamber at the Open University (OU), Milton Keynes, UK. This
facility permits multiple aspects of the martian environment to
be simulated, including temperature, UV radiation, atmospheric
pressure and composition. Analyses of the amino acids extracted
from the mineral surfaces after the experiments were performed
by gas chromatography-mass spectrometry (GC-MS). Our results
are particularly relevant for future in situ life-detection missions,
such as the ESA-Roscosmos ExoMars 2018 rover and the NASA
Mars 2020 mission, highlighting which minerals may be the most
suitable to protect amino acids from the harsh environmental
conditions found at the martian surface.
2. Materials and methods
2.1. Minerals and XRD characterization
Eleven mineral samples were used in this work: augite (A), en-
statite (E), goethite (G), gypsum (Gy), hematite (H), jarosite (J),
labradorite (L), montmorillonite (M), nontronite (N), olivine (O)
and saponite (S). Basaltic lava (B) was also used. They were all se-
lected as representing abundant mineral phases on Mars ( Ehlmann
and Edwards, 2014 ). Augite, jarosite, labradorite, nontronite, and
saponite were purchased from Richard Tayler ( http://richardtayler.
co.uk , Cobham, Surrey, UK). Enstatite, goethite and olivine were
obtained from the Natural History Museum collection (NHM, Lon-
don), all of them unregistered specimens in the NHM collection.
The basaltic lava is a specimen collected in Mauna Loa (Hawaii) at
the point of lava quenching and donated by Joe Michalski. Gypsum
and hematite were purchased from Sigma Aldrich. The montmoril-
lonite is SAz-1 (smectite-rich rock of volcanic origin) described in
Cuadros (2002) .
Minerals were ground to powder by hand with a mortar and
pestle and they were analyzed with X-ray diffraction (XRD) at
the NHM, in order to determine their purity and structure. They
were side-loaded to avoid preferred orientation of particles and
analyzed in the range 3–80 °2 θusing a PANanalytical X’Pert Pro
diffractometer operated at 45 kV and 40 mA, with Cu K αradiation,
divergence slit of 0.25 °, Soller slits of 1.146 °and a solid-state
X’Celerator detector covering an angle of 2.1 °. The basaltic lava
contains the following mineral phases in the estimated order
of abundance: volcanic glass, pyroxene, olivine, and labradorite.
Jarosite is of the natrojarosite variety. Olivine is forsterite. The
augite and enstatite contain some traces of amphibole; the non-
tronite and montmorillonite contain traces of quartz; the other
minerals are pure at the XRD detection level. Fig. 1 shows the
X-ray pattern of hematite as an example.
2.2. Chemicals and tools
The pipette tips and eppendorfs used in this work were bought
sterile. Hydrochloric acid (37 wt. %), and high performance liquid
chromatography (HPLC)-grade water were purchased from Sigma-
Aldrich. Sodium hydroxide was purchased from Riedel-de Haen.
Aluminium hydroxide and 2-aminoheptanoic acid ( > 97%) were
purchased from Fluka. AG 50W-X8 resin (10 0–20 0 mesh) was
acquired from Bio-Rad. HPLC-grade dichloromethane (DCM) was
344 R. dos Santos et al. / Icarus 277 (2016) 342–353
Fig. 2. Mars Chamber simulator located at the Open University (left) and the experimental setup inside the chamber showing the amino acid-spiked minerals (right).
purchased from Fisher Scientific. Copper turnings used for sulfur
removal were purchased from BDH. The 25 amino acids used
in the experiments were: α-aminoisobutyric acid ( α-AIB); D,L-
isovaline; D,L-alanine; D,L-valine; glycine; D,L-norvaline; D,L- β-
aminoisobutyric acid (D,L- β-AIB); D,L- β-aminobutyric acid (D,L- β-
ABA); β-alanine; D,L-leucine; D,L-norleucine; γ-aminobutyric acid
( γ-ABA); D,L-aspartic acid; D,L-glutamic acid and 6-aminohexanoic
acid (6-AHA). The amino acid L-2-aminoheptanoic acid (L-2-AHA)
was not subjected to the simulation experiments and was used as
internal standard for the GC-MS analysis. All the amino acid stan-
dards were purchased from Sigma-Aldrich, except D,L-isovaline,
which was bought from Acros Organics. The trifluoroacetic an-
hydride isopropanol (TFAA-IPA) derivatization kit was obtained
from Alltech. All glass tools and ceramics used were sterilized by
wrapping in aluminum foil and heating in a furnace for at least 3
h at 500 °C.
2.3. Spiking of amino acids
A stock solution of 0.005 M concentration was prepared for
each of the 25 amino acids. One milliliter of each amino acid
stock solution was used to prepare a spiking solution containing an
equimolar mixture of the 25 amino acids. Four of these solutions
were prepared with final concentrations of 50, 25, 10 and 1 μM
of each amino acid. The spiking solutions containing 1, 10, 25 and
50 μM concentrations of each amino acid were labeled as solution
1, 2, 3 and 4, respectively. Concentrations were chosen by adapt-
ing the protocols from Parbhakar et al (2007) and Cuadros et al.
(2009) . These authors show that at low amino acid concentrations
the mechanism of amino acid adsorption on smectite is a simple
exchange with interlayer cations, whereas at higher amino acid
concentration physical interaction between amino acid molecules
become important. In the present work we wanted to be in the
low-amino acid concentration (i.e. much lower than 0.025 M) in or-
der to avoid amino acid interaction with other amino acids.
Approximately 30 mg of each mineral sample described in
Section. 2.1 were weighed in Pyrex test tubes. Three milliliters of
each of the solutions described above (1–4) were transferred into
12 test tubes, each containing one of the minerals.
The experiments containing the minerals and the spiking so-
lutions were named by the respective mineral initial provided in
Section. 2.1 , followed by the number of the solution. For exam-
ple, augite spiked with solution 1 (i.e., 1 μM of each amino acid)
was labeled as A1, while augite samples labeled as A2, A3 and A4
were spiked with solution 2, 3 and 4, respectively. Using this la-
beling procedure, the experiments carried out include basaltic lava
(with experiments B1, B2, B3 and B4), enstatite (E1, E2, E3 and E4),
goethite (G1, G2, G3 and G4), gypsum (Gy1, Gy2, Gy3 and Gy4),
hematite (H1, H2, H3 and H4), montmorillonite (M1, M2, M3 and
M4), nontronite (N1, N2, N3 and N4), olivine (O1, O2, O3 and O4)
and saponite (S1, S2, S3 and S4). Jarosite was only used in exper-
iments 3 and 4, resulting in a total of two samples (J3 and J4).
Labradorite was only used in experiments 1, 2 and 3 (L1, L2 and
L3).
All the test tubes containing the mineral samples and the spik-
ing solutions were flame sealed and placed in an orbital shaker
(Heidolph Polymax 1040) for 24 h at 50 revolutions per minute
(rpm) in order to let amino acids adsorb onto the mineral surfaces.
The outside of the test tubes was rinsed with HPLC grade water
and cracked open. The content of the test tubes was dried under a
flow of nitrogen (i.e., the spiking solution was dried in contact with
the mineral). Thus, the 1, 10 , 25 and 50 μM solutions correspond
to 0.1, 1, 2.5 and 5 μmol/g of the amino acids on the minerals,
respectively.
Control experiments were prepared by repeating the same pro-
cedure described in this section with a second set of samples. The
first set of samples was used to perform the Mars chamber sim-
ulations, while the second set was used as controls (i.e., samples
that were spiked but not subjected to the Mars simulation).
2.4. Mars chamber simulations
The spiked mineral standards were transferred into 14 mm di-
ameter metallic sample cups and placed inside a Mars chamber
simulator at the Open University, Milton Keynes, UK ( Fig. 2 ). The
sample cups were pre-sterilized by heat at 500 °C for 4 h. The
thickness of the deposits was approximately 1 mm in order to
avoid any self-shielding issues. The sample cups were placed on
a custom-made cold plate, to enable the cooling of the samples to
Mars-relevant temperatures. Copper shielding was provided to the
edges of the plate to define a cold zone, and the external faces of
the plate and shields were insulated to provide an efficient sample
cooling zone. The cooling plate was connected to a liquid nitro-
gen supply, with thermal valves providing control over the sample
R. dos Santos et al. / Icarus 277 (2016) 342–353 345
Fig. 3. UV lamp spectrum and modeled UV spectrum expected at the martian surface.
temperature. Temp eratu re was monitored using an array of ther-
mocouples mounted on the sample plate. The resulting sample
configuration is shown in Fig. 2 (right). The chamber contained
a Xe light source at the top of the chamber using a fused silica
window (to ensure good UV transmission) providing direct illu-
mination of the sample area with a UV spectrum similar to that
encountered on the surface of Mars (e.g. Patel et al., 2002 ). The
lamp output, along with a typical modeled UV irradiance expected
at the surface of Mars at local noon (taken from Patel et al., 2002 )
is shown in Fig. 3 . After setting the samples in the chamber and
previous to the experiments, the pressure was reduced to a vac-
uum ( < 1 mbar) for > 10 min and at room temperature. This en-
sures that there is no air and no water vapor in the atmosphere.
Then, the chamber was pressurized at 6 mbar with a mixture of
95% CO
2
and 5% N
2
, mimicking the approximate Mars pressure en-
vironment. The very dry conditions established by the initial vac-
uum treatment and the simulated Mars atmosphere (water vapor
partial pressure is nominally zero) eliminated adsorbed water from
the mineral surfaces. Complete removal of adsorbed water is likely
not obtained, unless the samples are sufficiently heated. Thermal
cycling of the sample (to simulate the potential diurnal thermal
cycle of Mars, e.g. Kieffer et al., 1977 ) was performed, with a cy-
cle from –80 °C to + 20 °C of 2 h duration repeated throughout
the exposure. During thermal cycling the samples were exposed
to UV, and overnight the samples were maintained at room tem-
perature with no UV. The samples received a total of 28 h of real-
time continuous UV illumination. On Mars, the diurnal profile of
UV irradiance encountered at the surface exhibits a bell-shaped
profile (such as demonstrated in Patel et al., 2002 ), therefore the
local noon irradiance represents a peak irradiance and the UV lev-
els throughout the rest of the day are significantly lower. Given
the higher irradiance level of the lamp as shown in Fig. 3 , coupled
with the effect of a diurnal light curve profile, the lab irradiance of
28 h is calculated to correspond to a martian equivalent UV dose
of approximately 6.5 days. Upon completion, the chamber was re-
stored to ambient conditions before removal of the samples from
the chamber.
2.5. Extraction, derivatization and GC-MS analyses of amino acids
After the Mars simulation, amino acids were extracted from the
minerals and derivatized according to the procedure described by
Martins et al. (2011, 2015 and references therein). A step to remove
sulfur was performed between the desalting and derivatization,
by using copper turnings (activated in a 10% HCl solution). The
activated copper turnings were added to V-vials containing the
desalted amino acid sample residues, brought up with 1 mL of
HPLC grade water, and left overnight. The copper turnings were
then removed and the V-vials were dried under a flow of N
2
. The
derivatized amino acids were dissolved in 75 μL of DCM.
The GC-MS analyses were performed using a Perkin Elmer
Clarus 580 gas chromatograph/Clarus SQ 8S mass spectrometer.
The amino acids were separated using two Agilent Chirasil L-Val
capillary columns (each 25 m, inner diameter 0.25 mm, film thick-
ness 0.12 μm) connected by a zero dead-volume connector. Helium
was used as carrier gas with a 1 mL/min flow. GC injector temper-
ature was set at 220 °C. Automatic splitless mode was used for in-
jection and the oven programme was: 1) 35 °C for 10 min; 2) 2 °C
per minute increase until 80 °C, hold for 5 min; 3) 1 °C per minute
increase until 100 °C; and 4) 2 °C per min increase until 200 °C,
hold for 10 min (total run time 117. 5 min). Temperatures for the
transfer line and the MS ion source were set at 220 °C and 230 °C,
respectively.
346 R. dos Santos et al. / Icarus 277 (2016) 342–353
Fig. 4. Single ion GC-MS chromatograms (25 to 85 min) of the derivatized (N-TFA, O-isopropyl) amino acids extracted from control sample G4 (goethite spiked with solution
4, but not subjected to the Mars simulation; chromatograms pointing upwards) and corresponding sample G4 (goethite spiked with solution 4 and analyzed after the Mars
chamber simulations; chromatograms pointing downwards). All single ions chromatograms are in the same scale. 1)
α-AIB; 2) D,L-isovaline; 3) D-alanine; 4) L-alanine; 5)
D-valine; 6) L-valine; 7) glycine; 8) D-norvaline; 9) D-
β-AIB; 10) L- β-AIB; 11) D- β-ABA; 12) β-alanine; 13) L- β-ABA; 14) L-norvaline; 15) D-leucine; 16) D-norleucine; 17)
L-leucine; 18) L-norleucine; 19 ) D-2-aminoheptanoic acid (internal standard); 20)
γ-ABA; 21) L-2-aminoheptanoic acid (L-2-AHA, internal standard); 22) D-aspartic acid; 23)
L-aspartic acid; 24) 6-AHA; 25) D-glutamic acid; 26) L-glutamic acid.
The identification of amino acids was achieved by comparing
the retention times and mass fragmentation patterns of the amino
acids present in the samples with those obtained from known
amino acid standard mixtures. The amino acid detection limit of
the GC-MS was verified to be approximately 3 parts per billion
(ppb). Typical GC-MS chromatograms from simulated G4 sample
and respective control are provided in Fig. 4 . Amino acids were
quantified by peak area integration of the corresponding ion frag-
ment, which were then converted to abundances using calibration
curves. These were created by plotting the ratio of the amino acid
standard/internal standard target ion peak area versus the mass of
amino acid standard injected into the column.
2.6. Brunauer–Emmett–Teller (BET) analyses
Brunauer–Emmett–Teller (BET) analyses were performed to
measure the surface area and pore size of the 11 minerals and
basaltic lava used in this work. These two variables are likely to be
the most relevant for amino acid adsorption and protection from
UV radiation because they have an important control on amino
acid distribution and arrangement on the mineral surface and on
physical shielding. Prior to analysis, approximately 0.5 g of all
samples were outgassed overnight at 353 K, under high vacuum.
Measurements were performed using a Micrometrics TriStar 30 0 0
gas adsorption analyzer, using N
2
as adsorptive gas. Measurements
were made in the relative pressure (P/P
0
) range from 0.01 up to
0.99. Final results were calculated using 9 equilibrium points in
the P/P
0
range between 0.03 and 0.20 (all linear regressions had
a correlation coefficient higher than 0.999).
3. Results
3.1. Degradation of amino acids under simulated Mars conditions
The fraction of extractable amino acids preserved after expo-
sition to simulated Mars surface conditions was calculated as the
ratio A/A0 (%), where A is the amount of each amino acid that was
not degraded and successfully extracted after the Mars Chamber
experiment, and A0 is the total amount of amino acid extracted
from the correspondent control (i.e., equivalent samples, prepared
in the same conditions, but not exposed in the Mars Chamber). The
amount of the amino acids extracted from the controls is an effec-
tive way to ascertain whether a lack of detection of amino acids
in a tested sample is due to degradation or to low extraction. The
lack of amino acid detection in both exposed sample and corre-
spondent control suggests that the lack of detection in the former
cannot be attributed to degradation induced by the simulated mar-
tian environmental conditions.
The fractions of amino acids extracted from the control samples
(i.e. [A0]/initial amino acid in the spiking solution) were calculated.
They ranged from 0 (no amino acids were detected in augite A1,
basalt lava B1 and nontronite N1) to 86%, 13 to 100%, 0 to 96%,
and 0 to 92% for experiments 1, 2, 3, and 4, respectively. Fig. 5
shows the average A/A0 ratios (in %) obtained for the 25 amino
acids that were used in experiments 2, 3 and 4. These values were
calculated from the individual A/A0 (%) obtained for each of the 25
amino acids (individual A/A0 ratios are shown in Tables 1–3 ).
Results from experiment 1 (i.e., minerals spiked with solution
containing 1 μM of each amino acid; not presented) show that
no amino acids were detected in any of the exposed samples (the
amino acid detection limit of the GC-MS is 3 ppb). In the con-
R. dos Santos et al. / Icarus 277 (2016) 342–353 347
Fig. 5. Summary of the average A/A0 amino acid ratios (in %) obtained after the simulation experiments in the Mars Chamber, where A is the amount of amino acids that
were not degraded and extracted after the simulation, and A0 is the total amount of amino acids extracted from the corresponding controls. Average values presented in
this figure were calculated using all the A/A0 ratios obtained for each of the 25 amino acids that were spiked in a given experiment found in Tabl es 1–3 . The lack of bars in
basaltic lava and enstatite for experiment 2 means complete degradation of
amino acids. Labradorite and jarosite were not used in experiments 4 and 2, respectively.
Table. 1
Summary of the individual A/A0 ratios (in %) obtained for experiment 2 (spiking solution, 10
μM of each amino acid) where A is the amount of the remaining amino acids
detected in simulated samples and A0 is the amount of amino acids detected in the control.
Individual amino acid Augite Basaltic Enstatite Goethite Gypsum Hematite Labradorite Montmorillonite Nontronite Olivine Saponite
(A/A0) vs mineral (A2) lava (B2) (E2) (G2) (Gy2) (H2) (L2) (M2) (N2) (O2) (S2)
α-AIB 3 ±1 0
a 0
a 9 ±0 17 ±1 0
a 0
a 42 ±4 18 ±2 22 ±1 18 ±7
D,L-isovaline
b 3 ±1 0
a 0
a 7 ±0 12 ±1 5 ±2 0
a 34 ±3 12 ±1 14 ±1 14 ±5
D-alanine 0
a 0
a 0
a 0
a 24 ±2 0
a 0
a 40 ±4 25 ±2 16 ±1 19 ±6
L-alanine 0
a 0
a 0
a 0
a 20 ±2 0
a 0
a 34 ±3 22 ±2 16 ±2 15 ±5
D- valine 2 ±0 0
a 0
a 4 ±0 32 ±2 3 ±0 9 ±1 30 ±2 18 ±2 14 ±1 15 ±5
L- valine 2 ±0 0
a 0
a 5 ±0 30 ±1 4 ±1 8 ±0 31 ±2 19 ±1 13 ±1 12 ±4
glycine 0
a 0
a 0
a 0
a 54 ±4 0
a 0
a 68 ±5 51 ±5 22 ±1 18 ±5
D-
β- AIB 0
a 0
a 0
a 0
a 49 ±2 0
a 0
a 32 ±3 29 ±3 26 ±3 0
a
L-
β- AIB 0
a 0
a 0
a 0
a 48 ±5 0
a 0
a 35 ±5 24 ±4 26 ±2 0
a
D-
β- ABA 0
a 0
a 0
a 0
a 56 ±3 0
a 0
a 35 ±3 29 ±6 17 ±1 19 ±6
L-
β- ABA 0
a 0
a 0
a 0
a 54 ±5 0
a 0
a 39 ±3 30 ±4 19 ±2 14 ±4
D- norvaline 0
a 0
a 0
a 0
a 50 ±3 0
a 10 ±2 33 ±1 23 ±1 15 ±1 15 ±4
L- norvaline 0
a 0
a 0
a 0
a 53 ±3 0
a 11 ±2 36 ±2 24 ±1 18 ±1 15 ±4
β-alanine 0
a 0
a 0
a 0
a 52 ±4 0
a 0
a 38 ±2 27 ±1 19 ±2 13 ±4
D- leucine 0
a 0
a 0
a 0
a 51 ±4 2 ±0 3 ±1 34 ±1 22 ±1 11 ±1 10 ±3
L- leucine 0
a 0
a 0
a 0
a 45 ±3 2 ±0 5 ±1 32 ±1 22 ±1 11 ±1 10 ±3
D- norleucine 0
a 0
a 0
a 0
a 43 ±4 0
a 7 ±0 18 ±2 12 ±1 13 ±1 17 ±8
L- norleucine 0
a 0
a 0
a 0
a 41 ±1 0
a 5 ±0 21 ±1 15 ±1 13 ±1 14 ±7
γ-ABA 0
a 0
a 0
a 0
a 66 ±4 0
a 0
a 32 ±2 28 ±3 26 ±2 19 ±6
D- aspartic acid 13 ±1 0
a 0
a 12 ±1 55 ±2 9 ±0 7 ±1 22 ±2 31 ±2 24 ±1 12 ±3
L- aspartic acid 14 ±1 0
a 0
a 14 ±1 59 ±3 10 ±0 8 ±0 25 ±1 34 ±2 26 ±2 13 ±4
6-AHA 0
a 0
a 0
a 0
a 40 ±3 0
a 0
a 22 ±1 25 ±1 25 ±1 20 ±5
D- glutamic acid 0
a 0
a 0
a 6 ±0 51 ±4 0
a 9 ±0 29 ±2 29 ±1 31 ±3 15 ±3
L- glutamic acid 0
a 0
a 0
a 6 ±0 49 ±3 0
a 9 ±1 25 ±2 26 ±2 32 ±3 14 ±4
a Complete degradation (A/A0 = 0).
b Enantiomeric separation not possible under chromatographic conditions.
trols, no amino acids were detected in augite (A1), basalt lava (B1)
and nontronite (N1). Hence, the lack of amino acid detection in the
A1, B1 and N1 experiments cannot be unequivocally interpreted as
caused by degradation. For the remaining minerals of experiment
1, amino acid degradation was observed. In the case of enstatite
(E1) all amino acids suffered complete degradation.
In experiment 2 (spiking solution containing 10 μM of each
amino acid), gypsum (Gy2) was the mineral that, on average,
preserved a greater proportion of amino acids, whereas amino
acids on enstatite (E2) and basaltic lava (B2) were completely de-
graded ( Fig. 5 and Table 1 ). Gypsum, olivine, montmorillonite and
nontronite were the only minerals that preserved all amino acids
( Table 1 ). Saponite prevented degradation of all amino acids except
D, L- β-AIB ( Table 1 ).
Results from experiment 3 (minerals spiked with a solution
containing 25 μM of each amino acid) showed that amino acids
were preserved (to different degrees) in all minerals ( Fig. 5 ). The
percentage of surviving amino acids for augite (A3), basaltic lava,
(B3) enstatite (E3), hematite (H3) and labradorite (L3) were be-
low 10% ( Fig. 5 and Table 2 ). On average, amino acids were
348 R. dos Santos et al. / Icarus 277 (2016) 342–353
Table. 2
Summary of the individual A/A0 ratios (in %) obtained for experiment 3 (spiking solution, 25
μM of each amino acid) where A is the amount of the remaining amino acids
detected in simulated samples and A0 is the amount of amino acids detected in the control.
Individual amino acid Augite Basaltic Enstatite Goethite Gypsum Hematite Jarosite Labradorite Monmorillonite Nontronite Olivine Saponite
(A/A0) vs mineral (A3) lava (B3) (E3) (G3) (Gy3) (H3) (J3) (L3) (M3) (N3) (O3) (S3)
α-AIB 16 ±1 2 ±0 8 ±1 22 ±2 0
a 5 ±1 43 ±4 6 ±1 17 ±2 12 ±1 12 ±1 20 ±1
D,L-isovaline
b 13 ±2 2 ±0 6 ±1 21 ±2 0
a 5 ±1 44 ±4 5 ±1 13 ±1 11 ±1 8 ±1 22 ±2
D-alanine 6 ±1 3 ±1 5 ±1 21 ±3 11 ±1 6 ±1 32 ±2 7 ±1 24 ±2 15 ±2 13 ±1 17 ±1
L-alanine 5 ±0 2 ±0 4 ±0 18 ±2 7 ±1 5 ±1 28 ±2 6 ±1 21 ±1 11 ±1 12 ±1 15 ±1
D- valine 7 ±0 3 ±1 5 ±1 24 ±2 15 ±2 8 ±1 47 ±3 8 ±1 22 ±2 16 ±2 17 ±1 24 ±2
L- valine 7 ±0 3 ±0 5 ±0 24 ±1 12 ±1 8 ±1 46 ±2 7 ±0 20 ±1 13 ±1 15 ±1 20 ±2
Glycine 0
a 0
a 0
a 31 ±3 22 ±2 7 ±1 28 ±4 9 ±1 40 ±2 19 ±2 18 ±2 15 ±1
D-
β- AIB 0
a 0
a 0
a 29 ±2 23 ±3 0
a 42 ±5 19 ±2 25 ±2 31 ±3 20 ±2 18 ±2
L-
β- AIB 0
a 0
a 0
a 33 ±2 27 ±3 0
a 37 ±5 19 ±2 28 ±2 30 ±3 20 ±3 17 ±1
D-
β- ABA 0
a 0
a 0
a 28 ±1 20 ±1 7 ±0 32 ±4 9 ±1 24 ±2 24 ±2 20 ±2 15 ±1
L-
β- ABA 0
a 0
a 0
a 29 ±2 23 ±2 8 ±1 31 ±3 9 ±1 26 ±2 24 ±3 20 ±2 13 ±1
D- norvaline 6 ±0 3 ±1 5 ±1 29 ±2 20 ±2 9 ±0 50 ±3 8 ±0 26 ±2 20 ±1 23 ±1 24 ±2
L- norvaline 7 ±0 4 ±1 6 ±0 32 ±2 23 ±1 10 ±1 51 ±3 9 ±1 30 ±2 19 ±2 26 ±2 21 ±1
β-Alanine 0
a 0
a 0
a 28 ±2 24 ±2 5 ±0 24 ±2 10 ±1 31 ±2 24 ±2 17 ±1 12 ±1
D- Leucine 5 ±0 3 ±0 5 ±0 29 ±1 25 ±1 7 ±0 32 ±3 5 ±0 18 ±1 13 ±1 21 ±1 12 ±0
L- Leucine 5 ±0 3 ±0 5 ±0 28 ±1 24 ±1 7 ±0 29 ±1 5 ±0 19 ±1 12 ±1 19 ±1 11 ±0
D- norleucine 6 ±1 5 ±0 5 ±0 26 ±1 23 ±1 8 ±1 42 ±2 8 ±0 18 ±2 18 ±1 20 ±2 20 ±1
L- norleucine 9 ±1 5 ±0 5 ±0 29 ±1 25 ±1 7 ±1 45 ±1 7 ±0 22 ±2 17 ±1 23 ±1 20 ±1
γ-ABA 0
a 0
a 0
a 37 ±2 38 ±5 10 ±1 78 ±3 10 ±1 23 ±1 39 ±2 21 ±1 18 ±2
D- aspartic acid 5 ±0 5 ±0 6 ±0 35 ±3 33 ±2 12 ±1 32 ±3 10 ±0 16 ±1 32 ±3 26 ±1 16 ±1
L- aspartic acid 6 ±0 7 ±0 7 ±1 40 ±2 37 ±2 14 ±1 34 ±4 11 ±1 19 ±1 35 ±3 28 ±1 18 ±1
6-AHA 0
a 0
a 0
a 35 ±3 33 ±3 8 ±1 0
a 12 ±1 18 ±1 28 ±2 20 ±1 22 ±2
D- glutamic acid 0
a 5 ±0 7 ±0 40 ±4 32 ±2 8 ±1 40 ±4 11 ±0 15 ±1 32 ±2 26 ±1 23 ±2
L- glutamic acid 0
a 5 ±0 6 ±1 37 ±4 30 ±2 8 ±1 38 ±2 10 ±0 13 ±1 29 ±2 25 ±1 22 ±2
a Complete degradation (A/A0 = 0).
b Enantiomeric separation not possible under chromatographic conditions.
preserved most efficiently in jarosite (J3). Basaltic lava (B3) pre-
served the smallest amount of amino acids ( Fig. 5 ).
The simulations using the minerals that were spiked with the
50 μM solution (experiment 4) reveal that nontronite (N4) pre-
served, on average, the largest proportion of amino acids ( Fig. 5 ).
The lowest percentage of surviving amino acids were found in
augite (A4), basaltic lava (B4) and hematite (H4), with A/A0 values
of 11%, 9% and 9%, respectively ( Fig. 5 ).
In addition, our results indicate that amino acid enantiomers
are degraded in the same degree (individual preservation ra-
tios obtained for D- and L-amino acids enantiomers are pro-
vided in Tables 1–3 ). The average A/A0 calculated for all D and
L enantiomers preserved after experiment 4 were 20.0 ±1.1 % and
20.4 ±1.1%, respectively. In experiment 3, D-amino acids had an
average A/A0 of 16. 5 ±1.2%, while L enantiomers had an aver-
age A/A0 of 16. 7 ±1.2%. Similarly, the average A/A0 ratios for D
and L-amino acids obtained for experiment 2 were 17.3 ±1.9% and
17.1 ±1.9%, respectively.
3.2. BET analyses
The results obtained for the surface areas and pore sizes of the
11 minerals and basaltic lava used in the simulations are provided
in Table 4 . Surface area values range from 0.22 m
2
/g (for basaltic
lava) up to 129.01 m
2
/g (for montmorillonite). Pore size values
range from 5.17 nm in saponite up to 21.04 nm in olivine.
4. Discussion
The preservation from UV-induced degradation of amino acids
spiked onto minerals is likely dependant on multiple factors. Here
we analyze the results obtained from the simulations in light of
the effect of 1) the structure of amino acids and 2) the physi-
cal/chemical features of the minerals that were used, in particular
their ferric/ferrous iron content, surface area and pore size.
4.1. Effect of the amino acid structure
Decarboxylation induced by UV photolysis has been proposed
as one of the main destruction pathways of amino acids ( Bertrand
et al., 2015; Boillot et al., 2002; Ehrenfreund et al., 2001; Johns and
Seuret, 1970 ; ten Kate et al., 2005 ). Boillot et al. (2002) verified
that L-leucine was subjected to decarboxylation under UV radia-
tion. Furthermore, Ehrenfreund et al. (2001) suggested this mech-
anism to explain the destruction of amino acids such as glycine,
alanine, α-aminoisobutyric acid and β-alanine under simulated
conditions in interstellar gas and interstellar icy grains. Decarboxy-
lation of amino acids by UV radiation results in the formation of a
radical in the α-carbon atom ( Ehrenfreund et al., 2001 ). The stabil-
ity of the radical is dependent on the substituents bonded to the
α-carbon atom. Alkyl substituent groups attached to the α-carbon
atom contribute towards the stability of the resulting alkyl amine
radical that forms after UV-induced decarboxylation and prolong
the life of the amino acid ( ten Kate et al., 2005 ). Therefore, we
should expect that glycine (the simplest amino acid, with two
hydrogen atoms bonded to the α-carbon) would be more degraded
and have the lowest surviving ratios after the Mars-conditions sim-
ulation experiments. In fact, Li and Brill (2003) have shown that
glycine has the highest relative aqueous decarboxylation rate when
compared to the protein amino acids leucine, isoleucine, valine and
alanine. In addition, we would expect that the amino acids more
resistant to UV radiation and less prone to decarboxylation would
be α-aminoisobutyric and isovaline, which are doubly substituted
in the α-carbon. A group of our results agree with the overall
effect of substitution in the α-carbon described above ( Tables 1–3 ).
For instance, glycine was less preserved than α-aminoisobutyric
and isovaline in all augite, basaltic lava, enstatite and jarosite
experiments 2, 3 and 4 ( Tables 1–3 ). However, it is evident that
the alkyl substituent groups are not the only factor contributing
towards the stability of the amino acids. If that was the case, then
isovaline and α-aminoisobutyric would be the most stable amino
acids in our experiments and the ones with the highest A/A0
values, which is not observed in our results. Other amino acid
R. dos Santos et al. / Icarus 277 (2016) 342–353 349
Table. 3
Summary of the individual A/A0 ratios (in %) obtained for experiment 4 (spiking solution, 50
μM of each amino acid) where A is the amount of the remaining amino acids
detected in simulated samples and A0 is the amount of amino acids detected in the control.
Individual amino acid Augite Basaltic Enstatite Goethite Gypsum Hematite Jarosite Montmorillonite Nontronite Olivine Saponite
(A/A0) vs mineral (A4) lava (B4) (E4) (G4) (Gy4) (H4) (J4) (M4) (N4) (O4) (S4)
α-AIB 38 ±4 22 ±1 27 ±3 34 ±3 27 ±1 9 ±0 27 ±3 33 ±4 36 ±2 11 ±2 11 ±1
D,L-isovaline
b 34 ±5 23 ±1 26 ±2 33 ±3 27 ±1 9 ±0 31 ±3 24 ±3 40 ±2 8 ±1 8 ±0
D-alanine 21 ±3 10 ±1 22 ±2 26 ±2 21 ±2 9 ±0 26 ±2 34 ±3 31 ±2 11 ±1 15 ±1
L-alanine 17 ±2 8 ±1 19 ±2 22 ±2 17 ±2 9 ±1 22 ±2 30 ±3 27 ±2 9 ±1 12 ±2
D- valine 15 ±2 16 ±1 39 ±2 26 ±2 22 ±1 8 ±1 22 ±1 26 ±2 46 ±2 16