Manuscript Click here to view linked References
Extended Survival of Several Organisms and Amino Acid Biomarkers under
Simulated Martian Surface Conditions
Johnson, A.P.a, Pratt, L.M.b, Vishnivetskaya, T.c, Pfiffner, S.d, Bryan, R.A.e,
Dadachova, E.f, Whyte, L.g, Radtke, K.h, Chan, E.i, 1, Tronick, S.j, Borgonie, G.k,
Mancinelli, R.M.l, Rothshchild, L.J.m, Rogoff, D.A.n, Horikawa, D.D.o, Onstott,
T.C.p
aCorresponding Author Department of Molecular and Cellular Biochemistry Indiana University 1001 E. 10th Street Bloomington, IN 47405 Phone: (812)-606-6310 Fax: (812)-855-7961 Email: [email protected]
bDepartment of Geological Sciences Indiana University 1001 E. 10th Street Bloomington, IN 47405 Email: [email protected]
1
cBiosciences Division Oak Ridge National Laboratory Oak Ridge, TN 37831 Email: [email protected]
dCenter for Environmental Biotechnology Department of Microbiology University of Tennessee 676 Dabney Hall Knoxville, TN 37932 Email: [email protected]
eDepartment of Nuclear Medicine Albert Einstein College of Medicine 1575 Blondell Avenue, Room 103 Bronx, NY 10461 Email: [email protected]
fDepartment of Nuclear Medicine Department of Microbiology and Immunology Albert Einstein College of Medicine 1695A Eastchester Road Bronx, NY 10461 Email: [email protected]
gDepartment of Natural Resource Sciences McGill University Macdonald Campus 21,111 Lakeshore Ste. Anne de Bellevue, Quebec Canada H9X 3V9 Email: [email protected]
hDepartment of Natural Resource Sciences McGill University Macdonald Campus 21,111 Lakeshore Ste. Anne de Bellevue, Quebec Canada H9X 3V9 Email: [email protected]
2
iDepartment of Geosciences Princeton University B79 Guyot Hall Princeton, NJ 08544 Email: [email protected] 1Present Address: Department of Oceanography Texas A&M University MS 3146 College Station, TX 77843-3146
jDepartment of Geosciences Princeton University B79 Guyot Hall Princeton, NJ 08544 Email:[email protected]
kDepartment of Biology Nematology Section University of Ghent Ldeganckstraat 35 9000 Gent Belgium Email: [email protected]
lSETI Institute 515 N. Whisman Rd Mountain View, CA 94043 Email: [email protected]
mNASA Ames Research Center MS 239-20 Moffat Field, CA 94035 Email: [email protected]
nBAER Institute NASA Ames Research Center Mail Stop 239-20, Bldg N239 Rm 371 Moffett Field, CA 94035 Email: [email protected]
3 oNASA Ames Research Center Moffat Field, CA 94035 Email:[email protected]
pDepartment of Geosciences Princeton University B79 Guyot Hall Princeton, NJ 08544 Email:[email protected]
4
1 Abstract
2 Recent orbital and landed missions have provided substantial evidence for
3 ancient liquid water on the Martian surface as well as evidence of more recent
4 sedimentary deposits formed by water and/or ice. These observations raise
5 serious questions regarding an independent origin and evolution of life on Mars.
6 Future missions seek to identify signs of extinct Martian biota in the form of
7 biomarkers or morphological characteristics, but the inherent danger of space
8 craft-borne terrestrial life makes the possibility of forward contamination a
9 serious threat not only to the life detection experiments, but also to any extant
10 Martian ecosystem. A variety of cold and desiccation-tolerant organisms were
11 exposed to 40 days of simulated Martian surface conditions while embedded
12 within several centimeters of regolith simulant in order to ascertain the
13 plausibility of such organisms’ survival as a function of environmental parameters
14 and burial depth. Relevant amino acid biomarkers associated with terrestrial life
15 were also analyzed in order to understand the feasibility of detecting biomarker
16 evidence for previous biological activity. Results indicate that stresses due to
17 desiccation, oxidation, and UV-associated damage were the primary deterrent to
18 organism survival, and that the effect of diurnal temperature variations and
19 reactive atmospheric species were minimal. Organisms with resistance to
20 desiccation and radiation environments showed increased levels of survival after
21 the experiment compared to organisms characterized as psychrotolerant. Amino
1
22 acid biomarker analyses indicated the presence of an oxidation mechanism that
23 migrated downward through the samples during the course of the experiment and
24 likely represents the formation of various oxidizing species at mineral surfaces as
25 water vapor diffused through the regolith. Current sterilization protocols may
26 specifically select for organisms best adapted to survival at the Martian surface,
27 namely species that show tolerance to radical-induced oxidative damage and low
28 water activity environments. Additionally, any hypothetical Martian ecosystems
29 may have evolved similar physiological traits that allow sporadic metabolism
30 during periods of increased water activity.
31 Keywords: Exobiology, Mars, regoliths, search for extraterrestrial life,
32 photochemistry
2
33 1. Introduction
34 Landed and orbital Martian missions have provided significant evidence
35 for aqueous activity during the Noachian based on geomorphological (Schon et
36 al., 2009; Wood, 2006), mineralogical (Ehlmann et al., 2008; Gendrin et al., 2005;
37 Mustard et al., 2008; Osterloo et al., 2008; Poulet et al., 2005) and geochemical
38 (Haskin et al., 2005; Klingelhofer et al., 2004; Rao et al., 2009; Squyres et al.,
39 2004) analysis of surface materials. The evidence for an ancient liquid water
40 cycle supports previous research proposing that Noachian Mars was characterized
41 by a thicker atmosphere and relatively warmer and wetter conditions than those
42 found today (Kargel, 2004; Mckay and Davis, 1991; Pollack et al., 1987), raising
43 valid questions as to whether Mars might have independently developed life
44 (Nicholson et al., 2005). Recent modeling of the geochemistry of the sedimentary
45 strata analyzed by Opportunity at Meridiani Planum predicts substantial
46 difficulties for the proliferation and survival of any terrestrial-like microbial life
47 during this time period due to the inferred low water activity of an evaporating,
48 low latitude, Mg-and-sulfate-rich liquid water environment (Tosca et al., 2008).
49 However, if Meridiani Planum was formed as the result of ice deposition and
50 subsequent sublimation due to polar wander (Boynton et al., 2009), the water
51 activity would have been in the range for known terrestrial organisms to survive
52 (Tosca et al., 2008) and that, despite locally harsh conditions, liquid water
53 environments formed by saline solutions with water activities as high as 0.95 may
3
54 have been prevalent in the Noachian (Fairen et al., 2009). The fitness of past or
55 present Martian ecosystems may have depended upon adaptation to highly acidic
56 and low water-activity environmental conditions similar to those recorded by
57 sedimentary strata at Meridiani Planum (Knoll et al., 2005), although the
58 identification of phyllosilicates (Poulet et al., 2005) and carbonates (Ehlmann et
59 al., 2008) suggest that Mars may have also had more neutral environments in its
60 geologic past.
61 The present Martian surface is characterized by high levels of ultraviolet
62 radiation (Cockell et al., 2000), a 1 to 10 mbar CO2 atmosphere (Owen et al.,
63 1977), diurnal temperature variations of 80 °C at low latitudes, and a partial
64 pressure of atmospheric H2O of less than 10 μbar, maintained by the permanently
65 frozen H2O polar caps (Ingersol, 1970; Kuznetz and Gan, 2002), that results in
66 diurnal oscillations in H2O activity, aw, from 0.5 to 0.0001 (Beaty and Group,
67 2006; Kuznetz and Gan, 2002). These conditions, in addition to the apparent
68 paucity of organic carbon in the Martian regolith (Biemann et al., 1977; Boynton
69 et al., 2009), suggest that life is not prevalent on the Martian surface today. This
70 is especially disconcerting given that saline water may have existed at the Phoenix
71 landing site as recently as 4 million years ago and the regolith appears to have all
72 of the essential elements for life (Nicholson et al., 2005; Stoker et al., 2010). The
73 absence of organic carbon in the Martian regolith despite input from meteorites
74 and comets (Chun et al., 1978), modeling of the observed disappearance of
4
75 atmospheric methane (Lefevre and Forget, 2009), and the results from the Viking
76 life detection experiments (Oyama and Berdahl, 1977; Zent and McKay, 1994),
77 strongly suggest that a rapid organic oxidation mechanism is at work in the
78 Martian regolith. Such mechanisms of active, abiotic organic decomposition are
79 found terrestrially in the hyper-arid regions of the Atacama desert (Navarro-
80 Gonzalez et al., 2003) and would provide further physiological challenges to
81 microbial life living in extremely dry surface environments like that of Mars.
82 The recent discovery of possible seasonal methane releases into the
83 Martian atmosphere from two distinct regions on Mars indicates active venting of
84 subsurface methane (Mumma et al., 2009) or the first sign that active
85 biogeochemical carbon cycling is occurring in some regions just beneath the
86 Martian surface (Krasnopolsky, 2005; Krasnopolsky et al., 2004). The latter
87 hypothesis is supported by experiments reporting 90% survival of permafrost
88 methanogens under simulated Martian conditions (Morozova et al. 2007). In light
89 of this, upcoming missions will seek to identify hospitable near-surface
90 environments where aqueous fluids may support sheltered, extant Martian life or
91 geochemical evidence for extinct Martian ecosystems. A critical consideration
92 when planning to sample environments where water or evidence of Martian life
93 may exist is whether contamination of the site by terrestrial microorganisms
94 would not only undermine life detection experiments, but also endanger any
95 indigenous Martian ecosystem. Previous laboratory experiments have sought to
5
96 assess the fate of terrestrial microorganisms (Cockell et al., 2005; Diaz and
97 Schulze-Makuch, 2006; Fendrihan et al., 2009; Hansen et al., 2009; Newcombe et
98 al., 2005; Nicholson and Schuerger, 2005; Schuerger et al., 2003; Schuerger and
99 Nicholson, 2006; Schuerger et al., 2006; Smith et al., 2009a; Tauscher et al.,
100 2006) and relevant biomolecules (Garry et al., 2006; Schuerger et al., 2008;
101 Stoker and Bullock, 1997; ten Kate et al., 2006; ten Kate et al., 2005) under
102 simulated Martian surface conditions. These studies have determined that the flux
103 of UV-C at the Martian surface is the single most detrimental factor for
104 microorganism survival (Cockell et al., 2005; Schuerger et al., 2003; Schuerger et
105 al., 2006), causing extensive protein oxidation (Daly et al., 2007) and DNA
106 inactivation (Cockell et al., 2000) that results in loss of over 99% of viable cells
107 within seconds to minutes of direct exposure. However, burial to depths as
108 shallow as 1 mm within certain mineral substrates provides adequate protection
109 from UV radiation (Barbier et al., 1998; Barbier et al., 2002; Cockell et al., 2002;
110 Cockell et al., 2005; Hansen et al., 2009; Mancinelli and Klovstad, 2000;
111 Schuerger et al., 2003), allowing viable cells to exist for several hours. Organism
112 survivability improves significantly with even partial shielding of high energy
113 UV-C due to atmospheric dust and/or CO2 attenuation (Smith et al., 2009). If
114 atmospheric attenuation of UV or millimeter-scale burial within a Martian
115 regolith or evaporite-mineral matrix is adequate to prevent complete sterilization
116 of terrestrial organisms, then the likelihood that a shallow subsurface Martian
6
117 microbial ecosystem could exist becomes significantly greater, as does the danger
118 that spacecraft-born terrestrial microorganisms could compromise such an
119 ecosystem. Under such conditions, any terrestrial organisms capable of
120 withstanding the low temperature, low water activity environment of the Martian
121 surface may be able to propagate, although this has yet to be demonstrated
122 (Cockell et al., 2005; Nicholson et al., 2005).
123 Psychrophilic or psychrotolerant organisms are able to withstand
124 temperature ranges of -17 to 10 °C (Cavicchioli, 2002) and organisms such as
125 these collected from Siberian permafrost environments have shown sustained
126 growth at subzero temperatures (Bakermans et al., 2006; Rodrigues et al., 2006;
127 Vishnivetskaya et al., 2006; Vishnivetskaya et al., 2007). These organisms are
128 proposed to be likely candidates to survive extended periods of time at the
129 Martian surface (Cockell et al., 2005; Nicholson et al., 2005); however, the
130 psychrotolerant Psychrobacter cryohalolentis showed several orders of magnitude
131 decrease in cell populations due to desiccation (Smith et al., 2009) and studies of
132 permafrost microbial communities showed significant cell loss attributed to both
133 desiccation stresses and freeze/thaw cycling (Hansen et al., 2009).
134 Psychrotolerance alone may not be a sufficient adaptation for the Martian near-
135 surface environment and desiccation stresses due to low water activity may be a
136 more limiting factor to organism survival below the surface. However,
137 microorganisms that have shown increased levels of survival under Mars-like
7
138 surface conditions, such as halophiles (Kish et al., 2009; Kottemann et al., 2005),
139 radiotolerant microorganisms (Diaz and Schulze-Makuch, 2006), methanogens
140 (Morozova et al. 2007; Morozova and Wagner 2007) and endolithic phototrophic
141 microbial communities (Cockell et al., 2005), display a unique ability to resist
142 oxidation stresses associated with radiation and desiccation. Terrestrial organisms
143 possessing these types of adaptations could provide models for hypothetical
144 Martian life and a framework for understanding potential forward contamination
145 of the Martian surface.
146 In order to ascertain the long-term survival of a wide range of psychro-
147 and desiccation-tolerant microorganisms and biomarkers at simulated Mars
148 surface conditions, including UV flux, diurnal temperature variations, and
149 atmospheric pressure and composition, we exposed several strains of bacteria,
150 archaea and eukaryotes to 40 days of simulated Martian conditions while
151 embedded within a representative Mars analog substrate. Previous experiments
152 utilizing various Mars substrate analogs such as JSC-1 or other mineral mixtures
153 (Cockell et al., 2005; Garry et al., 2006; Schuerger et al., 2003; Stoker and
154 Bullock, 1997) broadly mimicked reported geochemical aspects of the Martian
155 regolith but took minimal precautions to remove in-situ organics and
156 microorganisms. A new Mars analog regolith substrate was developed
157 specifically for this experiment with the intention of accurately reproducing the
158 Martian mineralogy and elemental composition while providing a sterile and
8
159 organic-carbon free substrate. Real-time atmospheric water vapor concentrations
160 were measured during the entirety of the experiment, providing the first multi-
161 week monitor of diurnal water vapor concentrations during a Mars simulation
162 experiment. Organisms were selected based on organism-specific adaptations,
163 including desiccation and low-temperature resistance, and were comprised of
164 various psychrophilic chemotrophs, fungi, methanogens, species of nematode,
165 halophiles, and one species of tardigrade. Representative biomolecules included a
166 super-coiled DNA plasmid (pUC19) and a levorotary pool of amino acids.
167 Individual lifetimes at Mars surface conditions were determined by quantifying
168 the viable fraction of cells as a function of depth at ten-day intervals relative to
169 control samples run at room temperature in ambient atmosphere and low relative
170 humidity. Samples were analyzed for colony forming units per gram regolith
171 (CFU g-1 regolith), changes in concentration (ppm), or, with the case of the
172 methanogens, the methanogen activity. Results were treated statistically, reported
173 as a percent survival with respect to time and depth within the regolith, and half-
174 lives at Martian conditions extrapolated from best-fit regressions. Data were
175 interpreted in terms of environmental conditions and physiological characteristics
176 that would narrow the field for potential risks of forward contamination by
177 terrestrial organisms.
9
178 2. Experimental Methods
179 2.1 Development of the Indiana-Mars Analog Regolith (I-MARS)
180 A large batch of Mars analog regolith was prepared from 1.046 kg of
181 Collier Cone (44° 11’ 31.09” N, 121° 47’ 19.61” W) andesitic basalt recently
182 exposed in the Cascade Mountains and 1.964 kg of basalt taken from Miocene-
183 aged Steens Mountain basalt outcrops exposed during faulting of the Warner
184 Valley in eastern Oregon (42° 41' N, 118° 33' W). X-ray diffraction of the bulk
185 basaltic mixture revealed ordered and intermediately ordered sodian and calcian
186 plagioclase, although previous petrographic analysis of the Steens Mountain
187 basalts revealed minor amounts of olivine, clinopyroxene and titanomagnetites
188 (Gunn and Watkins, 1970). Basalt samples were ground in an agate swing mill,
189 sieved to <200 mesh, and baked at 400 °C for 12 hours in ambient atmosphere to
190 sterilize, de-gas and remove organic contaminants from the sample. After
191 cooling, the regolith was soaked in a solution of 61.6 g ACS-certified MgSO4- Table 1 192 7H20 (Fisher Scientific) per 750 mL of Millipore deionized water (18.2MΩ-cm at
193 25 °C); the saturated basalt was rapidly dried by freeze-drying in order to mimic
194 the formation of a sulfate rind during evaporation. Individual mineral constituents
195 thought to represent minerals identified on the Martian surface (Table 1) were
196 ground, sieved, and baked at 400 °C for three hours to sterilize and remove
197 organics prior to addition to the homogenized basalt. Samples were homogenized
198 using conventional flour sifter rinsed with hexane, methanol and
10
199 dichloromethane, pumped down under vacuum and Drierite©, and brought to
200 atmosphere with dry CO2 (+99.99%, Indiana Oxygen, Bloomington, IN). The
201 CO2-equilibrated regolith was stored under low-CO2 vacuum and desiccant to
202 minimize further exposure to water vapor and terrestrial atmosphere. Gravity
203 sedimentation coupled with x-ray monitoring of particle size distribution
204 indicated that more than 80% of cumulative fines were less than or equal to 64 μm
205 in diameter (Fig. 1A). Electron microprobe analysis was performed by melting
206 the sample for 48 hours at 1200 °C on a Fe-saturated Pt loop. The oxygen
207 fugacity was maintained at approximately the Ni-Ni oxide oxygen buffer using a
208 mixture of H2-CO2 gas that was passed through the furnace. After rapid
209 quenching of the sample the ensuing glass bead was mounted in an acrylic holder,
210 polished, and a total of 10 analyses were taken randomly by a Cameca SX50
211 electron microprobe. The final elemental composition of the Indiana Mars
212 Analog Regolith Simulant (I-MARS) mimics the high-silica andesitic basalts
213 thought to represent the low-latitude regolith compositions of the bulk Martian
214 dust as sampled by the Pathfinder and Mars Exploration Rover missions (Gellert Fig. 1
215 et al., 2004; McSween et al., 2003; Rieder et al., 2004; Wanke et al., 2001) (Fig.
216 1B).
11
217 2.2 Environmental Chamber Setup and Design
218 Experiments were conducted in a Mars Environmental Chamber at
219 TechShot Laboratories, which provides a fully automated environmental
220 simulator programmed to mimic the diurnal cycle of a low-latitude, low-elevation
221 region of present-day Mars during vernal equinox. The simulator is capable of
222 replicating Mars-like surface conditions including atmospheric composition, Table 2
223 pressure, daily temperature extremes, solar cycle, ultraviolet radiation flux, and
224 surface solar spectrum (Table 2). These conditions were attained in a silica glass
225 vacuum vessel contained within a cryogenic vault cooled by evaporating liquid N2
226 and heated by resistive heaters. The solar spectrum is simulated by an
227 automatically sparking a 1 kW xenon arc lamp with filters that yield spectra
228 characteristic of the light currently reaching the Martian surface. The xenon arc
229 lamp illuminates a 400 cm2 area of the vessel, providing UV radiation up to 400
230 nm (Fig. 2B). The overall intensity and absolute irradiance (in µW cm-2 nm-1) of
231 the xenon arc lamp was measured from 210 to 900 nm using a handheld Ocean
232 Optics, Inc. spectrometer (Model USB4000). Because of an instrument-
233 dependent error (Schuerger, pers. comm), UV irradiances at wavelengths between
234 210 nm and 280 nm were linearly extrapolated (r2=0.83) from the measured
235 values above 280 nm to modeled values for UV flux at the Martian surface (Patel
236 et al., 2002) and the previously determined UV spectra from xenon lamps
237 (Schuerger, pers. comm) (Fig. 2A).
12
238 The Martian atmospheric water vapor concentration was simulated using a
239 set of two custom-certified high-grade gas mixtures (Matheson). One gas was
240 composed of 0.04% H2O and balanced with Ar; the second was a mixture of 2.7%
241 N2, 0.13% O2, 0.07% CO, and 0.01% H2 with the remaining balance CO2. The
242 individual tanks were fed at 5 psi each into a 500 cm3 stainless steel, PTFE-coated
243 sample cylinder (Swagelok©), which acted as a mixing and equilibration chamber
244 prior to injection into the Mars Environmental Chamber (Table 2). To ensure the
245 correct number of moles of each constituent gas was available for biological Fig. 2 246 processes, the experimental atmospheric pressure was maintained near 15 mbar,
247 twice the average Martian atmospheric pressure of about 7 mbar, effectively
248 doubling the available moles of each atmospheric component. Mars simulated
249 atmosphere was injected for 15 seconds at 1-hour intervals to ensure uniform
250 atmospheric composition and to compensate for the active pumping used to
251 maintain the approximately 15 mbar atmospheric pressure (Table 2, Fig. 2B).
252 Accurate characterization of water vapor within the chamber during the
253 duration of the experiment was performed using a CR-1A chilled mirror
254 hygrometer (Buck Laboratories, Boulder, Colorado). The hygrometer recorded
255 the water vapor concentrations within the chamber as a function of temperature,
256 pressure, and/or solar insolation cycles (Fig. 2B), allowing valuable insight into
257 understanding the concentration of available water vapor for biological and
258 oxidative processes. Water vapor calculations were performed using a modified
13
259 saturation vapor pressure formula water vapor pressures as a function of
260 measured frost points during the experiment.
(18.678 - t / 234.5) t / (257.14 + t) 261 ew = 6.1121 e (1)
262 where ew is the saturation vapor pressure (mbar) at frost point temperature t in
263 Celsius. Partial pressure of water vapor was calculated from mole fraction of
264 water in the atmospheric gas mixture and the atmospheric pressure within the
265 chamber and was used to calculate hourly average relative humidities for the 40
266 days of experiment (Fig. 2B).
267 2.3 Biological Molecule and Organism Samples
268 Several dozen strains of organisms were screened at individual labs for
269 both desiccation resistance and tolerance to incubation in the I-MARS regolith
270 prior to inclusion in the experiment. The final experiment included species from
271 all three domains of life and represented both uni- and multi-cellular organisms
272 with varying degrees of metabolism, growth conditions, and physiological
273 characteristics that were thought to impart some beneficial survival under the
274 extreme Martian surface conditions.
275
276 2.3.1 Amino Acids
14
277 A solution of the levorotary amino acids glycine, alanine, valine, aspartic
278 and glutamic acids was inoculated into the sterile I-MARS regolith in order to
279 study the effects of regolith and Mars conditions on a suite of biologically
280 relevant molecules (Fig. 3). Approximately 0.1 g of each L-amino acid (>99%,
281 Sigma) was dissolved into 50 mL of Millipore deionized water (18.2 MΩ-cm at
282 25 °C) and diluted 10-fold to a final volume of 50 mL. The diluted volume was
283 then used to saturate 89.7 g of regolith analog and immediately freeze-dried.
284 Dried inoculate was re-homogenized and stored under CO2 such that final
285 concentrations of each amino acid were approximately 100 μg g-1regolith. After
286 exposure in the Mars Environmental Chamber, samples were sub-sampled at 1 cm
287 depth intervals, separated into sterile micro-centrifuge tubes, and kept at -20°C
288 until analysis at Indiana University. Amino acids were extracted from the regolith
289 at 1 cm depth intervals using a standard analytical protocol consisting of vapor-
290 phase acid hydrolysis and desalting prior to analysis (Aubrey et al., 2006). Amino
291 acids and their respective enantiomers were separated using high performance
292 liquid chromatography (HPLC) with elution performed on a Phenomenex Luna C-
293 18 (250 mm x 4.6 mm x 5 μm) reverse-phase column using a binary gradient
294 consisting of 50 mM sodium acetate (8% methanol, pH 8.1) and methanol
295 (Optima, HPLC grade). A Dionex U3000 UV/VIS Single Channel
296 Spectrophotometer (λabs=340 nm) was used to detect the primary amines
297 derivatized for 1 minute with 0.15 M o-phthaldialdehyde-N-acetyl-L-cysteine
15
298 (OPA-NAC). Sample concentrations were determined using intensities from a
299 known standard and corrected for desalting and derivatization efficiency, which
300 was determined by recovery of 5x10-9 moles of DL-Norleucine added prior to
301 desalting. Recoveries averaged 80-100% for the entire desalting and
302 derivatization procedure (N=6), with sample concentrations corrected based on
303 averaged calculated internal standard recoveries corrected to the 99% confidence
304 interval to remove outliers; final concentrations are reported as total hydrolyzable
305 amino acids (THAA) and normalized to an average initial starting concentration
306 of 500 μg g-1 regolith.
307 2.3.2 DNA Plasmid pUC19
308 In order to ascertain the lifetimes of DNA and the effects of Mars-like
309 conditions on complex DNA structures, we used DNA plasmid pUC19 to identify
310 various types of DNA damage. The double strand form of the plasmid is a super-
311 coiled 2686 base pair (bp) piece of DNA that, when exposed to UV, should
312 produce either single or double strand breaks that result in either open circle or
313 linear DNA morphologies, respectively. These variations in the morphology will
314 cause the DNA to run at different speeds on an agarose gel, thus double-, single-
315 strand or linear pieces can be detected when labeled with a nucleic acid stain.
316 Exposure to UV radiation may also cause thymine dimerization (TD), which is
317 tested using a TD antibody detection protocol.
16
318 The DNA plasmid pUC19 was suspended in Tris EDTA (1.0 μg mL-1) and
319 aseptically dried onto the I-MARS regolith in a 1:1 ratio. Additionally, 10 mL of
320 1.0 μg mL-1 pUC19 plasmid was also loaded onto sterile 9 cm Whatman GF/F
321 glass fiber filters. DNA loaded glass-fiber filters were cut into 3 mm squares and
322 placed at 1 cm depth intervals in DNA-free I-MARS regolith; the uppermost filter
323 paper sample was covered in only a thin dusting of regolith accumulated during
324 loading of the sample.
325 2.3.3 Exiguobacterium sibircum
326 Exiguobacterium sibircum strains 255-15 and 7-3 (Fig. 4) are novel
327 species of Gram-positive chemoheterotrophic bacterial isolates collected from
328 core samples taken in the Kolyma Lowland permafrost of Siberia (Rodrigues et
329 al., 2006; Vishnivetskaya et al., 2006; Vishnivetskaya et al., 2007) and were
330 grown and tested at Oak Ridge National Laboratory and the University of
331 Tennessee. These organisms are rod-shaped, motile, facultative aerobes with
332 growth ranges from -6 to +40 °C in 10% NaCl solution and showed tolerance to
333 repeated freeze-thaw cycling and exposure to UV radiation (Rodrigues et al.,
334 2006; Vishnivetskaya, 2010; Vishnivetskaya et al., 2006; Vishnivetskaya et al.,
335 2007). The strains were grown aerobically overnight on a trypticase soy agar
336 (TSA) at 24 °C and collected into a phosphate buffered saline (PBS) solution with
337 10 mM NaHCO3, washed twice and re-suspended in buffer at a cell concentration
17
338 of ~109 cells mL-1. Cell suspensions (85 mL) were dried overnight at room
339 temperature in a sterile laminar flow hood at initial cell concentrations of 4.2x1010
340 cfu mL-1 (strain 7-3) and 1.4x1010 cfu mL-1 (strain 255-15). Upon mixing with
341 the regolith, the number of aerobic cfu was determined by re-suspending the
342 regolith and cell mixture in 5 mL PBS buffer and incubating for 1 hour. Plates
343 with half-strength trypticase soy broth (1/2 TSB) supplemented with 1.5% agar
344 were inoculated in triplicate from serial dilution of the resuspended cells and
345 incubated at room temperature for 72 hours prior to quantification. As an
346 additional growth control, 5 mL of 1/2 TSB was inoculated with 0.5 mL of the
347 original resuspension and incubated at 4 °C for 2 weeks. After drying and mixing
348 with regolith, the number of culturable cells was reduced to 7.6x107 cfu g-1
349 regolith and 9.57x107 cfu g-1 regolith, respectively. The three orders of magnitude
350 loss is attributed to either formation of cell aggregates within the regolith upon
351 drying or stresses due to desiccation. After exposure in the Mars Environmental
352 Chamber, samples were sub-divided into above 3 mm and below 3 mm depths;
353 cell numbers were determined as described above and statistical differences
354 between UV, dark and control samples were analyzed using the Kruskal-Wallis
355 ANOVA on Ranks test of the SigmaPlot 11 software (Systat Software, Inc., San
356 Jose, CA) and reported as percent survival during the 40 days of experiment.
357 2.3.4 Methanobacterium veterum MK4
18
358 Methanobacterium veterum strain MK4 is a Gram-negative H2/CO2
359 utilizing methanogenic Archaea isolated from Pliocene-aged sediments collected
360 in northeastern Siberia (Rivkina et al., 2007). Cells are non-motile curved rods,
361 have growth temperatures between 10 °C and 45 °C at an optimal pH of 7.2-7.4
362 (Rivkina et al., 2007). M. veterum was grown anaerobically at Princeton
363 University on a modified Methanobacterium espanolae medium at 22 °C under an
364 80:20 H2/CO2 atmosphere (1.5 bar) for five weeks. After initial cell counts, 86.5
365 mL culture was centrifuged and re-suspended into 10 mL of 50 mg L-1 NaCl; 500
366 μL aliquots of culture were dried on sterile glass slides anaerobically in a glove
367 bag containing 80:20 N2 and H2 prior to introduction to 80.1 g I-MARS regolith.
368 Final cell concentrations were estimated to be 5.4x108 cells g-1 regolith. The
369 regolith was sealed in an anaerobic canister, placed on blue ice in a cooler and
370 driven to the TechShot facility.
371 Following exposure to the Mars Environmental Chamber, triplicate
372 samples were sub-divided into 1 cm depth profiles; approximately 100 mg
373 samples were placed into a sterile 1 mL centrifuge tube with 1 mL of the modified
374 Methanobacterium media, vortexed, and centrifuged for 3 minutes at 1500 rpm.
375 Supernatant and re-suspended cells were diluted 10-fold and placed in 30 mL of
376 media in sterile Balch tubes. Using MPN methods, four additional 10-fold
377 dilutions were made to an estimated final concentration of 5.4x102 cells mL-1. An
378 atmosphere of 80:20 H2/CO2 (1.5 bar) was added to the headspace of each
19
379 sample, stored in the dark at 22 °C for a minimum of 50 days, at which time the
380 headspace gas was analyzed for the production of methane as a method of
381 enumerating the relative viability of cells in the samples over time. A Kappa 5
382 gas chromatograph coupled with flame ionization detection (GC-FID, Peak
383 Laboratories) was used to analyze 15 mL of headspace gas from each tube; 5 mL
384 was used to flush the inlet into the GC-FID and 10 mL was injected. Calibration
385 was performed using a 5 – 1000 ppm serial dilution made from a 1000 ppm CH4
386 standard, and values significantly over the media blank background were criteria
387 for a positive tube. Cells were visualized using a UV microscope by staining 100
388 μL of the observed samples with 5 μL Sytox Green (Invitrogen) diluted to 250 μL
389 DI H2O. Cells were viewed under a teal filter at 1000x; pictures were obtained
390 with an Optronics camera using MangaFire 2.1C software, with 1 μm green
391 fluorescent beads (Polysciences, Inc.) used for scale. Live/dead stains were
392 performed using the BacLight Live/Dead stain kit (Molecular Probes, Invitrogen)
393 and were visualized as described above.
394 2.3.5 Arthrobacter psychrolactophilus
395 Arthrobacter psychrolactophilus (Fig. 5) is a Gram-positive, non-spore-
396 forming, aerobic chemoheterotrophic bacteria isolated from whey-enriched farm
397 soils (dePrada et al., 1996; Loveland-Curtze et al., 1999). Cells are non-motile,
398 capable of growth between 0 and 30 °C, undergo a distinct rod/coccus cycle
20
399 during growth and stationary phases(dePrada et al., 1996; Loveland-Curtze et al.,
400 1999), and have shown highly-specific growth and metabolic processes at sub-
401 zero temperatures (Nakagawa et al., 2003). A. psychrolactophilus was grown and
402 tested for viability at Princeton University in TSB media to a cell concentration of
11 -1 403 1.8x10 cells mL ; 500 μL aliquots were dried anaerobically under 80:20 N2:H2
404 and mixed with 80.1 g dry I-MARS regolith to a final cell concentration of 1.2x109
405 cells g-1 regolith. After exposure in the Mars Environmental Chamber, each
406 sample was divided into three 1 cm depth profiles, of which 100 mg sample was
407 used. Samples were re-suspended in 1 mL TSB media in sterile micro-centrifuge
408 tubes, vortexed, centrifuged for 3 minutes at 1500 rpm, and the resulting
409 supernatant and cells were collected. Serial dilutions were performed using Most
410 Probable Number (MPN) techniques and plated on TSA; plates were incubated at
411 room temperature for three days prior to quantification of cell numbers. Cells
412 were visualized microscopically as described above for Methanobacterium
413 veterum MK4
.
414 2.3.6 Halorubrum chaoviator
415 Halorubrum chaoviator is a Gram-negative, pleiomorphic, extremely
416 halophilic archaea isolated from a saltern in Baja California, Mexico (Mancinelli
417 et al., 2009); cells are motile and range in size from 2.0-4.0 μm in length.
418 Colonies are circular, pigmented red, and capable of sustaining growth in salt
21
419 concentrations of 12-30% NaCl at temperatures ranging from 28-50 °C.
420 Halorubrum was selected for the experiment due to previous studies in which
421 cells showed survival after 2 weeks exposure to space conditions aboard the
422 European Space Agency’s BIOPAN-1 mission (Mancinelli et al., 1998). Cell
423 samples were grown at the SETI Institute to mid-log phase, rinsed three times in
424 25% NaCl, centrifuged, and diluted to a final concentration of 5x106 cells μL-1.
425 Samples were inoculated by drying 200 mL of diluted cells in approximately 50 g
426 of I-MARS regolith, yielding a final cell concentration of about 1012 cells g-1
427 regolith. After exposure in the Mars Environmental Chamber, samples were
428 returned to the SETI Institute where they were stored at -20 °C prior to being sub-
429 sampled into two depth profiles: the upper layer, which represented the topmost
430 0.5 mm of regolith sample and was removed with sterile tweezers and gentle
431 tapping, and the lower layer, which represented the remaining regolith. Samples
432 were re-suspended in buffer and enumerated using a MPN method (Koch, 1994)
433 using up to 60+ dilutions per sample. Recovered cell concentrations were
434 compared to lab controls and percent survival at Mars conditions calculated from
435 recorded differences; percent deviation from the mean was a maximum of 10%
436 for the procedure.
437 2.3.7 Cryptococcus sp. NP33-C
22
438 Cryptococcus sp. NP33-C (Fig. 6) is a yeast-like eukaryote isolated from
439 the Gypsum Hill cold saline spring of the high Canadian Arctic (Perreault et al.,
440 2008). These springs are among the only known cold springs in thick permafrost
441 on Earth and are regarded as analogues to possible Martian liquid water habitats
442 (Andersen et al., 2002). The strain was isolated along with a bacterial species of
443 Hafnia (Perreault et al., 2008) and 16S rRNA gene sequencing of this strain
444 revealed 99% similarity to an unidentified eukaryotic clone (ACC # AY494432.1)
445 isolated from Pleistocene-aged tundra soil. NP33-C is a facultative anaerobic
446 chemoheterotroph capable of growth at -10 °C in up to 12.5% NaCl and showed
447 greater than 20% survival rates after 4 days of desiccation within the I-MARS
448 regolith. Cells were grown at McGill University in R2A broth to a cell
449 concentration of 1.58x1010 cells mL-1 and 1 mL aliquots were dried overnight on
450 sterile glass slides prior to mixing within the I-MARS regolith. The expected final
451 cell concentration was 1.58x1010 cells g-1 regolith, although the samples showed a
452 several order of magnitude decrease in viable organisms between drying on the
453 regolith and the initial time point at day 0. For post-experimental analysis, 1 g
454 sample was re-suspended in 1 mL PBS buffer and mixed thoroughly by gentle
455 pipetting. The resulting supernatant was serially diluted, plated onto R2A media
456 plates, and incubated at 25 °C for 1 week prior to counting; day 10 samples were
457 not successfully quantified due to contamination.
23
458 2.3.8 Wangiella dermatitidis
459 Wild-type Wangiella dermatitidis strain 8656 (Fig. 7A) and its
460 unmelanized mutant wdpks1Δ-1 (Fig. 7B) are polymorphic fungal pathogens that
461 are primarily yeast-like but can produce a number of morphological features
462 (Paolo et al., 2006). Both wild type and mutant strains, isolated and characterized
463 in the laboratory of Paul J. Szaniszlo, were provided by collaborative agreement
464 with the Albert Einstein College of Medicine. Wild type W. dermatitidis is
465 dematiaceous due to the deposition of melanin on the cell wall; unmelanized
466 mutant lack the gene WdPKS1, which controls production of a particular
467 polyketide synthase that is considered the primary enzyme in melanin production
468 (Feng et al., 2001). The production of melanin by organisms has been shown to
469 provide increased resistance to UV radiation (Cockell and Knowland, 1999),
470 freeze-thaw processes (Paolo et al., 2006) and desiccation (Zhdanova and
471 Pokhoden, 1973). Individual strains were grown at room temperature for 20 days
472 in 500 mL of a sterile minimal medium adjusted to pH 6.5. Cells were gravity-
473 collected, rinsed twice in sterile water, and re-suspended to a final volume of 25
474 mL. Cultured Wangiella and its mutant were combined and dried overnight in
8 7 475 CO2 atmosphere and added to the regolith to a final concentration of 10 and 10
476 cfu g-1 regolith, respectively, and, similar to other organisms experienced a several
477 order of magnitude reduction in cell numbers prior to the initial time point. After
478 exposure in the Mars Environmental Chamber, cells were suspended in 1, 5 or 10
24
479 mL aliquots of water per gram of regolith, vortexed, plated in duplicate on a
480 Sabauroud dextrose agar (SAB) (Difco, Becton Dickinson), and incubated at
481 room temperature four to five days prior to quantification.
482 2.3.9 Ramazzttius varieornatus
483 The multicellular tardigrade Ramazzttius varieornatus exhibits
484 anhydrobiosis, an ametabolic dry state induced by environmental desiccation;
485 during this state, R. varieornatus exhibits high tolerance to extreme environmental
486 conditions such as temperature and ionizing radiation (Horikawa et al., 2008),
487 making it an excellent specimen for the current study. Adult tardigrade
488 specimens were collected from agar culture plates containing distilled water and
489 the green alga Chlorella vulgaris and suspended in distilled water in a sterile Petri
490 dish at NASA Ames Research Facility. A drop of water containing 20-30 animals
491 was placed onto a piece of 4 mm square filter and kept under 34% relative
492 humidity at 22ºC for 2 days, during which, the tardigrades entered into
493 anhydrobiosis. Filter papers with dried tardigrades were embedded in samples of
494 regolith at depth of 5 mm below the surface immediately prior to exposure in the
495 Mars Environmental Chamber. Unlike other organisms, tardigrade samples were
496 not sampled at 10-day intervals but remained in the chamber until the final
497 sampling at day 40. After exposure, the tardigrades were rehydrated with distilled
498 water and transferred onto an agar plate containing Chlorococcum sp. and
25
499 distilled water. Culture plates were covered and incubated in the dark at 22 ºC.
500 Survival was determined by observing body motion on a daily basis for up to six
501 days after rehydration. Differences in survival between UV and dark groups were
502 compared using a 2-square test with Yeats’ correction for UV (N=20 organisms)
503 and dark (N=26 organisms) samples.
504 2.3.10 Nematodes
505 Caenorhabditis elegans, Rhabditis oxycerca, Acrobeloides maximus are
506 non-parasitic members of the phylum Nematoda (Fig. 8). These small (<1 mm)
507 round worms contain less than 1000 cells but contain complex anatomy,
508 physiology, and a simple nervous system (Riddle, 1997). This makes them an
509 excellent source of study for understanding the effects of photochemistry (Mills
510 and Hartman, 1998) and temperature (Wergin et al., 2000) on complex
511 biochemical systems. Individual nematode species were cultured on agar plates at
512 the University of Ghent (Brenner, 1974) and several large batches grown on S
513 Medium using concentrated Escherichia coli OP50 as a food source (Lewis,
514 1995) to obtain large amounts of various life stages. Nematodes of each species
515 were divided into individual samples and controls prior to air-drying in sterile
516 Eppendorf tubes. Preparatory experiments and SEM analysis had clearly
517 indicated that the I-MARS regolith analog damaged and cut the worms (Fig. 8A,B)
518 and, as a result, nematode samples were placed in the Mars Environmental
26
519 Chamber in Eppendorf tubes in the absence of regolith. Nematodes are not
520 known to be UV-tolerant, so nematode samples were exposed to only the diurnal
521 temperature and atmospheric conditions by isolating the nematode samples from
522 the UV irradiated samples. Upon return of the samples to the University of
523 Ghent, the dried worm pellet was put on an agar plate and E. coli OP50 was
524 spotted beside the rehydrating worms in an attempt to elicit exit from dauer stage.
525 Analysis of recovered nematode populations was investigated through scanning
526 electron microscopy (SEM) (Willems et al., 2005) using a stereomicroscope to
527 check whether any nematodes survived the treatment. Briefly, nematodes were
528 fixed in a 4% formaldehyde solution for 24 hrs and subsequently dehydrated
529 using an ethanol-rinse series (30%, 50%, 75%, and 95%) for an additional eight
530 hours. After initial dehydration, specimens were left overnight in 100% ethanol.
531 To avoid collapsing, the nematodes were critically point-dried with CO2 using a
532 Balzers CPD020. The dried nematodes were individually removed, sputter-
533 coated with gold using a Balzers SCD040, and observed using a Jeol JSM-840
534 Scanning Electron Microscope. Images were taken on Agfa APX120 film and
535 developed commercially in a local photography store.
536 2.4 Experimental Design, Setup, Sampling, and Half-Life Determination
537 Samples of each organism/organic to be studied (60 samples per
538 organism) were divided into three separate experimental groups. One set was
27
539 exposed to Mars atmospheric, temperature and UV and solar conditions (UV
540 samples); a second set was exposed to Mars atmospheric and temperature
541 conditions but shielded from the simulated UV and solar flux (dark samples), and
542 the final set was kept in the dark at room temperature (25.7 °C) and fixed
543 humidity (2.3% RH) in a sterile laminar flow hood (control samples).
544 Approximately 1 g of sample-containing regolith was placed in small (4.5 cm
545 long x 6 mm diam.) hand-blown silica-glass test tubes that had been previously
546 annealed at 500 °C overnight to ensure sterility and removal of organics; total
547 sample column depth upon sample loading and packing was approximately 2.8-
548 3.0 cm. Samples were placed into a sterile aluminum sample rack such that the
549 upper 0.5 to 1 cm of sample was exposed and the lower 1-2 cm was shadowed in
550 order to minimize diffuse UV exposure. Both UV and dark sample sets were
551 introduced into the sterilized environmental chamber and stored dark at 10 °C and
552 15 mbar of simulated Mars atmosphere for several days to ensure complete
553 equilibration between the atmosphere and regolith prior to exposure to simulated
554 Mars conditions. Selected Mars conditions included 12 hours of simulated solar
555 spectra per Martian sol, providing a temperature maximum of 24 °C during
556 illuminated “day” conditions and a programmed low temperature of -40 °C during
557 “night” conditions. The low temperature of -40 °C was selected based on the
558 chamber’s ability to maintain the 15 mbar Mars atmospheric pressure as described
559 above; decreasing the temperatures further caused a physical failure in the
28
560 chamber’s vacuum seals, causing low-pressure loss and the introduction of
561 external atmosphere to the chamber.
562 Samplings took place every 10 days from the end of the seven-day
563 equilibration period and continued for a total of 40 days. Sampling occurred by
564 first raising the chamber temperature to 20 °C for approximately 30 minutes and
565 then bringing the chamber to atmospheric pressure. Triplicate samples of UV and
566 dark samples were removed from the chamber and triplicate controls removed
567 from the sterile laminar flow hood using sterilized forceps and transferred to a
568 CO2-purged sterile glove bag. Remaining UV and dark samples were placed back
569 into the environmental chamber and returned to experimental conditions;
570 estimated time of sample exposure to ambient terrestrial atmospheric conditions
571 was less than 30 minutes and time outside Mars relevant temperature conditions
572 was less than 2 hours per sampling. Sample vials were then labeled, packed with
573 annealed (500 °C, 12 hrs) glass wool, sealed under CO2 with autoclaved 6 mm
574 rubber septa (to prevent shifting during transport), and shipped to each
575 investigator’s lab under CO2 in autoclaved glass packing jars on dry ice. Analysis
576 of the individual samples occurred at collaborating laboratories using the methods
577 outlined above.
578 Rate constants for the degradation of each organism or molecule were
579 calculated based on a linear fit to the data from each time point and a first order
580 rate constant k (day-1) (Eq. 2):
29
581 ln(Nt/No) = -k t (2)
582 where t is the experiment time in days, No is the number of organisms or
583 molecules at day 0, and Nt is the number of organisms or molecules at day t. Rate
584 constants were based on the change in viable cell numbers during exposure to the
585 Mars-like environmental conditions and do not include the loss of cells due to
586 drying on the regolith. The experimentally determined rate constant k was used to
587 calculate half-lives (in days) for each of the investigated species using Eq.3:
588 T1/2 = ln2/k (3)
589 where T1/2 represents the number of days for 50% of the population to be
590 removed. Errors in half-lives were calculated from the maximum deviation in
591 rate constant, k, based on the experimental error for each sample.
592 3. Results
593 3.1 Simulated Martian Environmental Conditions
594 Temperature ranges for the UV-exposed regolith ranged from 24 °C
595 during daytime hours to -40.4 °C for nighttime lows, with an average regolith
596 temperature of -17.6 °C (Table 2); no second temperature measurement of dark
30
597 regolith samples was possible due to limitations of the environmental chamber.
598 Atmospheric pressure within the chamber averaged 13.3 mbar and fluctuated
599 diurnally, reaching 20 mbar during maximum daytime temperatures and dropping
600 to around 12 mbar during night hours (Fig. 2B). Sampling points are identified in
601 the pressure profile (Fig. 2B, black arrows) by the sharp pressure spikes where the
602 chamber returned to ambient atmospheric conditions while samples were
603 removed.
604 Corrected UV measurements (Fig. 2A) yielded a total radiation dose for
605 the 40-day experiment of 3.6x108 J m-2. Shortwave UV-C radiation between 210
606 and 400 nm is 9.2% of this total dose, providing 3.3x107 J m-2 to UV-exposed
607 samples. Previously modeled and simulated Martian UV irradiance in a low-
608 atmospheric dust load (Patel et al., 2002; Schuerger et al., 2003) gives a UV flux
609 in the 200-400 nm wavelength range of 49.95 W m-2; total dose values from this
610 study in the same wavelength range are 19.3 W m-2, or about 39% of the expected
611 Martian UV values.
612 Frost point varied regularly depending on chamber pressures and
613 temperatures, reaching maximum values of 10 °C near the daytime high
614 temperatures and low temperatures of -65 to -75 °C during the cold night cycle,
615 with an average frost point of -46 °C, well below the average regolith temperature
616 of -17.6 °C (Fig. 2B). Comparisons of diurnal dew point and regolith temperature
617 variations indicate that the two parameters vary cyclically but rarely coincide at
31
618 the same value (Fig. 2B); this implies that during the course of the experiment,
619 most available water was in the vapor form and that processes associated with ice
620 formation were minimal. At several points during the experiment, larger
621 fluctuations in frost point occur, such as near day 5 and days 18-20 (Fig. 2B, grey
622 boxes). These represent points in the simulation when the available liquid N2 was
623 below the amount needed to keep the chilled mirror hygrometer operational or
624 where data errors occurred, giving erroneous values that do not represent actual
625 water vapor concentrations. Average relative humidity during the experiment was
626 18.2%; diurnal fluctuations in relative humidity varied inversely with dew point
627 and ranged from less than 1.0% to greater than 90%. The strong diurnal
628 fluctuations in relative humidity fits several lines of evidence indicating severe
629 diurnal and seasonal changes in relative humidity near the Martian surface (Smith,
630 2002; Smith et al., 2009; Whiteway et al., 2009) and the low average relative
631 humidity suggests minimal freezing of atmospheric water vapor during the
632 experiment.
633 3.2 Biomarker and Organism Survival
634 3.2.1 Amino Acids
32
635 Recovered amino acid concentrations at day 0 ranged between 400 to 600
636 μg THAA g-1 regolith. The relative concentrations for all samples were calculated
637 by normalizing to an initial concentration of 500 μg THAA g-1 regolith.
638 Recoveries of THAA’s from the upper two centimeters of UV exposed regolith
639 (Fig. 3A) show an immediate decrease of 15-20% in concentration by day 10, Fig. 3 640 indicating a rapid oxidation mechanism within the top 2 cm of regolith.
641 Subsequent concentrations in this layer are statistically identical, implying the
642 effects of UV were minimal after 10 days, most likely due to shielding effects or a
643 decrease in concentration of available oxidant. The bottom centimeter profile
644 shows a constant concentration of amino acids for the first 20 days experiment,
645 but nearly 20% loss by day 30, equivalent to the loss in the upper centimeter and
646 may represent a diffusion-controlled mechanism. Top and middle centimeter
647 profiles of the dark samples showed a similar THAA oxidation profile (Fig. 3B).
648 Concentrations of amino acids remained steady to slightly decreasing during the
649 first 20 days of experiment, but by day 30, recovered amino acid values had
650 sharply dropped by nearly 20%, similar to the concentrations seen in the UV
651 samples, and remained at this level until the end of the experiment. The bottom
652 centimeter of regolith in dark samples showed no change in concentration over
653 the 40 days of experimental conditions. Control samples were variable in their
654 recovered THAA concentrations and followed no clear trend, and in some cases
655 increased in value (Fig. 3C); however, recovered values averaged near the initial
33
656 values of 500 ppm within experimental error. Attempts to calculate half-lives for
657 the amino acids under these conditions gave values that were variable and fit
658 poorly to a first order decay constant due the nature of the depth-dependent
659 oxidation profile; best-fit of the data gave half-lives for these compounds of
660 140±8 days, 138±11 days, and 429±120 days for UV, dark and control samples,
661 respectively.
662 3.2.2 DNA Plasmid pUC19
663 All attempts to recover the DNA plasmid pUC19 from sample regolith
664 resulted in no banding morphology on agarose gels as an indication of single- or
665 double-strand breaks, instead yielding streaked DNA, which is usually indicative
666 of DNA shearing. Replicate analysis did not provide useful conclusive data, and
667 thymine dimer analyses were inconclusive, but seemed to indicate increased DNA
668 damage in controls relative to samples exposed to the experimental Martian
669 conditions. Secondary experiments attempting to remove the plasmid from
670 similarly prepared filter paper after exposure to varying amounts of UV radiation
671 also yielded inconclusive results. This irregularity would imply that the DNA
672 analysis protocols used were not suitable for the environmental and sample
673 matrices investigated in this experiment. Results from this study can only be said
674 to provide no measurable quantity of super-coiled, open circle or linear DNA,
34
675 yielding at best random fragments of the original plasmid that provide no
676 information on the type of DNA damage.
677 3.2.3 Exiguobacterium sibircum
678 Exiguobacterium sibircum, like many of the organisms in this study,
679 showed a several order of magnitude decrease in viable cells during desiccation
680 onto the I-MARS regolith prior to the sampling at day 0. This loss in cell number
681 is attributed to desiccation stresses on the organism, the formation of cellular
682 aggregates upon drying, or due to some unknown toxicity of the regolith.
683 However, between day 0 and day 40, percent survival in the bulk regolith for UV,
684 dark, and control samples did not show a significant difference in the strain E.
685 sibircum 7-3 (Fig. 4A), whereas the differences between the UV and dark Fig. 4 686 treatments were statistically significant for strain 255-15 (Fig. 4B) during that
687 same period. Controls for strain 255-15, however, did not differ significantly
688 from UV samples (p = 0.786). At continuous Martian conditions, strain 7-3
689 exhibits half-lives of 7.8±0.9 days for UV samples and 7.6±0.7 days for dark
690 samples relative to the 5.7±0.2 days for control samples (Table 3). Strain 255-15
691 was overall more resistant, with estimated half-lives of 10.2±1.7 days for UV
692 samples, 12.2±1.4 days for dark samples, and 8.2±0.3 days for control sample sets
693 (Table 3). Analysis of cells in the top 3 mm of regolith compared to deeper
694 depths indicated there was a statistically significant difference in survival rates
35
695 between the two layers for both strains (Fig. 4C, D). Exposure to the simulated
696 Martian environment yielded half-lives for strain 7-3 in the upper 3 mm of
697 regolith of 3.9±0.4 days compared to 7.8±0.7 days for deeper samples (Table 4).
698 Strain 255-15 had a half-life of 7.4±0.5 days in the upper 3 mm profile versus
699 10.2±1.2 days in the deeper layers (Table 4).
700 3.2.4 Methanobacterium veterum MK4
701 The first four time points of the experiment yielded CH4 concentrations
702 near 5 ppm regardless of exposure conditions or depth within the regolith (Fig. 4
703 J); this was slightly above the 4.7 ppm average found in the media blanks and
704 near the ambient air CH4 concentration of 1.8 ppm. Methane concentrations at
705 the final time point were elevated to 8 – 10 ppm for the samples, although these
706 values were not different from negative control media blanks containing the same
707 headspace and were the same for all samples regardless of experimental
708 conditions or depth within the regolith. The only exception was the dark middle
709 centimeter profile, which showed methane concentrations of 6.4 ppm and was
710 outside the standard deviations of both standards and controls (±0.2-0.8 ppm). As
711 a positive test for CH4 production, a live control yielded a concentration of
712 2100±1200 ppm CH4 after 101 days of incubation; these elevated values indicate
713 the reported values of 6-10 ppm CH4 were statistically well below what would be
714 expected from a mostly living sample.
36
715 Microscopic analysis of the pure M. veterum MK4 culture and the first
716 dilution of the uppermost 1 cm from the 40-day control sample detected cells with
717 morphologies similar to that of M. veterum MK4 in the control sample. BacLight
718 live/dead stain of day 40 bottom regolith sample contained both live and dead
719 cells indicating that some cell death had occurred during the course of the
720 experiment, severe enough to perforate the cell wall.
721 3.2.5 Arthrobacter psychrolactophilis
722 Initial cell concentrations of A. psychrolactophilis were approximately
723 1.2x109 cells g-1 regolith, and after inoculation and the equilibration period prior
724 to the experiment, remained near 109 cells g-1 regolith; this indicates A.
725 psychrolactophilis did not exhibit the same intolerance to drying or desiccation on
726 the I-MARS regolith as many other organisms in this study showed (Fig. 5). By
727 day 10, cell survival in all samples at all depths dropped by three orders of
728 magnitude and signified the largest overall drop in viable cells during the
729 experiment. Over the remaining 30 days of experiment, concentrations dropped
730 to less than 0.0001% of their original concentrations, although at day 30, cell
731 numbers increased back to near the day 10 values (Fig. 5A-C). Extrapolation and
732 best fit of a first-order rate of loss for UV, dark, and control samples show
733 average half-lives of 2.4 (±0.7) days for A. psychrolactophilis in the lower depth
734 profiles for all samples. In the top centimeter profile, shorter half-lives were seen
37
Fig. 5
735 for the UV samples (2.0±0.2 days) compared to dark and control samples (2.4±0.3
736 and 2.3±0.1 days, respectively) (Table 3, 4), although experimental error indicates
737 this difference may not be statistically significant. The similar expected half-lives
738 for UV, dark, and control samples at all depths and in UV samples implies that
739 Mars-like atmosphere, UV and temperature conditions are not necessarily a
740 detrimental factor to A. psychrolactophilis. More likely, the low water activity
741 and desiccation stresses within the regolith combined with the lack of available
742 organic carbon substrates are a more powerful deterrent to A. psychrolactophilis
743 survival.
744 The increase in cell concentrations at day 30 is intriguing as cell
745 concentrations increased to similar values in all samples and at all depths, with no
746 discernable change in chamber conditions that could explain this effect (Fig. 2B).
747 It is entirely possible that at 30 days enough soluble organic carbon from dead
748 cells had accumulated for spurts of A. psychrolactophilis metabolism to occur if a
749 lack of organic carbon substrate is indeed a primary deterrent for A.
750 psychrolactophilis survival in the I-MARS regolith. The identification of
751 exponential phase cell morphologies in day 40 control samples by fluorescent
752 staining and UV microscopy (Fig. 5D) may provide additional evidence for
753 active, albeit sporadic, metabolism in A. psychrolactophilis under Mars
754 conditions. This would require growth under a partial pressure of O2 of only 20
755 μbars and may partially explain the delayed increase in cell numbers at day 30.
38
756 This may not be an unreasonable conclusion given that de Vera et al. (de Vera et
757 al., 2010) have recently reported aerobic heterotrophic fungal respiration at Mars-
758 equivalent pressures and compositions.
759 3.2.6 Halorubrum chaoviator
760 Initial concentrations of H. chaoviator were approximately 1012 cells g-1
761 regolith and individual time points were analyzed with respect to depth in the
762 regolith. Analysis of the upper 0.5 mm of sample gave survival rates of 100% (±
763 10% deviation from the mean) in UV, dark, and control samples relative to
764 independent laboratory controls for all time points, indicating that H. chaoviator
765 is capable of long-term, sustained exposure to Mars-like conditions when
766 embedded within sub-millimeter depths of I-MARS regolith analog. Regolith
767 samples below 0.5 mm were not analyzed based on the assumption that increasing
768 burial depth should not have a negative effect on cell survival. Assuming the
769 maximum reported deviation from the mean, a minimum estimate of the half-life
770 for H. chaoviator under the diurnal Martian conditions is 70 days for both UV and
771 dark samples protected by a minimum of 0.5 mm of regolith material (Table 3).
772 3.2.7 Cryptococcus sp. NP-33C
39
773 Initial cell concentrations of Cryptococcus sp. NP-33C were
774 approximately 1.6x1010 cells g-1 regolith after mixing in the I-MARS regolith; cell
775 recoveries at day 0 were several orders of magnitude lower than this, indicating a
776 similar mechanism of cell loss as seen in several other species in this study and
777 indicative of some desiccation stress or unknown toxicity to the I-MARS regolith
778 (Fig.6). Percent survival and half-lives were based on relative changes in cell
779 concentration from day 0 (Fig. 6, inset). Samples at day 10 were compromised
780 during analysis due to issues with contamination and were not included in this
781 study, nor were samples from day 30, which provided no viable cells in any
782 sample regardless of experimental conditions. The surviving population of NP33-
783 C within the regolith dropped significantly by day 20; both UV and controls
784 showed less than 40% survival rates whereas dark samples showed 60% survival. Fig. 6
785 Day 40 samplings gave 7% survival for UV samples and 18% survival for dark
786 samples relative to initial day 0 values, while control samples showed 55%
787 survival. Linear regressions for UV and dark sample sets yielded half-lives at
788 Mars surface conditions of 10.5±0.1 days and 16.1±0.8 days for UV and dark
789 samples, respectively (Table 3). Control samples were more variable and gave
790 half-lives of approximately 46.2±4.4 days (Table 3), indicating a significant
791 impact from the Mars-like environmental conditions.
792 3.2.8 Wangiella dermatitidis
40
793 Mixing of the wild type and mutant W. dermatitidis strains with the I-
794 MARS regolith and drying under CO2 atmosphere lowered the number of viable
795 cells by 90% (Fig. 7A, B), again indicative of desiccation stresses associated with
796 the regolith drying process, the formation of cellular aggregates, or an unknown
797 toxicity to the regolith. However, the greatest drop in viability during the
798 experiment occurred during the initial equilibration period, when the simulation
799 chamber was held at 10 °C at Mars atmosphere and 15 mbar pressure (Fig. 7C,
800 D). Viable cell numbers dropped from 1.7x107 cells gram-1 regolith to 5x104 cells
801 gram-1 regolith for wild type W. dermatitidis and from 8x106 cells g-1 regolith to
802 103 cells g-1 regolith for the mutant wdpks1Δ-1. Subsequent time points did not
803 have as profound an effect on viability, with an additional order of magnitude
804 reduction in cell concentrations by day 40. Based on cell concentrations at day 0,
805 half-lives at Mars-like conditions for UV, dark and control samples of the wild-
806 type W. dermatitidis were 8.0±0.7 days, 8.5±0.8 days, and 8.2±0.4 days,
807 respectively; mutant wdpks1Δ-1 showed greater variability, with half-lives of
808 7.2±0.1 days, 8.0±0.2 days, and 10.8±10 days for the same sample conditions
809 (Table 3). Based on the average half-lives and error, it would appear that the
810 Martian environmental conditions had little to no effect on the survival capability
811 of W. dermatitidis in the bulk regolith and that some uniform mechanism for cell
812 loss was at work in all sample sets.
41
813 Wild type W. dermatitidis exhibited no dependence on depth in the UV-
814 irradiated samples, with statistically similar cell numbers at all time points (Fig.
815 7E). However, the lack of melanin in the W. dermatitidis mutant wdpks1Δ-1
816 appears to endow some physiological advantage to cells relative to depth in the
817 sample (Fig. 7F); cells in the uppermost 1 cm of sample show better survival rates
818 than those in the bottom centimeters. This trend is apparent in dark samples as Fig. 7
819 well but absent in control samples (data not shown) and wild type samples (Fig.
820 7E). This depth-dependent benefit imparts first order half-lives for mutant W.
821 dermatitidis of 16.0±2.0 days (Table 4) in the upper layers of regolith versus half-
822 lives of only 9.1±0.2 days in the bottom portions of the regolith. For comparison,
823 wild type W. dermatitidis shows half-lives of 3.8±0.0 days in the upper layer of
824 regolith and 8.3±0.4 days in the lower portion (Table 4), indicating an increased
825 susceptibility to UV-associated damages in the upper layers of regolith relative to
826 the unmelanized mutant.
827 3.2.9 Ramazzttius varieornatus
828 Percent survival of R. varieornatus after burial in 5 mm of I-MARS
829 regolith and exposure to 40 days of simulated Martian environmental conditions
830 was determined. Statistical analysis of R. varieornatus after burial in 5 mm of I-
831 MARS regolith and exposure to 40 days of simulated Martian environmental
832 conditions showed significant differences (p >0.05) in survival between UV
42
833 (70%) and dark samples (92.3%), indicating a UV-dependent effect on R.
834 varieornatus survival. However, at a burial depth of 5 mm, the direct effects of
835 UV radiation should be minimal, implying either secondary UV effects or an
836 alternate mechanism for R. varieornatus inactivation below the surface. Half-
837 lives for the UV and dark samples are 78 days and 350 days, respectively (Table
838 3) and, without day 0 data, must be interpreted as a minimum value. Unlike the
839 multicellular nematode populations (below), the R. varieornatus population
840 showed the ability to survive long-term exposure to Mars-like environmental
841 conditions at lifetimes significantly greater than most of their prokaryotic
842 counterparts. This indicates that eukaryotes with physiological adaptations
843 evolved to cope with extreme environments are as, if not more, capable of a
844 sustained presence as either an extant Martian ecosystem or as a potential source
845 of forward contamination.
846 3.2.10 Nematodes
847 Nematode species exposed to diurnal Mars temperature and atmospheric
848 conditions showed no recovery at any of the collected time points. Nematode
849 species were exposed without inoculation within the I-MARS regolith due to their Fig. 8 850 inability to survive the sharp basaltic fines of the regolith (Fig. 8B). Desiccation
851 stresses combined with the extreme temperature conditions of the simulated Mars
852 environment proved lethal for all three nematode strains, indicating that
43
853 biochemically complex organisms that are not specifically adapted to harsh
854 environmental conditions or capable of entering some inactive metabolic state are
855 incapable of survival at such conditions, even in the absence of UV exposure.
856 4. Discussion
857 4.1 Organism and Biomarker Survival
858 The current study indicates that several species of organisms seem
859 unaffected by the extreme Martian diurnal temperature variations and atmospheric
860 composition and pressures relative to room temperature controls, implying an
861 alternate mechanism for the large decrease in cell numbers during the course of
862 the experiment. In large part, the most pronounced decrease in microbial cell
863 populations came during the initial desiccation of the organism onto the I-MARS
864 regolith analog, yielding a several order of magnitude loss of viable cells prior to
865 exposure in the Mars Simulation Chamber with the notable exceptions of H.
866 chaoviator and R. varieornatus. This loss in viable cells may be a result of the
867 formation of cellular aggregates, stresses associated with desiccation on the
868 regolith, or an unknown toxicity within the regolith itself. The data from this
869 study indicates that organisms adapted to environments with low available water
870 or capable of withstanding desiccation had the highest probability of survival
871 under Mars-like conditions; additionally, these organisms showed minimal cell
872 loss between their inoculation on the regolith and the initial sampling. Therefore,
44
873 it is probable the mechanism of cell loss was driven primarily by desiccation
874 and/or oxidation, and that this loss in cell numbers occurs in addition to those
875 processes attributed to the simulated Martian environment.
876 Upon introduction into the Mars Simulation Chamber, most of the
877 exposed samples showed lifetimes similar to controls kept at room temperature
878 and ambient atmosphere, although several organisms showed susceptibility to the
879 effects of UV-associated damage relative to samples below the UV penetration
880 depth and/or dark samples. This indicates a correlation between estimated half-
881 lives and burial depth within the regolith substrate. For example, the permafrost
882 bacteria E. sibircum strains 7-3 and 255-15 showed a definite reduction in cell
883 survival (Fig. 4C, D) and expected half-lives (Table 4) in the upper 3 mm of
884 regolith compared to samples from below 3 mm, and the psychrotolerant
885 eukaryote Cryptococcus sp. NP-33C showed half-lives in UV-exposed bulk Table 3 886 regolith samples that were 35% less than the corresponding dark samples (Table
887 3). The non-melanized mutant W. dermatitidis a wdpks1Δ-1, however, actually
888 showed increased half-lives in the upper UV-exposed regolith horizon relative to
889 the dematiaceous wild type strain (Table 4). Melanin production is shown to
890 provide increased resistance to exposure at low temperatures due to stabilization
891 of the cell membrane (Paolo et al., 2006), and melanin deficient mutants would
892 most likely show increased cell numbers in areas of higher average temperatures.
893 Previous simulation studies show a strong thermal gradient in sediment profiles
45
894 exposed to Mars-like conditions (Hansen et al., 2009); increased survival at
895 shallow depths in UV-exposed samples may represent a preference for regions of
896 relatively elevated temperatures caused by radiative heating from the simulated
897 solar spectra. However, dark samples showed a similar depth profile (data not
898 shown), indicating either that a similar but less extreme thermal gradient caused
899 by ambient chamber temperature existed in dark regolith samples, or that some
900 simulated Martian atmospheric constituent or unknown process was imparting
901 some beneficial effect on the non-melanized Wangiella.
902 The similarity in half-lives and percent survival between samples exposed
903 to Mars-like temperatures and room temperature controls points to a lack of
904 temperature sensitivity in a number of species, including W. dermatitidis, A.
905 psychrolactophilis, E. sibircum strains 255-15 and 7-3, and H. chaoviator (Table
906 3). Species of Exiguobacterium actually showed increased half-lives under Mars-
907 like temperatures, and of the organisms studied, only the eukaryote Cryptococcus
908 sp. NP-33C showed a significant decrease in half-life between samples exposed to
909 the diurnal Mars temperature cycle and controls. Freeze-thaw cycling is inferred
910 to be one of the primary means of limiting survival at depths below direct UV
911 irradiation during long-term Martian simulation studies (Hansen et al., 2009);
912 however, if freeze-thaw cycling was a primary mechanism for decreasing viable
913 cell numbers, this trend would be expected to favor survival within the
914 psychrotolerant organisms. This trend is not apparent; the data indicate
46
915 temperature affects organisms to different extents, and that the psychrotolerant
916 microorganisms’ lifetimes are less than the lifetimes shown by organisms
917 displaying desiccation resistance.
918 The low availability of water during the course of the experiment also
919 supports minimal effects due to freeze-thaw processes. Diurnal temperature
920 variations rarely coincided with frost points (Fig. 2B), indicating that extracellular
921 water was inherently in the vapor form; however, intracellular water would be
922 susceptible to freezing unless some mechanism existed to prevent this, such as
923 increased salinity or organic secretion. The fact that extracellular water was
924 primarily in the form of water vapor would increase the osmotic pressure and
925 desiccation stresses on the cell; data from this study would indicate that processes
926 associated with freezing of intracellular water and mechanical destruction of the
927 cell appear to be secondary to desiccation stresses caused by osmotic pressure.
928 The extreme haloarchaea Halorubrum chaoviator showed complete
929 recovery in the top 0.5 mm of sample relative to laboratory controls after 40 days
930 of simulated Mars conditions (Table 3). The halophile showed no dependence on
931 the presence of UV despite sampling the uppermost portion (<0.5 mm) of
932 regolith, where the effects of UV damage would be expected to be at a maximum.
933 This is in agreement with previous work investigating similar halophilic
934 microorganisms (Fendrihan et al., 2009; Kish et al., 2009; Mancinelli et al., 1998;
935 Martin et al., 2000) showing viable cells after exposure to high-energy radiation
47
936 conditions. Studies showed that halophilic microorganisms such as H. chaoviator
937 and H. salinarum NRC-1 can utilize intracellular halides as a mechanism for
938 scavenging reactive radical species formed by UV damage and contain a high
939 Mn/Fe ratio similar to those found in radio-tolerant organisms (Kish et al., 2009).
940 The underlying mechanisms for prevention and repair of oxidative damage in
941 halophiles is thought to represent a secondary effect evolutionarily derived from
942 resistance to desiccation within their naturally hypersaline, low water activity
943 environments (Fredrickson et al., 2008). Additionally, the organism showed no
944 loss of sample due to temperature effects, indicating that freeze-thaw cycling has
945 no negative effect on cell survival despite optimal growth temperatures that are
946 well above freezing; this supports previous work showing minimal temperature
947 dependence on halophile survival (Mancinelli et al., 2004). This may be due to
948 the high intracellular concentration of solutes that normally compensate for
949 osmotic pressure, and in this case act additionally as a natural “anti-freeze” to
950 prevent cytoplasm and membrane freezing and minimize effects associated with
951 desiccation and freezing.
952 The multicellular R. varieonarnatus, which has previously shown extreme
953 resistance to high-energy radiation and extreme desiccation in its anhydrobiotic
954 state (Horikawa et al., 2008), had minimum half-lives approaching 80 days under
955 simulated Martian conditions in the presence of UV (Table 3); those minimum
956 half-lives approach hundreds of days when UV is absent. The increased half-lives
48
957 in an organism that shows resistance to oxidative damages associated with
958 radiation and desiccation relative to psychrophilic organisms would again imply
959 that temperature-related damages associated with survival under Mars-like
960 conditions are secondary to accumulated damage from desiccation and/or
961 radiation stresses.
962 Analysis of amino acid recovery from the I-MARS samples supports the
963 limited effect of UV below the uppermost few millimeters of regolith. The Table 4 964 immediate decrease in amino acid concentration by day 10 in the upper centimeter
965 of UV samples but minimal change in recovery during subsequent samplings
966 implies that there is a rapid oxidation of amino acids within the UV-exposed
967 upper centimeters of regolith that decreases amino acid concentrations to
968 approximately 75-80% of their original value. Photolytic oxidation alone cannot
969 explain the decrease in amino acid concentration at depths below the UV
970 penetration depth, where the effect of UV is negligible due to shielding effects
971 from the overlying regolith. Using a model for irradiation of a column of regolith
972 (Garry et al., 2006), we calculate that, based on average particle size, density, and
973 sample mass, only about 0.13% of the total I-MARS regolith surface area
974 experienced the full UV dose; this corresponds to a penetration depth of roughly
975 37 μm. Calculation of the extinction coefficient of the regolith from 200 to 400
976 nm puts the UV penetration depth at 56 μm. The limited UV penetration into the
977 regolith would minimize the effects of a strictly photolytic oxidation, implying an
49
978 alternate mechanism for the decrease in amino acid concentration. At depths
979 below 2 cm, the concentration was unchanged from initial values until day 30, at
980 which time amino acid concentrations dropped to values similar to those seen in
981 the upper centimeter profile. Dark samples showed a similar delay in
982 concentration loss, with no changes in concentration in the upper and middle
983 centimeter profiles until day 30 and no change in the bottom profile over the
984 course of the 40-day experiment. The delayed oxidation of amino acids in dark
985 and subsurface UV samples may imply a diffusion-controlled mechanism for the
986 oxidation of these compounds, visualized as a moving “oxidation front” through
987 the regolith with increasing experiment times. The rate of loss of these
988 compounds is on the same order for photolytic or radiolytic oxidation rates (10-7
989 to 10-8 s-1) (Kminek and Bada, 2006; ten Kate et al., 2005), implying the proposed
990 oxidation front acts in a similar reaction mechanism.
991 It has been proposed that condensation of water vapor at mineral grain
992 boundaries during low temperature cycles may allow the formation of hydroxyl
993 radicals through Fenton and photo-Fenton-like reaction pathways (Benner et al.,
994 2000; Garry et al., 2006; Mohlmann, 2005) by the following series of reactions
995 (Eq. 4-7) (Zhang et al., 2005)
2+ 3+ - 996 Fe + H2O2 Fe + OH + °OH (4)
997 Fe2+ + °OH Fe3+ + OH- (5)
998 R-H + °OH H2O + R° (6)
50
999 R° + Fe3+ R+ + Fe2+ (7)
1000 where R-H represents a reduced carbon species and R° is an organic carbon
1001 radical. The reaction of reactive basaltic surfaces with minor amounts of water
1002 can produce measurable quantities of H2O2 in the absence of UV radiation or gas
1003 phase reactions (Hurowitz et al., 2007). The destruction of amino acids in this
1004 study at rates similar to those reported for photolytic and radiolytic oxidation
1005 mechanisms may represent the formation of H2O2 and potential hydroxyl radicals
1006 by the diffusion and accumulation of water vapor that, during cold periods of the
1007 experiment, condenses at mineral grain boundaries. Recent data has indicated
1008 that presence of liquid water containing concentrated leached metals and salts in
1009 solution increases the oxidation rate of organics by several orders of magnitude
1010 through metal-catalyzed oxidation pathways (Johnson and Pratt, 2010). Rough
1011 calculations for adsorbed water vapor to the I-MARS mineral surface, based on an
1012 average regolith surface area of 650 cm2 and a water molecule radius of 1.4 Å
-1 1013 indicates an available surface area for some 8 μg H2O g regolith. Calculated
1014 average absolute humidities based on measured dew points for the Mars simulated
1015 atmosphere are about 7.3 μg H2O, and, based on available surface area of each
1016 sample, implies that the regolith surface may equilibrate with a uniform, single
1017 layer of water molecules. This is in good agreement with previous studies that
1018 predict a few monolayers of water adsorbed permanently to the Martian regolith
1019 (Mohlmann, 2005). Amino acid oxidation would then be dependent on the initial
51
1020 rate of water vapor diffusion into the regolith with time and the availability of
1021 reactive basaltic surfaces.
1022 4.2 Astrobiological Significance and the Potential of Forward Contamination
1023 Long-term studies investigating the survivability of complex terrestrial
1024 microorganism communities at Mars-like conditions have previously attributed
1025 the majority of oxidative damage to organisms and organic molecules to the direct
1026 effects of UV (Chun et al., 1978; Cockell et al., 2000; Oro and Holzer, 1979;
1027 Stoker and Bullock, 1997). The results from this study support previous analysis
1028 (Cockell et al., 2005; Garry et al., 2006; Hansen et al., 2009; Mancinelli and
1029 Klovstad, 2000; Schuerger et al., 2003; Smith et al., 2009) and in-depth review
1030 (Nicholson et al., 2005), which indicate that although direct UV exposure rapidly
1031 deactivates microbial populations, damage is attenuated with burial in mineral
1032 substrate (Table 4). Data from this study indicates that the effects of UV are
1033 limited to only the uppermost layers of regolith (<1 cm) (Table 4) and that below
1034 this depth organism survival is limited by stresses associated with desiccation
1035 (Diaz and Schulze-Makuch, 2006; Schuerger and Nicholson, 2006; Smith et al.,
1036 2009) and oxidative stresses that may arise from interactions of water with
1037 mineral surfaces and the formation of peroxides and radical species (Garry et al.,
1038 2006; Hurowitz et al., 2007).
52
1039 Previous studies have investigated the role of photolytic reactive species in
1040 the Martian atmosphere in oxidation of organic matter in the regolith (Chun et al.,
1041 1978; Garry et al., 2006; Oro and Holzer, 1979; Stoker and Bullock, 1997; ten
1042 Kate et al., 2006). Although results varied, studies showed no changes in organic
1043 matter oxidation rates due to the presence of Mars-equivalent atmosphere. The
1044 findings of this study also support an alternate mechanism for cell loss with
1045 increasing burial depth in the I-MARS regolith. Sample sets exposed to Mars-like
1046 conditions show similar bulk survival relative to controls in all organisms studied
1047 except Cryptococcus sp. NP-33C and indicate a uniform mechanism for cell loss
1048 in all sample sets that is not atmospheric or temperature dependent. These
1049 findings would appear to refute the atmospheric formation of reactive species
1050 such as H2O2 as the single primary source of active oxidant in this study.
1051 However, if the sample regolith utilized in this study was slowly equilibrated with
1052 a uniform layer of adsorbed water molecules by atmospheric diffusion and
1053 condensation of water vapor, then it is entirely plausible that H2O2 formation may
1054 be occurring on mineral grain boundaries rather than in the Martian atmosphere.
1055 The production of H2O2 would further drive the formation of reactive hydroxyl
1056 radical species through Fenton, and where applicable, UV-driven photo-Fenton
1057 reaction mechanisms, leading to cell death and biomarker oxidation by increasing
1058 the reactivity and oxidative potential of the system. This method of oxidation is
1059 supported by the distribution of amino acid concentrations with depth among the
53
1060 three sample sets; subsurface UV and dark samples show a delayed amino acid
1061 loss that may be attributed to slow diffusion and condensation of water vapor on
1062 cold mineral surfaces. This mechanism would eventually plateau at a fixed amino
1063 acid concentration as available reactive surfaces on the regolith are exhausted.
1064 Diffusion driven oxidant-producing reactions, in combination with
1065 desiccation stresses associated with initial drying processes on the regolith and
1066 low vapor pressure of water in the Mars chamber and control samples, is most
1067 likely the primary mechanism for loss of viable organisms and biomarker
1068 oxidation. In this study, the organisms capable of substantially controlling the
1069 rate of protein and DNA oxidation associated with desiccation and radical-
1070 formation show the greatest chance for surviving extended periods at the Martian
1071 surface, despite the lack of ability to metabolize in low temperature environments
1072 like their psychrophilic counterparts. The current study indicates that terrestrial
1073 organisms adapted to low-temperature environments would pose minimal threat
1074 for forward contamination. Instead, organisms capable of withstanding radiation
1075 and desiccation damage would have the greatest chance of survival; results from
1076 this study show that the most successful organisms at surviving were those which
1077 were capable of sustained survival in environments characterized by extreme
1078 desiccation and minimal water activity, and which possess moderate to high
1079 radiation tolerance.
54
1080 5. Conclusions
1081 The current study indicates that proliferation of terrestrial organism on the
1082 Martian surface is limited by stresses associated with desiccation and highly
1083 oxidizing conditions arising from interactions of water vapor in the Martian
1084 regolith. Decreased survival and corresponding half-lives for psychrophilic
1085 organisms relative to more desiccation and radiation resistant species indicate that
1086 mechanisms for cell death associated with low-temperature conditions would
1087 appear to be secondary to the effects of the low water vapor environment and the
1088 potential production of oxidants in the Martian regolith. Like previous studies,
1089 results indicate that, in addition to desiccation and oxidative stresses, UV
1090 radiation is a detrimental facet for organism survival while on the surface of Mars.
1091 Of the six microorganisms for which depth profile data exist, three exhibited
1092 statistically significant shorter survival half-lives in the upper few millimeters to
1093 centimeter of UV-exposed regolith versus in the >1-2 cm in depth and dark
1094 samples, one did not, one exhibited a longer survival half-life in the upper few
1095 millimeters and one exhibited no detrimental effects whatsoever from UV
1096 exposure. However, similar recoveries between UV, dark and control samples
1097 indicates the loss in viable cell numbers is primarily controlled by stresses
1098 associated with desiccation and the reactivity of the Martian regolith. Amino acid
1099 concentrations in regolith samples showed a diffusion controlled degradation
1100 mechanism that approaches concentrations of about 75%-80% of initial values in
55
1101 both UV-exposed and dark samples; the plateau in concentration is thought to
1102 represent the point where reactive surfaces on the regolith surfaces are passivated.
1103 Rate constants for this process are along the same order of magnitude as
1104 photolytic and radiolytic oxidation mechanisms and imply an oxidation
1105 mechanism driven by the formation of radical species through the interaction of
1106 diffusing water vapor that condenses on mineral grain surfaces; future work will
1107 focus on monitoring water diffusion through a regolith depth profile and attempts
1108 to quantify the rates for the proposed hydrogen peroxide formation.
1109 Of the studied organisms, the extremely halophilic Halorubrum
1110 chaoviator and the tardigrade Ramazzttius varieornatus showed the greatest
1111 lifetimes of survival under the simulated Martian conditions. These organisms
1112 showed previous resistance to oxidative damage in the form of radiation and
1113 desiccation resistance, indicating that a physiology evolved to cope with low
1114 water activity and high radiation environments may be more influential to
1115 survival than adaptations developed for psychrotolerance. The estimated
1116 minimum half-life for R. varieornatus in the upper 1 cm was nearly double the 40
1117 days of the simulated Martian conditions and nearly ten times the experimental
1118 run for the dark samples. Both H. chaoviator and R. varieornatus appear capable
1119 of surviving the Martian environment longer than most proposed mission
1120 durations of 90 days and potentially for a complete Martian seasonal cycle.
1121 Multicellular organisms such as the tardigrades in this study and symbiotic
56
1122 organisms such as lichens (de Vera et al., 2010) have been shown to survive under
1123 Mars-like conditions, indicating the possibility of forward contamination by
1124 eukaryotic organisms may still pose a significant threat.
1125 Current methods of sterilization in spacecraft assembly often utilize dry
1126 heat treatments and vaporous hydrogen peroxide in order to minimize the
1127 potential of contamination of electronics and spacecraft surfaces (Chung et al.,
1128 2008; Schubert et al., 2003). However, results of this study show that such
1129 sterilization methods may in fact be selecting for highly desiccation and radiation
1130 resistant organisms that may prove to have the greatest chance of extended
1131 survival on the Martian surface. Future protocols for life-detection instruments
1132 must ensure sterilization procedures account for these possibilities, since
1133 organisms capable of survival in those environments are the most likely to be able
1134 to cope with the extreme protein and DNA damage associated with highly
1135 desiccating and oxidizing surface environments. Additionally, any Martian
1136 organism capable of survival at the immediate to shallow subsurface must have
1137 evolved physiological characteristics that would impart similar tolerances to
1138 radical-induced damages and desiccation resistance as a minimal requirement for
1139 survival during the current period of minimal water activity at the Martian
1140 surface. Additional adaptations such as psychrotolerance would act to impart
1141 further strategies, such as increased freeze-thaw tolerance, for survival and
1142 potential proliferation under the less than ideal surface conditions of Mars.
57
1143 During periods of episodic liquid water or redistribution of polar ice during
1144 extreme obliquity, these organisms may be able thrive in phreatic zones and
1145 permafrost wetlands where seasonal thaw could generate relatively dilute brines
1146 and habitats more hospitable to life.
1147 6. Acknowledgements
1148 Special thanks to Dr. Paul Todd and Mr. Michael (Andy) Kurk of
1149 TechShot Laboratories, Greenville, IN, for their help in setup, running and
1150 sampling within the Mars Environmental Chamber. Additional thanks go to Dr.
1151 Christine Shriner, Indiana University Department of Geological Sciences, for her
1152 help in determining particle size distribution of the I-MARS regolith. A large
1153 thank you goes to Dr. James Brophy, Indiana University Department of
1154 Geological Sciences, for his help in obtaining samples of the Collier Cone basalt
1155 and electron microprobe analysis of the final elemental composition of the I-
1156 MARS regolith. This work was funded by the NASA Astrobiology Institute Grant
1157 NNA04CC03A S000018.
58
1158 7. References 1159 1160 Andersen, D. T., Pollard, W. H., McKay, C. P., Heldmann, J., 2002. Cold springs
1161 in permafrost on Earth and Mars. Journal of Geophysical Research-
1162 Planets. 107, -.
1163 Aubrey, A., Cleaves, H. J., Chalmers, J. H., Skelley, A. M., Mathies, R. A.,
1164 Grunthaner, F. J., Ehrenfreund, P., Bada, J. L., 2006. Sulfate minerals and
1165 organic compounds on Mars. Geology. 34, 357-360.
1166 Bakermans, C., Ayala-del-rio, H. L., Ponder, M. A., Vishnivetskaya, T.,
1167 Gilichinsky, D., Thomashow, M. F., Tiedje, J. M., 2006. Psychrobacter
1168 cryohalolentis sp nov and Psychrobacter arcticus sp nov., isolated from
1169 Siberian permafrost. International Journal of Systematic and Evolutionary
1170 Microbiology. 56, 1285-1291.
1171 Barbier, B., Chabin, A., Chaput, D., Brack, A., 1998. Photochemical processing
1172 of amino acids in Earth orbit. Planetary and Space Science. 46, 391-398.
1173 Barbier, B., Henin, O., Boillot, F., Chabin, A., Chaput, D., Brack, A., 2002.
1174 Exposure of amino acids and derivatives in the Earth orbit. Planetary and
1175 Space Science. 50, 353-359.
1176 Beaty, D. W., Group, T. M. S. R. S. A., 2006. Findings of the Mars Special
1177 Regions Science Analysis Group. Astrobiology. 6, 677-732.
59
1178 Benner, S. A., Devine, K. G., Matveeva, L. N., Powell, D. H., 2000. The missing
1179 organic molecules on Mars. Proceedings of the National Academy of
1180 Sciences of the United States of America. 97, 2425-2430.
1181 Biemann, K., Oro, J., III, P. T., Orgel, L. E., Nier, A. O., Anderson, D. M.,
1182 Simmonds, P. G., Flory, D., Diaz, A. V., Rushneck, D. R., Biller, J. E.,
1183 Lafleur, A. L., 1977. The Search for Organic Substances and Inorganic
1184 Volatile Compounds in the Surface of Mars. J. Geophys. Res. 82, 4641-
1185 4658.
1186 Boynton, W. V., Ming, D. W., Kounaves, S. P., Young, S. M. M., Arvidson, R.
1187 E., Hecht, M. H., Hoffman, J., Niles, P. B., Hamara, D. K., Quinn, R. C.,
1188 Smith, P. H., Sutter, B., Catling, D. C., Morris, R. V., 2009. Evidence for
1189 Calcium Carbonate at the Mars Phoenix Landing Site. Science. 325, 61-
1190 64.
1191 Brenner, S., 1974. Genetics of Caenorhabditis-Elegans. Genetics. 77, 71-94.
1192 Buck, A. L., 1981. New Equations for Computing Vapor-Pressure and
1193 Enhancement Factor. J. Appl. Meteorol. 20, 1527-1532.
1194 Buck, A. L., Buck Research Manual. 1996.
1195 Cavicchioli, R., 2002. Extremophiles and the search for extraterrestrial life.
1196 Astrobiology. 2, 281-292.
1197 Chun, S. F. S., Pang, K. D., Cutts, J. A., Ajello, J. M., 1978. Photocatalytic
1198 oxidation of organic compounds on Mars. Nature. 274, 875-876.
60
1199 Chung, S., Kern, R., Koukol, R., Barengoltz, J., Cash, H., 2008. Vapor hydrogen
1200 peroxide as alternative to dry heat microbial reduction. Advances in Space
1201 Research. 42, 1150-1160.
1202 Cockell, C. S., Catling, D. C., Davis, W. L., Snook, K., Kepner, R. L., Lee, P.,
1203 McKay, C. P., 2000. The ultraviolet environment of Mars: Biological
1204 implications past, present, and future. Icarus. 146, 343-359.
1205 Cockell, C. S., Knowland, J., 1999. Ultraviolet radiation screening compounds.
1206 Biol. Rev. Cambridge Philosophic. Soc. 74, 311-345.
1207 Cockell, C. S., Lee, P., Osinski, G., Horneck, G., Broady, P., 2002. Impact-
1208 induced microbial endolithic habitats. Meteoritics & Planetary Science.
1209 37, 1287-1298.
1210 Cockell, C. S., Schuerger, A. C., Billi, D., Friedmann, E. I., Panitz, C., 2005.
1211 Effects of a simulated martian UV flux on the cyanobacterium,
1212 Chroococcidiopsis sp 029. Astrobiology. 5, 127-140.
1213 Daly, M. J., Gaidamakova, E. K., Matrosova, V. Y., Vasilenko, A., Zhai, M.,
1214 Leapman, R. D., Lai, B., Ravel, B., Li, S. M. W., Kemner, K. M.,
1215 Fredrickson, J. K., 2007. Protein oxidation implicated as the primary
1216 determinant of bacterial radioresistance. Plos Biology. 5, 769-779.
1217 de Vera, J. P., Mohlmann, D., Butina, F., Lorek, A., Wernecke, R., Ott, S., 2010.
1218 Survival Potential and Photosynthetic Activity of Lichens Under Mars-
1219 Like Conditions: A Laboratory Study. Astrobiology. 10, 215-227.
61
1220 dePrada, P., LovelandCurtze, J., Brenchley, J. E., 1996. Production of two
1221 extracellular alkaline phosphatases by a psychrophilic Arthrobacter strain.
1222 Applied and Environmental Microbiology. 62, 3732-3738.
1223 Diaz, B., Schulze-Makuch, D., 2006. Microbial survival rates of Escherichia coli
1224 and Deinococcus radiodurans under low temperature, low pressure, and
1225 UV-Irradiation conditions, and their relevance to possible martian life.
1226 Astrobiology. 6, 332-347.
1227 Ehlmann, B. L., Mustard, J. F., Murchie, S. L., Poulet, F., Bishop, J. L., Brown,
1228 A. J., Calvin, W. M., Clark, R. N., Des Marais, D. J., Milliken, R. E.,
1229 Roach, L. H., Roush, T. L., Swayze, G. A., Wray, J. J., 2008. Orbital
1230 identification of carbonate-bearing rocks on Mars. Science. 322, 1828-
1231 1832.
1232 Fairen, A. G., Davila, A. F., Gago-Duport, L., Amils, R., McKay, C. P., 2009.
1233 Stability against freezing of aqueous solutions on early Mars. Nature. 459,
1234 401-404.
1235 Fendrihan, S., Berces, A., Lammer, H., Musso, M., Ronto, G., Polacsek, T. K.,
1236 Holzinger, A., Kolb, C., Stan-Lotter, H., 2009. Investigating the Effects of
1237 Simulated Martian Ultraviolet Radiation on Halococcus dombrowskii and
1238 Other Extremely Halophilic Archaebacteria. Astrobiology. 9, 104-112.
1239 Feng, B., Wang, X., Hauser, M., Kaufmann, S., Jentsch, S., Haase, G., Becker, J.
1240 M., Szaniszlo, P. J., 2001. Molecular cloning and characterization of
62
1241 WdPKS1, a gene involved in dihydroxynaphthalene melanin biosynthesis
1242 and virulence in Wangiella (Exophiala) dermatitidis. Infection and
1243 Immunity. 69, 1781-1794.
1244 Fredrickson, J. K., Li, S. M. W., Gaidamakova, E. K., Matrosova, V. Y., Zhai, M.,
1245 Sulloway, H. M., Scholten, J. C., Brown, M. G., Balkwill, D. L., Daly, M.
1246 J., 2008. Protein oxidation: key to bacterial desiccation resistance? Isme
1247 Journal. 2, 393-403.
1248 Garry, J. R. C., Ten Kate, I. L., Martins, Z., Nornberg, P., Ehrenfreund, P., 2006.
1249 Analysis and survival of amino acids in Martian regolith analogs.
1250 Meteoritics & Planetary Science. 41, 391-405.
1251 Gellert, R., Rieder, R., Anderson, R. C., Bruckner, J., Clark, B. C., Dreibus, G.,
1252 Economou, T., Klingelhofer, G., Lugmair, G. W., Ming, D. W., Squyres,
1253 S. W., d'Uston, C., Wanke, H., Yen, A., Zipfel, J., 2004. Chemistry of
1254 rocks and soils in Gusev crater from the alpha particle x-ray spectrometer.
1255 Science. 305, 829-832.
1256 Gendrin, A., Mangold, N., Bibring, J. P., Langevin, Y., Gondet, B., Poulet, F.,
1257 Bonello, G., Quantin, C., Mustard, J., Arvidson, R., LeMouelic, S., 2005.
1258 Sulfates in martian layered terrains: the OMEGA/Mars Express view.
1259 Science. 307, 1587-1591.
1260 Gunn, B. M., Watkins, N. D., 1970. Geochemistry of Steens-Mountain Basalts,
1261 Oregon. Geological Society of America Bulletin. 81, 1497-&.
63
1262 Hansen, A. A., Jensen, L. L., Kristoffersen, T., Mikkelsen, K., Merrison, J.,
1263 Finster, K. W., Lomstein, B. A., 2009. Effects of Long-Term Simulated
1264 Martian Conditions on a Freeze-Dried and Homogenized Bacterial
1265 Permafrost Community. Astrobiology. 9, 229-240.
1266 Haskin, L. A., Wang, A., Jolliff, B. L., McSween, H. Y., Clark, B. C., Des
1267 Marais, D. J., McLennan, S. M., Tosca, N. J., Hurowitz, J. A., Farmer, J.
1268 D., Yen, A., Squyres, S. W., Arvidson, R. E., Klingelhofer, G., Schroder,
1269 C., de Souza, P. A., Ming, D. W., Gellert, R., Zipfel, J., Bruckner, J., Bell,
1270 J. F., Herkenhoff, K., Christensen, P. R., Ruff, S., Blaney, D., Gorevan, S.,
1271 Cabrol, N. A., Crumpler, L., Grant, J., Soderblom, L., 2005. Water
1272 alteration of rocks and soils on Mars at the Spirit rover site in Gusev
1273 crater. Nature. 436, 66-69.
1274 Horikawa, D. D., Kunieda, T., Abe, W., Watanabe, M., Nakahara, Y., Yukuhiro,
1275 F., Sakashita, T., Hamada, N., Wada, S., Funayama, T., Katagiri, C.,
1276 Kobayashi, Y., Higashi, S., Okuda, T., 2008. Establishment of a rearing
1277 system of the extremotolerant tardigrade Ramazzottius varieornatus: A
1278 new model animal for astrobiology. Astrobiology. 8, 549-556.
1279 Hurowitz, J. A., Tosca, N. J., McLennan, S. M., Schoonen, M. A. A., 2007.
1280 Production of hydrogen peroxide in Martian and lunar soils. Earth and
1281 Planetary Science Letters. 255, 41-52.
1282 Ingersol, A., 1970. Mars - Occurance of Liquid Water. Science. 168, 972-973.
64
1283 Johnson, A. P., Pratt, L.M., 2010. Metal Catalyzed Degradation and Racemization
1284 of Amino Acids in Iron Sulfate Brines under Simulated Martian Surface
1285 Conditions. Icarus. 207, 124-132.
1286 Kargel, J. S., 2004. Mars : a warmer, wetter planet. Springer ;
1287 Praxis, London ; New York
1288 Chichester.delete returns in Endnote file.
1289 Kish, A., Kirkali, G., Robinson, C., Rosenblatt, R., Jaruga, P., Dizdaroglu, M.,
1290 DiRuggiero, J., 2009. Salt shield: intracellular salts provide cellular
1291 protection against ionizing radiation in the halophilic archaeon,
1292 Halobacterium salinarum NRC-1. Environmental Microbiology. 11,
1293 1066-1078.
1294 Klingelhofer, G., Morris, R. V., Bernhardt, B., Schroder, C., Rodionov, D. S., de
1295 Souza, P. A., Yen, A., Gellert, R., Evlanov, E. N., Zubkov, B., Foh, J.,
1296 Bonnes, U., Kankeleit, E., Gutlich, P., Ming, D. W., Renz, F., Wdowiak,
1297 T., Squyres, S. W., Arvidson, R. E., 2004. Jarosite and hematite at
1298 Meridiani Planum from Opportunity's Mossbauer spectrometer. Science.
1299 306, 1740-1745.
1300 Kminek, G., Bada, J. L., 2006. The effect of ionizing radiation on the preservation
1301 of amino acids on Mars. Earth and Planetary Science Letters. 245, 1-5.
1302 Knoll, A. H., Carr, M., Clark, B., Marais, D. J. D., Farmer, J. D., Fischer, W. W.,
1303 Grotzinger, J. P., McLennan, S. M., Malin, M., Schroder, C., Squyres, S.,
65
1304 Tosca, N. J., Wdowiak, T., 2005. An astrobiological perspective on
1305 Meridiani Planum. Earth and Planetary Science Letters. 240, 179-189.
1306 Koch, A. L., Growth Measurement. In: P. Gerhardt, (Ed.), Methods for Genearl
1307 and Molecular Bacteriology, 1994, pp. 248-277.
1308 Kottemann, M., Kish, A., Iloanusi, C., Bjork, S., DiRuggiero, J., 2005.
1309 Physiological responses of the halophilic archaeon Halobacterium sp
1310 strain NRC1 to desiccation and gamma irradiation. Extremophiles. 9, 219-
1311 227.
1312 Krasnopolsky, V. A., 2005. Some problems related to the origin of methane on
1313 Mars. Icarus. in press.in complete reference
1314 Krasnopolsky, V. A., Maillard, J. P., Owen, T. C., 2004. Detection of methane in
1315 the martian atmosphere: evidence for life? Icarus. 172, 537-547.
1316 Kuznetz, L. H., Gan, D. C., 2002. On the existence and stability of liquid water on
1317 the surface of Mars today. Astrobiology. 2, 183-195.
1318 Lefevre, F., Forget, F., 2009. Observed variations of methane on Mars
1319 unexplained by known atmospheric chemistry and physics. Nature. 460,
1320 720-723.
1321 Lewis, J. A., Fleming, J.T., 1995. Caenorhabditis elegans : modern biological
1322 analysis of an organism. Academic Press, San Diego.
1323 Loveland-Curtze, J., Sheridan, P. P., Gutshall, K. R., Brenchley, J. E., 1999.
1324 Biochemical and phylogenetic analyses of psychrophilic isolates
66
1325 belonging to the Arthrobacter subgroup and description of Arthrobacter
1326 psychrolactophilus, sp nov. Archives of Microbiology. 171, 355-363.
1327 Mancinelli, R. L., Fahlen, T. F., Landheim, R., Klovstad, M. R., 2004. Brines and
1328 evaporites: analogs for Martian life. Space Life Sciences: Search for
1329 Signatures of Life, and Space Flight Environmental Effects on the
1330 Nervous System. 33, 1244-1246.
1331 Mancinelli, R. L., Klovstad, M., 2000. Martian soil and UV radiation: microbial
1332 viability assessment on spacecraft surfaces. Planetary and Space Science.
1333 48, 1093-1097.
1334 Mancinelli, R. L., Landheim, R., Sanchez-Porro, C., Dornmayr-Pfaffenhuemer,
1335 M., Gruber, C., Legat, A., Ventosa, A., Radax, C., Ihara, K., White, M. R.,
1336 Stan-Lotter, H., 2009. Halorubrum chaoviator sp nov., a haloarchaeon
1337 isolated from sea salt in Baja California, Mexico, Western Australia and
1338 Naxos, Greece. International Journal of Systematic and Evolutionary
1339 Microbiology. 59, 1908-1913.
1340 Mancinelli, R. L., White, M. R., Rothschild, L. J., Biopan-survival I: Exposure of
1341 the osmophiles Synechococcus sp. (Nageli) and Haloarcula sp. to the
1342 space environment. In: G. Horneck, et al., Eds.), Life Sciences:
1343 Exobiology. Pergamon Press Ltd, Oxford, 1998, pp. 327-334.
1344 Martin, E. L., Reinhardt, R. L., Baum, L. L., Becker, M. R., Shaffer, J. J.,
1345 Kokjohn, T. A., 2000. The effects of ultraviolet radiation on the moderate
67
1346 halophile Halomonas elongata and the extreme halophile Halobacterium
1347 salinarum. Canadian Journal of Microbiology. 46, 180-187.
1348 Mckay, C. P., Davis, W. L., 1991. Duration of Liquid Water Habitats on Early
1349 Mars. Icarus. 90, 214-221.
1350 McSween, H. Y., Jr., Grove, T. L., Wyatt, M. B., 2003. Constraints on the
1351 composition and petrogenesis of the Martian crust. J. Geophys. Res. 108,
1352 9-1-9-9-19.
1353 Mills, D. K., Hartman, P. S., 1998. Lethal consequences of simulated solar
1354 radiation on the nematode Caenorhabditis elegans in the presence and
1355 absence of photosensitizers. Photochemistry and Photobiology. 68, 816-
1356 823.
1357 Mohlmann, D., 2005. Adsorption water-related potential chemical and biological
1358 processes in the upper martian surface. Astrobiology. 5, 770-777.
1359 Morozova, D. Mohlmann, D. and Wagner, D. 2007. Survival of Methanogenic
1360 Archaea from Siberian Permafrost under Simulated Martian Thermal
1361 Conditions. Orig. Life Evo. Biosph. 37:189-200.
1362 Morozova D. and Wagner, D. 2007. Stress response of methanogenic archaea
1363 from Siberian permafrost compared with metahnogens from
1364 nonpermafrost habitats. FEMS Microbial Ecol. 61:16-25.
68
1365 Mumma, M. J., Villaneuva, G. L., Novak, R. E., Hewagama, T., Bonev, B. P.,
1366 DiSanti, M. A., Mandell, A. M., Smith, M. D., 2009. Strong release of
1367 methane on Mars in northern summer 2003. Science. 1041-1045.
1368 Mustard, J. F., Murchie, S. L., Pelkey, S. M., Ehlmann, B. L., Milliken, R. E.,
1369 Grant, J. A., Bibring, J. P., Poulet, F., Bishop, J., Dobrea, E. N., Roach, L.,
1370 Seelos, F., Arvidson, R. E., Wiseman, S., Green, R., Hash, C., Humm, D.,
1371 Malaret, E., McGovern, J. A., Seelos, K., Clancy, T., Clark, R., Des
1372 Marais, D., Izenberg, N., Knudson, A., Langevin, Y., Martin, T., McGuire,
1373 P., Morris, R., Robinson, M., Roush, T., Smith, M., Swayze, G., Taylor,
1374 H., Titus, T., Wolff, M., 2008. Hydrated silicate minerals on mars
1375 observed by the Mars reconnaissance orbiter CRISM instrument. Nature.
1376 454, 305-309.
1377 Nakagawa, T., Fujimoto, Y., Uchino, M., Miyaji, T., Takano, K., Tomizuka, N.,
1378 2003. Isolation and characterization of psychrophiles producing cold-
1379 active beta-galactosidase. Letters in Applied Microbiology. 37, 154-157.
1380 Navarro-Gonzalez, R., Rainey, F. A., Molina, P., Bagaley, D. R., Hollen, B. J.,
1381 Rosa, J. d. l., Small, A. M., Quinn, R. C., Grunthaner, F. J., Ca'ceres, L.,
1382 Gomez-Silva, B., McKay, C. P., 2003. Mars-Like Soils in the Atacama
1383 Desert, Chile, and the Dry Limit of Microbial Life. Science. 302, 1018-
1384 1021.
69
1385 Newcombe, D. A., Schuerger, A. C., Benardini, J. N., Dickinson, D., Tanner, R.,
1386 Venkateswaran, K., 2005. Survival of spacecraft-associated
1387 microorganisms under simulated Martian UV irradiation. Applied and
1388 Environmental Microbiology. 71, 8147-8156.
1389 Nicholson, W. L., Schuerger, A. C., 2005. Bacillus subtilis spore survival and
1390 expression of germination-induced bioluminescence after prolonged
1391 incubation under simulated Mars atmospheric pressure and composition:
1392 Implications for planetary protection and lithopanspermia. Astrobiology.
1393 5, 536-544.
1394 Nicholson, W. L., Schuerger, A. C., Setlow, P., 2005. The solar UV environment
1395 and bacterial spore UV resistance: considerations for Earth-to-Mars
1396 transport by natural processes and human spaceflight. Mutation Research-
1397 Fundamental and Molecular Mechanisms of Mutagenesis. 571, 249-264.
1398 Oro, J., Holzer, G., 1979. Photolytic Degradation and Oxidation of Organic
1399 Compounds under Simulated Martian Conditions. Journal of Molecular
1400 Evolution. 14, 153-160.
1401 Osterloo, M. M., Hamilton, V. E., Bandfield, J. L., Glotch, T. D., Baldridge, A.
1402 M., Christensen, P. R., Tornabene, L. L., Anderson, F. S., 2008. Chloride-
1403 bearing materials in the southern highlands of Mars. Science. 319, 1651-
1404 1654.
70
1405 Owen, T., Biemann, K., Rushneck, D. R., Biller, J. E., Howarth, D. W., Lafleur,
1406 A. L., 1977. The composition of the atmosphere at the surface of Mars. J.
1407 Geophys. Res. 82, 4635-4639.
1408 Oyama, V. I., Berdahl, B. J., 1977. The Viking Gas Exchange Experiment Results
1409 From Chryse and Utopia Surface Samples. J. Geophys. Res. 82, 4669-
1410 4676.
1411 Paolo, W. F., Dadachova, E., Mandal, P., Casadevall, A., Szaniszlo, P. J.,
1412 Nosanchuk, J. D., 2006. Effects of disrupting the polyketide synthase gene
1413 WdPKS1 in Wangiella [Exophiala] dermatitidis on melanin production
1414 and resistance to killing by antifungal compounds, enzymatic degradation,
1415 and extremes in temperature. Bmc Microbiology. 6.
1416 Patel, M. R., Zarnecki, J. C., Catling, D. C., 2002. Ultraviolet radiation on the
1417 surface of Mars and the Beagle 2 UV sensor. Planetary and Space Science.
1418 50, 915-927.
1419 Perreault, N. N., Greer, C. W., Andersen, D. T., Tille, S., Lacrampe-Couloume,
1420 G., Lollar, B. S., Whyte, L. G., 2008. Heterotrophic and Autotrophic
1421 Microbial Populations in Cold Perennial Springs of the High Arctic.
1422 Applied and Environmental Microbiology. 74, 6898-6907.
1423 Pollack, J. B., Kasting, J. F., Richardson, S. M., Poliakoff, K., 1987. The Case for
1424 a Wet, Warm Climate on Early Mars. Icarus. 71, 203-224.
71
1425 Poulet, F., Bibring, J. P., Mustard, J. F., Gendrin, A., Mangold, N., Langevin, Y.,
1426 Arvidson, R. E., Gondet, B., Gomez, C., Team, O., 2005. Phyllosilicates
1427 on Mars and implications for early martian climate. Nature. 438, 623-627.
1428 Rao, M. N., Nyquist, L. E., Sutton, S. R., Dreibus, G., Garrison, D. H., Herrin, J.,
1429 2009. Fluid-evaporation records preserved in salt assemblages in
1430 Meridiani rocks. Earth and Planetary Science Letters. In Press, Corrected
1431 Proof.
1432 Riddle, D. L., Blumenthal, T., Meyer, B. J., Priess, J. R. (Ed.) 1997. C. elegans II.
1433 Cold Springs Harbor Laboratory Press, Cold Springs Harbor.
1434 Rieder, R., Gellert, R., Anderson, R. C., Bruckner, J., Clark, B. C., Dreibus, G.,
1435 Economou, T., Klingelhoffer, G., Lugmair, G. W., Ming, D. W., Squyres,
1436 S. W., d'Uston, C., Wanke, H., Yen, A., Zipfel, J., 2004. Chemistry of
1437 rocks and soils at Meridiani Planum from the alpha particle X-ray
1438 spectrometer. Science. 306, 1746-1749.
1439 Rivkina, E., Shcherbakova, V., Laurinavichius, K., Petrovskaya, L., Krivushin,
1440 K., Kraev, G., Pecheritsina, S., Gilichinsky, D., 2007. Biogeochemistry of
1441 methane and methanogenic archaea in permafrost. Fems Microbiology
1442 Ecology. 61, 1-15.
1443 Rodrigues, D. F., Goris, J., Vishnivetskaya, T., Gilichinsky, D., Thomashow, M.
1444 F., Tiedje, J. M., 2006. Characterization of Exiguobacterium isolates from
72
1445 the Siberian permafrost. Description of Exiguobacterium sibiricum sp nov.
1446 Extremophiles. 10, 285-294.
1447 Schon, S. C., Head, J. W., Fassett, C. I., 2009. Unique chronostratigraphic marker
1448 in depositional fan stratigraphy on Mars: Evidence for ca. 1.25 Ma gully
1449 activity and surficial meltwater origin. Geology. 37, 207-210.
1450 Schubert, W. W., Arakelian, T., Barengoltz, J. B., Chough, N. G., Chung, S. Y.,
1451 Law, J., Newlin, L. E., Morales, F., 2003. Microbial certification of the
1452 MER spacecraft. Abstracts of the General Meeting of the American
1453 Society for Microbiology. 103, Q-275.
1454 Schuerger, A. C., Ardo-Cavazos, P. F., Clausen, C. A., Moores, J. E., Smith, P.
1455 H., Nicholson, W. L., 2008. Slow degradation of ATP in simulated
1456 martian environments suggests long residence times for the biosignature
1457 molecule on spacecraft surfaces on Mars. Icarus. 194, 86-100.
1458 Schuerger, A. C., Mancinelli, R. L., Kern, R. G., Rothschild, L. J., McKay, C. P.,
1459 2003. Survival of endospores of Bacillus subtilis on spacecraft surfaces
1460 under simulated martian environments: implications for the forward
1461 contamination of Mars. Icarus. 165, 253-276.
1462 Schuerger, A. C., Nicholson, W. L., 2006. Interactive effects of hypobaria, low
1463 temperature, and CO2 atmospheres inhibit the growth of mesophilic
1464 Bacillus spp. under simulated martian conditions. Icarus. 185, 143-152.
73
1465 Schuerger, A. C., Richards, J. T., Newcombe, D. A., Venkateswaran, K., 2006.
1466 Rapid inactivation of seven Bacillus spp. under simulated Mars UV
1467 irradiation. Icarus. 181, 52-62.
1468 Smith, D. J., Schuerger, A. C., Davidson, M. M., Pacala, S. W., Bakermans, C.,
1469 Onstott, T. C., 2009. Survivability of Psychrobacter cryohalolentis K5
1470 Under Simulated Martian Surface Conditions. Astrobiology. 9, 221-228.
1471 Smith, M. D., 2002. The annual cycle of water vapor on Mars as observed by the
1472 Thermal Emission Spectrometer. Journal of Geophysical Research-
1473 Planets. 107.
1474 Smith, M. D., Wolff, M. J., Clancy, R. T., Murchie, S. L., 2009. Compact
1475 Reconnaissance Imaging Spectrometer observations of water vapor and
1476 carbon monoxide. Journal of Geophysical Research-Planets. 114.
1477 Squyres, S. W., Grotzinger, J. P., Arvidson, R. E., Bell, J. F., Calvin, W.,
1478 Christensen, P. R., Clark, B. C., Crisp, J. A., Farrand, W. H., Herkenhoff,
1479 K. E., Johnson, J. R., Klingelhofer, G., Knoll, A. H., McLennan, S. M.,
1480 McSween, H. Y., Morris, R. V., Rice, J. W., Rieder, R., Soderblom, L. A.,
1481 2004. In situ evidence for an ancient aqueous environment at Meridiani
1482 Planum, Mars. Science. 306, 1709-1714.
1483 Stan-Lotter, H., McGenity, T. J., Legat, A., Denner, E. B. M., Glaser, K., Stetter,
1484 K. O., Wanner, G., 1999. Very similar strains of Halococcus salifodinae
74
1485 are found in geographically separated Permo-Triassic salt deposits.
1486 Microbiology-Uk. 145, 3565-3574.
1487 Stoker, C. R., Bullock, M. A., 1997. Organic degradation under simulated Martian
1488 conditions. Journal of Geophysical Research-Planets. 102, 10881-10888.
1489 Stoker, C. R., Zent, A., Catling, D. C., Douglas, S., Marshal, J. R., Archer, D.,
1490 Clark, B., Kounaves, S. P., Lemmon, M. T., Quinn, R., Renno, N., Smith,
1491 P. H., Young, S. M. M., 2010. Habitability of the Phoenix landing site.
1492 Journal of Geophysical Research-Planets. 115, -.
1493 Tauscher, C., Schuerger, A. C., Nicholson, W. L., 2006. Survival and
1494 germinability of Bacillus subtilis spores exposed to simulated Mars solar
1495 radiation: Implications for life detection and planetary protection.
1496 Astrobiology. 6, 592-605.
1497 ten Kate, I. L., Garry, J. R. C., Peeters, Z., Foing, B., Ehrenfreund, P., 2006. The
1498 effects of Martian near surface conditions on the photochemistry of amino
1499 acids. Planetary and Space Science. 54, 296-302.
1500 ten Kate, I. L., Garry, J. R. C., Peeters, Z., Quinn, R., Foing, B., Ehrenfreund, P.,
1501 2005. Amino acid photostability on the Martian surface. Meteoritics &
1502 Planetary Science. 40, 1185-1193.
1503 Tosca, N. J., Knoll, A. H., McLennan, S. M., 2008. Water activity and the
1504 challenge for life on early Mars. Science. 320, 1204-1207.
1505 Vishnivetskaya, T., Personal Communication. 2010.
75
1506 Vishnivetskaya, T. A., Petrova, M. A., Urbance, J., Ponder, M., Moyer, C. L.,
1507 Gilichinsky, D. A., Tiedje, J. M., 2006. Bacterial community in ancient
1508 Siberian permafrost as characterized by culture and culture-independent
1509 methods. Astrobiology. 6, 400-414.
1510 Vishnivetskaya, T. A., Siletzky, R., Jefferies, N., Tiedje, J. M., Kathariou, S.,
1511 2007. Effect of low temperature and culture media on the growth and
1512 freeze-thawing tolerance of Exiguobacterium strains. Cryobiology. 54,
1513 234-240.
1514 Wanke, H., Bruckner, J., Dreibus, G., Rieder, R., Ryabchikov, I., 2001. Chemical
1515 composition of rocks and soils at the Pathfinder site. Space Science
1516 Reviews. 96, 317-330.
1517 Wergin, W. P., Yaklich, R. W., Carta, L. K., Erbe, E. F., Murphy, C. A., 2000.
1518 Effect of an ice-nucleating activity agent on subzero survival of nematode
1519 juveniles. Journal of Nematology. 32, 198-204.
1520 Whiteway, J. A., Komguem, L., Dickinson, C., Cook, C., Illnicki, M., Seabrook,
1521 J., Popovici, V., Duck, T. J., Davy, R., Taylor, P. A., Pathak, J., Fisher, D.,
1522 Carswell, A. I., Daly, M., Hipkin, V., Zent, A. P., Hecht, M. H., Wood, S.
1523 E., Tamppari, L. K., Renno, N., Moores, J. E., Lemmon, M. T., Daerden,
1524 F., Smith, P. H., 2009. Mars Water-Ice Clouds and Precipitation. Science.
1525 325, 68-70.
76
1526 Willems, M., Houthoofd, W., Claeys, M., Couvreur, M., Van Driessche, R.,
1527 Adriaens, D., Jacobsen, K., Borgonie, G., 2005. Unusual intestinal
1528 lamellae in the nematode Rhabditophanes sp KR3021 (Nematoda :
1529 Alloinematidae). Journal of Morphology. 264, 223-232.
1530 Wood, L. J., 2006. Quantitative geomorphology of the Mars Eberswalde delta.
1531 Geological Society of America Bulletin. 118, 557-566.
1532 Zent, A. P., McKay, C. P., 1994. The chemical reactivity of the martian soil and
1533 implications for future missions. Icarus. 108, 146-157.
1534 Zhang, H., Choi, H. J., Huang, C. P., 2005. Optimization of Fenton process for the
1535 treatment of landfill leachate. Journal of Hazardous Materials. 125, 166-
1536 174.
1537 Zhdanova, N. N., Pokhoden, V., 1973. Possible Participation of Melanin Pigment
1538 in Protection of Fungal Cell from Dehydration. Mikrobiologiya. 42, 848-
1539 853.
77
1540 Figure Captions:
1541 Fig.1: A) Particle size distribution determined by gravimetric sedimentation of the
1542 I-MARS regolith shows a cumulative finer mass percent of >80% particles less
1543 than 64 μm in diameter. B) Elemental analysis of bulk regolith compared to bulk
1544 Gusev soil (Gellert et al., 2004), bulk soil free rock (Wanke et al., 2001), and
1545 global dust (McSween et al., 2003) compositions. Error bars represent standard
1546 deviation in triplicate analysis.
1547 Fig. 2: A) Modeled UV irradiances for summer equatorial Mars with minimal
1548 dust attenuation (dashed line) (Patel et al., 2002) and instrument-error corrected
1549 values of UV irradiance used in the experiment (solid grey line) (see text). B)
1550 Hourly-averaged diurnal fluctuations in temperature, atmospheric pressure, dew
1551 point, and relative humidity within the chamber; relative humidity is calculated as
1552 the percent ratio of the partial pressure of water and the saturation pressure of
1553 water at temperature t in Celsius. Sampling points at Days 10, 20 and 30 are
1554 noted with black arrows and identified by sharp increases in chamber pressure.
1555 Grey areas mark points where either liquid N2 was insufficient to chill the
1556 mirrored surface of the hygrometer or where data errors corrupted reported
1557 values.
78
1558 Fig. 3: Average total hydrolyzable amino acid (THAA) concentrations as a
1559 function of depth in A) UV B) dark and C) control samples. Sample
1560 concentrations were normalized to 500 ppm, which represented the assumed
1561 initial concentrations at the time of sample preparation. Error bars represent
1562 experimental standard deviations based on N=3-6 individual samples adjusted for
1563 outliers based on a 99% confidence interval.
1564 Fig. 4: Percent survival for Exiguobacterium sibircum with respect to time at
1565 Mars simulated conditions are shown for A) strain 7-3 and B) strain 255-15; for
1566 UV, dark and control samples. Percent survival in UV-exposed samples at burial
1567 depths above and below 3 mm regolith shown are shown in C) and D) for strain 7-
1568 3 and strain 255-15, respectively. The 40+ day sampling represents a “caching”
1569 of sample that was held in the dark at -80 °C for several weeks to simulate long
1570 term storage at Mars-like temperatures * denotes a statistically significant P-
1571 value
1572 Fig. 5: Arthrobacter psychrolactophilis survival after exposure to simulated
1573 Martian surface conditions. A) Top, B) middle and C) bottom centimeter profiles
1574 showing percent survival for UV, dark and control samples; error bars represent
1575 standard deviation for the experiment from triplicate sample analysis. Best of fit
1576 (R2) values represent a linear regression to the data assuming a first order rate
1577 constant. D) Fluorescent imaging of Arthrobacter cells taken from day 40
79
1578 controls; the log phase morphology indicates either an active metabolism or the
1579 locking of cells in rod morphology upon desiccation. Scale bars represent 1 μm.
1580 Fig. 6: Percent survival for Cryptococcus sp. NP33-C in UV, dark, and control
1581 samples from initial concentrations and at 20-day sampling intervals; survival
1582 rates are based on concentrations at day 0 sampling (inset). Best of fit (R2) values
1583 for a first order linear regression are shown for each sample set.
1584 Fig. 7: A) Melanized wild type and B) non-melanized mutant wdpks1Δ-1
1585 Wangiella dermatitidis. Percent survival for C) wild-type Wangiella and D)
1586 mutant wdpks1Δ-1 after exposure to the simulated Mars conditions. The 80+-day
1587 sample represents survival in long-term sample “caches” that were stored in the
1588 dark at -80 °C. Survival rates for Wangiella and the non-melanized mutant with
1589 respect to 1 centimeter depth profiles in UV-irradiated samples are shown in E)
1590 and F), respectively. Error bars represent standard deviation from triplicate
1591 sample analysis. The differences between the top and bottom layers were
1592 significant at 10, (p=0.001), 20, (p=0.023), 30 (p=0.005), and 40 (p=0.065) days.
1593 Fig. 8: A) SEM of healthy nematode population showing overall cell size and
1594 morphology; nematode sample was critically point-dried to prevent collapsing
1595 during analysis. Scale bar represents 100 μm. B) SEM of nematode sample after
1596 exposure to the Mars analog regolith; sample was not critically point-dried due
1597 the presence of the I-MARS regolith. Inoculation within the regolith analog
80
1598 proved fatal for nematode species due to severe laceration of the soft-bodied
1599 worms by sharp basaltic fines of the regolith. Scale bar represents 20 μm.
1600 Table 1: Chemical formula, sampling location, and weight percent of individual
1601 mineral and bulk rock constituents added to the I-MARS regolith analog to
1602 simulate the mineralogy of the global Martian dust. Bulk basalt was sterilized for
1603 12 hours at 400 °C; individual minerals and chemicals were sterilized for 3 hours
1604 at 400 °C prior to homogenization.
1605 Table 2: Environmental parameters for low-latitude, low elevation surface of
1606 Mars as reported by Squyres, 2004 and Nair, 1994 compared to those utilized by
1607 the TechShot Mars Simulation Chamber during the 40 days of experimental
1608 conditions. Reported ranges are based on maximum and minimum recorded
1609 values taken at 5 minute intervals during the 40 days of experiment.
1610 Table 3: Bulk regolith survival and extrapolated half-lives, in days, for amino
1611 acids and organisms after exposure to 40 days of simulated Martian surface
1612 conditions; data is reported based on percent survival from initial sampling at day
1613 0 of the experiment. Error in percent survival represents deviations from the
1614 average based on triplicate sample analysis; errors in half-lives are a calculated
1615 based on the with respect to the maximum deviation in rate constant k (days-1)
1616 based on the experimental error for each sample.
81
1617 Table 4: Half-lives, in days, for amino acids and select organisms with respect to
1618 burial depth in the UV-exposed I-MARS regolith sample after 40 days of
1619 simulated Martian conditions. Organisms shown showed a statistical difference
1620 in percent survival and half-life based on burial depth in the regolith; organisms
1621 not displayed either were untested at different depth profiles or showed no
1622 variation with respect to depth in the regolith. Errors in half-lives are a calculated
1623 based on the with respect to the maximum deviation in rate constant k (days-1)
1624 based on the experimental error for each sample
82
Figure 1 Click here to download High Resolution Copy of Figure: Johnson_Figure 1.tif Figure 2 Click here to download High Resolution Copy of Figure: Johnson_Figure 2.tif Figure 3 Click here to download High Resolution Copy of Figure: Johnson_Figure 3.tif Figure 4 Click here to download High Resolution Copy of Figure: Johnson_Figure 4.tif Figure 5 Click here to download High Resolution Copy of Figure: Johnson_Figure 5.tif Figure 6 Click here to download High Resolution Copy of Figure: Johnson_Figure 6.tif Figure 7 Click here to download High Resolution Copy of Figure: Johnson_Figure 7.tif Figure 8 Click here to download High Resolution Copy of Figure: Johnson_Figure 8.tif Table 1 Click here to download high resolution image Table 2 Click here to download high resolution image Table 3 Click here to download high resolution image Table 4 Click here to download high resolution image