1. The Harsh Mars Surface Environment may Mitigate Against the Forward Contamination of Mars During Robotic and Human Missions Andrew C. Schuerger, Ph.D. Dept. of Plant Pathology, University of Florida Space Life Sciences Lab, Kennedy Space Center, FL Andrew C. Schuerger, Ph.D. Dept. of Plant Pathology, University of Florida Space Life Sciences Lab, Kennedy Space Center, FL

2. Environmental parameterEarthMars Solar constant1368 W m -2 590 W m -2 Solar UV irradiationλ ≥ 290 nmλ > 190 nm UVC + UVB flux at surface≈ 2 W m -2 8.6 W m -2 (tau = 0.3) 0.37 W m -2 (tau = 3.5) Solar particle events (SPE)noneup to 0.1 Sv/h Galactic cosmic radiation (GCR)1-2 mSv/yr0.1-0.2 Sv/yr Mean atmospheric pressure1013 mb (10 5 Pa)7.1 mb (710 Pa) Temperature range–50 to 45 o C–90 to –10 o C (Viking data) –90 to +20 o C (S. hemisphere) Atmosphere78.1 % N 2 95.3 % CO 2 20.9 % O 2 2.7 % N 2 0.0375 % CO 2 1.6% Ar variable % H 2 O0.1% O 2 UV-Glow discharge from blowing dustminimal effectlikely and prevalent Nightglow [N + + O – = NO] yields UVminimal effectprevalent Solar UV-induced volatile reactants [O 2 –, O –, H 2 O 2, O 3, etc.] minimal effectprevalent Globally distributed oxidizing soilnoprevalent Adapted from Applebaum and Flood (1990), Buhler and Calle (2003), Cockell and Andrady (1999), Horneck et al. (2001,2003), Kuhn and Atreya (1979), Owen (1992), and Schuerger et al. (2003, 2004).

3. Microbial Bioload at Launch is Finite: Limited Species Diversity and Biomass. Microbial Bioload at Launch is Finite: Limited Species Diversity and Biomass. Spore-formers ≈ 10% total biomass (range: 1-34%; Dillon et al., 1973). Culturable species are typically human-associated, airborne, and soil borne microbes. Non-culturable species are present but poorly defined at the moment. Average bioload launched: 3.0 x 10 5 spore-formers; + ≈ 1 decade higher non- spore forming species; + ≈ 2 decades higher non-culturable species. Spore-formers ≈ 10% total biomass (range: 1-34%; Dillon et al., 1973). Culturable species are typically human-associated, airborne, and soil borne microbes. Non-culturable species are present but poorly defined at the moment. Average bioload launched: 3.0 x 10 5 spore-formers; + ≈ 1 decade higher non- spore forming species; + ≈ 2 decades higher non-culturable species.

4. Effects of Vacuum on Survival of Endospores of Bacillus subtilis. Spirit at Gusev Crater

5. Mars Electrostatic Chamber (MEC), KSC. Schuerger et al., 2003, Icarus 165:253-276. Mars Simulation Chamber (MSC), KSC.

6. Effects of UV dosage on the Survival of Bacillus subtilis HA101 under Mars-Normal UV and Earth-Normal Environmental Conditions. Schuerger, Newcombe, Venkateswaran 2005, Icarus, submitted. Rapid inactivation kinetics under equatorial Mars UV simulations.

7. Survival of Bacteria on Sun-Exposed Spacecraft Surfaces. Lander Pad 3; Viking 1 Spirit Lander

8. Growth of Seven Bacillus spp. under Martian Conditions. Pressure: down to 15 mb Temp: 30, 20, 15, 10, & 5 C Gases: CO 2 vs ppO 2 /ppN 2 Pressure: down to 15 mb Temp: 30, 20, 15, 10, & 5 C Gases: CO 2 vs ppO 2 /ppN 2 Pressure: down to 0.1 mb Temp: -100 to +200 C (programmable) Gases: CO 2 ; O 2 /N 2 ; Mars mix (top 5 gases) UV-VIS-NIR: equatorial to polar fluence rates Dust loading from tau 0.1 to 3.5 Pressure: down to 0.1 mb Temp: -100 to +200 C (programmable) Gases: CO 2 ; O 2 /N 2 ; Mars mix (top 5 gases) UV-VIS-NIR: equatorial to polar fluence rates Dust loading from tau 0.1 to 3.5

9. Effects of Temperature on Growth of Vegetative Cells 7 Bacillus spp. Bacillus species30 o C20 o C15 o C10 o C5 o C B. pumilus (SAFR-032)430.950 + B. pumilus (FO-36B)42.80.650 + B. subtilis (HA-101)42.40.50 + B. subtilis (42HS-1)430.450 + B. megaterium (KL-197)42.81.10 + B. nealsonii (FO-092)41.60 + B. licheniformis (KL-196)41.10 + Bacillus species30 o C20 o C15 o C10 o C5 o C B. pumilus (SAFR-032)0.60.070 + B. pumilus (FO-36B)0.550 + B. subtilis (HA-101)10.070 + B. subtilis (42HS-1)0.750.090 + B. megaterium (KL-197)0.20.050 + B. nealsonii (FO-092)1.30.090 + B. licheniformis (KL-196)1.40.170 + Rating scale: 4 = large robust colonies > 5 mm in diameter; 3 = colonies 2-4 mm in diameter; 2 = colonies ≈ 1 mm in diameter; 1 = colonies ≈ 0.5 mm in diameter; 0.50 = colonies < 0.5 mm in diameter; 0.1 = smallest visually discernable growth; 0 = no growth ; 0 = no growth (n = 5). O 2 /N 2 atmosphere CO 2 atmosphere 48 hrs at 1013 mb

10. Effects of Pressure on Vegetative Cells of 7 Bacillus spp. O 2 /N 2 atmosphere Bacillus species1013 mb100 mb50 mb35 mb25 mb B. pumilus (SAFR-032)442.510.1 B. pumilus (FO-36B)442.50.50.1 B. subtilis (HA-101)43.52.210.1 B. subtilis (42HS-1)43.22.10.50.1 B. megaterium (KL-197)4320.30.1 B. nealsonii (FO-092)43.7311 B. licheniformis (KL-196)43.5311 CO 2 atmosphere Bacillus species1013 mb100 mb50 mb35 mb25 mb B. pumilus (SAFR-032)0.60.290.070.170.05 B. pumilus (FO-36B)0.50.070.050.120.02 B. subtilis (HA-101)10.90.850.350.2 B. subtilis (42HS-1)0.550.10.020.20.12 B. megaterium (KL-197)0.60.140.050.20 B. nealsonii (FO-092)1.61.71.81.40.75 B. licheniformis (KL-196)1.31.21.31.20.6 Rating scale: 4 = large robust colonies > 5 mm in diameter; 3 = colonies 2-4 mm in diameter; 2 = colonies ≈ 1 mm in diameter; 1 = colonies ≈ 0.5 mm in diameter; 0.50 = colonies < 0.5 mm in diameter; 0.1 = smallest visually discernable growth; 0 = no growth ; 0 = no growth (n = 5). 48 hrs at 30 C

11. Effects of Pressure on Germination and Growth of Endospores 7 Bacillus spp. O 2 /N 2 atmosphere Bacillus species1013 mb100 mb50 mb35 mb25 mb B. pumilus (SAFR-032)431.80 + B. pumilus (FO-36B)431.80 + B. subtilis (HA-101)43.32.30.50 + B. subtilis (42HS-1)4320 + B. megaterium (KL-197)43.72.30 + B. nealsonii (FO-092)4430 + B. licheniformis (KL-196)4430 + CO 2 atmosphere Bacillus species1013 mb100 mb50 mb35 mb25 mb B. pumilus (SAFR-032)0 + B. pumilus (FO-36B)0 + B. subtilis (HA-101)0.50 + B. subtilis (42HS-1)0.030 + B. megaterium (KL-197)0 + B. nealsonii (FO-092)1.91.3 10 + B. licheniformis (KL-196)110.80.70 + Rating scale: 4 = large robust colonies > 5 mm in diameter; 3 = colonies 2-4 mm in diameter; 2 = colonies ≈ 1 mm in diameter; 1 = colonies ≈ 0.5 mm in diameter; 0.50 = colonies < 0.5 mm in diameter; 0.1 = smallest visually discernable growth; 0 = no growth ; 0 = no growth (n = 3). 48 hrs at 30 C

12. Effects of Pressure on Growth of 6 Non-Spore Forming Species. O 2 /N 2 atmosphere CO 2 atmosphere Rating scale: 4 = large robust colonies > 5 mm in diameter; 3 = colonies 2-4 mm in diameter; 2 = colonies ≈ 1 mm in diameter; 1 = colonies ≈ 0.5 mm in diameter; 0.50 = colonies < 0.5 mm in diameter; 0.1 = smallest visually discernable growth; 0 = no growth ; 0 = no growth (n = 3). 48 hrs at 30 C BacteriaO 2 /N 2 1013 mb100 mb25 mb Escherichia coli (K12)3.53.00 + Streptomyces coelicolor (A3)2.82.00 + Deinococcus radiodurans (R1)2.52.00 + Acinetobacter caloaceticus (50V1)3.32.00 + Comamonas acidovorans (30V3)3.52.80 + Clavibacter michiganensis (27V1B)3.52.30 + Bacillus subtilis (HA101)4.03.30 + CO 2 1013 mb100 mb25 mb Escherichia coli (K12) 1.20.90.5 Streptomyces coelicolor (A3)0 + Deinococcus radiodurans (R1)0 + Acinetobacter caloaceticus (50V1)0 + Comamonas acidovorans (30V3)0 + Clavibacter michiganensis (27V1B)0 + Bacillus subtilis (HA101) 0.60.40 +

13. Mars Base-1: 2030+ Courtesy of Carter Emmart.

14. Conclusions  Spacecraft are assembled under strict conditions which constrain bioloads at launch to low numbers of species and low total biomass.  During cruise phase to Mars, spore-forming species are reduced about 1 order of magnitude; non-spore formers likely 2-3 orders of magnitude.  Once on Mars, UV irradiation rapidly reduces bioloads on sun-exposed surfaces by up to 6 orders of magnitude within a few tens of minutes to a few hours.  And for those lucky microbes to survive on UV shielded surfaces, the synergistic effects of low pressure, low temperature, and CO 2 atmospheres (+ other biocidal factors) create significant hurdles over which common spacecraft contaminants must cope with for continued survival and growth on Mars.  Spacecraft are assembled under strict conditions which constrain bioloads at launch to low numbers of species and low total biomass.  During cruise phase to Mars, spore-forming species are reduced about 1 order of magnitude; non-spore formers likely 2-3 orders of magnitude.  Once on Mars, UV irradiation rapidly reduces bioloads on sun-exposed surfaces by up to 6 orders of magnitude within a few tens of minutes to a few hours.  And for those lucky microbes to survive on UV shielded surfaces, the synergistic effects of low pressure, low temperature, and CO 2 atmospheres (+ other biocidal factors) create significant hurdles over which common spacecraft contaminants must cope with for continued survival and growth on Mars.