5
The Sterilization of Samples from Mars

The array of instrumentation and the number of personnel needed to carry out a definitive study of the Mars samples are both very large. It is not practical to construct a quarantine facility large enough to house them. It is essential that a means be found to safely remove aliquots of the samples from the quarantine facility and distribute them to qualified members of the international scientific community, so that the full complement of modern scientific instrumentation and individual talents can be brought to bear on the analysis and interpretation of the samples. The prospect of using the large and very sophisticated laboratory instruments available throughout the world for this purpose was the main justification for returning samples to Earth rather than trying to study Mars materials in situ on that planet. Even if life detection were the only scientific issue that needed to be addressed, not all of the required life-detection work could be carried out in the quarantine facility. Moreover, research beyond life detection cannot be ignored: Geochemical studies of the samples will be very important, to provide a sample context for the scientists searching for life, and also to expand current knowledge of the geologic history of Mars and allow a more informed choice of landing sites for subsequent sample-return missions.

COMPLEX recommends sterilization1 of a subset of the samples as a means of safely transferring them into the laboratories of the international scientific community. Commonly employed techniques of sterilization are reviewed in Table 5.1. The techniques that appear to be best suited for this application are gamma irradiation and dry-heat sterilization. Sterilization techniques that involve gases and liquids were not chosen because they lack the ability to penetrate to the centers of the samples. Electron-beam irradiation was excluded both because of its limited penetration and the size and expense of the equipment necessary. Both gamma-ray sterilization and dry heat have the advantages that they penetrate to the centers of samples, and they can be implemented on a relatively modest scale.

DAMAGE TO SCIENTIFIC INFORMATION IN SAMPLES AS A RESULT OF THEIR STERILIZATION

The sterilization techniques listed in Table 5.1 degrade samples to varying degrees, causing a loss of the scientific information in them. Some scientific studies are little affected by heat (less than 150 ºC) or gamma-ray

1  

Throughout this report, COMPLEX uses the words “sterilized” and “sterilization” as being synonymous with treatment by heat and/or gamma radiation to such a level as to kill any known terrestrial organism.



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The Quarantine and Certification of Martian Samples 5 The Sterilization of Samples from Mars The array of instrumentation and the number of personnel needed to carry out a definitive study of the Mars samples are both very large. It is not practical to construct a quarantine facility large enough to house them. It is essential that a means be found to safely remove aliquots of the samples from the quarantine facility and distribute them to qualified members of the international scientific community, so that the full complement of modern scientific instrumentation and individual talents can be brought to bear on the analysis and interpretation of the samples. The prospect of using the large and very sophisticated laboratory instruments available throughout the world for this purpose was the main justification for returning samples to Earth rather than trying to study Mars materials in situ on that planet. Even if life detection were the only scientific issue that needed to be addressed, not all of the required life-detection work could be carried out in the quarantine facility. Moreover, research beyond life detection cannot be ignored: Geochemical studies of the samples will be very important, to provide a sample context for the scientists searching for life, and also to expand current knowledge of the geologic history of Mars and allow a more informed choice of landing sites for subsequent sample-return missions. COMPLEX recommends sterilization1 of a subset of the samples as a means of safely transferring them into the laboratories of the international scientific community. Commonly employed techniques of sterilization are reviewed in Table 5.1. The techniques that appear to be best suited for this application are gamma irradiation and dry-heat sterilization. Sterilization techniques that involve gases and liquids were not chosen because they lack the ability to penetrate to the centers of the samples. Electron-beam irradiation was excluded both because of its limited penetration and the size and expense of the equipment necessary. Both gamma-ray sterilization and dry heat have the advantages that they penetrate to the centers of samples, and they can be implemented on a relatively modest scale. DAMAGE TO SCIENTIFIC INFORMATION IN SAMPLES AS A RESULT OF THEIR STERILIZATION The sterilization techniques listed in Table 5.1 degrade samples to varying degrees, causing a loss of the scientific information in them. Some scientific studies are little affected by heat (less than 150 ºC) or gamma-ray 1   Throughout this report, COMPLEX uses the words “sterilized” and “sterilization” as being synonymous with treatment by heat and/or gamma radiation to such a level as to kill any known terrestrial organism.

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The Quarantine and Certification of Martian Samples TABLE 5.1 Potential Sterilization Techniques     Applicability   Conditions Surface Interior Problems Heat   Drya 135 ºC, 24 h Yes Yes Alters organics, volatilizes Wet/steam 125 ºC, 24 h Yes Yes Alters organics, volatilizes Radiation   Gamma (60Co) >1 Mrad Yes Yes Alters organics Electron beam — Yes Limited Large facility; untestedb Alkylating chemicals   Formaldehyde Liquid, 80 ºC Yes No Residual organics Ethylene oxide Vapor, 60 ºC Yes No Residual organics Oxidizing chemicals   Hydrogen peroxide Vapor, 50 ºC Yes No Some residuals? Untestedb Chlorine dioxide Gas, 50 ºC Yes No Some residuals? Untestedb Ozone Gas, 50 ºC Yes No Some residuals? Untestedb Peracetic acid Liquid, 50 ºC Yes No Some residuals? Untestedb Hydrogen peroxide/plasma 50 ºC Yes No Some residuals? Untestedb Mixed chemical/plasma 50 ºC Yes No Some residuals? Untestedb aMethod used to reduce bioload on the Viking spacecraft, 1976. bUntested on rock-soil-microbial mixes. irradiation. These include most of the inorganic (geochemical, isotopic, and mineralogic) analyses. An exception to this is studies of volatile components such as adsorbed water. Water and other volatile components will certainly be lost by heat sterilization. Probably the most serious compromises occur for the organic compounds in the samples, which is unfortunate because these include the biomarkers that researchers hope will yield information about life processes on Mars. Many of the organic compounds are very labile and are destroyed by heat or radiation. Table 5.2 lists the compounds that are of interest and indicates current understanding of the effect of different types of sterilization on them. As can be seen, many of the compounds will either be destroyed by the process or at least compromised to the point that the analyses may be hard to interpret. It is also apparent that there is much uncertainty about the extent of damage caused to organic compounds by sterilizing procedures. One mitigating circumstance is that many of the biomarker compounds can be extracted by thermal desorption and supercritical fluids within the quarantine facility and, pending verification that the procedure effectively sterilizes the extracts produced, the latter can be distributed to laboratories for studies without further treatment (Figure 4.1; see also “The Study of Extracts” in Chapter 4). Recommendation. A program of research should be initiated to determine the effects on organic compounds in rocky matrices, and also on microscopic morphological evidence of life, of varying degrees of application of heat and gamma irradiation. This research should be started well in advance of the return of the Mars samples, so that treatment protocols can be designed intelligently and data obtained from analyses of treated samples can be interpreted with minimal ambiguity. INTENSITY OF STERILIZATION The question of intensity of sterilization is crucial, but the choice of actual parameters is an issue of implementation and is beyond the scope of this report. Qualitatively, very vigorous sterilization measures should be used, as

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The Quarantine and Certification of Martian Samples TABLE 5.2 Effect of Sterilization Treatments on Biomarker Compounds (and Kerogens)   Heating in Dry Inert Atmosphere 60Co γ-irradiation   Compound Class 12 h at 125 ºC 3 d at 125 ºC 7 d at 150 ºC 0.3 Mrad 300 Mrad Extractabilitya Desorption Thermal Amino acids   √   Decompositionb √ √ √ √ X   Racemizationc √ √ √ √ X Sugars Xd Xd Xd X? X? √ e Nucleotides √? √? X? X X √ e Unsaturated lipids √? √? √? ? ?   √ Aliphatic hydrocarbons √ √ √ X? X? √ Tetrapyrroles X? X X ? ? e Carotenoids X? X X ? ? e Geobiopolymers (kerogen) √ √ √ √? √? f NOTE: √, survives; X, destroyed. aCapacity for being extracted, at least partially, by washing the sample with polar solvents. bStable (?) at 150 ºC under wet conditions. cRacemization is the destruction of chirality: numbers of right- and left-handed molecules are randomized. Under wet conditions, racemization occurs rapidly at 125 ºC. dDehydrated to form secondary products. eCharacteristic secondary products released. fPresence detectable by release of simple products at high temperatures. it will be difficult to assess the effectiveness of any more modest sterilization conditions. For the case of dry heat sterilization, one plausible standard is that commonly used in the surgical suites of hospitals. This is a stringent standard because the tools sterilized have come from the hospital environment, a very dirty environment with respect to microbial contamination, and after sterilization they will be inserted into patients with weakened immune systems. The typical conditions used in a hospital steam autoclave are 121 ºC for 15 minutes or 134 ºC for 4 to 5 minutes. The contact time may be lengthened for substantial loads. For dry heat sterilization the temperature and contact times vary from 170 ºC for 1 hour to 140 ºC for 3 hours. A more conservative treatment may be considered necessary for the martian samples. Gamma irradiation devices small enough to be incorporated in a quarantine facility are obtainable (Figure 5.1). Gamma irradiation is sometimes used to eliminate specific classes of organisms from foodstuffs, although total sterilization is not attempted because the doses needed can make food rancid and unpalatable. Table 5.3 lists some useful and/or critical gamma irradiation doses and their effects. However, the target organisms in food sterilization are relatively vulnerable to the effects of ionizing radiation, and the possibility must be entertained that organisms with much greater resistance may have survived on Mars. Attention has been drawn to the terrestrial bacterium Deinococcus radiodurans (Figure 5.2), which is in this category. Comparison of Figure 5.3 with Table 5.3 shows that D. radiodurans is vastly more resistant to radiation than are other familiar organisms. The case has been made that if life has formed and survived on Mars, it is probably in the form of species that have the same exceptional ability to repair genetic damage that D. radiodurans has (see Appendix A). Note that the radiation dose required to kill D. radiodurans is sufficient to kill viruses (Table 5.3). One factor that should temper speculation about organisms able to survive radiation sterilization is that if they are present on or near the surface of Mars (a necessary condition for them to be included in a returned sample), then similar material has already been transported to Earth in the form of martian meteorites (see Box 5.1). Mars

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The Quarantine and Certification of Martian Samples FIGURE 5.1 Example of a small gamma-irradiation sterilizer: the Gammacell 220 Excel research irradiator, marketed by MDS™ Nordion™, Kanata, Ont., Canada. The unit is 2.1 m (7 ft) tall and weighs 4,000 kg (8,818 lb). SOURCE: Figure courtesy of MDS™ Nordion™. TABLE 5.3 Some Gamma-ray Irradiation Doses and Their Effects or Applications Dose (Mrad) Effect or Application 0.0003-0.0004 50% killing of human beings exposed and not given medical treatmenta 0.005 Will promptly kill human beings by central nervous system damagea 0.005-0.015 Used to inhibit sprouting of white potatoesb 0.02-0.05 Used for control of mold in wheat flourb 0.02 90% killingc of Salmonella typhimurium (potentially pathogenic bacterium)d 0.024-0.031 90% killing of Escherichia coli O157:H7 (a pathogenic bacterium)e 0.04-0.08 90% killing of Salmonella typhimuriumf 0.03-0.1 Used to control Trichinella spiralis (parasites) in porkb 0.05 90% killing of a typical yeast or fungusd 0.07 90% killing of Salmonella typhimuriumg 0.08 90% killing of a typical growing bacterial cultureh 0.1 Used to control insects and increase shelf life of fruits and vegetablesb 0.1-0.3 Equivalent to 10,000 years in space

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The Quarantine and Certification of Martian Samples Dose (Mrad) Effect or Application 0.1-0.5 90% killing of the insects in a populationd 0.15 90% inactivation of Ebola virusi 0.15-0.3 Used to reduce bacterial pathogens (especially Salmonella) in poultryb 0.2-2 Used for control of Salmonella in animal feed and pet foodj 0.24-0.33 90% killing of Gram-positive anaerobic spore-forming bacteriad 0.33 90% inactivation of Polio virusk 0.37 90% killing of the cells in a deep-frozen bacterial culture 0.45 Used to reduce bacterial pathogens in red meatb 0.53 90% inactivation of hoof-and-mouth disease virusl 1.04 90% killing of Deinococcus radiodurans cells (one of the most radiation-tolerant organism known)h 1.07 90% inactivation of minute virus of micel 2–5 90% inactivation of the molecules of a typical enzymed 2.5 Inactivation of HIV-I by a factor of 10–5 to 10–6 m,n 2.5 Dose recommended by International Atomic Energy Agency for the sterilization of medical products 3.0 Sufficient to destroy HIV-I DNA as assayed by PCRo 3.0 Used to kill insects and decontaminate dry herbs and spicesb 4.4 Used to sterilize meat frozen and packaged for NASAp NOTE: Some of the values given can serve only as guidelines, since the values for the radiation sensitivity of specific organisms vary significantly in the literature (see van Gerwen, S.J., Rombouts, F.M., van’t Riet, K., and Zwietering, M.H. 1999. A data analysis of the irradiation parameter D10 for bacteria and spores under various conditions. J. Food Prot. 62:1024-1032). For example, it is very commonly stated and published that Deinococcus radiodurans has “100% survival at 1.5 Mrad,” yet published values for 90% killing range from 0.5 to 1 Mrad. aSee <http://www.jlab.org/div_dept/train/rad_guide/effects.html>. bSee <http://www.cdc.gov/ncidod/dbmd/diseaseinfo/foodirradiation.htm>. cThe dose at which 90% of the individuals in a population are killed is called D10 (dose of 10% survival). dSee <http://www.agen.ufl.edu/~chyn/age4660/lect/lect_27/radiatio.htm>. But see also f and g. eClavero, M.R., Monk, J.D., Beuchat, L.R., Doyle, M.P., and Brackett, R.E. 1994. Inactivation of Escherichia coli O157:H7, salmonellae, and Campylobacter jejuni in raw ground beef by gamma irradiation. Appl. Environ. Microbiol. 60:2069-2075. fU.S. Food and Drug Administration. 1997. Irradiation in the production, processing and handling of food. Federal Register 62(232): 64107-64121. But see also d and g. gSee <http://ans.neep.wisc.edu/~ans/meetings/98-99/mathews.html>. But see also d and f. hvan Gerwen, S.J., Rombouts, F.M., van’t Riet, K., and Zwietering, M.H. 1999. A data analysis of the irradiation parameter D10 for bacteria and spores under various conditions. J. Food Prot. 62:1024-1032. iElliott, L.H., McCormick, J.B., and Johnson, K.M. 1982. Inactivation of Lassa, Marburg, and Ebola viruses by gamma irradiation. J. Clin. Microbiol. 16:704-708. jSee <http://www.foodsafety.org/ga/ga022.htm>. kKaupert, N., Burgi, E., and Scolaro, L. 1999. Inactivation of poliovirus by gamma irradiation of wastewater sludges. Rev. Argent. Microbiol. 31:49-52. lHouse, C., House, J.A., and Yedloutschnig, R.J. 1990. Inactivation of viral agents in bovine serum by gamma irradiation. Can. J. Microbiol. 36:737-740. mSalai, M., Vonsover, A., Pritch, M., von Versen, R., and Horoszowski, H. 1997. Human immunodeficiency virus (HIV) inactivation of banked bone by gamma irradiation. Ann. Transplant. 2:55-56. nHiemstra, H., Tersmette, M., Vos, A.H., Over, J., van Berkel, M.P., and de Bree, H. 1991. Inactivation of human immunodeficiency virus by gamma radiation and its effect on plasma and coagulation factors. Transfusion 31:32-39. oFideler, B.M., Vangsness, C.T., Jr., Moore, T., Li, Z., and Rasheed, S. 1994. Effects of gamma irradiation on the human immunodeficiency virus. A study in frozen human bone-patellar ligament-bone grafts obtained from infected cadavera. J. Bone Jt. Surg., Am. 76:1032-1035. pSee <http://www.wisc.edu/fri/foodirrd.htm>.

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The Quarantine and Certification of Martian Samples FIGURE 5.2 A pair of Deinococcus radiodurans cells, in the act of dividing into a tetrad. Transmission electron microscope image of a cross section of the cells, approximately 3 micrometers wide. SOURCE: Image courtesy of M.C. Henk and J.R. Battista. FIGURE 5.3 “Killing curve” for Deinococcus radiodurans in a state of desiccation, when it is most resistant to gamma radiation. Colonies of D. radiodurans were dried on glass plates at 5% relative humidity for a week, irradiated in the dry state, and then rehydrated and cultured. Exposure to 17.5 Mrad of radiation left no detectable viable organisms. SOURCE: Unpublished data of J. Battista, Louisiana State University. See also Auda, H., and Emborg, C. 1973. Studies on postirradiation degradation in Micrococcus radiodurans, Strain RII5. Rad. Res. 5:273-280.

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The Quarantine and Certification of Martian Samples Box 5.1 Meteorites from Mars Most meteorites that fall to Earth are fragments of asteroids. However, about 0.5% of the meteorites that fall, the SNC category of meteorites, are pieces of Mars. The young radiometric ages of SNC meteorites require that they come from a planet large enough to retain some geologic activity, not from asteroids, which cooled and became inactive soon after they were formed. Mars as a source of the SNCs was confirmed when one of them was found to contain noble gases in the same proportions and with the same isotopic compositions as noble gases in the martian atmosphere (which had been analyzed by a Viking lander in 1976).1 Some of the debris from cratering impacts on the surface of Mars, derived from a range of depths in the planet, is launched into space. The heliocentric orbits of the debris fragments evolve with time because of gravitational perturbations and random collisions they experience. Some eventually become Earth-crossing orbits, and the fragments in these orbits may fall to Earth as SNC meteorites.2 The abundances of cosmic-ray-produced isotopes in known SNC meteorites show that they wandered in space, absorbing cosmic rays, for 0.5 to 15 million years between the time they were broken out of Mars and the time when they fell to Earth.3 1   Bogard, D.D., and Johnson, P. 1983. Martian gases in an Antarctic meteorite? Science 221:651-654. 2   Gladman, B.J., Burns, J.A., Duncan, M., Lee, P., and Levinson, H.F. 1996. The exchange of impact ejecta between Terrestrial Planets. Science 271:1387-1392. 3   Eugster, O., Weigel, A., and Polnau, E. 1997. Ejection times of Martian meteorites. Geochim. Cosmochim. Acta 61:2749-2757. meteorites that were 0.5 to 15 million years in transit to Earth (Box 5.1) have absorbed 10 to 300 Mrad of cosmic radiation.2 The high end of this range of doses almost certainly would be adequate to kill any organisms they might contain. However, the low end of the range, 10 Mrad, is not enough to kill some known strains of terrestrial organisms (see Figure 5.3). And the SNC meteorites that have been collected and studied represent only a tiny fraction of the Mars material that has been transported to Earth; some of this material is certain to have come to Earth within less than 0.5 million years, and it received a smaller radiation dose than 10 Mrad. (Indeed, orbital simulations show that some martian ejecta immediately enter Earth-crossing orbits, and a portion of that almost certainly is delivered to Earth in less than a few years.3) If this material contained organisms sufficiently resistant to radiation to survive the relatively small dose they received, then Earth already has been infected by those organisms.4 In practice the sterilization dose used should be the minimum dose that will kill everything known, with some extrapolation factor for the possibility of unknown, more resistant, life forms. The key question is the size of the extrapolation factor. Before D. radiodurans was discovered, the most radiation-resistant organisms known were about a factor of 10 less resistant than it is. Thus extrapolation of a sterilizing irradiation dose by a factor of 2, or even 5, from that adequate to kill the previous record holder might easily have led to a dose that would not effectively sterilize a sample containing D. radiodurans. (Radiation resistance of D. radiodurans appears to have been a consequence of adaptation to conditions of severe dessication.5) However, in spite of previous bad 2   Clark, B.C. 2001. Planetary interchange of bioactive material: Probability factors and implications. Origins of Life and Evolution of the Biosphere 31:185-197. 3   Gladman, B. 1997. Destination: Earth. Martian meteorite delivery. Icarus 130:228-246. 4   Gladman, B., and Burns, J.A. 1996. Mars meteorite transfer: Simulation. Science 274:161-162. 5   Mattimore, V., and Battista, J.R. 1996. Radioresistance of Deinococcus radiodurans: Functions necessary to survive ionizing radiation are also necessary to survive prolonged desiccation. J. Bacteriol. 177:5232.

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The Quarantine and Certification of Martian Samples extrapolations, the strategy is sound. There must be a dose at which sufficient chemical damage is done to a cell for recovery to be impossible. VERIFICATION OF STERILIZATION The verification and certification that a sample does not constitute a biohazard is a difficult issue. Just as there are substantial limitations on researchers’ ability to detect life (Chapter 2), there are corresponding limitations on their ability to verify that sterilization has been successful. In essence, it is not logical to expect to verify that an organism has been killed if researchers are incapable of growing it. However, it is possible to certify that samples have been effectively sterilized even without the detection of life and subsequent killing. Three cases can be considered: (1) abundant life is detected before sterilization, (2) little life is detected before sterilization, and (3) no life is detected before sterilization. However, it will be seen that in many respects, all samples will need to be treated as in the third case. If abundant viable life is detected (“viable” meaning than it can be cultivated, or at least it can perform reactions that require the input of metabolic energy), then preliminary experiments can be performed to produce a “killing curve,” quantifying the efficiency of killing as a function of the sterilization dose (see, for example, Figure 5.3). Extrapolation of the killing curve to doses for which substantially less than one survivor would be expected in the total sample provides a lower bound on the sterilizing dose. (In practice, it is more conservative to assume that the sample might contain an undetected more resistant organism, and that the actual dose needed will be greater than this. However, it is important to demonstrate that the treatment really is killing all the types of organisms known to exist in the sample.) As part of verifying the sterilization of a given subsample, a small portion is tested after sterilization to verify that the organisms initially present are no longer viable. The test aliquot can be removed after sterilization, or it can be removed before sterilization, packaged in the same manner as the balance of the sample, and sterilized simultaneously in the same device. This test is only meaningful if the organisms in the presterilized samples are sufficiently abundant that the test sample is expected to have a large number of viable organisms prior to sterilization, so killing them makes an observable difference. However, if life is not very abundant in the sample, a small subsample might lack life simply because of sampling statistics, not as a result of the sterilization treatment. In this case, measuring loss of life is impractical. The same would be true if no life at all were detected to begin with. In these cases, and in the first case as well, it is appropriate to assume that the sample contains some small amount of an undetected, sterilization-resistant organism at the outset. Then the main issue is devising a model for the organism that is “realistic.” For example, scientists know the thermal tolerance of many organisms and do not know of any that comes close to surviving sterilization at 150 ºC for several hours. Similarly, Deinococcus radiodurans is the most radiation-resistant organism known, and researchers know what dose is required to kill a particular number of cells with a given confidence. Whatever sterilization regime is chosen for the samples, it is necessary to physically demonstrate that it does kill the most resistant organisms known. So if we want 99.99% confidence that we can kill all of the 108 D. radiodurans cells in a Mars sample (the expected number of surviving cells is less than 10–4 per 108 input cells, which is equivalent to <10–12 per input cell), we should demonstrate that there are no surviving cells out of the ~1012 cells initially present in an irradiated test sample. The preceding section in this chapter argued that if there are Mars organisms sufficiently robust to survive a realistic sterilization treatment in the quarantine facility, then some of these resistant organisms also would have survived transit to Earth in meteorites, and our planet already has been infected by them. Thus sample certification as “effectively sterilized” is appropriately based on verifying that the treatment used kills the most resistant known terrestrial organisms, and that the treatment is at least as harsh as that experienced by recent meteorites in Mars to Earth transit. Being substantially harsher than this will not be necessary.