Spacecraft Assembly Cleanrooms
Cleanrooms are environments with controlled levels of contaminants used for assembling sensitive equipment and compounds including pharmaceuticals, electronics and spacecraft. Since objects that leave Earth’s atmosphere are assembled here, contamination of these objects with terrestrial life could cause spread to extraterrestrial environments[9]. In preparation for the Viking expeditions (1967), guidelines were established by the United Nations to govern the exploration of space[9]. The Outer Space Treaty has a section about contamination of spacecraft, stating that all spacefaring nations must “… pursue studies of outer space … [and] conduct exploration … so as to avoid their [celestial bodies’] harmful contamination…”[11]. In accordance with this agreement, spacefaring nations undertake measures to ensure the decontamination of all rooms, personnel, and equipment that will directly or indirectly come into contact with extraterrestrial or extraterrestrial-bound objects.
Decontamination Procedures
Depending on the type of mission, different decontamination procedures and certification levels are required by NASA. The certification of cleanrooms is described by the number of particles in the air (see Table 1). In addition to air filtration, with high efficiency particulate air (HEPA) or ultra-low penetration air (ULTA) filters removing 99.97% or 99.999% of particulates ≥ 0.3 μm respectively, the temperature, humidity and pressure are controlled[8]; the cleanroom air is exchanged every 7 hours[3]. Spacecraft assembly cleanrooms range in certification from ISO 4 (Johnson Space Center Genesis Curation Laboratory [JSC]) to ISO 8 (Jet Propulsion Laboratory Spacecraft Assembly Facility [JPL]). Decontamination procedures include desiccation, UV irradiation, and cleaning with H2O2[6]. Cleaning includes vacuuming and mopping floors with disinfectants and replacing tacky mats at entrances; prevention of novel contamination is accomplished through the use of cleanroom certified suits[8]. Decontamination inhibits the growth of most microbes, but selects for resistant bacteria. Additionally, disinfection techniques used on spacecraft surfaces must be gentle so as not to damage sensitive equipment; only moderate levels of dry heat and certain chemical disinfectants can be used on these surfaces and these measures are not strong enough to kill off robust bacteria and most endospores [2].
Environments
Primary Environments
Due to the extensive decontamination procedures in cleanrooms, these environments are considered to be extreme. Organic matter is kept out of cleanrooms by these procedures, but a significant amount of human skin cells are shed and used as carbon and energy sources by heterotrophs; nonetheless, cleanrooms are oligotrophic environments [5][6][7][8]. Cleanrooms are kept at low humidity and moderately high temperatures, inhibiting the growth of many organisms[5][6]. Particles from the environment are present in cleanrooms, therefore the cleanrooms are representative of the surrounding area; JPL and Kennedy Space Centre (KSC) cleanrooms have desert and swamp microenvironments, respectively[7]. These microenvironments have anaerobic regions, but most inhabited surfaces are aerobic[2].
Secondary Environments
These environments, including Mars and the International Space Station (ISS), are similar to cleanrooms: oligotrophic, hot/cold, and low in moisture. However, many of these environments are anaerobic; Mars has only 0.13% O2 levels and is exposed to ultraviolet (UV) and gamma radiation[3]. Additionally, Mars has regions resembling permafrost biotopes on Earth; permafrost has shown the ability to support life[3].
Microbial Community
Numerous surveys of spacecraft assembly facilities have been performed and many thousands of species, including novel species, have been discovered in these environments. Depending on assay used (culture-dependent or -independent), different types of microbes were found to be dominant. In culture-independent assays, a near-equal mixture of Gram-positive and -negative bacteria, Actinobacteria, and fungi was found[5]. Culture-dependent assays, however, show a bias towards members of Bacillus, likely due to the techniques used to culture isolates, and underestimates the total microbial community[5]. Of all cultivable bacteria, 85% are gram positive (25% Bacillus licheniformis, 16% Bacillus subtilis) [7]. Though some bacteria are common throughout cleanrooms, each facility has its own flora: JPL has desert-like organisms and KSC has swamp-like organisms[4]. Cyanobacteria from the desert surrounding JPL have been found in their cleanrooms and are photosynthetic chemoautotrophs, an adaptation unique within cleanroom flora[2]. Extremophiles are common in cleanrooms; bacteria were found that thrived from pH 3 to pH 10.6, at NaCl concentrations of 20% w/v, at temperatures between 4°C and 65°C, and at gamma and UV radiation levels of 2500 J/m2 [4][6][7][9]. The density of microbes also varied with the area sampled; the floor of KSC is 1-2 orders of magnitude more densely populated than other surfaces[5].
Secondary environments have different microbial makeups; most isolates from the ISS are the result of human occupation: Staphylococcus aureus, Staphylococcus pasteuri, Propionibacterium acnes and Micrococcus luteus[1]. Anaerobic and opportunistic pathogens were also found, including Clostridium spp. In all surveys so far, no Archaea have been found[1].
Metabolic Processes
Since surveys of microbes in spacecraft-related facilities began in the 1980’s, most microbes found have been spore-formers[6]. This is due to the fact that many tests for microbes culture specifically spores[6], which are resistant to pressures present in cleanrooms; spores of Bacillus Odyssei are 100% resistant to desiccation, 26% resistant to 5% H2O2, 10% resistant to UV radiation at 660 J/m2, and 0.4% resistant to gamma radiation[4]. Many non-sporulating bacteria produce similar enzymes to spore-formers, such as catalase, but lack others, including cytochrome C oxidase and β-galactosidase[5][9][10]. Until recently, most assays for bacterial presence have been exclusively oxic, selecting for aerobes. Since anaerobic bacteria can now be cultured, many anaerobic, carbon-, and nitrogen-fixing microbes have been discovered[3]. Most bacteria cultivated have been chemoheterotrophs, though some chemolithotrophs (Variovorax sp. and Cupriavidus sp.) have been cultivated whose respiration include dehalorespiration and iron reduction[7].
Forward Contamination
Forward contamination of celestial objects is a major reason that cleanrooms are used for assembly of spacecraft. Extraterrestrial microbiologists stress that microorganisms are present on spacecraft that have been, and are being, sent to Mars: a pristine extraterrestrial system[5]. Since these organisms can withstand decontamination in cleanrooms, they can likely survive the conditions present on the way to, and on the surface of, Mars: oxidizing UV and gamma radiation, oligotrophic environments, and extreme temperatures[4]. While many assembly surfaces show no evidence of spores[6], other surfaces such as circuit boards[4] prove that bacteria are being transported off the planet. The environments of extraterrestrial bodies are harsh; 200 J/m2 of UV254nm are absorbed on the surface of Mars in 30-60 minutes[7], enough to kill most bacteria[2]. Bacillus pumilis SAFR-032, however, requires almost 2500 J/m2 for populations to decrease two orders of magnitude[7]. Now that anaerobic bacteria have been cultivated from cleanrooms, there is evidence that “pioneer” lithoautotrophic bacteria, such as Paenibacillus sp., could arrive on Mars and live off of gaseous CO2 and N2, providing organic material for future bacteria to survive[3]. In addition, there is evidence that there have been natural exchanges of organic material between Earth and Mars through meteorites; if Earth-like organisms are found on Mars that are similar to those found in cleanrooms, doubt about the microbes’ origins could cause the findings to be discredited[7].
References
[1] Castro, V.A., Thrasher, A.N., Healy, M., Ott, C.M., and Pierson, D.L. 2004. Microbial characterization during the early habitation of the international space station. Microb. Ecol. 47: 119-126.
[2] Crawford, R.L. 2005. Microbial diversity and its relationship to planetary protection. Appl. Envrion. Microbiol. 71: 4162-4168.
[3] Stieglmeier, M., Wirth, R., Kminek, G., and Moissl-Eichinger, C. 2009. Cultivation of anaerobic and facultatively anaerobic bacteria from spacecraft-associated clean rooms. Appl. Environ. Microbiol. 75: 3484-3491.
[4] La Duc, M.T., Satomi, M., and Venkateswaran, K. 2004. Bacillus odyssey sp. nov., a round-spore-forming bacillus isolated from the Mars Odyssey spacecraft. Int. J. Syst. Evol. Microbiol. 54: 195-201.
[5] La Duc, M.T., Kern, R., and Venkateswaran, K. 2004. Microbial monitoring of spacecraft and associated environments. Microb. Ecol. 47: 150-158.
[6] La Duc, M.T., Dekas, A., Osman, S., Moissl, C., Newcombe, D., and Venkateswaran, K. 2007. Isolation and characterization of bacteria capable of tolerating the extreme conditions of clean room envrionments. Appl. Envrion. Microbiol. 73: 2600-2611.
[7] Link, L., Sawyer, J., Venkatesaran, K., and Nicholson, W. 2004. Extreme spore UV resistance of Bacillus pumilus isolates obtained from an untraclean spacecraft assembly facility. Microb. Ecol. 47: 159-163.
[8] Newcombe, D.A., La Duc, M.T., Vaishampayan, P., and Venkateswaran K. 2008. Impact of assembly, testing and launch operations on the airborne bacterial diversity within a spacecraft assembly facility clean-room. Astrobiology. 7: 223-236.
[9] Newcombe, D., Dekas, A., Mayilraj, S., and Venkateswaren, K. 2009. Bacillus canaveralius sp. nov., an alkali-tolerant bacterium isolated from a spacecraft assembly facility. Int. J. Syst. Evol. Microbiol. 59: 2015-2019.
[10] Vaishampayan, P., Probst, A., Krishnamurthi, S., Ghosh, S., Shariff, O., McDowall, A., Ruckmani, A., Mayilraj, S., and Venkateswaran, K. 2010. Bacillus horneckiae sp. nov., isolated from a spacecraft-assembly clean room. Int. J. Syst. Evol. Microbiol. 60: 1031-1037.
[11] UN Treaty. 1967. Treaty on the principles governing the activities of states in the exploration and use of outer space, including the moon and other celestial bodies. Article IX, UN Doc. A/RES/222/(XXI), TIAS No. 6347.
