New detection methods reinforce protocols for planetary protection

01/01/2008

NASA planetary protection programs charged with keeping biocontamination out of space.

By Patrick Hogue, Johns Hopkins University, Applied Physics Laboratory

National Aeronautics and Space Administration (NASA) missions to solar system bodies with the potential to sustain life, or that could potentially contain life in a fundamental evolutionary state, have stringent requirements on the maximum spore count permissible on spacecraft surfaces; and these levels are likely to become lower as cleanroom protocols become more efficient. Several promising technologies can help contractors reduce spore counts to acceptable levels, provide for the rapid determination of microorganisms, and determine the genomic inventory of spacecraft microorganisms.

Establishing planetary protection policy

The need for planetary protection, and protection of Earth by sample return missions, was recognized at the dawn of the Space Age through the Committee on Space Research (COSPAR).¹ Article IX of the Outer Space Treaty of 1967 states, in part, “...parties to the Treaty shall pursue studies of outer space including the Moon and other celestial bodies, and conduct exploration of them so as to avoid their harmful contamination and also adverse changes in the environment of the Earth resulting from the introduction of extraterrestrial matter and, where necessary, shall adopt appropriate measures for this purpose.”²

NASA Policy Directive (NPD) 8020.7 establishes NASA policy for implementing planetary protection (PP), which includes protection of planetary bodies for future exploration and of Earth from extraterrestrial sources of contamination. Depending upon the target, implementation may range from obtaining a letter from NASA approving mission design as adequate planetary protection (e.g., New Horizons Pluto fly-by, category II) all the way to full implementation (e.g., Mars sample return, category V). For samples returned from a solar system body with the potential for life (e.g., Europa) mission design must “break the chain of contact”; furthermore, “for un-sterilized samples returned to Earth, a program of life detection and biohazard testing or a proven sterilization process shall be undertaken as an absolute precondition for the controlled distribution of any portion of the sample.”³ Depending upon the nature of samples returned to Earth quarantine up to Biosafety Level (BSL) 4 may be required.

In reaching these decisions, NASA seeks the opinion of scientific advisory groups such as the Space Studies Board of the National Academy of Sciences. NASA Procedural Requirement (NPR) 8020.12 delineates a uniform set of planetary protection requirements for all NASA robotic extraterrestrial missions and references NPR 5340.1, which provides a uniform set of procedures for performing microbial assays for enumerating bioburden levels of spacecraft and facilities used to assemble, test, and launch spacecraft with planetary protection requirements (it is written primarily for use by microbiologists). This year, NPR 5340.1 will re-release as NASA HDBK 6022, which will not set requirements but will list approved protocols. The two Viking Landers, which received dry heat sterilization at 125°C, are considered the gold standard for planetary protection.

Detailed microbial burden requirements

NPR 8020.12 allows alternatives to 125°C dry heat sterilization provided that procedures and quality controls are approved by the NASA Planetary Protection Officer (PPO). These methods are then spelled out in the approved PP plan. Flight hardware drawings may call for these unique microbial reduction methods by citing their specification numbers. Microbial barriers may be used to prevent recontamination of previously sterilized areas; a pressure of at least 1,244 Pa (5 inches H₂O) is considered satisfactory to prevent the entry of microorganisms. High efficiency particulate air (HEPA) filters (99.97 percent efficient for 0.3 µm) are also considered effective microbial barriers. NASA requires that spacecraft assembly occur in a cleanroom meeting ISO class 8 (Fed. Std. 209E Class 100,000) as a minimum.

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For a Mars Lander mission, the maximum spore count is 300,000 for the entire spacecraft (or <300 bacterial spores/m²); all other targets still have a probabalistic requirement. The quantity of 300,000 spores per total spacecraft surface area applies to non-special regions of Mars (most of the surface); the total allowed, including organisms inside hardware (e.g., encapsulated in potting) is 500,000. The Vikings, and spacecraft accessing “special regions” or looking for life, met/must meet 300,000 and then reduce the surface bioburden by 10,000 with dry heat sterilization–meaning no more than 30 viable spores total on the surface. Alternatives to dry heat are discussed later. Based upon Viking experience, NPR 8020.12 assumes spore content as shown in the table. Possible reduction methods are noted and also will be explained in another section.

Assessing microorganism levels

Most aerospace cleanrooms have unknown microbial deposition rates and surface microbial populations and usually do not have a microbiology laboratory readily available. While a proper microbiology laboratory is being created to implement NPR 5340.1, PP programs may choose to get a head start in making their cleanrooms as aseptic as possible. To this end an interim laboratory can be constructed, using a Class 100 (ISO class 5) flow bench with table-top incubator. Initial assays of the cleanrooms (including thermal vacuum chambers, acoustic, and vibration facilities) and associated equipment can be performed using commercially available settling plates (to capture microbial fall-out) and contact plates (to assay cleanroom surfaces) based on Tryptic soy agar (TSA). These procedures are designed primarily for the detection and enumeration of heterotrophic, mesophilic, aerobic, and anaerobic microorganisms; consult NPR 5340.1 for details. Microorganisms likely to survive space and planetary environments are halophiles, certain Bacillus species and extremophiles.

It is recommended that these initial assessment techniques, and others that may be recommended by local microbiology or pharmaceutical laboratory suppliers, be included in the PP plan and that they, along with the full NPR 5340.1 implementation plan, be approved by the NASA PP officer.

During this initial phase of assessing cleanrooms for fall-out and “hot spots,” a portable aerosol particle counter may be used to scan HEPA filter outlets for leaks and any repairs or replacements made before the start-up of PP program work. A rotary centrifugal impactor equipped with TSA strips can be used to validate the biological effectiveness of each HEPA outlet in the overall cleanroom. An alternative would be to place one settling plate for each HEPA outlet at a distance of approximately 1 m; of course, timely retrieval and incubation are essential.

If the trial-and-error methods described previously are not satisfactory for characterizing the cleanroom, then recourse can be made to real-time microbial particle detection technology. Most of these are based upon the fluorescence of energetic metabolic chemicals (e.g., adenosine triphosphate, or ATP, riboflavin) induced by ultraviolet light^4,5, although immunoaffinity-based phosphorescent sensors are being developed for detection of bacterial spores.⁶ Recently individual bacterial cells have been detected using Raman spectroscopy.⁷

Spot-checks of surfaces can be accomplished using commercially available portable systems based on the luciferin-luciferase reaction produced in the common firefly. These methods usually consist of reagent-dampened sterile swabs that lyse (chemically open) living cells and then react with ATP, the energy currency of living cells, to produce a brightly colored species that can be quantitatively measured with a luminometer. This kind of spot-check was recently performed on the Space Station surfaces using a NASA-developed variant called LOCAD-PTS, which is expected to become an approved protocol in NASA HDBK 6022 (see Fig. 1).⁸

Rapid microbial detection using filter-based media with ATP bioluminescence enumeration by a CCD camera equipped with a microchannel plate photomultiplier can reduce assay times from 72 hours to 5 hours or less as demonstrated by NASA/JPL.⁹ A recent addition to this technology is the use of a 16-base RNA probe that can be used to identify specific organisms after the non-specific fluorescence enumeration measurement is accomplished.¹⁰

Implementation of NPR 5340.1

The cost of a fully equipped microbiology laboratory to support a spacecraft program with full PP requirements–a Mars Lander, for example–is estimated to be $50,000 and will require at least two technicians, preferably three, to provide 24/7 coverage.¹¹ The cost associated with PP implementation is estimated to have added 2 months to an 18-month assembly, test, and launch operations program for Mars Polar Lander (MPL) and required 1,200 assays.¹² Viking program PP cost was 10 percent or about $7 million.¹³ A successful PP implementation hinges on the following:

1. Careful and thorough integration of PP into all aspects of the program
2. Management buy-in and support of the PP engineer
3. Universal personnel training (including purchasing and support groups)
4. Pre-planned bio-assay database that parallels the assembly flow
5. Lessons learned from the MPL program:
   • Daily change of cleanroom garments
   • Purchase of 200 new sets
   • Strict enforcement of aseptic techniques–a departure from the usual
   • Sterile, powder-free cleanroom gloves
   • Daily janitorial service (IPA used instead of ammonia for floors and walls)
   • Facility modifications to better isolate anteroom
   • Special cleaning and isolation techniques for vibration, acoustics, and thermal vacuum facilities
   • A standard assembly drawing note: “Perform bio-assay sampling prior to close-out”
   • Launch site processing facilities needed similar steps to maintain spacecraft biocleanliness; assessment of these facilities may occur early in the program
   • A portable clean tent was needed for the launch site
   • It is important to flow PP requirements to subcontractors–anticipate helping them understand and properly implement PP requirements for their hardware elements
   • Integration and test planning staff should strive to have parallel paths so that a delay associated with bio-assay could be accommodated by a shift in tasking
6. MPL lessons learned that were successfully applied to the Phoenix program:
   • Daily management briefing that includes PP lab lead
   • PP assay database must be robust and web-based for easy access
   • Program PP engineer should coordinate with JPL or NASA counterpart
   • Phoenix robotic arm (complex geometry) was sheathed in easily cleaned/verified biobarrier (it tears through this barrier when deployed)
   • Be prepared: NASA’s independent bio-assay testing might require re-cleaning or re-sterilization of spacecraft–could impact launch date

Reducing microorganism levels

Several technologies are available that have the potential to reduce airborne and surface bioburden levels.

Cleanrooms and other facilities used for PP hardware can be sterilized using commercially available fogging systems based on hydrogen peroxide (H₂O₂) or a combination of H₂O₂ and peracetic acid (CH₃COOOH); chlorine dioxide is also very effective. Compatibility of these chemicals with materials of construction should be verified with the supplier prior to use. Spore strips are used to determine the effectiveness of the kill. H₂O₂ fogging of spacecraft followed by immediate vacuum bake-out is one possible method of meeting the 10,000 spore reduction for probes accessing “special” regions or that search for life.

Various sterilization techniques exist for air streams: free radicals with scrubber; glow discharge; non-thermal corona discharge; ultraviolet light to lyse organisms (254 nm) and oxidize residue (185 nm); water impingement to mechanically entrain particles; electrostatic precipitation; biocidal filters; and ozone infusion.

Figure 1. Astronaut Suni Williams, Expedition 14/15 flight engineer, works with the Lab-on-a-Chip Application Development???Portable Test System (LOCAD-PTS). Williams is placing the sample mixed with water from the swabbing unit into the LOCAD-PTS cartridge. Photos courtesy of NASA.8Click here to enlarge image

Cold plasma has been shown to destroy microorganisms and their component proteins, including misfolded proteins called prions.^14,15

Daily janitorial service with proven antiseptic solutions (e.g., 10 percent IPA in deionized [DI] water) can also be effective in reducing surface bioburden levels.

A word of caution is in order regarding DI water systems: Their internal wetted surfaces are almost always coated with biofilm. Sterilization, and verification of effectiveness, may be performed prior to the start of a PP program.

Genomic inventory of spacecraft

A widely known but unfortunate fact is that between 0.1 percent and 10 percent of viable microorganisms grow when cultured on standard agar media. Identifying and cataloging what microbes survive sterilization is important in interpreting results of life detection missions to other planets. If similar microbes turn up in alien samples, researchers could disregard the results as contamination and not evidence of extraterrestrial life.

A recent paper describes the first of its kind sampling of three spacecraft assembly cleanrooms (JPL, KSC, and JSC) using gene sequencing technology; despite the highly desiccated, nutrient-bare conditions within these cleanrooms, a broad diversity of bacteria was detected, covering all the main bacterial phyla.¹⁶ Ribosomal RNA sequence analysis allowed the researchers to identify a greater number and diversity of bacteria than previously detected in the rooms. Although a few microorganisms, like those common on human skin, were found at all three sites, the study discovered that each room had a bacterial community largely unique to itself. Many factors could be responsible for this diversity, the researchers said, including the differing types of air filters and cleaning agents used and the facilities’ different geographic locations. Current cleaning techniques kill most common microbes and the resulting lack of competition could contribute to the number and diversity of the durable survivors found by the genetic testing approach.

Figure 2. Genome Sequencer FLX from 454 Life Sciences with “hood up,” showing picotiterplate drawer and optical system aperture to left and nucleotide sequencing reagents and fluidics system to right. Image courtesy of APL/454 Life Sciences.Click here to enlarge image

Future PP missions are expected to include genomic inventory.¹⁷ A good primer on this subject, with links to automated sequencing suppliers, is given on the Wikipedia web site: http://en.wikipedia.org/wiki/Gene_sequencing. Figure 2 shows one such gene sequencer.

Conclusion

Missions with PP requirements demand careful and thorough integration of aseptic manufacturing into every aspect of training, manufacture, purchase, integration, test, and launch operations. Several technologies exist to reduce airborne and surface bioburden, cold plasma being one of the more promising. Rapid methods of microbial detection and enumeration are advancing; PP programs that want to use them may obtain NASA PP officer approval. In the not-too-distant future, genomic inventory is likely to become a PP mission requirement. Many of the protocols common to pharmaceutical cleanrooms are expected to become common in aerospace cleanrooms with PP requirements.

Patrick Hogue is senior member of the professional staff in the Applied Physics Laboratory at Johns Hopkins University in Maryland.

References

1. Astrobiology, Vol. 6, No. 5, 2006, “The Astrobiology Primer: An Outline of General Knowledge–Version 1,” 2006 section 7C.
2. Treaty on Principles Governing the Activities of States in the Exploration and Use of Outer Space, Including the Moon and Other Celestial Bodies (entered into force Oct. 10, 1967).
3. NPR 8020.12C, Appendix A, paragraph 3.2.
4. V. Agranovski, Z. Ristovski, “Real-time Monitoring of Viable Bioaerosols: Capability of the UVAPS to Predict the Amount of Individual Microorganisms in Aerosol Particles,” J. Aerosol Sci., Vol. 36, 2005.
5. C. Bolotin, “Instantaneous Microbial Detection” (white paper), BioVigilant, Tucson, AZ, 2006.
6. P. Scholl et al., “Immunoaffinity Based Phosphorescent Sensor Platform for the Detection of Bacterial Spores,” Proc. SPIE, Vol. 3913, 2006.
7. J. Popp, “Identification of Micro-organisms by Raman Spectroscopy,” Proc. SPIE Biomedical Imaging, 2007.
8. Space.com, “Astronaut Suni Williams, Expedition 14/15 flight engineer, works with the Lab-on-a-Chip Application Development-Portable Test System (LOCAD-PTS, Charles River Laboratories, Charleston, SC),” June 26, 2007.
9. F. Chen et al., “A Rapid Micro-Detection System for the Enumeration of Bacterial Endospores,” IEEEAC, Paper 1501, Version 2, updated Nov. 28, 2005.
10. Private communication with W. Miller at Millipore Corp. re: “Milliprobe.”
11. Private communication with A. Baker of Technical Administrative Services, during the 2007 NASA PP Policy and Practices Workshop, Santa Cruz, CA.
12. As presented by D. Tisdale, Lockheed Martin Corp., during session 10 of the 2007 NASA PP Policy and Practices Workshop, Santa Cruz, CA.
13. As presented by P. Stabekis, Windmere Consulting, during session 10 of the 2007 NASA PP Policy and Practices Workshop, Santa Cruz, CA.
14. “Plasma Sterilization,” The Futurist, p. 17, September/October 2003.
15. Q. Yu et al., “Sterilization Effects of Atmospheric Cold Plasma Brush,” Appl. Phys. Lett., 88, 013903, 2006.
16. C. Moissl et al., “Molecular Bacterial Community Analysis of Cleanrooms where Spacecraft are Assembled,” FEMS Microbiology Ecology, 61, pp. 509???521, 2007.
17. As presented by Dr. Catherine Conley, acting NASA PP Officer, during the 2007 NASA PP Policy and Practices Workshop, Santa Cruz, CA.