NASA uses a variety of methods to measure, control, and reduce spacecraft microbial contamination for planetary protection purposes. Compliance with planetary protection is mandatory for solar system missions, as stipulated in the NASA Policy Directive (NPD) 8020.7G. Many of the implementation practices used to achieve compliance are described below, and are further detailed in the NASA Procedural Requirements (NPR) 8020.12.
In order to define and communicate the mission-specific planetary protection requirements, a set of documents must be reviewed and approved by the NASA Planetary Protection Officer (PPO). For all missions, this initially involves a letter to the NASA PPO requesting categorization of the mission (Categories I-V). Assignment to Category I relieves the mission of all further documentation requirements. For Categories II-V, several additional documents are required using a submission timeline that correlates with the pre- and post-launch phases of mission.
The required documents for Category II-V missions include a Planetary Protection Plan that describes how a planetary flight project will meet its planetary protection requirements; a Pre-Launch Report that specifically describes the practices used to achieve and verify compliance; a Post-Launch Report that provides the final analysis of the planetary protection practices, such as the measurements of impact probabilities, contamination probabilities, and spacecraft bioburden; an Extended Mission Report (as needed) that details the new timeline and/or purpose of the proposed extended mission, along with any new planetary protection constraints; and an End-of-Mission Report that provides a comprehensive analysis and assessment of the final disposition and/or decommissioning of the spacecraft. For missions involving strict orbital requirements and/or bioburden control, additional documents such as the Implementation Plan, which provides step-wise descriptions of the practices used to achieve compliance, and the Contamination Analysis Plan, Microbiology Plan, and Microbial Reduction Plan may be required (each of these Plans are subsidiary to the Planetary Protection Plan and precede the Pre-Launch Report). For all Category V ‘restricted’ sample return missions, several documents that detail the safeguards for protecting Earth are also required. More information regarding the format of each of these documents is provided in NPR 8020.12 (Section 2.7 of NPR 8020.12D) and can be obtained from the NASA PPO.
Clean Rooms and Microbial Barriers
The use of cleanroom facilities is a significant component to controlling the bioburden and contamination of spacecraft. Through the use of laminar flow systems and advanced filtration systems, cleanroom environments control factors such as airborne particle density (particles/cm3 at specific particle sizes), temperature, humidity, and pressure. As indicated in NPR 8012.12 (Appendix D of NPR 8012.12D), all Category II, III, and IV missions shall assemble and maintain spacecraft and payloads in Class 100,000 (or ISO class 8) cleanrooms in the operational mode. Control of the particulate levels is also facilitated through proper garmenting procedures, which are determined on a mission-specific basis, and typically include hoods, masks, surgical gloves, booties, and protective suits (commonly referred to as bunny suits).
For reference purposes, ISO class 8 (Class 100,000) cleanrooms maintain particle densities of 3,520,000 particles/cm3 (or 100,000 particles/ft3) for particles ≥ 0.5 µm in size; whereas ISO class 5 (Class 100) cleanrooms maintain densities of 3520 particles/cm3 (100 particles/ft3) for particles ≥ 0.5 µm in size, and densities of 100,000 particles/cm3 (2840 particles/ft3) for particles ≥ 0.1 µm in size.1
Cleaning and Sterilization
For many missions, it is essential to control and reduce the bioburden (and organic contaminants) of cleanroom facilities and spacecraft. Methods used to control particulate levels, which then tends to reduce microbial contamination, include the cleaning of surfaces and floors with compatible organic solvents (e.g., wiping with ethanol or isopropanol) and cleanroom-appropriate detergents, respectively. However, when more stringent bioburden levels are required, spacecraft and components are subjected to well-established sterilization procedures. Currently, the NASA-approved sterilization modalities include dry heat microbial reduction (DHMR) and vapor phase hydrogen peroxide (VHP). The Mars Viking landers were subjected to DHMR at the sub-system and full-system levels, with the Viking-1 lander (as a terminal sterilization step) being heated at 111.7 ˚C (233.1 ˚F) for 30.23 h at a specified humidity of 1.3 mg/mL. The quantitative impacts of the Viking-based treatments, on the surface, encapsulated, and mated materials, serve as the basis for today’s Category IVa-c bioburden constraints. The DHMR specifications were recently expanded beyond the original Viking specifications, allowing for an increase in the degree of bioburden reduction from 4 to 6-orders of magnitude with a greater range of acceptable humidity control.2 NASA recently certified VHP as a suitable means to reduce the bioburden on exposed surfaces (at the terminal or component levels); however, as a chemical sterilant, VHP does not impact the enclosed areas of the spacecraft or the mated and encapsulated materials. Several other modalities have been used at the sub-system or component level, with relevant examples including autoclaving for tubing and cleanroom materials, gamma radiation for the sterilization of the parachute for the Beagle-2 Mars probe, and a low-temperature hydrogen peroxide plasma for the batteries and electronic assemblies of the Beagle-2 Mars probe. Together, these examples serve to demonstrate that the specific implementation procedures for reducing spacecraft bioburden are determined on a mission-specific and category-dependent basis, and are subject to approval by the NASA PPO.
The Office of Planetary Protection actively promotes research and development of new or improved methods, technologies, and procedures for spacecraft sterilization that are compatible with spacecraft materials and assemblies. More information regarding current research and future funding opportunities can be obtained from the NASA PPO.
Prevention of Recontamination
Preventing the recontamination of spacecraft and their components is a significant concern to planetary protection. Once cleaned or sterilized, spacecraft and spacecraft components are typically packaged or protected in ways that prevent recontamination from other sections of the spacecraft or from the surrounding environment (e.g., the cleanroom or atmospheric conditions of the launch pad). Example recontamination prevention strategies include draping, deployable biobarriers, filtration systems that reduce particulate transport, overpressure approaches, and packaging/assembly techniques. For the Mars Phoenix lander, a material known as Tedlar® was to used protect the robotic arm from recontamination and was only retracted once, just before the arm was deployed for the trenching and sampling on Mars. During the differing assembly phases for the Mars Curiosity rover, components of the assembled spacecraft were protected from recontamination using strategies including draping in antistatic materials and packaging in clean storage bags or containers. For the Mars Pathfinder lander, recontamination from the launch pad was minimized by temporarily covering the depressurization vent on the aeroshell of the spacecraft (the aeroshell covered the lander during the launch and voyage to Mars). These examples are provided to demonstrate that the specific procedures used to prevent recontamination are dependent upon mission needs, as well as the differing phases of the assembly, testing, and launch operations. Further information on preventing recontamination can be obtained from the NASA PPO.
Bioburden Assay Methods
Reliable measurement of the bioburden, or abundance of microorganisms, on spacecraft, spacecraft components, and within cleanroom facilities is essential to understanding the potential for contaminating solar system bodies, and for demonstrating planetary protection compliance. Among the methods most commonly used to measure spacecraft-associated bioburden are the NASA Standard Assay, the Total Adenosine Triphosphate (ATP) assay, and the Limulus Amebocyte Lysate (LAL) assay.
The NASA Standard Assay provides the enumeration of cultivable heat-tolerant microorganisms; where the survivors of this assay, typically dominated by spore-formers such as Bacilli, are used as a proxy for total bioburden. The NASA Standard Assay method derives from the Viking project, for which ~20 people conducted more than 6000 assays, averaging a minimum of 1000 assays on each landed spacecraft. In contrast, for the Mars Science Laboratory, 7 people or fewer collected more than 47,997 samples. In general, the procedures for the NASA Standard Assay include sampling of the surface by swab or wipe methods, heat shock of the extracted samples at 80°C for 15 min, followed by plating of the samples onto Trypticase Soy Agar, and enumeration of the colony forming units after incubation at 32˚C for 72 h. For simplicity, the term ‘spores’ (for Mars missions, as per Section 5.3 of NPR 8020.12D) refers to the aerobic microorganisms that survive a heat shock of 353 K (80°C) for 15 minutes and are cultured on Trypticase Soy Agar at 305 K (32°C) for 72 hours.
The ATP and LAL assays are molecular-based approaches that measure the respective abundances of ATP and lipopolysaccharides, which are biochemicals common to microorganisms. The biochemical fuel, ATP, is a component in the metabolism of all living cells, although it can also be found in dead cells. The ATP assay was pioneered by the food industry to assess bacterial contamination, and is based upon the bioluminescence of the luciferase reaction3, which can be measured and quantitated with a portable luminometer, thus yielding results within approximately one hour. The level of bioluminescence is directly proportional to the amount of ATP in the sample; however, because the amount of ATP per cell varies based on metabolic state (ranging from trace amounts for spores, to high amounts for yeast), direct correlation between the amount of ATP and the total cell number is often not feasible. This means that the Total ATP assay provides a good threshold of biological contamination, but is not quantitative for viable organisms.4
As originally developed for the pharmaceutical industry, the LAL assay provides a proxy for bacterial abundance through quantification of lipopolysaccharides (endotoxins), which are lipids found in high abundance in the outer membrane of Gram-negative bacteria.5 However, due to an additional sensitivity towards (1,3)-β-D-glucans, found in fungal cell walls, the LAL assay can also measure the presence of yeast and mold. For these reasons, the LAL assay can be used as a proxy for spacecraft bioburden.6 The LAL assay utilizes extracts from blood cells (amebocytes) of the horseshoe crab Limulus polyphemus, which contain a proteolytic enzyme cascade that is sensitive to lipopolysaccharides and ‘LAL reactive materials’ such as β-D-glucans (or glucose polysaccharides).7 The cascade is initiated by binding of the lipopolysaccharides or β-D-glucans to enzymes within the extract (Factor C and G, respectively7) and ultimately detected through proteolysis of a chromogenic substrate. Among the considerations when evaluating the LAL assay are the high sensitivity and rapid processing times (15-30 minutes), as well as the reactivity with the lipopolysaccharides from live, dead, and non-cultivable organisms, and β-D-glucans from non-microbial sources. Like the Total ATP assay, the LAL assay is not quantitative for viable cells.
The Office of Planetary Protection is actively engaged in advancing and streamlining the technologies for bioburden measurement. Among the issues of interest to planetary protection are life detection in returned samples, the identification of live versus dead cells when using genetic methods, the detection of cultivable and non-cultivable microorganisms, and the development of new rapid and cost-effective bioburden measurement methods. Example methods currently under consideration include meta-genomics, advanced genomic sequencing, and fluorescent cell sorting; with advanced physical methods, micro-culturing, alternative biomolecular detection methods, and other molecular microbiological techniques having potential future applications. More information regarding current research and future funding opportunities can be obtained from the NASA PPO.
Preventing Impacts and Contamination of Solar System Bodies
All solar systems missions are subject to constraints designed to minimize the unintentional impacts of Mars and, when applicable, the contamination of icy satellites such as Europa, Enceladus, and Ganymede. Practices that ensure low probabilities of impact include trajectory corrections, trajectory biasing, and accurate mathematical modeling and statistical analyses of the flight plan, injection maneuvers, and orbital dynamics. For missions that may encounter icy satellites, the calculation of the probability of contamination for a liquid water body is required. These calculations for the probability of contamination include (but are not limited to) factors such as (A) an estimation of bioburden at launch; (B) survival of contaminating organisms during the cruise phase; (C ) survival of contaminating organisms in the radiation environment adjacent to the target; (D) probability of encountering or landing on target; (E) probability of surviving landing or impact on the target; (D) mechanisms and timescales of transport to the subsurface; and (F) survival of contaminating organisms before, during, and after subsurface transfer. More information on these constraints and the methods needed to achieve compliance can be obtained from the NASA PPO, or are stipulated in NPR 8012.12.
- (A) ISO 14644-1: Cleanrooms and associated controlled environments — Part 1: Classification of air cleanliness by particle concentration. (B) ISO 14644-2: Cleanrooms and associated controlled environments — Part 2: Specifications for monitoring and periodic testing to prove continued compliance with ISO 14644-1.
- Frick, A. F., Mogul, R., Stabekis, P., Conley, C. A., Ehrenfreund, P. Overview of Current Capabilities and Research and Technology Developments for Planetary Protection. Adv. Space Res. 54, 221-240 (2014).
- Lundin, A. Use of firefly luciferase in ATP-related assays of biomass, enzymes, and metabolites. Methods Enzymol. 305, 346-370 (2000).
- Venkateswaran, K., Hattori, N., La Duc, M. T. & Kern, R. ATP as a biomarker of viable microorganisms in clean-room facilities. J. Microbiol. Methods 52, 367-377 (2003).
- Guidance for Industry Pyrogen and Endotoxins Testing: Questions and Answers; Federal Drug Administration, 2012
- (A) Morris, H. C., Monaco, L. A., Steele, A. & Wainwright, N. Setting a standard: the limulus amebocyte lysate assay and the assessment of microbial contamination on spacecraft surfaces. Astrobiology 10, 845-852 (2010). (B) Obayashi, T. et al. Plasma (1→3)-β-D-glucan measurement in diagnosis of invasive deep mycosis and fungal febrile episodes. Lancet 345, 17-20 (1995). (C ) Foto, M et al. Modification of the Limulus amebocyte lysate assay for the analysis of glucan in indoor environments. Anal. Bioanal. Chem. 379, 156-162 (2004).
- (A) Magalhães, Pérola O., et al. Methods of endotoxin removal from biological preparations: a review. J. Pharm. Pharm. Sci. 10, 388-404 (2007). (B) Roslansky, P.F. & Novitsky, T. J. Sensitivity of Limulus amebocyte lysate (LAL) to LAL-reactive glucans. J. Clin. Microbiol. 29, 2477-2483 (1991).
- Iwanaga, S. The limulus clotting reaction. Curr. Opin. Immunol. 5, 74-82 (1993).