Implementing Planetary Protection Measures on the Mars Science Laboratory

Abstract

The Mars Science Laboratory (MSL), comprising a cruise stage; an aeroshell; an entry, descent, and landing system; and the radioisotope thermoelectric generator–powered Curiosity rover, made history with its unprecedented sky crane landing on Mars on August 6, 2012. The mission's primary science objective has been to explore the area surrounding Gale Crater and assess its habitability for past life. Because microbial contamination could profoundly impact the integrity of the mission and compliance with international treaty was required, planetary protection measures were implemented on MSL hardware to verify that bioburden levels complied with NASA regulations. By applying the proper antimicrobial countermeasures throughout all phases of assembly, the total bacterial endospore burden of MSL at the time of launch was kept to 2.78×10⁵ spores, well within the required specification of less than 5.0×10⁵ spores. The total spore burden of the exposed surfaces of the landed MSL hardware was 5.64×10⁴, well below the allowed limit of 3.0×10⁵ spores. At the time of launch, the MSL spacecraft was burdened with an average of 22 spores/m², which included both planned landed and planned impacted hardware. Here, we report the results of a campaign to implement and verify planetary protection measures on the MSL flight system. Key Words: Planetary protection—Spore—Bioburden—MSL—Curiosity—Contamination—Mars. Astrobiology 14, 27–32.

1. Introduction

Because microbial contamination could potentially compromise the scientific integrity of extraterrestrial experiments, most missions to Mars must ensure that the number of contaminant bacterial endospores present on a spacecraft is kept to a minimum and that this bioburden level complies with maximum allowable requirements agreed upon internationally. To this end, planetary protection (PP) requirements for United States space missions are imposed by the National Aeronautics and Space Administration (NASA) in accordance with basic PP policy and international treaty (NASA, 2005). Such regulations are reviewed by the Committee on Space Research (COSPAR), an international body charged with the oversight of compliance with the treaty (COSPAR, 2011). From initial conceptualization, all spacecraft destined to land on Mars are subjected to continuous PP oversight and quality control measures. These include extensive bioassays that help verify that the level of bioburden associated with the spacecraft complies with NASA requirements for flight hardware destined to land on Mars (NASA, 2005). These regulations mandate that, at launch, “Spacecraft that do not meet impact avoidance constraints shall limit their total [surface-associated+mated (i.e., between adjoining surfaces)+encapsulated] bioburden level to≤5×10⁵ spores.” Furthermore, “PP Category IVa lander systems not carrying instruments for the investigations of extant martian life shall be restricted to a surface biological burden level of≤3×10⁵ spores in total and an average of≤300 spores per square meter of exposed external and internal spacecraft surfaces” (NASA, 2005).

The Mars Science Laboratory (MSL) launched on November 26, 2011, from the Cape Canaveral Air Force Station in Florida and landed flawlessly at Mars on August 6, 2012. Highlighting the MSL mission, the Curiosity rover continues to perform unprecedented scientific investigations in appraising the area surrounding the selected Gale Crater landing site as a potential habitat for extant or extinct life. The rover's suite of scientific instruments is gathering extensive data that pertain to the geochemistry and atmospheric conditions within Gale Crater. Since none of the instruments in its payload are intended to perform life-detection experiments, Curiosity's activities in searching for habitable regions at Gale Crater were ultimately classified a NASA Category IVa mission.

Perhaps contrary to logical assumption, the goal of PP is not sterilization but rather a reasonable assurance of the cleanliness of the spacecraft. All spacecraft destined to land on Mars are required to meet or exceed the cleanliness levels mentioned above. Thus, the entire MSL flight system was subjected to a diverse suite of microbial reduction regimens. Prior to final design, MSL engineers conducted analyses to demonstrate that components of the flight system had a low probability (<10⁻⁴) of inadvertently contaminating Mars. Prior to obtaining final approval for launch, the MSL project demonstrated compliance with all the requirements in the approved PP documents.

2. Materials and Methods

The general approach to achieving compliance with PP-imposed bioburden requirements for the MSL mission was straightforward. Facility personnel, engineers, and technicians went about meticulously cleaning the surfaces of the spacecraft hardware, ground support equipment, and clean room facilities following conventional clean room good housekeeping techniques. This included wiping surfaces with 70% isopropyl alcohol and mopping facility floors with mild detergents (e.g., Kleenol-30). The PP team then carried out a suite of bioburden reduction strategies (detailed below) and conducted an extensive bioassay-based cleanliness verification campaign. All the while necessary precautions were taken and countermeasures imposed to minimize the extent of biological and particulate contamination being shed from the facility onto MSL flight hardware. This included the double-bagging of components until deployment, capping off of air duct ends appropriately, placing hardware in proximity to air-conditioning outlets, and engineering airflow patterns to promote the movement of clean air from clean MSL hardware toward dirtier surfaces—and not the other way around. To further aid in the prevention of re-contamination, all MSL flight hardware was assembled, tested, and maintained under no less stringent conditions than those of ISO Class 8 clean room environments.

2.1. Bioburden reduction strategies

2.1.1. Dry heat microbial reduction (DHMR)

The MSL project took advantage of previously calculated surface-associated and embedded-bioburden DHMR specifications. In general, heat sterilization for surface-associated and embedded bioburden was achieved by subjecting hardware to temperatures at, or in excess of, 110°C for a minimum of 50 or 250 h, respectively (at a pressure of ≤1 torr). Other temperature/time/pressure combinations were utilized on a case-by-case basis as the NASA planetary protection officer approved them.

2.1.2. High-efficiency particulate air (HEPA) filtration

According to the standards of the United States Department of Energy, a HEPA filter must remove 99.97% of the particles larger than 0.3 microns in diameter as air passes through it. As such, bacterial endospores (∼1 micron in diameter) cannot pass through such a filter, which makes HEPA filtration very attractive to those interested in minimizing contamination when assembling and testing spacecraft hardware. An assortment of HEPA filters or other suitable devices (e.g.,0.2 μm pleated microfiber glass filters) were used to maintain low levels of airborne particulates in the spacecraft assembly clean rooms (SACs) in which MSL assembly, testing, and launch operations were carried out.

In addition, several HEPA filters were incorporated into the structure of the spacecraft to exempt the interior surfaces of particular structural modules from PP bioburden accounting. After having ensured that the structural joints and other penetrations were sealed, the HEPA-barricaded interior surfaces of the module were then omitted from PP bioburden accounting. Several elements on the rover utilized this approach. A collection of HEPA filters worked to ensure that the interior of the rover chassis, which was chock-full of cabling and critical circuitry, remained protected from particulate recontamination while simultaneously allowing for outgassing and depressurization. All the HEPA filters associated with the MSL project were successfully tested with a particle counter such as Met One (Grants Pass, OR).

2.2. Recontamination countermeasures

2.2.1. Transporting flight hardware

Transporting flight hardware increases the risk of biological recontamination because it threatens (A) the introduction of dirty hardware components into the controlled SAC environment and (B) the contamination of hardware during transport arising from improper containment, engineering, or logistical controls. To prevent recontamination during transportation from one SAC environment to another, hardware was housed in doubled-up Amerstat bags. In addition, once hardware was cleaned, it was protected from recontamination by bagging and/or taping over surface areas that were particularly challenging to clean (e.g., placing Kapton tape over the end caps of tubular structures). With respect to recontamination avoidance, the MSL project placed particular emphasis on the drill bits and rover wheels. The drill bits were cleaned, subjected to the DHMR process, and enclosed in the flight bit boxes to prevent recontamination prior to launch [with the exception of the one that was flown in its ready position (a project-requested and NASA PPO deviation)]. The rover wheel treads were covered and protected from recontamination after having been subjected to extensive DHMR.

2.2.2. Airflow and positioning of flight hardware

Proper airflow and placement of MSL hardware in the assembly facility clean rooms was also important. Throughout assembly, test, and launch operations (ATLO), necessary precautions were taken and countermeasures were imposed to minimize the extent of biological and particulate contamination transported from the assembly facilities onto the MSL spacecraft. This included the double-bagging of MSL hardware until deployment, capping off air duct ends appropriately, placing hardware in proximity to air-conditioning outlets, and engineering airflow patterns to promote the movement of clean air from clean MSL hardware toward dirtier surfaces (and not the other way around).

2.2.3. Certified SAC environments

Throughout the lifetime of the MSL project, all ATLO activities were performed in ISO Class 8 (or cleaner) clean room environments. These facilities were continually monitored and typically equipped with HEPA and/or ULPA filters and air-lock entryways to control the influx of particulates upon ingress and egress of personnel. Collectively, these safeguards worked to promote air quality at, or cleaner than, 100,000 particles≥0.5 microns in size per cubic foot.

2.2.4. Gowning practices

Throughout MSL ATLO, personnel adhered to strict gowning practices upon entering the clean room assembly facilities. Prior to entering the airlock to gain access to the clean room environment, all personnel were required to don a full clean room–certified bunny suit, clean room boots, facial masks, headgear, and clean room gloves taped to the sleeves of the bunny suits.

2.3. Sampling strategy and bioassays

Each and every piece of MSL hardware was the responsibility of a cognizant engineer (CogE). CogEs were responsible for completing hardware review and certification records (HRCR), which recorded the types of PP treatment received (e.g., DHMR) for each piece of hardware prior to its installation on the MSL spacecraft. PP personnel worked with CogEs in devising suitable periodicity and procedures for sampling the surfaces of MSL hardware during the course of ATLO.

The microbiological protocols and procedures used to conduct bioassays of surface-associated spore burden for MSL hardware were similar to those employed to assay spore abundance on missions from the early 1970s through the 2003 Mars Exploration Rovers (MER). The sample collection strategy included both sterile cotton swabs and polyester wipes, which were dampened with pre-sterilized distilled water, as has been described in detail previously (NASA, 2005; Kwan et al., 2011). Polyester wipes were used preferentially when sampling large, flat surfaces, which helped generate more meaningful data. In accordance with standard procedures detailed in NASA handbooks (NASA, 2005, 2010) and described in previous studies (La Duc et al., 2007; Cooper et al., 2011), microbiological spore assays typically consisted of sonication of the sample in sterile distilled water to remove the spores from the swab and wipes, heat shock (80°C, 15 min) to kill the vegetative cells and thus select for endospores, and finally aerobic incubation (32°C, 3 days) in trypticase soy agar (TSA). The number of colony-forming units (CFU) arising in and on TSA following incubation was interpreted to represent the number of intact, viable bacterial endospores present at the time of sample collection.

2.4. Management and statistical treatment of data

Data collected during the MSL PP campaign were recorded and managed in two master files. A Microsoft (MS) Excel file referred to as the Planetary Protection Equipment List (PPEL) was used to monitor spore burden and metadata (e.g., dimensions, material composition, cleaning practice) that pertain to each and every piece of MSL hardware—down to the resistor. The final version had approximately 4500 rows of data, one for each hardware item. In addition, an MS Access relational database file was used to archive and categorize all the information that pertains to each of the bioassays conducted. The data file was accessed through a second MS Access program, colloquially referred to as the “barcode program,” that was linked to the data file. This barcode program simplified the processing of samples and agar plates by providing organized barcode labels that were placed on the sample tubes and plates to identify them and properly record the results after CFU were enumerated at 24, 48, and 72 h. At each of these time points, the CFU tally corresponding to each agar plate was manually entered into the data file upon scanning the barcode label on the bottom of each plate.

The statistical methods applied to the analysis of MSL samples were greatly improved over those used for the MER analysis. Standard Gaussian and Poisson statistical analyses were invoked depending on the distribution of the raw data generated. Because each wipe typically covered a surface area of between 0.25 and 1.0 m² and each swab covered a mere 0.0025 m², a weighted statistical method was developed. In this approach, each swab was given a weight of one, while each wipe was treated as multiple swabs by weighting them according to the ratio of the wipe area divided by the swab area. A detailed description of this statistical methodology and its derivation have previously been reported (Beaudet, 2013).

In contrast to the practices of previous PP campaigns (e.g., MER), where all the analyses were conducted exclusively by exporting all the data into multiple Excel spreadsheets, the MSP PP team benefited from developing a novel MS Access “PPAnalysis” program that was written to compute the statistical analysis and generate necessary reports. Since the PPAnalysis program was directly linked to the original data file, results could be generated without exporting any data out of the data file. The program automatically calculated the bioburden densities and total bioburdens for each set of assays that could be grouped together. To be conservative, as in past missions, the average bioburden values plus three standard deviations, called the 3-sigma bioburden values, were what was reported. The 3-sigma bioburden values provided approximately a 99.9% confidence limit. The program also generated automatically the final assay tables that appeared in the MSL prelaunch report. The assays on all hardware items that had been exposed to a similar environment were grouped together in the statistical analysis to reduce the standard deviation and obtain a more representative value for the bioburdens.

2.5. Volumetric accounting of encapsulated bioburden

The bacterial endospores entrained within some spacecraft materials, such as plastics, carbon fibers, and paints, have been shown to contribute to the flight system's total bioburden (Stam et al., 2012). When no bioassays were performed to appraise the extent of encapsulated bioburden empirically, a NASA specification that presumes 30 spores/cm³ was applied to these materials (NASA, 2005). Thus, for every liter of paint used on the spacecraft, 3×10⁴ spores were factored into the total bioburden tally, unless bioassays could experimentally demonstrate scarcer spore densities. When pursuing the experimental route, studies were carried out in which materials were first mechanically ground into very small shards and particles, from which endospores were extracted, assayed, and accounted per cubic centimeter of material. An extensive set of controls was assayed in parallel with the actual samples to interpolate and calibrate the results accurately.

3. Results

At the time of MSL launch, members of the MSL PP campaign had conducted more than 500 sampling events, and 3541 swab samples and 1312 wipe samples had been assayed and analyzed. The total spore burden of MSL at the time of launch was 2.78×10⁵ spores, which is well within the required launch specification of <5.0×10⁵ spores (55.6%). Upon landing on Mars, MSL's total spore burden was 5.64×10⁴, only 18.8% of the allowed landed limit of 3.0×10⁵ spores (Table 1). Requirements also dictate that the average spore burden on the exposed surfaces of any landed hardware shall not exceed 300 spores/m² at the time of launch. The MSL system was well within this specification, hosting an average launched burden of 22 spores/m² (7.3% of the allowable amount) for the combined planned landed and planned impacted hardware.

Table 1.

Summary of Bioburden Allocation by MSL Subsystem

	Accountable
Subsystem	Surface area (m²)	Volume (cm³×10⁴)	Maximum spore burden at launch (spores×10⁴)
Rover	127.4	0.41	1.57
Planned landed hardware	127.4	0.41	1.57
Planned impacted hardware	—	—	—
Descent stage	1129.48	65.6	11.5
Planned landed hardware	206.32	—	1.84
Planned impacted hardware	923.16	65.6	9.67
Aeroshell	2173.05	111	4.88
Planned landed hardware	377.54	—	1.43
Planned impacted hardware	1796	111	3.44
Other EDL	741.89	0	0.79
Planned landed hardware	741.89	—	0.79
Planned impacted hardware	—	—	—
Cruise stage	1354	0	5
Planned landed hardware	—	—	—
Planned impacted hardware	1354	—	5
Total accountable surface area	4172	—	—
Total accountable volume	—	177	—
Small miscellaneous surface area	—	—	4
Requirement			<50.0
Total			27.8
Reserve			22.2

Exceedingly stringent engineering handling constraints and PP-imposed anticontamination countermeasures were key to maintaining clean flight hardware surfaces. In contrast to previous missions where a much greater percentage of the flight hardware's total surface area was subjected only to standard cleaning regimens, ∼89% of MSL's total surface area and ∼61% of MSL's total volume were subjected to DHMR (data not shown). In addition, another ∼11% and ∼1% of the total surface area was treated with isopropyl alcohol (either rinsed or wiped) and subjected to precision cleaning regimens, respectively. In the end, less than 0.01% (0.66 m²) of MSL's total accountable surface area (4.17×10³ m²) and less than 0.15% (1.68×10³ cm³) of MSL's total accountable volume (1.77×10⁶ cm³) went without any microbial reduction treatment.

Spore burden values were calculated for the various components of the MSL spacecraft at the time of launch and are presented in Table 1. As mentioned above, the results must be ascribed to either planned landed or planned impacted hardware. For example, all the rover-associated bioburden (1.57×10⁴ spores, in toto) was recorded as planned landed hardware, and as such, this subsystem yielded no values for planned impacted hardware. By the time MSL launched, its planned landed hardware constituted a total surface area of 1453.15 m², while its planned impacted hardware constituted a total surface area of 4076.16 m².

4. Discussion

Many factors contributed to making MSL the cleanest NASA spacecraft launched since the 1970s Viking landers. Perhaps nothing was more valuable in achieving this outcome than the streamlined feedback loop maintained between PP personnel and MSL engineers. Effective and efficient communication and willingness to cooperate enabled the execution of the most stringent engineering handling constraints and PP-imposed anticontamination countermeasures to date. Measures such as gowning with full bunny suits throughout the ATLO process, rigorous cleaning of hardware and associated ground support equipment and clean room surfaces, and ideal positioning of flight hardware in facilities with respect to airflow were critical in ensuring the cleanliness of MSL. These and a multitude of thoroughly planned and executed precautionary practices, in addition to the relentless attention to detail paid by countless engineers, technicians, and scientists alike, helped deliver a launch-ready MSL flight system whose cleanliness and scant spore counts drastically eclipsed (55.6% margin; reserve=2.22×10⁵ spores) all PP bioburden requirements.

The MSL flight system comprised a rover, descent stage, cruise stage, and an aeroshell, which consisted of a heat shield and backshell (Fig. 1). The aeroshell enclosed the entry, descent, and landing (EDL) system (colloquially referred to as the “sky crane”), and the Curiosity rover. To be readily available for entry braking, the parachute and retrorocket motors were incorporated into the backshell. In launch configuration, the MSL cruise stage was attached to a Centaur motor, which boosted this payload to escape velocity shortly after launch via the payload launch fairing that protected the spacecraft during transportation and launch. The entire MSL flight system was assembled in an ISO Class 8 clean room facility. Once assembled and integrated with the launch vehicle, all the EDL, rover, and interior surfaces of the aeroshell were protected from ambient recontamination by the aeroshell itself and an environmentally controlled Atlas V payload fairing.

FIG. 1.

Exploded view (A) and folded-up view (B) of the MSL flight system.

4.1. MSL hardware surface accounting

There are several factors to consider when discussing the various types of MSL hardware. Hardware that is planned to land optimally on the surface of Mars is referred to as “planned landed,” while components intended to impact the surface, more so than land, are deemed “planned impacted” hardware. In turn, each of these hardware types consists of both accountable and non-accountable surface areas and volumes, with regard to PP bioburden accounting.

When considering MSL flight hardware, external and internal surface types must be segregated into two distinct categories. Surfaces in the first category, accountable surfaces, are those whose spore burdens must be determined and factored into the total spacecraft bioburden calculation. Conversely, non-accountable surfaces are those whose spore burdens do not factor into the total spacecraft bioburden tally. Accountable surfaces are referred to as either mated (i.e., inaccessible due to the nature of surface adjoining) or exposed. An exposed surface is characterized as either (a) external in flight or deployed configuration or (b) contained within no more than a single enclosure, in either configuration. For example, design modules with single enclosures were vented through HEPA filters. This definition allows spacecraft modules to be partially enclosed but vented (via, e.g., HEPA filters), with the exception of hermetically sealed modules and internal free surfaces. All the surfaces of the EDL system, including the interior of the aeroshell and its internal thermal blankets, the parachute, the parachute canister and bridle, the backshell interface plate, the rocket motors and their multilayer insulation, and other miscellaneous surfaces, were deemed “exposed.” Conversely, surfaces in the second category, non-accountable surfaces, are exempt from spore burden accounting due to (a) their enclosed state within the particular subsystem and (b) the incorporation of HEPA filters to facilitate the aseptic exchange of gases (to mitigate fluctuations in pressure).

With this in mind, there are also accountable surfaces for which special exemptions from bioburden accountability were granted. The cruise stage, for instance, was exempted based on modeling analyses that demonstrated that, upon entry into the martian atmosphere, it would be heated sufficiently to achieve sterility. In a similar vein, analyses predicted that the external heat shield surfaces would be heated to sterilizing temperatures upon entering the martian atmosphere. However, the inboard side of the heat shield and at least part of the backshell did not reach such temperatures.

The Curiosity rover was the only subsystem that contributed planned landed hardware exclusively. However, the internal surfaces of the external rover payload, cameras, and warm electronics box (i.e., the inside of the chassis) formed self-contained enclosures that were vented by HEPA filters. Thus, these were considered to be non-accountable surfaces, and their spore burdens were exempted from consideration. Only the surfaces of the rover and associated instrument payload that were external to these HEPA filter–vented enclosures contributed to the total planned hardware spore burden. Entry heating analysis indicated that, with the exception of the propulsion fuel tank and cruise balance mass, the cruise stage would completely burn up if a sufficient amount of fuel remained to dissipate the heat. However, because the cruise stage had the potential to contaminate the backshell during launch, samples were collected and processed, and any spores detected on the cruise stage were accounted for in the total planned impacted hardware spore burden tally.

Both the aeroshell and backshell contributed spore burden from both planned landed and planned impacted hardware surfaces. Even though calculations estimated that the heat shield would impact the martian surface at a velocity that would heat its exterior surface to sterilizing temperatures, the spore burden associated with the surfaces of its inboard sensors and instrumentation had to be factored into the total planned impacted hardware bioburden. After lowering the rover to the surface, the sky crane was to propel itself away from the landing site and crash-land; thus, its associated bioburden contributed exclusively to the total planned impacted hardware spore tally. Finally, a small category of small miscellaneous hardware was included in the total spacecraft bioburden calculations. This category included items such as “threaded fasteners” (screws), shims, and similar small items installed during ATLO that, while cleaned, were too small to be meaningfully sampled. A very conservative spore burden estimate of 4×10⁴ was allocated for these items, in toto.

5. Conclusion

Ultimately, MSL flight hardware was extremely clean, and its associated spore indices easily met all PP bioburden requirements at the time of launch. At no time was the MSL landing package burdened with endospores at a level that approached the maximum allowable limit—neither in terms of total spores per spacecraft nor in terms of average density of spores per unit of hardware surface area. These results demonstrate that the campaign conducted by the MSL project was of sufficient rigor to meet the NASA PP requirements for a NASA Category IVa Mars rover mission.

Footnotes

Acknowledgments

Part of the research described in this paper was carried out by the Jet Propulsion Laboratory, California Institute of Technology, under contract with NASA. The authors acknowledge the contributions of C. Conley and P. Stabekis of the NASA planetary protection office. A significant amount of sample collection, processing, and analysis was carried out by F. Morales and G. Kazarians. The authors are indebted to W. Schubert for assistance in sample collection and processing, as well as the initial setup of the PP laboratory at Kennedy Space Center. Finally, we extend thanks to S. Bergstrom for logistical support at Kennedy Space Center and J.A. Spry and K. Buxbaum for managerial oversight.

Abbreviations

ATLO, assembly, test, and launch operations; CFU, colony-forming units; CogE, cognizant engineer; DHMR, dry heat microbial reduction; EDL, entry, descent, and landing; HEPA, high-efficiency particulate air; MER, Mars Exploration Rovers; MS, Microsoft; MSL, Mars Science Laboratory; PP, planetary protection; SAC, spacecraft assembly clean room; TSA, trypticase soy agar.

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