Mars 2020 Mission Biological Return Sample Contamination Control Approach and Verification.

The Mars 2020 Perseverance rover is equipped with a Sample Caching System (SCS) designed to collect and cache martian core and regolith samples for potential return to Earth. To ensure the integrity of these samples, the mission requirements for each encapsulated sample for return is less than one Earth-sourced viable organism (VO) and more than a 99.9% probability of being free of any Earth-sourced VO. To satisfy the stringent biological contamination requirements in support of return sample science investigations, special bioburden mitigation and reduction approaches were developed and implemented for SCS hardware that would directly contact or be in close proximity to the martian samples. In this study, we describe the implemented approaches for microbiological contamination reduction and mitigation, detail the processes of the SCS aseptic assembly, and report the estimated VO for each returned sample. We found that our conservative estimate of the computed probability of a single VO in the returned sample is more than one order of magnitude lower than the biological contamination requirement while the best estimate exceeds two orders of magnitude.

Planetary Protection Implementation and Verification Approach for the Mars 2020 Mission.

The Mars 2020 Flight System comprises a Cruise Stage; Aeroshell; Entry, Descent, and Landing system; Perseverance rover; and the Ingenuity helicopter. The Perseverance rover was successfully delivered to Jezero Crater on February 18, 2021. Among its science objectives, Perseverance is meant to search for rocks that are capable of preserving chemical traces of ancient life, if it existed, and to core and cache rock and regolith samples. The Perseverance rover is gathering samples for potential return to Earth as part of a Mars Sample Return campaign. Thus, controlling the presence of Earth-sourced biological contamination is important to protect the integrity of the scientific results as well as to comply with international treaty and NASA requirements governing Planetary Protection prior to launch. An unprecedented campaign of sampling and environmental monitoring occurred, which resulted in over 16,000 biological samples collected throughout spacecraft assembly. Engineering design, microbial reduction measures, monitoring, and process controls enabled the mission to limit the total spore bioburden to 3.73 × 105 spores, which provided 25.4% margin against the required limit. Furthermore, the total spore bioburden of all landed hardware was 3.86 × 104, which provided 87% margin against the required limit. This manuscript outlines the Planetary Protection implementation approach and verification methodologies applied to the Mars 2020 flight system and its surrounding environments.

Applied Approach for Assessing Bioburden of the Mars 2020 Parachute Assembly.

The Mars 2020 mission delivered the Perseverance rover to the surface of Mars using a supersonic parachute manufactured at Airborne Systems, California. The Mars 2020 spacecraft, including the flight parachute, was subject to Planetary Protection spore bioburden compliance. Many previous missions with similar parachutes applied manufacturing specifications for calculating bioburden. Although the Mars 2020 parachute was manufactured in an uncontrolled environment, preliminary sampling of a flight-like parachute manufactured in the same facility suggested that the actual spore bioburden levels were potentially orders of magnitude lower than specification values for uncontrolled manufacturing (100,000 spores/m2). Several experiments were designed and carried out throughout the project timeline in an effort to estimate a representative bioburden for the flight parachute. Tests were performed on various parachute materials, including direct sampling and destructive assays of proxy materials. Different bioburden densities were applied to large continuous areas of the canopy, which experienced minimal handling, and seamed areas of the parachute that were likely to experience more handling during the stitching process. In addition, an approach to account for various thermal zones was developed and applied toward calculating log reduction for the parachute assembly. These various methods that were used toward different areas and materials of the Mars 2020 flight parachute provided a nuanced and data-backed estimate of spore bioburden density that can be adopted by future missions.

Comparison of innovative molecular approaches and standard spore assays for assessment of surface cleanliness.

A bacterial spore assay and a molecular DNA microarray method were compared for their ability to assess relative cleanliness in the context of bacterial abundance and diversity on spacecraft surfaces. Colony counts derived from the NASA standard spore assay were extremely low for spacecraft surfaces. However, the PhyloChip generation 3 (G3) DNA microarray resolved the genetic signatures of a highly diverse suite of microorganisms in the very same sample set. Samples completely devoid of cultivable spores were shown to harbor the DNA of more than 100 distinct microbial phylotypes. Furthermore, samples with higher numbers of cultivable spores did not necessarily give rise to a greater microbial diversity upon analysis with the DNA microarray. The findings of this study clearly demonstrated that there is not a statistically significant correlation between the cultivable spore counts obtained from a sample and the degree of bacterial diversity present. Based on these results, it can be stated that validated state-of-the-art molecular techniques, such as DNA microarrays, can be utilized in parallel with classical culture-based methods to further describe the cleanliness of spacecraft surfaces.

Nonthermal dielectric-barrier discharge plasma-induced inactivation involves oxidative DNA damage and membrane lipid peroxidation in Escherichia coli.

Oxidative stress leads to membrane lipid peroxidation, which yields products causing variable degrees of detrimental oxidative modifications in cells. Reactive oxygen species (ROS) are the key regulators in this process and induce lipid peroxidation in Escherichia coli. Application of nonthermal (cold) plasma is increasingly used for inactivation of surface contaminants. Recently, we reported a successful application of nonthermal plasma, using a floating-electrode dielectric-barrier discharge (FE-DBD) technique for rapid inactivation of bacterial contaminants in normal atmospheric air (S. G. Joshi et al., Am. J. Infect. Control 38:293-301, 2010). In the present report, we demonstrate that FE-DBD plasma-mediated inactivation involves membrane lipid peroxidation in E. coli. Dose-dependent ROS, such as singlet oxygen and hydrogen peroxide-like species generated during plasma-induced oxidative stress, were responsible for membrane lipid peroxidation, and ROS scavengers, such as α-tocopherol (vitamin E), were able to significantly inhibit the extent of lipid peroxidation and oxidative DNA damage. These findings indicate that this is a major mechanism involved in FE-DBD plasma-mediated inactivation of bacteria.