NASA Procedural Requirement 8020.12C entitled “Planetary Protection Provisions for Robotic Extraterrestrial Missions” states that the source-specific encapsulated microbial density for encapsulated organisms (div(0)) in nonmetallic materials ranges from 1-30 spores/cm3. The standard laboratory procedure, NASA Standard Procedures for the Microbial Examination of Space Hardware, NHB 5340.1B, does not provide any direction into the methodologies to understand the bioburden within such a fluid as CFC-11 (Freon). This general specification value for the Freon would be applicable to the Freon charged within the Mars Science Laboratory’s (MSL’s) Heat Rejection System. Due to the large volume required to fill this system, MSL could not afford to conservatively allocate 55.8% of the total spore budget of the entire laboratory system (rover, descent stage, cruise stage, and aeroshell) of 5.00 × 105 spores at launch.
A novel filtration approach was developed to analyze the Freon employing a 50 kDa molecular weight cutoff (MCO) filter, followed by 0.22-μm pore-size filter to establish a calculated microbial bioburden.
Filtration of microorganisms from liquid matrices is a standard laboratory approach. Due to the volatility of Freon, a standard vacuum filtration unit would not suffice because of the lack of a cold trap on the vacuum unit. A more economical approach had to be devised. The two-pronged concentration approach is advantageous due to the fact that it initially concentrates the Freon from liters to milliliters where it can then be feasibility filtered and microbes extracted from the filter. This is a technology improvement over prior art as it defines the specific parameters to concentrate microbial organisms from a low-boiling-point fluid such as Freon.
This work relates to the current MSL mission but also has implications for future NASA missions that will utilize the same or similar heat rejection fluids. If the same lot of material is utilized on a future mission, then the experimentally derived value can be directly used based on this study (MSL-heritage). If a new lot or similar material composition is used in a future mission, then this technology can be employed or modified accordingly to accommodate such a fluid. This technology development will allow for a heritage-based starting point for fluids on other missions in which a calculated microbial bioburden is necessary.
This work was done by James N. Benardini, Robert C. Koukol, Gayane A. Kazarians, and Fabian Morales of Caltech for NASA’s Jet Propulsion Laboratory. NPO-48303
This Brief includes a Technical Support Package (TSP).
Development of a Centrifugal Technique for the Microbial Bioburden Analysis of Freon (CFC-11)
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Overview
The document outlines research conducted at NASA's Jet Propulsion Laboratory (JPL) focused on developing a centrifugal technique for analyzing microbial bioburden in Freon (CFC-11). This work is significant due to the stringent planetary protection requirements outlined in NASA Procedural Requirement 8020.12C, which mandates specific microbial density limits for encapsulated organisms in nonmetallic materials, including Freon used in spacecraft systems.
The study employs a two-step filtration process to assess microbial contamination in Freon. Step A utilizes a Millipore 50kDa Molecular Weight Cutoff (MWCO) filter to concentrate microbial spores from the Freon samples. The process involves centrifugation, where the Freon is filtered through the MWCO filter, and the resulting fluids are collected for further analysis. Positive and negative controls are implemented to validate the assay's efficacy, with Bacillus atrophaeus spores used as a positive control to calculate recovery percentages.
In Step B, the decanted fluids from Step A undergo a secondary filtration using a 0.22 µm pore-size nitrocellulose membrane. This step aims to further purify the samples and extract any remaining microbial content. The filters are sonicated and vortexed to ensure thorough extraction, followed by incubation of the samples on tryptic soy agar (TSA) plates to enumerate total culturable bioburden at intervals of 24, 48, and 72 hours.
The research highlights the importance of ensuring that the Freon used in the Mars Science Laboratory (MSL) heat rejection system meets the specified microbial limits to prevent contamination of extraterrestrial environments. The findings contribute to the broader field of planetary protection and microbial analysis, with potential applications in pharmaceutical development and chemical manufacturing.
Overall, this document serves as a technical support package detailing the methodologies and significance of the research, emphasizing the collaboration between JPL and NASA in advancing aerospace-related technologies while adhering to strict planetary protection protocols. The work is credited to a team of researchers at JPL and is part of NASA's efforts to ensure safe and responsible exploration of other planets.
