Engineering topics
Publications and source records attributed to Benardini, James.
Using the EB approach to generate a probabilistic bioburden distribution as demonstrated through mission use cases provides a valid approach for use in the end-to-end requirements verification process.
In accordance with NASA Planetary Protection (PP) policy requirements, flight project hardware may be required to undergo microbial reduction processes to prevent the forward contamination of target planetary bodies with Earth organisms. Heat microbial reduction (HMR) is the most commonly employed modality used at JPL for reducing the microbial bioburden on flight hardware. In 2013, longstanding HMR specifications were abandoned, and revised specifications were developed which integrated the latest findings on bacterial spore heat resistivity. Revised decimal reduction values (D-values) for time-temperature lethality curves (110 °C to 200 °C) were developed to account for “hardy” bacterial spores that exhibit greater heat resistance than previously understood. Presented here is a comparative analysis of the revised NASA HMR specifications against empirical data compiled from recent JPL studies, and peer-reviewed, published literature. Bacillus sp. strain ATCC 29669 displayed high heat resistance, and this strain’s 4-log heat lethality curve was comparable to the revised 4-log specification. Spores of Bacillus atrophaeus ATCC 9372 displayed less heat resistance, and exhibited D-values which were less than the revised 3-log microbial reduction specifications. Extrapolations indicate that the current 6-log reduction credit applied to 350 °C for 1 hr. and 500 °C for 0.5 sec. is highly conservative. Projections indicate that a 10- to 18-log reduction of both hardy and non-hardy spores may be achievable at bakeouts of 350 °C for 1 hr. The findings reported here indicate the revised NASA HMR specifications from 110 °C to 200 °C are appropriate for achieving 4-log and 6-log reductions with hardy spore populations; however, for non-hardy spore populations, or for temperatures above 200 °C, the specifications are exceedingly conservative.
Spacecraft surfaces that are destined to land on potential life-harboring celestial bodies are required to be rigorously cleaned and continuously monitored for spore bioburden as a proxy for spacecraft cleanliness. The NASA standard spore assay (NSA), used for spacecraft bioburden estimates, specifically measures spores that are cultivable, aerobic, resistant to heat shock, and grow at 30˚C in a nutrient-rich medium. Since the vast majority of microorganisms cannot be cultivated using the NSA assay, it is necessary to utilize state-of-the art molecular techniques to better understand the presence of all viable microorganisms, not just those measured with the NSA. In this study, the nutrient-deprived low biomass cleanrooms, where spacecraft are assembled, were used as a surrogate to spacecraft surfaces to measure the ratio of NSA spores in relation to the total viable microorganism population to compare with a 2006 space studies report that estimates that for every 1 spore there is approximately 50,000 viable organisms. Ninety-eight surface wipe samples were collected from the spacecraft assembly facility (SAF) cleanroom at the Jet Propulsion Laboratory (JPL) over a 6-month period. The samples were processed and analyzed using classical microbiology along with molecular assays. Traditional microbiology plating methods were used to determine the cultivable bacterial, fungal, and spore populations. Molecular assays were used to determine the total organisms (TO, dead and live) and the viable organisms (VO, live). The TO was measured using adenine triphosphate (ATP) and quantitative polymerase chain reaction (qPCR) assays. The VO was measured using internal ATP, propidium monoazide (PMA)-qPCR, and flow cytometry (after staining for viable microorganisms) assays. Based on the results, it was possible to establish a ratio between spore counts and VO for each viability assay. The ATP based spore to VO ratio ranged from 149 – 746 and the bacterial PMA-qPCR assay based ratio ranged from 314 – 1491 VO. The most conservative estimate came from FACS, which estimated the ratio to be 12,091 VO per 1 NSA spore. Since archaeal (<1%) and fungal (~2%) populations were negligible, the spore to VO ratios were based on bacterial population estimates. The most conservative ratio from this study can be used as a replacement for the SSB estimate on nutrient-deprived (oligotrophic) desiccated spacecraft surfaces, to estimate the VO from NSA measurements without utilizing state-of-the art molecular methods that are costly and require more biomass than is typically found of spacecraft surfaces.
This innovation is derived from a proprietary amplification scheme that is based upon random fragmentation of the genome into a series of short, overlapping templates. The resulting shorter DNA strands (<400 bp) constitute a library of DNA fragments with defined 3 and 5 termini. Specific primers to these termini are then used to isothermally amplify this library into potentially unlimited quantities that can be used immediately for multiple downstream applications including gel eletrophoresis, quantitative polymerase chain reaction (QPCR), comparative genomic hybridization microarray, SNP analysis, and sequencing. The standard reaction can be performed with minimal hands-on time, and can produce amplified DNA in as little as three hours. Post-fragmentation whole genome amplification-based technology provides a robust and accurate method of amplifying femtogram levels of starting material into microgram yields with no detectable allele bias. The amplified DNA also facilitates the preservation of samples (spacecraft samples) by amplifying scarce amounts of template DNA into microgram concentrations in just a few hours. Based on further optimization of this technology, this could be a feasible technology to use in sample preservation for potential future sample return missions. The research and technology development described here can be pivotal in dealing with backward/forward biological contamination from planetary missions. Such efforts rely heavily on an increasing understanding of the burden and diversity of microorganisms present on spacecraft surfaces throughout assembly and testing. The development and implementation of these technologies could significantly improve the comprehensiveness and resolving power of spacecraft-associated microbial population censuses, and are important to the continued evolution and advancement of planetary protection capabilities. Current molecular procedures for assaying spacecraft-associated microbial burden and diversity have inherent sample loss issues at practically every step, particularly nucleic acid extraction. In engineering a molecular means of amplifying nucleic acids directly from single cells in their native state within the sample matrix, this innovation has circumvented entirely the need for DNA extraction regimes in the sample processing scheme.