Multimission Metagenomics Technology Development for Planetary Protection
UNKNOWN
Venkateswaran, Kasthuri↗
Engineering topics
Benardini, James N.
Publications and source records attributed to Benardini, James N..
Organic and inorganic contamination control approaches for return sample investigation on Mars 2020
The Mars 2020 Rover mission will have the capability to collect and cache samples for potential Mars sample return. Specifically, the sample caching system (SCS) is designed for coring Mars samples and acquiring regolith samples as well as handling, sealing and caching on Mars. As the potential first Martian samples that could be returned to Earth, assuring low levels of terrestrial contamination is of the utmost concern. In developing the SCS, the project prioritizes limiting sample contamination in organic, inorganic and biological areas. The focus of this paper is on the strategies being implemented to limit terrestrial organic and inorganic contamination in the samples.
Steltzner, Adam↗
Targeted gene(s) amplification-based NGS analysis of low biomass samples from SAF/Spacecraft surfaces
UNKNOWN
Venkateswaran, Kasthuri↗
Characterization of Biological Fallout Particles of Cleanrooms to Measure Spacecraft Cleanliness
NASA cleanrooms are humidity, temperature-regulated, and particle contaminant-controlled environments used for spacecraft assembly. Microorganisms, which are crucial contaminants shed from humans and transported through the environment, can adapt to the oligotrophic cleanroom conditions. These biogenic particles need to be identified to extend our knowledge of biological contamination for future NASA mission use. Here, we devised an aluminum coupon with polycarbonate filter setup to collect fallout particles from an ISO 5 and an ISO 7 cleanroom. We demonstrated the presence of microbial particles, as standalone entities and associated with inert particles in cleanroom environments. The contamination was confirmed as biogenic in virtue of estimating the elemental composition of individual and aggregated particles using an epifluorescence and Field Emission Scanning Electron Microscopy (FESEM) coupled with an energy dispersive X-ray analysis (EDX). Epifluorescence microscopy showed that the particles were stained less (25%) with SYTO 9 and more (75%) with propidium iodide, suggesting that the suspected biogenic particles are membrane-compromised. Remarkably, biological particles such as bacteria and fungal spores/hyphae were observed at sizes ranging from 2 to 70 µm, with abundant C and O elemental signatures and essential tracers (Na, Mg, K, Ca and Cl). Inert particles (2 to 500 µm) that were characterized included compositions such as silica (SiO2), aluminosilicates (Al, Si), and carbonaceous fibers. Furthermore, we designed and illustrated a unique correlative epifluorescence and FE-SEM method to strengthen this approach by acquiring the same biogenic particles in the same filter coupon for a one-to-one comparison. Interestingly, the SYTO 9 stained particles showed abundant C and O elemental signature implying that they are viable microbial particles. Constant evaluation of cleanroom biological and inert particles will help to characterize biological contamination for future NASA missions (e.g. Mars 2020).
Malli Mohan, Ganesh Babu↗
The Evolution of Planetary Protection Implementation on Mars Landed Missions
NASA has developed requirements dedicated to the prevention of forward and backward contamination during space exploration. Historically, international agreements provided guidelines to prevent contamination of the Moon and other celestial bodies, as well as the Earth (e.g., sample return missions). The UN Outer Space Treaty was established in 1967 and the Committee on Space Research (COSPAR) maintains a planetary protection policy complying with Article IX of this treaty. By avoiding forward contamination, the integrity of scientific exploration is preserved. Planetary Protection mission requirements are levied on missions to control contamination. These requirements are dependent on the science of the mission and on the celestial bodies encountered or targeted along the way. Consequently, categories are assigned to missions, and specific implementation plans are developed to meet the planetary protection requirements. NASA missions have evolved over time with increasingly more demanding scientific objectives and more complex flight systems to achieve those objectives and, thus, planetary protection methods and processes used for implementation have become much more intricate, complicated, and challenging. Here, we will portray the evolution of planetary protection implementation at JPL in several important areas throughout the course of NASA sponsored robotic Mars lander or rover missions, starting from Mars Pathfinder through the beginning of Mars 2020. Highlighted in the discussion will be process changes in planetary protection requirements development and flow down. Development and implementation of new and improved methods used in the reduction of spacecraft bioburden will be discussed as well as approaches and challenges that come along with setting up remote laboratories to perform bioassays. The consequences and forward planning of delays on missions will be highlighted as well as lessons learned on the impact of communication and training in achieving planetary protection requirements. The evolution of methods used for the detection of microbial bioburden on spacecraft hardware will be considered. These methods use standard microbiology as well as the adaptation of advances in biotechnology, molecular biology, and bioinformatics. Technical approaches developed for the prevention of contamination and recontamination of hardware during Assembly, Test, and Launch Operations will be discussed.
Kazarians, Gayane A.↗
Using a Blender to Assess the Microbial Density of Encapsulated Organisms
There are specific NASA requirements for source-specific encapsulated microbial density for encapsulated organisms in non-metallic materials. Projects such as the Mars Science Laboratory (MSL) that use large volumes of non-metallic materials of planetary protection concern pose a challenge to their bioburden budget. An optimized and adapted destructive hardware technology employing a commercial blender was developed to assess the embedded bioburden of thermal paint for the MSL project. The main objective of this optimization was to blend the painted foil pieces in the smallest sizes possible without excessive heating. The small size increased the surface area of the paint and enabled the release of the maximum number of encapsulated microbes. During a trial run, a piece of foil was placed into a blender for 10 minutes. The outside of the blender was very hot to the touch. Thus, the grinding was reduced to five 2-minute periods with 2-minute cooling periods between cycles. However, almost 20% of the foil fraction was larger (>2 mm). Thus, the largest fractions were then put into the blender and reground, resulting in a 71% increase in particles less than 1 mm in size, and a 76% decrease in particles greater than 2 mm in size. Because a repeatable process had been developed, a painted sample was processed with over 80% of the particles being <2 mm. It was not perceived that the properties (i.e. weight and rubber-like nature) of the painted/foil pieces would allow for a finer size distribution. With these constraints, each section would be ground for a total of 10 minutes with five cycles of a 2-minute pulse followed by a 2-minute pause. It was observed on several occasions that a larger blade affected the recovery of seeded spores by approximately half an order of magnitude. In the standard approach, each piece of painted foil was aseptically removed from the bag and placed onto a sterile tray where they were sized, cut, and cleaned. Each section was then weighed and placed into a sterile Waring Laboratory Blender. Samples were processed on low speed. The ground-up samples were then transferred to a 500-mL bottle using a sterile 1-in. (.2.5-cm) trim brush. To each of the bottles sterile planetary protection rinse solution was added and a modified NASA Standard Assay (NASA HBK 6022) was performed. Both vegetative and spore plates were analyzed.
Benardini, James N.↗
Development of a Centrifugal Technique for the Microbial Bioburden Analysis of Freon (CFC-11)
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/cubic cm. 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 fs (MSL fs) 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 X 10(exp 5) 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-micron pore-size filter to establish a calculated microbial bioburden.
Benardini, James N.↗
Adaptation of a Filter Assembly to Assess Microbial Bioburden of Pressurant Within a Propulsion System
A report describes an adaptation of a filter assembly to enable it to be used to filter out microorganisms from a propulsion system. The filter assembly has previously been used for particulates greater than 2 micrometers. Projects that utilize large volumes of nonmetallic materials of planetary protection concern pose a challenge to their bioburden budget, as a conservative specification value of 30 spores per cubic centimeter is typically used. Helium was collected utilizing an adapted filtration approach employing an existing Millipore filter assembly apparatus used by the propulsion team for particulate analysis. The filter holder on the assembly has a 47-mm diameter, and typically a 1.2-5 micrometer pore-size filter is used for particulate analysis making it compatible with commercially available sterilization filters (0.22 micrometers) that are necessary for biological sampling. This adaptation to an existing technology provides a proof-of-concept and a demonstration of successful use in a ground equipment system. This adaptation has demonstrated that the Millipore filter assembly can be utilized to filter out microorganisms from a propulsion system, whereas in previous uses the filter assembly was utilized for particulates greater than 2 micrometers.
Benardini, James N.↗
Purifying, Separating, and Concentrating Cells From a Sample Low in Biomass
Frequently there is an inability to process and analyze samples of low biomass due to limiting amounts of relevant biomaterial in the sample. Furthermore, molecular biological protocols geared towards increasing the density of recovered cells and biomolecules of interest, by their very nature, also concentrate unwanted inhibitory humic acids and other particulates that have an adversarial effect on downstream analysis. A novel and robust fluorescence-activated cell-sorting (FACS)-based technology has been developed for purifying (removing cells from sampling matrices), separating (based on size, density, morphology), and concentrating cells (spores, prokaryotic, eukaryotic) from a sample low in biomass. The technology capitalizes on fluorescent cell-sorting technologies to purify and concentrate bacterial cells from a low-biomass, high-volume sample. Over the past decade, cell-sorting detection systems have undergone enhancements and increased sensitivity, making bacterial cell sorting a feasible concept. Although there are many unknown limitations with regard to the applicability of this technology to environmental samples (smaller cells, few cells, mixed populations), dogmatic principles support the theoretical effectiveness of this technique upon thorough testing and proper optimization. Furthermore, the pilot study from which this report is based proved effective and demonstrated this technology capable of sorting and concentrating bacterial endospore and bacterial cells of varying size and morphology. Two commercial off-the-shelf bacterial counting kits were used to optimize a bacterial stain/dye FACS protocol. A LIVE/DEAD BacLight Viability and Counting Kit was used to distinguish between the live and dead cells. A Bacterial Counting Kit comprising SYTO BC (mixture of SYTO dyes) was employed as a broad-spectrum bacterial counting agent. Optimization using epifluorescence microscopy was performed with these two dye/stains. This refined protocol was further validated using varying ratios and mixtures of cells to ensure homogenous staining compared to that of individual cells, and were utilized for flow analyzer and FACS labeling. This technology focuses on the purification and concentration of cells from low-biomass spacecraft assembly facility samples. Currently, purification and concentration of low-biomass samples plague planetary protection downstream analyses. Having a capability to use flow cytometry to concentrate cells out of low-biomass, high-volume spacecraft/ facility sample extracts will be of extreme benefit to the fields of planetary protection and astrobiology. Successful research and development of this novel methodology will significantly increase the knowledge base for designing more effective cleaning protocols, and ultimately lead to a more empirical and true account of the microbial diversity present on spacecraft surfaces. Refined cleaning and an enhanced ability to resolve microbial diversity may decrease the overall cost of spacecraft assembly and/or provide a means to begin to assess challenging planetary protection missions.
Benardini, James N.↗
Employing a Grinding Technology to Assess the Microbial Density for Encapsulated Organisms
Projects that utilize large volumes of nonmetallic materials of planetary protection concern pose a challenge to their bioburden budget, as the most conservative value of 30 spores/cubic cm is typically used. The standard laboratory procedures do not provide any direction into the methodologies to understand the embedded bioburden within such nonmetallic components such as adhesives, insulation, or paint. A tailored, novel, destructive hardware technology employing a household box grater was developed to assess the embedded bioburden within the adhesives, insulation, and paint for the Mars Science Laboratory (MSL) project.
Benardini, James N.↗
Isolation of the Paenibacillus phoenicis, a Spore-Forming Bacterium
A microorganism was isolated from the surfaces of the cleanroom facility in which the Phoenix lander was assembled. The isolated bacterial strain was subjected to a comprehensive polyphasic analysis to characterize its taxonomic position. Both phenotypic and phylogenetic analyses clearly indicate that this isolate belongs to the genus Paenibacillus and represents a novel species. Bacillus spores have been utilized to assess the degree and level of microbiological contamination on spacecraft and their associated spacecraft assembly facilities. Spores of Bacillus species are of particular concern to planetary protection due to the extreme resistance of some members of the genus to space environmental conditions such as UV and gamma radiation, vacuum, oxidation, and temperature fluctuation. These resistive spore phenotypes have enhanced potential for transfer, and subsequent proliferation, of terrestrial microbes on another solar body. Due to decreased nutrient conditions within spacecraft assembly facility clean rooms, the vegetative cells of Bacillus species and other spore-forming Paenibacillus species are induced to sporulate, thereby enhancing their survivability of bioreduction
Benardini, James N.↗
Survival of spacecraft-associated microorganisms under simulated Martian UV irradiation
Spore-forming microbes recovered from spacecraft surfaces and assembly facilities were exposed to simulated Martian UV irradiation. The effects of UVA (315 to 400 nm), UVA_B (280 to 400 nm), and the full UV spectrum (200 to 400 nm) on the survival of microorganisms were studied at UV intensities expected to strike the surfaces of Mars. Microbial species isolated from the surfaces of several spacecraft, including Mars Odyssey, X-2000 (avionics), and the International Space Station, and their assembly facilities were identified using 16S rRNA gene sequencing. Forty-three Bacillus spore lines were screened, and 19 isolates showed resistance to UVC irradiation (200 to 280 nm) after exposure to 1,000 J m_2 of UVC irradiation at 254 nm using a low-pressure mercury lamp. Spores of Bacillus species isolated from spacecraft-associated surfaces were more resistant than a standard dosimetric strain, Bacillus subtilis 168. In addition, the exposure time required for UVA_B irradiation to reduce the viable spore numbers by 90% was 35-fold longer than the exposure time required for the full UV spectrum to do this, confirming that UVC is the primary biocidal bandwidth. Among the Bacillus species tested, spores of a Bacillus pumilus strain showed the greatest resistance to all three UV bandwidths, as well as the total spectrum. The resistance to simulated Mars UV irradiation was strain specific; B. pumilus SAFR-032 exhibited greater resistance than all other strains tested. The isolation of organisms like B. pumilus SAFR-032 and the greater survival of this organism (sixfold) than of the standard dosimetric strains should be considered when the anitation capabilities of UV irradiation are determined.
Venkateswaran, Kasthuri↗