Technology Development

Planetary Protection addresses microbial contamination of the solar system by spacecraft that we launch from Earth (forward contamination). This contamination must be prevented in order to preserve the integrity of exploring the solar system; celestial bodies that may have once held an environment suitable for life (e.g., Mars and outer planet icy bodies) are especially vulnerable. Likewise, extraterrestrial contamination of the Earth and Moon (backward contamination), by way of sample return missions, must be prevented. We must approach with caution and preparedness in bringing unknown and potentially dangerous biological materials back to Earth.

While searching for life on the surface of a solar system body (via life-detection instruments) or in future samples returned to Earth, contamination could result in the “false-positive” indication of life. Thus, Planetary Protection’s primary strategy to prevent contamination is to confirm that spacecraft launched from Earth are clean. This precaution ensures that planets, and any life that might be there, remain in their original pristine state for scientific analysis. After hardware is treated with various forms of microbial reduction, technicians assembling the spacecraft frequently wipe hardware surfaces with an alcohol solution to keep the spacecraft clean. Planetary Protection engineers carefully sample the surfaces and perform microbiology tests to show that the spacecraft meets the specified requirements for biological cleanliness.

In addition to this mission implementation role, the Biotechnology and Planetary Protection Group seeks to develop or adapt modern molecular analytical methods to rapidly detect, classify, and/or enumerate the widest possible spectrum of Earth microbes carried by spacecraft (on surfaces and/or in bulk materials, especially at low densities) before, during, and after assembly, test, and launch operations. Additionally, the group aims to identify new or improved methods, technologies, and procedures for spacecraft sterilization that are compatible with spacecraft materials and assemblies.

First and Largest Custom Database of MALDI-TOF MS Profiles

Since the Viking missions (1970s), JPL’s Biological and Organic Materials Archive has maintained the largest collection of bacterial isolates from spacecraft-associated surfaces. 16S ribosomal RNA (rRNA) gene sequencing is a common and well-established method used to identify and compare bacteria present within a given sample. This industry standard technique, however, is expensive and time consuming. An alternative and widely used method is Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry, which can obtain a high probability match to organisms in the Bruker Daltonics database. The database primarily targets clinical isolates and lacks bacterial isolates commonly found in spacecraft assembly cleanrooms.

At JPL, Planetary Protection engineers routinely sample spacecraft surfaces to verify biological cleanliness. Thus, JPL has developed a custom comprehensive database of profiles (Main Spectral Profile or MSP) using isolates obtained from spacecraft-associated surfaces and cleanroom environments. The isolates used to create the MSP were previously identified using 16S rRNA sequencing. Planetary Protection scientists grouped the sequences based on 99% similarity and selected one representative from each group to populate the MSP. As additional isolates are recovered from routine sampling events, they are identified using MALDI-TOF, which can obtain a high probability match to organisms in the MSP database. For more information about the Biological Materials Archive, click here.

Viability-Linked Metagenomics Analysis of Cleanroom Environments

Spacecraft hardware is assembled in controlled cleanroom environments to keep spacecraft clean and ultimately prevent contamination of other solar system bodies. As a proactive measure to monitor cleanliness and ensure mission integrity, researchers have been diligently cataloging the diverse microbial populations detected on spacecraft and associated surfaces. Cultivation-based analyses lack the resolution required to capture the entire breadth of microbial diversity in cleanroom environments; the selective nature of laboratory media and incubation conditions, as well as competition from other organisms during incubation, are limitations. Metagenomics methods overcome these limitations by extracting DNA or RNA from entire assemblages of microorganisms and sequencing them simultaneously.

Metagenomics analysis, however, does not distinguish viable from dead microbial cells in the samples. To understand the genetic potential of living biological communities in cleanroom environments, researchers utilized PMA-based chemistry with metagenomics sequencing. In this study, samples were collected from spacecraft assembly cleanroom (including gowning area). Samples were split into two equal fractions, where one fraction was treated with PMA. Propidium monoazide (PMA) is an agent able to penetrate and access the DNA of membrane-compromised (dead) cells. Under visible light, PMA integrates with the DNA by covalently bonding with the DNA double-helix structure. Because of DNA modification, follow-up molecular tools will only select for viable cells (intact membranes). Treating cells with PMA prior to metagenomics analysis provides a meaningful understanding of the metabolic and/or functional characteristics of living microorganisms in cleanrooms.

Weinmaier, Thomas, et al. "A viability-linked metagenomic analysis of cleanroom environments: eukarya, prokaryotes, and viruses." Microbiome 3.1 (2015): 62

Sensitive, Rapid Detection of Bacterial Spores

Planetary Protection engineers use the NASA Standard Assay to assess the microbial bioburden on spacecraft in cleanrooms. As spacecraft assembly, testing, and launch operations progress, a rapid endospore assay is necessary to reduce sample-processing times. A Rapid Microbiology Detection System (RMDS) developed by Millipore has been used to rapidly quantify microbial contamination (it can detect as little as one colony-forming unit per sample). It was modified and flight qualified by NASA PPO to detect endospores as a pivotal alternative for Planetary Protection applications.

The RMDS technique combines membrane filtration, adenosine triphosphate (ATP) bioluminescence reagents, and image detection and analysis based upon photon detection. One major change to this technique is the addition of a heat shock treatment at 80°C for 15 minutes to eliminate non-spores. By combining ATP detection with a simple heat-shock sample preparation strategy, spore-forming microbes can be rapidly monitored. Different luciferases and formulations were also tested in order to reduce the typical 18-24-hour incubation time required by the RMDS process to ~5 hours.

Adenosine Monophosphate-Based Detection of Bacterial Spores

Planetary Protection engineers use the NASA Standard Assay to monitor the cleanliness of spacecraft. The detection of spores using this assay follows a 3-day process. A rapid detection method, as a proxy for the NASA Standard Assay, is often needed to meet demanding spacecraft assembly schedules. As such, projects can make a decision to proceed on risk that the rapid assay will be in alignment with the results of the NASA Standard Assay.

The traditional rapid adenosine triphosphate (ATP) assay – an effective sampling tool approved by NASA as a rapid screening tool for biological cleanliness – is not very sensitive for spore detection; in contrast to ATP, adenosine monophosphate (AMP) levels are much higher in spores. The developed method for detecting AMP in spores involves two heat-shock procedures. First, a sample of spores suspended in aqueous solution is heated to 80°C for 15 minutes. All non-spore-forming bacteria and vegetative cells are killed and removed from the solution via a low-acceleration centrifugation wash. Re-suspension in adenosine phosphate deaminase eliminates extracellular ATP and AMP. A second heat-shock is performed at 100°C for 10 minutes, causing the release of AMP from spores. AMP is measured as light that is generated from Kikkoman's CheckLite-SP (TM) bioluminescence reagent with pyruvate orthophosphate dikinase (PPDK).

Novel Live versus Dead Nucleic Acid Technology

To prevent forward contamination, spacecraft are assembled and tested in controlled cleanroom environments. To assess the threat of forward contamination it is important to understand the diverse microbial populations that grow in these cleanrooms and on spacecraft surfaces, abundant or otherwise.

Polymerase Chain Reaction (PCR) favors dominant bacterial populations and therefore does not provide a true representation of the cleanroom bacterial environment. During amplification, the majority of the reagents opt for the predominant DNA present in the sample. The technique developed to reduce this bias relies on PMA-based chemistry. Propidium monoazide (PMA) can bind to free DNA or access the DNA in membrane-compromised (dead) cells. Using sub-optimum concentration levels of PMA, the PMA dye has a higher probability of binding to DNA from abundant species. Therefore, the unhindered “minority population” becomes available for PCR amplification and a better representation of the bacterial population.

PMA-Linked Fluorescence for Rapid Detection of Viable Bacterial Endospores

This method gives researchers the ability to distinguish viable microbes from non-viable microbes by direct visualization. This novel technology involves the coupling of PMA-based chemistry and FISH-based microscopy. The outcome of such a method is that living cells can both be enumerated visually and be differentiated from dead cells without the need for heavily biased cultivation based methodologies. This PMA-FISH method is a tool to detect viable endospores in spacecraft-associated environments, which is of crucial importance for Planetary Protection implementation requirements, aimed at reducing the abundance of spacecraft-borne microbial contaminants.

Propidium monoazide (PMA) is an agent able to penetrate and access the DNA of membrane-compromised (dead) cells. Under visible light, PMA integrates with the DNA by covalently bonding with the DNA double-helix structure. Because of DNA modification, follow-up molecular tools will only select for viable cells (intact membranes). PMA-based selective chemistry has never been coupled with FISH-based microscopy. Fluorescence in situ hybridization (FISH) relies on the ability of fluorescently labeled oligonucleotide probes to hybridize with target specific nucleic acid sequences within cells. Because of initial treatment with PMA, only living, viable cells and spores are detected via the oligonucleotide probes. PMA-treated cells have DNA that is inaccessible during the FISH assay, which allows viable cells to be differentiated from non-viable cells and simultaneously viewed via fluorescence microscopy.

PMA-PhyloChip DNA Microarray to Elucidate Viable Microbial Community Structure

Measuring spacecraft cleanliness by enumerating microbes remains challenging when trying to separate viable microbes from dead cells whose DNA remains stable. PMA-phyloChip DNA microarray selectively estimates viable microbes, and, unlike traditional culture based assays, which can take days to complete, takes just a few hours.

Propidium monoazide (PMA) is an agent able to penetrate and access the DNA of membrane-compromised (dead) cells. Under visible light, PMA integrates with the DNA by covalently bonding with the DNA double-helix structure. Because of DNA modification, follow-up molecular tools will only select for viable cells (intact membranes). Following PMA treatment, only the 16S rRNA gene fragments of viable cells are amplified by PCR, to screen the total microbial community using PhyloChip DNA microarray analysis.

Purifying, Separating, and Concentrating Cells from a Sample Low in Biomass

In order to prevent forward contamination, it is important to understand the full spectrum of microorganisms present in the cleanroom environments and on spacecraft surfaces. Identifying their presence is useful for Planetary Protection contamination-prevention measures. Due to limiting amounts of biomaterial present on spacecraft surfaces, measuring the full breadth of bacterial diversity in samples can be difficult. This limitation is further complicated by the fact that samples often contain interfering/inhibitory materials such as humic acid.

Fluorescence-activated cell-sorting (FACS) provides a method for sorting a heterogeneous mixture of biological cells. The instrument is able to record the fluorescent signal from individual cells and physically separate the cells of interest. Applying dyes (e.g., SYTO dyes) to a microbial community prior to downstream assessment technologies, such as hybridization or PCR, allows portions of the population to be discriminated (e.g., live versus dead). In this study, researchers coupled FACS technology with fluorescent staining methodologies to purify, separate, and concentrate bacterial cells and endospores from an environmental sample. Thus, downstream molecular techniques will result in a far more robust and accurate assessment of the microbial community.

Ultraviolet-Resistant Bacterial Spores

Biological indicators such as B. subtilis are used to verify sterilization methods in hospitals and government facilities. However, spores of B. pumilus SAFR-032 are shown to survive greater proportions of ultraviolet (UV) radiation, γ radiation, and hydrogen peroxide, suggesting that B. pumilus SAFR-032 is a superior model indicator for sterilization procedures.

Discrimination of Spore-Forming Bacilli Using spoIVA

An important tool for understanding a bacterial population present in an environment of interest, is one that is able to discriminate the more problematic spore-forming bacteria from non-spore forming bacteria. Such a method provides valuable information for improved Planetary Protection contamination-prevention and sterilization efforts.

This method includes “sporulation-specific primers” for the bacterial gene spoIVA (sporulation gene). During PCR, these primers are added to the reagent mix, and following several cycles of DNA amplification, the types of bacterial cells present within the sample can be determined based on the intensity of amplification. If the resulting amplicon is strong, then it is concluded that the bacterial population in the sample consists of predominantly, or entirely, spore-forming cells.

Airborne Endospore Bioburden as an Indicator of Spacecraft Cleanliness

There is an abundance of bacterial endospores on Earth that can survive harsh environments. Studies suggest that Airborne Endospore Bioburden (AEB) is a suitable indicator of spacecraft cleanliness: airborne endospore counts can be correlated to surface microbial contamination. The trend in the settling of airborne spores can be tracked with standard microbial monitoring methods. The trend helps to predict surface bioburden on critical spacecraft hardware and helps to expedite sterilization measures.

Cryogenic Grinding for Mechanical Abrasion for Hardy Endospores

The extraction of DNA from bacterial endospores typically involves mechanical abrasion strategies for degrading and breaking open the exosporia and spore coat. These methods involve pretreatment regimes that often degrade naked DNA molecules upon spore rupture. The cryogenic grinding method, however, proves to be a superior method for optimal DNA recovery. A SamplePrep 6870 cryogenic grinder is used which employs liquid nitrogen. Following optimum grinding conditions (e.g., cycle time and cycle periodicity) results reveal only the slightest 1-log reduction.

Vapor Hydrogen Peroxide (VHP) Sterilization

Modern spacecraft with thermally sensitive electronics and hardware materials are not compatible with heat microbial reduction (HMR). Therefore, several vapor phase sterilization methods were considered as a low temperature alternative; Ethylene oxide & Methyl bromide and Formaldehyde & Paraformaldehyde leave organic residue, which is not ideal for organic sensitive hardware. Hydrogen peroxide (H2O2) on the other hand, does not leave organic residue. Its only by-products are oxygen and water. Additionally, the technique is cheaper, ideal for heat sensitive parts, more efficient, and takes a shorter amount of time to process than HMR.

To pursue vapor phase hydrogen peroxide (VHP) as an alternative sterilization technique, the Biotechnology and Planetary Protection Group at JPL conducted literature review and research work to define process specifications. During research work, microbes were selected to test the lethality of the technique, including Bacillus stearothermophilus, Bacillus subtilis var. niger, Bacillus pumilus and Bacillus circulans. The microbes were deposited on different materials including aluminum, polymer, paint, and epoxied graphite. Following the research work and results, NASA PPO granted approval to use this technique for spacecraft subsystems and systems.

The initial stage for VHP sterilization involves a vacuum chamber where water is evacuated from the environment. In the next stage, H2O2 is injected into the chamber. In the third stage, sterile filtered air is injected into the chamber, which allows H2O2 vapor to penetrate packaging and diffusion restricted areas to enhance the efficacy of the sterilization process. In the final stages of the process, the chamber is evacuated once more, followed by venting with sterile filtered air: the concentration of hydrogen peroxide vapor returns to ambient levels and the enclosure can be opened to retrieve contents.

NASA PPO has approved use of VHP as a low-temperature sterilization modality, for simple surfaces, with specifications for time, rate, and H2O2 concentration levels. Complex geometries (e.g., vented boxes and electronic chassis inside the warm electronics box of a rover), however, require further directions. Thus, an additional study aims to describe the effect of VHP on electronic materials, materials with different configurations, solder joints, etc. Work has also begun for developing a portable VHP system that can be used locally (e.g., at the site of hardware integration in a cleanroom or on the launch pad).

Plasma + Microwave Sterilization Technology

To eliminate risk of backward contamination, any Earth-return samples that have been exposed to the extraterrestrial environment are subject to Category V (restricted) Planetary Protection requirements. This requirement necessitates a sealing or sterilization of the sample return container in order to prevent contact between the extraterrestrial environment and Earth’s biosphere. Several concepts and technologies are currently in development to address the transfer and storage of the samples whilst preventing contact between the two environments. One of these concepts is a Dielectric Barrier Discharge (DBD) + Microwave discharge configuration, which acts as an in-orbit implemented sterilization technology.

Dielectric Barrier Discharge (DBD) plasma is generated by applying an alternating high voltage across two electrodes separated by an insulating dielectric barrier. Charges accumulate on the dielectric surface and generate an electric field that opposes the applied field. Plasma is a gas-like substance consisting of particles such as positive ions (atoms that have lost their neutrality). These positive ions are destructive free radicals that cause injury to cells by way of chemical reactions (hydrogen abstraction and double bond cleavage). Plasma in combination with UV radiation causes further destruction to cells by damaging DNA and RNA, and ultimately leads to cell death. Moreover, UV radiation induces additional free radicals, perpetuating the process. Thus, plasma can kill spores, bacteria and other microorganisms at low temperature without thermal and oxidative damage to the treated surfaces. The addition of microwaves changes the plasma system from a surface-only phenomenon to volumetric. Areas in this system yet to be defined are optimal chamber design (e.g., material and geometry), microbial reduction capability (determining the rate at which a microbial population has been reduced), gas delivery to the plasma chamber, and sterilization temperature.

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