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All sourcesNASA NTRSDOE OSTI
NASA NTRS2011

PMA-PhyloChip DNA Microarray to Elucidate Viable Microbial Community Structure

Since the Viking missions in the mid-1970s, traditional culture-based methods have been used for microbial enumeration by various NASA programs. Viable microbes are of particular concern for spacecraft cleanliness, for forward contamination of extraterrestrial bodies (proliferation of microbes), and for crew health/safety (viable pathogenic microbes). However, a "true" estimation of viable microbial population and differentiation from their dead cells using the most sensitive molecular methods is a challenge, because of the stability of DNA from dead cells. The goal of this research is to evaluate a rapid and sensitive microbial detection concept that will selectively estimate viable microbes. Nucleic acid amplification approaches such as the polymerase chain reaction (PCR) have shown promise for reducing time to detection for a wide range of applications. The proposed method is based on the use of a fluorescent DNA intercalating agent, propidium monoazide (PMA), which can only penetrate the membrane of dead cells. The PMA-quenched reaction mixtures can be screened, where only the DNA from live cells will be available for subsequent PCR reaction and microarray detection, and be identified as part of the viable microbial community. An additional advantage of the proposed rapid method is that it will detect viable microbes and differentiate from dead cells in only a few hours, as opposed to less comprehensive culture-based assays, which take days to complete. This novel combination approach is called the PMA-Microarray method. DNA intercalating agents such as PMA have previously been used to selectively distinguish between viable and dead bacterial cells. Once in the cell, the dye intercalates with the DNA and, upon photolysis under visible light, produces stable DNA adducts. DNA cross-linked in this way is unavailable for PCR. Environmental samples suspected of containing a mixture of live and dead microbial cells/spores will be treated with PMA, and then incubated in the dark. Thereafter, the sample is exposed to visible light for five minutes, so that the DNA from dead cells will be cross-linked. Following this PMA treatment step, the sample is concentrated by centrifugation and washed (to remove excessive PMA) before DNA is extracted. The 16S rRNA gene fragments will be amplified by PCR to screen the total microbial community using PhyloChip DNA microarray analysis. This approach will detect only the viable microbial community since the PMA intercalated DNA from dead cells would be unavailable for PCR amplification. The total detection time including PCR reaction for low biomass samples will be a few hours. Numerous markets may use this technology. The food industry uses spore detection to validate new alternative food processing technologies, sterility, and quality. Pharmaceutical and medical equipment companies also detect spores as a marker for sterility. This system can be used for validating sterilization processes, water treatment systems, and in various public health and homeland security applications.

Venkateswaran, Kasthuri J.↗
NASA NTRS2012

Molecular Technique to Reduce PCR Bias for Deeper Understanding of Microbial Diversity

Current planetary protection policies require that spacecraft targeted to sensitive solar system bodies be assembled and readied for launch in controlled cleanroom environments. A better understanding of the distribution and frequency at which high-risk contaminant microbes are encountered on spacecraft surfaces would significantly aid in assessing the threat of forward contamination. However, despite a growing understanding of the diverse microbial populations present in cleanrooms, less abundant microbial populations are probably not adequately taken into account due to technological limitations. This novel approach encompasses a wide spectrum of microbial species and will represent the true picture of spacecraft cleanroom-associated microbial diversity. All of the current microbial diversity assessment techniques are based on an initial PCR amplification step. However, a number of factors are known to bias PCR amplification and jeopardize the true representation of bacterial diversity. PCR amplification of a minor template appears to be suppressed by the amplification of a more abundant template. It is widely acknowledged among environmental molecular microbiologists that genetic biosignatures identified from an environment only represent the most dominant populations. The technological bottleneck overlooks the presence of the less abundant minority population and may underestimate their role in the ecosystem maintenance. DNA intercalating agents such as propidium monoazide (PMA) covalently bind with DNA molecules upon photolysis using visible light, and make it unavailable for DNA polymerase enzyme during polymerase chain reaction (PCR). Environmental DNA samples will be treated with suboptimum PMA concentration, enough to intercalate with 90 99% of the total DNA. The probability of PMA binding with DNA from abundant bacterial species will be much higher than binding with DNA from less abundant species. This will increase the relative DNA concentration of previously "shadowed" less abundant species available for PCR amplification. These PCR products obtained with and without PMA treatment will then be subjected to downstream diversity analyses such as sequencing and DNA microarray. It is expected that PMA-coupled PCR will amplify the "minority population" and help in understanding microbial diversity spectrum of an environmental sample at a much deeper level. This new protocol aims to overcome the major potential biases faced when analyzing microbial 16S rRNA gene diversity. This study will lead to a technological advancement and a commercial product that will aid microbial ecologists in understanding microbial diversity from various environmental niches. Implementation of this technique may lead to discoveries of novel microbes and their functions in sustenance of the ecosystem.

Vaishampayan, Parag A.↗
NASA NTRS2012

Hardy Bacterium Isolated From Two Geographically Distinct Spacecraft Assembly Cleanroom Facilities

Earlier studies have confirmed that a tenacious hardy bacterial population manages to persist and survive throughout a spacecraft assembly process. The widespread detection of these organisms underscores the challenges in eliminating them completely. Only comprehensive and repetitive microbial diversity studies of geographically distinct cleanroom facilities will bolster the understanding of planetary protection relevant microbes. Extensive characterizations of the physiological traits demonstrated by cleanroom microbes will aid NASA in gauging the forward contamination risk that hardy bacteria (such as Tersicoccus phoenicis) pose to spacecraft. This study reports on the isolation and identification of two gram-positive, non-motile, non-spore-forming bacterial strains from the spacecraft assembly facilities at Kennedy Space Center, Florida, USA and Centre Spatial Guyanais, Kourou, French Guiana. DNA-DNA relatedness values between the novel strains indicates that these novel strains were indeed members of a same species. Phylogenetic evidence derived from a 16S ribosomal DNA analysis indicated that both the novel strains are less closely related to all other Arthrobacter species.

Vaisham-payan, Parag A.↗
NASA NTRS2012

Methods for determining the genetic affinity of microorganisms and viruses

Selecting which sub-sequences in a database of nucleic acid such as 16S rRNA are highly characteristic of particular groupings of bacteria, microorganisms, fungi, etc. on a substantially phylogenetic tree. Also applicable to viruses comprising viral genomic RNA or DNA. A catalogue of highly characteristic sequences identified by this method is assembled to establish the genetic identity of an unknown organism. The characteristic sequences are used to design nucleic acid hybridization probes that include the characteristic sequence or its complement, or are derived from one or more characteristic sequences. A plurality of these characteristic sequences is used in hybridization to determine the phylogenetic tree position of the organism(s) in a sample. Those target organisms represented in the original sequence database and sufficient characteristic sequences can identify to the species or subspecies level. Oligonucleotide arrays of many probes are especially preferred. A hybridization signal can comprise fluorescence, chemiluminescence, or isotopic labeling, etc.; or sequences in a sample can be detected by direct means, e.g. mass spectrometry. The method's characteristic sequences can also be used to design specific PCR primers. The method uniquely identifies the phylogenetic affinity of an unknown organism without requiring prior knowledge of what is present in the sample. Even if the organism has not been previously encountered, the method still provides useful information about which phylogenetic tree bifurcation nodes encompass the organism.

Fox, George E.↗
NASA NTRS2013

Characterization of Microbial Communities Found in Bioreactor Effluent

The purpose of this investigation was to examine microbial communities of simulated wastewater effluent from hollow fiber membrane bioreactors collected from the Space Life Science Laboratory and Texas Technical University. Microbes were characterized using quantitative polymerase chain reaction where a total count of bacteria and fungi were determined. The primers that were used to determine the total count of bacteria and fungi were targeted for 16S rDNA genes and the internal transcribed spacer, respectively. PCR products were detected with SYBR Green I fluorescent dye and a melting curve analysis was performed to identify unique melt profiles resulting from DNA sequence variations from each species of the community. Results from both the total bacteria and total fungi count assays showed that distinct populations were present in isolates from these bioreactors. This was exhibited by variation in the number of peaks observed on the melting curve analysis graph. Further analysis of these results using species-specific primers will shed light on exactly which microbes are present in these effluents. Information gained from this study will enable the design of a system that can efficiently monitor microbes that play a role in the biogeochemical cycling of nitrogen in wastewater on the International Space Station to assist in the design of a sustainable system capable of converting this nutrient.

Flowe, Candice↗
NASA NTRS2014

Salt Tolerance and Polyphyly in the Cyanobacterium Chroococcidiopsis (Pleurocapsales)1

Chroococcidiopsis Geitler (Geitler 1933) is a genus of cyanobacteria containing desiccation and radiation resistant species. Members of the genus live in habitats ranging from hot and cold deserts to fresh and saltwater environments. Morphology and cell division pattern have historically been used to define the genus. To better understand the genetic and phenotypic diversity of the genus, 15 species were selected that had been previously isolated from different locations, including salt and freshwater environments. Four markers were sequenced from these 15 species, the 16S rRNA, rbcL, desC1 and gltX genes. Phylogenetic trees were generated which identified two distinct clades, a salt-tolerant clade and a freshwater clade. This study demonstrates that the genus is polyphyletic based on saltwater and freshwater phenotypes. To understand the resistance to salt in more details, species were grown on a range of sea salt concentrations which demonstrated that the freshwater species were salt-intolerant whilst the saltwater species required salt for growth. This study shows an increased resolution of the phylogeny of Chroococcidiopsis and provides further evidence that the genus is polyphyletic and should be reclassified to improve clarity in the literature.

Genus↗
NASA NTRS2011

Deep Diversity: Novel Approach to Overcoming the PCR Bias Encountered During Environmental Analysis of Microbial Populations for Alpha-Diversity

Alpha-diversity studies are of crucial importance to environmental microbiologists. The polymerase chain reaction (PCR) method has been paramount for studies interrogating microbial environmental samples for taxon richness. Phylogenetic studies using this technique are based on the amplification and comparison of the 16S rRNA coding regions. PCR, due disproportionate distribution of microbial species in the environment, increasingly favors the amplification of the most predominant phylotypes with every subsequent reaction cycle. The genetic and chemical complexity of environmental samples are intrinsic factors that exacerbate an inherit bias in PCR-based quantitative and qualitative studies of microbial communities. We report that treatment of a genetically complex total genomic environmental DNA extract with Propidium Monoazide (PMA), a DNA intercalating molecule capable of forming a covalent cross-linkage to organic moieties upon light exposure, disproportionally inactivates predominant phylotypes and results in the exponential amplification of previously shadowed microbial ?-diversity quantified as a 19.5% increase in OUTs reported via phylogenetic screening using PhyloChip.

phylogenetic microarray technologies↗
NASA NTRS2015

Inflight Microbial Monitoring- An Alternative Method to Culture Based Detection Currently Used on the International Space Station

Previous research has shown that potentially destructive microorganisms and human pathogens have been detected on the International Space Station (ISS). The likelihood of introducing new microorganisms occurs with every exchange of crew or addition of equipment or supplies. Microorganisms introduced to the ISS are readily transferred between crew and subsystems (i.e. ECLSS, environmental control and life support systems). Current microbial characterization methods require enrichment of microorganisms and at least a 48-hour incubation time. This increases the microbial load while detecting only a limited number of the total microorganisms. The culture based method detects approximately 1-10% of the total organisms present and provides no identification. To identify and enumerate ISS microbes requires that samples be returned to Earth for complete analysis. Therefore, a more expedient, low-cost, in-flight method of microbial detection, identification, and enumeration is warranted. The RAZOR EX, a ruggedized, commercial off the shelf, real-time PCR field instrument was tested for its ability to detect microorganisms at low concentrations within one hour. Escherichia coli, Salmonella enterica Typhimurium, and Pseudomonas aeruginosa were detected at low levels using real-time DNA amplification. Total heterotrophic counts could also be detected using a 16S gene marker that can identify up to 98% of all bacteria. To reflect viable cells found in the samples, RNA was also detectable using a modified, single-step reverse transcription reaction.

Inflight Microbial↗
NASA NTRS2015

In-Flight Microbial Monitor

Previous research has shown that potential human pathogens have been detected on the International Space Station (ISS). New microorganisms are introduced with every exchange of crew and cargo. Microorganisms introduced to the ISS are readily transferred between crew and subsystems (i.e., ECLSS, environmental control and life support systems). Current microbial characterization methods require a culture-based enrichment of microorganisms and at least a 48-hour incubation time. This increases the microbial load while detecting only a limited number of microorganisms. The culture-based method detects approximately 1-10% of the total organisms present and provides no identification. To identify and enumerate ISS samples requires that the microbes be returned to Earth for complete analysis. Therefore, a more expedient, low-cost, inflight method of microbial detection, identification, and enumeration is needed. The RAZOR EX, a ruggedized, commercial off the shelf, real-time PCR field instrument was tested for its ability to detect microorganisms at low concentrations within one hour. Escherichia coli, Salmonella enterica Typhimurium, and Pseudomonas aeruginosa were detected at low levels using real-time DNA amplification. Total heterotrophic counts could also be detected using a 16S gene marker that can identify up to 98% of all bacteria. To reflect viable cells found in the samples, RNA was also detectable using a modified, single-step reverse transcription reaction.

Technology Portfolio System↗
NASA NTRS2015

Inflight Microbial Monitoring-An Alternative Method to Culture Based Detection Currently Used on International Space Station

Previous research has shown that microorganisms and potential human pathogens have been detected on the International Space Station (ISS). The potential to introduce new microorganisms occurs with every exchange of crew or addition of equipment or supplies. Previous research has shown that microorganisms introduced to the ISS are readily transferred between crew and subsystems and back (i.e. ECLSS, environmental control and life support systems). Current microbial characterization methods require enrichment of microorganisms and a 48-hour incubation time. This increases the microbial load while detecting a limited number of microorganisms. The culture based method detects approximately 1-10% of the total organisms present and provides no identification, To identify and enumerate ISS samples requires that samples to be returned to Earth for complete analysis. Therefore, a more expedient, low-cost, in-flight method of microbial detection, identification, and enumeration is warranted. The RAZOR EX, a ruggedized, commercial off the shelf, real-time PCR field instrument was tested for its ability to detect microorganism at low concentrations within one hour. Escherichia coli, Salmonella enterica Typhimurium, and Pseudomonas aeruginosa were detected at low levels using real-time DNA amplification. Total heterotrophic counts could also be detected using a 16S gene marker that can identify up to 98% of all bacteria. To reflect viable cells found in the samples, RNA was also detectable using a modified, single-step reverse transcription reaction.

Inflight Microbial↗
NASA NTRS2017

Identification of Fungal Colonies on Ground Control and Flight Veggie Plant Pillows

The Veggie system focuses on growing fresh produce that can be harvested and consumed by astronauts. The microbial colonies in each Veggie experiment are evaluated to determine the safety level of the produce and then differences between flight and ground samples. The identifications of the microbial species can detail risks or benefits to astronaut and plant health. Each Veggie ground or flight experiment includes six plants grown from seeds that are glued into wicks in Teflon pillows filled with clay arcillite and fertilizer. Fungal colonies were isolated from seed wicks, growth media, and lettuce (cv. 'Outredgeous') roots grown in VEG-01B pillows on ISS and in corresponding ground control pillows grown in controlled growth chambers. The colonies were sorted by morphology and identified using MicroSeq(TM) 500 16s rDNA Bacterial Identification System and BIOLOG GEN III MicroPlate(TM). Health risks for each fungal identification were then assessed using literature sources. The goal was to identify all the colonies isolated from flight and ground control VEG-01B plants, roots, and rooting medium and compare the resulting identifications.

Scotten, Jessica E.↗
NASA NTRS2016

Permanent Draft Genome of Strain ESFC-1: Ecological Genomics of a Newly Discovered Lineage of Filamentous Diazotrophic Cyanobacteria

The nonheterocystous filamentous cyanobacterium, strain ESFC-1, is a recently described member of the order Oscillatoriales within the Cyanobacteria. ESFC-1 has been shown to be a major diazotroph in the intertidal microbial mat system at Elkhorn Slough, CA, USA. Based on phylogenetic analyses of the 16S RNA gene, ESFC-1 appears to belong to a unique, genus-level divergence; the draft genome sequence of this strain has now been determined. Here we report features of this genome as they relate to the ecological functions and capabilities of strain ESFC-1. The 5,632,035 bp genome sequence encodes 4914 protein-coding genes and 92 RNA genes. One striking feature of this cyanobacterium is the apparent lack of either uptake or bi-directional hydrogenases typically expected within a diazotroph. Additionally, a large genomic island is found that contains numerous low GC-content genes and genes related to extracellular polysaccharide production and cell wall synthesis and maintenance.

R Craig Everroad↗
NASA NTRS2017

Comparison of Two Bioinformatics Tools Used to Characterize the Microbial Diversity and Predictive Functional Attributes of Microbial Mats from Lake Obersee, Antarctica

In this study, using NextGen sequencing of the collective 16S rRNA genes obtained from two sets of samples collected from Lake Obersee, Antarctica, we compared and contrasted two bioinformatics tools, PICRUSt and Tax4Fun. We then developed an R script to assess the taxonomic and predictive functional profiles of the microbial communities within the samples. Taxa such as Pseudoxanthomonas, Planctomycetaceae, Cyanobacteria Subsection III, Nitrosomonadaceae, Leptothrix, and Rhodobacter were exclusively identified by Tax4Fun that uses SILVA database; whereas PICRUSt that uses Greengenes database uniquely identified Pirellulaceae, Gemmatimonadetes A1-B1, Pseudanabaena, Salinibacterium and Sinibacteraceae. Predictive functional profiling of the microbial communities using Tax4Fun and PICRUSt separately revealed common metabolic capabilities, while also showing specific functional IDs not shared between the two approaches. Combining these functional predictions using a customized R script revealed a more inclusive metabolic profile, such as hydrolases, oxidoreductases, transferases; enzymes involved in carbohydrate and amino acid metabolisms; and membrane transport proteins known for nutrient uptake from the surrounding environment. Our results present the first molecular-phylogenetic characterization and predictive functional profiles of the microbial mat communities in Lake Obersee, while demonstrating the efficacy of combining both the taxonomic assignment information and functional IDs using the R script created in this study for a more streamlined evaluation of predictive functional profiles of microbial communities.

Hyunmin Koo↗
NASA NTRS2018

Monitoring Astronaut Health with DNA Sequencing

In recent years microbe a plethora of microbe populations have been identified onboard the ISS (International Space Station). Approaches for real-time tracking of microbes for routine housekeeping and food/water safety monitoring will be critical for mission safety and crew health on future longer duration missions to the Moon or Mars. This work is a proof-of-concept study demonstrating an end-to-end phylogenetic identification and full genome sequencing effort of multiple microbial populations. Our methodology utilized the ISS flight-certified WetLab-2 molecular toolbox and the Biomolecule Sequencer projects for real-time end-to-end on-orbit microbial biological samples processing and molecular analysis with real time results generated utilizing only field "offline" analytic software. For this experiment we colony-cultured several ISS isolated microorganisms before generation of the pre-sequencing library via the automated VolTRAX device which enabled high library turnover with little wet-bench activity or potential future costly astronaut time. The pre-sequencing library is diluted in loading buffer and injected into the MinION sample port, drawn into the nanopore window by capillary action, and sequenced using the MinKnown. 16S and full genome alignment, nucleotide matching, gene identification, and phylogenetic sorting was accomplished utilizing the Epi2me software and the offline NCBI Blast viral, microbiome, and human somatic databases. In short, the methodologies developed herein replace the myriad of specific, often highly targeted microbiological tests used in the clinical laboratory, which would be difficult if not impossible to currently implement aboard the ISS or in deep space, with a single metagenomics test.

genomics↗
NASA NTRS2019

A Lab with a View: Findings from NASA's Aircraft Bioaerosol Collector

Airborne microorganisms in the upper troposphere and lower stratosphere remain elusive due to a lack of reliable sample collection systems. To address this problem, we designed, installed, and flight-validated a novel Aircraft Bioaerosol Collector (ABC) for NASA's C-20A that can make collections for microbiological research investigations up to altitudes of 13.7 km. Herein we report results from the first set of science flights-four consecutive missions flown over the United States (US) from 30 October to 2 November, 2017. To ascertain how the concentration of airborne bacteria changed across the tropopause, we collected air during aircraft Ascent/Descent (0.3 to 11 km), as well as sustained Cruise altitudes in the lower stratosphere (~12 km). Bioaerosols were captured on DNA-treated gelatinous filters inside a cascade air sampler, then analyzed with molecular and culture-based characterization. Several viable bacterial isolates were recovered from flight altitudes, including Bacillus sp., Micrococcus sp., Arthrobacter sp., and Staphylococcus sp. from Cruise samples and Brachybacterium sp. from Ascent/Descent samples. Using 16S V4 sequencing methods for a culture-independent analysis of bacteria, the average number of total OTUs was 305 for Cruise samples and 276 for Ascent/Descent samples. Surprisingly, our results revealed a homogeneous distribution of bacteria in the atmosphere up to 12 km. The observation could be due to atmospheric conditions producing similar background aerosols across the western US, as suggested by modeled back trajectories and satellite measurements. However, the influence of aircraft-associated bacterial contaminants could not be fully eliminated and that background signal was reported throughout our dataset. Considering the tremendous engineering challenge of collecting biomass at extreme altitudes where contamination from flight hardware remains an ever-present issue, we note the utility of using the stratosphere as a proving ground for planned life detection missions across the solar system.

bioaerosols↗
NASA NTRS2019

Plant Microbiomes May Provide Vital Information to Plant Success

Plant associated microbiomes, the rhizosphere and phyllosphere, are composed of communities of bacteria and fungi that may be mutualistic or pathogenic. These communities have the potential to influence plant health and development and can affect plant growth. Crop plants are being investigated as a fresh and safe supplement to astronauts’ diet and it is critical to understand and characterize these microbial communities. Multi-species crops, Mizuna mustard (Brassica rapa var japonica), ‘Outredgeous’ red romaine lettuce (Lactuca sativa), and Waldman’s Green lettuce (Lactuca sativa) were grown in two Veggie units on the International Space Station (ISS) for three grow outs in various combinations of plant types. Upon harvest, plant and pillow samples were frozen and returned to Earth for analysis. Bacterial and fungal community analyses for plant leaf and root, as well as pillow components, wick and media, were completed using next generation sequencing with the goal of surveying the composition of the entire community and identifying any potential pathogens. Bacteria were identified using the 16S rRNA gene whereas, fungi were identified with the internal transcribed spacer (ITS). The community composition for these three crops was compared between crop types and between plant tissue types. It is vital to mission success for the short term and long term to add nutritious, safe to eat vegetables providing a supplement to the crew members’ dietary requirements as well as to develop planning for deep space missions as we reach for the moon and on to Mars. Veggie technology validation tests were supported by NASA’s Space Biology Program.

Khodadad, Christina L.↗
NASA NTRSDate not supplied

A METHOD TO REDUCE BIOBURDEN IN ASTROMATERIALS CURATION FACILITIES WITHOUT INTRODUCING UNWANTED CONTAMINATION

Introduction: NASA curates its Astromaterials collections in cleanrooms that are carefully monitored for particulate, inorganic and trace metal contamination. Current sample collections are not particularly susceptible to organic contamination or biological alteration. However, new collections like those from the OSIRIS-REx and Hayabusa2 missions will have organic contamination requirements and are susceptible to biodegradation. It will be necessary sterilize or at least disinfect curation labs, as well as tools and equipment in a manner that does not introduce additional contamination and does not affect the samples 1. Current curation cleaning procedures utilize isopropyl alcohol which offers some bioburden reduction, but is not effective against spore-forming bacteria or fungal spores 2. We present a modified disinfection method that uses ultrapure hydrogen peroxide to reduce bioburden inside curation labs and glove boxes without introducing contamination or damaging curation equipment. We tested this method in the meteorite processing lab as well as on a glovebox being cleaned for use in processing ANGSA (Apollo Next Generation Sample Analysis) samples and present the results of those tests. We discuss the limitations of this method and describe potential situations in which it will not be applicable. The CDC guidelines for disinfection andsterilization in healthcare facilities discusses over 15different methods for reducing bioburden in hospitalsettings 3. The most common method, steamsterilization, is well suited to sterilizing curationprocessing tools but cannot easily be used to sterilizecleanroom surfaces or large equipment likegloveboxes. Chemical sterilization with bleach(NaOCl) is also a common strategy in healthcare andpharmaceutical settings that presents materialcompatibility issues as well as serious inorganiccontamination concerns for curation facilities.Introducing a new source of Na and Cl into curationlabs is not acceptable. Other chemical methods likeethylene oxide, formaldehyde, iodophors andquaternary ammonium compounds could introduceorganic and inorganic contamination. We chose tofocus on hydrogen peroxide because it is generallycompatible with commonly used curation materialslike stainless steel, aluminum and Teflon and becauseit decomposes to oxygen and water. The CDCguidelines for hydrogen peroxide specify using a 7.5wt% solution at 25 ̊C with a contact time of 30 minutesfor high level disinfection and 6 hours for sterilization.High level disinfection is defined as a technique thatwill kill all microorganisms except large numbers ofbacterial spores 3. Methods: We prepared a solution of 7.5 wt%hydrogen peroxide from a stock solution of ultrapure30 wt% peroxide (JT Baker) and curation gradeultrapure water. This ultrapure water is already used incuration cleaning procedures and thus is not consideredand additional source of contamination. We conducteda materials compatibility test by exposing unanodizedand anodized 6061 T6 Al alloy to the peroxide solutionfor up to six hours and periodically inspecting thesurfaces for visible defects. We used this peroxide todisinfect the floor of the meteorite processing lab andthe interior of a curation glovebox by exposing thesesurfaces to the peroxide solution for 30 min. Thesurfaces were swabbed with a dry macrofoam swabbefore (Puritan Brand 2518051PFRNDFD) and afterperoxide treatment to collect microbes present on thesurfaces. Microbes were extracted by sonication fromthe swab into 15 ml of PBS (phosphate buffered saline)and inoculated onto the following media: TSA (trypticsoy agar) BA (blood agar), R2A (Reasoners 2 agar),Potato Dextrose Agar, Saboraud Dextrose Agar andSaboraud Dextrose Agar with 0.1 mg/ mlchloramphenicol. Four TSA plates and two BA plateswere inoculated with 0.1 ml of PBS each andincubated at 35 and 37 for 48 hours. Two R2A°C°Cplates (0.1 ml of PBS each) were incubated at 25 .°CThe remaining plates were inoculated with 0.2ml ofPBS and incubated at 30 ̊C for seven days. Afterincubation bacterial and fungal isolates were countedand transferred to new plates for identification usingthe VITEK24 automated system or by sequencing aportion of the barcode gene (16S rRNA for bacteria,small subunit gene for fungi) on an ABI 3500 Sangersequencer. Negative controls consisted of swabs thatwere opened in the sampling environment andanalyzed alongside the experimental samples.Results: A 6 hour exposure to hydrogen peroxideresulted in visible pitting on un-anodized 6061 Al, butnot on anodized surfaces. No visible pitting occurredafter a 30 minute exposure. Therefore, we decided tolimit our experimental tests to 30 min. exposures. 17bacterial CFU (colony forming units) representing 4distinct organisms were isolated from the meteorite processing lab floor prior to hydrogen peroxidetreatment. We were unable culture any organisms afterperoxide treatment. In the glovebox we were able toculture three bacterial CFU representing three distinctspecies, including a spore forming bacterium prior todisinfection with peroxide. After the peroxidetreatment we were unable to culture any organisms.Routine monitoring of the meteorite processing lab andthe glovebox did not indicate any increase in unwantedinorganic contamination after these peroxidetreatments. Discussion: A 30 minute treatment with 7.5 wt%peroxide appears to be an effective method forreducing bioburden on typical cleanroom surfaces. Themethod does not introduce unwanted organic orinorganic contamination and is compatible withcommonly used curation materials like stainless steel,Teflon and anodized aluminum alloys. Special careshould be taken with un-anodized aluminum.Prolonged exposure to hydrogen peroxide can causepitting on this material. We recommend using thismethod to disinfect curation labs and equipment whenbiological alteration is a concern. This method iseffective at room temperature and cannot be used todisinfect labs and equipment where the ambienttemperature is < 0 ̊C. Astromaterials samples shouldbe removed from the area where disinfection is tooccur. Hydrogen peroxide is a powerful oxidizingagent and will react with any organic carbon present inthe sample. References: [1.] Mccubbin, F. M. et al.Sp. Sci Rev(2019) doi:10.1007/s11214-019-0615-9. [2.] Mogul, R.et al.Astrobiology 18, ast.2017.1814 (2018). [3.]Rutala, W. A. & Weber, D. J. Guideline for Disinfection and Sterilization in Healthcare Facilities, 2008. [4.] Pincus, D. H. in Encyclopedia of Rapid Microbiological Methods (2005).

A. B. Regberg↗
NASA NTRS2005

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↗