How Many Particles are Present in The Air? Bioaerosol Detection Using an Air Particle Counter

Relative cleanliness in terms of particle abundance in spacecraft assembly facilities is determined by particle counts carried out in clean rooms during resting conditions. Particle counters assess total particles and particle size distribution, but do not distinguish inert particles from biological particles, which may include bacterial spores that are resistant to standard cleanroom sterilization procedures. Current cleanroom certifications do not fully assess the effects of human presence on spacecraft contamination since humans are known symbionts to enumerate microorganisms and assessments are performed at rest when there is no human presence. In this study, contamination risks and bioburden in spacecraft assembly facilities were determined by simultaneous detection of total biological and inert particle content and particle size distribution in JPL cleanrooms during working and resting conditions using BioVigilant IMD-350A, and metadata collection. The findings of this study clearly demonstrate a correlation between human activity levels and elevated levels of biological particles primarily of 0.5 to 1 micron size, contrary to the current literature, as well as elevated total particle counts compared to baseline resting conditions. The results of this study will serve as a model for reassessing current standards for bio-aerosol transport and serve as a feasibility assay to Mars 2020

Molecular Technique to Understand Deep Microbial Diversity

Current sequencing-based and DNA microarray techniques to study microbial diversity are based on an initial PCR (polymerase chain reaction) amplification step. However, a number of factors are known to bias PCR amplification and jeopardize the true representation of bacterial diversity. PCR amplification of the minor template appears to be suppressed by the exponential amplification of the 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 has overlooked the presence of the less abundant minority population, and underestimated their role in the ecosystem maintenance. To generate PCR amplicons for subsequent diversity analysis, bacterial l6S rRNA genes are amplified by PCR using universal primers. Two distinct PCR regimes are employed in parallel: one using normal and the other using biotinlabeled universal primers. PCR products obtained with biotin-labeled primers are mixed with streptavidin-labeled magnetic beads and selectively captured in the presence of a magnetic field. Less-abundant DNA templates that fail to amplify in this first round of PCR amplification are subjected to a second round of PCR using normal universal primers. These PCR products are then subjected to downstream diversity analyses such as conventional cloning and sequencing. A second round of PCR amplified the minority population and completed the deep diversity picture of the environmental sample

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

Extreme Ionizing-Radiation-Resistant Bacterium

There is a growing concern that desiccation and extreme radiation-resistant, non-spore-forming microorganisms associated with spacecraft surfaces can withstand space environmental conditions and subsequent proliferation on another solar body. Such forward contamination would jeopardize future life detection or sample return technologies. The prime focus of NASA s planetary protection efforts is the development of strategies for inactivating resistance-bearing microorganisms. Eradification techniques can be designed to target resistance-conferring microbial populations by first identifying and understanding their physiologic and biochemical capabilities that confers its elevated tolerance (as is being studied in Deinococcus phoenicis, as a result of this description). Furthermore, hospitals, food, and government agencies frequently use biological indicators to ensure the efficacy of a wide range of radiation- based sterilization processes. Due to their resistance to a variety of perturbations, the non-spore forming D. phoenicis may be a more appropriate biological indicator than those currently in use. The high flux of cosmic rays during space travel and onto the unshielded surface of Mars poses a significant hazard to the survival of microbial life. Thus, radiation-resistant microorganisms are of particular concern that can survive extreme radiation, desiccation, and low temperatures experienced during space travel. Spore-forming bacteria, a common inhabitant of spacecraft assembly facilities, are known to tolerate these extreme conditions. Since the Viking era, spores have been utilized to assess the degree and level of microbiological contamination on spacecraft and their associated spacecraft assembly facilities. Members of the non-spore-forming bacterial community such as Deinococcus radiodurans can survive acute exposures to ionizing radiation (5 kGy), ultraviolet light (1 kJ/sq m), and desiccation (years). These resistive phenotypes of Deinococcus enhance the potential for transfer, and subsequent proliferation, on another solar body such as Mars and Europa. These organisms are more likely to escape planetary protection assays, which only take into account presence of spores. Hence, presences of extreme radiation-resistant Deinococcus in the cleanroom facility where spacecraft are assembled pose a serious risk for integrity of life-detection missions. The microorganism described herein 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. This bacterium exhibits very low 16SrRNA similarity with any other environmental isolate reported to date. Both phenotypic and phylogenetic analyses clearly indicate that this isolate belongs to the genus Deinococcus and represents a novel species. The name Deinococcus phoenicis was proposed after the Phoenix spacecraft, which was undergoing assembly, testing, and launch operations in the spacecraft assembly facility at the time of isolation. D. phoenicis cells exhibited higher resistance to ionizing radiation (cobalt-60; 14 kGy) than the cells of the D. radiodurans (5 kGy). Thus, it is in the best interest of NASA to thoroughly characterize this organism, which will further assess in determining the potential for forward contamination. Upon the completion of genetic and physiological characteristics of D. phoenicis, it will be added to a planetary protection database to be able to further model and predict the probability of forward contamination

Identification of Bacteria and Determination of Biological Indicators

The ultimate goal of planetary protection research is to develop superior strategies for inactivating resistance bearing micro-organisms like Rummeli - bacillus stabekisii. By first identifying the particular physiologic pathway and/or structural component of the cell/spore that affords it such elevated tolerance, eradication regimes can then be designed to target these resistance-conferring moieties without jeopardizing the structural integrity of spacecraft hardware. Furthermore, hospitals and government agencies frequently use biological indicators to ensure the efficacy of a wide range of sterilization processes. The spores of Rummelibacillus stabekisii, which are far more resistant to many of such perturbations, could likely serve as a more significant biological indicator for potential survival than those being used currently

Phylogenetic Diversity of Microbial Isolates from the Mars Pathfinder

As spacecraft are sent to different planets, they take with them microscopic pieces of life from Earth. It is the task of the Biotechnology and Planetary Protection Group to keep as much of this life off other planets as possible as well as document any life that may have been sent. During the construction of the Mars Pathfinder, samples were collected from various locations on the spacecraft to test for contamination. These samples were then isolated, grown, documented, preserved and their 16S rRNA genes were sequenced for identification. The 16S rRNA gene sequence is utilized because it is a highly conserved portion of the transcriptional machinery of bacteria but also has known variable regions allowing it to be and amplified and used for distinguishing different genera and species of microbial life. All of the bacterial strains analyzed from this study were members of the genus Bacillus. Seventeen strains were sequenced and identified at greater than 98% homology to known type strains. After identifying the bacterial contaminant types, the National Aeronautics and Space Administration’s Jet Propulsion Laboratory will be able to better determine cleanliness protocols to maintain the international standard and protect Mars from Earth contamination on future missions

Microorganisms Associated with Mars-Bound Spacecraft: Preservation, Identification, Characterization

The Biotechnology and Planetary Protection Group (BPPG) at the Jet Propulsion Lab (JPL) focuses on avoiding forward and backward contamination between Earth and extraterrestrial bodies, ensuring that planetary bodies can be studied in their natural state in the future. This endeavor involves sampling organisms from Mars bound spacecraft during assembly, testing, and launch operations, archiving the organisms for long-term storage, and identifying the organisms through MALDI-TOF (matrix-assisted laser desorption and ionization time of flight) mass spectrometry. Because the MALDI-TOF database is primarily composed of clinical samples, it is necessary to continuously update the database with isolates collected from Marsbound spacecraft to make future identification efforts at BPPG easier. During this internship, efforts were made to continue updating the in-house MALDI-TOF database by studying microbial samples taken from spacecraft hardware and flight facilities. In addition, novel organisms from spacecraft required further biochemical and taxonomical identification, 16S rRNA sequencing, and addition of new spectral profiles to the MALDI database. These efforts culminated in a more comprehensive JPL in-house database, the characterization of several novel organisms, and the proper identification and storage of numerous microbial samples in accordance to BPPG standards

Collecting Diverse Microorganisms from Rover Spacecraft

. The Planetary Protection discipline at NASA’s Jet Propulsion Laboratory develops and implements procedures to prevent both forward and backward contamination between the Earth and solar system bodies. However, there will always be some microorganisms that will be resistant to the strictest of sterilization methods. In order understand the microorganisms found on spacecraft during assembly, and to rapidly identify them, a mass spectrometry approach was developed. As an experimental approach, a custom database was created for a subset of microorganisms in the Planetary Protection Archive. In order to make the database as accurate and efficient as possible, several different procedures have been developed on how to identify and classify each isolate within the database. Building upon previous research in the area, we designed a method characterizing revived isolates with known 16SrRNA gene sequence OTUs (Operational Taxonomy Units) to create MSPs (Mass Spectral Profiles) and RTCs (Real Time Classifications) using MALDI-TOF Mass Spectroscopy. We will use these profiles to enhance the Planetary Protection custom classification database, for immediate and future investigations. This work was carried out at NASA’s Jet Propulsion Laboratory, California Institute of Technology

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 bioreductio

Microbial Community Structures of Novel Icelandic Hot Spring Systems Revealed by PhyloChip G3 Analysis

Microbial community profiles of recently formed hot spring systems ranging in temperatures from 57°C to 100°C and pH values from 2 to 4 in Hveragerði (Iceland) were analyzed with PhyloChip G3 technology. In total, 1173 bacterial operational taxonomic units (OTUs) spanning 576 subfamilies and 38 archaeal OTUs covering 32 subfamilies were observed. As expected, the hyperthermophilic (100°C) spring system exhibited both low microbial biomass and diversity when compared to thermophilic (60°C) springs. Ordination analysis revealed distinct bacterial and archaeal diversity in geographically distinct hot springs. Slight variations in temperature (from 57°C to 64°C) within the interconnected pools led to a marked fluctuation in microbial abundance and diversity. Correlation and PERMANOVA tests provided evidence that temperature was the key environmental factor responsible for microbial community dynamics, while pH, H_(2)S, and SO_2 influenced the abundance of specific microbial groups. When archaeal community composition was analyzed, the majority of detected OTUs correlated negatively with temperature, and few correlated positively with pH. Key Words: Microbial diversity—PhyloChip G3—Acidophilic—Thermophilic—Hot springs—Iceland. Astrobiology 14, xxx–xxx