Quantitative and Qualitative Microbiological Profiles of the Apollo 10 and 11 Spacecraft

Microbiological profiles were determined for surfaces of the command module, lunar module (ascent and descent stages), instrument unit, Saturn S-4B stage, and the spacecraft lunar module adapter of the Apollo 10 and 11 spacecraft. Average levels of contamination of the command module were 2.1 x 10(4) and 2.7 x 10(4) microorganisms per ft(2) for Apollo 10 and 11, respectively. With the exception of the exterior surfaces of the ascent stage of the lunar module and the interior surfaces of the command module, average levels of microbial contamination on all components of the Apollo 11 were found to be lower than those observed on Apollo 10. For each Apollo mission, approximately 2,000 colonies were picked from a variety of media and identified. The results showed that approximately 95% of all isolates were those considered indigenous to humans; the remaining were associated with soil and dust in the environment. However, the ratio of these two general groups varied depending on the degrees of personnel density and environmental control associated with each module.     

Microbiological Profiles of Four Apollo Spacecraft

Selected surfaces from the Command Module, Lunar Module (ascent and descent stages), Instrument Unit, Saturn S-4B engine, and Spacecraft Lunar Module Adapter comprised the various components of four Apollo spacecraft which were assayed quantitatively and qualitatively for microorganisms. In addition, the first Lunar Roving Vehicle was assayed. Average levels of microbial contamination (10(4) per square foot of surface) on the Command Module, Instrument Unit, and Saturn S-4B engine were relatively consistent among spacecraft. The first postflight sampling of interior surfaces of the Command Module was possible due to elimination of the 21-day back-contamination quarantine period. Results of the pre- and postflight samples revealed increases in the postflight samples of 3 logs/inch(2). A total of 5,862 microbial isolates was identified; 183 and 327 were obtained from the Command Module at preflight and postflight sampling periods, respectively. Although the results showed that the majority of microorganisms isolated were those considered to be indigenous to humans, an increase in organisms associated with soil and dust was noted with each successive Apollo spacecraft.     

Comparative levels and types of microbial contamination detected in industrial clean rooms

The primary objective of this study was to determine quantitatively and qualitatively the predominant types of microbial contamination occurring in conventional and laminar flow clean rooms. One horizontal laminar flow, three conventional industrial clean rooms, and three open factory areas were selected for microbiological tests. The results showed that as the environment and personnel of a clean room were controlled in a more positive manner with respect to the reduction of particulate contamination, the levels of airborne and surface microbial contaminants were reduced accordingly. The chief sources of microbial contamination were associated with the density and activity of clean room personnel. In addition, the majority of microorganisms isolated from the intramural air by air samplers were those indigenous to humans. Studies on the fallout and accumulation of airborne microorganisms on stainless-steel surfaces showed that, although there were no significant differences in the levels of microbial contamination among the conventional clean rooms, the type of microorganism detected on stainless-steel surfaces was consistently and significantly different. In addition, the "plateau phenomenon" occurred in all environments studied. It was concluded that the stainless-steel strip method for detecting microbial accumulation on surfaces is efficient and sensitive in ultra-clean environments and is the most reliable and practical method for monitoring microbial contamination in future class 100 clean rooms to be used for the assembly of spacecraft which will be sterilized.     

Evaluation of Membrane Filter Field Monitors for Microbiological Air Sampling

Due to area constraints encountered in assembly and testing areas of spacecraft, the membrane filter field monitor (MF) and the National Aeronautics and Space Administration-accepted Reyniers slit air sampler were compared for recovery of airborne microbial contamination. The intramural air in a microbiological laboratory area and a clean room environment used for the assembly and testing of the Apollo spacecraft was studied. A significantly higher number of microorganisms was recovered by the Reyniers sampler. A high degree of consistency between the two sampling methods was shown by a regression analysis, with a correlation coefficient of 0.93. The MF samplers detected 79% of the concentration measured by the Reyniers slit samplers. The types of microorganisms identified from both sampling methods were similar. Variables in the MF samplers, such as pore size, relative humidity, and flow rates, have been studied, but no effect was noted on recovery. The results show that the MF method could be used to estimate the number and types of microorganisms found in the air.     

Microbial Monitoring of Spacecraft and Associated Environments

Rapid microbial monitoring technologies are invaluable in assessing contamination of spacecraft and associated environments. Universal and widespread elements of microbial structure and chemistry are logical targets for assessing microbial burden. Several biomarkers such as ATP, LPS, and DNA (ribosomal or spore-specific), were targeted to quantify either total bioburden or specific types of microbial contamination. The findings of these assays were compared with conventional, culture-dependent methods. This review evaluates the applicability and efficacy of some of these methods in monitoring the microbial burden of spacecraft and associated environments. Samples were collected from the surfaces of spacecraft, from surfaces of assembly facilities, and from drinking water reservoirs aboard the International Space Station (ISS). Culture-dependent techniques found species of Bacillus to be dominant on these surfaces. In contrast, rapid, culture-independent techniques revealed the presence of many Gram-positive and Gram-negative microorganisms, as well as actinomycetes and fungi. These included both cultivable and noncultivable microbes, findings further confirmed by DNA-based microbial detection techniques. Although the ISS drinking water was devoid of cultivable microbes, molecular-based techniques retrieved DNA sequences of numerous opportunistic pathogens. Each of the methods tested in this study has its advantages, and by coupling two or more of these techniques even more reliable information as to microbial burden is rapidly obtained.     

Terrestrial contamination in Apollo lunar samples

The Apollo lunar samples were seen to offer a unique opportunity in the search for extraterrestrial organic matter without the ambiguity surrounding meteorite analysis due to their unknown contamination histories. The recognition that only a small amount of indigenous organic material was likely to be present in lunar samples combined with the extreme sensitivity of organic analysis methods made it clear that this opportunity could be realized only by carefully controlling the collection, processing, and analysis of the samples in order that they might remain free of significant levels of contamination. The contamination control procedures adopted are described and the analytical evidence obtained throughout the program on potential contamination sources is presented. The organic contaminants actually found in the lunar samples by the various investigators are summarized. It is shown that the program succeeded in providing investigators with samples containing less than 0.1 ppm total contamination.     

Microbial biomass and activity distribution in an anoxic, hypersaline basin

The evaluation of an improved wipe-rinse technique for the bioassay of large areas was undertaken due to inherent inadequacies in the cotton swab-rinse technique to which assay of spacecraft is currently restricted. Four types of contamination control cloths were initially tested. A polyester-bonded cloth (PBC) was selected for further evaluation because of its superior efficiency and handling characteristics. Results from comparative tests with PBC and cotton swabs on simulated spacecraft surfaces indicated a significantly higher recovery efficiency for the PBC than for the cotton (90.4 versus 75.2%). Of the sampling areas sites studied, PBC was found to be most effective on surface areas not exceeding 0.74 m2 (8.0 feet 2).     

Organic contamination problems in the Viking molecular analysis experiment

The combination of analytical instrumentation selected for the molecular analysis experiment can carry out a survey of the organic compounds present on Mars regardless of their origin. The high sensitivity of this analysis, the limited number of samples which can be analyzed, the close proximity to the landed spacecraft on the surface of Mars which is accessible to the sampling device, the implications of the positive detection of indigenous organic matter in the Martian soil, and our previous experience with meteorites and lunar samples point to the need for a carefully designed program to maintain the inteprity of the analyzed Martian surface samples. A principal problem in interpreting the results of an organic analysis of an extraterrestrial sample is that of distinguishing contaminating material from indigenous material when unknown types and amounts of contaminants make their way into the sample being analyzed. An approach for control of sample integrity in the Viking molecular analysis experiment has been devised which we believe will eliminate such problems. Basically this involves (1) placing an upper limit on the amount of terrestrial contamination that can be tolerated and still allow scientifically meaningful analyses, (2) identifying the potential sources of contamination and analyzing their relative significance, (3) establishing methods to control these sources, and (4) obtaining complete information on the chemical composition of potential contaminants. Our previous experience in the Apollo mission has been of great value in developing the Viking program, perhaps the most important carryover being the recognition of the importance of establishing a comprehensive contamination control program in the early stages of mission planning and hardware design. The upper limit of total allowable organic contamination has been established as 1 μg g^?1. The principal source types, or modes, which contribute to the contamination load have been identified, each requiring a different approach to control. Spacecraft outgassing is controlled by materials selection to minimize outgassing and hermetic sealing whenever possible. Particulate fallout is controlled by selection of materials, particulate seals, cleaning of the spacecraft exterior, and clean room handling. The cleanliness of the direct sample path is controlled by severe materials limitations, ultracleaning, and pressurized sealing of the assembled hardware. Analysis of the relative probabilities of the sources contributing to the allowable contamination and consideration of the practical aspects of achieving a desired level of control for a particular source has resulted in an allocation ‘tree’ whereby fractions of the total allowable contamination are distributed to the various individual sources. These efforts have pointed out the need for more information concerning some of these sources and have actually dictated certain design changes in the spacecraft. Additional information was obtained experimentally on descent engine exhaust characteristics which led to the use of an organically cleaner fuel. In summary, the early recognition in the Viking mission of the importance of organic contamination control has allowed the evolution of a complete contamination control program encompassing spacecraft design, mission operations, flight operations, and the design of the science instrumentation for the molecular analysis experiment.     

Microbiological Burden on the Surfaces of Explorer XXXIII Spacecraft

The Explorer XXXIII Spacecraft (Anchored Interplanetary Monitoring Platform, or AIMP) was decontaminated to prevent gross contamination of the moon with terrestrial microorganisms. Assay of the total spacecraft surface before and after decontamination showed that the decontamination procedure reduced the viable microbiological burden from 1.40 x 10(6) to 3.60 x 10(4). However, assembly of parts which were not decontaminated for engineering reasons or were not assembled under cleanroom conditions increased the viable microbial burden at the time of launch to 2.62 x 10(5).     

The solar UV environment and bacterial spore UV resistance: considerations for Earth-to-Mars transport by natural processes and human spaceflight

The environment in space and on planets such as Mars can be lethal to microorganisms because of the high vacuum and high solar radiation flux, in particular UV radiation, in such environments. Spores of various Bacillus species are among the organisms most resistant to the lethal effects of high vacuum and UV radiation, and as a consequence are of major concern for planetary contamination via unmanned spacecraft or even natural processes. This review focuses on the spores of various Bacillus species: (i) their mechanisms of UV resistance; (ii) their survival in unmanned spacecraft, space flight and simulated space flight and Martian conditions; (iii) the UV flux in space and on Mars; (iv) factors affecting spore survival in such high UV flux environments.     

Potential effects of recent findings on spacecraft sterilization requirements

An important task related to the formulation of planetary quarantine standards is the achievement of an acceptable compromise between (1) the prevention of planetary contamination and (2) the impact of quarantine requirements on the conduct of planetary missions. Such a task is a continuing effort, which must take all pertinent new information into account as it becomes available. This paper provides an analytical framework for the assessment of data which have become available during the past year or which are currently being evolved. In particular an evaluation is made of the probability of release of viable organisms from the spacecraft as a function of: (1) impact velocity magnitudes and the probability of their occurrence; (2) the degree of equipment fracturing at impact velocities; and (3) the number of viable organisms in spacecraft materials. Work being done to quantify each of three types of contamination, i.e. that on open surfaces, mated surfaces and buried contamination, is described in the context of seeking an approach to spacecraft sterilization that would be most compatible with the implementation of planetary missions. It is concluded that the results of work now in progress on spacecraft-material fracturing, on the estimation of buried contamination loads, and on microbial resistance on mated surfaces, may lead to less severe dry-heat sterilization of planetary spacecraft than had been considered necessary in the past.Work reported herein by Exotech Inc. authors has been supported under contract NASw-1558 with the NASA Office of Planetary Program and under contract NASw-1666 with the NASA Office of Biosciences.     

Metabolism of Di- and Mono-n-Butyl Phthalate by Soil Bacteria

Samples for mycological analysis were collected from surfaces in the Skylab spacecraft before launch and during flight for each manned mission. Fungal contamination levels were low during the first two flights; however, the species recovered were different for each mission. On the third mission, widespread contamination of the Skylab spacecraft with Aspergillus and Pencillium spp. was detected. This contamination was traced to several contaminated space suit undergarments.     

Research for amino acids in lunar samples

Water extracts of lunar fines were analyzed for amino acids by a gas-liquid chromatographic technique whereby amino acids were converted to the N-trifluoroacetyln-butyl, esters prior to analysis. The lunar material studied included both Apollo 14 (14240 SESC and 14298) and Apollo 12 (12023) samples. The water extract of the special Apollo 14 sample (14240 SESC) was analyzed both for free and bound amino acids (hydrolysis with 6 N hydrochloric acid). In both the hydrolyzed and unhydrolyzed extracts, the amino acids were not observed above background levels.The analysis of Apollo 12 and 14 samples (12023 14298) yielded similar results. Detection limits were established at 300 pg to 1 ng for different amino acids. A large chromatographic peak with a retention temperature of 126°C was observed on analysis of sample, (12023); it was identified as oxalic acid by GC-MS. The concentration of amino acids in the Apollo 14 SESC samples processed and analyzed in the joint experiments at Ames by GLC and IEC were found to be extremely low (glycine at 3 to 4 ng g^–1). As the quantities were so minute, these identifications could not be confirmed by GLC-MS and therefore should still be considered as tentative. Other studies included the analysis of performance standards at the 2 to 6 ng level of each of 17 amino acids, and the analysis of 5 ml of H₂O containing 2 ppb of each amino acid. Recovery of amino acids added to lunar fines were conducted at the 10, 50, and 70 ng level of each amino acid with 50 to 70 mg of lunar material. The recoveries varied from as high as 80% for some of the aliphatics to complete loss of the amino acids ornithine and lysine.Contributed from Missouri Agricultural Experiment Station Journal Series No. 6255. Approved by the Director. Supported in part by grants from the National Aeronautics and Space Administration (NGR 26-004-011) and the Experiment Station Chemical Laboratories.     

Comparison of innovative molecular approaches and standard spore assays for assessment of surface cleanliness

A bacterial spore assay and a molecular DNA microarray method were compared for their ability to assess relative cleanliness in the context of bacterial abundance and diversity on spacecraft surfaces. Colony counts derived from the NASA standard spore assay were extremely low for spacecraft surfaces. However, the PhyloChip generation 3 (G3) DNA microarray resolved the genetic signatures of a highly diverse suite of microorganisms in the very same sample set. Samples completely devoid of cultivable spores were shown to harbor the DNA of more than 100 distinct microbial phylotypes. Furthermore, samples with higher numbers of cultivable spores did not necessarily give rise to a greater microbial diversity upon analysis with the DNA microarray. The findings of this study clearly demonstrated that there is not a statistically significant correlation between the cultivable spore counts obtained from a sample and the degree of bacterial diversity present. Based on these results, it can be stated that validated state-of-the-art molecular techniques, such as DNA microarrays, can be utilized in parallel with classical culture-based methods to further describe the cleanliness of spacecraft surfaces.     

Changes in the Fungal Autoflora of Apollo Astronauts

Specimens were repeatedly obtained for mycological examination from the skin, throat, urine, and feces of the six astronauts who conducted the Apollo 14 and Apollo 15 lunar exploration missions. Analysis of preflight data demonstrates that the process of severely restricting opportunities from colonization for 3 weeks before flight resulted in a 50% reduction in the number of isolated species. Postflight data indicate that exposure to the space flight environment for up to 2 weeks resulted in an even greater reduction with a relative increase in the potential pathogen Candida albicans. No incidences of microbial shock were observed when crewmembers were quarantined for 16 days after completion of the space flight. Intercrew transfer of particular species could not be demonstrated because most species were not consistently recovered.     

Microbiological profiles of the Viking spacecraft.

Planetary quarantine requirements associated with the launch of two Viking spacecraft necessitated microbiological assessment during assembly and testing at Cape Canaveral and the Kennedy Space Center. Samples were collected from selected surface of the Viking Lander Capsules (VLC), Orbiters, (VO), and Shrouds at predetermined intervals during assembly and testing. Approximately 7,000 samples were assayed. Levels of bacterial spores per square meter on the VLC-1 and VLC-2 were 1.6 x 10(2) and 9.7 x 10(1), respectively, prior to dry-heat sterilization. The ranges of aerobic mesophilic microorganisms detected on the VO-1 and VO-2 at various sampling events were 4.2 x 10(2) to 4.3 x 10(3) and 2.3 x 10(2) to 8.9 x 10(3)/m2, respectively. Approximately 1,300 colonies were picked from culture plates, identified, lypholipized, and stored for future reference. About 75% of all isolates were microorganisms considered indigenous to humans; the remaining isolates were associated with soil and dust in the environment. The percentage of microorganisms of human origin was consistent with results obtained with previous automated spacecraft but slightly lower than those observed for manned (Apollo) spacecraft.     

Pyrosequencing-derived bacterial, archaeal, and fungal diversity of spacecraft hardware destined for Mars

Spacecraft hardware and assembly cleanroom surfaces (233 m(2) in total) were sampled, total genomic DNA was extracted, hypervariable regions of the 16S rRNA gene (bacteria and archaea) and ribosomal internal transcribed spacer (ITS) region (fungi) were subjected to 454 tag-encoded pyrosequencing PCR amplification, and 203,852 resulting high-quality sequences were analyzed. Bioinformatic analyses revealed correlations between operational taxonomic unit (OTU) abundance and certain sample characteristics, such as source (cleanroom floor, ground support equipment [GSE], or spacecraft hardware), cleaning regimen applied, and location about the facility or spacecraft. National Aeronautics and Space Administration (NASA) cleanroom floor and GSE surfaces gave rise to a larger number of diverse bacterial communities (619 OTU; 20 m(2)) than colocated spacecraft hardware (187 OTU; 162 m(2)). In contrast to the results of bacterial pyrosequencing, where at least some sequences were generated from each of the 31 sample sets examined, only 13 and 18 of these sample sets gave rise to archaeal and fungal sequences, respectively. As was the case for bacteria, the abundance of fungal OTU in the GSE surface samples dramatically diminished (9× less) once cleaning protocols had been applied. The presence of OTU representative of actinobacteria, deinococci, acidobacteria, firmicutes, and proteobacteria on spacecraft surfaces suggests that certain bacterial lineages persist even following rigorous quality control and cleaning practices. The majority of bacterial OTU observed as being recurrent belonged to actinobacteria and alphaproteobacteria, supporting the hypothesis that the measures of cleanliness exerted in spacecraft assembly cleanrooms (SAC) inadvertently select for the organisms which are the most fit to survive long journeys in space.     

Release of microbial contamination from fractured solids

Estimation of the probability of release of microbial contamination from the interior of solids upon fracture due to impact is essential to the formulation of planetary quarantine and spacecraft sterilization requirements. A model system was designed in which known concentrations of bacterial spores were incorporated in methyl methacrylate plastic. Pieces of plastic were fractured in a uniform manner exposing interior surface areas of consistent and measurable size. Known surface areas were incubated in sets of 20 culture tubes containing liquid growth medium. The subsequent occurrence of visible growth expressed as percent of tubes positive was interpreted as an estimate of the probability of release of at least one viable micro-organism.From these experiments probability of release as a function of microbial concentration in plastic was estimated for exposed interior surface areas of 30.6, 61.2, 91.8 or 122.4 mm². Good agreement of the empirical results with a theoretical mathematical model of the probability of release of contamination from solids was demonstrated. Analysis of the data using the maximum likelihood procedure provided a means of calculating a proportionality constant representing the effective thickness of the exposed area and the characteristics of the recovery procedure.     

Search for amino acids in Apollo returned lunar soil.

The lunar samples from Apollo flights 11 through 17 provided the students of chemical evolution with an opportunity of examining extraterrestrial materials for evidence of early prebiological chemistry in the solar system. Our search was directed to water-extractable compounds with emphasis on amino acids. Gas chromatography, ion-exchange chromatography and gas chromatography combined with mass spectrometry were used for the analysis. It is our conclusion that amino acids are not present in the lunar regolith above the background levels of our investigations.     

A quest for porphyrins in lunar soil: Samples from Apollo 11, 12 and 14

Pigments exhibiting porphyrin-like behavior were detected in samples from Apollo 11 and 12. Those from Apollo 11 appeared to be directly related to the rocket exhaust of the descent engine; those from Apollo 12 from indigenous lunar material. Porphyrin-like pigments were not detected in a sample from Apollo 14.