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NASA NTRS2002

Composition and physiological profiling of sprout-associated microbial communities

The native microfloras of various types of sprouts (alfalfa, clover, sunflower, mung bean, and broccoli sprouts) were examined to assess the relative effects of sprout type and inoculum factors (i.e., sprout-growing facility, seed lot, and inoculation with sprout-derived inocula) on the microbial community structure of sprouts. Sprouts were sonicated for 7 min or hand shaken with glass beads for 2 min to recover native microfloras from the surface, and the resulting suspensions were diluted and plated. The culturable fraction was characterized by the density (log CFU/g), richness (e.g., number of types of bacteria), and diversity (e.g., microbial richness and evenness) of colonies on tryptic soy agar plates incubated for 48 h at 30 degrees C. The relative similarity between sprout-associated microbial communities was assessed with the use of community-level physiological profiles (CLPPs) based on patterns of utilization of 95 separate carbon sources. Aerobic plate counts of 7.96 +/- 0.91 log CFU/g of sprout tissue (fresh weight) were observed, with no statistically significant differences in microbial cell density, richness, or diversity due to sprout type, sprout-growing facility, or seed lot. CLPP analyses revealed that the microbial communities associated with alfalfa and clover sprouts are more similar than those associated with the other sprout types tested. Variability among sprout types was more extensive than any differences between microbial communities associated with alfalfa and clover sprouts from different sprout-growing facilities and seed lots. These results indicate that the subsequent testing of biocontrol agents should focus on similar organisms for alfalfa and clover, but alternative types may be most suitable for the other sprout types tested. The inoculation of alfalfa sprouts with communities derived from various sprout types had a significant, source-independent effect on microbial community structure, indicating that the process of inoculation alters the dynamics of community development regardless of the types of organisms involved.

NASA Center KSC↗

NASA NTRS2004

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. Copyright 2004 Springer-Verlag.

Environmental Monitoring/methods↗

DOE OSTI2022

Comparison of Microbial Profiling and Tracer Testing for the Characterization of Injector-Producer Interwell Connectivities

Insufficient understanding of the microbial communities and associated microbial processes in geological reservoirs hinders the utilization of this rich data source for improved resource management. In this study, along with four interwell tracer tests at a 1478-m deep fractured crystalline-rock aquifer, we analyzed the microbial communities in the injected and produced water by high-throughput sequencing. The microbial community similarities across boreholes during an interwell flow scenario frequently encountered in reservoir development was explored. Despite the significant tracer recoveries (~30%) in all tracer tests and the cumulatively >100,000 L of exogenous water (carrying exogenous microbes) injected into the 10-m-scale reservoir, the overall structure of produced-fluid microbiome did not increasingly resemble that of the injectate. However, producers with better connectivity with the injector (based on tracer test results) did have more amplicon sequence variants (ASVs) that overlapped with those in the injectate. We identified possible drivers behind our observations and verified the practicality of repeated microbial sampling in the context of reservoir characterization and long-term monitoring. We highlight that injector-producer microbial profiling could provide insights on the relative connectivities across different producers with a given injector, and that the associated logistical needs may be comparable or even less than that of classic tracer tests.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2020

Soil microbial EPS resiliency is influenced by carbon source accessibility

The adaptability of soil microbial communities to prolonged periods of drought is influenced by their ability to produce extracellular polymeric substances (EPS) with sufficient water retention properties. Microbial EPSs as water reservoirs during drought have been extensively investigated, but it remains unknown how carbon substrate accessibility to soil microbial communities will affect the chemical properties of the EPS they generate, and whether this in turn will alter their water retention ability. In this work, we observed that the accessibility of carbon substrates influenced microbial community structure and, consequently, the chemical properties of EPS produced by the microbial communities. Further, our results demonstrated that an insoluble carbon substrate (i.e., chitin), stimulated microbial communities to produce EPS with better water retention properties in comparison to a soluble carbon substrate (i.e., N-acetylglucosamine; NAG). In all, this study demonstrates the importance of carbon substrate accessibility by soil microorganisms in regulating the community structure and consequently, the EPS carbon chemistry, which in turn can greatly influence the adaptability of soil microbial communities to drought.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2025

Drought increases microbial allocation to stress tolerance but with few tradeoffs among community-level traits

Climate change will increase soil drying, altering microbial communities via increasing water stress and decreasing resource availability. The responses of these microbial communities to changing environments could be governed by physiological tradeoffs between high yield, resource acquisition, and stress tolerance (YAS framework). We leveraged a unique field experiment that manipulates both drought and carbon availability across two years and three land uses, and we measured both physiological (with bioassays) and genetic (with metagenomics) microbial traits at the community level to test the following hypotheses: 1. Drought increases microbial allocation to stress tolerance functions, at both physiological and genetic levels. 2. Because microbes are resource-limited under drought, increased carbon will enable greater expression of stress tolerance. 3. All three key life history traits described in the YAS framework will trade off. Drought did increase microbial physiological investment in stress tolerance (measured via trehalose production), but we saw few other changes in microbial communities under drought. Adding carbon to plots increased resource acquisition (measured via enzyme activity and resource acquisition gene abundance) and stress tolerance (trehalose assay), but did so in both drought and average rainfall environments. Here, we found little evidence of trait tradeoffs, as a negative correlation between rRNA copy number and resource acquisition gene abundance was the only significant negative correlation between traits that we found that was consistent across years (for physiological or genetic traits). In summary, we found C addition, and to a lesser extent, drought, altered microbial community function and functional genes. However, resources did not alter drought response in a way that was consistent with theory of life history tradeoffs.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Microbial inoculum effects on the rumen epithelial transcriptome and rumen epimural metatranscriptome in calves

Manipulation of the rumen microbial ecosystem in early life may affect ruminal fermentation and enhance the productive performance of dairy cows. The objective of this experiment was to evaluate the effects of dosing three different types of microbial inoculum on the rumen epithelium tissue (RE) transcriptome and the rumen epimural metatranscriptome (REM) in dairy calves. For this objective, 15 Holstein bull calves were enrolled in the study at birth and assigned to three different intraruminal inoculum treatments dosed orally once weekly from three to six weeks of age. The inoculum treatments were prepared from rumen contents collected from rumen fistulated lactating cows and were either autoclaved (control; ARF), processed by differential centrifugation to create the bacterial-enriched inoculum (BE), or through gravimetric separation to create the protozoal-enriched inoculum (PE). Calves were fed 2.5 L/d pasteurized waste milk 3x/d from 0 to 7 weeks of age and texturized starter until euthanasia at 9 weeks of age, when the RE tissues were collected for transcriptome and microbial metatranscriptome analyses, from four randomly selected calves from each treatment. The different types of inoculum altered the RE transcriptome and REM. Compared to ARF, 9 genes were upregulated in the RE of BE and 92 in PE, whereas between BE and PE there were 13 genes upregulated in BE and 114 in PE. Gene ontology analysis identified enriched GO terms in biological process category between PE and ARF, with no enrichment between BE and ARF. The RE functional signature showed different KEGG pathways related to BE and ARF, and no specific KEGG pathway for PE. We observed a lower alpha diversity index for RE microbiome in ARF (observed genera and Chao1 (p < 0.05)). Five microbial genera showed a significant correlation with the changes in host gene expression: Roseburia (25 genes), Entamoeba (two genes); Anaerosinus, Lachnospira, and Succiniclasticum were each related to one gene. sPLS-DA analysis showed that RE microbial communities differ among the treatments, although the taxonomic and functional microbial profiles show different distributions. Co-expression Differential Network Analysis indicated that both BE and PE had an impact on the abundance of KEGG modules related to acyl-CoA synthesis, type VI secretion, and methanogenesis, while PE had a significant impact on KEGGs related to ectoine biosynthesis and D-xylose transport. Our study indicated that artificial dosing with different microbial inocula in early life alters not only the RE transcriptome, but also affects the REM and its functions.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

The Q 10 of in situ microbial soil respiration varies with mean annual temperature, precipitation, pH, and plant cover: a meta-analysis and spatial prediction of Q 10

The temperature sensitivity of soil microbial respiration, commonly quantified using the Q 10 coefficient, is a key parameter in carbon cycle models. Uncovering how environmental factors affect in situ Q 10 values can therefore provide critical insight into potential shifts in global carbon stocks under climate change. We collected data from previously published field experiments that measured soil microbial respiration across a range of temperatures. We hypothesized that the Q 10 coefficient of in situ soil microbial respiration would vary based on environmental factors including mean annual temperature (MAT), mean annual precipitation (MAP), plant cover type, pH, soil C:N, and latitude. Linear regression revealed that Q 10 correlates negatively with MAT and MAP and positively with pH and absolute latitude. Additionally, average Q 10 varied significantly across different plant cover types; it was highest in mountain grasslands and lowest in tropical moist forests. Variation in microbial Q 10 across environmental factors may arise from underlying mechanisms such as enzyme kinetics, substrate availability and complexity, and microbial adaptation. To capture patterns in Q 10 more comprehensively, we developed a multiple linear regression model of Q 10 based on the most individually significant environmental drivers and applied it to public datasets to generate a global map of predicted Q 10 . Q 10 was higher in high-latitude and high-altitude regions, where large permafrost carbon stores are vulnerable to thawing and decomposition. We also compared fits between the Q 10 equation and a model produced from macromolecular rate theory (MMRT). We found that the MMRT model had the superior fit and may be better suited to model temperature sensitivity of complex biological reactions. Overall, our results emphasize that relationships between microbial Q 10 and environmental variables should be accounted for in climate models. Incorporating these variations in the Q 10 parameter, rather than using a fixed value, will help predict whether CO 2 emissions will be buffered or exacerbated by soil microbial respiration under climate change.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2021

Direct evidence for the role of microbial community composition in the formation of soil organic matter composition and persistence

The largest terrestrial carbon sink on earth is soil carbon stocks. As the climate changes, the rate at which the Earth’s climate warms depends in part on the persistence of soil organic carbon. Microbial turnover forms the backbone of soil organic matter (SOM) formation and it has been recently proposed that SOM molecular complexity is a key driver of stability. Despite this, the links between microbial diversity, chemical complexity and biogeochemical nature of SOM remain missing. Here we tested the hypotheses that distinct microbial communities shape the composition of SOM, and microbial-derived SOM has distinct decomposition potential depending on its community of origin. We inoculated microbial communities of varying diversities into a model soil matrix amended with simple carbon (cellobiose) and measured the thermal stability of the resultant SOM. Using a Rock-Eval ® ramped thermal analysis, we found that microbial community composition drives the chemical fingerprint of soil carbon. While diversity was not a driver of SOM composition, bacteria-only communities lead to more thermally labile soil C pools than communities with bacteria and fungi. Our results provide direct evidence for a link between microbial community structure, SOM composition, and thermal stability. This evidence demonstrates the relevance of soil microorganisms in building persistent SOM stocks.

Domeignoz-Horta, Luiz A. (ORCID:0000000346186253)↗

DOE OSTI2021

Microbial community composition controls carbon flux across litter types in early phase of litter decomposition

Leaf litter decomposition is a major carbon input to soil, making it a target for increasing soil carbon storage through microbiome engineering. We expand upon previous findings to show with multiple leaf litter types that microbial composition can drive variation in carbon flow from litter decomposition and specific microbial community features are associated with synonymous patterns of carbon flow among litter types. Although plant litter type selects for different decomposer communities, within a litter type, microbial composition drives variation in the quantity of dissolved organic carbon (DOC) measured at the end of the decomposition period. Bacterial richness was negatively correlated with DOC quantity, supporting our hypothesis that across multiple litter types there are common microbial traits linked to carbon flow patterns. Variation in DOC abundance (i.e. high versus low DOC) driven by microbial composition is tentatively due to differences in bacterial metabolism of labile compounds, rather than catabolism of non-labile substrates such as lignin. The temporal asynchrony of metabolic processes across litter types may be a substantial impediment to discovering more microbial features common to synonymous patterns of carbon flow among litters. Overall, our findings support the concept that carbon flow may be programmed by manipulating microbial community composition.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Robust measurement of microbial reduction of graphene oxide nanoparticles using image analysis

ABSTRACT Shewanella oneidensis ( S. oneidensis ) has the capacity to reduce electron acceptors within a medium and is thus used frequently in microbial fuel generation, pollutant breakdown, and nanoparticle fabrication. Microbial fuel setups, however, often require costly or labor-intensive components, thus making optimization of their performance onerous. For rapid optimization of setup conditions, a model reduction assay can be employed to allow simultaneous, large-scale experiments at lower cost and effort. Since S. oneidensis uses different extracellular electron transfer pathways depending on the electron acceptor, it is essential to use a reduction assay that mirrors the pathways employed in the microbial fuel system. For microbial fuel setups that use nanoparticles to stimulate electron transfer, reduction of graphene oxide provides a more accurate model than other commonly used assays as it is a bulk material that forms flocculates in solutions with a large ionic component. However, graphene oxide flocculates can interfere with traditional absorbance-based measurement techniques. This study introduces a novel image analysis method for quantifying graphene oxide reduction, showing improved performance and statistical accuracy over traditional methods. A comparative analysis shows that the image analysis method produces smaller errors between replicates and reveals more statistically significant differences between samples than traditional plate reader measurements under conditions causing graphene oxide flocculation. Image analysis can also detect reduction activity at earlier time points due to its use of larger solution volumes, enhancing color detection. These improvements in accuracy make image analysis a promising method for optimizing microbial fuel cells that use nanoparticles or bulk substrates. IMPORTANCE Shewanella oneidensis ( S. oneidensis ) is widely used in reduction processes such as microbial fuel generation due to its capacity to reduce electron acceptors. Often, these setups are labor-intensive to operate and require days to produce results, so use of a model assay would reduce the time and expenses needed for optimization. Our research developed a novel digital analysis method for analysis of graphene oxide flocculates that may be utilized as a model assay for reduction platforms featuring nanoparticles. Use of this model reduction assay will enable rapid optimization and drive improvements in the microbial fuel generation sector.

Bennett, Danielle T. (ORCID:0009000188748827)↗

DOE OSTI2024

Genomic insights into redox-driven microbial processes for carbon decomposition in thawing Arctic soils and permafrost

Climate change is rapidly transforming Arctic landscapes where increasing soil temperatures speed up permafrost thaw. This exposes large carbon stocks to microbial decomposition, possibly worsening climate change by releasing more greenhouse gases. Understanding how microbes break down soil carbon, especially under the anaerobic conditions of thawing permafrost, is important to determine future changes. Here, we studied the microbial community dynamics and soil carbon decomposition potential in permafrost and active layer soils under anaerobic laboratory conditions that simulated an Arctic summer thaw. The microbial and viral compositions in the samples were analyzed based on metagenomes, metagenome-assembled genomes, and metagenomic viral contigs (mVCs). Following the thawing of permafrost, there was a notable shift in microbial community structure, with fermentative Firmicutes and Bacteroidota taking over from Actinobacteria and Proteobacteria over the 60-day incubation period. The increase in iron and sulfate-reducing microbes had a significant role in limiting methane production from thawed permafrost, underscoring the competition within microbial communities. We explored the growth strategies of microbial communities and found that slow growth was the major strategy in both the active layer and permafrost. Our findings challenge the assumption that fast-growing microbes mainly respond to environmental changes like permafrost thaw. Instead, they indicate a common strategy of slow growth among microbial communities, likely due to the thermodynamic constraints of soil substrates and electron acceptors, and the need for microbes to adjust to post-thaw conditions. The mVCs harbored a wide range of auxiliary metabolic genes that may support cell protection from ice formation in virus-infected cells.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2026

Exploring life’s hidden majority: microbial dark matter symposium highlights

The Microbial Dark Matter Symposium held on August 28–29, 2025, in Laguna Beach, Orange County, CA, convened a multidisciplinary group of scientists to address the vast unknowns in microbial life—from uncultured taxa and uncharacterized proteins to elusive viruses and spacefaring microbes. Set against a scenic coastal backdrop, the symposium highlighted advances in single-cell genomics, proximity ligation sequencing, and artificial intelligence-ready bioinformatics, while also probing the limits of microbial persistence, metabolism, and ecological distribution. Sessions explored microbial dark matter from multiple dimensions: cultivability, where new strategies are enabling recovery of elusive microbes; functional ambiguity, where metagenomic dark zones are illuminated by computational annotation; and genomic representation, where single-cell methods bridge gaps left by shotgun community sequencing. Researchers shared breakthroughs in identifying atmospheric microbiomes, “dark oxygen” production in groundwater ecosystems, and microbial survival on the International Space Station. The symposium emphasized integration of methods, disciplines, and ecosystems, advancing a collective push to illuminate the microbial dark matter on Earth and beyond. By highlighting emerging tools, pressing questions, and cross-domain insights, the symposium underscored the need for collaborative, open, and adaptive approaches to study the microbial unknown. The meeting marks a pivotal moment in microbiology, where cultivating knowledge of the uncultivated promises transformative understanding of life, everywhere.

Podar, Mircea [ORNL] (ORCID:0000000327760205)↗

DOE OSTI2022

Microbial extracellular enzyme activity with simulated climate change

It is critical to understand the consequences of environmental change for the microbial regulation of carbon and nutrient cycling. Specifically, understanding microbial community traits, such as extracellular enzyme activity, can help inform nutrient cycling models and address knowledge gaps. We analyzed data on extracellular enzyme activities and litter decomposition from an 18-month experiment in which microbial communities were reciprocally transplanted along a climate gradient in Southern California. Communities were from desert, scrubland, grassland, pine–oak, and subalpine ecosystems. We aimed to test how enzyme activities responded to climate change following transplantation and how those responses related to decomposition rates. We hypothesized that microbial communities would specialize on their native climate conditions, resulting in higher enzyme activities when transplanted back into their native climate. We investigated the relationship between extracellular enzyme Vmax values, substrate mass loss, and microbial biomass as well as variation in these variables across the climate gradient. We found little evidence for climate specialization, and there was rarely a reduction in enzyme functioning after microbial communities were transplanted into new climate conditions. Moreover, observed differences in decomposition were not related to changes in extracellular enzyme potential, although there were significant differences in enzyme activities and decomposition rates across sites. These results suggest that direct, physiological impacts of climate are likely to be important for enzyme-mediated decomposition, but climate specialization will not constrain the microbial response to climate change in our system.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2022

microTrait: A Toolset for a Trait-Based Representation of Microbial Genomes

Remote sensing approaches have revolutionized the study of macroorganisms, allowing theories of population and community ecology to be tested across increasingly larger scales without much compromise in resolution of biological complexity. In microbial ecology, our remote window into the ecology of microorganisms is through the lens of genome sequencing. For microbial organisms, recent evidence from genomes recovered from metagenomic samples corroborate a highly complex view of their metabolic diversity and other associated traits which map into high physiological complexity. Regardless, during the first decades of this omics era, microbial ecological research has primarily focused on taxa and functional genes as ecological units, favoring breadth of coverage over resolution of biological complexity manifested as physiological diversity. Recently, the rate at which provisional draft genomes are generated has increased substantially, giving new insights into ecological processes and interactions. From a genotype perspective, the wide availability of genome-centric data requires new data synthesis approaches that place organismal genomes center stage in the study of environmental roles and functional performance. Extraction of ecologically relevant traits from microbial genomes will be essential to the future of microbial ecological research. Here, we present microTrait , a computational pipeline that infers and distills ecologically relevant traits from microbial genome sequences. microTrait maps a genome sequence into a trait space, including discrete and continuous traits, as well as simple and composite. Traits are inferred from genes and pathways representing energetic, resource acquisition, and stress tolerance mechanisms, while genome-wide signatures are used to infer composite, or life history, traits of microorganisms. This approach is extensible to any microbial habitat, although we provide initial examples of this approach with reference to soil microbiomes.

Karaoz, Ulas↗

DOE OSTI2022

Characterizing Natural Organic Matter Transformations by Microbial Communities in Terrestrial Subsurface Ecosystems: A Critical Review of Analytical Techniques and Challenges

Determining the mechanisms, traits, and pathways that regulate microbial transformation of natural organic matter (NOM) is critical to informing our understanding of the microbial impacts on the global carbon cycle. The capillary fringe of subsurface soils is a highly dynamic environment that remains poorly understood. Characterization of organo-mineral chemistry combined with a nuanced understanding of microbial community composition and function is necessary to understand microbial impacts on NOM speciation in the capillary fringe. We present a critical review of the popular analytical and omics techniques used for characterizing complex carbon transformation by microbial communities and focus on how complementary information obtained from the different techniques enable us to connect chemical signatures with microbial genes and pathways. This holistic approach offers a way forward for the comprehensive characterization of the formation, transformation, and mineralization of terrestrial NOM as influenced by microbial communities.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1995

PCR Based Microbial Monitor for Analysis of Recycled Water Aboard the ISSA: Issues and Prospects

The monitoring of spacecraft life support systems for the presence of health threatening microorganisms is paramount for crew well being and successful completion of missions. Development of technology to monitor spacecraft recycled water based on detection and identification of the genetic material of contaminating microorganisms and viruses would be a substantial improvement over current NASA plans to monitor recycled water samples that call for the use of conventional microbiology techniques which are slow, insensitive, and labor intensive. The union of the molecular biology techniques of DNA probe hybridization and polymerase chain reaction (PCR) offers a powerful method for the detection, identification, and quantification of microorganisms and viruses. This technology is theoretically capable of assaying samples in as little as two hours with specificity and sensitivity unmatched by any other method. A major advance in probe-hybridization/PCR has come about in a technology called TaqMan(TM), which was invented by Perkin Elmer. Instrumentation using TaqMan concepts is evolving towards devices that could meet NASA's needs of size, low power use, and simplicity of operation. The chemistry and molecular biology needed to utilize these probe-hybridization/PCR instruments must evolve in parallel with the hardware. The following issues of chemistry and biology must be addressed in developing a monitor: Early in the development of a PCR-based microbial monitor it will be necessary to decide how many and which organisms does the system need the capacity to detect. We propose a set of 17 different tests that would detect groups of bacteria and fungus, as well as specific eukaryotic parasites and viruses; In order to use the great sensitivity of PCR it will be necessary to concentrate water samples using filtration. If a lower limit of detection of 1 microorganism per 100 ml is required then the microbes in a 100 ml sample must be concentrated into a volume that can be added to a PCR assay; There are not likely to be contaminants in ISSA recycled water that would inhibit PCR resulting in false-negative results; The TaqMan PCR product detection system is the most promising method for developing a rapid, highly automated gene-based microbial monitoring system. The method is inherently quantitative. NASA and other government agencies have invested in other technologies that, although potentially could lead to revolutionary advances, are not likely to mature in the next 5 years into working systems; PCR-based methods cannot distinguish between DNA or RNA of a viable microorganism and that of a non-viable organism. This may or may not be an important issue with reclaimed water on the ISSA. The recycling system probably damages the capacity of the genetic material of any bacteria or viruses killed during processing to serve as a template in a PCR desinged to amplify a large segment of DNA (less than 650 base pairs). If necessary, vital dye staining could be used in addition to PCR, to enumerate the viable cells in a water sample; The quality control methods have been developed to insure that PCR's are working properly, and that reactions are not contaminated with PCR carryover products which could lead to the generation of false-positive results; and The sequences of the small rRNA subunit gene for a large number of microorganisms are known, and they consititue the best database for rational development of the oligonucleotide reagents that give PCR its great specificity. From those gene sequences, sets of oligonucleotide primers for PCR and Taqman detection that could be used in a NASA microbial monitor were constructed using computer based methods. In addition to space utilization, a microbial monitior will have tremendous terrestrial applications. Analysis of patient samples for microbial pathogens, testing industrial effluent for biofouling bacteria, and detection biological warfare agents on the battlefield are but a few of the diverse potential uses for this technology. Once fully developed, gene-based microbial monitors will become the fundamental tool in every lab that tests for microbial contaminants, and serve as a powerful weapon in mankind's war with the germ world.

Cassell, Gail H.↗

NASA NTRS1999

A Survey of Environmental Microbial Flora During Closed Chamber Studies

Services, Inc. and NASA Johnson Space Center, Houston, TX As NASA prepares for long-term missions aboard the International Space Station and the eventual exploration of Mars, closed-environment chambers on Earth have become important test beds for systems evaluations. During 2 separate studies of a selfcontained ecosystem containing 4 crewmembers, microbial surveys of samples from 13 surface and 3 air sites were performed. Microbial concentration of samples from surface sites with frequent water contact (e.g., urinal, sink) did not indicate significantly higher levels of contamination than drier areas, though surface cleaning by the crew may have influenced this conclusion. Changes in bacterial diversity on surface sites implied that the number of transient species was high, suggesting movement by crew activities, aerosols, or both. A non-linear relationship between bacterial diversity and enumeration from surface samples indicated that a rapid increase occurred in the number of species as cell concentration increased to 5 CFU/sq cm. Above this concentration, the number of different bacterial species varied between 11 and 16. Airborne bacteria and fungi averaged only 160 and 1 CFU/m3, respectively. Microbial contamination of the potable water system primarily consisted of 3 species of Gram negative bacteria; however, after 60 days during one study, several species of Bacillus became the dominant flora. This study suggests that under these conditions, microbial contamination in the air and water was suppressed by the life-support systems, though contamination was possible. Conversely, the crew and their activities controlled microbial levels on surfaces. Understanding the factors that affect microbial control will improve the design of microbial testing both during space flight and in analogous Earth-based environments.

Ott, C. Mark↗

NASA NTRS2000

Modern Microbial Ecosystems are a Key to Understanding Our Biosphere's Early Evolution and its Contributions To The Atmosphere and Rock Record

The survival of our early biosphere depended upon efficient coordination anion- diverse microbial populations. Microbial mats exhibit a 3.46-billion-year fossil record, thus they are the oldest known ecosystems. Photosynthetic microbial mats were key because, today, sunlight powers more than 99 percent of global primary productivity. Thus photosynthetic ecosystems have affected the atmosphere profoundly and have created the most pervasive, easily-detected fossils. Photosynthetic biospheres elsewhere will be most detectible via telescopes or spacecraft. As a part of the Astrobiology Institute, our Ames Microbial Ecosystems group examines the roles played by ecological processes in the early evolution of our biosphere, as recorded in geologic fossils and in the macromolecules of living cells: (1) We are defining the microbial mat microenvironment, which was an important milieu for early evolution. (2) We are comparing mats in contrasting environments to discern strategies of adaptation and diversification, traits that were key for long-term survival. (3) We have selected sites that mimic key environmental attributes of early Earth and thereby focus upon evolutionary adaptations to long-term changes in the global environment. (4) Our studies of gas exchange contribute to better estimates of biogenic gases in Earth's early atmosphere. This group therefore directly addresses the question: How have the Earth and its biosphere influenced each other over time Our studies strengthen the systematics for interpreting the microbial fossil record and thereby enhance astrobiological studies of martian samples. Our models of biogenic gas emissions will enhance models of atmospheres that might be detected on inhabited extrasolar planets. This work therefore also addresses the question: How can other biospheres be recogniZed" Our choice of field sites helps us explore Earth's evolving early environment. For example, modern mats that occupy thermal springs and certain freshwater environments experience conditions such as low O2 and sulfate and high inorganic carbon and sulfide levels that resemble those of ancient marine environments. Later in history, both biologically-induced carbonate precipitation and the trapping and binding of suspended grains of carbonate became a dominant mechanism for carbonate deposition. Modern marine carbonate platforms and alkaline offer good examples of microbiologically-induced calcification. Both marine platforms and solar salterns illustrate microbially-driven trapping and binding. We are also exploring the effects of water composition upon the exchange of biogenic gases with the atmosphere.

DesMarais, David J.↗