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All sourcesNASA NTRSDOE OSTI
DOE OSTI2026

Genome-resolved insights into microbial diversity and elemental cycling in Winogradsky columns

We retained 18 MAGs with ≥50% completion and <10% contamination (i.e., at least medium quality). Of these, 10 had >90% completion and <5% contamination; however, only one (Paceibacteria Bin.003_MG) can be described as high-quality, as the others lacked a full suite of 5S, 16S, and 23S rRNA genes. To maximize the diversity of our recovered MAGs, we also retained one MAG (Chromatiaceae Bin.008_AM) with >40% (but less than 50%) completion and <5% contamination, as well as one (Rhodopseudomonas Bin.015_MK) with >90% completion and <20% (but>10%) contamination. Interestingly, significant chimerism was not detected in this MAG (40) , suggesting that the elevated contamination (20%) may instead reflect two closely related strains collapsing into a single bin. Consistent with this, contig coverage was bimodal, with roughly 17% of the assembly at ~115x and the remaining 83% at ~282x, while GC content remained uniform across both groups (~64%), arguing against contamination from a taxonomically distinct source.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2010

Microbial Diversity in Surface Iron-Rich Aqueous Environments: Implications for Seeking Signs of Life on Mars

The success of selecting future landing sites on Mars to discover extinct and/or extant extraterrestrial life is dependent on the correct approximation of available knowledge about terrestrial paleogeochemistry and life evolution to Martian (paleo) geology and geochemistry. It is well known that both Earth and Mars are Fe rich. This widespread occurrence suggests that Fe may have played a key role in early life forms, where it probably served as a key constituent in early prosthetic moieties in many proteins of ancient microbes on Earth and likely Mars. The second critical idea is the premise that Life on Mars could most likely have developed when Mars experienced tectonic activity [1] which dramatically decreased around 1 bin years after Martian creation. After that Martian life could have gone extinct or hibernated in the deep subsurface, which would be expensive to reach in contrast to the successful work of Martian surface rovers. Here we analyze the diversity of microbes in several terrestrial Fe rich surface environments in conjunction with the phylogeny and molecular timing of emergence of those microbes on Earth. Anticipated results should help evaluate future landing sites on Mars in searches for biosignatures.

Brown, I. I.↗
NASA NTRS2021

Characterization of Spacesuit Associated Microbial Communities and their Implications for NASA Missions

Crewed National Aeronautics and Space Administration (NASA) missions to other solar system bodies are currently being planned. One high-profile scientific focus during such expeditions would be life detection, specifically the discovery of past or present microbial life, if they exist. However, both humans and associated objects typically carry a high microbial burden. Thus, it is essential to distinguish between microbes brought with the expedition and those present on the exploring planets. Modern spacesuits are unique, customized spacecraft which provide protection, mobility and life support to crew during spacewalks, yet they vent continuously, and the mobility of microbes through spacesuits has not been studied. To evaluate the microbial colonization of spacesuits, NASA used an Extravehicular Activity swab kit to examine viable microbial populations of 48 samples from spacesuits using both traditional microbiological methods and molecular sequencing methods. The cultivable microbial population ranged from below the detection limit to 9 x 102 colony forming units per 25 cm2 of sample and also significantly varied by the location. The cultivable microbial diversity was dominated by members of Bacillus, Arthrobacter, and Ascomycota. However, 16S rRNA-based viable bacterial burden ranged from 105 to 106 copies per 25 cm2 of sample. Shotgun metagenome sequencing revealed the presence of a diverse microbial population on the spacesuit surfaces, including Curtobacterium and Methylobacterium from across all sets of spacesuits in high abundance. Among bacterial species identified, higher abundance of Cutibacterium acnes, Methylobacterium oryzae, and M. phyllosphaerae reads were documented. The results of this study provide evidence that identical microbial strains may live on the wrist joint, inner gauntlet, and outer gauntlet of spacesuits. This raises the possibility, but does not confirm, that viable microbes are able to migrate between the interior and exterior of spacesuits. Overall, these data provide the first estimate of microbial dispersion from internal to external spacesuit surfaces, which will help future mission planners develop effective planetary protection strategies.

spacesuit↗
NASA NTRS2012

Genetic Inventory Task Final Report

Contaminant terrestrial microbiota could profoundly impact the scientific integrity of extraterrestrial life-detection experiments. It is therefore important to know what organisms persist on spacecraft surfaces so that their presence can be eliminated or discriminated from authentic extraterrestrial biosignatures. Although there is a growing understanding of the biodiversity associated with spacecraft and cleanroom surfaces, it remains challenging to assess the risk of these microbes confounding life-detection or sample-return experiments. A key challenge is to provide a comprehensive inventory of microbes present on spacecraft surfaces. To assess the phylogenetic breadth of microorganisms on spacecraft and associated surfaces, the Genetic Inventory team used three technologies: conventional cloning techniques, PhyloChip DNA microarrays, and 454 tag-encoded pyrosequencing, together with a methodology to systematically collect, process, and archive nucleic acids. These three analysis methods yielded considerably different results: Traditional approaches provided the least comprehensive assessment of microbial diversity, while PhyloChip and pyrosequencing illuminated more diverse microbial populations. The overall results stress the importance of selecting sample collection and processing approaches based on the desired target and required level of detection. The DNA archive generated in this study can be made available to future researchers as genetic-inventory-oriented technologies further mature.

microbial diversity↗
DOE OSTI2025

Diversity and Distribution of Hydrocarbon-Degrading Genes in the Cold Seeps from the Mediterranean and Caspian Seas

Marine cold seeps are unique ecological niches characterized by the emergence of hydrocarbons, including methane, which fosters diverse microbial communities. This study investigates the diversity and distribution of hydrocarbon-degrading genes and organisms in sediments from the Caspian and Mediterranean Seas, utilizing 16S rRNA and metagenomic sequencing to elucidate microbial community structure and functional potential. Our findings reveal distinct differences in hydrocarbon degrading gene profiles between the two seas, with pathways for aerobic and anaerobic hydrocarbon degradation co-existing in sediments from both basins. Aerobic pathways predominate in the surface sediments of the Mediterranean Sea, while anaerobic pathways are favored in the surface sediments of the anoxic Caspian Sea. Additionally, sediment depths significantly influence microbial diversity, with variations in gene abundance and community composition observed at different depths. Aerobic hydrocarbon-degrading genes decrease in diversity with depth in the Mediterranean Sea, whereas the diversity of aerobic hydrocarbon-degrading genes increases with depth in the Caspian Sea. These results enhance our understanding of microbial ecology in cold seep environments and have implications for bioremediation practices targeting hydrocarbon pollutants in marine ecosystems.

Microbiology↗
DOE OSTI2025

Expanded Diversity of Microbial Groups Capable of Anaerobic Pyrite Reduction and Assimilation of Dissolution Products

Pyrite, the most abundant iron sulfide mineral in the Earth's crust, has traditionally been considered as a sink for iron and sulfur in the absence of oxygen. Recent research, however, has shown that anaerobic methanogenic archaea can reductively dissolve pyrite and assimilate its products as sources of iron and sulfur. This study explores whether other anaerobic bacteria, including fermentative, nitrate-, iron oxide-, fumarate-, and sulfate-respiring bacteria, can also reduce pyrite and use its dissolution products as sources of iron and sulfur. Results indicate that heterotrophic bacteria respiring fumarate or sulfate, or fermenting organic carbon, can reduce pyrite and assimilate released iron and sulfur. In contrast, nitrate- or iron oxide-respiring cells did not reduce pyrite, suggesting that microbial pyrite reduction is metabolism-specific. All strains capable of reducing pyrite could also use mackinawite as an iron and sulfur source. With the exception of fermentative Bacteroides, strains did not require direct contact with pyrite to reduce the mineral, indicating extracellular electron transfer via electron shuttles. These findings expand the known diversity of microbial groups capable of pyrite reduction and highlight the mineral's lability in various anaerobic environments, with potential implications for the biogeochemical cycles of iron, sulfur, carbon, and oxygen.

58 GEOSCIENCES↗
NASA NTRSDate not supplied

The Pheno-Evo Model: Evolution of Microbial Phenotypic Diversity in 2D Space

Biologists appreciate microbes for their simplicity and predictability: we assume that a population of genetically identical cells in a uniform environment will all behave the same way. While this assumption is often useful, it is also often wrong. Not only might microbes in a clonal population act differently from one another, the differences may be categorical (growing v. non-growing; toxin-sensitive v. tolerant), and this diversity might be an evolved trait conferring increased fitness on the population. How does such phenotypic heterogeneity evolve? And how does a population find the optimal distribution of phenotypes for a given environment? Efforts at modeling microbial phenotypic heterogeneity often focus on populations with two discrete phenotypic types; phenotypes in continuous distributions remain poorly explored. To address this gap and to explore the role of spatial relationships, we use agent-based modeling to simulate a phenotypically diverse population of microbial cells evolving in the presence of periodic toxic stress. Cells on patches in a 2D grid may degrade toxin, suffer damage from toxin, switch phenotype, and reproduce. An individual's phenotype its toxin degradation rate-- for which there is a tradeoff with reproduction-- and the genotype encodes the distribution of phenotypic values in the population. The rate of toxin diffusion determines how individuals influence their neighbors' environments. We examine the effects of toxin concentration, diffusion rate, and environmental predictability on the survival success of populations with different phenotype distributions, and populations' evolutionary trajectories when phenotype distribution is allowed to evolve. We conduct all simulations on the platform NetLogo, which provides a friendly interface allowing users of any experience level to tweak parameters and run their own simulations. We have also created custom tools in R for analyzing and visualizing the results of multiple runs.

microbial evolution↗
NASA NTRS2004

Evidence of pathogenic microbes in the International Space Station drinking water: reason for concern?

Molecular analyses were carried out on four preflight and six postflight International Space Station (ISS)-associated potable water samples at various stages of purification, storage, and transport, to ascertain their associated microbial diversities and overall microbial burdens. Following DNA extraction, PCR amplification, and molecular cloning procedures, rDNA sequences closely related to pathogenic species of Acidovorax, Afipia, Brevundimonas, Propionibacterium, Serratia, and others were recovered in varying abundance. Retrieval of sequences arising from the iodine (biocide)-reducing Delftia acidovorans in postflight waters is also of concern. Total microbial burdens of ISS potable waters were derived from data generated by an ATP-based enumeration procedure, with results ranging from 0 to 4.9 x 10(4) cells/ml. Regardless of innate biases in sample collection and analysis, such circumstantial evidence for the presence of viable, intact pathogenic cells should not be taken lightly. Implementation of new cultivation approaches and/or viability-based assays are requisite to confirm such an occurrence.

STS-113 Shuttle Project↗
NASA NTRS2012

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

Frequently there is an inability to process and analyze samples of low biomass due to limiting amounts of relevant biomaterial in the sample. Furthermore, molecular biological protocols geared towards increasing the density of recovered cells and biomolecules of interest, by their very nature, also concentrate unwanted inhibitory humic acids and other particulates that have an adversarial effect on downstream analysis. A novel and robust fluorescence-activated cell-sorting (FACS)-based technology has been developed for purifying (removing cells from sampling matrices), separating (based on size, density, morphology), and concentrating cells (spores, prokaryotic, eukaryotic) from a sample low in biomass. The technology capitalizes on fluorescent cell-sorting technologies to purify and concentrate bacterial cells from a low-biomass, high-volume sample. Over the past decade, cell-sorting detection systems have undergone enhancements and increased sensitivity, making bacterial cell sorting a feasible concept. Although there are many unknown limitations with regard to the applicability of this technology to environmental samples (smaller cells, few cells, mixed populations), dogmatic principles support the theoretical effectiveness of this technique upon thorough testing and proper optimization. Furthermore, the pilot study from which this report is based proved effective and demonstrated this technology capable of sorting and concentrating bacterial endospore and bacterial cells of varying size and morphology. Two commercial off-the-shelf bacterial counting kits were used to optimize a bacterial stain/dye FACS protocol. A LIVE/DEAD BacLight Viability and Counting Kit was used to distinguish between the live and dead cells. A Bacterial Counting Kit comprising SYTO BC (mixture of SYTO dyes) was employed as a broad-spectrum bacterial counting agent. Optimization using epifluorescence microscopy was performed with these two dye/stains. This refined protocol was further validated using varying ratios and mixtures of cells to ensure homogenous staining compared to that of individual cells, and were utilized for flow analyzer and FACS labeling. This technology focuses on the purification and concentration of cells from low-biomass spacecraft assembly facility samples. Currently, purification and concentration of low-biomass samples plague planetary protection downstream analyses. Having a capability to use flow cytometry to concentrate cells out of low-biomass, high-volume spacecraft/ facility sample extracts will be of extreme benefit to the fields of planetary protection and astrobiology. Successful research and development of this novel methodology will significantly increase the knowledge base for designing more effective cleaning protocols, and ultimately lead to a more empirical and true account of the microbial diversity present on spacecraft surfaces. Refined cleaning and an enhanced ability to resolve microbial diversity may decrease the overall cost of spacecraft assembly and/or provide a means to begin to assess challenging planetary protection missions.

Benardini, James N.↗
DOE OSTI2025

Limited effects of tannin supplementation on the dairy cattle fecal microbiome with modulation of metabolites

Tannins are plant secondary metabolites that bind organic carbon (C) and nitrogen (N), potentially altering substrate bioavailability for enteric fermentation in ruminants. This interaction may reduce greenhouse gas (GHG) emissions and influence nitrogen partitioning. Given tannins' resistance to ruminal degradation and persistence through the gastrointestinal tract, this study investigated the effects of a tannin-based feed additive on fecal microbial diversity, fecal chemical composition, and GHG emissions. Twenty-four early- to mid-lactation dairy cows were randomized to receive either a tannin-based feed additive (TRT; containing condensed and hydrolyzable tannins from Schinopsis quebracho-colorado [Schltdl.]) or a control diet (CON) for 64 days. Cows were blocked by parity, dry matter intake, milk yield, body weight, and days in milk. Fecal samples were collected on days 0, 16, 32, and 64 and analyzed using 16S rRNA gene amplicon sequencing. Fecal C, N, and indole-3-lactate were measured, and GHG emissions (N2O, CH4, CO2) were assessed via 14-day laboratory incubation. A total of 1,538 amplicon sequence variants were identified, with Firmicutes as the dominant phylum. Fecal phylogenetic diversity showed a significant treatment × day interaction (p < 0.01), with TRT cows exhibiting reduced microbial diversity from day 16 to 64. Fecal C and N concentrations were significantly lower (p < 0.01) in TRT cows on day 16, while indole-3-lactate levels were higher on day 64 (p = 0.02). GHG emissions did not differ significantly between treatments. The tannin-based feed additive influenced fecal microbial community structure and select chemical parameters but did not significantly affect GHG emissions from feces. These findings suggest that dietary tannins may modulate gut microbial ecology with minimal impact on downstream manure-related emissions.

Klein, Matthew L↗
DOE OSTI2025

Identifying microbial functional guilds performing cryptic organotrophic and lithotrophic redox cycles in anaerobic granular biofilms

Granular biofilms used in anaerobic digester systems contain diverse microbial populations that interact to hydrolyze organic matter and produce methane within controlled environments. Prior research investigated the feasibility of utilizing granular biofilms obtained from an anaerobic digester to remove nitrate without the addition of exogenous electron donors. These granules possessed a unique structure of alternating light and dark iron sulfide and pyrite rich layers that potentially served as both an electron source and sink, linking carbon, nitrogen, sulfur, and iron cycles. To characterize the functional roles of diverse microbial populations enriched within these layered biofilms, we analyzed metagenomes obtained from three different granules. Comparisons between the functional gene content of forty metagenome assembled genomes (MAGs) identified phylogenetically cohesive functional guilds. Each of these functional MAG clusters was assigned to specific steps in anaerobic digestion (hydrolysis, acidogenesis, acetogenesis, and methanogenesis) and anaerobic respiration (denitrification and sulfate reduction). Comparisons with metagenomes derived from a variety of natural and engineered ecosystems confirmed that the enriched denitrifying bacteria were similar to populations typically found in wetlands and biological nitrogen removal systems. Analysis of read alignments to individual genes within the forty MAGs identified conserved genomic features that were representative of the functions that distinguished functional guilds. Overall, this research illustrates the utility of functional based classification of microorganisms for characterizing ecosystem functions and highlights the potential application of engineered ecosystems to serve as experimental models for complex natural ecosystems.

Ecosystem engineering↗
NASA NTRS2008

Purifying Nucleic Acids from Samples of Extremely Low Biomass

A new method is able to circumvent the bias to which one commercial DNA extraction method falls prey with regard to the lysing of certain types of microbial cells, resulting in a truncated spectrum of microbial diversity. By prefacing the protocol with glass-bead-beating agitation (mechanically lysing a much more encompassing array of cell types and spores), the resulting microbial diversity detection is greatly enhanced. In preliminary studies, a commercially available automated DNA extraction method is effective at delivering total DNA yield, but only the non-hardy members of the bacterial bisque were represented in clone libraries, suggesting that this method was ineffective at lysing the hardier cell types. To circumvent such a bias in cells, yet another extraction method was devised. In this technique, samples are first subjected to a stringent bead-beating step, and then are processed via standard protocols. Prior to being loaded into extraction vials, samples are placed in micro-centrifuge bead tubes containing 50 micro-L of commercially produced lysis solution. After inverting several times, tubes are agitated at maximum speed for two minutes. Following agitation, tubes are centrifuged at 10,000 x g for one minute. At this time, the aqueous volumes are removed from the bead tubes and are loaded into extraction vials to be further processed via extraction regime. The new method couples two independent methodologies in such as way as to yield the highest concentration of PCR-amplifiable DNA with consistent and reproducible results and with the most accurate and encompassing report of species richness.

La Duc, Myron↗
DOE OSTI2025

Rhizosphere Microbiome Diversity Potentially Supports Robust Nature of Field Pennycress ( Thlaspi arvense L.) in Dryland Cropping Systems of Eastern Washington

ABSTRACT Field pennycress ( Thlaspi arvense L.) is an annual in the Brassicaceae family and is currently being developed as an oilseed intermediate crop suitable for renewable biodiesel and jet fuel. It displays many desirable characteristics for this role including cold tolerance, a rapid life cycle, and a seed fatty acid profile conducive to bioenergy generation. These traits make field pennycress favorable for winter oilseed cultivation in the inland Pacific Northwest (iPNW). Simultaneously, intermediate crops are an increasingly recognized component of both agronomic sustainability and soil health management. Intermediate crops enhance soil microbial diversity, which benefits both soil and plant health. To understand the impact of field pennycress on soil microbial diversity, two natural accessions and seven experimental accessions were grown at three sites in Eastern Washington. Aboveground biomass and rhizosphere soil were then collected. Soil genomic DNA was extracted from rhizosphere samples and used to generate an amplicon library for bacterial (16S) and fungal (ITS) rRNA sequences. The resulting libraries were analyzed in QIIME2, which revealed that not only did the fad2 deficient line from the Spring32‐10 background have significantly increased aboveground biomass production compared to other pennycress genotypes, but also displayed significantly higher β‐diversity in the rhizosphere community specifically at the site experiencing the driest conditions. ANCOM analysis showed that multiple sequences similar to beneficial plant and soil health enhancing organisms such as Trichoderma spirale , Pseudomonas spp., and Methylobacterium goesingense were found to be enriched in the microbiome of the fad2 Spring32‐10 background also at that site. To add additional context to rhizosphere community data, root exudates from two pennycress genotypes were captured in magenta boxes and analyzed using HPLC. Future work will expand our understanding of the mechanisms by which field pennycress creates diversity in the rhizosphere, thus expanding our ability to cultivate this crop in the iPNW.

54 ENVIRONMENTAL SCIENCES↗
NASA NTRS2004

Evaluation of Performance of Five Parallel Biological Water Proce

The objective of the work entitled Molecular Characterization of Eubacteria in a Biological Water Processor was to gain an understanding of the microbial diversity and species stability of the consortia that inhabit an anoxic bioreactor and to correlate those factors with functional performance, mechanical reliability, and stability. The evaluation was divided into four studies. During Study 1, replicate biological water processor (BWP) systems were operated to evaluate variability in the microbial diversity over time as a function of the initial consortia used for inoculation of the BWP reactors. Study 2 was designed to investigate the impact of an inoculum source on BWP performance. Study 3 was a modification of Study 2 where the impact of inoculum on BWP performance from inoculation until steady state operations was monitored. In Study 4, the reactors were divided into three different operational periods, based on the operational periods of the integrated water recovery test at the Johnson Space Center (JSC) in 2001.

Vega, Leticia M.↗
NASA NTRS2004

Microbial structural diversity estimated by dilution-extinction of phenotypic traits and T-RFLP analysis along a land-use intensification gradient

The present work tested whether the relationship between functional traits and inoculum density reflected structural diversity in bacterial communities from a land-use intensification gradient applying a mathematical model. Terminal restriction fragment length polymorphism (T-RFLP) analysis was also performed to provide an independent assessment of species richness. Successive 10-fold dilutions of a soil suspension were inoculated onto Biolog GN(R) microplates. Soil bacterial density was determined by total cell and plate counts. The relationship between phenotypic traits and inoculum density fit the model, allowing the estimation of maximal phenotypic potential (Rmax) and inoculum density (KI) at which Rmax will be half-reduced. Though Rmax decreased with time elapsed since clearing of native vegetation, KI remained high in two of the disturbed sites. The genetic pool of bacterial community did not experience a significant reduction, but the active fraction responding in the Biolog assay was adversely affected, suggesting a reduction in the functional potential. c2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

NASA Center KSC↗