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All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

From wolves to humans: oral microbiome resistance to transfer across mammalian hosts

The mammalian mouth is colonized by complex microbial communities, adapted to specific niches, and in homeostasis with the host. Individual microbes interact metabolically and rely primarily on nutrients provided by the host, with which they have potentially co-evolved along the mammalian lineages. The oral environment is similar across mammals, but the diversity, specificity, and evolution of community structure in related or interacting mammals are little understood. Here, we compared the oral microbiomes of dogs with those of wild wolves and humans. In dogs, we found an increased microbial diversity relative to wolves, possibly related to the transition to omnivorous nutrition following domestication. This includes a larger diversity of Patescibacteria than previously reported in any other oral microbiota. The oral microbes are most distinct at bacterial species or strain levels, with few if any shared between humans and canids, while the close evolutionary relationship between wolves and dogs is reflected by numerous shared taxa. More taxa are shared at higher taxonomic levels including with humans, supporting their more ancestral common mammalian colonization followed by diversification. Phylogenies of selected oral bacterial lineages do not support stable human-dog microbial transfers but suggest diversification along mammalian lineages (apes and canids). Therefore, despite millennia of cohabitation and close interaction, the host and its native community controls and limits the assimilation of new microbes, even if closely related. Higher resolution metagenomic and microbial physiological studies, covering a larger mammalian diversity, should help understand how oral communities assemble, adapt, and interact with their hosts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Direct root contact among neighboring plants influences activity of soil extracellular enzymes

Composition and diversity of vegetation systems can influence soil microbial activity and extracellular enzyme (EE) dynamics, which are crucial for soil carbon (C) accrual and nutrient cycling. Yet, the impact of plant interactions and competition on EE activities remains a notable knowledge gap. This study examines how direct root contact and neighboring plant identity affect the activity and spatial distribution of four key soil EEs: β-glucosidase (BGlu), chitinase, acid phosphatase (AcidP), and alkaline phosphatase (AlkP). Using three-compartment rhizoboxes with switchgrass (Panicum virgatum L.) grown alongside bush clover (Lespedeza capitata Michx.), and black-eyed Susan (Rudbeckia hirta L.), we assessed enzyme activities using zymography under conditions that either allowed or restricted direct root contact by root barriers. Results show that root proliferation and species interactions significantly influenced EE activity. While BGlu and AcidP activities were strongly correlated with root biomass, AlkP activity was consistently higher in the absence of root barriers, indicating a pronounced microbial response to plant interactions via direct/close root contacts. Additionally, soil phosphorus availability modulated enzyme activity, with higher phosphatase activities in low-P soils. Furthermore, these findings highlight the importance of root-root interactions and plant species composition in shaping soil biochemical processes.

enyzme activity↗
DOE OSTI2024

MIBiG 4.0: advancing biosynthetic gene cluster curation through global collaboration

Specialized or secondary metabolites are small molecules of biological origin, often showing potent biological activities with applications in agriculture, engineering and medicine. Usually, the biosynthesis of these natural products is governed by sets of co-regulated and physically clustered genes known as biosynthetic gene clusters (BGCs). To share information about BGCs in a standardized and machine-readable way, the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard and repository was initiated in 2015. Since its conception, MIBiG has been regularly updated to expand data coverage and remain up to date with innovations in natural product research. Here, we describe MIBiG version 4.0, an extensive update to the data repository and the underlying data standard. In a massive community annotation effort, 267 contributors performed 8304 edits, creating 557 new entries and modifying 590 existing entries, resulting in a new total of 3059 curated entries in MIBiG. Particular attention was paid to ensuring high data quality, with automated data validation using a newly developed custom submission portal prototype, paired with a novel peer-reviewing model. MIBiG 4.0 also takes steps towards a rolling release model and a broader involvement of the scientific community. MIBiG 4.0 is accessible online at https://mibig.secondarymetabolites.org/.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Mineralogy of microbially induced calcium carbonate precipitates formed using single cell drop-based microfluidics

Microbe-mineral interactions are ubiquitous and can facilitate major biogeochemical reactions that drive dynamic Earth processes such as rock formation. One example is microbially induced calcium carbonate precipitation (MICP) in which microbial activity leads to the formation of calcium carbonate precipitates. A majority of MICP studies have been conducted at the mesoscale but fundamental questions persist regarding the mechanisms of cell encapsulation and mineral polymorphism. Here, we are the first to investigate and characterize precipitates on the microscale formed by MICP starting from single ureolytic E. coli MJK2 cells in 25 µm diameter drops. Mineral precipitation was observed over time and cells surrounded by calcium carbonate precipitates were observed under hydrated conditions. Using Raman microspectroscopy, amorphous calcium carbonate (ACC) was observed first in the drops, followed by vaterite formation. ACC and vaterite remained stable for up to 4 days, possibly due to the presence of organics. The vaterite precipitates exhibited a dense interior structure with a grainy exterior when examined using electron microscopy. Autofluorescence of these precipitates was observed possibly indicating the development of a calcite phase. The developed approach provides an avenue for future investigations surrounding fundamental processes such as precipitate nucleation on bacteria, microbe-mineral interactions, and polymorph transitions.

58 GEOSCIENCES↗
DOE OSTI2022

Soil organic carbon accrual due to more efficient microbial utilization of plant inputs at greater long-term soil moisture

High long-term soil moisture may either stimulate or inhibit soil organic carbon (SOC) losses through changes to mineral and chemical composition, and resultant organo-mineral interactions. Yet, the trade-off between mineralization and accrual of SOC under long-term variation in unsaturated soil moisture remains uncertain. We tested the underexplored relationships between long-term soil moisture and organo-mineral chemical composition and its implications for SOC persistence in an experimental field in New York, USA, with differences in long-term mean soil volumetric water content (0–0.15 m depth) ranging from 0.40 to 0.63 (v/v) during the growing season. Long-term soil moisture across 20 subplots on four fallow plots were positively correlated with SOC (R 2 = 0.23; P = 0.019, n = 20), mineral-associated organic matter (MAOM) content (g fraction/g soil) (R 2 = 0.44; P = 0.001; n = 20) and occluded particulate organic matter (oPOM) content (R 2 = 0.18; P = 0.033; n = 20). Higher long-term soil moisture was associated with a decrease in the relative content of sodium pyrophosphate extractable Fe (R 2 = 0.33; P < 0.005; n = 20), an increase in sodium dithionite extractable Fe (R 2 = 0.44; P < 0.001; n = 20), and an increase in SOC retention by non-crystalline Al pools (R 2 = 0.51; P = 0.0002 for sodium pyrophosphate extracts, R 2 = 0.41; P = 0.0014 for hydroxylamine hydrochloride extracts; n = 20 for both). Increasing long-term soil moisture was associated with a four-fold increase in microbial biomass C (per unit SOC) and lower metabolic quotient (R 2 = 0.56, P < 0.001). MAOM fractions of high-moisture soils had lower C:N (from C:N 9.5 to 9.0, R 2 = 0.27, P = 0.011, n = 20). Consistent with decreasing C:N, increasing decomposition with increasing moisture was reflected by a 15% and 10% greater proportion of oxidized carboxylic-C to aromatic-C and O-alkyl C, respectively, as measured with 13 C NMR, and a more pronounced FTIR signature of N-containing proteinaceous compounds in high-moisture MAOM fractions, indicative of microbial metabolites and transformation products. A partial least squares regression showed that SOC content increased with greater long-term moisture (P = 0.019), pyrophosphate-extractable Al (P = 0.0001), and exchangeable Ca (P = 0.013). Here our results show that higher long-term soil moisture resulted in SOC accrual by enhancing conversion of plant inputs into microbial biomass that interacts with reactive minerals.

58 GEOSCIENCES↗
DOE OSTI2024

Species-specific ribosomal RNA-FISH identifies interspecies cellular-material exchange, active-cell population dynamics and cellular localization of translation machinery in clostridial cultures and co-cultures

ABSTRACT The development of synthetic microbial consortia in recent years has revealed that complex interspecies interactions, notably the exchange of cytoplasmic material, exist even among organisms that originate from different ecological niches. Although morphogenetic characteristics, viable RNA and protein dyes, and fluorescent reporter proteins have played an essential role in exploring such interactions, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to further investigate interactions in synthetic or semisynthetic consortia. Despite its maturity, several challenges exist in using rRNA-FISH as a tool to quantify individual species population dynamics and interspecies interactions using high-throughput instrumentation such as flow cytometry. In this work, we resolve such challenges and apply rRNA-FISH to double and triple co-cultures of Clostridium acetobutylicum, Clostridium ljungdahlii, and Clostridium kluyveri . In pursuing our goal to capture each organism’s population dynamics, we demonstrate dynamic rRNA, and thus ribosome, exchange between the three species leading to the formation of hybrid cells. We also characterize the localization patterns of the translation machinery in the three species, identifying distinct, dynamic localization patterns among them. Our data also support the use of rRNA-FISH to assess the culture’s health and expansion potential, and, here again, our data find surprising differences among the three species examined. Taken together, our study argues for rRNA-FISH as a valuable and accessible tool for quantitative exploration of interspecies interactions, especially in organisms which cannot be genetically engineered or in consortia where selective pressures to maintain recombinant species cannot be used. IMPORTANCE Though dyes and fluorescent reporter proteins have played an essential role in identifying microbial species in co-cultures, we hypothesized that ribosomal RNA-fluorescence in situ hybridization (rRNA-FISH) could be adapted and applied to quantitatively probe complex interactions between organisms in synthetic consortia. Despite its maturity, several challenges existed before rRNA-FISH could be used to study Clostridium co-cultures of interest. First, species-specific probes for Clostridium acetobutylicum and Clostridium ljungdahlii had not been developed. Second, “state-of-the-art” labeling protocols were tedious and often resulted in sample loss. Third, it was unclear if FISH was compatible with existing fluorescent reporter proteins. We resolved these key challenges and applied the technique to co-cultures of C. acetobutylicum , C. ljungdahlii , and Clostridium kluyveri . We demonstrate that rRNA-FISH is capable of identifying rRNA/ribosome exchange between the three organisms and characterized rRNA localization patterns in each. In combination with flow cytometry, rRNA-FISH can capture sub-population dynamics in co-cultures.

Hill, John D.↗
DOE OSTI2022

Removal of primary nutrient degrading members severely reduces growth of soil microbial communities even when additional degraders are present

Understanding how microorganisms within a soil community interact to support collective respiration and growth remains challenging. Here we used a model substrate, chitin, and a Model Soil Consortium, MSC-2, to investigate how individual members of a microbial community contribute to decomposition and community growth. While MSC-2 can grow using chitin as the sole carbon source, we do not yet know how the growth kinetics or final biomass yields of MSC-2 vary when certain chitin degraders, or other important members, are absent. To characterize specific roles within this representative community, we carried out experiments leaving out members of MSC-2 and measuring biomass yields and CO2 production. We chose two members to iteratively leave out (referred to by genus name): Streptomyces, as it is predicted via gene expression analysis to be a major chitin degrader in the community, and Rhodococcus as it is predicted via species co-abundance analysis to interact with several other members. Our results showed that when MSC-2 lacked Streptomyces, growth and respiration of the community was severely reduced. Removal of either Streptomyces or Rhodococcus led to major changes in abundance for several other species, pointing to a comprehensive shifting of the microbial community when important members are removed as well as alterations in the metabolic profile, especially when Streptomyces was removed. These results show that when keystone, chitin degrading members are removed, other members, even those with the potential to degrade chitin, do not fill the same metabolic niche to promote community growth. In addition, highly connected members may be removed with similar or even increased levels of growth and respiration. Our findings are critical to a better understanding of soil microbiology, specifically in how communities maintain activity when biotic or abiotic factors lead to changes in biodiversity in soil systems.

McClure, Ryan S↗
DOE OSTI2025

Spatially structured bacterial interactions alter algal carbon flow to bacteria

Phytoplankton account for nearly half of global photosynthetic carbon fixation, and the fate of that carbon is regulated in large part by microbial food web processing. We currently lack a mechanistic understanding of how interactions among heterotrophic bacteria impact the fate of photosynthetically fixed carbon. Here, we used a set of bacterial isolates capable of growing on exudates from the diatom Phaeodactylum tricornutum to investigate how bacteria-bacteria interactions affect the balance between exudate remineralization and incorporation into biomass. With exometabolomics and genome-scale metabolic modeling, we estimated the degree of resource competition between bacterial pairs. In a sequential spent media experiment, we found that pairwise interactions were more beneficial than predicted based on resource competition alone, and 30% exhibited facilitative interactions. To link this to carbon fate, we used single-cell isotope tracing in a custom cultivation system to compare the impact of different "primary" bacterial strains in close proximity to live P. tricornutum on a distal "secondary" strain. We found that a primary strain with a high degree of competition decreased secondary strain carbon drawdown by 51% at the single-cell level, providing a quantitative metric for the "cost" of competition on algal carbon fate. Additionally, a primary strain classified as facilitative based on sequential interactions increased total algal-derived carbon assimilation by 7.6 times, integrated over all members, compared to the competitive primary strain. Our findings suggest that the degree of interaction between bacteria along a spectrum from competitive to facilitative is directly linked to algal carbon drawdown.

genome-scale metabolic model↗
DOE OSTI2023

Rapid remodeling of the soil lipidome in response to a drying-rewetting event

Abstract Background Microbiomes contribute to multiple ecosystem services by transforming organic matter in the soil. Extreme shifts in the environment, such as drying-rewetting cycles during drought, can impact the microbial metabolism of organic matter by altering microbial physiology and function. These physiological responses are mediated in part by lipids that are responsible for regulating interactions between cells and the environment. Despite this critical role in regulating the microbial response to stress, little is known about microbial lipids and metabolites in the soil or how they influence phenotypes that are expressed under drying-rewetting cycles. To address this knowledge gap, we conducted a soil incubation experiment to simulate soil drying during a summer drought of an arid grassland, then measured the response of the soil lipidome and metabolome during the first 3 h after wet-up. Results Reduced nutrient access during soil drying incurred a replacement of membrane phospholipids, resulting in a diminished abundance of multiple phosphorus-rich membrane lipids. The hot and dry conditions increased the prevalence of sphingolipids and lipids containing long-chain polyunsaturated fatty acids, both of which are associated with heat and osmotic stress-mitigating properties in fungi. This novel finding suggests that lipids commonly present in eukaryotes such as fungi may play a significant role in supporting community resilience displayed by arid land soil microbiomes during drought. As early as 10 min after rewetting dry soil, distinct changes were observed in several lipids that had bacterial signatures including a rapid increase in the abundance of glycerophospholipids with saturated and short fatty acid chains, prototypical of bacterial membrane lipids. Polar metabolites including disaccharides, nucleic acids, organic acids, inositols, and amino acids also increased in abundance upon rewetting. This rapid metabolic reactivation and growth after rewetting coincided with an increase in the relative abundance of firmicutes, suggesting that members of this phylum were positively impacted by rewetting. Conclusions Our study revealed specific changes in lipids and metabolites that are indicative of stress adaptation, substrate use, and cellular recovery during soil drying and subsequent rewetting. The drought-induced nutrient limitation was reflected in the lipidome and polar metabolome, both of which rapidly shifted (within hours) upon rewet. Reduced nutrient access in dry soil caused the replacement of glycerophospholipids with phosphorus-free lipids and impeded resource-expensive osmolyte accumulation. Elevated levels of ceramides and lipids with long-chain polyunsaturated fatty acids in dry soil suggest that lipids likely play an important role in the drought tolerance of microbial taxa capable of synthesizing these lipids. An increasing abundance of bacterial glycerophospholipids and triacylglycerols with fatty acids typical of bacteria and polar metabolites suggest a metabolic recovery in representative bacteria once the environmental conditions are conducive for growth. These results underscore the importance of the soil lipidome as a robust indicator of microbial community responses, especially at the short time scales of cell-environment reactions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Microbiome assembly in thawing permafrost and its feedbacks to climate

Abstract The physical and chemical changes that accompany permafrost thaw directly influence the microbial communities that mediate the decomposition of formerly frozen organic matter, leading to uncertainty in permafrost–climate feedbacks. Although changes to microbial metabolism and community structure are documented following thaw, the generality of post‐thaw assembly patterns across permafrost soils of the world remains uncertain, limiting our ability to predict biogeochemistry and microbial community responses to climate change. Based on our review of the Arctic microbiome, permafrost microbiology, and community ecology, we propose that Assembly Theory provides a framework to better understand thaw‐mediated microbiome changes and the implications for community function and climate feedbacks. This framework posits that the prevalence of deterministic or stochastic processes indicates whether the community is well‐suited to thrive in changing environmental conditions. We predict that on a short timescale and following high‐disturbance thaw (e.g., thermokarst), stochasticity dominates post‐thaw microbiome assembly, suggesting that functional predictions will be aided by detailed information about the microbiome. At a longer timescale and lower‐intensity disturbance (e.g., active layer deepening), deterministic processes likely dominate, making environmental parameters sufficient for predicting function. We propose that the contribution of stochastic and deterministic processes to post‐thaw microbiome assembly depends on the characteristics of the thaw disturbance, as well as characteristics of the microbial community, such as the ecological and phylogenetic breadth of functional guilds, their functional redundancy, and biotic interactions. These propagate across space and time, potentially providing a means for predicting the microbial forcing of greenhouse gas feedbacks to global climate change.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

Viral infections likely mediate microbial controls on ecosystem responses to global warming

Abstract Climate change is affecting how energy and matter flow through ecosystems, thereby altering global carbon and nutrient cycles. Microorganisms play a fundamental role in carbon and nutrient cycling and are thus an integral link between ecosystems and climate. Here, we highlight a major black box hindering our ability to anticipate ecosystem climate responses: viral infections within complex microbial food webs. We show how understanding and predicting ecosystem responses to warming could be challenging—if not impossible—without accounting for the direct and indirect effects of viral infections on different microbes (bacteria, archaea, fungi, protists) that together perform diverse ecosystem functions. Importantly, understanding how rising temperatures associated with climate change influence viruses and virus-host dynamics is crucial to this task, yet is severely understudied. In this perspective, we (i) synthesize existing knowledge about virus-microbe-temperature interactions and (ii) identify important gaps to guide future investigations regarding how climate change might alter microbial food web effects on ecosystem functioning. To provide real-world context, we consider how these processes may operate in peatlands—globally significant carbon sinks that are threatened by climate change. We stress that understanding how warming affects biogeochemical cycles in any ecosystem hinges on disentangling complex interactions and temperature responses within microbial food webs.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

Gallionellaceae in rice root plaque: metabolic roles in iron oxidation, nutrient cycling, and plant interactions

On the roots of wetland plants such as rice, Fe(II) oxidation forms Fe(III) oxyhydroxide-rich plaques that modulate plant nutrient and metal uptake. The microbial roles in catalyzing this oxidation have been debated and it is unclear if these iron-oxidizers mediate other important biogeochemical and plant interactions. To investigate this, we studied the microbial communities, metagenomes, and geochemistry of iron plaque on field-grown rice, plus the surrounding rhizosphere and bulk soil. Plaque iron content (per mass root) increased over the growing season, showing continuous deposition. Analysis of 16S rRNA genes showed abundant Fe(II)-oxidizing and Fe(III)-reducing bacteria (FeOB and FeRB) in plaque, rhizosphere, and bulk soil. FeOB were enriched in relative abundance in plaque, suggesting FeOB affinity for the root surface. Gallionellaceae FeOB Sideroxydans were enriched during vegetative and early reproductive rice growth stages, while a Gallionella was enriched during reproduction through grain maturity, suggesting distinct FeOB niches over the rice life cycle. FeRB Anaeromyxobacter and Geobacter increased in plaque later, during reproduction and grain ripening, corresponding to increased plaque iron. Metagenome-assembled genomes revealed that Gallionellaceae may grow mixotrophically using both Fe(II) and organics. The Sideroxydans are facultative, able to use non-Fe substrates, which may allow colonization of rice roots early in the season. FeOB genomes suggest adaptations for interacting with plants, including colonization, plant immunity defense, utilization of plant organics, and nitrogen fixation. Taken together, our results strongly suggest that rhizoplane and rhizosphere FeOB can specifically associate with rice roots, catalyzing iron plaque formation, with the potential to contribute to plant growth.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2016

Risk of Adverse Health Effects Due to Host-Microorganism Interactions

Numerous spaceflight experiments have been conducted to investigate alterations in microbial responses resulting from culture during spaceflight and spaceflight-analogs. However, recent studies investigating spaceflight-associated alterations in microbial virulence have initiated the review and production of evidence to better understand the impact these alterations would have on the incidence of infectious disease during a spaceflight exploration mission. The preponderance of evidence indicates that alterations in microbial gene expression and phenotype (including virulence) are occurring; however, the clinical implications of such changes are still unclear. Greater knowledge is required including a better understanding of the mechanism behind unique spaceflight-associated microbial responses to determine how this environmental stimulus impacts various microorganisms, their diversity and concentration in the spacecraft and crew microbiome, their impact on the vehicle and crew, and their resistance to current mitigation and antibiotic regimens. This knowledge will enable us to determine requirements, guidelines, and processes for design and monitoring of the next generation vehicles.

Ott, C. Mark↗
DOE OSTI2021

Kinetic, metabolic, and statistical analytics: addressing metabolic transport limitations among organelles and microbial communities

Microbial organisms engage in a variety of metabolic interactions. A crucial part of these interactions is the exchange of molecules between different organelles, cells, and the environment. The main forces mediating this metabolic exchange are transporters. This transport can be difficult to measure experimentally because several transport mechanisms remain opaque. However, theoretical calculations about the inputs and outputs of cells via metabolic exchanges have enabled the successful inference of the workings of intra-organismal and inter-organismal systems. Kinetic, metabolic, and statistical modeling approaches in combination with omics data are enhancing our knowledge and understanding about metabolic exchange and mass resource allocation. Furthermore, this model-driven analytics approach can guide effective experimental design and yield new insights into biological function and control.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Potential virus-mediated nitrogen cycling in oxygen-depleted oceanic waters

Viruses play an important role in the ecology and biogeochemistry of marine ecosystems. Beyond mortality and gene transfer, viruses can reprogram microbial metabolism during infection by expressing auxiliary metabolic genes (AMGs) involved in photosynthesis, central carbon metabolism, and nutrient cycling. While previous studies have focused on AMG diversity in the sunlit and dark ocean, less is known about the role of viruses in shaping metabolic networks along redox gradients associated with marine oxygen minimum zones (OMZs). Here, we analyzed relatively quantitative viral metagenomic datasets that profiled the oxygen gradient across Eastern Tropical South Pacific (ETSP) OMZ waters, assessing whether OMZ viruses might impact nitrogen (N) cycling via AMGs. Identified viral genomes encoded six N-cycle AMGs associated with denitrification, nitrification, assimilatory nitrate reduction, and nitrite transport. The majority of these AMGs (80%) were identified in T4-like Myoviridae phages, predicted to infect Cyanobacteria and Proteobacteria, or in unclassified archaeal viruses predicted to infect Thaumarchaeota. Four AMGs were exclusive to anoxic waters and had distributions that paralleled homologous microbial genes. Together, these findings suggest viruses modulate N-cycling processes within the ETSP OMZ and may contribute to nitrogen loss throughout the global oceans thus providing a baseline for their inclusion in the ecosystem and geochemical models.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Investigating Bacterial-Fungal Interactions using Fungal Highway Columns in Diverse Environments and Substrates

Bacterial-fungal interactions (BFIs) play an integral role in shaping microbial community composition, biogeochemical functions, spatial dynamics, and microbial dispersal. Mycelial networks created by filamentous fungi or other filamentous microorganisms (e.g., Oomycetes) act as 'fungal highways' that can be utilized by bacteria for transport throughout heterogeneous environments, greatly facilitating their mobility and granting them access to regions that may be challenging or impossible to reach on their own (e.g., due to air pockets within the soil). Several devices and experimental protocols have been created to study these fungal highways, including fungal highway columns. The fungal highway column designed by our group can be used for a variety of in situ or in vitro applications, as well as with diverse environmental and host-associated sample types. Herein, we describe the methods for performing experiments with these columns, including designing, printing, sterilizing, and preparing the devices. The options for analyzing data obtained from the use of these devices are also discussed here, and troubleshooting advice regarding potential pitfalls associated with experiments using fungal highway columns is offered. These devices can be used to gain a more comprehensive understanding of the diversity, mechanisms, and dynamics of fungal highway BFIs to provide valuable insights into the structural and functional dynamics within complex environments (e.g., soils) and across diverse habitats in which bacteria and fungi co-exist.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2011

Microbiological Lessons Learned from the Space Shuttle

After 30 years of being the centerpiece of NASA s human spacecraft, the Space Shuttle will retire. This highly successful program provided many valuable lessons for the International Space Station (ISS) and future spacecraft. Major microbiological risks to crewmembers include food, water, air, surfaces, payloads, animals, other crewmembers, and ground support personnel. Adverse effects of microorganisms are varied and can jeopardize crew health and safety, spacecraft systems, and mission objectives. Engineering practices and operational procedures can minimize the negative effects of microorganisms. To minimize problems associated with microorganisms, appropriate steps must begin in the design phase of new spacecraft or space habitats. Spacecraft design must include requirements to control accumulation of water including humidity, leaks, and condensate on surfaces. Materials used in habitable volumes must not contribute to microbial growth. Use of appropriate materials and the implementation of robust housekeeping that utilizes periodic cleaning and disinfection will prevent high levels of microbial growth on surfaces. Air filtration can ensure low levels of bioaerosols and particulates in the breathing air. The use of physical and chemical steps to disinfect drinking water coupled with filtration can provide safe drinking water. Thorough preflight examination of flight crews, consumables, and the environment can greatly reduce pathogens in spacecraft. The advances in knowledge of living and working onboard the Space Shuttle formed the foundation for environmental microbiology requirements and operations for the International Space Station (ISS) and future spacecraft. Research conducted during the Space Shuttle Program resulted in an improved understanding of the effects of spaceflight on human physiology, microbial properties, and specifically the host-microbe interactions. Host-microbe interactions are substantially affected by spaceflight. Astronaut immune functions were found to be altered. Selected microorganisms were found to become more virulent during spaceflight. The increased knowledge gained on the Space Shuttle resulted in further studies of the host-microbe interactions on the ISS to determine if countermeasures were necessary. Lessons learned from the Space Shuttle Program were integrated into the ISS resulting in the safest space habitat to date.

Pierson, Duane L.↗
DOE OSTI2020

A Systems Biology Approach to Energy Flow in H 2 Producing Microbial Communities: Microbial Mats

This report describes the results of work conducted at NASA Ames Research Center, in collaboration with investigators at Lawrence Livermore National Laboratory, during the time period 12/15/2014 to 12/15/2017. The work was an extension of a collaboration with LLNL that began in 2008. Work this reporting period focused primarily on tasks related to microbial mats from both the Elkhorn Slough and Baja California field sites, including the analysis and publication of data produced prior to 12/15/2014. During this reporting period, in addition to the microbial mat work, we also conducted a series of experiments with a particular algal-bacterial association (C. sorokiniana and A. brasilense). A number of aspects of this well-characterized algal-bacterial association enabled us not only to further our understanding of interactions between these particular organisms, but to apply that knowledge and techniques to our collection of microorganisms isolated from microbial mats. Microbial mat experiments focused both on intact mat communities and on cultures of organisms isolated from the mats (the Microbial Mat Living Library). In a series of co-culture experiments, we documented both positive and negative interactions amongst heterotrophic and autotrophic isolates of microbial mat microorganisms. One particularly important and novel mat cyanobacterium (ESFC-1) was studied in detail. We developed techniques to pursue an investigation of functional redundancy (the idea that closely related organisms perform identical roles in a community) in cyanobacterial autotrophs in the Elkhorn Slough microbial mat. In those experiments, we documented numerous instances of increases in growth rate between co-cultures of two organisms (relative to their growth rate in isolation) performing what would seem to be identical functional roles. Simplified microbial mat communities consisting of organisms from the Microbial Mat Living Library were constructed and studied in laboratory experiments. The “constructed mats” were grown in standard laboratory glassware and plasticware, but cultures were also combined with artificial materials in an effort to re-create the three-dimensional structure of naturally occurring microbial mats. These constructed mats re-created a number of functions attributable to natural microbial mats (N-fixation, H2-production) and simplified their study using molecular ecological techniques due to the smaller number of microbial “species” present. Cyanobacteria which migrated in response to vertical variations in light intensity (irradiance) obtained characteristic positions in constructed mats that were similar to their positions in natural mats. A detailed investigation of nitrogen cycling in both the Baja California and Elkhorn Slough microbial mats was initiated during this reporting period. The nitrogen cycle in the microbial mats has not been studied in depth, despite the fact that nitrogen usually limits growth in marine environments. The organisms conducting nitrogen cycling in both microbial mats were studied using the genomes available from earlier work on these mats, as well as with a targeted approach using molecular ecological methods developed here. Quantitative measurements of rates of nitrogen transformations were made using stable isotopically labeled nitrogen. The processes quantified and examined using molecular ecological methods included: nitrogen fixation, ammonification, ammonium and nitrate assimilation, denitrification, anaerobic oxidation of ammonium, and dissimilatory nitrate reduction to ammonium. In a series of experiments designed to examine a potential application for microbial mats, we demonstrated that both Elkhorn Slough and Baja California microbial mats were very effective in removing large quantities of both nitrate and ammonium from waste streams containing these compounds at levels where they would be considered to be pollutants. Considerable effort was expended during this reporting period to analyze, and prepare for publication, metagenomes and metatranscriptomes collected from two experimental manipulations of microbial mats. The first, a diel study, was conducted in November 2011, while the second, an investigation of the effects of sulfate on community composition, was conducted during this reporting period. For the earlier study, reference-based and reference-free methods were used to assess microbial ecology and genetic partitioning in these complex microbial systems, and that work is available as a pre-print. Four metagenomes from the same experiment were published. In the second, longer (nearly 200 day) manipulation, rRNA iTag libraries for cDNA and DNA were analyzed to determine the effects of sulfate concentration on microbial mat community composition over the course of the experiment. Work on the algal-bacterial association (Chlorella sorokiniana and Azospirillum brasilense) focused initially on demonstrating the transfer of carbon and nitrogen between the partners, using stable isotopic labeling and the LLNL NanoSIMS. Subsequently, the potential role of diazotrophy (nitrogen fixation) by the bacterial partner in supporting the growth of the association was assessed in a series of experiments. It appears to be the case that, although the bacterial partner demonstrably increases the fitness of the algae under nitrogen limitation, and cells of C. sorokiniana receive nitrogen originally fixed from atmospheric nitrogen by A. brasilense, the amount of nitrogen obtained by diazotrophy is too small to explain the increase in fitness.

08 HYDROGEN↗