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At least 109 records · Page 6
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

The Study of Microbial Physiology Under Microoxic Conditions Is Critical but Neglected

ABSTRACT During the early evolution of life on Earth, the environment was largely free of molecular oxygen, and only anaerobic life existed. With the subsequent oxidation of oceans and the atmosphere, a wide range of environmental niches, ranging from anoxic to microoxic/hypoxic and oxic, developed. Despite this broad range of natural environments, microbiology as a field has focused on the physiology, metabolism, and genetics of aerobic microorganisms, with less attention paid to anaerobes and much less attention paid to microaerophiles. The disparity in studies between aerobic and anaerobic conditions is rampant in host‐associated systems, particularly in human health, and studies of microorganisms in intermediate oxygen conditions between fully aerobic and fully anoxic conditions are exceedingly rare. Studies on the physiological behaviour, metabolism, growth response, and drug susceptibility patterns of commensal and pathogenic organisms are almost totally neglected in microoxic conditions. Furthermore, microorganisms from microaerobic and microoxic ecosystems have been less robustly explored in terms of physiology, growth, and metabolism. In this work, we highlight the importance of understanding the physiological and metabolic behaviours of microorganisms under hypoxic or microoxic conditions.

Environmental Sciences & Ecology↗
DOE OSTI2022

Balancing Trade-Offs Imposed by Growth Media and Mass Spectrometry for Bacterial Exometabolomics

The bacterial exometabolome consists of a vast array of specialized metabolites, many of which are only produced in response to specific environmental stimuli. For this reason, it is desirable to control the extracellular environment with a defined growth medium composed of pure ingredients. However, complex (undefined) media are expected to support the robust growth of a greater variety of microorganisms than defined media. Here, we investigate the trade-offs inherent to a range of complex and defined solid media for the growth of soil microorganisms, production of specialized metabolites, and detection of these compounds using direct infusion mass spectrometry. We find that complex media support growth of more soil microorganisms, as well as allowing for the detection of more previously discovered natural products as a fraction of total m/z features detected in each sample. However, the use of complex media often caused mass spectrometer injection failures and poor-quality mass spectra, which in some cases resulted in over a quarter of samples being removed from analysis. Defined media, while more limiting in growth, generated higher quality spectra and yielded more m/z features after background subtraction. These results inform future exometabolomic experiments requiring a medium that supports the robust growth of many soil microorganisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Community Structure and Microbial Associations in Sediment-Free Methanotrophic Enrichment Cultures from a Marine Methane Seep

We report Syntrophic consortia of anaerobic methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB) consume large amounts of methane and serve as the foundational microorganisms in marine methane seeps. Despite their importance in the carbon cycle, research on the physiology of ANME-SRB consortia has been hampered by the slow growth and complex physicochemical environment the consortia inhabit. Here, we report successful sediment-free enrichment of ANME-SRB consortia from deep-sea methane seep sediments in the Santa Monica Basin, California. Anoxic Percoll density gradients and size-selective filtration were used to separate ANME-SRB consortia from sediment particles and single cells to accelerate the cultivation process. Over a 3-year period, a subset of the sediment-associated ANME and SRB lineages, predominantly comprised of ANME-2a/2b (“Candidatus Methanocomedenaceae”) and their syntrophic bacterial partners, SEEP-SRB1/2, adapted and grew under defined laboratory conditions. Metagenome-assembled genomes from several enrichments revealed that ANME-2a, SEEP-SRB1, and Methanococcoides in different enrichments from the same inoculum represented distinct species, whereas other coenriched microorganisms were closely related at the species level. This suggests that ANME, SRB, and Methanococcoides are more genetically diverse than other members in methane seeps. Flow cytometry sorting and sequencing of cell aggregates revealed that Methanococcoides, Anaerolineales, and SEEP-SRB1 were overrepresented in multiple ANME-2a cell aggregates relative to the bulk metagenomes, suggesting they were physically associated and possibly interacting. Overall, this study represents a successful case of selective cultivation of anaerobic slow-growing microorganisms from sediments based on their physical characteristics, introducing new opportunities for detailed genomic, physiological, biochemical, and ecological analyses. Biological anaerobic oxidation of methane (AOM) coupled with sulfate reduction represents a large methane sink in global ocean sediments. Methane consumption is carried out by syntrophic archaeal-bacterial consortia and fuels a unique ecosystem, yet the interactions in these slow-growing syntrophic consortia and with other associated community members remain poorly understood. The significance of this study is the establishment of sediment-free enrichment cultures of anaerobic methanotrophic archaea and sulfate-reducing bacteria performing AOM with sulfate using selective cultivation approaches based on size, density, and metabolism. By reconstructing microbial genomes and analyzing community composition of the enrichment cultures and cell aggregates, we shed light on the diversity of microorganisms physically associated with AOM consortia beyond the core syntrophic partners. These enrichment cultures offer simplified model systems to extend our understanding of the diversity of microbial interactions within marine methane seeps.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Biomining Critical Elements and Metals

The US government has identified 17 critical elements, including most rare earth elements (REEs), and metals, which are used extensively in consumer electronics as well as military and national security hardware. We are developing techniques for biomining these elements, the process of using microorganisms to extract critical elements from water, ores, and mine waste to fill these needs. Bioextraction is focusing on: 1) building on our first-year successes of identifying microbes that generate exudates that enhance the extraction of multivalent cations, 2) evaluating available phosphate solubilizing microbes to dissolve apatite and monazite, and 3) and biosurfactant producing microbes. Bioaccumulation efforts involves testing microbes that: 1) naturally release complex exudates that contain organic chelating agents, and 2) naturally accumulate or hyperaccumulate (wt-% levels) metals. Results from year one includes proof of concept that biosurfactant producing microorganisms can release REEs from select ores and that release of REEs is increased by the addition of glucose. Molecular results for microorganism profiling proved a population shift after addition of microorganisms. The study will culminate in a proof-of-concept demonstration, manuscripts, and the data needed for scaledup biomining of REEs.

36 MATERIALS SCIENCE↗
DOE OSTI2024

Impact of intense sanitization procedures on bacterial communities recovered from floor drains in pork processing plants

Pork processing plants in the United States (US) cease operations for 24–48 h every six or twelve months to perform intense sanitization (IS) using fogging, foaming, and further antimicrobial treatments to disrupt natural biofilms that may harbor pathogens and spoilage organisms. The impact such treatments have on short-term changes in environmental microorganisms is not well understood, nor is the rate at which bacterial communities return. Swab samples were collected from floor drains to provide representative environmental microorganisms at two US pork processing plants before, during, and after an IS procedure. Samples were collected from four coolers where finished carcasses were chilled and from four locations near cutting tables. Each sample was characterized by total mesophile count (TMC), total psychrophile count (TPC), and other indicator bacteria; their biofilm-forming ability, tolerance of the formed biofilm to a quaternary ammonium compound (300 ppm, QAC), and ability to protect co-inoculated Salmonella enterica. In addition, bacterial community composition was determined using shotgun metagenomic sequencing. IS procedures disrupted bacteria present but to different extents depending on the plant and the area of the plant. IS reduced TPC and TMC, by up to 1.5 Log 10 CFU only to return to pre-IS levels within 2–3 days. The impact of IS on microorganisms in coolers was varied, with reductions of 2–4 Log 10 , and required 2 to 4 weeks to return to pre-IS levels. The results near fabrication lines were mixed, with little to no significant changes at one plant, while at the other, two processing lines showed 4 to 6 Log 10 reductions. Resistance to QAC and the protection of Salmonella by the biofilms varied between plants and between areas of the plants as well. Community profiling of bacteria at the genus level showed that IS reduced species diversity and the disruption led to new community compositions that in some cases did not return to the pre-IS state even after 15 to 16 weeks. The results found here reveal the impact of using IS to disrupt the presence of pathogen or spoilage microorganisms in US pork processing facilities may not have the intended effect.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Reveal of Uranium Bioremediation Mechanisms by Bacillus Species through Proteomics Studies

Radionuclides, such as Uranium (U) and heavy metals continue to pose threats to the ecosystem health and processes at the Department of Energy (DOE) managed, Savannah River Site (SRS), located along the Savannah River near Aiken, SC. Such co-contaminated environments are difficult to remediate using conventional excavation and disposal or pump-and-treat approaches. Globally, more than 109 tons of uranium contaminated areas pose a long term threat to human and ecological health. Even with presented low concentrations in the brain, central nervous effects are still observed. Uranium and depleted uranium (DU) have long term effects on the kidneys. Some small general health effects include severe headaches and breathing problems. Uranus ions are toxic to living cells because they inhibit metabolism of carbohydrates by blocking ATP binding sites. Bioremediation by microorganisms represents an alternative solution, which is advantageous because of the possibility of biosorbent regeneration, cost-effectiveness, increased metal removal and easy recovery of some valuable metals. Bacillus sp. bacterium was previously used in the bioremediation of heavy metals in coal mine run off waters of SRS. However, its ability to bioremediate uranium was unknown so far. Hence, in the present study, uranium bioremediation by Bacillus sp. bacterium was investigated. The mechanism of bioremediation was also revealed through proteomics studies. Heavy metals contamination poses a serious threat to water, soil and human health. Soil and water are contaminated due to excessive exploitation of uranium mines for generation of nuclear energy and weaponry. It is not degradable easily and persist in soil and water for a long period of time due to its long half- life. Savannah river site (SRS) is one of the uranium contaminated sites. The physical or chemical remediation techniques are costly and complex. Microbial system approaches with competent bacteria has received increased attention due to its adaptability in various environmental matrices and cost effectiveness. However, even though there are multiple suggested pathways (F1), the specific mechanisms that drive this behavior are still unclear, especially with popular microorganisms such as Bacillus species. In a previous research (Ibeanusi et al, 2003) Bacillus sp. was shown to detoxify and precipitate a variety of heavy metals in coal pile runoff waters of SRS site. Additionally, genomic analysis demonstrated that this microorganism posses multiple attributes for chemical transport regulation and metabolic pathways. U remediation occurs during the first 20 hours of exposure. During this time period, Bacillus sp. may work primarily under two mechanisms - sorption and accumulation. These two mechanisms simultaneously work to protect the microorganism from high concentrations. Figure 10 demonstrates that certain proteins are up regulated and down regulated under extreme stressful conditions. Membrane fraction proteins were up regulated. Cytosolic fraction proteins were down regulated. This protein information coincides with the adsorption behavior. Bacillus sp. is a good candidate for U remediation at various concentrations.

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗
DOE OSTI2022

Phosphate Availability Modulates Root Exudate Composition and Rhizosphere Microbial Community in a Teosinte and a Modern Maize Cultivar

Domestication and breeding have affected interactions between plants and their microbiomes in ways that are only beginning to be understood but may have important implications for recruitment of rhizosphere microorganisms, particularly under stress conditions. We investigated the responses of a modern maize (Zea mays subsp. mays) cultivar and its wild relative, teosinte (Z. mays subsp. parviglumis), to different phosphate availabilities. We appraised responses of the plant-microbial holobiont to phosphate stresses by profiling root exudate metabolomes, and microbial communities in the root endosphere and rhizosphere. We also performed plate assays to quantify phosphate-solubilizing microorganisms from the rhizosphere. Although root exudate metabolite profiles were distinct between the teosinte and modern maize under high phosphate, both plants shifted exudate compositions in response to phosphate stress toward a common metabolite profile. Root and rhizosphere microbial communities also responded significantly to both plant type and the phosphate availability. A subset of bacterial and fungal taxa were differentially abundant under the different phosphate conditions, with each of the three conditions favoring different taxa. Both teosinte and maize rhizospheres harbored phosphate-solubilizing microorganisms under all growth conditions. These results suggest that the root exudation response to phosphate stress was conserved through the domestication of maize from teosinte, shifting exudation levels of specific metabolites. Although microbial communities also shifted, plate-based assays did not detect selective recruitment of phosphate solubilizers in response to phosphate availability.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Microbial potentiometric sensor technology for real-time detecting and monitoring of toxic metals in aquatic matrices

Considering that toxic metals can affect metabolic processes in microorganisms adversely, it can be hypothesized that these metals in water matrices would induce a decrease in metabolic activity of the biofilm microorganisms populating the surface of a sensing electrode, which could be registered as a change in the open-circuit potential (OCP) generated by the biofilm microorganisms. The goal of this study was to test this hypothesis and demonstrate the underlying principle that microbial potentiometric sensor (MPS) technology could be used for long-term and real-time monitoring and detection of rapid changes in metal concentrations in realistic aquatic environments. To address the goal, four objective were addressed: (1) a batch reactor with three graphite-based MPS electrodes was fabricated; (2) a set of single-ion solutions and one multiple ion solution were prepared reflecting realistic concentrations of metals found in electroplating wastewaters; (3) the responses of the MPS to the simultaneous presence of multiple toxic metal ions in a single solution were measured; and (4) the changes of the MPS signals to the presence of individual metal ion solutions were examined. While the hypothesis was validated, the study also revealed that the MPS was sufficiently sensitive to not only detect, but also quantify, toxic metal ion concentrations in aqueous solutions. The coefficients of determination, which were R 2 > 0.995, and responsiveness of <1 μmol/L for some toxic metal cations, strongly support the performance of MPS technology in the echelons of expensive analytical tools capable detecting and measuring trace elements.The magnitude of the MPS response was toxic metal specific. When the molar concertation normalizes the inhibition portion of the signal area, the assessed sensitivity order was: Se>Cd>Pb>Ag>Ni> Zn. The study provides valuable information for enforcement agents, environmental professionals, and wastewater treatment operators, so toxic metal pollution and its detrimental impacts can be prevented and mitigated.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

BONCAT-FACS-Seq reveals the active fraction of a biocrust community undergoing a wet-up event

Determining which microorganisms are active within soil communities remains a major technical endeavor in microbial ecology research. One promising method to accomplish this is coupling bioorthogonal non-canonical amino acid tagging (BONCAT) with fluorescence activated cell sorting (FACS) which sorts cells based on whether or not they are producing new proteins. Combined with shotgun metagenomic sequencing (Seq), we apply this method to profile the diversity and potential functional capabilities of both active and inactive microorganisms in a biocrust community after being resuscitated by a simulated rain event. We find that BONCAT-FACS-Seq is capable of discerning the pools of active and inactive microorganisms, especially within hours of applying the BONCAT probe. The active and inactive components of the biocrust community differed in species richness and composition at both 4 and 21 h after the wetting event. The active fraction of the biocrust community is marked by taxa commonly observed in other biocrust communities, many of which play important roles in species interactions and nutrient transformations. Among these, 11 families within the Firmicutes are enriched in the active fraction, supporting previous reports indicating that the Firmicutes are key early responders to biocrust wetting. We highlight the apparent inactivity of many Actinobacteria and Proteobacteria through 21 h after wetting, and note that members of the Chitinophagaceae, enriched in the active fraction, may play important ecological roles following wetting. Based on the enrichment of COGs in the active fraction, predation by phage and other bacterial members, as well as scavenging and recycling of labile nutrients, appear to be important ecological processes soon after wetting. To our knowledge, this is the first time BONCAT-FACS-Seq has been applied to biocrust samples, and therefore we discuss the potential advantages and shortcomings of coupling metagenomics to BONCAT to intact soil communities such as biocrust. In all, by pairing BONCAT-FACS and metagenomics, we are capable of highlighting the taxa and potential functions that typifies the microbes actively responding to a rain event.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Advancing biological processing for valorization of plastic wastes

We report the useful applications of plastics are as significant as the problems associated with their disposal. Current waste management strategies are ineffective at preventing the flow of plastics into landfills, wastewater treatment, and the environment. Despite the importance of recycling technologies, most plastics are used for consumables and are discarded after limited usage. Moreover, only certain plastics are recycled, and those that are generally yield lower value products. Discovering alternative routes for valorizing plastic wastes is essential. Emerging research involves the use of biocatalysts (i.e., enzymes and microorganisms) to produce valuable products like specialty polymers, biosurfactants, and drug precursors from these feedstocks. These bioprocesses commonly integrate physio-chemical pretreatments (to generate biologically reactive intermediates) and biological upgrading (to convert intermediates to products). Herein, publications that explore plastic deconstruction technologies and bioprocesses are reviewed with an emphasis on plastic waste valorization. Understanding how microorganisms metabolize the intermediates of plastic degradation and manage their inhibitory effects is a prerequisite for engineering bioprocesses. For these heterogeneous wastes, a paradigm shift from axenic cultivation of microorganisms to cultivation of microbial consortia may benefit conversion efficiency and stress resilience. Thus, a summary is provided for metabolic pathways and constituent enzymes required to synthesize biofuels, biomaterials, and chemicals from biologically reactive intermediates. Colossal challenges associated with the use of plastics as feedstocks for direct biological processing highlight the need for integrating physical, chemical, and biological technologies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Carbon acquisition ecological strategies to connect soil microbial biodiversity and carbon cycling

Soil carbon feedbacks to global change are uncertain, and the biological processes that govern soil organic matter decomposition are not resolved in current ecosystem models. Though it is recognized that microbial biodiversity influences decomposition rates, incorporating this relationship into ecosystem models is challenging because microbial communities are prohibitively diverse. It is likely necessary to distill microbial biodiversity by focusing on functional groups or ecological strategies. The ecological strategies that currently dominate the microbial ecology literature derive from macroecological theory, have clear weaknesses, and have had limited success when applied to predict soil carbon dynamics. Here, we present a new framework for soil microorganisms: Carbon Acquisition Ecological Strategies (CAES), and we outline a path toward incorporating microbial biodiversity into ecosystem models using this framework to enhance predictions of soil carbon feedbacks to global change. Because a microorganism's diet is central to its ecological niche and likely to covary with other ecologically significant traits, we posit that carbon acquisition may serve as a tractable foundation for developing ecological strategies. Further, we describe four candidate ecological strategies for soil microorganisms: 1° decomposers that assimilate complex plant polymers, 2° decomposers that assimilate microbial necromass, passive consumers that assimilate dissolved organic carbon, and predatory microbes that assimilate live microbial biomass. These strategies are directly linked to soil carbon pools currently represented in ecosystem models and may provide a foundation for greater integration of microbial community dynamics into ecosystem models.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

A genomic view of Earth’s biomes

Microorganisms are essential to all life on Earth through critical roles in key biological processes and diverse interactions with other organisms that shape ecosystems, drive biogeochemical cycles and influence both human health and environmental health. High-throughput sequencing from environmental samples has revolutionized the understanding of microbial diversity and functions. With vast amounts of genomes now available across Earth’s biomes, these data provide a blueprint of microbial life that can be harnessed for a more holistic understanding of microbiome structure and function across the various ecosystems on Earth. Here we review the application of genome-centric approaches, including recent advances in single-cell sequencing and functional profiling, to survey microbial and viral diversity. Furthermore, we highlight some of the most impactful evolutionary and functional discoveries, explore the spatial diversity and temporal dynamics of microorganisms across diverse environments, and discuss genome-enabled insights into host-associated microorganisms.

Ecology↗
DOE OSTI2022

Life and death in the soil microbiome: how ecological processes influence biogeochemistry

We report soil microorganisms shape global element cycles in life and death. Living soil microorganisms are a major engine of terrestrial biogeochemistry, driving the turnover of soil organic matter — Earth’s largest terrestrial carbon pool and the primary source of plant nutrients. Their metabolic functions are influenced by ecological interactions with other soil microbial populations, soil fauna and plants, and the surrounding soil environment. Remnants of dead microbial cells serve as fuel for these biogeochemical engines because their chemical constituents persist as soil organic matter. This non-living microbial biomass accretes over time in soil, forming one of the largest pools of organic matter on the planet. In this Review, we discuss how the biogeochemical cycling of organic matter depends on both living and dead soil microorganisms, their functional traits, and their interactions with the soil matrix and other organisms. With recent omics advances, many of the traits that frame microbial population dynamics and their ecophysiological adaptations can be deciphered directly from assembled genomes or patterns of gene or protein expression. Thus, it is now possible to leverage a trait-based understanding of microbial life and death within improved biogeochemical models and to better predict ecosystem functioning under new climate regimes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Microbial dispersal into surface soil is limited on a meter scale

Abstract Dispersal shapes microbial communities, yet it is largely unknown how fast or how far free-living microorganisms move in the environment. Here, we deployed microbial traps along transects spanning a grassland and neighboring shrubland to quantify the rate and distance at which microorganisms disperse into the soil surface. We found that bacteria disperse at a similar rate across the two ecosystems, and both bacteria and fungi exhibit a signature of dispersal limitation at a meter scale, indicating highly heterogeneous dispersal of microorganisms into soil.

Environmental Sciences & Ecology↗
DOE OSTI2025

CABO-16S—a Combined Archaea, Bacteria, Organelle 16S rRNA database framework for amplicon analysis of prokaryotes and eukaryotes in environmental samples

Abstract Identification of both prokaryotic and eukaryotic microorganisms in environmental samples is currently challenged by the need for additional sequencing to obtain separate 16S and 18S ribosomal RNA (rRNA) amplicons or the constraints imposed by “universal” primers. Organellar 16S rRNA sequences are amplified and sequenced along with prokaryote 16S rRNA and provide an alternative method to identify eukaryotic microorganisms. CABO-16S combines bacterial and archaeal sequences from the SILVA database with 16S rRNA sequences of plastids and other organelles from the PR2 database to enable identification of all 16S rRNA sequences. Comparison of CABO-16S with SILVA 138.2 results in equivalent taxonomic classification of mock communities and increased classification of diverse environmental samples. In particular, identification of phototrophic eukaryotes in shallow seagrass environments, marine waters, and lake waters was increased. The CABO-16S framework allows users to add custom sequences for further classification of underrepresented clades and can be easily updated with future releases of reference databases. Addition of sequences obtained from Sanger sequencing of methane seep sediments and curated sequences of the polyphyletic SEEP-SRB1 clade resulted in differentiation of syntrophic and non-syntrophic SEEP-SRB1 in hydrothermal vent sediments. CABO-16S highlights the benefit of combining and amending existing training sets when studying microorganisms in diverse environments.

Eitel, Eryn M. (ORCID:0009000723919297)↗
DOE OSTI2021

Decreased growth of wild soil microbes after 15 years of transplant‐induced warming in a montane meadow

Abstract The carbon stored in soil exceeds that of plant biomass and atmospheric carbon and its stability can impact global climate. Growth of decomposer microorganisms mediates both the accrual and loss of soil carbon. Growth is sensitive to temperature and given the vast biological diversity of soil microorganisms, the response of decomposer growth rates to warming may be strongly idiosyncratic, varying among taxa, making ecosystem predictions difficult. Here, we show that 15 years of warming by transplanting plant–soil mesocosms down in elevation, strongly reduced the growth rates of soil microorganisms, measured in the field using undisturbed soil. The magnitude of the response to warming varied among microbial taxa. However, the direction of the response—reduced growth—was universal and warming explained twofold more variation than did the sum of taxonomic identity and its interaction with warming. For this ecosystem, most of the growth responses to warming could be explained without taxon‐specific information, suggesting that in some cases microbial responses measured in aggregate may be adequate for climate modeling. Long‐term experimental warming also reduced soil carbon content, likely a consequence of a warming‐induced increase in decomposition, as warming‐induced changes in plant productivity were negligible. The loss of soil carbon and decreased microbial biomass with warming may explain the reduced growth of the microbial community, more than the direct effects of temperature on growth. These findings show that direct and indirect effects of long‐term warming can reduce growth rates of soil microbes, which may have important feedbacks to global warming.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Quantitative Stable-Isotope Probing (qSIP) with Metagenomics Links Microbial Physiology and Activity to Soil Moisture in Mediterranean-Climate Grassland Ecosystems

The growth and physiology of soil microorganisms, which play vital roles in biogeochemical cycling, are shaped by both current and historical soil environmental conditions. Here, we developed and applied a genome-resolved metagenomic implementation of quantitative stable isotope probing (qSIP) with an H 2 18 O labeling experiment to identify actively growing soil microorganisms and their genomic capacities. qSIP enabled measurement of taxon-specific growth because isotopic incorporation into microbial DNA requires production of new genome copies. We studied three Mediterranean grassland soils across a rainfall gradient to evaluate the hypothesis that historic precipitation levels are an important factor controlling trait selection. We used qSIP-informed genome-resolved metagenomics to resolve the active subset of soil community members and identify their characteristic ecophysiological traits. Higher year-round precipitation levels correlated with higher activity and growth rates of flagellar motile microorganisms. In addition to heavily isotopically labeled bacteria, we identified abundant isotope-labeled phages, suggesting phage-induced cell lysis likely contributed to necromass production at all three sites. Further, there was a positive correlation between phage activity and the activity of putative phage hosts. Contrary to our expectations, the capacity to decompose the diverse complex carbohydrates common in soil organic matter or oxidize methanol and carbon monoxide were broadly distributed across active and inactive bacteria in all three soils, implying that these traits are not highly selected for by historical precipitation.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

A standardized quantitative analysis strategy for stable isotope probing metagenomics

ABSTRACT Stable isotope probing (SIP) facilitates culture-independent identification of active microbial populations within complex ecosystems through isotopic enrichment of nucleic acids. Many DNA-SIP studies rely on 16S rRNA gene sequences to identify active taxa, but connecting these sequences to specific bacterial genomes is often challenging. Here, we describe a standardized laboratory and analysis framework to quantify isotopic enrichment on a per-genome basis using shotgun metagenomics instead of 16S rRNA gene sequencing. To develop this framework, we explored various sample processing and analysis approaches using a designed microbiome where the identity of labeled genomes and their level of isotopic enrichment were experimentally controlled. With this ground truth dataset, we empirically assessed the accuracy of different analytical models for identifying active taxa and examined how sequencing depth impacts the detection of isotopically labeled genomes. We also demonstrate that using synthetic DNA internal standards to measure absolute genome abundances in SIP density fractions improves estimates of isotopic enrichment. In addition, our study illustrates the utility of internal standards to reveal anomalies in sample handling that could negatively impact SIP metagenomic analyses if left undetected. Finally, we present SIPmg , an R package to facilitate the estimation of absolute abundances and perform statistical analyses for identifying labeled genomes within SIP metagenomic data. This experimentally validated analysis framework strengthens the foundation of DNA-SIP metagenomics as a tool for accurately measuring the in situ activity of environmental microbial populations and assessing their genomic potential. IMPORTANCE Answering the questions, “who is eating what?” and “who is active?” within complex microbial communities is paramount for our ability to model, predict, and modulate microbiomes for improved human and planetary health. These questions can be pursued using stable isotope probing to track the incorporation of labeled compounds into cellular DNA during microbial growth. However, with traditional stable isotope methods, it is challenging to establish links between an active microorganism’s taxonomic identity and genome composition while providing quantitative estimates of the microorganism’s isotope incorporation rate. Here, we report an experimental and analytical workflow that lays the foundation for improved detection of metabolically active microorganisms and better quantitative estimates of genome-resolved isotope incorporation, which can be used to further refine ecosystem-scale models for carbon and nutrient fluxes within microbiomes.

54 ENVIRONMENTAL SCIENCES↗