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At least 271 records · Page 15
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Data from: Niche differentiation of bacterial versus archaeal soil nitrifiers induced by ammonium inhibition along a management gradient

Soil nitrification, mediated mainly by ammonia oxidizing archaea (AOA) and bacteria (AOB), converts ammonium (NH4+) to nitrite (NO2−) and thence nitrate (NO3−). To better understand ecological differences between AOA and AOB, we investigated the nitrification kinetics of AOA and AOB under eight replicated cropped and unmanaged ecosystems (including two fertilized natural systems) along a long-term management intensity gradient in the upper U.S. Midwest. For five of eight ecosystems, AOB but not AOA exhibited Haldane kinetics (inhibited by high NH4+ additions), especially in perennial and successional systems. In contrast, AOA predominantly exhibited Michaelis-Menten kinetics, suggesting greater resistance to high nitrogen inputs than AOB. These responses suggest the potential for NH4+-induced niche differentiation between AOA and AOB. Additionally, long-term fertilization significantly enhanced maximum nitrification rates (Vmax) in the early successional systems for both AOA and AOB, but not in the deciduous forest systems. This was likely due to pH suppression of nitrification in the acidic forest soils, corroborated by a positive correlation of Vmax with soil pH but not with amoA gene abundance. Results also demonstrated that soil nitrification potentials were relatively stable, as there were no seasonal differences. Overall, results suggest that (1) NH4+ inhibition of AOB but not AOA could be another factor contributing to niche differentiation between AOA and AOB in soil, and (2) nitrification by both AOA and AOB can be significantly promoted by long-term nitrogen inputs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Evidence of cryptic methane cycling and non-methanogenic methylamine consumption in the sulfate-reducing zone of sediment in the Santa Barbara Basin, California

The recently discovered cryptic methane cycle in the sulfate-reducing zone of marine and wetland sediment couples methylotrophic methanogenesis to anaerobic oxidation of methane (AOM). Here we present evidence of cryptic methane cycling activity within the upper regions of the sulfate-reducing zone, along a depth transect within the Santa Barbara Basin, off the coast of California, USA. The top 0–20 cm of sediment from each station was subjected to geochemical analyses and radiotracer incubations using 35 S–SO$^{2-}_{4}$, 14 C–mono-methylamine, and 14 C–CH 4 to find evidence of cryptic methane cycling. Methane concentrations were consistently low (3 to 16 µM) across the depth transect, despite AOM rates increasing with decreasing water depth (from max 0.05 nmol cm -3 d -1 at the deepest station to max 1.8 nmol cm -3 d -1 at the shallowest station). Porewater sulfate concentrations remained high (23 to 29 mM), despite the detection of sulfate reduction activity from 35 S–SO$^{2-}_{4}$ incubations with rates up to 134 nmol cm -3 d -1 . Metabolomic analysis showed that substrates for methanogenesis (i.e., acetate, methanol and methylamines) were mostly below the detection limit in the porewater, but some samples from the 1–2 cm depth section showed non-quantifiable evidence of these substrates, indicating their rapid turnover. Estimated methanogenesis from mono-methylamine ranged from 0.2 to 0.5 nmol cm -3 d -1 . Discrepancies between the rate constants (k) of methanogenesis (from 14 C–mono-methylamine) and AOM (from either 14 C–mono-methylamine-derived 14 C–CH 4 or from directly injected 14 C–CH 4 ) suggest the activity of a separate, concurrent metabolic process directly metabolizing mono-methylamine to inorganic carbon. We conclude that the results presented in this work show strong evidence of cryptic methane cycling occurring within the top 20 cm of sediment in the Santa Barbara Basin. The rapid cycling of carbon between methanogenesis and methanotropy likely prevents major build-up of methane in the sulfate-reducing zone. Furthermore, our data suggest that methylamine is utilized by both methanogenic archaea capable of methylotrophic methanogenesis and non-methanogenic microbial groups. We hypothesize that sulfate reduction is responsible for the additional methylamine turnover, but further investigation is needed to elucidate this metabolic activity.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2025

The stable carbon isotope fractionation of methanogenesis products at complete carbon consumption

The stable carbon isotope signature (δ 13 C) of methane (CH 4 ) is used to discriminate between biological, thermogenic, and abiotic sources. Methanogens, or methane producing archaea, inhabit a broad range of chemical conditions. Many of these environments are replete in dissolved inorganic carbon (DIC), causing isotopically depleted δ 13 C biogenic CH 4 . However, some extreme environments inhabited by methanogens, such as serpentinising systems, exhibit low carbon dioxide (CO 2 ) availability, replete H 2 , and isotopically enriched δ 13 C CH 4 that is outside the known biogenic range. We measured the δ 13 C of CO 2 , biomass, lipids, and CH 4 during hydrogenotrophic methanogenesis under hydrogen replete conditions with a limited carbon pool to investigate carbon isotope dynamics at complete DIC consumption. As theory predicts, we found that the final, accumulated methane δ 13 C values closely reflect the δ 13 C of the initial DIC supply, and that methane is more 13 C enriched than biomass and lipids. This provides the first experimental evidence that methanogens can achieve complete carbon consumption and thus can produce accumulated CH 4 products that isotopically reflect the initial CO 2 . These data show that the range of possible δ 13 C values from biogenic methane needs to be expanded for natural environments impacted by extreme carbon limitation.

biomass↗
DOE OSTI2024

Impact of storage and extraction methods on peat soil microbiomes

Recovered microbial community structure is known to be influenced by sample storage conditions and nucleic acid extraction methods, and the impact varies by sample type. Peat soils store a large portion of soil carbon and their microbiomes mediate climate feedbacks. Here, we tested three storage conditions and five extraction protocols on peat soils from three physicochemically distinct habitats in Stordalen Mire, Sweden, revealing significant methodological impacts on microbial (here, meaning bacteria and archaea) community structure. Initial preservation method impacted alpha but not beta diversity, with in-field storage in LifeGuard buffer yielding roughly two-thirds the richness of in-field flash-freezing or transport from the field on ice (all samples were stored at −80 °C after return from the field). Nucleic acid extraction method impacted both alpha and beta diversity; one method (the PowerSoil Total RNA Isolation kit with DNA Elution Accessory kit) diverged from the others (PowerMax Soil DNA Isolation kit-High Humic Acid Protocol, and three variations of a modifiedPowerMax Soil DNA/RNA isolation kit), capturing more diverse microbial taxa, with divergent community structures. Although habitat and sample depth still consistently dominated community variation, method-based biases in microbiome recovery for these climatologically-relevant soils are significant, and underscore the importance of methodological consistency for accurate inter-study comparisons, long-term monitoring, and consistent ecological interpretations.

Science & Technology - Other Topics↗
DOE OSTI2020

The IsoGenie database: an interdisciplinary data management solution for ecosystems biology and environmental research

Modern microbial and ecosystem sciences require diverse interdisciplinary teams that are often challenged in “speaking” to one another due to different languages and data product types. Here we introduce the IsoGenie Database, a de novo developed data management and exploration platform, as a solution to this challenge of accurately representing and integrating heterogenous environmental and microbial data across ecosystem scales. The IsoGenieDB is a public and private data infrastructure designed to store and query data generated by the IsoGenie Project, a ~10 year DOE-funded project focused on discovering ecosystem climate feedbacks in a thawing permafrost landscape. The IsoGenieDB provides (i) a platform for IsoGenie Project members to explore the project’s interdisciplinary datasets across scales through the inherent relationships among data entities, (ii) a framework to consolidate and harmonize the datasets needed by the team’s modelers, and (iii) a public venue that leverages the same spatially explicit, disciplinarily integrated data structure to share published datasets. The IsoGenieDB is also being expanded to cover the NASA-funded Archaea to Atmosphere (A2A) project, which scales the findings of IsoGenie to a broader suite of Arctic peatlands, via the umbrella A2A Database (A2A-DB). The IsoGenieDB’s expandability and flexible architecture allow it to serve as an example ecosystems database.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

Diazotroph diversity across a land-management gradient

Soil diazotrophs are ubiquitous bacteria and archaea responsible for converting atmospheric nitrogen into plant-available ammonium. This process, if properly harnessed, can ameliorate excessive fertilizer inputs in conventional agricultural systems. However, the effect of diazotroph biodiversity on free-living nitrogen fixation (FLNF) is poorly understood. Here, we characterized diazotroph biodiversity across a land-management intensity gradient, determining the interactive effects of land management and diazotroph biodiversity on their ecosystem functioning (FLNF). Subsequently, we evaluated the functional redundancy of diazotroph biodiversity on FLNF in-vitro by performing a biodiversity reduction assay with chloroform fumigation, incubating soils with chloroform for either 24 or 72 hours. We estimated diazotroph biodiversity for both in-situ and chloroform-fumigated samples by preparing and sequencing nifH gene libraries. The raw sequencing data of our nifH library is deposited here.

Kristy, Brandon↗
DOE OSTI2023

Biomethanation to Upgrade Biogas to Pipeline Grade Methane

NREL is working closely with DOE, Electrochaea GmbH, and Southern California Gas Company (SoCalGas) to reduce costs of a biomethanation process capable of megawatt-scale deployment that upgrades organic biogas waste streams to produce pipeline quality renewable natural gas (RNG). Biomethanation is a two-step process using a single-celled methanogenic archaea that converts low-carbon low-cost hydrogen (H2) and waste carbon dioxide (CO2) to produce renewable methane (CH4). The process upgrades the biogenic CO2 - while allowing the CH4 to pass through - from biogas sources like dairies, wastewater treatment plants, and landfills. The CH4 produced is a drop-in direct replacement fuel and producers can participate in the growing number of carbon markets; like California's Low Carbon Fuel Standard and the Federal Renewable Fuel Standard. NREL and Argonne National Laboratory have completed a life cycle analysis using the GREET model to show that the biomethanation process produces RNG that is carbon negative even when H2 production via low-temperature water electrolysis is driven by the existing carbon intensity of California's electricity grid. And of course, even further carbon negative (-233 kg CO2e/kWh) when the electricity is produced from low-carbon sources like wind and solar. Leveraging lessons learned from operating SoCalGas' 700L 18-bar bioreactor system, NREL is designing and building a flexible RD&D platform that will enable field trials at biogas and other CO2 sources. A custom 16' long trailer will house a 20L 18-bar bioreactor, 3 - 25 kW proton exchange membrane electrolyzer, and dosing, thermal, and controls systems to support operations with only power, biogas, and water feedstocks required by the field locations. The end-of-project goal is to demonstrate pipeline quality RNG production (> 95% CH4, < 4% H2, <1% CO2, < 0.2% O2 and < 4 parts per million H2 sulfide) using real biogas feedstocks - thereby recycling both greenhouse gases for injection into the natural gas network or to be used onsite.

biogas upgrading↗
DOE OSTI2025

Response of Subsurface Nitrogen-Cycling Microbial Communities to Environmental Fluctuations (Final Technical Report)

Riparian floodplains are dynamic ecosystems linking terrestrial and riverine systems. These floodplains experience hydrological shifts such as changes in water table height, flooding, and drought and can be ‘hotspots’ of biogeochemical cycling due to shifting sediment moisture (and saturation) and subsurface exchanges of water, nutrients, and other compounds across different sediment layers. Subsurface microbial communities are the primary drivers of biogeochemical processes in floodplains, and thus their structure and function can directly influence both surface and groundwater quality. The microbial nitrogen (N) cycle is particularly important in floodplains as it affects nutrient availability and removal. Two functional guilds of chemoautotrophic (i.e. CO2-fixing) microorganisms are responsible for the first oxidative step of the N cycle, nitrification: ammonia-oxidizing archaea (AOA) and bacteria (AOB) catalyze the oxidation of ammonia to nitrite, while nitrite-oxidizing bacteria (NOB) oxidize nitrite to nitrate. Despite the critical role nitrification plays in N-cycling in both terrestrial and aquatic ecosystems, our understanding of the diversity, ecophysiology, and activity of nitrifying organisms in subsurface floodplain soils/sediments is extremely limited. To help address this critical knowledge gap, the overarching goal of this project was to determine how shifts in key environmental parameters and gradients impact microbial N-cycling communities/processes, with particular emphasis on nitrification, within hydrologically-variable floodplain sediments in the Wind River Basin near Riverton, Wyoming. The three specific objectives of this project were to: (1) to associate in situ environmental drivers of N cycling with distinct functional guilds; (2) determine the guild response to variation in key ecosystem drivers; and (3) develop a dynamic ecosystem model of the microbial N cycle with the Riverton subsurface using community genomic and biogeochemical data collected in the first two objectives. Over the course of this project, we employed both 16S rRNA gene amplicon sequencing and genome-resolved metagenomics to examine the phylogenetic diversity and metabolic potential of subsurface nitrifier communities within 68 samples collected across multiple sites, depths, and time points within the Riverton floodplain, allowing for both spatial and temporal investigations at different scales. This project benefitted tremendously from recent advances in high-throughput sequencing technologies coupled with dramatic improvements in the computational tools and algorithms available for analyzing such large, complex genomic datasets. By pairing these cutting-edge genomic approaches with depth-resolved sampling and detailed geochemical analyses of the Riverton floodplain, we have gained novel insights into the structure and function of subsurface nitrifier communities in relation to both hydrology and biogeochemistry. This project resulted in the most detailed and comprehensive characterization of N-cycling floodplain microbial communities to date and will hopefully inspire and pave the way for future studies using similar approaches in other floodplains. Indeed, such information is critical for understanding subsurface biogeochemical cycling and how elemental stores are altered from perturbations initiated by the water cycle within floodplains. Finally, because of the terrestrial-aquatic nature of the Riverton floodplain, results from this project are also of relevance to disciplines such as soil science, estuarine science, limnology & oceanography, biogeochemistry, geobiology, environmental engineering, as well as genomics and data science.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2021

INSIGHTS INTO MARINE UNICELLULAR CYANOBACTERIAL AND NON-CYANOBACTERIAL DIAZOTROPHS THROUGH SINGLE-CELL ANALYSES

Nutrient availability affects primary productivity, and nitrogen (N) is the limiting nutrient over large regions of the ocean. Biological N fixation (BNF) is the conversion of atmospheric dinitrogen (N2) to bioavailable forms of N (ammonium and amino acids). A select group of Bacteria and Archaea, known as diazotrophs, are the only organisms capable of this conversion. BNF is a critical source of N in nutrient-depleted (oligotrophic) marine waters. As such, BNF studies are a fundamental component for better understanding primary productivity and carbon cycling, as well as parameterizing predictive future global ocean models. Unicellular diazotrophs are widespread in marine systems and, in some cases, important contributors of BNF. This dissertation investigates unicellular diazotrophs at the single-cell level, including: UCYN-A, a cyanobacterial diazotroph that lives in symbiosis with an algal host, non-cyanobacterial diazotrophs (NCDs), which are putative heterotrophs, as well as a specific NCD known as Gamma A, a commonly occurring organism throughout the tropics and subtropics that belongs to the class Gammaproteobacteria. The single-cell investigations used in these studies can provide high-resolution insights that are not possible from community-level analyses of BNF, such as the N2 fixation rate of individual cells and cell morphology. Singlecell nanoscale secondary ion mass spectrometry (nanoSIMS) analyses were used in chapter 2 and 3 to investigate the cell-specific N2 fixation rates of (2) UCYN-A in the viii cold waters of the Bering and Arctic Seas where N2 fixation was not previously believed to be possible, and (3) NCDs in the North Pacific Subtropical Gyre where we demonstrated the first direct evidence of NCD N2 fixation on open ocean particles. Chapter 4 focused on the uncultivated NCD, Gamma A, using geneFISH to visualize single cells in natural seawater samples. We successfully identified Gamma A by developing a fluorescent in situ hybridization (FISH) probe targeting the diazotrophic functional marker gene nifH, revealing Gamma A cells are most often attached to particles. Together these studies provide new insights into the distributions, activities, and physiologies of geographically widespread, unicellular diazotrophs, thus improving our understanding of their roles as sources of fixed N in the surface ocean.

Harding, K.↗
DOE OSTI2021

The tree of life describes a tripartite cellular world

The canonical view of a 3-domain (3D) tree of life was recently challenged by the discovery of Asgardarchaeota encoding eukaryote signature proteins (ESPs), which were treated as missing links of a 2-domain (2D) tree. Here we revisit the debate. We discuss methodological limitations of building trees with alignment-dependent approaches, which often fail to satisfactorily address the problem of ‘‘gaps.’’ In addition, most phylogenies are reconstructed unrooted, neglecting the power of direct rooting methods. Alignment-free methodologies lift most difficulties but require employing realistic evolutionary models. We argue that the discoveries of Asgards and ESPs, by themselves, do not rule out the 3D tree, which is strongly supported by comparative and evolutionary genomic analyses and vast genomic and biochemical superkingdom distinctions. Given uncertainties of retrodiction and interpretation difficulties, we conclude that the 3D view has not been falsified but instead has been strengthened by genomic analyses. In turn, the objections to the 2D model have not been lifted. The debate remains open. Also see the video abstract here: https://youtu.be/-6TBN0bubI8

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Discovery, characterization, and application of chromosomal integration sites in the hyperthermophilic archaeon Sulfolobus islandicus

Sulfolobus islandicus , an emerging archaeal model organism, offers unique advantages for metabolic engineering and synthetic biology applications owing to its ability to thrive in extreme environments. Although several genetic tools have been established for this organism, the lack of well-characterized chromosomal integration sites has limited its potential as a cellular factory. Here, in this work, we systematically identified and characterized 13 artificial CRISPR RNAs targeting eight integration sites in S. islandicus using the CRISPR-COPIES pipeline and a multi-omics-informed computational workflow. We leveraged the endogenous CRISPR-Cas system to integrate the reporter gene lacS and validated heterologous expression through a β-galactosidase assay, revealing significant positional effects. As a proof of concept, we utilized these sites to genetically manipulate lipid ether composition by overexpressing glycerol dibiphytanyl glycerol tetraether (GDGT) ring synthase B (GrsB). This study expands the genetic toolbox for S. islandicus and advances its potential as a robust platform for archaeal synthetic biology and industrial biotechnology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Methanogenesis and Acetogenesis in Hydrogenotrophy with Carbonate Minerals: Dependence on Mineral Surface Area, Biofilm Growth, and Microbial Community

The production, storage, and use of hydrogen are anticipated to grow substantially to achieve energy and climate goals. Consequently, microbial communities in many terrestrial and subsurface Earth environments could be exposed to elevated hydrogen concentrations. Hydrogen stimulates metabolic processes that reduce aqueous chemical species, such as bicarbonate or sulfate, that can exchange with solid mineral phases, but the controls on microbial hydrogenotrophy with mineral sources of electron acceptors are not fully understood. Herein, we applied laboratory experiments and biogeochemical modeling to study the response of a natural microbial community to an elevated partial pressure of hydrogen in the presence of carbonate minerals of varying composition, solubility, and size. Experimental incubations and simulation results showed that hydrogen consumption by microbial communities was initially dominated by sulfate reduction and, subsequently, transitioned to acetogenesis and methanogenesis. The rates of acetogenesis and methanogenesis were not correlated with the solubility of carbonate minerals. Instead, we observed strong linear correlations between the rates and surface area of carbonate minerals. Methane and acetate production slowed down in all incubations after about 2 weeks of incubation, although biogeochemical modeling predicted that the metabolic processes were not thermodynamically limited. Electron microscopy and infrared spectroscopy showed that biofilms with diverse microorganisms grew on the carbonates during this period. The methane δ13C value significantly increased, consistent with slower growth at elevated pH. This work highlights that microbial communities form biofilm on carbonate mineral surfaces as a response to hydrogen and that biofilm formation could pose a strong kinetic limitation to hydrogenotrophic metabolism utilizing carbonate minerals.

Acetobacterium↗
DOE OSTI2025

Conformational Dynamics and Catalytic Backups in a Hyper-thermostable Engineered Archaeal Protein Tyrosine Phosphatase

Protein tyrosine phosphatases (PTPs) are a family of enzymes that play important roles in regulating cellular signaling pathways. The activity of these enzymes is regulated by the motion of a catalytic loop that places a critical conserved aspartic acid side chain into the active site for acid–base catalysis upon loop closure. These enzymes also have a conserved phosphate-binding loop that is typically highly rigid and forms a well-defined anion-binding nest. The intimate links between loop dynamics and chemistry in these enzymes make PTPs an excellent model system for understanding the role of loop dynamics in protein function and evolution. In this context, archaeal PTPs, which have often evolved in extremophilic organisms, are highly understudied, despite their unusual biophysical properties. We present here an engineered chimeric PTP (ShufPTP) generated by shuffling the amino acid sequence of five extant hyperthermophilic archaeal PTPs. Despite ShufPTP’s high sequence similarity to its natural counterparts, it presents a suite of unique properties, including high flexibility of the phosphate binding P-loop, facile oxidation of the active-site cysteine, mechanistic promiscuity, and, most notably, hyperthermostability, with a denaturation temperature likely >130 °C (>8 °C higher than the highest recorded growth temperature of any archaeal strain). Our combined structural, biochemical, biophysical, and computational analysis provides insight both into how small steps in evolutionary space can radically modulate the biophysical properties of an enzyme and showcases the tremendous potential of archaeal enzymes for biotechnology, to generate novel enzymes capable of operating under extreme conditions.

archaea↗
DOE OSTI2023

The minimal SUF system is not required for Fe–S cluster biogenesis in the methanogenic archaeon Methanosarcina acetivorans

Abstract Iron–sulfur (Fe–S) proteins are essential for the ability of methanogens to carry out methanogenesis and biological nitrogen fixation (diazotrophy). Nonetheless, the factors involved in Fe–S cluster biogenesis in methanogens remain largely unknown. The minimal SUF Fe–S cluster biogenesis system (i.e., SufBC) is postulated to serve as the primary system in methanogens. Here, the role of SufBC in Methanosarcina acetivorans , which contains two sufCB gene clusters, was investigated. The CRISPRi-dCas9 and CRISPR-Cas9 systems were utilized to repress or delete sufC1B1 and sufC2B2 , respectively. Neither the dual repression of sufC1B1 and sufC2B2 nor the deletion of both sufC1B1 and sufC2B2 affected the growth of M. acetivorans under any conditions tested, including diazotrophy. Interestingly, deletion of only sufC1B1 led to a delayed-growth phenotype under all growth conditions, suggesting that the deletion of sufC2B2 acts as a suppressor mutation in the absence of sufC1B1 . In addition, the deletion of sufC1B1 and/or sufC2B2 did not affect the total Fe–S cluster content in M. acetivorans cells. Overall, these results reveal that the minimal SUF system is not required for Fe–S cluster biogenesis in M. acetivorans and challenge the universal role of SufBC in Fe–S cluster biogenesis in methanogens.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗