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At least 577 records · Page 32
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DOE OSTI2020

Appendix Q: Recommendations for Developing Molecular Assays for Microbial Pathogen Detection Using Modern In Silico Approaches

We describe the use of in silico approaches to improve the process of molecular assay development and reduce time and cost by utilizing available databases of whole genome pathogen sequences combined with modern bioinformatics and physical modeling tools. Well-characterized assays are needed for accurately detecting pathogens in environmental and patient samples and also for evaluation of the efficacy of a medical countermeasure that may be administered to patients. The polymerase chain reaction (PCR) remains the gold standard for pathogen detection due to the simplicity of its instrumentation, low cost of reagents, and outstanding limit of detection (LOD), sensitivity, and specificity. However, creation of such PCR assays often involves iterations of design, preliminary testing, and thorough validation with clinical isolates and testing in relevant matrices, which can be time consuming, costly, and result in suboptimal assays. Since formal validation (e.g., for Emergency Use Authorization [EUA] or Food and Drug Administration [FDA] licensure) of an infectious disease assay can be very expensive and can require extensive time of development, having a well-designed assay up front is a critical first step. Yet, many assays described in the literature utilized limited design capabilities and many initially promising assays fail the validation process, resulting in increased costs and timelines for successful product development. While the computational approaches outlined in this document by no means obviate the need for wet lab testing, they can reduce the amount of effort wasted on empirical optimization and iterative redesigns and also guide validation studies. The proposed computational approaches also result in higher performing assays with better sensitivity, specificity, and lower LOD and reduce the possibility of assay failure due to signature erosion. To provide clarity, an extensive glossary of defined terms is provided.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Linking Hydrology and Dissolved Organic Matter Characteristics in a Subtropical Wetland: A Long-Term Study of the Florida Everglades

Dissolved organic matter (DOM) acts as an important biogeochemical component of aquatic ecosystems that controls nutrient cycling, influences water quality, and links terrestrial and oceanic carbon pools, yet long-term studies of how changing environmental drivers alter its abundance and composition are rare. Using a ten-year, spatially explicit dataset from Everglades National Park (ENP), a globally significant wetland, we investigated the relationships between DOM quality/quantity and hydrologic/climatic drivers along two contrasting marsh-estuarine transects based on generalized linear modeling and a cumulative sums analysis. Analyses revealed distinct spatial, seasonal, and interannual patterns in variability of DOC and optical properties. Landscape-scale seasonal patterns showed an enrichment in microbial-like and protein-like DOM during the dry season relative to the wet season. We found that, while some compositional constituents varied with the solar calendar, responsive to temperature and photoperiod, others varied with the hydrologic calendar. Independent water level and discharge effects indicated strong hydrologic control on DOM quality that differed between the two transects, evidencing differences in their connectivity to areas of high agricultural activity. Across all sites, a significant long-term increasing trend in the fluorescence index was observed, associated with a positive correlation with precipitation and also potential changes in agricultural inputs, with other features associated with drought and hurricanes. Lastly, the cumulative sums analysis revealed differences between the two transects in the sensitivity of DOM composition to decreased water levels associated with 30-year climate scenarios, with the less hydrologically dynamic transect exhibiting greater potential sensitivity.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

Impact of crowding on the diversity of expanding populations

Crowding effects critically impact the self-organization of densely packed cellular assemblies, such as biofilms, solid tumors, and developing tissues. When cells grow and divide, they push each other apart, remodeling the structure and extent of the population’s range. Recent work has shown that crowding has a strong impact on the strength of natural selection. However, the impact of crowding on neutral processes, which controls the fate of new variants as long as they are rare, remains unclear. Here, we quantify the genetic diversity of expanding microbial colonies and uncover signatures of crowding in the site frequency spectrum. By combining Luria–Delbrück fluctuation tests, lineage tracing in a novel microfluidic incubator, cell-based simulations, and theoretical modeling, we find that the majority of mutations arise behind the expanding frontier, giving rise to clones that are mechanically “pushed out” of the growing region by the proliferating cells in front. These excluded-volume interactions result in a clone-size distribution that solely depends on where the mutation first arose relative to the front and is characterized by a simple power law for low-frequency clones. Our model predicts that the distribution depends on a single parameter—the characteristic growth layer thickness—and hence allows estimation of the mutation rate in a variety of crowded cellular populations. Combined with previous studies on high-frequency mutations, our finding provides a unified picture of the genetic diversity in expanding populations over the whole frequency range and suggests a practical method to assess growth dynamics by sequencing populations across spatial scales.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Combining Random Mutagenesis and Metabolic Engineering for Enhanced Tryptophan Production in Synechocystis sp. Strain PCC 6803

Tryptophan (Trp) is an essential aromatic amino acid that has value as an animal feed supplement, as the amount found in plant-based sources is insufficient. An alternative to production by engineered microbial fermentation is to have tryptophan biosynthesized by a photosynthetic microorganism that could replace or supplement both the plant and industrially used microbes. We selected Synechocystis sp. strain PCC 6803, a model cyanobacterium, as the host and studied metabolic engineering and random mutagenesis approaches. Previous work on engineering heterotrophic microbes for improved Trp titers has targeted allosteric feedback regulation in enzymes 3-deoxy-d-arabinoheptulosonate 7-phosphate synthase (DAHPS) and anthranilate synthase (AS) as major bottlenecks in the shikimate pathway. In this work, the genes encoding feedback-resistant enzymes from Escherichia coli, aroG fbr and trpE fbr , were overexpressed in the host wild-type (WT) strain. Separately, the WT strain was subjected to random mutagenesis and selection using an amino acid analog to isolate tryptophan-overproducing strains. The randomly mutagenized strains were sequenced in order to identify the mutations that resulted in the desirable phenotypes. Interestingly, the tryptophan overproducers had mutations in the gene encoding chorismate mutase (CM), which catalyzes the conversion of chorismate to prephenate. The best tryptophan overproducer from random mutagenesis was selected as a host for metabolic engineering where aroG fbr and trpE fbr were overexpressed. The best strain developed produced 212 ± 23 mg/liter of tryptophan after 10 days of photoautotrophic growth under 3% (vol/vol) CO2. We demonstrated that a combination of random mutagenesis and metabolic engineering was superior to either individual approach.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Lower soil moisture and deep soil temperatures in thermokarst features increase old soil carbon loss after 10 years of experimental permafrost warming

Almost half of the global terrestrial soil carbon (C) is stored in the northern circumpolar permafrost region, where air temperatures are increasing two times faster than the global average. As climate warms, permafrost thaws and soil organic matter becomes vulnerable to greater microbial decomposition. Long-term soil warming of ice-rich permafrost can result in thermokarst formation that creates variability in environmental conditions. Consequently, plant and microbial proportional contributions to ecosystem respiration may change in response to long-term soil warming. Natural abundance δ 13 C and Δ 14 C of aboveground and belowground plant material, and of young and old soil respiration were used to inform a mixing model to partition the contribution of each source to ecosystem respiration fluxes. We employed a hierarchical Bayesian approach that incorporated gross primary productivity and environmental drivers to constrain source contributions. We found that long-term experimental permafrost warming introduced a soil hydrology component that interacted with temperature to affect old soil C respiration. Old soil C loss was suppressed in plots with warmer deep soil temperatures because they tended to be wetter. When soil volumetric water content significantly decreased in 2018 relative to 2016 and 2017, the dominant respiration sources shifted from plant aboveground and young soil respiration to old soil respiration. The proportion of ecosystem respiration from old soil C accounted for up to 39% of ecosystem respiration and represented a 30-fold increase compared to the wet-year average. Our findings show that thermokarst formation may act to moderate microbial decomposition of old soil C when soil is highly saturated. However, when soil moisture decreases, a higher proportion of old soil C is vulnerable to decomposition and can become a large flux to the atmosphere. Furthermore, as permafrost systems continue to change with climate, we must understand the thresholds that may propel these systems from a C sink to a source.

permafrost↗
DOE OSTI2025

Coupled Hydrological-Thermal-Biogeochemical Modeling for Predicting Arctic Carbon Emissions (CH4PACE)

The Arctic, with nearly 1,700 billion metric tons of carbon stored in its soils, is experiencing significant warming trends that raise concerns about the potential release of greenhouse gases, particularly methane. This report investigates the conversion of carbon stored in Arctic soils and permafrost into methane and its subsequent atmospheric release over the coming decades. Recent data indicate a rise in global methane concentrations, highlighting the importance of understanding the mechanisms of methane generation under anaerobic conditions prevalent in thawing permafrost. Our study employs a multi-faceted approach that integrates fieldwork, benchtop experiments, and numerical simulations to explore the subsurface hydrological-thermal-biogeochemical systems influencing methane emissions. We focus on the relationship between microbial activity in decomposing organic matter under oxygen-limited conditions and the transport of methane out of these saturated wetland soils. Key hydrological parameters, including permeability, porosity, and thermal conductivity, were measured on soil samples from Fairbanks, Alaska, and used to parameterize PFLOTRAN, a coupled hydrological-thermal-reactive transport simulator. Our model enables prediction of the evolution of the active layer and its impact on methane production and release. Our findings underscore the critical need for a mechanistic understanding of methane dynamics in permafrost environments to improve predictions of methane emissions in Earth systems models.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2022

Quantifying pH buffering capacity in acidic, organic-rich Arctic soils: Measurable proxies and implications for soil carbon degradation

Dynamic pH change promoted by biogeochemical reactions in Arctic tundra soils can be a major control on the production and release of CO 2 and CH 4 , which contribute to rising global temperatures. Large quantities of soil organic matter (SOM) in these soils are susceptible to microbial decomposition, leading to pH changes during permafrost thaw. Soil pH buffering capacity (β) modulates the extent of pH change but has not been thoroughly studied and represented in predictive ecosystem scale biogeochemical models in Arctic tundra soils. In this study, we generated titration curves for 21 acidic tundra soils from three Arctic sites across northern Alaska, United States of America. Geochemical and hydrological soil properties were evaluated, and correlations with β were developed. Strong correlations between β and both gravimetric water content (Θ g ) (R 2 = 0.847, p < 0.001) and soil water retention (SWR) (R 2 = 0.849, p = 0.001) indicate that the ability of soil to retain water could be associated with its buffering properties. Correlations between β and soil organic carbon (SOC) and cation exchange capacity (CEC) were also explored, and relationships to SWR are discussed. These correlations were then used with existing soil databases reporting SOC, CEC, and SWR to estimate β across Alaska soils. We further demonstrated the quantitative relationships between β and the simulated rates of biogeochemical reactions and show that lower β leads to higher soil pH and more CH 4 production. Overall, our study provides simple proxies for β in Arctic soils and highlights the importance and implications of representing soil buffering in predictive models, thereby enabling quantitative coupling between pH dynamics associated with biogeochemical reactions. Integrating β into predictive models of Arctic biogeochemical cycling may reduce model uncertainty and further our understanding of permafrost SOM degradation accelerated by warming.

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

Evaluation of Nanaerobic Digestion as a Mechanism to Explain Surplus Methane Production in Animal Rumina and Engineered Digesters

Nanaerobes are a newly described class of microorganisms that use a unique cytochrome bd oxidase to achieve nanaerobic respiration at <2 μM dissolved oxygen (~1% of atmospheric oxygen) but are not viable above this value due to the lack of other terminal oxidases. Although sharing an overlapping ecological niche with methanogenic archaea, the role of nanaerobes in methanogenic systems has not been studied so far. To explore their occurrence and significance, we re-analyzed published meta-omic datasets from animal rumina and waste-to-energy digesters, including conventional anaerobic digesters and anaerobic digesters with ultra-low oxygenation. Results show that animal rumina share broad similarities in the microbial community and system performance with oxygenated digesters, rather than with conventional anaerobic digesters, implying that trace levels of oxygen drive the efficient digestion in ruminants. The rumen system serves as an ideal model for the newly named nanaerobic digestion, as it relies on the synergistic co-occurrence of nanaerobes and methanogens for methane yield enhancement. The most abundant ruminal bacterial family Prevotellaceae contains many nanaerobes, which perform not only anaerobic fermentation but also nanaerobic respiration using cytochrome bd oxidase. These nanaerobes generally accompany hydrogenotrophic methanogens to constitute a thermodynamically and physiologically consistent framework for efficient methane generation. Our findings provide new insights into ruminal methane emissions and strategies to enhance methane generation from biomass.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Positive correlations between soil C stabilization and loss are regulated by roots, minerals, and microbes

Modern conceptual models of soil organic carbon (SOC) cycling place a strong focus on the microbe-mineral interactions that regulate C stabilization. However, the formation of ‘stable’ soil organic matter, which consists mainly of microbial residues bonded to mineral surfaces, is inextricably linked to C loss through microbial respiration. Therefore, what is the net impact of microbial metabolism on the total quantity of C held in the soil? To address this question, we constructed artificial root-soil systems to identify controls on C cycling across the plant-microbe-mineral continuum, simultaneously quantifying the formation of persistent mineral-associated C and C losses to respiration. Roots and minerals interacted to regulate these processes: while roots stimulated respiratory C losses and depleted mineral-associated C pools in low-activity clays, roots triggered formation of stable C in high-activity clays. Moreover, we observed a strong positive correlation between the formation of mineral-associated C and respiration. This suggests that the growth of persistent C pools which can resist subsequent disturbance comes at the expense of C loss from the system.

58 GEOSCIENCES↗
DOE OSTI2025

Five PMI Isolates from Populus Deltoides and Populus Trichocarpa

Five bacterial isolates were isolated from the roots of poplar trees (Populus deltoides and P. trichocarpa), which are model organisms and a main focus of the Plant-Microbe Interfaces (PMI) project at ORNL. These strains belong to genera are not well represented and give a more complete view of the microbial community and bacterial interactions with poplar trees. These strains will support future studies and contribute to the broader PMI goal of understanding microbe-microbe and plant-microbe interactions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Diet outperforms microbial transplant to drive microbiome recovery in mice

A high-fat, low-fibre Western-style diet (WD) induces microbiome dysbiosis characterized by reduced taxonomic diversity and metabolic breadth, which in turn increases risk for a wide array of metabolic, immune and systemic pathologies. Recent work has established that WD can impair microbiome resilience to acute perturbations such as antibiotic treatment, although little is known about the mechanism of impairment and the specific consequences for the host of prolonged post-antibiotic dysbiosis. Here, in this study, we characterize the trajectory by which the gut microbiome recovers its taxonomic and functional profile after antibiotic treatment in mice on regular chow (RC) or WD, and find that only mice on RC undergo a rapid successional process of recovery. Metabolic modelling indicates that a RC diet promotes the development of syntrophic cross-feeding interactions, whereas in mice on WD, a dominant taxon monopolizes readily available resources without releasing syntrophic byproducts. Intervention experiments reveal that an appropriate dietary resource environment is both necessary and sufficient for rapid and robust microbiome recovery, whereas microbial transplant is neither. Furthermore, prolonged post-antibiotic dysbiosis in mice on WD renders them susceptible to infection by the intestinal pathogen Salmonella enterica serovar Typhimurium. Our data challenge widespread enthusiasm for faecal microbiota transplant (FMT) as a strategy to address dysbiosis, and demonstrate that specific dietary interventions are, at a minimum, an essential prerequisite for effective FMT, and may afford a safer, more natural and less invasive alternative.

Kennedy, M. S. [University of Chicago, IL (United ↗
DOE OSTI2021

Non-additive microbial community responses to environmental complexity

Environmental composition is a major, though poorly understood, determinant of microbiome dynamics. Here we ask whether general principles govern how microbial community growth yield and diversity scale with an increasing number of environmental molecules. By assembling hundreds of synthetic consortia in vitro, we find that growth yield can remain constant or increase in a non-additive manner with environmental complexity. Conversely, taxonomic diversity is often much lower than expected. To better understand these deviations, we formulate metrics for epistatic interactions between environments and use them to compare our results to communities simulated with experimentally-parametrized consumer resource models. We find that key metabolic and ecological factors, including species similarity, degree of specialization, and metabolic interactions, modulate the observed non-additivity and govern the response of communities to combinations of resource pools. Our results demonstrate that environmental complexity alone is not sufficient for maintaining community diversity, and provide practical guidance for designing and controlling microbial ecosystems.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Sampling Microbial Dynamics in the Salish Sea Estuary: Evaluating Methods to Capture Cyanobacteria and Cyanophage

Introduction: Picocyanobacteria from the genera Prochlorococcus and Synechococcus thrive across the globe in aquatic environments, have relatively small genomes, and have growth dynamics regulated by both viral interactions and abiotic conditions, making them excellent model organisms for exploring host-pathogencoevolution. Methods: We developed and refined methods to sample and sequence cyanobacteria, cyanophages, and measured features of their abiotic environment. Results: The protocol described herein can successfully discriminate large-cell eukaryotic organisms, but size fractionation of picocyanobacteria appears to be affected by the presence of free DNA, multicellular structures, and abundant tycheposons. Our preferred final protocol from this exploratory effort included a combination of in-line and single vacuum flask filtrations, which reduced filtration processing time by over threefold in some cases compared to other tested methods, such as a fully in-line sequence or in-site filtrations. We successfully extracted an average of approximately 400–1200 ng for all filter fractions, with some variations between kits. Discussion: The protocol described herein can successfully discriminate large-cell eukaryotic organisms, but size fractionation of picocyanobacteria appears to be affected by the presence of free DNA, multicellular structures, and abundant tycheposons.

Salish Sea↗
DOE OSTI2025

Temperature and Water Levels Collectively Regulate Methane Emissions From Subtropical Freshwater Wetlands

Abstract Wetlands are the largest and most climate‐sensitive natural sources of methane. Accurately estimating wetland methane emissions involves reconciling inversion (“top‐down”) and process‐based (“bottom‐up”) models within the global methane budget. However, estimates from these two model types are inherently interdependent and often reveal substantial discrepancies. To enhance the reliability of both approaches, we need a comprehensive understanding of wetland methane emissions and an independent high‐resolution long‐term flux data set. Here, we employed a data‐driven random forest approach to identify key variables influencing methane emissions from subtropical freshwater wetlands in the Southeastern United States. The model‐estimated monthly mean methane fluxes fit well with measured methane fluxes ( R 2 = 0.67) at four representative FLUXNET‐CH4 wetland sites across the region. Variable importance analysis highlighted the sensitivity of subtropical freshwater wetland methane emissions to variations in both temperature and water levels. High temperatures facilitate methanogenesis by enhancing microbial activities, while elevated water levels maintain anaerobic conditions necessary for methane production. Notably, the response of methane emissions to water level fluctuations is contingent on temperature conditions, and vice versa. Moreover, we constructed the first high‐spatial‐resolution (∼1 km × 1 km) and long‐term (1982–2010) gridded regional wetland methane flux product for the Southeastern United States, estimating annual methane emissions from subtropical freshwater wetlands in the region at 4.93 ± 0.11 Tg CH 4 yr −1 for 1982–2010. This new benchmark product holds promise for validating and parameterizing uncertain wetland methane emission processes in bottom‐up models and provides improved prior information for top‐down models.

He, Keqi [Earth and Climate Sciences Nicholas Scho↗
DOE OSTI2021

Atomic Force Microscopy to Characterize Antimicrobial Peptide-Induced Defects in Model Supported Lipid Bilayers

Antimicrobial peptides (AMPs) interact with bacterial cell membranes through a variety of mechanisms, causing changes extending from nanopore formation to microscale membrane lysis, eventually leading to cell death. Several AMPs also disrupt mammalian cell membranes, despite their significantly different lipid composition and such collateral hemolytic damage hinders the potential therapeutic applicability of the AMP as an anti-microbial. Elucidating the mechanisms underlying the AMP–membrane interactions is challenging due to the variations in the chemical and structural features of the AMPs, the complex compositional variations of cell membranes and the inadequacy of any single experimental technique to comprehensively probe them. (1) Background: Atomic Force Microscopy (AFM) imaging can be used in combination with other techniques to help understand how AMPs alter the orientation and structural organization of the molecules within cell membranes exposed to AMPs. The structure, size, net charge, hydrophobicity and amphipathicity of the AMPs affect how they interact with cell membranes of differing lipid compositions. (2) Methods: Our study examined two different types of AMPs, a 20-amino acid, neutral, α-helical (amphipathic) peptide, alamethicin, and a 13-amino acid, non-α-helical cationic peptide, indolicidin (which intramolecularly folds, creating a hydrophobic core), for their interactions with supported lipid bilayers (SLBs). Robust SLB model membranes on quartz supports, incorporating predominantly anionic lipids representative of bacterial cells, are currently not available and remain to be developed. Therefore, the SLBs of zwitterionic egg phosphatidylcholine (PC), which represents the composition of a mammalian cell membrane, was utilized as the model membrane. This also allows for a comparison with the results obtained from the Quartz Crystal Microbalance with Dissipation (QCM-D) experiments conducted for these peptides interacting with the same zwitterionic SLBs. Further, in the case of alamethicin, because of its neutrality, the lipid charge may be less relevant for understanding its membrane interactions. (3) Results: Using AFM imaging and roughness analysis, we found that alamethicin produced large, unstable defects in the membrane at 5 µM concentrations, and completely removed the bilayer at 10 µM. Indolicidin produced smaller holes in the bilayer at 5 and 10 µM, although they were able to fill in over time. The root-mean-square (RMS) roughness values for the images showed that the surface roughness caused by visible defects peaked after peptide injection and gradually decreased over time. (4) Conclusions: AFM is useful for helping to uncover the dynamic interactions between different AMPs and cell membranes, which can facilitate the selection and design of more efficient AMPs for use in therapeutics and antimicrobial applications.

Swana, Kathleen W.↗
DOE OSTI2021

Organic sulfur fluxes and geomorphic control of sulfur isotope ratios in rivers

Pyrite oxidation plays a critical role in the relationship between weathering and climate, and its impact on the global carbon cycle has previously been constrained through inversion models utilizing observations of river sulfate ($SO^{2–}_{4}$) and its 34 S/ 32 S isotope ratio (δ 34 S SO4 ). However, measurements from some rivers have suggested that SSO4 can be substantially impacted by processes such as microbial sulfate reduction and/or sulfur assimilation and cycling, rather than simply reflecting a weighted mixture of lithologic sulfur sources. To study the prevalence and controls on $SO^{2–}_{4}$ transformations, in this study we measured dissolved major element concentrations and δ 34 S SO4 in river water samples from throughout western Iceland. Our analyses focused on samples from a small catchment hosting the Efri Haukadalsá river, a system with relatively uniform and isotopically constrained basaltic bedrock. We also measured sediment δ 34 S and sulfur speciation using sulfur K-edge X-ray absorption spectroscopy on sediment and vegetation samples from this catchment. Values of dissolved δ 34 S SO4 in the Efri Haukadalsá ranged from 2.5‰ to 23.7‰ and had a linear relationship with Cl – /$SO^{2–}_{4}$ ratios, indicating that $SO^{2–}_{4}$ predominantly derived from basalt weathering and meteoric precipitation. The lower δ 34 S SO4 values were found in fluvial valleys with V-shaped cross sections, while higher values of δ 34 S SO4 occurred in U-shaped, glacially eroded valleys with thick alluvial fills blanketing the valley floor. Spectroscopic observations identified organic sulfur phases in suspended river sediment, floodplain deposits, and vegetation. Mass balance calculations quantified the organic sulfur flux as less than 10% of $SO^{2–}_{4}$ export, and sediment δ 34 S values were comparable to river δ 34 S SO4 . We interpreted these isotopic and chemical patterns as reflecting differences in the availability of unweathered bedrock across the Efri Haukadalsá catchment, with V-shaped valleys having greater access to fresh sulfide-bearing minerals than alluviated U-shaped valleys; this interpretation is in contrast to one in which the elevated δ 34 S SO4 values reflect fractionation during sulfur transformations along alluvial reaches. These results validated the application of river inversion models for constraining weathering fluxes and affirmed that pyrite oxidation globally, even in the presence of river sulfur cycling, modulates the abundance of atmospheric carbon dioxide.

54 ENVIRONMENTAL SCIENCES↗