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At least 343 records · Page 19

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DOE OSTI2023

Risk Assessment of Industrial Microbes Using a Terrestrial Mesocosm Platform

Abstract Industrial microbes and bio-derived products have emerged as an integral component of the bioeconomy, with an array of agricultural, bioenergy, and biomedical applications. However, the rapid development of microbial biotechnology raises concerns related to environmental escape of laboratory microbes, detection and tracking thereof, and resultant impact upon native ecosystems. Indeed, though wild-type and genetically modified microbes are actively deployed in industrial bioprocesses, an understanding of microbial interactivity and impact upon the environment is severely lacking. In particular, the persistence and sustained ecosystem impact of industrial microbes following laboratory release or unintentional laboratory escape remains largely unexplored. Herein, we investigate the applicability of soil-sorghum mesocosms for the ecological risk assessment of the industrial microbe, Saccharomyces cerevisiae . We developed and applied a suite of diagnostic and bioinformatic analyses, including digital droplet PCR, microscopy, and phylogenomic analyses to assess the impacts of a terrestrial ecosystem perturbation event over a 30-day time course. The platform enables reproducible, high-sensitivity tracking of S. cerevisiae in a complex soil microbiome and analysis of the impact upon abiotic soil characteristics and soil microbiome population dynamics and diversity. The resultant data indicate that even though S. cerevisiae is relatively short-lived in the soil, a single perturbation event can have sustained impact upon mesocosm soil composition and underlying microbial populations in our system, underscoring the necessity for more comprehensive risk assessment and development of mitigation and biocontainment strategies in industrial bioprocesses.

09 BIOMASS FUELS↗

NASA NTRS1996

Biological degradation and composition of inedible sweetpotato biomass

Many challenges are presented by biological degradation in a bioregenerative Controlled Ecological Life Support System (CELSS) as envisioned by the U.S. National Aeronautics and Space Administration (NASA). In the studies conducted with biodegradative microorganism indigenous to sweetpotato fields, it was determined that a particle size of 75 microns and incubation temperature of 30 degrees C were optimal for degradation. The composition of the inedible biomass and characterization of plant nutrient solution indicated the presence of potential energy sources to drive microbial transformations of plant waste. Selected indigenous soil isolates with ligno-cellulolytic or sulfate-reducing ability were utilized in biological studies and demonstrated diversity in ability to reduce sulfate in solution and to utilize alternative carbon sources: a lignin analog--4-hydroxy, 3-methoxy cinnamic acid, cellulose, arabinose, glucose, sucrose, mannitol, galactose, ascorbic acid.

Vegetables/chemistry/metabolism↗

DOE OSTI2022

Practical and thermodynamic constraints on electromicrobially accelerated CO 2 mineralization

By the end of the century, tens of gigatonnes of CO 2 will need to be removed from the atmosphere every year to maintain global temperatures. Natural weathering of ultramafic rocks and subsequent mineralization reactions can convert CO 2 into ultra-stable carbonates. Although this will draw down all excess CO 2 , it will take thousands of years. CO 2 mineralization could be accelerated by weathering ultramafic rocks with biodegradable lixiviants. We show that if these lixiviants come from cellulosic biomass, this demand could monopolize the world’s biomass supply. We demonstrate that electromicrobial production technologies (EMP) that combine renewable electricity and microbial metabolism could produce lixiviants for as little as $\$200$ to $\$400$ per tonne at solar electricity prices achievable within the decade. We demonstrate that EMP could make enough lixiviants to sequester a tonne of CO 2 for less than $\$100$. This work highlights the potential of this approach and the need for extensive R&D.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Trophic interactions shape the spatial organization of medium-chain carboxylic acid producing granular biofilm communities

Abstract Granular biofilms producing medium-chain carboxylic acids (MCCA) from carbohydrate-rich industrial feedstocks harbor highly streamlined communities converting sugars to MCCA either directly or via lactic acid as intermediate. We investigated the spatial organization and growth activity patterns of MCCA producing granular biofilms grown on an industrial side stream to test (i) whether key functional guilds (lactic acid producing Olsenella and MCCA producing Oscillospiraceae) stratified in the biofilm based on substrate usage, and (ii) whether spatial patterns of growth activity shaped the unique, lenticular morphology of these biofilms. First, three novel isolates (one Olsenella and two Oscillospiraceae species) representing over half of the granular biofilm community were obtained and used to develop FISH probes, revealing that key functional guilds were not stratified. Instead, the outer 150–500 µm of the granular biofilm consisted of a well-mixed community of Olsenella and Oscillospiraceae, while deeper layers were made up of other bacteria with lower activities. Second, nanoSIMS analysis of 15N incorporation in biofilms grown in normal and lactic acid amended conditions suggested Oscillospiraceae switched from sugars to lactic acid as substrate. This suggests competitive-cooperative interactions may govern the spatial organization of these biofilms, and suggests that optimizing biofilm size may be a suitable process engineering strategy. Third, growth activities were similar in the polar and equatorial biofilm peripheries, leaving the mechanism behind the lenticular biofilm morphology unexplained. Physical processes (e.g., shear hydrodynamics, biofilm life cycles) may have contributed to lenticular biofilm development. Together, this study develops an ecological framework of MCCA-producing granular biofilms that informs bioprocess development.

09 BIOMASS FUELS↗

DOE OSTI2025

Integrating catalytic fractionation and microbial funneling to produce 2-pyrone-4,6-dicarboxylic acid and ethanol

Replacing biorefinery designs that use stepwise fractionation, depolymerization, and conversion processes with designed tandem process steps can greatly reduce a biorefinery's operating costs and environmental impact. Reductive Catalytic Fractionation (RCF) is a highly efficient lignin-first approach that combines biomass fractionation and lignin depolymerization to generate a hydrogenolysis oil and pulp. The oil is composed of a complex mixture of phenolic monomers, dimers, and oligomers. Intergrating this chemical deconstruction process with microbial funneling of phenolics can simplify the product mixture and make high-value products. We applied RCF to poplar biomass in a biomass-to-bioproduct processing chain in which the phenolics were funneled to 2-pyrone-4,6-dicarboxylic acid (PDC) by an engineered strain of Novosphingobium aromaticivorans DSM12444. The pulp was enzymatically digested and the glucose and xylose was funneled to ethanol by an engineered strain of Saccharomyces cerevisiae GLBRCY945. By combining biomass fractionation and lignin depolymerization we removed a costly processing step that directly translated into a 29% reduction in the minimum selling price of PDC. This work combines experimentation with process modeling of an integrated biorefinery design to show how utilizing tandem process steps can significantly reduce operating expenses and environmental impact of upgrading lignocellulosic biomass to a portfolio of high value products.

Sener, Canan [Great Lakes Bioenergy Research Cente↗

DOE OSTI2023

Development and implementation of an in situ high-resolution isotopic microscope for measuring metabolic interactions in soil mesocosms (Final Report)

Many of our planet’s ecosystems rely on the activities of soil microbial communities. These microbes have significant and wide-ranging effects: they metabolize carbon and other nutrients, interact with plants and fungi, and perform other processes important to soil health. However, our ability to directly observe the enzymatic and metabolic activities of microbes within soil is currently limited, not only by the complexity of soil microbial communities themselves, but also by the lack of experimental tools to study them and their molecular interactions in situ. These challenges hinder our understanding of the life-sustaining processes of biomass decomposition and the manner in which it contributes to the movement of freed carbon within soil ecosystems. Here we aimed to develop a novel ultrahigh-resolution isotopic microscope that combines complementary imaging modalities to gain insights into metabolic cycling in soil. This final report covers both the portion of this work that was initially completed at the University of North Carolina at Chapel Hill from 2018 to 2019 (as award DE-SC0019012) and then from 2020 to 2022 (2023 in NCE) at the University of Massachusetts Chan Medical School. This goal was to build an integrated platform consisting of fluorescence microscopy, Raman microspectroscopy, and nanospray desorption electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (nanoDESI-FTICR-MS) to directly investigate microbial activities and molecular transformations occurring in soil by exploiting the use of both fluorescent labels and stable isotope probing. Specifically, our instrument was conceived as integrating: (A) fluorescence detection to localize soil microbes, identify bacteria taking up polysaccharides, and monitor gene expression of enzymes involved in decomposition; (B) Raman microspectroscopy to determine which microbes incorporate decomposition products into their biomass, as well as which decomposers are metabolically active; and (C) nanoDESI-FTICR-MS imaging to spatially probe, in real-time, the metabolites in the surrounding area, which we expect will reveal the distributions of the products of the enzymatic breakdown of polysaccharides, as well as specialized metabolites acting as cell-cell signals between decomposers. To enable these measurements, we exploited fluorescence-based probes to map the microbes that are enzymatically active (per A) and utilized stable isotope-labeled substrates to visualize both the microbial and molecular fate of decomposed biomass (B and C, respectively). To accomplish our goal, we combined the expertise of a multidisciplinary group of scientists to pursue the construction of this microscope and to investigate scientific questions that would be facilitated by such a capability. We aimed to employ this spatially informative, high-resolution isotopic microscope to visualize the critical steps of biomass degradation and the molecular fate of other environmentally relevant substrates within soil mesocosms. Accordingly, this technology will enhance our understanding of the microbial and metabolic interactions occurring within soil communities that are relevant to carbon degradation and other soil processes. The instrument has been developed and housed at EMSL, where it will be available to the entire EMSL User Base, enabling a variety of related DOE-relevant systems to be interrogated in the future by diverse scientific research groups.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2022

Drought impacts on microbial trait distribution and feedback to soil carbon cycling

Abstract Quantifying the impact of drought on microbial processes and its consequences for soil carbon cycling is hindered by the lack of underlying mechanistic understanding. Therefore, there is a need to scale up the physiological response to changing water status from individual soil microbes to collective communities across different ecosystems. Here we propose the use of a framework that incorporates trait‐based ecology to link drought‐impacted microbial processes to rates of soil carbon decomposition and stabilisation. We briefly synthesise existing knowledge on the effects of drought on microbial physiology at the individual to community scale, before integrating this understanding within a framework incorporating life‐history strategy, ecological strategy and biochemistry. This framework highlights a dynamic allocation to high yield (Y), resource acquisition (A) and stress tolerance (S) pathways as environmental conditions change. Y‐A‐S strategies represent sets of traits that tend to correlate due to physiological or evolutionary trade‐offs. This framework enables assessment of microbial processes along two key environmental gradients of water and resource availability, both of which are constrained by drought. The variable chemistry of biomass and necromass produced under different physiological strategies in response to drying–rewetting impacts organic matter decomposition and stabilisation in soils, and should also be considered when quantifying soil carbon balance. We highlight that diversion of resources away from microbial growth can alter soil organic matter chemistry and its persistence depending on the kind of microbial compounds produced. To advance such a framework, we highlight avenues of research that would enable the further identification and quantification of traits linked to Y‐A‐S strategies and the physiological outcomes at the community level under drought and rewetting, and conclude by hypothesising how ecosystem‐level changes might feedback on to the soil carbon cycle. A scalable understanding of microbial drought‐response mechanisms affecting soil carbon cycling will transform the way microbial physiology is represented in ecosystem studies. Read the free Plain Language Summary for this article on the Journal blog.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Principles and practice of designing microbial biocatalysts for fuel and chemical production

Abstract The finite nature of fossil fuels and the environmental impact of its use have raised interest in alternate renewable energy sources. Specifically, nonfood carbohydrates, such as lignocellulosic biomass, can be used to produce next generation biofuels, including cellulosic ethanol and other nonethanol fuels like butanol. However, currently there is no native microorganism that can ferment all lignocellulosic sugars to fuel molecules. Thus, research is focused on engineering improved microbial biocatalysts for production of liquid fuels at high productivity, titer, and yield. A clear understanding and application of the basic principles of microbial physiology and biochemistry are crucial to achieve this goal. In this review, we present and discuss the construction of microbial biocatalysts that integrate these principles with ethanol-producing Escherichia coli as an example of metabolic engineering. These principles also apply to fermentation of lignocellulosic sugars to other chemicals that are currently produced from petroleum.

09 BIOMASS FUELS↗

DOE OSTI2023

Oxic–anoxic cycling promotes coupling between complex carbon metabolism and denitrification in woodchip bioreactors

Denitrifying woodchip bioreactors (WBRs) are increasingly used to manage the release of non-point source nitrogen (N) by stimulating microbial denitrification. Woodchips serve as a renewable organic carbon (C) source, yet the recalcitrance of organic C in lignocellulosic biomass causes many WBRs to be C-limited. Prior studies have observed that oxic–anoxic cycling increased the mobilization of organic C, increased nitrate (NO 3 – ) removal rates, and attenuated production of nitrous oxide (N 2 O). Here, in this work, we use multi-omics approaches and amplicon sequencing of fungal 5.8S-ITS2 and prokaryotic 16S rRNA genes to elucidate the microbial drivers for enhanced NO 3 – removal and attenuated N 2 O production under redox-dynamic conditions. Transient oxic periods stimulated the expression of fungal ligninolytic enzymes, increasing the bioavailability of woodchip-derived C and stimulating the expression of denitrification genes. Nitrous oxide reductase (nosZ) genes were primarily clade II, and the ratio of clade II/clade I nosZ transcripts during the oxic–anoxic transition was strongly correlated with the N 2 O yield. Analysis of metagenome-assembled genomes revealed that many of the denitrifying microorganisms also have a genotypic ability to degrade complex polysaccharides like cellulose and hemicellulose, highlighting the adaptation of the WBR microbiome to the ecophysiological niche of the woodchip matrix.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Abiotic Stress Reorganizes Rhizosphere and Endosphere Network Structure of Sorghum bicolor

Sorghum bicolor is a promising bioenergy feedstock with high biomass production and unusual tolerance for stresses, such as water and nutrient limitation. Although the membership of the sorghum microbiome in response to stress has been explored, relatively little is known about how microbe–microbe networks change under water- or nutrient-limited conditions. This is important because network changes can indicate impacts on the functionality and stability of microbial communities. We performed network-based analysis on the core bacterial and archaeal community of an agronomically promising high biomass bioenergy genotype, Grassl, grown under nitrogen and water stress. Stress caused relatively minor changes in bacterial abundances within soil, rhizosphere, and endosphere communities but led to significant changes in bacterial network structure and modularity. We found a complete reorganization of network roles in all plant compartments, as well as an increase in the modularity and proportion of positive associations, which potentially could represent coexistence and cooperation in the sorghum bacterial/archaeal community under stress. Although stressors are often believed to be destabilizing, we found stressed networks were as or more stable than non-stressed networks, likely due to their redundancy and compartmentalization. Together, these findings support the idea that both sorghum and its bacterial/archaeal community can be resilient to future environmental stressors.

09 BIOMASS FUELS↗

DOE OSTI2020

Electricity from lignocellulosic substrates by thermophilic Geobacillus species

Given our vast lignocellulosic biomass reserves and the difficulty in bioprocessing them without expensive pretreatment and fuel separation steps, the conversion of lignocellulosic biomass directly into electricity would be beneficial. Here we report the previously unexplored capabilities of thermophilic Geobacillus sp. strain WSUCF1 to generate electricity directly from such complex substrates in microbial fuel cells. This process obviates the need for exogenous enzymes and redox mediator supplements. Cyclic voltammetry and chromatography studies revealed the electrochemical signatures of riboflavin molecules that reflect mediated electron transfer capabilities of strain WSUCF1. Proteomics and genomics analysis corroborated that WSUCF1 biofilms uses type-II NADH dehydrogenase and demethylmenaquinone methyltransferase to transfer the electrons to conducting anode via the redox active pheromone lipoproteins localized at the cell membrane.

09 BIOMASS FUELS↗

DOE OSTI2021

Topographical Controls on Hillslope-Scale Hydrology Drive Shrub Distributions on the Seward Peninsula, Alaska

Observations indicate shrubs are expanding across the Arctic tundra, mainly on hillslopes and primarily in response to climate warming. However, the impact topography exerts on hydrology, nutrient dynamics, and plant growth can make untangling the mechanisms behind shrub expansion difficult. We examined the role topography plays in determining shrub expansion by applying a coupled transect version of a mechanistic ecosystem model (ecosys) in a tundra hillslope site in the Seward Peninsula, Alaska. Modeled biomass of the dominant plant functional types agreed well with field measurements (R 2 = 0.89) and accurately represented shrub expansion over the past 30 years inferred from satellite observations. In the well-drained crest position, canopy water potential and plant nitrogen (N) uptake was modeled to be low from plant and microbial water stress. Intermediate soil water content in the mid-slope position enhanced mineralization and plant N uptake, increasing shrub biomass. The deciduous shrub growth in the mid-slope position was further enhanced by symbiotic N 2 fixation primed by increased root carbon allocation. The gentle slope in the poorly drained lower-slope position resulted in saturated soil conditions that reduced soil O 2 concentrations, leading to lower root O 2 uptake and lower nutrient uptake and plant biomass. Additionally, a simulation that removed topographical interconnectivity between grid cells resulted in (1) a 28% underestimate of mean shrub biomass and (2) over or underestimated shrub productivity at the various hillslope positions. Our results indicate that land models need to account for hillslope-scale coupled surface and subsurface hydrology to accurately predict current plant distributions and future trajectories in Arctic ecosystems.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2021

BETO 2021 Peer Review - Waste Carbon Gas Upgrading via Acetogens 2.3.2.106

Waste carbon gas represents a large and diverse set of feedstocks that could be captured and turned into useful products. This includes waste gas emitted from industrial activity, syngas from burned plant biomass or processed municipal waste, and electrochemical reduction of CO2. Currently, carbon gas is being microbially converted to ethanol as a main product. However, ethanol is a lower value product with a limited market size. While these microbes can make other higher value products, there are no commercial processes for generating these other products, leaving a gap in understanding potential implementation for commercialization. Expanding the products microbially produced from waste carbon gas requires several steps before commercial implementation. We are studying the acetogen Clostridium ljungdahlii as a biocatalyst to convert waste carbon gas to the chemical 3-hydroxybutyrate (3HB), a plastic monomer and fuel precursor. For that, we are studying and engineering microbial characteristics for novel 3HB product formation from waste gas streams. This includes metabolic characterization, genetic engineering, gas fermentation scaling, as well as technoeconomic and life cycle analysis.

29 ENERGY PLANNING, POLICY, AND ECONOMY↗

DOE OSTI2023

Dissecting the Division of Labor in Microbial Consortia for the Production of Biofuels and Chemicals (Final Technical Report)

The overarching objective of this project is to elucidate the fundamental design rules for microbial division of labor (DOL), the core of ecosystem organization, in the context of artificial yeast—lactic acid bacteria consortia that produce advanced biofuel and chemical from cellulosic biomass. Over the course of four years, we have successfully achieved the objective through three lines of research that combines experiment with mathematical modeling. Specifically, we have shown that compositional and temporal DOL is effective in modulating mixed sugar fermentation by an engineered consortium and uncovered general quantitative criteria for conditions under which microbial DOL outperforms single superbugs for simultaneous utilization of mixed substrates. We also demonstrated, in synthetic consortia of yeast and lactic acid bacteria, that partitioning the labor for substrate breakdown and end-product synthesis can allow an efficient production of 2-butanol that is difficult otherwise for a single yeast strain. Additionally, by systematically probing the stability of an engineered cooperative consortium, we found that the stability of microbial symbiosis is governed by the topological structure of the underlying cellular interactions rather than specific microbial species and that rational modulation of the interactions may facilitate the restoration of collapsed consortia as well as the intervention of target communities. Together, this project has advanced the fundamental knowledge of microbial DOL in terms of its strength in mixed substrate fermentation, production of complex chemicals, and ecological system stability in the context of microbial consortia. It also provides valuable insights into the design, construction and optimization of artificial consortia for the utilization of cellulosic biomass and economic production of biofuel and chemicals.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

Using Novosphingobium aromaticivorans for Concurrent Production of Intracellular and Extracellular Products from Aromatics Extracted from Poplar Biomass

Achieving high biochemical production in biotransformations of renewable resources requires using concentrated cultures that not only generate the product of interest but also produce abundant microbial cell waste. We explored the concept of gaining value from microbial cells by producing intracellular products in tandem with a desired extracellular product. Specifically, we engineered a strain ofNovosphingobium aromaticivorans to extracellularly produce 2-pyrone-4,6-dicarboxylic acid (PDC) from aromatic substrates and to intracellularly accumulate astaxanthin along with coenzyme Q 10 , all of which are products of industrial interest. Achieving the goal of concurrent production of intracellular and extracellular products required the creative application of bioreactor engineering principles. Although a continuously fed membrane bioreactor (MBR) maximized extracellular product biosynthesis, it had a negative effect on intracellular product accumulation. However, operating the MBR as a sequencing batch reactor (MBR-SBR) with a step-feed resulted in stable concurrent production of both extracellular and intracellular products. With aromatics extracted from poplar biomass, we achieved productivities of 1.14 g of PDC/L-h for the extracellular product and 0.04 mg of astaxanthin/L-h and 0.64 mg of CoQ 10 /L-h for intracellular products, respectively. Our findings demonstrate that the mode of operation of a bioreactor impacts the simultaneous production of intracellular and extracellular products byN. aromaticivorans.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2023

A new approach of microbiome monitoring in the built environment: feasibility analysis of condensation capture

Background: Humans emit approximately 30 million microbial cells per hour into their immediate vicinity. However, sampling of aerosolized microbial taxa (aerobiome) remains largely uncharacterized due to the complexity and limitations of sampling techniques, which are highly susceptible to low biomass and rapid sample degradation. Recently, there has been an interest in developing technology that collects naturally occurring water from the atmosphere, even within the built environment. Here, we analyze the feasibility of indoor aerosol condensation collection as a method to capture and analyze the aerobiome. Methods: Aerosols were collected via condensation or active impingement in a laboratory setting over the course of 8 h. Microbial DNA was extracted from collected samples and sequenced (16S rRNA) to analyze microbial diversity and community composition. Dimensional reduction and multivariate statistics were employed to identify significant (p < 0.05) differences in relative abundances of specific microbial taxa observed between the two sampling platforms. Results: Aerosol condensation capture is highly efficient with a yield greater than 95% when compared to expected values. Compared to air impingement, aerosol condensation showed no significant difference (ANOVA, p > 0.05) in microbial diversity. Among identified taxa, Streptophyta and Pseudomonadales comprised approximately 70% of the microbial community composition. Conclusion: The results suggest that condensation of atmospheric humidity is a suitable method for the capture of airborne microbial taxa reflected by microbial community similarity between devices. Future investigation of aerosol condensation may provide insight into the efficacy and viability of this new tool to investigate airborne microorganisms.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

Data from TropiRoot 1.0 database: tropical root characteristics across environments

TropiRoot 1.0 is a new tropical root database with root characteristics across environment gradients. It has data extracted from 104 new sources, resulting in more than 8000 rows of data (either species or community data). Most of the data in TropiRoot 1.0 includes root characteristics such as root biomass, morphology, root dynamics, mass fraction, architecture, anatomy, physiology and root chemistry. This initiative represents an approximately 30% increase in the currently available data for tropical roots in the Fine Root Ecology Database (FRED). TropiRoot 1.0, contains root characteristics from 25 different countries where seven are located in Asia, six in South America, five in Central America and the Caribbean, four in Africa, two in North America, and 1 in Oceania. Due to the volume of data, when ancillary data was available, including soil data, these data was either extracted and included in the database or their availability was recorded in an additional column. Multiple contributors checked the entries for outliers during the collation process to ensure data quality. For text-based observations, we examined all cells to ensure that their content relates to their specific categories. For numerical observations, we ordered each numerical value from least to greatest and plotted the values, checking apparent outliers against the data in their respective sources and correcting or removing incorrect or impossible values. Some data (soil and aboveground) have different columns for the same variable presented in different units, including originally published units, but root characteristics data had units converted to match the ones reported in FRED. By filling a gap from global databases, TropiRoot 1.0 expands our knowledge of otherwise so far underrepresented regions, and our ability to assess global trends. This advancement can be used to improve tropical forest representation in vegetation models.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2026

Fine-Root Ecology Database (FRED): A Global Collection of Root Trait Data with Coincident Site, Vegetation, Edaphic, and Climatic Data, Version 4.

To address the need for a centralized root trait database, we compiled the Fine-Root Ecology Database (FRED) from published and unpublished data sources. We have continued to add to the FRED database since the release of FRED 1.0 in 2017, followed by 2.0 in 2018, and 3.0 in 2021. This new release of FRED 4.0 now has 213,941 observations of 238 root traits, for a combined total of roughly 3.4 million data fields for root traits and ancillary data together. FRED 4.0 has 39.8% more root trait observations than FRED 3.0 and a 34.4% increase in unique data sources. This release of FRED 4.0 also includes significant increases in geographic regions that have long been underrepresented in global datasets, notably in the tropical low latitudes. Ancillary data on associated site, vegetation, edaphic, and climatic conditions from across the globe have also increased concurrently with root trait observations. FRED is focused on fine roots (traditionally defined as roots less than 2 mm in diameter), as coarse roots are studied using different methodology, often at very different scales, and have different traits and trait interpretations. Despite this fine-root focus, FRED accepts data collected from roots of all sizes and contains observations of many root classes including coarse roots. Data collection will continue for the foreseeable future. The FRED4_Entire_Database_2026.csv file is the flat csv data file for FRED 4.0, and the FRED4_dd.csv file is the data dictionary of all columns available in FRED, including column IDs, column names, definitions, and unit (where applicable).

54 ENVIRONMENTAL SCIENCES↗