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DOE OSTI2024

Scarcity of fixed carbon transfer in a model microbial phototroph–heterotroph interaction

Although the green alga Chlamydomonas reinhardtii has long served as a reference organism, few studies have interrogated its role as a primary producer in microbial interactions. Here, we quantitatively investigated C. reinhardtii’s capacity to support a heterotrophic microbe using the established coculture system with Mesorhizobium japonicum, a vitamin B 12 -producing α-proteobacterium. Using stable isotope probing and nanoscale secondary ion mass spectrometry (nanoSIMS), we tracked the flow of photosynthetic fixed carbon and consequent bacterial biomass synthesis under continuous and diurnal light with single-cell resolution. We found that more 13 C fixed by the alga was taken up by bacterial cells under continuous light, invalidating the hypothesis that the alga’s fermentative degradation of starch reserves during the night would boost M. japonicum heterotrophy. 15 NH 4 assimilation rates and changes in cell size revealed that M. japonicum cells reduced new biomass synthesis in coculture with the alga but continued to divide—a hallmark of nutrient limitation often referred to as reductive division. Despite this sign of starvation, the bacterium still synthesized vitamin B 12 and supported the growth of a B 12 -dependent C. reinhardtii mutant. Finally, we showed that bacterial proliferation could be supported solely by the algal lysis that occurred in coculture, highlighting the role of necromass in carbon cycling. Collectively, these results reveal the scarcity of fixed carbon in this microbial trophic relationship (particularly under environmentally relevant light regimes), demonstrate B 12 exchange even during bacterial starvation, and underscore the importance of quantitative approaches for assessing metabolic coupling in algal–bacterial interactions.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRSDate not supplied

Microbial Characterization of Heat Melt Compaction for Treatment of Space Generated Solid Wastes

One treatment process in development for solid waste management in space has been the Trash Compaction Processing System (TCPS). Human space mission wastes typically contain large percentages of contaminated wet solid waste. The Heat Melt Compactor (HMC) is being developed to be a multi-function means of water recovery, volume reduction, and the making safe of contaminant-rich trash with the potential for waste stabilization and/or sterilization. To determine the efficacy of the HMC treatment to kill microorganisms in solid waste and remain biologically stable, testing was conducted on three tiles produced by HMC-Gen 2 at Ames Research Center. Samples were shipped to Kennedy Space Center to test for microbial viability after compaction, determine the bio-stability of the HMC disks during storage (43 days), and assess potential airborne contaminate microbial growth on surfaces at low and high humidity conditions. In addition to the products of solid waste processing technologies, there is a concern that the crew might come into contact with hardware surfaces that have been contaminated by microorganisms during waste processing. The extent of microbial surface contamination of waste processing hardware was determined by surface sample swabbing and analysis for total bacterial and yeast counts and cultivable counts of aerobic and anaerobic bacteria, spore-forming bacteria, and fungi. Results indicate that trash processing increased bacterial counts on the surfaces of the compacter. All but one biological indicator spore strip imbedded in the HMC produced tiles were negative for growth after incubation for five days indicating effective sterilization through the heat melt compaction process. Analysis of core samples as well as surface growth of tiles inoculated with Aspergillus niger fungal spores incubated at three levels of humidity indicate that HMC created tiles did not support the proliferation of bacterial and fungal growth.

Mary E Hummerick↗

DOE OSTI2023

Comparison of metagenomes from fermentation of various agroindustrial residues suggests a common model of community organization

The liquid residue resulting from various agroindustrial processes is both rich in organic material and an attractive source to produce a variety of chemicals. Using microbial communities to produce chemicals from these liquid residues is an active area of research, but it is unclear how to deploy microbial communities to produce specific products from the different agroindustrial residues. To address this, we fed anaerobic bioreactors one of several agroindustrial residues (carbohydrate-rich lignocellulosic fermentation conversion residue, xylose, dairy manure hydrolysate, ultra-filtered milk permeate, and thin stillage from a starch bioethanol plant) and inoculated them with a microbial community from an acid-phase digester operated at the wastewater treatment plant in Madison, WI, United States. The bioreactors were monitored over a period of months and sampled to assess microbial community composition and extracellular fermentation products. We obtained metagenome assembled genomes (MAGs) from the microbial communities in each bioreactor and performed comparative genomic analyses to identify common microorganisms, as well as any community members that were unique to each reactor. Collectively, we obtained a dataset of 217 non-redundant MAGs from these bioreactors. This metagenome assembled genome dataset was used to evaluate whether a specific microbial ecology model in which medium chain fatty acids (MCFAs) are simultaneously produced from intermediate products (e.g., lactic acid) and carbohydrates could be applicable to all fermentation systems, regardless of the feedstock. MAGs were classified using a multiclass classification machine learning algorithm into three groups, organisms fermenting the carbohydrates to intermediate products, organisms utilizing the intermediate products to produce MCFAs, and organisms producing MCFAs directly from carbohydrates. This analysis revealed common biological functions among the microbial communities in different bioreactors, and although different microorganisms were enriched depending on the agroindustrial residue tested, the results supported the conclusion that the microbial ecology model tested was appropriate to explain the MCFA production potential from all agricultural residues.

60 APPLIED LIFE SCIENCES↗

NASA NTRSDate not supplied

Characterization of Microbe Resistant Coatings for Use in the ISS Water System

The ISS is a highly controlled environment; however, there are still microbes on it that astronauts bring from their own microbiome. Once within the ISS water treatment system, these microbes not only pose a health risk for astronauts, but they can form biofilms, causing material degradation and system failure. Due to their protective structure, biofilms are notoriously resistant to disinfectants and antibiotics. One proposed solution is applying polymer coatings to the metal parts of the water system to prevent biofilms from forming. Two types of polyampholyte polymers with previously demonstrated nonfouling properties were sent to the ISS in 2021 to test their viability for preventing microbial adhesion in space. However, the only microbial assays performed were microscopy. To enhance this ongoing research project, I developed procedures for culturing, applying, and quantifying Ralstonia pickettii on these polymers. R. pickettii was chosen because it is one of the most prevalent microbes found in the ISS water treatment system. Optical density data was recorded to obtain a growth curve demonstrating a doubling time between 2.83 and 4.88 hours in 30°C, not shaken conditions. Optical density was also correlated to colony forming units in a plating experiment. Another result was design (and pilot testing) of a surface colonization experiment. These results provided vital knowledge about R. pickettii, which will be used for a 2023 payload of antimicrobial polymer coatings on stainless steel, and improved methods for biofilm analysis upon its return to evaluate the efficacy of the coatings.

microbiology↗

DOE OSTI2020

Microbial Community Composition in Deep‐Subsurface Reservoir Fluids Reveals Natural Interwell Connectivity

Abstract The identification of natural fractures and the wells they connect is crucial for the development of geological reservoirs because it may have an important impact on reservoir model construction and hydraulic fracture propagation. In this study we investigated the use of a novel data source, the microbial community composition in the reservoir formation fluids, for identification of interwell connectivity caused by natural fractures. We verified this concept at a newly developed mesoscale enhanced geothermal system (EGS) field testbed located 4,850 ft (1,478 m) beneath ground surface at the Sanford Underground Research Facility in Lead, SD. Fluids produced at or near the EGS testbed were sampled and subjected to high‐throughput 16S rRNA gene amplicon sequencing to analyze the microbial community profile therein. Despite the typically substantial heterogeneity across the community profiles of samples spatially distributed (10 m to 1.9 km apart) throughout the site, samples from two wells at the EGS testbed showed highly similar microbial community composition, suggesting the two wells intersected the same natural fracture. This evidence of natural connectivity between the two wells at the EGS testbed was later corroborated by core log analysis and sewer camera surveys into the boreholes. Besides the field case described in this study, microbial community composition as a reservoir diagnostic tool would be applicable in a much broader context such as unconventional hydrocarbon exploitation, groundwater reservoir characterization, and environmental remediation, adding valuable “hard” data capable of pinpointing the origins of fluids unambiguously.

Zhang, Yuran↗

NASA NTRSDate not supplied

Precision Fermentation: Relieving the Crabtree Effect in S. cerevisiae through Genetic Engineering

Nutrient degradation in the current NASA pre-packaged food system poses significant challenges to crew health in missions beyond low earth orbit as certain vitamins and nutrients have been shown to degrade during extended storage, and resupply and fresh food items cannot be readily provided. BioNutrients(BN) uses synthetic biology in an effort to supplement the NASA food system by delivering in situ production of nutrients and therapeutics produced by genetically engineered microbes. One yeast S. cerevisiae strain utilized in BN is genetically modified to produce two types of antioxidants: zeaxanthin and β-carotene. Zeaxanthin is a non-provitamin A carotenoid and has been implicated in eye health and vision performance, and β-carotene is a provitamin A carotenoid. Microbial production of high-value nutrients can aid our understanding and address the issue of nutrient degradation and loss in the NASA food system. Although S. cerevisiae is an excellent candidate for a long shelf life, on-demand nutrient production system, its space applications are limited by the Crabtree effect where respiration is shut down in favor of fermentative metabolism when glucose is present in high concentrations, causing the release of unwanted ethanol. Current and likely future life support systems are negatively impacted by ethanol and ethanol release is strictly limited.To improve feasibility of using S. cerevisiae, we explored ways to reduce the Crabtree effect, thereby limiting its ethanol production. Specifically, we used CRISPR-cas9 to genetically modify various genes in this pathway

E Zaroff↗

DOE OSTI2020

The Model System Saccharomyces cerevisiae Versus Emerging Non-Model Yeasts for the Production of Biofuels

Microorganisms are effective platforms for the production of a variety of chemicals including biofuels, commodity chemicals, polymers and other natural products. However, deep cellular understanding is required for improvement of current biofuel cell factories to truly transform the Bioeconomy. Modifications in microbial metabolic pathways and increased resistance to various types of stress caused by the production of these chemicals are crucial in the generation of robust and efficient production hosts. Recent advances in systems and synthetic biology provide new tools for metabolic engineering to design strategies and construct optimal biocatalysts for the sustainable production of desired chemicals, especially in the case of ethanol and fatty acid production. Yeast is an efficient producer of bioethanol and most of the available synthetic biology tools have been developed for the industrial yeast Saccharomyces cerevisiae. Non-conventional yeast systems have several advantageous characteristics that are not easily engineered such as ethanol tolerance, low pH tolerance, thermotolerance, inhibitor tolerance, genetic diversity and so forth. Currently, synthetic biology is still in its initial steps for studies in non-conventional yeasts such as Yarrowia lipolytica, Kluyveromyces marxianus, Issatchenkia orientalis and Pichia pastoris. Therefore, the development and application of advanced synthetic engineering tools must also focus on these underexploited, non-conventional yeast species. Herein, we review the basic synthetic biology tools that can be applied to the standard S. cerevisiae model strain, as well as those that have been developed for non-conventional yeasts. In addition, we will discuss the recent advances employed to develop non-conventional yeast strains that are efficient for the production of a variety of chemicals through the use of metabolic engineering and synthetic biology.

09 BIOMASS FUELS↗

DOE OSTI2022

Ecosystems and Networks Integrated with Genes and Molecular Assemblies (ENIGMA). Final report

The goal of the Stahl lab is to use a combination of isotopic, genomic, and activity-based analyses in the field, of selected field isolates, and defined synthetic communities to identify sources and sinks of different nitrogen species in the field, focusing on the FRC at the ORNL. The most general research objective is to characterize variants in the pathway for denitrification among field isolates and synthetic microbial assemblies as a foundation for predicting the biotic and abiotic controls of nitrogen and carbon transformations in the field. Specifically, we have focused on characterizing environmental controls of nitrous oxide emissions, an important greenhouse gas and ozone depleting molecule. Studies have mapped the flux of nitrous oxide and controlling genetic elements within the FRC subsurface, indicating the importance of different enzyme variants in nitrous oxide production and consumption in controlling surface emissions. We have developed methods to quantify and catalog the isotopic signatures imparted on nitrous oxide by different nitrous oxide reductase variants in isolated organisms and model synthetic communities. We have developed methods to quantify and catalog the nitrous oxide affinity of field relevant isolates and their biomass yield when respiring nitrous oxide on different carbon substrates. We continue to expand the collection of field-relevant microbial isolates within ENIGMA by focused enrichment and isolation from field material. These objectives have been facilitated by the development of new technologies, including: 1) the development of a multiplexed automated optical density measurement system for quantifying growth kinetics of microorganisms dependent on gaseous substrates in monoculture or gas exchange in synthetic communities and 2) the development of a microcalorimetric analysis format to quantify the impact of environmental stress on microbial maintenance energy. Since field relevant processes are mediated by assemblies of organisms interacting with each other and their local environment, we have also collaborated with other ENIGMA investigators on the assembly and characterization of synthetic communities. These studies have evaluated the role of organisms lacking a complete pathway for denitrification in the control of field processes, measuring process rates of assemblies of organisms that individually lack the full denitrification pathway but mediate the complete denitrification when growing together. These efforts have so far resulted in the publication of 18 peer reviewed papers ranging from ecological and physiological drivers of pathway optimization to field-scale process characterization.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2022

Long-Term Cellulose Enrichment Selects for Highly Cellulolytic Consortia and Competition for Public Goods

Microbial communities are a key driver of the carbon cycle through the breakdown of complex polysaccharides in diverse environments including soil, marine systems, and the mammalian gut. However, due to the complexity of these communities, the species-species interactions that impact community structure and ultimately shape the rate of decomposition are difficult to define.

microbial interactions↗

DOE OSTI2025

Bacterial nitrite production oxidizes Fe(II) bioremediating acidic abandoned coal mine drainage

Passive remediation systems (PRSs) treating either acidic or neutral abandoned coal mine drainage (AMD) are colonized by bacteria that can bioremediate iron (Fe) through chemical cycling. Due to the low pH in acidic AMD, iron oxidation from soluble Fe(II) to precipitated Fe(III) is mainly directed by microbial oxidation. Less well described are biotic reactions that lead to iron remediation through abiotic secondary reactions. We describe here iron oxidation in acidic AMD that is mediated by the bacterial reduction of nitrate to nitrite followed by the geochemical oxidation of Fe(II). Within an acidic PRS, 4,560 bacteria cultured from the microbial community were screened for their ability to oxidize iron and to perform nitrate-dependent iron oxidation (NDFO). Iron oxidation in the culturable community was observed in every pond of the system, ranging from 2.1% to 11.4%, and NDFO was observed in every pond, ranging from 1.4% to 6.0% of the culturable bacteria. Five NDFO isolates were purified and identified as Paraburkholderia spp. One of our isolates, Paraburkholderia sp. AV18 was shown to drive NDFO through the bacterial production of nitrite that in turn chemically oxidizes Fe(II) (nitrate reduction-iron oxidation; NRIO). AV18 expressed nitrate reductase, napA, concurrent to nitrite production. Burkholderiales are found by 16S rRNA gene sequencing in every pond of the PRS. The frequency of NDFO metabolism in the culturable microbial community and abundance of Burkholderiales in the PRS suggest nitrite producers contribute to the bioremediation of iron in acidic AMD and may be an unharnessed opportunity to increase iron bioremediation in acidic conditions.

(NDFO)↗

DOE OSTI2026

Data from a four-day long microcosm experiment addressing the destabilization of artificial mineral-associated organic matter by model root exudates embedded in a soil matrix from the Rocky Mountain Biological Laboratory (Gothic, CO, USA), 2019

This dataset provides data collected during a four-day long laboratory soil microcosm experiment testing the efficacy of root exudate-driven mineral-associated organic matter destabilization. This dataset contains four data files in comma-separate values (*.csv). The files provide the metadata and the experimental results on microbial respiration, MAOM-derived respiration, and sequential mineral-extractions. This data was used to produce the figures in Bölscher et al., 2026. The results of the experiment can be found in the open access article Bölscher et al., 2026 (https://doi.org/10.1016/j.soilbio.2026.110276). Abstract: Mineral-associated organic matter (MAOM) is often considered stable, but root exudates can destabilize MAOM via various pathways. Theory and model system studies suggest that direct MAOM destabilization by strong ligands, like oxalic acid, or reducing agents, like catechol, is more effective than indirect, microbial-mediated MAOM destabilization, stimulated by less reactive compounds like glucose. Here, we demonstrate that the presence of a soil matrix alters the efficacy of exudate-driven MAOM destabilization pathways. Glucose and catechol destabilized significantly greater amounts of MAOM from ferrihydrite and aluminum hydroxide (Al (OH)3) embedded in a soil matrix than oxalic acid. Our findings indicate that indirect, microbial-mediated MAOM destabilization may play a larger role than direct MAOM destabilization in soil environments.

Destabilization↗

NASA NTRSDate not supplied

Autonomous Science: Simulated Solar System Mission to Enceladus, Icy Ocean Moon of Saturn

Future NASA missions to icy ocean worlds such as Europa, Titan, or Enceladus will collect mass spectrometry (MS) data from exospheres, atmospheres, and plume volatiles to assess their geochemistry and potential for microbial life1. These remote missions face challenges related to limited bandwidth, communication, and power, highlighting the need for science autonomy2 to prioritize data for timely downlink. We extend our previous work on machine learning (ML) biosignature detection from isotope ratio mass spectrometry (IRMS) data to distributed systems missions (DSM) by incorporating the ML and autonomous data quality control and prioritization code into an onboard decision-making platform. We use simulated orbital telemetry for eight orbiters around Enceladus with a mothership to illustrate an automated biosignature and novel seawater chemistry detection with data prioritization from MS analyses of volatile CO2.

Conor Williams↗

DOE OSTI2024

Emergence of Orchestrated and Dynamic Metabolism of Saccharomyces cerevisiae

Microbial metabolism is a fundamental cellular process that involves many biochemical events and is distinguished by its emergent properties. While the molecular details of individual reactions have been increasingly elucidated, it is not well understood how these reactions are quantitatively orchestrated to produce collective cellular behaviors. Here we developed a coarse-grained, systems, and dynamic mathematical framework, which integrates metabolic reactions with signal transduction and gene regulation to dissect the emergent metabolic traits of Saccharomyces cerevisiae. Our framework mechanistically captures a set of characteristic cellular behaviors, including the Crabtree effect, diauxic shift, diauxic lag time, and differential growth under nutrient-altered environments. It also allows modular expansion for zooming in on specific pathways for detailed metabolic profiles. Furthermore, this study provides a systems mathematical framework for yeast metabolic behaviors, providing insights into yeast physiology and metabolic engineering.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Genome‐enabled exploration of microbial ecology and evolution in the sea: a rising tide lifts all boats

Summary As a young bacteriologist just launching my career during the early days of the ‘microbial revolution’ in the 1980s, I was fortunate to participate in some early discoveries, and collaborate in the development of cross‐disciplinary methods now commonly referred to as "metagenomics". My early scientific career focused on applying phylogenetic and genomic approaches to characterize ‘wild’ bacteria, archaea and viruses in their natural habitats, with an emphasis on marine systems. These central interests have not changed very much for me over the past three decades, but knowledge, methodological advances and new theoretical perspectives about the microbial world certainly have. In this invited ‘How we did it’ perspective, I trace some of the trajectories of my lab's collective efforts over the years, including phylogenetic surveys of microbial assemblages in marine plankton and sediments, development of microbial community gene‐ and genome‐enabled surveys, and application of genome‐guided, cultivation‐independent functional characterization of novel enzymes, pathways and their relationships to in situ biogeochemistry. Throughout this short review, I attempt to acknowledge, all the mentors, students, postdocs and collaborators who enabled this research. Inevitably, a brief autobiographical review like this cannot be fully comprehensive, so sincere apologies to any of my great colleagues who are not explicitly mentioned herein. I salute you all as well!

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Non-destructive quantification of anaerobic gut fungi and methanogens in co-culture reveals increased fungal growth rate and changes in metabolic flux relative to mono-culture

Abstract Background Quantification of individual species in microbial co-cultures and consortia is critical to understanding and designing communities with prescribed functions. However, it is difficult to physically separate species or measure species-specific attributes in most multi-species systems. Anaerobic gut fungi (AGF) (Neocallimastigomycetes) are native to the rumen of large herbivores, where they exist as minority members among a wealth of prokaryotes. AGF have significant biotechnological potential owing to their diverse repertoire of potent lignocellulose-degrading carbohydrate-active enzymes (CAZymes), which indirectly bolsters activity of other rumen microbes through metabolic exchange. While decades of literature suggest that polysaccharide degradation and AGF growth are accelerated in co-culture with prokaryotes, particularly methanogens, methods have not been available to measure concentrations of individual species in co-culture. New methods to disentangle the contributions of AGF and rumen prokaryotes are sorely needed to calculate AGF growth rates and metabolic fluxes to prove this hypothesis and understand its causality for predictable co-culture design. Results We present a simple, microplate-based method to measure AGF and methanogen concentrations in co-culture based on fluorescence and absorbance spectroscopies. Using samples of < 2% of the co-culture volume, we demonstrate significant increases in AGF growth rate and xylan and glucose degradation rates in co-culture with methanogens relative to mono-culture. Further, we calculate significant differences in AGF metabolic fluxes in co-culture relative to mono-culture, namely increased flux through the energy-generating hydrogenosome organelle. While calculated fluxes highlight uncertainties in AGF primary metabolism that preclude definitive explanations for this shift, our method will enable steady-state fluxomic experiments to probe AGF metabolism in greater detail. Conclusions The method we present to measure AGF and methanogen concentrations enables direct growth measurements and calculation of metabolic fluxes in co-culture. These metrics are critical to develop a quantitative understanding of interwoven rumen metabolism, as well as the impact of co-culture on polysaccharide degradation and metabolite production. The framework presented here can inspire new methods to probe systems beyond AGF and methanogens. Simple modifications to the method will likely extend its utility to co-cultures with more than two organisms or those grown on solid substrates to facilitate the design and deployment of microbial communities for bioproduction and beyond.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

An automated multiplexed turbidometric and data collection system for measuring growth kinetics of anaerobes dependent on gaseous substrates

Standard methods of monitoring the growth kinetics of anaerobic microorganisms are generally impractical when there is a protracted or indeterminate period of active growth, and when high numbers of samples or replications are required. As part of our studies of the adaptive evolution of a simple anaerobic syntrophic mutualism, requiring the characterization of many isolates and alternative syntrophic pairings, here we developed a multiplexed growth monitoring system using a combination of commercially available electronics and custom designed circuitry and materials. This system automatically monitors up to 64 sealed, and as needed pressurized, culture tubes and reports the growth data in real-time through integration with a customized relational database. The utility of this system was demonstrated by resolving minor differences in growth kinetics associated with the adaptive evolution of a simple microbial community comprised of a sulfate reducing bacterium, Desulfovibrio vulgaris, grown in syntrophic association with Methanococcus maripaludis, a hydrogenotrophic methanogen.

59 BASIC BIOLOGICAL 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↗

DOE OSTI2025

Particulate organic matter (POM) transport and transformation at the terrestrial-aquatic interface (Final Report)

This project investigates the input, transport, and degradation of particulate organic matter (POM) in near-surface riverbed sediments at the Hanford 300 Area of the Columbia River, a dynamic, regulated river system influenced by upstream dam operations. Riverbed sediments are biogeochemical hot spots where organic carbon inputs stimulate intense microbial activity, affecting nutrient cycling and redox transformations in the hyporheic zone (HZ). While dissolved organic matter (DOM) cycling has been studied extensively, little is known about the infiltration and transformation of POM—particularly under variable flow regimes common in large, regulated rivers.

54 ENVIRONMENTAL SCIENCES↗