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Revisiting “Additional Carbon”: Tracking Atmosphere–Ecosystem Carbon Exchange to Establish Mitigation and Negative Emissions From Bio-Based Systems

Climate stabilization plans rely heavily on advanced bioenergy and bioproducts for substitution of fossil-based energy sources and materials, and increasingly, for negative emissions via the direct sequestration of biogenic carbon. Yet, there remain persistent, largely unresolved critiques of bioenergy assessment methodology, particularly in the areas of land use and biogenic carbon accounting. The concept of “additional carbon” calls for evaluating the climate performance of bio-based systems by whether feedstock production creates measurable new local agro-ecosystem uptake of carbon from the atmosphere. This concept is challenging to operationalize for first-generation biofuels, and has largely been advanced as a negative critique. However, carbon additionality is more straightforward to establish—and less critical to overall system mitigation performance—in advanced bioenergy systems. In this Perspective, I review the additional carbon critique, and why it is analytically challenging to address in first-generation biofuel systems based on conventional food crops with large existing markets. Next, I make a case that carbon additionality (1) is more readily achievable with cellulosic feedstocks, (2) is more directly observable for dedicated biomass crops, and (3) is not a strict requirement for achieving net mitigation in carbon-negative bio-based systems. I end by discussing how centering atmosphere–ecosystem carbon exchanges in bio-based system assessment could create new opportunities for enterprise-scale performance monitoring and verification, augmenting and diversifying the current reliance on model-based life-cycle assessment approaches.

54 ENVIRONMENTAL SCIENCES↗

Blue Rock CropSpanPV - Low Cost Racking for Agricultural Solar Photovoltaics

In this work, a total of four solar panel layouts, applied to Agrivoltaics, are investigated. This study presents two hypotheses: 1. Prefabrication - Lower total cost can be achieved by constructing a pre-assembled, pre-wired solar array in a factory, with automation, then rapidly deploying it in the field. 2. Tension Structures - Using tension structures in a way to suspend an overhead solar array also reduces costs when compared to conventional support methods. Designs are presented for frames to hold the panels. Configurations are advanced to connect the frames into arrays. Supports and foundations to hold the arrays in the desired layout are calculated, selected and presented. Cost models are prepared for the four layouts, which include definition of the business enterprise, the manufacturing operation, and the required facility for production at scale. Cost models include, detail material and task/labor take-offs for both manufacturing and installation. Comparisons with conventional system adapted to Agrivoltaics reveal that the two hypotheses do not hold true. A prefabricated array requires additional material that is not needed in the base system and the cost of this additional material is not outweighed by the cost savings of reduced field installation. The cost of wire rope for a tension structure does result in an economy of material, however, the cost of end connections and tensioning devices adds significantly to the cost of the overall tension structure. Foundation loads with tension structures, especially the large wind uplift seen with a fixed tilt solar array, impose significant constraints and high costs. The following conclusions are recommended for further consideration: 1. Best Case Agrivoltaic Configuration - The best value/lowest cost configuration for agrivoltaics is the installation of solar panels mounted on a single axis tracker in either a 2-in-portrait or 2-in landscape arrangement. This is useful for grazing lands and for staple crops which require high light levels. These applications represent the vast majority of the potential agrivoltaic market. Important elements in this configuration are the specific geometric layout coordinated with the various field operations and a control system linked between the farm equipment and single axis tracker. The control system is to monitor location and orientation, then actively tilt the solar panels or brake the field equipment to provide clearance and avoid collisions. 2. Long Span Applications - The use of tension structures for an overhead fixed tilt array is beneficial where long spans are a necessity, especially where the base support in the project location has consolidated rock near the surface or some other solid structure. One application is spanning wide irrigation canals. Another may be as an installation at the top deck of parking structures. 3. Direct Ground Mount Configuration – Having skids at the base of a pre-fabricated array that extend when the array is deployed, could be a successful design. This could be a useful strategy for dense, flat, ground-mount configurations like that employed by a couple of successful commercial operations (5G Maverick and Erthos). Arrays would be orientated north and south, deployed very low on prepared grade. Arrays would be aligned closely next to each other and anchored with small ground screws or simply with ballast. This same product arrangement could be very useful for rapid deployment and set-up of solar arrays for temporary deployments, disaster response and the military operations

14 SOLAR ENERGY↗

Leafweb: Dataset in Support of Coupled Modeling of Photophysics, Photochemistry, and Biochemistry of Photosynthesis, December 2022 Release

This data set contains measurements of leaf gas exchange and Pulse-Amplitude Modulated (PAM) fluorometry of light, carbon dioxide (CO2), oxygen (O2), and temperature responses from 26 C3 photosynthetic and four C4 photosynthetic species measured by independent researchers in Canada, China, Finland, Netherlands, and United States in the field, garden, or greenhouse. Data were collected between 1987 and 2021, however data for individual species only provide coverage over a few hours to one year. Species include three lianas, three shrubs, two boreal deciduous trees, two boreal evergreen needle-leaf tree, three temperate deciduous trees, four tropical deciduous trees, three tropical evergreen trees, one C3 grass, three C4 grasses, and six crop varieties. These measurements are conducted according to standard protocols in gas exchange (Long and Bernacchi 2003) and PAM fluorometry (Baker 2008). The Scots pine (Pinus sylvestris) dataset contains one-year continuous fluorometry observations made at intervals of 10 or 30 minutes in the field under natural environments using Walz monitoring PAM. Measurements from all other 29 species include simultaneous PAM fluorometry and gas exchange observations. Among these 29 species, seven species were measured with light response curves only (i.e., light intensity varied systematically with ambient CO2 concentration controlled at a constant level, e.g., 400 ppm). All the other 22 species were measured with both the light response and CO2 response (i.e., ambient CO2 concentration varied systematically with light intensity controlled at a constant level, e.g., 1200 µmolm-2s-1). For most species, measurements were made with temperature controlled at ~ 25 degrees Celsius (C) with the exceptions of Scots pine (natural diurnal and seasonal variations), tomato cultivar Basket Vee (~ 21 degrees C), and cotton which contained temperature stress experiments (9 to 40 degrees C). All measurements were made at ambient O2 concentration except for the tomato cultivar Basket Vee and cotton which used two O2 levels (2 and 21%) and rice and tomato cultivar Growdena which used five O2 levels (2, 10, 21, 35, and 50%). This dataset supports the complete modeling of photosynthesis at the leaf scale, including photophysical, photochemical, and biochemical reactions. It will support research in the applications of remotely sensed solar induced chlorophyll fluorescence and the responses of energy dissipation pathways to environmental variations across species and climates. This dataset contains 260 data files in comma separate (.csv) format contained inside 11 compressed (*.zip) files. Additional metadata are provided: one data dictionary, a species information table, and a file-level metadata file in comma separate (.csv) format and a user guide in PDF (*.pdf) format.

air temperature↗

Monitoring agroecosystem productivity and phenology at a national scale: A metric assessment framework

Effective measurement of seasonal variations in the timing and amount of production is critical to managing spatially heterogeneous agroecosystems in a changing climate. Although numerous technologies for such measurements are available, their relationships to one another at a continental extent are unknown. Using data collected from across the Long-Term Agroecosystem Research (LTAR) network and other networks, we investigated correlations among key metrics representing primary production, phenology, and carbon fluxes in croplands, grazing lands, and crop-grazing integrated systems across the continental U.S. Metrics we examined included gross primary productivity (GPP) estimated from eddy covariance (EC) towers and modelled from the Landsat satellite, Landsat NDVI, and vegetation greenness (Green Chromatic Coordinate, GCC) from tower-mounted PhenoCams for 2017 and 2018. Overall, our analysis compared production dynamics estimated from three independent ground and remote platforms using data for 34 agricultural sites constituting 51 site-years of co-located time series. Pairwise sensor comparisons across all four metrics revealed stronger correlation and lower root mean square error (RMSE) between end of season (EOS) dates (Pearson R ranged from 0.6 to 0.7 and RMSE from 32.5 to 67.8) than start of season (SOS) dates (0.46 to 0.69 and 40.4 to 66.2). Overall, moderate to high correlations between SOS and EOS metrics complemented one another except at some lower productivity grazing land sites where estimating SOS can be challenging. Growing season length estimates derived from 16-day satellite GPP (179.1 days) were significantly longer than those from PhenoCam G CC (70.4 days, p adj < 0.0001) and EC GPP (79.6 days, p adj < 0.0001). Landscape heterogeneity did not explain differences in SOS and EOS estimates. Annual integrated estimates of productivity from EC GPP and PhenoCam G CC diverged from those estimated by Landsat GPP and NDVI at sites where annual production exceeds 1000 gC/m –2 yr –1 . Based on our results, we developed a “metric assessment framework” that articulates where and how metrics from satellite, eddy covariance and PhenoCams complement, diverge from, or are redundant with one another. The framework was designed to optimize instrumentation selection for monitoring, modeling, and forecasting ecosystem functioning with the ultimate goal of informing decision-making by land managers, policy-makers, and industry leaders working at multiple scales.

54 ENVIRONMENTAL SCIENCES↗

Combining organic amendments with enhanced rock weathering shifts soil carbon storage in croplands

Enhanced rock weathering (ERW) involves applying crushed silicate minerals to cropland soils to remove carbon dioxide and stabilize the global climate. If practiced widely, ERW has the potential to mitigate climate change and improve soil health and crop productivity. However, most ERW studies emphasize inorganic carbon (IC) chemistry, using model-based estimates and short-term mesocosms. Limited field data exist on how ERW interacts with organic amendments to affect organic carbon (C) cycling in soils. In a three-year field study in conventionally managed, irrigated maize fields, we monitored how key soil variables responded to crushed rock-alone, and in combination with compost and/or biochar. We measured weathering indicators (pH, major cations, and IC contents) and organic fractions, including particulate organic matter (POM), mineral-associated organic matter (MAOM), microbial biomass C, and water-extractable organic C. Rock-alone treatments increased weathering proxies (pH and IC) and showed an increasing trend in POM and MAOM, relative to control. In contrast, combining crushed rock with organic amendments resulted in lower soil organic C and nitrogen (N) concentrations (in both POM and MAOM) compared to organic amendments alone, though IC increased in the rock+compost treatment. Combining rock with both compost and biochar (compost/biochar) significantly lowered MAOM-N compared to compost/biochar alone. Overall, co-applying rock with organic inputs may promote weathering and C accrual but slow the accrual rate of organic C and N relative to organic amendments alone. Quantifying these trade-offs over multiple years and scales is critical to integrating ERW with existing soil health practices and climate mitigation strategies.

Biological and medical sciences↗

Mini-Canister Radiolysis Testing of ASNF Materials and Surrogates

An experimental irradiation campaign to investigate radiolysis behavior of ASNF was conducted using in situ gas monitoring of small, sealed stainless-steel vessels (mini-canisters) containing aluminum samples with adherent (oxy)hydroxide films under helium backfill. The samples were irradiated with gamma radiation from a Co-60 irradiator. The samples tested included aluminum plate assemblies with lab-grown (oxy)hydroxides as surrogates for fuel as well as an end cropping from an actual ASNF assembly retrieved from long-term wet storage. These experiments enabled investigation of the impacts of various fuel drying approaches on the radiolytic generation rate and measurement of the H 2 yield associated with a reactor exposed sample with reactor-formed (oxy)hydroxide. The resulting data can be incorporated into model development for ASNF in dry storage. This report presents the cumulative results from four surrogate assemblies tested after application of different preparation (drying) conditions as well as the ASNF cropping; some post-irradiation testing was included. The mini-canister results are compared to data from related experimental campaigns that also tested the impact of drying conditions using samples irradiated in glass ampoules and discusses implications of the combined data

12 MANAGEMENT OF RADIOACTIVE AND NON-RADIOACTIVE W↗

Bacterial Community Members Increase Bacillus subtilis Maintenance on the Roots of Arabidopsis thaliana

Plant-growth-promoting bacteria (PGPB) are used to improve plant health and promote crop production. However, because some PGPB (including Bacillus subtilis) do not maintain substantial colonization on plant roots over time, it is unclear how effective PGPB are throughout the plant growing cycle. A better understanding of the dynamics of plant root community assembly is needed to develop and harness the potential of PGPB. Although B. subtilis is often a member of the root microbiome, it does not efficiently monoassociate with plant roots. We hypothesized that B. subtilis may require other primary colonizers to efficiently associate with plant roots. We utilized a previously designed hydroponic system to add bacteria to Arabidopsis thaliana roots and monitor their attachment over time. We inoculated seedlings with B. subtilis and individual bacterial isolates from the native A. thaliana root microbiome either alone or together. We then measured how the coinoculum affected the ability of B. subtilis to colonize and maintain on A. thaliana roots. We screened 96 fully genome-sequenced strains and identified five bacterial strains that were able to significantly improve the maintenance of B. subtilis. Three of these rhizobacteria also increased the maintenance of two strains of B. amyloliquefaciens commonly used in commercially available bioadditives. These results not only illustrate the utility of this model system to address questions about plant–microbe interactions and how other bacteria affect the ability of PGPB to maintain their relationships with plant roots but also may help inform future agricultural interventions to increase crop yields.

59 BASIC BIOLOGICAL SCIENCES↗

Science–based maize stover removal can be sustainable

Maize (Zea mays L.) stover can be harvested for multiple uses or left in the field to sustain soil organic carbon (SOC), cycle essential plant nutrients, and protect soil health. This 13-yr field study quantified effects of no (0 Mg ha –1 yr –1 ), low (1.0–1.4 Mg ha –1 yr –1 ), moderate (3.5–4.0 Mg ha –1 yr –1 ), or high rates (4.7–5.4 Mg ha –1 yr –1 ) of stover harvest from either continuous maize or maize–soybean [Glycine max (L.) Merr.] rotation on grain yield, plant nutrient concentrations, and multiple soil properties at two sites in Iowa. Stover harvest increased plant macro- and micro-nutrient removal, but did not affect average grain yields of either crops. Soil inorganic carbon (IC), SOC, bulk density, pH, and cation exchange capacity (CEC) showed no significant differences due to stover harvest. Plant tissue and soil-test nutrient concentration effects were also minor and site-specific. Stover harvest significantly (p < .05) decreased exchangeable K and Ca concentrations by 8.3–23.8% and 0.3–22.5% but overall soil health indicator effects were minimal. Overall, based on crop yields, plant nutrient and soil-test concentrations, soil health indicators, and carbon sequestration estimates, maize stover harvest can be sustainable provided: (a) grain yields consistently exceed 11 Mg ha –1 , (b) stover removal does not exceed 40% of the aboveground biomass (i.e., 3.5–4.0 Mg ha –1 yr –1 ), and (c) plant nutrients (especially K) are closely monitored.

54 ENVIRONMENTAL SCIENCES↗

Structuring Nutrient Yields throughout Mississippi/Atchafalaya River Basin Using Machine Learning Approaches

To minimize the eutrophication pressure along the Gulf of Mexico or reduce the size of the hypoxic zone in the Gulf of Mexico, it is important to understand the underlying temporal and spatial variations and correlations in excess nutrient loads, which are strongly associated with the formation of hypoxia. This study’s objective was to reveal and visualize structures in high-dimensional datasets of nutrient yield distributions throughout the Mississippi/Atchafalaya River Basin (MARB). For this purpose, the annual mean nutrient concentrations were collected from thirty-three US Geological Survey (USGS) water stations scattered in the upper and lower MARB from 1996 to 2020. Eight surface water quality indicators were selected to make comparisons among water stations along the MARB over the past two decades. Principal component analysis (PCA) was used to comprehensively evaluate the nutrient yields across thirty-three USGS monitoring stations and identify the major contributing nutrient loads. The results showed that all samples could be analyzed using two main components, which accounted for 81.6% of the total variance. The PCA results showed that yields of orthophosphate (OP), silica (SI), nitrate–nitrites (NO 3 -NO 2 ), and total suspended sediment (TSS) are major contributors to nutrient yields. It also showed that land-planted crops, density of population, domestic and industrial discharges, and precipitation are fundamental causes of excess nutrient loads in MARB. These factors are of great significance for the excess nutrient load management and pollution control of the Mississippi River. It was found that the average nutrient yields were stable within the sub-MARB area, but the large nitrogen yields in the upper MARB and the large phosphorus yields in the lower MARB were of great concern. t-distributed stochastic neighbor embedding (t-SNE) revealed interesting nonlinear and local structures in nutrient yield distributions. Clustering analysis (CA) showed the detailed development of similarities in the nutrient yield distribution. Moreover, PCA, t-SNE, and CA showed consistent clustering results. This study demonstrated that the integration of dimension reduction techniques, PCA, and t-SNE with CA techniques in machine learning are effective tools for the visualization of the structures of the correlations in high-dimensional datasets of nutrient yields and provide a comprehensive understanding of the correlations in the distributions of nutrient loads across the MARB.

54 ENVIRONMENTAL SCIENCES↗

Matching high resolution satellite data and flux tower footprints improves their agreement in photosynthesis estimates

Mapping canopy photosynthesis in both high spatial and temporal resolution is essential for carbon cycle monitoring in heterogeneous areas. However, well established satellites in sun-synchronous orbits such as Sentinel-2, Landsat and MODIS can only provide either high spatial or high temporal resolution but not both. Recently established CubeSat satellite constellations have created an opportunity to overcome this resolution trade-off. In particular, Planet Fusion allows full utilization of the CubeSat data resolution and coverage while maintaining high radiometric quality. In this study, we used the Planet Fusion surface reflectance product to calculate daily, 3-m resolution, gap-free maps of the near-infrared radiation reflected from vegetation (NIRvP). We then evaluated the performance of these NIRvP maps for estimating canopy photosynthesis by comparing with data from a flux tower network in Sacramento-San Joaquin Delta, California, USA. Overall, NIRvP maps captured temporal variations in canopy photosynthesis of individual sites, despite changes in water extent in the wetlands and frequent mowing in the crop fields. When combining data from all sites, however, we found that robust agreement between NIRvP maps and canopy photosynthesis could only be achieved when matching NIRvP maps to the flux tower footprints. In this case of matched footprints, NIRvP maps showed considerably better performance than in situ NIRvP in estimating canopy photosynthesis both for daily sum and data around the time of satellite overpass (R2 = 0.78 vs. 0.60, for maps vs. in situ for the satellite overpass time case). This difference in performance was mostly due to the higher degree of consistency in slopes of NIRvP-canopy photosynthesis relationships across the study sites for flux tower footprint-matched maps. Our results show the importance of matching satellite observations to the flux tower footprint and demonstrate the potential of CubeSat constellation imagery to monitor canopy photosynthesis remotely at high spatio-temporal resolution.

54 ENVIRONMENTAL SCIENCES↗

Systematic approaches to C-lignin engineering in Medicago truncatula

Abstract Background C-lignin is a homopolymer of caffeyl alcohol present in the seed coats of a variety of plant species including vanilla orchid, various cacti, and the ornamental plant Cleome hassleriana . Because of its unique chemical and physical properties, there is considerable interest in engineering C-lignin into the cell walls of bioenergy crops as a high-value co-product of bioprocessing. We have used information from a transcriptomic analysis of developing C. hassleriana seed coats to suggest strategies for engineering C-lignin in a heterologous system, using hairy roots of the model legume Medicago truncatula . Results We systematically tested strategies for C-lignin engineering using a combination of gene overexpression and RNAi-mediated knockdown in the caffeic acid/5-hydroxy coniferaldehyde 3/5-O-methyltransferase ( comt ) mutant background, monitoring the outcomes by analysis of lignin composition and profiling of monolignol pathway metabolites. In all cases, C-lignin accumulation required strong down-regulation of caffeoyl CoA 3- O -methyltransferase (CCoAOMT) paired with loss of function of COMT. Overexpression of the Selaginella moellendorffii ferulate 5-hydroxylase ( SmF5H ) gene in comt mutant hairy roots resulted in lines that unexpectedly accumulated high levels of S-lignin. Conclusion C-Lignin accumulation of up to 15% of total lignin in lines with the greatest reduction in CCoAOMT expression required the strong down-regulation of both COMT and CCoAOMT, but did not require expression of a heterologous laccase, cinnamyl alcohol dehydrogenase (CAD) or cinnamoyl CoA reductase (CCR) with preference for 3,4-dihydroxy-substituted substrates in M. truncatula hairy roots. Cell wall fractionation studies suggested that the engineered C-units are not present in a heteropolymer with the bulk of the G-lignin.

09 BIOMASS FUELS↗

Rapid denitrification of nitrate-contaminated groundwater in a low-gradient blackwater stream valley

Leaching of excess nitrogen (N) to groundwater in fertilized landscapes can overwhelm natural biogeochemical processes and cause long-term eutrophication of aquatic systems. We investigated N fate and transport from an intensively managed short-rotation woody crop (Pinus taeda) plantation through the riparian zone of an intermittent, low-gradient blackwater stream. Fertilization of the P. taeda plantation on the uplands resulted in contamination of groundwater with nitrate concentrations between 0.9 and 1.9 mg N L -1 . No corresponding increase in nitrate was observed in stream water or shallow groundwater in the riparian zone. Groundwater travel-time modeling predicted that N from near-stream, upland plantation areas should have reached streams during the monitoring period. Two years of measuring N species in well water in contrasting landscape positions (within the plantation, swale, riparian edge, forested hillslope, and valley), indicated rapid nitrate transformation and denitrification within the forested wetland valleys. Denitrification in the shallow groundwater system within the toeslopes and the riparian zone was estimated to have removed > 90% of nitrate. Finally, these results highlight the importance of riparian zones as pathways for the removal of N and for controlling downstream N loads.

54 ENVIRONMENTAL SCIENCES↗

Two-Stage Wildlife Event Classification for Edge Deployment

Camera-based wildlife monitoring is often overwhelmed by non-target triggers and slowed by manual review or cloud-dependent inference, which can prevent timely intervention for high stakes human–wildlife conflicts. Our key contribution is a deployable, fully offline edge vision sensor that achieves near-real-time, highly accurate wildlife event classification by combining detector-based empty-image suppression with a lightweight classifier trained with a staged transfer-learning curriculum. Specifically, Stage 1 uses a pretrained You Only Look Once (YOLO)-family detector for permissive animal localization and empty-trigger suppression, and Stage 2 uses a lightweight EfficientNet-based binary classifier to confirm puma on detector crops and gate downstream actions. Our design is robust to low-quality nighttime monochrome imagery (motion blur, low contrast, illumination artifacts, and partial-body captures) and operates using commercially available components in connectivity-limited settings. In field deployments running since May 2025, end-to-end latency from camera trigger to action command is approximately 4 s. Ablation studies using a dataset of labeled wildlife images (pumas, not pumas) show that the two-stage approach substantially reduces false alarms in identifying pumas relative to a full-image classifier while maintaining high recall. On the held-out test set (N = 1434 events), the proposed two-stage cascade achieves precision 0.983, recall 0.975, F1 0.979, accuracy 0.986, and balanced accuracy 0.983, with only 8 false positives and 12 false negatives. The system can be easily adapted for other species, as demonstrated by rapid retraining of the second stage to classify ringtails. Downstream responses (e.g., notifications and optional audio/light outputs) provide flexible actuation capabilities that can be configured to support intervention.

58 GEOSCIENCES↗

Improved Biofuel Production through Discovery and Engineering of Terpene Metabolism in Switchgrass

Project Objectives - Of the myriad specialized metabolites that plants deploy to adapt to environmental challenges, terpenes form the largest group. In many major crops, unique terpene blends serve as key stress defenses that directly impact plant fitness and yield. In addition, terpenes, such as bisabolene and pinene, are used for producing renewable biofuels. Essential to advancing a broader use of terpenes for biofuel feedstock engineering is a system-wide knowledge of the diverse biosynthetic machinery and defensive potential of often species-specific terpene blends. The proposed project would merge genome-wide enzyme discovery with comparative –omics, protein structural and plant microbiome studies to define the biosynthesis and stress-defensive functions of the switchgrass (Panicum virgatum) terpene network. These insights would be combined with developing and applying non-transgenic genome editing tools to design plants with desirable terpene blends for higher productivity and biofuel production on marginal lands. As a dedicated lignocellulosic feedstock for U.S. biofuel production with high net energy yield, stress tolerance, and available genome resources, switchgrass is well-suited for devising new avenues for biofuel production. Project Description – The diversity of plant terpene defenses is governed by species-specific families of terpene synthase (TPS) and cytochrome P450 monooxygenase (P450) enzymes. Mining of the switchgrass genome (genotype Alamo) identified ~100 TPS and P450 candidate genes, and combinatorial biochemical analysis of synthesized TPSs and P450s revealed more than a dozen enzymes with common and novel activities. In addition, several identified terpene metabolites and the corresponding transcripts were up-regulated in response to abiotic stressors. These findings demonstrate a unique switchgrass terpene network with probable importance to abiotic stress tolerance, thus providing a large chemical portfolio for optimizing crop resistance, yield, and biofuel composition. Leveraging these preliminary data, we propose to generate a genome-wide map of the switchgrass terpene metabolic network through multi-gene co-expression analyses that allow the efficient cross-validation of TPS and P450 functions. Key enzymes would further be applied to structure-function studies via X-ray protein crystallography, homology modeling and site-directed mutagenesis to gain mechanistic insight into the catalytic specificity of switchgrass terpene metabolism and provide gene and amino acid targets for genome editing. In tandem with terpene pathway discovery, system-wide metabolomics, transcriptomics and proteomics studies in switchgrass accessions of contrasting drought tolerance would define the role of switchgrass terpene metabolism in conferring abiotic stress resilience. Metabolic changes would be assessed in a combined approach of targeted (terpenes) and untargeted metabolite profiling using a high-resolution LC-MS/MS approach, differential gene expression analyses through multiplexed Illumina RNA sequencing, and quantitative analysis of high-priority pathway enzymes using multiple reaction monitoring (MRM). Drawing on these insights, knock-down/out mutants of stress-associated pathway nodes would be generated by optimizing transient virus-induced gene silencing (VIGS) and CRISPR/Cas9 systems under control of the Tobacco Rattle Virus (TRV). The resulting mutant lines would then be analyzed for stress susceptibility and the impact on the root microbiome to define gene functions in planta. Knowledge of terpene pathways, enzyme mechanisms and bioactivities would be applied to enhance switchgrass stress resilience and to tailor-make terpene blends for biofuel production. Here, TRV-enabled CRISPR/Cas9 genome editing, including allele-specific knock-out of redundant genes, engineering of enzyme specificity via structure-guided point mutations, and overexpression of terpene genes relevant to stress-protection or biofuel production, would be used to increase metabolic flux toward desired pathways. Broader Impacts - Integrating the system-wide discovery, mechanistic analysis and non-transgenic genome engineering of the switchgrass terpene network aligns the required steps to unlock the chemical potential of this important metabolite class to generate crops that are more resistant to stress and provide advanced biofuel production in light of rising climate pressures as foreseeable challenges for bioenergy crop cultivation. The proposed project would further offer interdisciplinary student training through active involvement in the project and integration of research concepts and outcomes into newly-developed graduate and undergraduate courses on Plant Biotechnology.

09 BIOMASS FUELS↗

AmeriFlux FLUXNET-1F US-CF1 CAF-LTAR Cook East

This is the AmeriFlux Management Project (AMP) created FLUXNET-1F version of the carbon flux data for the site US-CF1 CAF-LTAR Cook East. This is the FLUXNET version of the carbon flux data for the site US-CF1 CAF-LTAR Cook East produced by applying the standard ONEFlux (1F) software. Site Description - CF1 has operated since May 2017 at the R.J. Cook Agronomy Farm outside of Pullman, Washington, and monitors the no-till side of a paired-catchment study that is part of the Longterm Agroecosystem Research (LTAR) site common experiment. CF1 is located in “Cook East,” a field that has been in no-till management since 1998, and represents the Alternative treatment of the LTAR common experiment. It is paired with the CF2 tower location in “Cook West,” which has been in conventional tillage since prior to 1998 and represents the Prevalent treatments of the common experiment. Cook Agronomy Farm is in the high precipitation agroecological zone of the Columbia Plateau’s dryland cropping region. Crop rotations are wheat-based and include winter wheat, spring wheat, chickpea, spring canola, and winter peas. Wheat is the principal cash crop, with other crops grown in rotation for diversity, nutrient, and pest management. Soils are predominantly silt loam texture Mollisols in the Palouse, Thatuna, Naff soil series. The CF1 tower replaced US-RC1, which operated 2012-2016 in a neighboring field that was also in no-till management since 1998. CF1 and RC1 have distinct footprints, aspects, and soil series compositions.

Phillips, Claire L.↗

Linking Plant and Microbial Traits to Soil Carbon for Reliable and Resilient Bioenergy Systems

Bioenergy systems in the United States offer a dual opportunity to supply renewable feedstocks while enhancing ecosystem services such as hydrologic regulation, erosion control, and soil carbon (C) storage. National assessments highlight the potential to grow perennial energy crops to improve soil function and ecosystem resilience. Realizing this potential requires understanding the ecological mechanisms that govern how C is added, transformed, and stabilized in soils. Plant traits determine the quantity, depth, and chemistry of organic inputs, while microbial processes—including carbon use efficiency, necromass formation, and trophic interactions—mediate their transformation and partitioning among soil carbon pools. These biological pathways are shaped by soil physical and chemical properties, including aggregation, texture, and mineralogy, and by environmental drivers such as temperature, moisture, and disturbance, leading to context-dependent outcomes across landscapes. Management practices that diversify feedstocks, minimize disturbance, and maintain soil cover can promote both biomass production and C retention, while microbial amendments and rhizosphere engineering offer emerging, but often context-dependent, tools to optimize plant–microbe interactions. Trade-offs between biomass yield and soil carbon storage may arise when systems favor rapid aboveground productivity at the expense of belowground inputs and microbial processing, underscoring the importance of trait combinations that support both functions. Advances in monitoring, reporting, and verification—spanning precision agriculture, remote sensing, and biosensing—are improving predictive capacity through microbial-explicit process models and model–experiment (ModEx) frameworks. By connecting soil, plant, and microbial processes with advances in modeling and biosensing, this review outlines research priorities focused on trait-based parameterization and ModEx integration. These priorities will support the design of bioenergy systems that are both reliable and resilient, enhancing renewable energy production and ecosystem sustainability.

bioenergy systems↗

AmeriFlux FLUXNET-1F US-CF2 CAF-LTAR Cook West

This is the AmeriFlux Management Project (AMP) created FLUXNET-1F version of the carbon flux data for the site US-CF2 CAF-LTAR Cook West. This is the FLUXNET version of the carbon flux data for the site US-CF2 CAF-LTAR Cook West produced by applying the standard ONEFlux (1F) software. Site Description - CF2 has operated since May 2017 at the R.J. Cook Agronomy Farm outside of Pullman, Washington, and monitors the conventionally tilled side of a paired-catchment study that is part of the Longterm Agroecosystem Research (LTAR) site common experiment. CF2 is located in “Cook West,” a field that has been in conventional tillage since prior to 1998 and constitutes the Prevalent treatment of the common experiment. In this case, conventional tillage involves chisel plowing after the winter wheat phase of rotations (approximately every 3-4 years) and occasional harrowing. CF2 is paired with the CF1 tower location in “Cook East,” which has been in no-till since 1998 and represents the Alternative treatment of the common experiment. Both fields are planted in the same crops every year. Cook Agronomy Farm is in the high precipitation agroecological zone of the Columbia Plateau’s dryland cropping region. Crop rotations are wheat-based and include winter wheat, spring wheat, chickpea, spring canola, and winter peas. Wheat is the principal cash crop, with other crops grown in rotation for diversity, nutrient, and pest management. Soils are predominantly silt loam texture Mollisols in the Palouse, Thatuna, Naff soil series. The CF2 tower replaced US-RC2, which operated 2012-2016 in a neighboring field that was also conventionally tilled. CF2 and RC2 have distinct footprints, aspects, and soil series compositions.

Huggins, Dave↗

Voucher Opportunity 5-15: Independent Assessment of Monitoring, Reporting, and Verification (MRV) Technologies and Practices for Enhanced Rock Weathering (CRADA 718) Abstract

Development of robust, transparent, and precise monitoring, reporting, and verification (MRV) technologies and practices is critical for carbon dioxide removal (CDR) project developers to comply with regulatory and permitting requirements, voluntary carbon market (VCM) protocols, and to ensure safety while reducing environmental impacts. Enhanced rock weathering (ERW)-based CDR technologies focus on removing atmospheric carbon through conversion into thermodynamically stable solid or aqueous carbonate forms for permanent storage (i.e., mineralization). This highly durable form of CDR enhances naturally occurring silicate rock weathering cycles by optimizing application of finely-ground silicate rock particles (i.e., from basalt) on terrestrial agricultural lands to accelerate natural silicate rock weathering and mineralization. Enhanced rock weathering may also provide improved crop yields and enhance soil health. A critical aspect for commercialization of these technologies is the development of MRV to quantify the net removal and durable storage of atmospheric CO 2 . For ERW systems, it is essential to accurately characterize the mineral feedstock selected for application to establish the baseline geochemical composition, mineral dissolution rates, and carbon removal potential of the feedstocks to estimate overall net removal. Given the difficulty with conducting MRV for ERW in diverse soil/environment types, over large application areas, and due to complex chemical reaction networks, this project will accelerate understanding towards consensus on best practices for MRV. The overall objectives of the proposed voucher project are to: 1) Characterize and analyze feedstock(s) intended for ERW field application by Lithos Carbon (“Voucher Recipient”/ “CRADA Participant”) to determine overall mineralization potential; 2) Facilitate knowledge transfer and documentation of experimental protocols, instrumentation, and other relevant best practices; and 3) Support the Voucher Recipient’s broader technology commercialization and ERW Research Facility development plans. This work will align with the Voucher Recipient’s MRV plans for field sites and build upon complementary efforts conducted by PNNL on mineralization MRV.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗