ECOLOGICAL INTENSIFICATION FOR SOIL HEALTH, CARBON SEQUESTRATION, AND GHG MITIGATION IN CASH-GRAIN AGROECOSYSTEMS OF THE NORTH CENTRAL US

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: ACTIVE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1032212
• Grant No.: 2024-67019-42335
• Cumulative Award Amt.: $749,830.00
• Proposal No.: 2023-10209
• Project Start Date: Sep 1, 2024
• Project End Date: Aug 31, 2028
• Grant Year: 2024
• Program Code: [A1401]- Foundational Program: Soil Health

Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218

Performing Department
(N/A)

Non Technical Summary
Several farming practices (e.g., cover crops) are promoted to improve soil health, sequester carbon (SOC), and reduce greenhouse gas (GHG) emissions. However, the efficacy of such practices is highly variable and remains the subject of considerable debate. We believe the aspirational goal of building soil health and mitigating climate change through SOC sequestration and greenhouse gas (GHG) abatement can be met, but only when approached holistically through ecological intensification (EI).We will work at the Wisconsin Integrated Cropping Systems Trial (WICST) where a suite of EI interventions was established in 2019. Our goal, to provide a comprehensive evaluation of the soil health, carbon sequestration, and GHG mitigation potential of EI in Midwestern grain systems will be met by 1) Quantifying medium-term changes in soil health and SOC; 2) Tracking the fate and persistence of C inputs from manure and cover crops, and 3) Assessing the net ecosystem carbon and GHG balance of each system.Knowledge generated by this research will enhance our understanding of how EI can improve "overall soil health and the resilience and sustainability of agricultural production systems", in addition to "directly evaluating the effects of management practices on soil microbial community function and their contribution to healthy soils, carbon sequestration, and greenhouse gas mitigation" as specified under Program Area Priority A1401-Soil Health.Regular engagement with WICST stakeholders will ensure the relevance of our EI interventions and ensure high impact for our results, which will be shared at field days, through our website, and in several UW-Madison courses.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

0%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
102	0110	1000	80%
132	0199	1000	10%
131	0199	1060	10%

Knowledge Area
102 - Soil, Plant, Water, Nutrient Relationships; 132 - Weather and Climate; 131 - Alternative Uses of Land;

Subject Of Investigation
0110 - Soil; 0199 - Soil and land, general;

Field Of Science
1060 - Biology (whole systems); 1000 - Biochemistry and biophysics;

Keywords

soil

health

Goals / Objectives
Approach-Specific objectives:To provide a comprehensive assessment of the ecological intensification management practices at Wisconsin Integrated Cropping Systems Trial (WICST), we propose the following three-tiered approach:We will measure an improved suite of soil health metrics including SOC, POM-C, PLFA, aggregate stability, and 24-h mineralizable C following the sixth consecutive year of ecological intensification practices.We will track the fate and persistence of carbon additions from manure and cover crops in tilled and no-till system using 13C labeling techniques.We will quantify the net ecosystem carbon balance (NECB) and GHG balance (N2O and CH4) in grain systems with ecological intensification management practices.Activities proposed and their sequence:Direct assessment of SOC & soil health: We will assess cumulative changes in SOC and key soil health indicators (POM-C, PLFA, Aggregate Stability, 24 h C mineralization, and ACE protein) following 6 full seasons of ecological intensification interventions at WICST.Rationale: SOC changes slowly making direct measurement of ΔSOC a challenge. Soil and climate variability, as well as experimental design constraints (limitations on replication and therefore power) can complicate the matter. Nevertheless, direct measurement of SOC stocks via dry combustion coupled with adjustments for equivalent soil mass remains the "gold" standard for accurately quantifying ΔSOC. Past work has demonstrated the robust statistical nature of WICST's experimental design (Sanford et al., 2012), and recent results have shown that ecological intensification is making a difference in sensitive soil health parameters at the trial. While changes in SOC were not discernable at year 3, we believe that we will be able to detect management-induced changes in SOC at year 6. Moreover, a recent publication has indicated that aggregate stability and 24 h C mineralization are core components of the "minimum suite of soil health indicators for North American Agriculture" (Bagnall et al., 2023), but these indicators were not measured as part of the original ecological intensification project at WICST. We will add these indicators to the six-year measurement suite, thereby providing a comprehensive, medium-term soil health assessment of ecological intensification practices.Expected outcome: At the end of six years, SOC and soil health parameters will have all improved relative to the control treatments as a result of ecological intensification interventions. In line with our early soil health findings, we anticipate the manure and combined interventions (EI-3, EI-4, EI-5) showing the most significant gains in SOC and soil health. While SOC improvements in cover crop (EI-1) and no-till (EI-2) interventions will be roughly equivalent, we expect them to differentially affect soil health parameters with cover cropping resulting in higher POM-C levels than no-till and no-till leading to greater aggregate stability and 24 h mineralization than cover cropping.Fate and persistence of C additions: Carbon from manure and cover crops will be tracked into soil POM, MOAM, and microbial "guilds" in both tilled and no-till systems at three timepoints across one growing season.Rationale: POM is made of partially decomposed plant material, while MAOM is derived from microbial residues. Recent work at WICST has shown that adding manure or integrating manure, cover crops, and no-till generally increases POM, microbial biomass, and AMF fungi. However, no changes have been detected when no-till and cover crop practices have been implemented individually. Furthermore, no changes to MAOM have been detected under any interventions, yet MAOM is the largest and most stable store of soil C and N (Rocci et al., 2021; Schrumpf et al., 2013). It is unclear why manure C is incorporated into the soil more efficiently than cover crop residue, why no-till practices have not enhanced C storage, and whether changes in MAOM are simply too small to detect using non-isotopic methods. This work will provide novel mechanistic detail about the pathways of POM and MAOM formation under various soil health interventions. This information will serve as the schematic for designing new systems that enhance SOC storage and will fill critical voids in current mechanistic SOC models.Expected outcome: Compared to cover crop-C, manure-derived C will have a longer POM residence time, be acquired more efficiently by fungi, and more quickly become stabilized as MAOM (Wang et al., 2022). No-till will slow the transfer of C into POM, microbes, and MAOM, but a greater proportion of C will be stabilized to MAOM compared to tilled systems.NECB and GHG footprint: The annual net ecosystem carbon balance and greenhouse gas footprint of six EI interventions will be quantified over three years.Rationale: The SOC impact of ecological intensification practices such as cover cropping, no-till, and manure can take several years to fully quantify, confounding adaptive management decisions. A NECB approach provides valuable "real time" information on the flux of carbon in an agroecosystem which can be used to validate direct measures of SOC stock change and assist farmers in management decisions. While soil carbon sequestration is of critical importance for climate mitigation and the provisioning of valuable ecosystems services (e.g. crop water regulation), ecological intensification interventions may unintentionally enhance the loss of climate warming GHGs, potentially negating the climate mitigating effects of gains in SOC (Lawrence et al., 2021). While soil organic carbon is the primary focus of this project, any effort to understand the climate mitigation potential of ecological intensification must account for the impacts of EI interventions on N2O or CH4. This work will help to validate the findings from our long-term direct assessment of SOC change and provide a comprehensive picture of the potential for ecological intensification interventions to serve as natural climate solutions.Expected outcome: Consistent with the soil health findings from the first three years of ecological intensification research, we anticipate a net flux of carbon into the EI systems at WICST, with the greatest carbon gains in manured (EI-3) and integrated interventions (EI-4 and EI-5). We further anticipate that systems receiving manure rather than synthetic N will emit less soil N2O than other systems, thus having the smallest GHG footprint.

Project Methods
Methods (abridged to meet character limit)Unabridged methods presented in full proposalDirect assessment of ΔSOC & soil health:Soil sampling: Samples (0 to 15 cm) will be collected from three locations within each 9 x 15-m EI plot, avoiding an area of 1.5 m from each plot edge. At each location a set of six 2 x 15-cm cores will be taken (2 in row, 2 between row, 2 intermediate), thoroughly homogenized, and composited.Bulk density: To relate all soil parameters to an area basis, calculate SOC stocks, and perform equivalent soil mass corrections, soil bulk density will be estimated in the surface 30 cm (0 to 15 and 15 to 30 cm) using a hand-held 7.6 x 15-cm hammer core. Soil cores will be carefully extracted to avoid compaction and dried at 60° C.Total C and N: Soil will be sieved to 2 mm, picked clean of all visible plant material, pulverized, and analyzed for CN using a Thermo-Finnigan EA 1112 flash combustion analyzer (Robertson et al., 2009).POM-C: POM will be separated by dispersing a 5-g soil subsample in sodium hexametaphosphate, shaking for 18 h, then poured onto a 53-µm sieve (Cotrufo et al. 2019). Material that remains in the sieve, which is classified as POM, will be thoroughly rinsed with DI water, dried at 60 °C, and weighed. The POM mass fraction will be determined by dividing the mass of POM by the mass of soil analyzed (Cates et al., 2016).PLFA: Phospholipid fatty-acid analysis (PLFA) will be used to quantify microbial groups as outlined by Herzberger et al. (2014). Lipids will be related to total microbial biomass as well as bacterial and fungal functional groups. PLFA has been shown to provide robust information on microbial community composition (Liang et al., 2016) and is very sensitive to shifts in management (Oates et al., 2012).Aggregate stability: Aggregate stability will be determined by wet sieving with an apparatus modified from the design of Yoder (1936) with 4 sieve mesh sizes: 2mm, 1mm, 500µm, 53µm. 24-h C mineralization (C-min): C-min will be determined by measuring the flush of carbon dioxide released by air-dried soil rewetted to 50% water filled pore space and incubated for 24-hours at 25°C (Franzluebbers, 1999; Franzluebbers et al., 2000).ACE protein: ACE protein will be measured on air-dried soils sieved to 2mm (Hurisso et al., 2018) following the method described in (Stott, 2019). Briefly, sodium citrate is used to extract 3 g air-dried soil. The extractant is mixed with bicinchoninic acid reagent, incubated for 60 minutes, and absorbance is quantified using a plate reader at 562 nm and a standard curve.Fate and persistence of C additions13C-labeled manure: Alfalfa will be 13C-labeled in a non-research field during the 2025 growing season. In July 2025, a Holstein heifer located at the USDA Dairy Forage Research Center will be fed unlabeled alfalfa for one day followed by the 13C-labeled alfalfa the next day. All liquid and solid excrement will be collected in separate batches for labeled and unlabeled material. Subsamples from this collection will be taken for δ13C analysis via IRMS to ensure sufficient 13C incorporation. The remaining manure will be frozen for use during the following field season. In April 2026, the labeled manure will be adjusted to 97% water to emulate typical dairy slurry manure, and a subsample will again be taken for δ13C analysis via IRMS. The labeled and unlabeled manure will then be applied by hand in the 13C-manure and unlabeled manure subplots (1 x 1 m), respectively, at a rate of 5,000 gallons per acre.13C-labeled cereal rye: Cereal rye will be 13C-labeled within designated 1 x 1 m subplots during April and early May 2026 when the rye plants are actively growing. The 13C-labeling procedure will follow the previously described procedure used to label the alfalfa. To ensure uniform 13C label, rye will be pulse labeled once per week. Using twelve labeling chambers, all 13C rye subplots will be labeled on the same days.Plant and soil sampling: Aboveground rye will be sampled for δ13C endmembers one day after the first cover crop labeling event (April 2026) and one day after the final cover crop labeling event (May 2026). Belowground samples will be collected in all subplots (including manure treatments) 6 months after cover crop termination (November 2026) in addition to the two aboveground biomass sampling dates. Above- and below-ground biomass will be dried at 60 °C, weighed, and analyzed δ13C analysis via IRMS. The remaining soil will be sieved to 2 mm, frozen, and saved for the 13C-POM, 13C-MAOM, and 13C-PLFA procedures.13C-POM and 13C-MAOM tracing: 13C will be traced from manure and the rye cover crop into POM and MAOM at the three timepoints corresponding to the aforementioned belowground sample collection (April, May, November). The contribution of 13C-labeled rye and manure to POM-C and MOAM-C will be calculated using a two-source isotope mixing model (e.g., Austin et al., 2017)13C-PLFA stable isotope probing: 13C will be traced from the manure and rye cover crops into the phospholipid fatty acids (PLFA) of gram-negative, gram-positive, saprotrophic fungi, arbuscular mycorrhizal fungi, actinomycetes, and anaerobic bacteria. PLFA biomarker abundance and δ13C will be quantified using a coupled GC-IRMS system, and the contribution of 13C-labeled rye and manure to each microbial functional group will be calculated using a two-source isotope mixing model (e.g., Kong et al. 2011).NECB and GHG balanceANPP: Above ground biomass will be collected from both corn and cereal rye each year. Corn biomass will be collected as close to peak standing biomass as possible (~R6) in two 1 m2 areas (2 corn rows x 0.66 m each). Cereal rye biomass will be collected using two 0.5 m2 quadrats in the spring just prior to cover crop termination (~May 5). Biomass will be dried at 60 °C, weighed, pulverized, and analyzed for total C and N.BNPP: Root biomass will be sampled in six locations within the center of each experimental unit following cereal rye termination and corn harvest in spring and fall, respectively.RH: Heterotrophic CO2 respiration (RH) will be measured using a LI-COR LI-7810 trace gas analyzer equipped with an LI-8200-01S smart chamber. To avoid the CO2 contribution from roots (autotrophic respiration), which is not soil-derived and therefore does not contribute to the NECB, a 1.5 m x 1.5 m section of each experimental unit will be kept free of all plant and weed biomass. Two RH measurements will be collected near the center of each plant-free area at 10-day intervals during the growing season (April 15 to Oct. 31), and at monthly intervals during the off-season (Nov. 1 to April 14). Annual RH will be assessed by linear interpolation and summation of measured instantaneous RH fluxes.GHGs: Trace gas fluxes of soil N2O, CO2, and CH4 will be measured using a LI-COR LI-7810 coupled with a LI-7820 and equipped with an LI-8200-01S smart chamber. Two trace gas measurements will be collected in each experimental unit at 10-day intervals during the growing season (April 15 to Oct. 31), and at monthly intervals during the off-season (Nov. 1 to April 14). Additional measurements will be made following application of manure and N fertilizer to capture ephemeral N2O pulses. Annual soil GHG exchange will be assessed by linear interpolation and summation of measured instantaneous GHG fluxes.Yield: Corn yields (grain & silage) will be collected using field scale agricultural equipment.

Progress 09/01/24 to 08/31/25

Outputs
Target Audience:The primary target audience reached during this firstreporting period has been scientific colleagues, extension agents, and graduate students, as well as farmers during field days and field tours. We have had a lot of interest in the agronomic systems management from extension personnel, as well as scientific colleagues and graduate students looking to leverage the work we have begun at WICST, to ask novel questions that were not previously possible. Field days at the trial have also provided an excellent opportunity to interact with farmers, extensionagents, and students. Changes/Problems:We have not encountered any signficant problems during our first year fo funding. There are two key changes to report. 1. Timeline: Our timeline has shifted relative to the originally submitted proposal. This is because the dates included in the original proposal presumed a late 2023 or early 2024 start date. As the project officially began on September 1, 2024, our timeline has been shifted to reflect this. We do not anticipate any issues completing the project in the time allotted for the grant (9/1/2024 to 8/31/2028). 2. While the project is directly funding one graduate student (Katelin Hermanson, MS candidate), Drs. Sanford and Jackson have leveraged additional funding sources (startup and foundation)to add two additional graduate students (Gideon Fynaardt, Jess Mehre) to the project. This has been an incredible boon to the project and will ensure its success and impact. What opportunities for training and professional development has the project provided?While the current project directly supports one graduate student (Kateline Hermanson, MS candidate, Sanford), we have used other support (PI Sanford) and (Co-PI Jackson,) to bring on two additional graduate students to support this project:Gideon Fynaardt (MS candidate, Sanford) and Jess Mehre (PhD candidate, Jackson). This has effectively tripled our capacity to train graduate students in critical aspects of agroecology, bioegeochemistry, and applied agricultural economics. Beyond the incredible training and professional development being provided to our three graduate research assistants we've brought on a team of undergraduate research technicians to support field work, sample collection, and sample processing. This team of students works directly with the graduate research assistants on the project to learn critical field and laboratory skills and gain invaluable research experience that will benefit them as they navigate choices about majors and future career paths. One noteable training opportunity that our team took advantage of was sending Jess Mehre to IsoCamp (https://isocamp.org/). The idea behind IsoCamp, which has been held annually since 1996,is to bring students and researchers together from around the world, teach them about stable isotopes, and help develop careers through lectures, field- and laboratory-based experiences. The camp is currently held atUniversity of New Mexico Center for Stable Isotopes. Jess is the PhD candidate that is taking on the second research tier ("fate and persistence of carbon inputs") in which he will using the stable isotope 13C to trace the fate of manure and cover carbon crop as it enters and flows through the soil system. PI Sanford and co-PI Jackson provided support to send Jess to this fantastic "camp" in direct support of her contributions to the current project. How have the results been disseminated to communities of interest?We have had several opportunities to discuss the project and report preliminary findings via presentations, fields days, and poster sessions. Presentations: Fynaardt, G., Dairy Symposium, "Ecological Intensification at the Wisconsin Integrated Cropping Systems Trial," Dairy Innovation Hub, Memorial Union, Madison, WI, United States. (May 14, 2025). Sanford, G.R., KBS Spring Seminar Series, "Carbon farming, and the promise of ecological intensification in the Northcentral US," Michigan State University's Kellogg Biological Station, Kellogg Biological Station, Hickory Corners, MI, United States. (April 19, 2025). Sanford, G.R., Tri-Societies Annual Meeting, "Enhancing soil health & SOC in Midwestern agroecosystems via ecological intensification," The American Society of Agronomy, the Crop Science Society of America, and the Soil Science Society of America, Henry B. Gonzalez Convention Center, San Antonio, TX, United States. (November 11, 2024). Sanford, G.R., RED Symposium, "Soil organic carbon sequestration: possibilities, pitfalls, and the arc of scientific inquiry," Research and Sponsored Programs, UW-Madison, Varsity Hall, Union South, Madison, WI, United States. (November 7, 2024). Sanford, G.R., Soils Science Seminar, "Back to the future: ecological intensification and a vision for 21st century farming," Department of Soil & Environmental Sciences, UW-Madison, Madison, WI, United States. (October 30, 2024). Field Days: Harvest of Ideas - WICST site visit, Arlington Agricultural Research Station, Arlington, WI, United States. (April 10, 2025). Dairy Soil and Water Regeneration Project - Annual meeting, WICST site visit, Arlington Agricultural Research Station, Arlington, WI, United States. (August 19, 2025). Posters: Fynaardt, G., von Haden, A., Read, H., Sanford, G.R., Dairy Symposium, "Back to the Future: Optimizing Carbon Capture and Crop Productivity via Ecological Intensification," Dairy Innovation Hub, Memorial Union, Madison, WI, United States. (May 14, 2025). Fynaardt, G., von Haden, A., Read, H., Sanford, G.R., Center for Ecology and the Environment Research Symposium, "Back to the Future: Optimizing Carbon Capture and Crop Productivity via Ecological Intensification," Center for Ecology and the Environment, Memorial Union, Madison, WI, United States. (April 14, 2025). Fynaardt, G., von Haden, A., Sanford, G.R., Professional Dairy Producers of Wisconsin - Business Meeting, "Ecological intensification for productivity, profitability, and soil health in Wisconsin Cropping Systems," Professional Dairy Producers of Wisconsin, Alliant Energy Center, Madison, WI, United States. (February 12, 2025). Fynaardt, G., Sanford, G.R., Dairy Summit, "Back to the Future: Optimizing Carbon Capture and Crop Productivity via Ecological Intensification," Dairy Innovation Hub, Varsity Hall, Union South, Madison, WI, United States. (November 20, 2024). What do you plan to do during the next reporting period to accomplish the goals?Our next reporting period will be one of the busiest for the duration of the grant. Planned activities include: 2025 - Q4 Objective 1 - 6y soil health assessment Collection of fall ANPP (cover crops) Fall Harvest (corn, soybean, wheat, barley) Soil health Sampling Soil health processing and analysis (UW & UMN, Cates) Curation of yield & econ data Objective 2 - fate & persistence of C additions Generate 13C labeled manure feed dairy heifers 13C labeled alfalfa/grass forage Objective 3 - NECB & GHG balance ANPP/BNPP data collection (crops & cover crops) Weekly GHG measurments (through Oct) Monthly GHG measurements (Nov - Dec) 2026 - Q1 Objective 1 - 6y soil health assessment Analysis of yield & econdata Soil health processing and analysis (UW & UMN, Cates) Objective 2 - fate & persistence of C additions Apply 13C labeled manure to field trials Objective 3 - NECB & GHG balance Monthly GHG measurements (Jan-March) Processing and analysis of ANPP/BNPP data Curation and analysis of GHG data 2026 - Q2 Objective 1 - 6y soil health assessment Analysis of yield & econdata Soil health processing and analysis (UW & UMN, Cates) Cover crop ANPP Planting etc. Objective 2 - fate & persistence of C additions Label CC biomass using 13C-CO2 Sample soil for 13C tracing work Objective 3 - NECB & GHG balance Weekly GHG measurement (April - June) Curation of GHG data ANPP/BNPP collection (cover crops) 2026 - Q3 Objective 1 - 6y soil health assessment Analysis of yield & econdata Soil health processing and analysis (UW & UMN, Cates) Objective 2 - fate & persistence of C additions Process soils for 13C labeled POM-C, PLFA, etc. Analysis of soil fractions for 13C label (UMN, Gutknecht) Objective 3 - NECB & GHG balance Weekly GHG measurements (July-Sept) Curation and analysis of GHG, ANPP, and BNPP data Additional details about completed (Y1) and pending work (Y2+) can be viewed in our project grant chart (see link below). Sanford progress tracker - 2025-08-12

Impacts
What was accomplished under these goals? For the first year, Dr. Sanford's team has had several opportunitiesto engage with farmers, extension agents, and policymakers through field days, tours, and conferences over the past year. These events have engendered productive discussion about the productivity, profitability, and environmentaltradeoffs of various agriculturalmanagement approaches. We anticipate even more conversation in the coming year. As data are generated from this project, we anticipate great adoption of ecological intensification as an agricultural management strategy with the ultimate impact of improving farmer productivity and profitability while enhancing our natural resources (soil, water, air). We have made significant progress towards meeting the goals and objectives outlined in our proposal (above). In addition to funding an MS student (Katelin Hermanson) directly with project funds, PI Sanford used other support to bring on a second MS student (Gideon Fynaardt) to work on the project. A third graduat student (Jess Mehre) working on her PhD has joined the project as well. Mehre is funded by co-PI Jackson. Togther this team of three graduate students with support from the larger team have made considerable progress since funding started on 9/2/2024. These collective efforts (below) have kept us on target to meet our goals/timelines. Tier 1: Agronomic & Economic Analysis Gideon Fynaardt, MS Student Agronomic Calendar Standardization: Ongoing work to standardize all EI agronomic calendar data entries for ease of future management and analysis Aboveground Net Primary Production (ANPP): In Fall and Spring, 1m x 1m quadrat of cover crop in all systems with cover were hand-harvested, dried, sorted by plant type, and weighed for assessment of cover crop productivity 3 x Canopeo photos were taken ~1m above the soil for assessment of photosynthetic cover Fall 2024 collected from the following treatment plots before killing frost: CS1: E1, E4, E5 CS2 (corn and soybeans): E1, E4, E5 CS3 (corn): E1, E4, E5 CS3 (wheat/barley): all EI treatment plots Spring 2025 collected from the following treatment plots before termination: CS1: E4, E5 CS2 (corn and soybeans): E1, E4, E5 CS3 (corn): E2, E4, E5 CS3 (soybeans): E5 Crop Yields Yield weight and moisture taken for all of the following: Wheat and barley grain harvested 7/18/2024 Wheat and barley straw baled 8/8/2024 Corn silage chopped 9/5 and 9/19 of 2024 Soybeans harvested 9/30/24 Corn grain harvested 10/22/24 Gross Margins Approximate commodity prices were determined for all outputs from EI systems for assessment of revenue Costs associated with all elements of agronomic management were determined Gaps in commodity prices and management costs will be addressed with the superintendent of Arlington Research Station Mike Bertram Tier 2: Fate and Persistence of C inputs Jess Mehre, PhD student Construction of Labeling Chambers: 12-1m3 labeling chambers were constructed in April 2025 Airtight seals of vinyl chambers confirmed with IRGA gas analyzer. Production of 13C-enriched forage: 12 m2 of pasture grass was labeled 4 times to produce 2.32 kg DM of 13C-enriched grass hay. 20 m2 of alfalfa was labeled 4 times to produce 7.95 kg DM of 13C-enriched alfalfa hay. Alfalfa and pasture biomass samples submitted for δ13C analysis to confirm level of enrichment. Production of 13C-enriched manure: IACUC animal use permit has been drafted with UW veterinary input. Feeding trial will be completed in December 2025. Tier 3: GHG Balance and NECB Measurements Katelin Hermanson, MS Student Greenhouse Gas (GHG) Flux Measurements: Weekly sampling began on April 11, 2025. As of June 21, 2025, a total of 17 sampling days have been completed. Measurements were taken from 54 collars each week, totaling 918 individual observations. Aboveground Net Primary Production (ANPP): Spring 2025 aboveground biomass collected from CS1 EI plots 4 and 5 3 one-meter quadrats per EI plot, 18 quadrats in total. Rye cover crop biomass is scheduled for grinding this week in preparation for C:N analysis. Belowground Net Primary Production (BNPP): Root biomass sampled from within each ANPP quadrat in CS1 EI plots 4 and 5. Two soil cores (to 30 cm depth) were collected per ANPP quadrat, one in-row and one out-row, totaling 36 soil cores. Roots were sieved to 4 mm, washed, sorted, dried, and pulverized in preparation for C:N analysis. Heterotrophic Respiration (Rh): Rh collars are maintained weekly by weeding a 1 m² area to clear living plants and roots. Weekly sampling began on April 11, 2025. Measurements were taken from 36 collars each week, totaling 612 individual observations to date. Crop Yield: No harvest or yield measurements for 2025 have been taken yet. Data Processing: Flux data processed weekly using Soil Flux Pro. Corrections include: Deleting duplicate or re-run samples Importing and aligning N2O data with water vapor offsets Adjusting deadband and observation end times

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