Source: COLORADO STATE UNIVERSITY submitted to
THE FUNCTIONAL NATURE OF SOIL ORGANIC MATTER POOLS: IDENTIFYING MECHANISTIC INDICATORS FOR SOIL HEALTH AND AGRONOMIC OUTCOMES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1025120
Grant No.
2021-67019-34241
Cumulative Award Amt.
$499,780.00
Proposal No.
2020-04275
Multistate No.
(N/A)
Project Start Date
May 1, 2021
Project End Date
Apr 30, 2025
Grant Year
2021
Program Code
[A1401]- Foundational Program: Soil Health
Recipient Organization
COLORADO STATE UNIVERSITY
(N/A)
FORT COLLINS,CO 80523
Performing Department
Natural Resource Ecology Lab
Non Technical Summary
Healthy soils are the foundation of resilient, productive, sustainable agroecosystems because they perform essential functions such as nutrient cycling, carbon storage, water retention, and cation exchange. This is a broadly accepted concept, but when it comes to defining and quantifying soil health, fundamental questions remain unanswered. For example, what are the best indicators of soil functioning that we can use to quantify soil health and predict agronomic sustainability, productivity, and resilience? This question is especially relevant for soil organic matter, which is one of the most commonly measured soil health properties but does not consistently correlate with agronomic performance. Solving this dilemma requires the development of quantitative soil health indices for soil organic matter that are mechanistically linked to soil functioning and therefore to agronomic outcomes such as increased yield, yield stability, and reduced inputs. Total soil organic matter is used across virtually all soil health studies and common soil health test protocols because it is widely accepted that it underlies multiple soil functions, including water purification, nutrient cycling, and habitat provision for soil organisms. Yet, soil organic matter is not consistently related to agronomic performance, and the mechanisms behind this paradox remain unexplored. We propose to resolve this problem by separating soil organic matter into two functional pools, particulate (POM) and mineral-associated organic matter (MAOM), and exploring their use as new soil health indicators. Our goals are to advance understanding of the roles of POM and MAOM in soil functioning and agronomic performance and facilitate their widespread use and measurement. Our objectives and related activities are: 1) compare effects of POM and MAOM on soil functioning and plant performance through a controlled greenhouse study; 2) evaluate POM and MAOM as new soil health indicators using over 300 soil samples and related data from a variety of agroecosystems under varied management; and 3) determine best protocols for POM and MAOM quantification through rigorous methods testing. Further, our activities will allow us to quantitatively assess soil organic matter regeneration and soil carbon sequestration potential across US agricultural soils in response to common management practices. The proposed work will enhance understanding of biogeochemical processes involved in soil health and develop soil health indicators to enhance measurement and management of soil organic matter pools to improve soil health and agricultural sustainability. Linking the new, emerging understanding of measurable, functional soil organic matter pools to indicators and drivers of soil health will allow for increased adoption and efficacy of agricultural practices that increase soil organic matter stocks, which can ultimately increase the performance and resilience of US cropping systems.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1020110106070%
1020110200030%
Knowledge Area
102 - Soil, Plant, Water, Nutrient Relationships;

Subject Of Investigation
0110 - Soil;

Field Of Science
1060 - Biology (whole systems); 2000 - Chemistry;
Goals / Objectives
The major goals of this project are to: 1) advance fundamental understanding of the functional role of particulate organic matter (POM) and mineral-associated organic matter (MAOM) in agricultural soils; 2) characterize POM and MAOM and evaluate their use as new soil health indicators in a variety of US agroecosystems under varied management; and 3) determine best practices for effective and efficient POM and MAOM quantification across diverse soil types.Objective 1: Compare effects of POM and MAOM quantities and ratios on plant yield, plant N content, and common soil health indicators in agricultural soilsDisentangling the functional roles of POM and MAOM, and the results for plant performance (i.e. agronomic outcomes) requires manipulating their quantities and qualities in a controlled environment. We will achieve this in a controlled greenhouse experiment with treatments to manipulate (1) total soil organic matter content, (2) the relative proportions of POM and MAOM, (3) the quality (i.e. C:N) of POM, and (4) the nutrient status of the soil (i.e. fertilizer). We will measure the effects of our manipulations on soil functions including plant-available nitrogen, water retention, CEC, soil pH, and soil respiration. We will also measure plant traits including total biomass, root:shoot ratio, N content, and grain mass. This will allow us to directly test the effects of total soil organic matter, POM:MAOM ratio, POM quality, and nutrient status of the soil functioning and plant performance.Objective 2: Evaluate the utility of POM and MAOM as indicators of soil health and agronomic performanceIdentifying useful soil health indicators requires broadscale testing of the relationships between those potential indicators and agronomic outcomes across diverse soil types and cropping systems. To achieve this, we will isolate POM and MAOM from soils of long-term agricultural research sites and working farms across the US. We will collect all available metadata associated with these fields, including yields, fertilizer inputs, climate, and soil properties. We will then perform statistical analyses, including structural equation modelling, to determine which properties of POM and MAOM are most strongly and consistently related to agronomic outcomes. We will use the results of the greenhouse experiment (Objective 1) to inform us on which properties of POM and MAOM are most promising for the analysis based on mechanistic links between POM and MAOM and soil functioning. Objective 3: Quantify soil organic matter regeneration and C sequestration potential across US agricultural soils in response to different management practicesWe will use data from Objective 2 to explore how soil organic matter quantity and character vary with management, using a space-for-time approach where possible to understand the soil organic matter regeneration potential resulting from changes in management practices. To the extent possible, we will also collect soils from time series that include management changes, for the same purpose. Using all of the soils measured for this project, we will construct a mathematical saturation curve for MAOM according to soil texture, pH, and other soil and ecosystem properties which we discover to be key moderators of MAOM saturation limits. We will use the saturation curve (or curves) to quantify long-term soil organic matter storage potential (saturation deficit) across agricultural soils of the US.Objective 4: Compare protocols for effective and efficient POM and MAOM quantification across soil typesWe aim to provide scientifically grounded guidance to scientists and soil health practitioners on how to successfully and efficiently separate POM and MAOM. We will perform methods testing of common soil fractionation schemes on 10 diverse soils with contrasting texture, soil organic matter content, metal oxide content, and pH, to determine which methods most successfully separate soil organic matter pools with contrasting properties, and whether their success depends on soil properties. We will also characterize the chemistry of the soils and resulting fractions using diffuse reflectance mid-infrared Fourier transform (DRIFT) spectroscopy to further understand the results of the different fractionation schemes and how they vary by soil type. Objective 5: Quantify POM and MAOM across diverse soils using DRIFT spectroscopyWe will add measured DRIFT spectra from Objective 4 to a larger database we are currently compiling of DRIFT spectra on hundreds of soils. We will use this database to test whether DRIFT spectroscopy, in combination with partial least-squares regression, is a viable tool for quantifying POM and MAOM without performing a physical procedure to isolate them. If successful, this high-throughput, low-cost method of quantifying POM and MAOM could greatly increase their use by labs around the world.
Project Methods
Soil function measurementsFor the greenhouse experiment and field soils, we will quantify several aspects of soil functioning that we expect to be most closely related to POM and MAOM, and to have direct impacts on plant performance. These are plant-available N, soil respiration, CEC, and water retention. Plant-available N will be extracted from soil samples according to a well-established method using 2.0 M KCl and measured on an AlpKem N AutoAnalyzer within 72 hours of extraction. Soil respiration, or short-term C mineralization, will be measured according to a procedure modified from the NRCS Soil Health Technical Note No. 450-03 (NRCS, 2019). We will incubate soil subsamples at 60% field capacity in airtight jars for 4 days as recommended, but we will replace KOH traps and titrations for CO2 measurement with direct measurements of jar headspace on a benchtop infrared gas analyzer (LI-COR Ll-7810).We will measure CEC, or CEC-7, according to the Kellogg Soil Survey Laboratory Methods Manual (4B1a1a1a1, Soil Survey Staff, 2014). Briefly, each sample will leached using 1 N ammonium acetate and a mechanical vacuum extractor. The NH4+-saturated soil will be rinsed with ethanol to remove the NH4+ that is not adsorbed, and then leached with 2 M KCl. This leachate will be analyzed by steam distillation and titration to determine the NH4+ adsorbed on the soil exchange complex. Water retention will be measured by the pressure desorption method (3C1a-e1a and 3C2a1a, Soil Survey Staff, 2014) at 33 kPa and 1500 kPa, and oven drying at 110°C to obtain gravimetric water content.Soil FractionationFor the purpose of this project, we will use physical fractionation to determine the distribution of soil C and N in the primary physical soil organic matter pools, POM and MAOM, and their subcomponents. Briefly, 2 mm sieved soil samples will be fully dispersed by shaking for 18 hours in dilute sodium hexametaphosphate with beads to break all aggregates. Dispersed soil samples will beseparated using a combined size and density approach. First, dissolved organic matter will be extracted by shaking soil samples in deionized water, followed by filtration at 0.45 µm. Next, the light POM (L-POM) will be floated off using a sodium polytungstate (SPT) solution of 1.8 g/cm3 density. The remaining heavy fraction will be separated according to size (53 µm) to isolate heavy POM (H-POM) and MAOM. Next, MAOM will be further separated to isolate fast-cycling MAOM, according to the results of our methods testing activities. For example, if we conclude that sequential density separation using a density cutoff of 2.0 g/cm3 produces the greatest contrast in accessibility of the resulting fractions, we will use that method to separate fast-cycling MAOM. Bulk soils and fractions obtained from the residue labelling experiments will be analyzed for %C and %N on a CHN analyzer.Statistical MethodsWe will use a combination of statistical techniques to analyze relationships between POM and MAOM properties (quantity, C:N, relative ratios), soil functions, and agronomic performance (yield, yield:input ratios, yield stability), including multiple regression and structural equation modeling (SEM). SEM provides a powerful framework for assessing direct versus indirect causal linkages between variables in complex systems, such as climate-vegetation-soil systems. SEM also allows for the representation of complex, multifaceted concepts, such as soil type, through the use of latent and composite variables (Grace and Bollen 2008). We will use latent and composite variables to represent climate and soil properties, which allow simultaneous inclusion of multiple indicators for each (e.g., "soil properties" can include pH and texture). Prior to SEM, we will inspect the data for interactive effects between indicators, non-linear relationships and heterogeneity of variance. Composite variables can be specified to handle these challenges, in addition to decreasing overall model complexity. For the greenhouse experiment and US agroecosystem soil survey, we will begin from our most complex metamodel, and use information gained through both traditional multivariate statistics and composite variable specification to decrease model complexity and design SEMs to test the structure of controls using multigroup analysis if necessary (e.g., in irrigated vs. non-irrigated fields). The metamodels are theoretical starting points, but additional drivers may also be considered, such as soil mineralogy, where data are available. Our large number of soils (300+) is sufficient to reliably fit this relatively complex SEM. We will fit the SEM using the "lavaan" package in R.To analyse the spectral data produced by DRIFT spectroscopy analyses, we will use a distance-based redundancy analysis (dbRDA) to assess of the overall differences in DRIFT spectral chemistry of how bulk soil, POM and MAOM among different sites, contrasting for soil taxa, land use, climate, etc. using the R: Vegan package. We plan to use the dbRDA analysis over other multivariate statistical approaches due to its non-linear distance-metric options, which have robust multi-dimensional resolution to assess categorical variables. Differences in the soil, POM or MAOM chemistry due to soil taxa, management and climate will be visualized via Canonical Analysis of Principle Coordinates.EffortsWe will use the results of our methods testing experiment to produce a white paper and outreach materials to aid in outreach to farmers/producers and soil health practitioners. We will maximize our reach using our existing networks through the Colorado State University Extension and Agricultural Experiment Stations, as well as the networks of our project collaborators.Evaluation To measure the success of this project, we will track:· -- Completion of proposed experiments and data analysis activities· -- The number of peer-reviewed publications resulting directly from the project activities, and their citations in the scientific literature· -- The number of farmers/producers and soil health practitioners with whom we communicate directly about SOM functional pools and methods of fractionation, including the number that use their quantification to inform management decisions, through personal communication· -- The number of students who graduate as a result of this project· -- The number of undergraduate workers who receive training while working on this project· -- The number of collaborative products (e.g. peer-reviewed papers, scientific conference sessions organized, grant proposals) that result from collaborations initiated during this project

Progress 05/01/23 to 04/30/24

Outputs
Target Audience:As in previous years, our efforts target the academic scientific community, including undergraduate students, across multiple fields including agricultural science, soil science, soil ecology, soil biogeochemistry, and other ecological fields. We also targetschool students and teachers at multiple levels, from elementary to high school. Finally, we communicated findings from this project toboth the non-profit (e.g., Environmental Defense Fund) and private industry (e.g. Indigo Ag) sectors. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?In addition to previously reported training, during this reporting period Dr. Leuthold was accepted into the School of Global Environmental Sustainability (CSU SoGES) Sustainability Leadership Fellowship program in 2023 which has provided him a series of different training opportunities. This included workshops focused on scientific communication, connections between science and policy, and other means of scientific outreach. A highly selective program within CSU, overall, the program provided training via 7 different intensive workshops. He has also attended workshops outside the university related to his work and professional development, including a workshop titled, "Challenges and Opportunities related to Soil Spectroscopy," led by the Soil Spectroscopy for Global Good group at the 2023 Soil Science Society Annual Meeting. This workshop aimed to produce a peer-reviewed article around the future of soil spectroscopy to be submitted to Geoderma during summer of 2024.In addition to this training, continuingtraining and development of labmethods and classwork, Dr. Leuthold mentored three undergraduate assistants under his direct supervision during his time at CSU. All three of these students have gone on to continue working in agricultural sciences, including in other labs and withing the USDA at the NRCS. During his time mentoring these students, Leuthold fostered important research skills in his mentees, including working directly with one of them to develop research results which were presented at the American Association for the Advancement of Science conference in February 2024. Co-PIMachmuller has mentored and supervised six undergraduate students and three research associates who have all learned (or are currently learning) how to measure and evaluate several aspects of soil health, including soil organic matter fractionation. As co-director of the CSU soil health center (IN-RICHES; Integrated Rocky Mountain Region center for Healthy Soils), Dr. Machmuller has led several outreach and professional development efforts. These include a Soil Health Workshop held at CSU Spur campus in February 2024 for over twenty practitioners and producers. Topics covered included soil health principles and management, soil health test interpretation, soil ecology, and the soil food web. She helped curate The Exploring Soils Curriculum, which is an extensive collection of over 100 educational links featuring soil health educational resources. Dr. Machmuller also presented at a webinar for the Colorado Department of Agriculture and conservation districts in June 2024 on how to interpret soil health testing and assessment. How have the results been disseminated to communities of interest?The results produced so far have been disseminated through peer-reviewed publications, conference, webinars and workshop presentations, including to industrial, producers and policy audiences, and they were included in classroom materials (Dr. Cotrufo classes on Soils and Global Change for seniors, graduate students and high school teachers). What do you plan to do during the next reporting period to accomplish the goals?Over the course of the next and final year of the project, we will complete the analyses of samples and data from the incubation experiment and prepare the results for publication in a peer-reviewed publications, satisfying Objective 1. We will also make progress with the lab work and analysis of the data relating fraction characteristics to agronomic outcomes at the regional scale, bolstering our results under Objectives 2. This includes leveraging the new understanding of MAOM N dynamics towards and exchangeable MAOM fraction towards questions around agronomic optimum N rate, using a unique dataset in collaboration with researchers at Ohio State University. A major goal for this final reporting year is to create an FTIR model for the prediction of the light POM fraction and refine our understanding and protocols for the separation of MAOM fractions (i.e., free vs occluded and stable vs. dynamic). Finally, we will explore the application of FTIR to the prediction of agronomic outcomes. We also plant to write a fact sheet and white paper to more broadly communicate our project's findings.

Impacts
What was accomplished under these goals? We conducted an incubation experiment that studied the changes in the relative contribution of mineral associated organic matter (MAOM) and particulate organic matter (POM) to plant available nitrogen (N) pools under varied POM stoichiometries (C:N) and POM:MAOM ratios C. This experiment leveraged isotopic tracing tools to understand the conditions under which MAOM N is mineralized and made available for plant uptake, while allowing for more rigorous quantification and inference around the causal mechanisms. We sampled two soils to isolate POM fractions with contrasting C:N ratios, one from a managed grassland ecosystem in Central KS, USA (high C:N), and one from a long-term alfalfa field in Eastern NE, USA (low C:N). We separated the POM from these two soils by density and characterized its C & N elemental and isotopic composition by EA-IRMS. To isolate an isotopically enriched MAOM fraction that realistically represented MAOM dynamics, we used a soil that had been previously fertilized with 15N-enriched ammonium nitrate (15NH415NO3-) as part of a long-term experiment located near Ames, IA. Soils were fractionated into MAOM using a one-step size fractionation at 53 mm. Our experiment was designed as a full factorial, randomized complete block design, with three treatments: SOC concentration (%), POM C:N, and POM:MAOM C ratio. Each treatment factor had two levels (typically high and low). For the SOC concentration treatment, our target SOC concentration for the low treatment was 0.50% C, and the target SOC concentration for the high treatment was 2.50% C. The two POM C:N ratio treatment levels were 33.25 and 17.78. For our POM:MAOM ratio, our low treatment mimicked the typical distribution of C in agricultural systems (20% POM - 80% MAOM), whereas the high treatment represented an extreme, with much more POM than is typically present even in regenerative management treatments (60% POM - 40% MAOM). Each treatment was replicated four times. During the course of the 8-week incubation, we monitored the amount of N mineralized at five time points: week 0, 2, 4, 6 and 8 by leaching the inorganic N out of the soils. At the end of the 8-week incubation period, the incubations units were destructively harvested. Two subsamples were taken; microbial biomass was measured on one subsample using the chloroform fumigation method, and one subsample are currently being fractionated using a combined size density fractionation into POM, MAOM, and organic matter associated with the coarse heavy fraction of soil (CHAOM) to assess C and N mass balances. All extracts up to the 6-week extractions have been analyses for mineral N and 15N, while all the 8week time harvest sample analyses are ongoing. We developed the methodology first described by Altabet et al. (2019; https://doi.org/10.1002/rcm.8454 ) to determine the 15N isotopic composition of the NO3- in the soil extracts (NH4+ was negligible) and applied to our samples. Preliminary datasuggest that MAOM-N is available to microbial communities as a N source, and the relative proportion of N sourced from this fraction is partially driven by the availability of C and N in other, more easily depolymerized fractions (i.e., POM). This experiment and preliminary data have been reported in a Chapter of Dr. Leuthold's PhD Dissertation. Data will be further processed once the week 8 harvest is complete and plans are to communicate these findings through a peer-reviewed publication and a conference presentation.We have completed and published (Leuthold et al., 2024 https://doi.org/10.5194/soil-10-307-2024 ) the work conducted in collaboration with researchers at Michigan State University to understand the utility of POM and MAOM as indicators of subfield-scale yields and yield stability. Our results show that, at the subfield scale, POM may be a useful indicator of yield stability and that, while SOM tends to increase as yield increase, increasing SOM may not directly confer increased yield stability. Instead, in fields with high spatiotemporal yield heterogeneity, SOM stocks may be determined by interactive effects of topography, weather, and soil characteristics on crop productivity and SOM decomposition. These findings highlight the value of considering these factors during soil sampling campaigns, especially when attempting to quantify farm-scale soil C stocks. Working toward the regional-scale analysis, we have obtained > 300 additional individual soil samples with associated agronomic data such as yield, nutrient inputs, and management activities from both industry partners and academic collaborators. Over 200 of these samples have already been fractionated using a combined size-density fractionation following the recommendations based on our work (see below), and all samples have been analyzed for spectral absorbance via diffuse reflectance infrared Fourier transformed (DRIFT) mid-infrared spectroscopy. We are in the process of fractionating the remaining samples and intend to finish fractionation by the year-end. We will also leverage the same data described above on an additional ~350 soil samples being collected under the USDA Climate Smart Commodities project of which Dr. Machmuller is a PI. We further worked for the publication and presentation of our findings on SOM fractionation protocols, which indicated that chemical, isotopic, and spectral composition of the POM fraction are dependent on fractionation scheme, with fractionation methodologies that employ a density separation step isolating a different, and more unique, POM fraction than those which employ only a size separation. These are now published in Geoderma (Leuthold et al., 2024, https://doi.org/10.1016/j.geoderma.2024.116870), contributed to a chapter of Dr. Leuthold's PhD Dissertation, and were presented at the Ecological Society of America conference in August of 2023, the American Geophysical Union conference in December of 2023, at the Indigo Science Policy Forum in April of 2024, and as part of the Lawrence Livermore National Lab Soil Biogeochemistry Group lab meeting series in April of 2024. We have continued scanning with FTIR any relevant bulk soil and fraction sample to establish a broad library for the effective and efficient estimation of SOM fractions. To date we have scanned over 4500 samples. We are specifically interested in establishing an FTIR model for the prediction of the light POM fraction, since published methods are for size fractions, but our comparison study mentioned above demonstrated that POM is better separated by density. Additionally, we have explored the use of FTIR to separate the stable from the dynamic MAOM fraction, leveraging soils from long term (52 years) fallow experiments in Sweden. The bare fallow MAOM lost aliphatic compounds while increased in phenolic and polysaccharidal compounds. These analyses enabled identifying organic functional groups potentially associated with a dynamic MAOM pool. These findings contributed to a Chapter of Dr. Leuthold PhD Dissertation and are being further processed for peer review publication. We have also extended the comparison of protocols for analyses of POM and MAOM to the free and the macro and microaggregate occluded fractions, which demonstrated that microaggregate occluded MAOM has typically a higher C loading, heavier 13C and at depth lower C:N than the free MAOM. This work contributed to a manuscript under review (Fulton-Smith & Cotrufo) in Geoderma. We are following up from these observations, expanding the analyses of micro-aggregate occluded MAOM fractions to more agricultural soils, also under contrasting tillage and depths, to understand the robustness of these findings and identify best protocols for the most effective separation of MAOM.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2024 Citation: Fulton-Smith, S., Even R., Cotrufo M.F. (in review) Depth impacts on the aggregate-mediated mechanisms of root carbon stabilization in soil: trade-off between MAOM and POM pathways. Geoderma.
  • Type: Theses/Dissertations Status: Accepted Year Published: 2024 Citation: Leuthold, S.J. (2024) ISOLATION, INTERPRETATION, AND IMPLICATIONS OF PHYSICAL SOIL ORGANIC MATTER FRACTIONS IN SOIL SYSTEMS. [Doctoral dissertation, Colorado State University]. ProQuest Dissertation Publishing (currently under embargo)
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Leuthold, S.J. (2024) Taking SOC a step further: Understanding measurement and function of physical soil organic matter fractions. 3rd Annual Indigo Science and Policy Forum. 16 April, Boston, MA
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Lavallee, J.M., Leuthold, S.J., Cotrufo, M.F. The Functional Nature of Soil Organic Matter Pools: Identifying Mechanistic Indicators for Soil Health and Agronomic Outcomes. Oral Presentation at the FY2024 (A1401) Soil Health Project Director Meeting, Kansas City, MO. April 10, 2024.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Machmuller, M.B., Zelikova, T.J., Ritten, J. Unpacking the carbon credit conundrum, Carbon Markets 101. Oral Presentation for webinar series hosted by Quivira Coalition & CSU Soil Carbon Solutions Center. February 20, 2024.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Machmuller, M.B., Zelikova, T.J. Soil Carbon Quantification & Markets. Oral Presentation, Webinar for The Nature Conservancy. October 30, 2023. Machmuller, M.B. Navigating the Soil Health Frontier. Oral Presentation, Webinar for The Colorado Soil Health Advisory Committee. December 6, 2023.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Machmuller, M.B., Firth, A., Trujillo, W., Mason, L., Choppennig, C. Soil Health Analysis and Interpretation. Webinar for the Colorado Department of Agriculture. June 10, 2023.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2024 Citation: Cotrufo (2024) Identifying mechanisms and controls of POM and MAOM formation and persistence. Invited presentation at the Webinar SPP2322 SoilSystems.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Leuthold, S.J., Lavallee, J.M, Haddix, M.H., Cotrufo, M.F. Reconciling the conceptual and procedural definitions of physical soil organic matter fractions. Oral presentation at the American Geophysical Union Annual Meeting. December 2023
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Leuthold, S.J., Soong, J., Even, R.E., Cotrufo, M.F. Litter decomposition traced over a decade confirms divergent mechanisms of POM and MAOM formation and persistence. Poster presentation at the American Geophysical Union Annual Meeting. December 2023.
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Leuthold, S. J., Lavallee, J. M., Haddix L.H., Cotrufo M. F. (2024). Contrasting properties of soil organic matter fractions isolated by different physical separation methodologies. Geoderma, 445, 116870
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: Leuthold, S. J., Lavallee, J. M., Basso, B., Brinton, W. F., Cotrufo M. F. (2024). Shifts in controls and abundance of particulate and mineral-associated organic matter fractions among subfield yield stability zones. Agriculture, Ecosystems and Environment, 10 (1), 307-319


Progress 05/01/22 to 04/30/23

Outputs
Target Audience:Our efforts primarely targeted the academic scientific community across multiple fields includingagricultural science, soil science, soilecology, soil biogeochemistry, and other ecological fields, through presentations at conferences, seminars and publications. We also targeted school students at multiple levels, specifically conducted work inhigh schools. We also targeted audiences in both the non-profit (The Nature Conservancy, Soil Health Institute, Woodwell Climate Research Center, and Environmental Defense Fund) and private industry (Target, Cargill, McDonald's, Indigo Ag) sectors. In general, we aim to describe soil organic matter fractionation to a broad audience and provide the information necessary to compare and make decisions about appropriate methodologies, because this information can be extremely difficult to find and decipher for non-experts. We targeted these audiences because general interest in soil carbon cycling and soil organic matter fractionation are surgingacross many soil-related disciplines and sectors, but detailed communication in these circles about what soil fractions are,why they are important, and the appropriate methods to separate them is often lacking. We aimed to provide importantbackground information and results of methods comparisons, translated for non-experts who are interested in the possibility. Changes/Problems:We are proud to report that we have made significant advancements toward the accomplishment of each of our three main objectives, as reported here. While maintaining productivity, we were able to save a significant amount of our initial budget, mostly because Dr. Lavallee moved to the Environmental Defense Fund where her salary is fully covered, and because Dr. Cotrufo instrumented her Soil Innovation laboratory and did not have to rely entirely on the EcoCore service facility for sample processing and analyses, saving in lab use fees and analytical costs. We will thus ask for a one-year no cost extension to complete and further expand the scope of our three objectives. What opportunities for training and professional development has the project provided?PhD student on the project Sam Leuthold has been trained across a variety of methods, including physical SOM fractionation methods, laboratory analyses of soil pH, Fe and Al concentrations, texture analysis, stable isotope analyses and other soil processing and characterization methodologies. He has been trained in the preparation of samples for both EA-IRMS and MID-IR spectral analysis and is capable of running both of those instruments independently. In addition, he has received training in advanced statistical methodologies such as structural equation modeling and multiple linear regression from co-PD Lavallee. During his time at CSU, he has taken 12 graduate level courses through the CSU Graduate Degree Program in Ecology, including a course on responsible research conduct. One course, "Environmental Data Science Applications: Food and Agriculture" culminated in a publication co-led by Leuthold, published in Environmental Research Letters in November 2022. He has also attended workshops related to his work and professional development, including a workshop titled, "Challenges and Opportunities related to Soil Spectroscopy," led by the Soil Spectroscopy for Global Good group at the 2023 Soil Science Society Annual Meeting. This workshop aims to produce a peer-reviewed article around the future of soil spectroscopy to be submitted to SOIL in February 2024. In addition to the methods and classwork, Leuthold has had three undergraduate assistants under his direct supervision during his time at CSU. Two of these students have gone on to continue working in agricultural sciences. The student that remains under his supervision works closely with Leuthold and is developing her independent research results that she can present at the AAAS conference in February 2024 with Leuthold's consultation. Leuthold also assisted in the teaching of the undergraduate capstone class at CSU, "Soils and Global Change," creating and administering laboratory activities and assignments in collaboration with fellow PhD candidates in the lab. Outside of CSU, Leuthold and PD Cotrufo have given several lectures to high-school students around soil organic matter, soil respiration, and the role of soils in global biogeochemical cycles. Leuthold has also been involved with the "Skype a Scientist" organization, which has led to him giving talks to elementary, middle, and high-school students across the United States and Canada about his research and career path. How have the results been disseminated to communities of interest?The results produced so far have been disseminated primarily to the scientific community, with plans for further dissemination upon the acceptance and dissemination of the peer-reviewed results. In particular, we have submitted peer-reviewed manuscripts related to the work in Objectives 4, 2, and 5 to Geoderma, SOIL, and Soil Biology and Biochemistry, respectively. We have also presented this work at a number of international conferences including the 2022 SSSA Annual Conference (Objective 2), the Ecological Society of America 2023 Annual Conference (Objective 4 and Objective 2), the 2023 SSSA Annual Conference (Objective 2 and Objective 5), and the 2023 American Geophysical Union Annual Conference (Objective 4). We have communicated with industry partners around methodology as well, including a presentation by Leuthold at an event hosted by the Soil Carbon Solution Center at CSU that aimed to identify areas of collaboration and knowledge transfer between multiple industry partners and academics. What do you plan to do during the next reporting period to accomplish the goals?Over the course of the next reporting year, we will perform the incubation experiment, but will require an extension to complete the data analyses and disseminate the results in a peer-reviewed publication, satisfying Objective 1. We will also make progress with the lab work and analysis of the data relating fraction characteristics to agronomic outcomes at the regional-scale, bolstering our results under Objectives 2 and 3. Building on these analyses, we will also continue to develop a mid-IR spectral library that allows for the prediction of POM separated by density fractionation (in addition to our current capabilities to predict POM separated by size fractionation) which will be valuable to the community at large.

Impacts
What was accomplished under these goals? The above five objectives were consolidated within the following three, accordingto the project narrative. Objective 1: Compare effects of POM and MAOM quantities and ratios on plant yield, plant N content, and common soil health indicators in agricultural soils.We have redesigned the experiment to address our hypothesis in a more targeted way and more effectively account for potential confounding factors. We are in the process of setting up an incubation that looks directly at the relationships between mineral-associated organic matter (MAOM) nitrogen contribution to cropping systems as influenced by both the amount of particulate organic matter (POM) and the quality of POM. This experiment will leverage isotopic tracing tools to understand the conditions under which MAOM nitrogen is made available for plant uptake, while allowing for rigorous quantification and inference around causality. We have collected the soils from which we will collect the POM for this experiment (a grassland in Central KS, and an alfalfa field in NE) and have begun to fractionate these soils to obtain the POM necessary for the incubation, which is a significant undertaking. We are in contact with collaborators who manage long-term agricultural 15N addition studies to obtain the soil from which we will derive our MAOM. Once both soils have been fractionated, they will be recombined in known ratios of POM and MAOM and incubated for 8 weeks. During that period, we will assess the nitrogen mineralized from the soil organic matter and use a two-pool isotopic mixing model to determine the proportion derived from MAOM. After the incubation, we will also examine the microbial biomass N retention, and the overall change in N concentrations in the POM and MAOM fractions. We expect this experimentto result in a peer-reviewed publication by the spring of 2025. Objective 2: Evaluate the utility of POM and MAOM as indicators of soil health and agronomic performance. We have made significant progress under Objective 2. In collaboration with researchers at Michigan State University, we obtained soil samples from various sub-field yield stability zones at 10 farms across the upper Midwestern United States. We fractionated these soils using a combined-size density fractionation and analyzed the resulting fractions for carbon and nitrogen concentrations. We observed a significant difference in the amount of POM carbon among different sub-field yield stability zones, with unstable yielding areas having a higher POM carbon concentration than stable yielding zones, regardless of average crop productivity. We further found that POM carbon was well-correlated with landscape position and the topographic position index, suggesting that POM may be able to act as an indicator of areas of the field that undergo periodic flooding conditions have a direct impact on cropping system productivity. Our results on this experiment present a novel finding that adds important nuance to current discussions around crop yield stability and soil organic matter concentrations. These findings were presented at the annual Soil Science Society of America conference in 2022, and developed into a manuscript submitted to SOIL in September 2023 which is currently undergoing peer review. This experiment represents an addition to our originally proposed approach, which we saw as a necessary bridging of scales between the laboratory experiment described above, and the large-scale regional analysis we originally proposed. By comparing our findings at the sub-field scale to the findings of the regional-scale study, we can better understand whether the observed functional roles of POM and MAOM depend on the scale of inquiry, which in turn gives additional insight into the mechanisms at play. Working toward the regional-scale analysis, we have obtained > 300 additional individual soil samples with associated agronomic data such as yield, nutrient inputs, and management activities from both industry partners and academic collaborators. Over 200 of these samples have already been fractionated using a combined size-density fractionation following the recommendations based on our work associated with Objective 3 (see below), and all samples have been analyzed for spectral absorbance via diffuse reflectance infrared Fourier transformed (DRIFT) mid-infrared spectroscopy (See Objective 3). We are in the process of fractionating the remaining samples and intend to finish fractionation by the year-end. Preliminary results associated with Objective 2 were presented at the annual Soil Science Society of America conference in November 2023. Objective 3: Compare protocols for effective and efficient POM and MAOM quantification across soil types. We have created a set of resources to help guide SOM fractionation procedure decisions. We published a book chapter entitled "Physical Fractionation Methods," in the Encyclopedia of Soils in the Environment in early 2022. This chapter provides an overview of the current state of the science regarding physical fractionation methods and acts as a resource for scientists considering various methods of separating organic matter fractions and has been cited six times since its publication. We also performed an experiment to understand the effect of soil physicochemical properties and fractionation methodology on the characteristics of the resulting fractions based on what was originally proposed. To this end, we obtained 12 soils from research farms across the continental United States. These soils were chosen to represent a range of soil physicochemical properties, including texture, pH, and iron and aluminum oxide content. We fractionated these soils using 4 different physical fractionation methods and analyzed the resulting fractions for carbon and nitrogen concentration and isotopic composition, as well as mid-infrared spectral absorbance. Our major findings indicate that chemical and spectral composition of the POM fraction are dependent on fractionation scheme, with fractionation methodologies that employ a density separation step isolating a different, and more unique, POM fraction than those which employ only a size separation. In addition, we found that each of the methods performed consistently across soil physicochemical characteristics, such that pH, mineralogy, and texture did not have a significant impact on fraction characteristics. These data were summarized and presented in brief at the Ecological Society of American annual conference in August 2023, and will be presented in full at the American Geophysical Union annual conference in December 2023. Additionally, a peer-reviewed manuscript fully detailing the results was submitted in May 2023 and currently remains under review at Geoderma. Since the installation of the spectrometer in PD Cotrufo's lab, PhD student Leuthold has been able to become acquainted with the methodology and has scanned nearly all soils that he has received throughout the course of his dissertation work. In addition to the SOM fraction characterization work related to Objective 4, he has scanned isolated MAOM samples from two time-points in a long-term bare fallow experiment in an attempt to identify spectral markers of unstable portions of the MAOM fraction. This work, in collaboration with researchers at the Swedish University of Agricultural Sciences, will be submitted to Soil Biology and Biochemistry as a peer-reviewed short communication in December 2023. Additionally, using the spectral library created through the scanning of samples that had been previously fractionated for Objective 2, preliminary work predicting POM and MAOM C content has been undertaken. Preliminary results were shared as a poster presentation at the annual Soil Science Society of America conference in November 2023 and further development of predictive models, including machine learning approaches, are underway.

Publications

  • Type: Journal Articles Status: Published Year Published: 2022 Citation: Driscoll, A. W., Leuthold, S. J., Choi, E., Clark, S. M., Cleveland, D. M., Dixon, M., Hsieh, M., Sitterson, J., and Mueller, N. D. 2022. Divergent impacts of crop diversity on caloric and economic yield stability, Environ. Res. Lett., 17, 124015. doi: 10.1088/1748-9326/aca2be.
  • Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Leuthold, S.J., Lavallee, J.M., Haddix, M.H., Cotrufo, M.F. Contrasting properties of soil organic matter fractions isolated by different physical separation methodologies. Geoderma.
  • Type: Journal Articles Status: Under Review Year Published: 2023 Citation: Leuthold, S.J., Lavallee, J.M., Basso, B., Brinton, W.F., Cotrufo, M.F. In-review. Shifts in controls and abundance of particulate and mineral-associated organic matter fractions among subfield yield stability zones. SOIL.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Leuthold, S.J., Lavallee, J.M., Basso, B., Brinton, W.F., Cotrufo, M.F. In search of a mechanistic link between soil organic matter and crop yield variability. Oral presentation at the Soil Science Society of America Annual Meeting.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Leuthold, S.J. Soil organic matter: a means for decreasing yield variability? Poster Presentation at the Colorado State University, Graduate Student Showcase.
  • Type: Journal Articles Status: Submitted Year Published: 2023 Citation: Leuthold, S.J., K�tterer, T., Bolinder, M., Cotrufo, M.F. Submitted. Chemical and spectral characterization of a dynamic mineral associated organic matter fraction. Soil Biology and Biochemistry.
  • Type: Conference Papers and Presentations Status: Accepted Year Published: 2023 Citation: Cotrufo, M.F., Leuthold, S.J.* Insight into molecular, spatial, and temporal dynamics gained via organic matter fractionation. Oral presentation at the Ecological Society of America Annual Meeting. August 2023. * S. Leuthold presenting author.
  • Type: Other Status: Accepted Year Published: 2022 Citation: Leuthold S.J., Even, R.E., Cotrufo, M.F. Efficient. Rigorous. Effective. Field and laboratory methods for quantifying soil organic carbon across scales. Oral presentation at CSU Soil Carbon Solution Center Investor Information Session. December 2022.


Progress 05/01/21 to 04/30/22

Outputs
Target Audience:During this reporting period, our efforts targeted the academic scientific community across multiple fields including agricultural science, soil science, soil ecology, soil biogeochemistry, and other ecological fields. We also targeted school students at multiple levels, from elementary to high school. We also targeted audiences in both the non-profit (The Nature Conservancy, Soil Health Institute, Woodwell Climate Research Center, and Environmental Defense Fund) and private industry (Target, Cargill, McDonald's, Indigo Ag) sectors. During this reporting period, we aimed to describe soil organic matter fractionation to a broad audience and provide the information necessary to compare and make decisions about appropriate methodologies, because this information can be extremely difficult to find and decipher for non-experts. We targeted these audiences because general interest in soil carbon cycling and soil organic matter fractionation are surging across many soil-related disciplines and sectors, but detailed communication in these circles about what soil fractions are, why they are important, and the appropriate methods to separate them is often lacking. We aimed to provide important background information and results of methods comparisons, translated for non-experts who are interested in the possibility of working with soil carbon or soil organic matter. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?PhD student Leuthold is being trained in multiple disciplines and methods as part of this project. Leuthold has been trained in soil fractionation methods including density fractionation, size fractionation, and combined methods. Leuthold has also been trained in the following soil-related laboratory procedures: particle size flotation, the micro-pipette method for texture analysis, ammonium oxalate iron and aluminum extractions, citrate dithionite iron and aluminum extractions, sample preparation for EA-IRMS analysis, and acid fumigation methods for soil carbonate removal. PhD student Leuthold is also taking part in professional development activities. Leuthold has taken 9 graduate-level courses in the Graduate Degree Program in Ecology at CSU. He has also participated in workshops moderated by the program (e.g., Inkscape and R Data Visualization workshop). He has conducted a hiring search and interviewed several candidates for an undergraduate research position and managed the student worker who was eventually hired for that position in collaboration with another PhD student in the lab. In addition to the work performed for his own project, he has also been able to participate in and learn from research activities for other, related experiments. This has included traveling to the Konza Prairie LTER and learning new soil sampling and field processing techniques, and the deconstruction and processing of soil microcosms. He applied and received an internal graduate student grant to support research complementary to the goals of this funding. In addition to the outreach discussed below, he has also had the opportunity to be active in cultivating younger scientists, speaking with elementary school students about his path to science, as well as judging the environmental science division of the Colorado Science fair. Leuthold has also been able to present his research to his peers within the Soil and Crop Sciences department, receiving valuable feedback and spurring collaboration. How have the results been disseminated to communities of interest?We (led by PhD student Leuthold) published a Chapter on soil organic matter fractionation methods in the Encyclopedia of Soils in the Environment, Second Edition, which features accessible content designed for a broad readership. We (led by PhD student Leuthold) also presented results of our methods comparison work at the Front Range Graduate Student Ecology Symposium, attended by hundreds of graduate students and academic faculty from the Mountain West spanning many ecological and related disciplines. Further, PhD student Leuthold and PD Cotrufo led outreach activities at local schools (high school and elementary) introducing students to key concepts in soil ecology and carbon cycling, as well as careers in soil-related disciplines. Co-PD Lavallee also presented the ideas behind the project to a broad range of stakeholders including academics, non-profits, and private industry through multiple webinars. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: We will finalize the experimental design of the greenhouse experiment, collect the soils to be used, and begin the greenhouse experiment in summer or fall of 2022. Upon completion of the greenhouse experiment, we will complete data analysis and being to prepare a manuscript for publication in a peer-reviewed journal, with intention to publish it by Fall 2023. Objectives 2 and 3: We will continue to receive soil samples to support these objectives and continue fractionation procedures as soils are received. A pilot study in collaboration with Drs. Basso and Brinton will be completed and a manuscript prepared with intention to publish by Winter 2022. The results of this study will also be disseminated at the American Geophysical Union annual conference. Objective 4: We plan publish a peer-reviewed manuscript presenting the results of the methods comparison, with submission of the manuscript slated for Summer 2022. We will also disseminate the results through webinars, a white paper, and a fact sheet designed for multiple audiences. Objective 5: We will complete set-up of the new spectrometer and training of PhD student Leuthold in the basics of DRIFT spectroscopy and related statistical methodology. We will scan all soil fraction samples for this project currently in our possession and continue to scan new samples as they arrive and are fractionated.

Impacts
What was accomplished under these goals? Objective 1: Compare effects of POM and MAOM quantities and ratios on plant yield, plant N content, and common soil health indicators in agricultural soils We initiated contact with land managers to obtain soils for the greenhouse experiment, and tested soils to ensure their appropriateness. We further refined the proposed methodology for this experiment, including experimental design and addition of a nitrogen labelling component (which will not add to our proposed budget). Objective 2: Evaluate the utility of POM and MAOM as indicators of soil health and agronomic performance We have obtained over 50 soil samples to support this objective, and have secured confirmation from multiple partners (Dr. Jordon Wade, Dr. Bruno Basso, Indigo Ag, Soil Health Institute, The Nature Conservancy) that they will provide hundreds more. We have met with these partners and discussed their available soil samples in detail to ensure they will meet our needs and provide appropriate means to test our hypotheses in our proposed statistical framework. We have completed fractionation procedures on the soils we have already procured. Objective 3: Quantify SOM regeneration and C sequestration potential across US agricultural soils in response to different management practices In addition to the efforts under Objective 2, which directly support Objective 3 since they rely on similar datasets, we have investigated possibilities for obtaining samples from time-series that include management changes. We have identified over 20 sites where this may be possible, and are following up to better understand details of the soil archives, management changes, and overall appropriateness for our framework. As part of this effort, we have built partnerships with researchers from the Long-Term Agroecosystem Research (LTAR) Network and Agricultural Research Service (USDA-ARS) to obtain archived soil samples or data for soil fractions already measured on such soils. Objective 4: Compare protocols for effective and efficient POM and MAOM quantification across soil types This has been the primary focus of our efforts during this reporting period. We obtained 15soil samples from diverse systems across the US and performed three different common fractionation procedures on all of them. We measured key properties of the resulting soil fractions, including C and N contents, δ13C and δ15N, texture, pH, and iron and aluminum oxide content . We then investigated the differences in these properties across the fractions from the different procedures and identified the key areas where these methods produce different results. Our results indicate that the physical fractionation method used to separate mineral-associated organic matter (MAOM) and particulate organic matter (POM) can have significant impacts on the chemical composition of a given fraction. POM separated via density floatation is distinct compared to POM separated by size, which resembles the heavy, coarse organic matter (hcOM) fraction in terms of chemical and isotopic composition.Based on this analysis, we recommend researchers use a density method whenever possible, however we are currently performing additional tests to confirm this result. PhD Student Leuthold presented these results at the Front Range Student Ecology Symposium in February 2022, and is currently writing a manuscript for publication in a peer-reviewed journal. Objective 5: Quantify POM and MAOM across diverse soils using DRIFT spectroscopy The lab of PD Cotrufo is in the process of obtaining a new Spectrometer which will be used to measure midIR spectra of all samples from this project. PhD Student Leuthold will be involved in the initial set up and calibration.

Publications

  • Type: Book Chapters Status: Awaiting Publication Year Published: 2022 Citation: Leuthold, S.J., Haddix, M. L., Lavallee, J. M., Cotrufo, M.F., 2022. Physical fractionation techniques, in: Staunton, S. (Ed.), Encyclopedia of Soils in the Environment, Second Edition. Elsevier.
  • Type: Conference Papers and Presentations Status: Other Year Published: 2022 Citation: Leuthold, S., Lavallee, J., Haddix, M., King, A., Machmuller, M., Cotrufo M.F. (2022). Exploring shifts in the character and distribution of soil organic matter fractions across a physiochemical soil gradient. Poster presentation at the Front Range Student in Ecology Seminar. February 23. Fort Collins, CO.