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Abstract: Non-symbiotic nitrogen fixation (NSNF) occurs in many environments, but the magnitude of its inputs, along with controls on NSNF rates, are unknown. We measured NSNF by applying a 15N2 tracer to field mesocosms that contained an intact soil profile (75 cm deep) with mature switchgrass (Panicum virgatum) at a site in southwest Michigan, USA. We measured NSNF both early and mid-growing season and scaled those measurements to an annual rate. We expressed NSNF rates per gram of individual plant tissues and soil, as well as per meter squared. We also calculated NSNF via mass balance. During the incubations, we measured weather conditions within the mesocosms (temperature, humidity, dew point, light). We also collected data on soil total carbon and nitrogen and soil inorganic nitrogen. This dataset includes all the raw and derived data necessary to complete the calculations described in Roley et al. (in review). TechnicalInfo: # Data from: In situ evidence for quantitative nitrogen fixation in a perennial grass ecosystem Dataset DOI: [10.5061/dryad.b5mkkwhsr](https://doi.org/10.5061/dryad.b5mkkwhsr) ## Description of the data and file structure This dataset includes all data necessary used in Roley et al. (in review). In short, we deployed mesocosms to measure non-symbiotic nitrogen fixation in a switchgrass plants and soils. We added ^15^N~2~ gas and then traced it into plant tissues and soil. We used the isotope data, along with data on soil and plant mass and nitrogen content, to calculate nitrogen fixation. We also included meteorological data and soil carbon and nitrogen data, which we used as explanatory variables. We have also included code where the calculations were not easily described. ## Files and variables ## Environmental Variables #### File: light.csv **Description:** These data are from HOBO Pendant light and temperature sensors (HOBO, Bourne, Massachusetts, USA), which were deployed within each individual mesocosm and in the surrounding switchgrass field. ##### Variables * Date_Time: Date and time of sample collection in GMT-5 * Intensity_Lux: Light intensity measured in lux or lumens per square meter * Mesocosm: Location of sensor, corresponding to the mesocosm replicate number or "Outside" for the sensor in the ambient air * Incub_Num: Incubation date where "Early" is early June 2022 and "Late" is late June 2022 * Lid: Indicates whether the lid was open or closed * date_time: Date and time of sample collection in GMT-5 #### File: CN_soil.csv **Description:** We sampled soils before and after the incubation, from four depths: 0-10 cm (0_10), 10-25 cm (10_25), 25-50 cm (25_50), and 50-75 cm (50_75). After the incubation was complete, we also sampled rhizosphere soil, which we defined as soil clinging to the roots after a good shake. The rhizosphere soil is labeled as a depth  = “rhiz.”  Percent carbon, percent nitrogen, and the carbon: nitrogen ratio were determined via combustion on a Vario ISOTOPE cube Elemental Analyzer (Elementar, Langenselbold, Germany). ##### Variables * Mesocosm: Unique identity of mesocosm with each replicate having its own number * Treatment: Indicates whether the mesocosm received 15N (15N2) or not (CTL) * Time: Indicates whether the sample was collected before the incubation (Initial) or after the incubation (Final) * Depth: Depth of sample in cm * CN ratio: Carbon to nitrogen ratio of dry soil * pct_N: The percent of soil mass as nitrogen * pct_C: The percent of soil mass as carbon #### File: T_VPD_RH_DP.csv **Description:** We deployed HOBO Temperature/RH data loggers (HOBO, Bourne, Massachusetts, USA) sensors in each mesocosm and in the surrounding switchgrass field. The sensors measured temperature, relative humidity, and dewpoint. We calculated vapor pressure deficit from temperature and humidity. ##### Variables: * date_time5: Date and time of sample collection in GMT-5 rounded to nearest five minutes * Mesocosm: Location of sensor, corresponding to the mesocosm replicate number or "Outside" for the sensor in the ambient air * Incub_Num: Incubation date where "Early" is June 2022 and "Late" is July 2022 * temp_c: Temperature in degrees Celcius * Date_Time_EST_GMT_5: Date and time of sample collection in GMT-5 * Temp_F: Temperature in degrees Fahrenheit * RH_pct: Relative humidity in percent * DewPt_F: Dew point in degrees Fahrenheit * Lid: Indicates whether the lid was open or closed * dewpt_c: Dew point in degrees Celcius * VPD: Vapor pressure deficit, calculated as the saturation vapor pressure minus the actual vapor pressure * time: time in GMT-5 * seconds: time in seconds * day: Indicates whether the sample was collected after sunrise and before sunset (day) or not (night) * photo: Indicates the suitability of the tor photosynthesis according to Wagle and Kakani (2014). * date: date as mm/dd/yyyy * Jday: Julian date #### File: DIN.csv **Description:** This file contains data on soil inorganic nitrogen from within the mesocosms. The data were collected via extraction with 1 M KCl (Robertson et al. 1999). The depth-resolved data were collected with a 2-cm diameter push corer and the rhizosphere samples were collected after the incubation by removing soil from the roots after shaking. ##### Variables: * Mesocosm: Unique identity of mesocosm with each replicate having its own number * Depth: Depth into soil in centimeters or "rhiz" = rhizosphere soil * NH3_mgNL:Concentration of nitrogen as ammonium in mg per L * NO3_mgNL: Concentration of nitrogen as nitrate in mg per L * NH3_ugN_gsoil: Concentration of nitrogen as ammonium in micrograms of N per g of soil * NO3_ugN_gsoil: Concentration of nitrogen as nitrate in micrograms of N per g of soil * DIN_ugN_gsoil: Concentration of dissolved inorganic nitrogen in micrograms of N per g of soil ## Gas Data #### File: gas_raw.csv **Description:** This file includes the gas raw isotope and sampling data. The gas samples were obtained by removing a gas sample from individual soil layers with a needle and syringe, injecting the sample into a vacuumed Exetainer. Samples with expected high ^15^N enrichment were diluted with ^14^N~2~. This file also includes relevant soil data for calculating the dilution factor. #### Variables: * Mesocosm: Unique identity of mesocosm with each replicate having its own number * Layer: Depth into soil in centimeters or headspace (HS) * Port: Location of sample, where the number indicates depth into soil and the letter indicates sub-replicate * Treatment: 15N2 =received 15N2 and CTL = control, which received no 15N2 * Period: Categorical sampling time period * V_Sampled_ml: Volume sampled in milliliters * V_Diluted_ml: Volume of N2 used to dilute the sample in milliliters * Date_Time: date and time of sample collection in GMT-4 * X15N: Raw delta 15N value of the gas from the IRMS * X30.28.ratio: Raw 30 to 28 N ratio from the IRMS * R_15N: R ^15^N calculated with standard equations (Robinson 2001) * R_30.28: R of the 30: 28 ratio calculated with standard equations * R_sample: R of the whole sample, calculated as the sum of R_15N and R_30.28 * atom_pct: ^15^N atom percent, as measured on the IRMS * X14N2_uL_Exetainer: The volume of ^14^N~2~ gas in the sample from the IRMS * pctN2_postflush: The percent of gas present as N~2~ after flushing, based on IRMS data * gas_vol_L: Volume of gas in the soil layer in liters, based on the soil bulk density and volume of objects in headspace * prop_layer: The proportion of the total gas volume represented in that layer. This variable is used to estimate the volume of N2 in this layer by assuming it dispersed * N2vol_postflush: The volume of N~2~ present in the soil layer or headspace after flushing * volN2_added: The volume of ^15^N~2~ gas added * prop_N2: The proportion of gas volume present as N~2~ * dilution_factor: The ratio of total N~2~ in vial to the amount of N~2~ sampled * sample_atpct: ^15^N atom percent in the sample with sample dilution accounted for by multiplying the measured ^15^N atom percent by the dilution factor #### File: gas_AE_all.csv **Description:** This file contains the ^15^N atom excess for each individual gas sample, including data needed for those calculations. This file is also input for calculating the ^15^N atom excess at the midpoint of the incubation (AE_atm_layer). Sub-replicates (from gas_raw) have been averaged and the control samples placed in their own column to facilitate calculation of ^15^N atom excess. #### Variables: * Period: Categorical sampling time period * Layer: Depth into soil in centimeters or headspace (HS) * Incub_Num: Incubation date where "Early" is early June 2022 and "Late" is late June 2022 * Mesocosm: Unique identity of mesocosm with each replicate having its own number * ENR_atpct: ^15^N atom percent from enriched mesocosm, where we assume the gas added is causes an increase in pressure; in other words, the added gas does not displace the gas already present * ENR_atpct_dis: ^15^N atom percent from enriched mesocosm, where we assume the gas added displaces existing gas. We used this column, because the numbers were more reasonable than when we assumed no displacement. * Time_Elapsed_h: Time elapsed in hours since incubation begun * Inc_Total_d: Total length of incubation in days * CTL_atpct: The ^15^N atom percent of the gas in the corresponding control mesocosm (no ^15^N added). In cases where there wasn't a matching CTL sample, we used the sample from the closest time period from the corresponding layer in the CTL mesocosm. * AE: ^15^N atom excess calculated as the difference in ^15^N atom percent between the enriched and the control mesocosms (ENR_atpct minus CTL_atpct) * AE_dis: ^15^N atom excess calculated as the difference in ^15^N atom percent between the enriched and the control mesocosms (ENR_atpct_dis minus CTL_atpct). We used this value in our subsequent calculations. #### File: AE_atm_layer.csv **Description:** This file contains the ^15^N atom excess (AE_atm) of the gas at each sampling location for each mesocosm. At each sampling location (headspace, 10 cm, 20 cm, 40 cm, and 60 cm) we collected multiple samples over the course of the incubation. The data here represent the 15N atom excess of the gas at the midpoint of the incubation (see Methods in Roley et al. (in review) for details). The belowground (BG) value is the weighted average of all belowground samples, with the samples weighted by root density. #### Variables: * Mesocosm: Unique identity of mesocosm with each replicate having its own number * Layer: Depth into the soil in centimeters or headspace (HS) or belowground (BG) * AE_atm: ^15^N atom excess of gas at the midpoint of the incubation. This value is used for the nitrogen fixation calculations. #### File: AEatm_layer.R **Description:** This file includes the R code for each calculation of AE_atm. For each layer and mesocosm, we plotted the AE_atm through time. If there was an increase or decrease, we used the midpoint of the incubation. If there was no trend through time, we used the mean. In some cases, we eliminated data points, such as unreasonably high AE_atm values (>100%). This file contains the code and notes on each layer and mesocosm so that the reader can see the calculations and decisions that led to each AE_atm value. ## Soil Data #### File: soil_iso_raw.csv **Description:** This file includes the raw isotope data from soil samples. #### Variables: * Name: sample name * Mesocosm: Unique identity of mesocosm with each replicate having its own number * Treatment: 15N2 = received 15N2 and CTL = control, which received no 15N2 * Time: Indicates whether the sample was collected before the incubation (Initial) or after the incubation (Final) * Depth: Depth into soil, in centimeters, or rhizosphere soil ("rhiz") which is not depth-resolved * Subrep: Subreplicate number where subreplicates were collected * mass: mass of sample packed for isotopic analysis * N_pct: Percent N of the sample * d_15N: Delta ^15^N value of the sample * atpct_15N: ^15^N atom percent of the sample * notes: Notes on samples where relevant * Incub_Num: Incubation date where "Early" is June 2022 and "Late" is July 2022 #### File: soil_fix_layers.csv **Description:** This file contains the data used to calculate soil fixed N accumulation in each layer (i.e., by depth). The isotope data come from soil cores taken prior to and immediately after each incubation at four depths: 0-10 cm, 10-25 cm, 25-50 cm, and 50-75 cm. The soil dry mass and moisture data come from samples taken after the incubation was complete. This file also includes the total mass of soils from each layer, collected via destructive sampling after the incubation. Fixed N accumulation in grams of N per g of soil per day (Nfix_gN_g_d) was calculated as: Nfix_gN_g_d = ((AE*TN_g)/ (AE_atm*total_DM*incub_tim_d). Nfix_gN_mes_d = ((AE*TN_g)/(AE_atm*incub_time)) #### Variables: * Mesocosm: Unique identity of mesocosm with each replicate having its own number * Depth: Depth of sample in centimeters * Treatment: Indicates whether the mesocosm received 15N (15N2) or not (CTL) * Incub_Num: Incubation date where "Early" is early June 2022 and "Late" is late June 2022 * N_pct.Initial: Percent N of the samples taken prior to the incubation * d_15N.Initial: Delta ^15^N value of the samples taken prior to the incubation * atpct_15N.Initial: ^15^N atom percent of the samples taken prior to the incubation * N_pct.Final: Percent N of the samples taken after the incubation * d_15N.Final: Delta ^15^N value of the samples after the incubation * atpct_15N.Final: ^15^N atom percent of the samples taken prior to the incubation * d_15N.Diff: The change in delta ^15^N after the incubation, calculated as d_15N.Final-d_15N.Initial * atpct_15N.Diff: The change in ^15^N atom percet after the incubation calculated as atpct_15N.Final - atpct_15N.Initial * DL_15N: Detection limit expressed as delta ^15^N and calculated as the absolute value of the change in delta ^15^N during the incubation in the control mesocosm * DL_atpct: Detection limit expressed as ^15^N atom percent and calculated as the absolute value of the change in ^15^N atom percent during the incubation in the control mesocosm * AEsoil: ^15^N atom excess in the soil calculated as the post-incubation ^15^N atom percent minus the pre-incubation atom percent * decon_DM_g: Dry mass of bulk soil in the mesocosm, collected after the incubation, during deconstruction * core_DM: Dry mass of cores removed after the incubation * total_DM_g: Total soil dry mass from deconstruction and from cores (decon_DM_g + core_DM), but excluding rhizosphere soil. * incub_time_d: Incubation time in days * incub_time_h: Incubation time in hours * TN_g: Total nitrogen in grams in bulk soil * Layer: Depth of gas samples associated with this layer * AE_atm: ^15^N atom excess in the gas (from AE_atm_layer) * Nfix_gN_g_d: Soil fixed nitrogen accumulation rate in grams of nitrogen per gram of dry soil per day * Nfix_ugN_g_d: Soil fixed nitrogen accumulation rate in micrograms of nitrogen per gram of dry soil per day * Nfix_gN_mes_d: Soil fixed nitrogen accumulation rate in grams of nitrogen per mesocosm per day * Nfix_ugN_mes_d: Soil fixed nitrogen accumulation rate in micrograms of nitrogen per mesocosm per day #### File: rhiz_fix.csv **Description:** This dataset contains fixed nitrogen accumulation rate data and input data values for calculating fixed nitrogen accumulation rates in rhizosphere soils. 15N atom excess was calculated as the difference in 15N atom percent in the rhizosphere soils that received 15N minus the 15N atom percent in the control mesocosm. Fixed N accumulation in grams of N per g of soil per day(Nfix_gN_g_d) was calculated as: Nfix_gN_g_d = ((rhiz_AE*TN_g)/ (AE_atm*total_DM*incub_time_d). Nfix_gN_mes_d = ((rhiz_AE*TN_g)/ (AE_atm*incub_time)) #### Variables: * Mesocosm: Unique identity of mesocosm with each replicate having its own number * AE_atm: ^15^N atom excess of the gas in the rhizosphere calculated as the average of the atom excess of the top two layers of soil (sampling depths = 10 cm and 20 cm) * rhiz_AE: ^15^N atom excess of the rhizosphere soil * total_DM_g: Total dry mass of rhizosphere soil * TN_g: Total nitrogen of the rhizosphere soil in grams, calculated as average percent nitrogen in the rhizosphere soil multiplied by the total dry mass * incub_time_d: Incubation time in days * Nfix_gN_g_d: Rhizosphere fixed nitrogen accumulation rate in grams of nitrogen per gram of dry soil per day * Nfix_ugN_g: Rhizosphere fixed nitrogen accumulation rate in micrograms of nitrogen per gram of dry soil per day * Nfix_gN_mes_d: Rhizosphere fixed nitrogen accumulation rate in grams of nitrogen per mesocosm per day * Nfix_ugN_mes_d: Rhizosphere fixed nitrogen accumulation rate in micrograms of nitrogen per mesocosm per day ## Plant Data #### File: plant_iso_raw.csv **Description:** This file contains the raw isotope data from the IRMS for switchgrass leaves, stems, flowers, and roots. #### Variables: * Name: sample name * Mesocosm: Unique identity of mesocosm with each replicate having its own number * Treatment: 15N2 = received ^15^N~2~ and CTL = control, which received no ^15^N~2~ * Time: Indicates whether the sample was collected before the incubation (Initial) or after the incubation (Final) * Tissue: Plant tissue type * Depth: Depth into soil (roots) or "AG" for aboveground and "bulk" for bulk roots that were not depth-resolved * mass: mass of sample packed for isotopic analysis * N_pct: Percent N of the sample * d_15N: Delta ^15^N value of the sample * atpct_15N: ^15^N atom percent of the sample * notes: Notes on samples where relevant #### File: AE_plants.csv **Description:** This file contains the data needed to calculate the ^15^N atom excess of each sample, which is the difference between the pre-incubation 15N atom percent and the post-incubation atom percent. #### Variables: * Tissue: Plant tissue type * Depth: Depth into soil (roots) or "AG" for aboveground and "bulk" for bulk roots that were not depth-resolved * Incub_Num: Incubation date where "Early" is early June 2022 and "Late" is late June 2022 * Mesocosm: Unique identity of mesocosm with each replicate having its own number * Treatment: 15N2 = received ^15^N~2~ and CTL = control, which received no ^15^N~2~ * d_15N.Initial: Delta ^15^N value of the pre-incubation sample * atpct_15N.Initial: ^15^N atom percent of the pre-incubation sample * N_pct.Final: Percent of the sample, by mass, present as nitrogen * d_15N.Final: Delta ^15^N value of the post-incubation sample * atpct_15N.Final: ^15^N atom percent of the post-incubation sample * DL_15N: The detection limit, in delta 15N, calculated as the absolute value of the difference between the pre- and post-incubation delta 15N value in the control mesocosm. See DL_plants.csv. * DL_atpct: The detection limit, in ^15^N atom percent, calculated as the absolute value of the difference between the pre- and post-incubation ^15^N atom percent value in the control mesocosm. See DL_plants.csv * CTL_d15N.Final: Delta ^15^N value of the post-incubation control sample * CTL_atpct.Final: ^15^N atom percent of the post-incubation control sample * d_15N.Initial1: Delta ^15^N value of the pre-incubation sample or, if there was no pre-incubation sample, the value from the control * atpct_15N.Initial1: ^15^N atom percent of the pre-incubation sample, or the control value, if there was no pre-incubation sample * d_15N.Diff: The difference in delta ^15^N, calculated as d_15N.Final - d_15N.Initial1 * atpct_15N.Diff: The difference in ^15^N atom percent, calculated as atpct_15N.Final - atpct_15N.Initial1 * AE: The atom excess of the plant tissue, which equal to atpct_15N.Diff, unless atpct_15N.Diff is below the detection limit, in which case it is 0 #### File: plant_fix.csv **Description:** This file contains the data used to calculate fixed N accumulation rates in plant tissues. The isotope data come from plant samples taken prior to and immediately after each incubation. The root samples were depth-resolved, taken at four depths (0-10 cm, 10-25 cm, 25-50 cm, and 50-75 cm) via a 2 cm push corer. Bulk roots are not depth-resolved and come from isotope values from the root ball that was removed during mesocosm deconstruction. This file includes data from AE_plants and from AE_atm_layer. #### Variables: * Mesocosm: Unique identity of mesocosm with each replicate having its own number * Tissue: Plant tissue type * Treatment: 15N2 = received ^15^N~2~ and CTL = control, which received no ^15^N~2~ * Depth: Depth into soil in centimeters or aboveground (AG) or root total (total_root) * AE: 15N atom excess calculated as the post-incubation 15N atom percent minus the pre-incubation atom percent (see AE_plants) * dry_mass_g: Dry mass in grams * N_pct.Final: Percent nitrogen by mass after the incubation * root_density: Density of roots based on depth-resolved post-incubation soil cores, in grams per milliliter, calculated as root biomass divided by the volume * total_root_g: Total root biomass, in grams, calculated as root density multiplied by volume in that layer * total_root_plugs_g: Mass of roots retrieved via post-incubation soil cores * total_density: sum of root_density for all layers in a mesocosm, in grams per milliliter * prop_layer: The proportion of the total roots present in that layer * DM_total_g: Total dry mass in grams * TN_g: Total nitrogen in grams * AE_atm.10: ^15^N atom excess of gas at 10 cm depth * AE_atm.20: ^15^N atom excess of gas at 20 cm depth * AE_atm.40: ^15^N atom excess of gas at 40 cm depth * AE_atm.60: ^15^N atom excess of gas at 60 cm depth * AE_atm.HS: ^15^N atom excess of gas in the headspace * AE_atm.BG: ^15^N atom excess of gas belowground, calculated as a weighted average of all layers weighted by root density * incub_time_d: Incubation time in days * incub_time_h: Incubation time in hours * AE_atm: 15N atom excess of gas appropriate for the layer (see AE_atm_layer) * Nfix_gN_g_d: Fixed nitrogen accumulation rate in grams of nitrogen per gram of dry plant material per day * Nfix_ugN_g: Fixed nitrogen accumulation rate in micrograms of nitrogen per gram of dry plant material per day * Nfix_gN_mes_d: Fixed nitrogen accumulation rate in grams of nitrogen per mesocosm per day * Nfix_ugN_mes_d: Fixed nitrogen accumulation rate in micrograms of nitrogen per mesocosm per day #### File: DL_plants.csv **Description:** This file contains the data used the calculate the detection limit on plant samples. The detection limit was calculated on data from the control mesocosms and was equal to the absolute value of the difference between the post-incubation and pre-incubation 15N atom percent. We calculated detection limits for each tissue type, depth, and incubation time. In some cases, there was insufficient mass to obtain isotope data for a particular tissue type and/or depth. In those cases, we made the following substitutions: For roots in the 50-75 cm layer, we used the detection limit for the 25-50 cm layer. For the flowers in the early incubation, we used the detection for the later incubation. Bulk roots were not sampled at all until after the incubation, because the process is destructive. Instead, we used the mean detection limit from the 0-10 and 10-25 layers, because most roots are in those layers. #### Variables: * Mesocosm: Unique identity of mesocosm with each replicate having its own number * Tissue: Plant tissue type * Treatment: 15N2 = received ^15^N~2~ and CTL = control, which received no ^15^N~2~ * Depth: Depth into soil in centimeters or aboveground (AG) or root total (total_root) * Incub_Num: Incubation date where "Early" is early June 2022 and "Late" is late June 2022 * N_pct.Initial: Percent of the pre-incubation sample, by mass, present as nitrogen * d_15N.Initial: Delta ^15^N value of the pre-incubation sample * atpct_15N.Initial: ^15^N atom percent of the pre-incubation sample * N_pct.Final: Percent of the post-incubation sample, by mass, present as nitrogen * d_15N.Final: Delta ^15^N value of the post-incubation sample * atpct_15N.Final:^15^N atom percent of the post-incubation sample * d_15N.Diff: The difference in delta ^15^N, calculated as d_15N.Final - d_15N.Initial * atpct_15N.Diff: The difference in ^15^N atom percent, calculated as atpct_15N.Final - atpct_15N.Initial * DL_15N: The detection limit, in delta ^15^N, calculated as the absolute value of the difference between the pre- and post-incubation delta ^15^N value in the control mesocosm or as in the Description, above. * DL_atpct: The detection limit, in ^15^N atom percent, calculated as the absolute value of the difference between the pre- and post-incubation ^15^N atom percent value in the control mesocosm or as in the Description, above. ## Scaling Data #### File: rain22.csv **Description:** This file contains weather data for 2022 at our field site, the Kellogg Biological Station Long-term Ecological Research site. This file was used to scale mesocosm N fixation rates to an annual input. #### Variables: * date: date as mm/dd/yyyy * precip_mm: total daily preciption in millimeters * mean_temp: daily mean temperature in degrees Celcius * max_temp: daily maximum temperature in degrees Celcius * min_temp: daily minimum temperature in degrees Celcius * year: year of sampling * event: characterizes the date as either an event (at least 10 mm of rain within past 3 days) or baseline * event_size_mm: the size of the rain event in millimeters #### File: areal_Nfix.csv **Description:** This file contains areal N fixation rates, expressed for each compartment (soil, roots, and aboveground plant tissues). It is used to estimate annual inputs using the script titled “annual_scaling.R”. #### Variables: * Mesocosm: Unique identity of mesocosm with each replicate having its own number * Nfix_ugN_mes_d: Fixed nitrogen accumulation rate as micrograms of nitrogen per mesocosm per day, from plant_fix * Nfix_gN_mes_d: Fixed nitrogen accumulation rate as grams of nitrogen per mesocosm per day, from plant_fix * Compartment: Location of fixed nitrogen * Incub_Num: Incubation date where "Early" is early June 2022 and "Late" is late June 2022 * Nfix_gN_m2_d: Fixed nitrogen accumulation rate as grams of nitrogen per square meter per day, calculated by dividing Nfix_gN_mes_d by the area of the mesocosm * Nfix_ugN_m2_d: Fixed nitrogen accumulation rate as micrograms of nitrogen per square meter per day, calculated by dividing Nfix_gN_mes_d by the area of the mesocosm #### File: annual_scaling.R **Description:** This file includes the R code for the annual scaling calculations. #### Citations: Robertson, G. P., Sollins, P., Ellis, B. G., Lajtha, K. 1999. Exchangeable ions, pH, and cation exchange capacity. Pages 106-114 in Standard soil methods for long-term ecological research, edited by Robertson, G. P., Coleman, D. C., Bledsoe, C. S., and Sollins, P. New York: Oxford University Press. Robinson, D. 2001. d15N as an integrator of the nitrogen cycle. Trends in Ecology and Evolution. 16: 153-162. Wagle, P., Kakani, V.G. 2014. Environmental control of daytime net ecosystrem exchange of carbon dioxide in switchgrass. Agric. Ecosyst. Environ. 186: 170-177. ## Code/software We completed all calculations within R (version 4.4.2) and RStudio (RStudio 2025.09.2+418 "Cucumberleaf Sunflower"). We used the following packages: ggplot2, plyr, dplyr, tidyr, and lubridate. We have included two scripts: AEatm_layer.R and annual_scaling.R The scripts are described with the Files and Variables. ## Access information Other publicly accessible locations of the data: * Rainfall and temperature data in rain22.csv are from [https://lter.kbs.msu.edu/datatables/7](https://lter.kbs.msu.edu/datatables/7) Data was derived from the following sources: * NA 

Abstract Biological nitrogen (N) fixation is an important terrestrial ecosystem N input that occurs in both symbiotic and non-symbiotic associations with plants. Non-symbiotic nitrogen fixation (NSNF) has been documented in many places, including soils, but the magnitude of its inputs, along with drivers of NSNF rates, remains uncertain due to methodological challenges. We measured NSNF by applying a¹⁵N₂tracer to field mesocosms that contained a reassembled soil profile (75 cm deep) with mature switchgrass (Panicum virgatum) at a site in southwest Michigan, USA. We measured NSNF both early and mid-growing season. We found fixed N throughout the entire soil profile, but fixed N declined with depth as did the soil C:N ratio. During the early growing season, most recently fixed N accumulated in the roots, but in the later incubation, fixed N accumulated equally in roots and in aboveground tissues. Annually scaled NSNF rates agreed with those from mass balance within an order of magnitude. Based on our mesocosm measurements and mass balance calculations, we estimate that NSNF inputs to switchgrass at our study site were 2–12 kg N ha⁻¹ yr⁻¹, which represented 27–68% of annual plant N needs. 

Abstract ‘Marginal lands’ are low productivity sites abandoned from agriculture for reasons such as low or high soil water content, challenging topography, or nutrient deficiency. To avoid competition with crop production, cellulosic bioenergy crops have been proposed for cultivation on marginal lands, however on these sites they may be more strongly affected by environmental stresses such as low soil water content. In this study we used rainout shelters to induce low soil moisture on marginal lands and determine the effect of soil water stress on switchgrass growth and the subsequent production of bioethanol. Five marginal land sites that span a latitudinal gradient in Michigan and Wisconsin were planted to switchgrass in 2013 and during the 2018–2021 growing seasons were exposed to reduced precipitation under rainout shelters in comparison to ambient precipitation. The effect of reduced precipitation was related to the environmental conditions at each site and biofuel production metrics (switchgrass biomass yields and composition and ethanol production). During the first year (2018), the rainout shelters were designed with 60% rain exclusion, which did not affect biomass yields compared to ambient conditions at any of the field sites, but decreased switchgrass fermentability at the Wisconsin Central–Hancock site. In subsequent years, the shelters were redesigned to fully exclude rainfall, which led to reduced biomass yields and inhibited fermentation for three sites. When switchgrass was grown in soils with large reductions in moisture and increases in temperature, the potential for biofuel production was significantly reduced, exposing some of the challenges associated with producing biofuels from lignocellulosic biomass grown under drought conditions. 

Growing season drought can be devastating to crop yields. Soil microbial communities have the potential to buffer yield loss under drought through increasing plant drought tolerance and soil water retention. Microbial inoculation on agricultural fields has been shown to increase plant growth, but few studies have examined the impact of microbial inoculation on plant and soil microbial drought tolerance. We conducted a rainout shelter experiment and subsequent greenhouse experiment to explore 3 objectives. First, we evaluated the performance of a large rainout shelter design for studying drought in agricultural fields. Second, we tested how crop (corn vs. soybean) and microbial inoculation alter the response of soil microbial composition, diversity, and biomass to drought. Third, we tested whether field inoculation treatments and drought exposure altered microbial communities in ways that promote plant drought tolerance in future generations. In our field experiment, the effects of drought on soil bacterial composition depended on crop type, while drought decreased bacterial diversity in corn plots and drought decreased microbial biomass carbon in soybean plots. Microbial inoculation did not alter overall microbial community composition, plant growth, or drought tolerance despite our efforts to address common barriers to inoculation success. Still, a history of inoculation affected growth of future plant generations in the greenhouse. Our study demonstrates the importance of plant species in shaping microbial community responses to drought and the importance of legacy effects of microbial inoculation. 

'Marginal lands' are low productivity sites abandoned from agriculture for reasons such as low or high soil water content, challenging topography, or nutrient deficiency. To avoid competition with crop production, cellulosic bioenergy crops have been proposed for cultivation on marginal lands, however on these sites they may be more strongly affected by environmental stresses such as low soil water content. In this study we used rainout shelters to induce low soil moisture on marginal lands and determine the effect of soil water stress on switchgrass growth and the subsequent production of bioethanol. Five marginal land sites that span a latitudinal gradient in Michigan and Wisconsin were planted to switchgrass in 2013 and during the 2018-2021 growing seasons were exposed to reduced precipitation under rainout shelters in comparison to ambient precipitation. The effect of reduced precipitation was related to the environmental conditions at each site and biofuel production metrics (switchgrass biomass yields and composition and ethanol production). During the first year (2018), the rainout shelters were designed with 60% rain exclusion, which did not affect biomass yields compared to ambient conditions at any of the field sites, but decreased switchgrass fermentability at the Wisconsin Central - Hancock site. In subsequent years, the shelters were redesigned to fully exclude rainfall, which led to reduced biomass yields and inhibited fermentation for three sites. When switchgrass was grown in soils with large reductions in moisture and increases in temperature, the potential for biofuel production was significantly reduced, exposing some of the challenges associated with producing biofuels from lignocellulosic biomass grown under drought conditions.