The field used in the study was similar to the marginally productive fields expected to be used for switchgrass biomass production and is in the western part of the main maize production area of the USA. It is located on the University of Nebraska’s Agricultural Research and Development Center (ARDC), Ithaca, Nebraska, USA (latitude 41.151, longitude 96.401 which is 50 km west of Omaha, NE. The field has Yutan silty clay loam (fine-silty, mixed, superactive, mesic Mollic Hapludalf) and Tomek silt loam (fine, smectitic, mesic Pachic Argiudoll) soils. The ranges in the surface three depths of the Yutan soil for water pH and for CEC were 6.2 to 6.8 and 26 to 32 cmoles kg−1, respectively. The corresponding range in the surface three depths of the Tomek soil were water pH values of 6.1 to 7.0 and a CEC range of from 24.4 to 32.3 cmoles kg−1. It is one of the least productive fields of the ARDC and is typical of marginally productive cropland fields that might be used for switchgrass production for bioenergy. The field used in the study was previously in sorghum (Sorghum bicolor (L.) Moench) in 1996 and in soybeans (Glycine max (L.) Merr.) in 1997.
The study is a randomized (r = 3) complete block split-split plot experimental design. Large plots were used so that field scale equipment could be used. Main plot lengths are the width of the field (150 m) and are 18 m wide. Main plot treatments were two cultivars of switchgrass, Trailblazer and Cave-in-Rock, and no-till maize. The maize hybrid used was a commercial glyphosate (Roundup®) tolerant hybrid adapted to the region. The experiment was established in 1998 with the planting of the switchgrass plots. In 1998, plots designated for the no-till maize treatments were planted to glyphosate tolerant soybeans and grown using no-till management. No-till maize production began in 1999.
Main plots were subdivided into three subplots which were used for N fertility treatments. Subplots are 30 m long × 18 m wide and are separated by 15 m wide alleys. Nitrogen (N) fertilizer rates were randomly assigned to the subplots within species main plots. No fertilizer was applied during the establishment year for the switchgrass. In 1999, N fertilizer treatments were N1 = 0, N2 = 80, N3 = 180, and N4 = 240 kg N ha−1. From 2000 on, they were N1 = 0, N2 = 60, N3 = 120, and N4 = 180 kg N ha−1. Fertilizer rates were reduced for 2000 and thereafter because of the 1999 results on maize and the summarization of previous fertility research on switchgrass . Rates on the switchgrass were N1, N2, and N3. Rates used on no-till maize were N2, N3, and N4. Ammonium nitrate fertilizer was broadcast with a bulk spreader throughout the duration of the study. The 0 N-rate for switchgrass was used as a low input treatment only for switchgrass. In 2001, the switchgrass and corn subplots were split lengthwise into 9 m wide sub-subplots for harvest treatments.
Switchgrass plots were seeded directly into the soybean stubble from the previous year using a no-till drill with a planting rate of 6.7 kg ha−1 (pure live seed basis). A pre-emergence application of 2 kg ha−1 atrazine [Aatrex 4 L®; 6-chloro-N-ethyl-N′-(1-methylethyl)-1, 3, 5-triazine-2, 4-diamine] was applied for weed control. There were no other management inputs the establishment year. The 60 and 120 kg N ha−1 rates represent the low to high rates recommended switchgrass grown for bioenergy  with the 0 rate representative of a no-input system. A previous study  showed that switchgrass harvested after a killing frost had significantly less N in the biomass than switchgrass harvested at anthesis indicating N was being recycled to the roots of switchgrass late in the growing season. Beginning in 2001, harvest treatments were applied to the sub-subplots within switchgrass cultivar N-fertility subplots to determine if harvest date might affect SOC. One harvest treatment (H1) was a mid-August harvest and the other (H2) was a harvest in October or November, following a killing frost. Plots were harvested only once a year. A 4.6 × 0.9 m (4.2 m2) area was harvested in each subplot with a flail-type plot harvester in 1998 and the following April, all remaining biomass from the previous year was removed with a field harvester prior to spring green-up. In 1999 and thereafter, switchgrass yield harvests were made with flail harvesters and associated weighing equipment by harvesting a 0.9 to 1.8 m wide swath (varied with harvester used) the full 30 m length of the plots. At time of harvest, subsamples were collected from each sub-subplot, weighed for moisture content, dried at 50°C for 48 h, and reweighed to determine dry matter content. Yields were adjusted to a dry weight basis. The C concentration of the switchgrass samples was determined using near infrared spectrometer (NIRS) procedures and calibrations . A field flail harvester was used to remove all remaining biomass from the plots immediately following the yield harvests using the same harvest height of 10 cm.
Maize seed was planted directly into soybean stubble of the previous year in 1999 with a no-till drill and the maize plots of the previous year thereafter. The maize was grown in 0.76 m wide rows. The N rates that were used represent the low-to-high rates for maize grown under rainfed conditions in the region. Nitrogen fertilizer was applied using the same equipment as for switchgrass plots. Glyphosate herbicide was applied after the maize had emerged and was about 40 cm in height. No other management inputs were applied until grain harvest. Aboveground samples (one row 4.4 m long) were collected soon after physiological maturity in each N rate subplot and later from each sub-subplot for total biomass yields. Ears were removed and stalks were then cut at ground level, chopped and weighed. A representative subsample was collected, dried and weighed for gravimetric moisture determination to calculate stover dry matter production. Ears were dried and weighed, added to the calculated stover weight to obtain total biomass yields on a dry weight basis. Maize grain yields were determined with a plot combine equipped with a weighing unit, subsamples were collected for moisture determination, and yields were adjusted to oven dry weight basis. Because of the emerging interest in using maize stover for biomass energy, in 2001 stover harvest treatments were applied to the sub-subplots. The harvest treatments were no residue harvested (H1) and approximately 50 % of the stover remaining after grain harvest (H2). Stover was harvested from the H2 treatments after grain harvest using the flail forage harvesters that were used to harvest switchgrass plots. Harvested stover yields were determined by harvesting the stover from two non-border rows of each sub-subplot its entire 30 m length with a plot-flail harvester. The remaining rows were harvested with a field scale flail harvester set at the same 10 cm height as the plot harvester. All stover weights were converted to a dry-weight basis (50°C oven for 48 h). Maize grain and stover samples were analyzed for total C by dry combustion .
Soil Sampling and Analysis
Baseline soil samples were obtained in July 1998 and plots were thereafter re-sampled at approximately 3-year intervals in May 2001, April 2004, and in May 2007. The initial sampling location was in the center of each subplot. Subsequent soil samples were offset a fixed distance from each subplot or sub-subplot center to prevent re-sampling of a previous sampling site from which soil had been removed. Sample collection was done using the procedures described by Follett et al. . In brief, the plant material was removed from the soil surface and then, using a flat-bladed shovel, undercutting and removing the soil from the 0–5, 5–10, and 10–30 cm depths. Samples were also collected from the 30–60, 60–90, 90–120, and 120–150 cm depths at the July 1998 and May 2007 sampling dates using a hydraulic probe. Soil bulk densities were determined using the USDA-NRCS National Soils Laboratory methods . The standardized procedure (Soil Survey Laboratory method 3B1) to measure bulk density requires collection of field occurring fabric (clods), coating them with Saran F-310 in the field (NRCS 2004; Soil Survey Laboratory method 3B), transport to the laboratory, and desorption to 33 kPa (1/3 bar). After reaching equilibrium, the clod is weighed in air to measure mass and in water to measure its volume, and next dried at 110°C (230°F) with its mass and volume again determined. A correction is made for mass and volume of rock fragments and the plastic coating with the BD value reported for <2 mm (0.079in) soil fabric.
Once samples were collected they were sieved through a 2 mm sieve and <2 mm plant material picked from the soil, air dried (room temperature), subsampled, mechanically ground to pass through a 0.2-mm sieve, and the subsamples were stored in sealed glass containers with screw type lids. All soils were checked for carbonates and in the very few cases where carbonates existed they were removed prior to analyses for organic C using accepted procedures [35,36]. All analyses were on an oven dry weight (55°C). The methodology is such that both the isotopic C analyses and the analyses for the total SOC are done at the same time for the same sample.
A subsample of soil from each layer was sieved (2 mm sieve size) and picked free of remaining recognizable plant and root fragments under ×20 magnification. Soil samples were oven dried (55°C), finely ground, and then analyzed for total SOC and 13C/12C isotope ratio. All samples were analyzed for total SOC and 13C/12C isotope ratio using a continuous-flow Europa Scientific 20–20 Stable Isotope Analyzer (isotope ratio mass spectrometer) interfaced with Europa Scientific ANCA-NT system (automated nitrogen carbon analyzer) Solid/Liquid Preparation Module (Dumas combustion sample preparation system) (Europa Scientific, Crewe Cheshire, UK—Sercon Ltd.). Soil organic C was calculated using the C concentration (%), soil bulk density (g cm3), and thickness for each individual sampled soil layer and then summed over layers. Soil organic C on an equivalent masses basis (SOC-EMB) also was calculated using the method of Ellert et al. . The 120 cm soil depth data were used to make direct comparisons between both calculation methods in Table 1 and Supplementary Figs. S1 and S2. Procedures to calculate the C3 and C4 components of the SOC using δ13C (‰) are described by Deines  and Follett et al. [33,39]. For the purposes of this report, results from the 0–5, 5–10, and 10–30 cm depths were combined.
Switchgrass and maize are both C4 plants that utilize the Hatch-Slack enzymatic pathway that is dominated by PEP-carboxylase which produces a C isotope fractionation between the CO2 in the air (currently about −8 mil (‰ = 1/1,000 th) , and about −6.5‰ from the pre-industrial period back 10,000 years  and the plant of about −4‰. The instrumental measurement of δ13C values are expressed relative to a calcium carbonate standard known as PDB from the Cretaceous Pee Dee formation in South Carolina . Sign of δ13C indicates whether a sample has higher or lower 13C/12C isotope ratio than PDB. This pathway fractionates the isotopic composition of the plant so that a typical δ13C of C4 (warm season) plants relative to PDB is about −11 to −13‰ [38,39]. In contrast, the dominant photosynthetic pathway of C3 (or cool-season plants) is Calvin–Benson, whereby the enzyme RuBP carboxylase produces a carbon isotope fractionation between the air and the plant of about −18 ‰ so that the δ13C of C3 plants relative to PDB is about −26 to −27 [36,38]. These differences make it feasible to determine the plant source (C4 or C3) of SOC using mass-spectrometric analyses.
Equation 1 expresses 13C/12C ratio as δ13C, which has “per mil” (‰) units. By convention, δ13C values are expressed relative to the PDB calcium carbonate standard . The sign of δ13C indicates whether a sample has a higher or lower 13C/12C isotope ratio than does PDB.
Besides measurements of total C and δ13C, fraction and weight of C originating from C3 plants and C4 plants were calculated based upon the mass of SOC and measured δ13C values (Eq. 1) of soil samples collected at the first and last soil sample collections, using Eqs. 2 and 3, and the δ13C of C3 and C4 plant material .
The data were analyzed using Mixed Model analysis procedures of SAS  to determine the effects of N and harvest (H) treatments on the grain and biomass yields and on changes in soil C. By using the Mixed Model analyses, it was possible to use a t test to test if the change in soil C was significant for each N × H treatment for both switchgrass and maize.