Quantifying the effects of switchgrass (Panicum virgatum) on deep organic C stocks using natural abundance 14C in three marginal soils

Perennial bioenergy crops have been shown to increase soil organic carbon (SOC) stocks, potentially offsetting anthropogenic C emissions. The effects of perennial bioenergy crops on SOC are typically assessed at shallow depths (< 30 cm), but the deep root systems of these crops may also have substantial effects on SOC stocks at greater depths. We hypothesized that deep (> 30 cm) soil organic carbon (SOC) stocks would be greater under bioenergy crops relative to stocks under shallow-rooted conventional crop cover. To test this, we sampled soils to between 1- and 3-meters depth at three sites in Oklahoma with 10-20 year old switchgrass (Panicum virgatum) stands, and collected paired samples from nearby fields cultivated with shallow rooted annual crops. We measured root biomass, total organic C, 14C, 13C, and other soil properties in three replicate soil cores in each field and used a mixing model to estimate the proportion of recently fixed C under switchgrass based on 14C. The subsoil C stock under switchgrass (defined over 500-1500 kg m-2 equivalent soil mass, approximately 30-100 cm depth) exceeded the subsoil stock in neighboring fields by 1.5 kg C m-2 at a sandy loam site, 0.6 kg C m-2 at a site with loam soils, and showed no significant difference at a third site with clay soils. Using the mixing model, we estimated that additional SOC introduced after switchgrass cultivation comprised 31% of the subsoil C stock at the sandy loam site, 22% at the loam site, and 0% at the clay site. These results suggest that switchgrass can contribute significantly to subsoil organic C—but also indicated that this effect varies across sites. Our analysis shows that agricultural strategies that emphasize deep-rooted grass cultivars can increase soil C relative to conventional crops while expanding energy biomass production on marginal lands.


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Inorganic C was quantified at Lawrence Livermore National Laboratory by treating finely-2 2 1 ground subsamples of each sample with 1 M phosphoric acid in a sealed jar and measuring CO 2 2 2 2 evolved using a LI-850 infrared gas analyzer (Robertson, 1999). Where carbonates were present, 2 2 3 total organic C was obtained by subtracting inorganic C from total C. C isotopes were quantified on a subset of the soil that was ground to a fine power by hand. Soils that contained carbonates were treated with 1 M HCl to remove inorganic C before isotope analysis. Direct addition of dilute (~ 1 M) HCl has measurable but relatively small (< 1‰) 2 2 7 effects on 13 C and 14 C in soils and sediments (Brodie et al., 2011;Komada et al., 2008) and 2 2 8 appears to be no more biased than alternative treatment approaches (Brodie et al., 2011). HCl was added to each sample until effervescence ceased and then was allowed to evaporate to 2 3 0 prevent leaching of acid-soluble C. Acid-treated soil was analyzed for 13 C at the University of Van de Graaff AMS at the Center for AMS at Lawrence Livermore National Laboratory. Samples were prepared for 14 C measurement by sealed-tube combustion to CO 2 in the presence 2 3 7 of CuO and Ag and then reduced onto iron powder in the presence of H 2 (Vogel et al., 1984). The 14 C content of each sample (∆ 14 C) was reported in ‰ relative to the absolute atmospheric 2 3 9 14 C activity in 1950. We report ∆ 14 C here rather than mean residence times because reporting  in Supplementary Table 1). Because collection and analysis occurred within a short period, no 2 4 6 correction was performed for decay of 14 C between sampling and analysis. The average 2 4 7 instrument uncertainty for ∆ 14 C was ± 4 ‰, and the average precision estimated from a set of six 2 4 8 duplicate samples was ± 5 ‰. We used measured C stocks to directly estimate the net difference in C between the 2 5 1 switchgrass and reference fields. We also used 14 C measurements to develop an indirect estimate 2 5 2 that was independent of the measured C stock in the reference field. The C stock calculations were carried out on an equivalent soil mass (ESM) basis using the cumulative coordinate approach (Gifford and Roderick, 2003;Rovira et al., 2015). We used this approach because it is 2 5 5 1 2 robust to differences in bulk density, and thus better suited to comparing C stocks under different 2 5 6 land uses (Wendt and Hauser, 2013). Calculations were performed separately on the surface soil 2 5 7 layers-which we defined as the top 500 kg m -2 of soil-and the subsoil-which we defined as 2 5 8 the 1000 kg m -2 of soil directly below the uppermost 500 kg m -2 of soil.

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We obtained C stocks by using linear interpolation to predict cumulative C mass from 2 6 0 cumulative soil mass (Gifford and Roderick, 2003). The mineral mass of each depth interval was 2 6 1 used as the basis for developing mass coordinates (Rovira et al., 2015). Mineral mass was Where C(t) is the cumulative C mass at the target cumulative soil mass M(t), C(z a ), and C(z b ) are 2 6 8 the cumulative C masses at the upper and lower a boundaries of the sampling interval containing values associated with each sampling layer by the contribution of that layer to the C stock. When the lower boundary of the topsoil or subsoil occurred within a layer, isotopic values from that 2 7 5 layer were weighted by the C mass that contributed to the topsoil or subsoil. We initially explored the use of 13 C as a quantitative tracer of switchgrass inputs in our 2 7 8 system. The mixed history of C 3 and C 4 vegetation at all three sites-and in particular the recent 2 7 9 history of periodic C 4 cropping at the Sandy Loam and Loam sites-suggested that our sites did for the subsoil (defined over 500-1500 kg m -2 ESM) in the reference plots at our sites ranged 2 8 2 between 16.1 and -14.9 ‰, which is at the higher end of the C 4 plant range (O'Leary, 1988). We ‰-indicating that the difference between isotopic end-members in a potential 13 C-based mixing 2 8 5 model in the subsoil was only 2-3 ‰. This range is comparable to ~2 ‰ fractionation effects that 2 8 6 apply to plant-tissue end members in isotopic mixing models and are a possible source of our sites-could not be used for identifying switchgrass contributions to SOC quantitatively.

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We did not carry out 14 C based calculations for the uppermost 500 kg m -2 of soil suggests that 14 C may only be a useful tracer of increased root inputs at depth, where SOC tends 3 0 7 to be 14 C depleted and contrasts strongly with recent inputs.

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We divided the subsoil SOC stock under switchgrass (C s , kg C m -2) into two parts: (1) a 3 0 9 component equal to the C stock under the reference plot (C r , kg C m -2 ), representing the initial C since 1998 or 2008 (C n , kg C m -2 ). By definition C s = C r + C n . Each of these components was  This mixing relationship was used to obtain the fraction (f n ) of the C stock under switchgrass comprised by C n and to solve for C n : The 14 C-based isotopic mixing model thus provided an estimate of the C stock difference based 3 2 2 on the observed C stock in the switchgrass plot and the shift in 14 C values between the two plots.  To constrain this range, we modeled the ∆ 14 C of SOC produced since 1998 or 2008 using a one- The one-pool C model was implemented in SoilR (Sierra et al., 2012) using the function 3 3 2 "OnepModel14" and a published atmospheric CO 2 record for northern hemisphere, extended to varying rate and the effect of varying inputs was small (e.g. halving litter inputs for the first four   We evaluated C stock differences between the reference and switchgrass plots by obtain distributions for each estimate of the difference in C stocks between plots given the 3 4 7 uncertainties in the input parameters. We obtained 95% confidence intervals from the Monte 1984), and we obtained empirical p-values from the Monte Carlo intervals to test the hypothesis 3 5 0 that the difference in stocks was greater than zero. P-values were obtained using the formula p = 3 5 1 (r + 1)/(n + 1), where r was the number of Monte Carlo replicates less than zero and n was the assumed a uniform distribution that ranged between the limiting cases defined in section 2.5 3 5 9 above. Parameter sets were drawn from the distributions 100,000 times. For each parameter set, 3 6 0 we calculated one of two quantities: an estimate of C n from the observed stock difference (C s -3 6 1 C r ) or the 14 C-based stock difference (f n *C s ). The three sites varied in texture, pH, and exchange properties ( were below detection, or less than 1% of the total cation pool at all sites, and thus not reported.   switchgrass and paired "reference" annual crops at three sites in Oklahoma characterized by deviations are listed in parentheses. Loam and Clay sites (Fig 1). Root biomass was much greater under switchgrass at all sites ( Fig   3  8  3 1). However, the reference plots were sampled after harvest, and the small number of cores representative of growing season conditions. a buried profile, which is shown with a dashed gray line. Total organic C concentrations were lowest throughout the soil at the Sandy Loam site, 3 9 7 intermediate at the Loam site, and highest at the Clay site (Fig 2). At the Sandy Loam site, the uppermost 200 cm of soil (Fig 2a). At the Loam site organic C concentrations were higher in the top 30 cm (Fig 2b). We also observed a substantial "bulge" in organic C below 200 cm at the 4 0 2 Loam site, which matched the top of the buried paleosol that we identified both in the soil series description and in our field observations. The organic C content of the buried soil was higher in 4 0 4 the cores sampled under the reference vegetation (Fig 2b). In contrast to the Sandy Loam and 4 0 5 Loam sites, at the Clay site organic C concentrations were generally similar under both 4 0 6 vegetation types (Fig 2c). In general, the 13 C signature of organic C varied with sampling depth across sites. At the 4 ‰ over 30-90 cm depth, and declined at greater depths (Fig 3a). This pattern appeared under  (Fig 3a). At the Loam site, δ 13 C values were also depleted at the surface and comparatively 4 2 2 higher at greater depths in a pattern similar to the Sandy Loam site (Fig 3b). The δ 13 C signature 4 2 3 was also comparatively higher in cores taken under switchgrass, but this difference attenuated 4 2 4 with depth (Fig 3b). At the Clay site, δ 13 C values were highest at the surface and declined with 4 2 5 depth (Fig 3c). Patterns under the two plant covers at the Clay site were similar, with slightly 4 2 6 higher isotopic values under switchgrass (Fig 3c). Radiocarbon values declined with depth at all sites (Fig 3d-f). At the Sandy Loam site other two sites (Fig 3f). The broadly similar under the two vegetation types (Fig 3f).   Table 3. While we focused on ESM 4 5 5 estimates when comparing plots to factor out bulk density differences between plots and sites, 4 5 6 we also report total estimates to a depth of 1.2 m-which was the greatest depth at which we 4 5 7 were able to collect samples across all sites-and to a depth of 2.4 m, which was attained at the 4 5 8 Sandy Loam and Loam sites (Table 3). All soil chemical data and C-isotope values are reported 4 5 9 in Supplementary Table 1.
We compared the C stocks under switchgrass and reference plots (Fig 4). At the Sandy 4 6 1 Loam site, direct comparison of the C stocks suggests that there was slightly more C under   sampling to this depth was not possible there, possibly due to calcium cementation in the subsoil.  At two out of the three sites we sampled, we observed significant differences in SOC these two sites, differences in subsoil C were in the range of 0.6-1.5 kg C m -2 . This range is systems. This range of rates is typical of switchgrass systems evaluated to a comparable depth Both 13 C and 14 C were sensitive to land use at the three sites, and in general 14 C confirmed 5 2 3 that larger C stocks under switchgrass at these sites (or lack thereof) can be attributed to recently fixed C in the subsoil. We did, however, discover some disagreement between the directly 5 2 5 measured C stocks and the difference estimated using 14 C: the directly-measured difference in 5 2 6

Discussion
subsoil C stocks was largest at the Sandy Loam, but the shift in 14 C values at this site was too 5 2 7 2 6 small to fully accommodate this difference. The simplest interpretation of this result is that the 5 2 8 initial C stocks were greater under the switchgrass field before planting-highlighting the limits 5 2 9 of the small sample size (n=3) plus the spatially pseudo-replication inherent to the paired 5 3 0 sampling design. This interpretation is supported by texture analysis of deeper soil horizons at and switchgrass fields at this site, the reference plot had a higher profile-averaged sand to silt 5 3 3 ratio than switchgrass at depths exceeding 90 cm (mean sand/silt = 14 ± 0.7 versus 2 ± 0.4 at a 5 3 4 depth of 120 cm, Table S1). This indicates that soil physical characteristics did not match 5 3 5 perfectly at this site below a certain depth. At the other two sites where the plots were more 5 3 6 closely paired, direct and 14 C-based methods agreed.

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Intriguingly, we observed less total C and comparatively depleted 14 C values in the buried depleted under the reference plot-and were actually slightly less depleted than the overlying 5 4 0 soil (Fig 3e). Given that roots were not observable in the paleosol, we think it is unlikely that 5 4 1 patterns in total C and 14 C at the depth are driven by modern plant cover. Instead, we think it is 5 4 2 most likely that the soil under the switchgrass and reference plots-while similar now- switchgrass plot was eroded prior to burial-which would explain its lower C concentrations and 5 4 7 14 C values relative to the reference plot. The material that was subsequently deposited over both