Impact of Phosphorus Fertilization Rates on Nitrous Oxide Emissions in Switchgrass: Nonlinear Response Reveals Emission Reduction at Low Phosphorus Rates

Impact of Phosphorus Fertilization Rates on Nitrous Oxide Emissions in Switchgrass: Nonlinear Response Reveals Emission Reduction at Low Phosphorus Rates | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Impact of Phosphorus Fertilization Rates on Nitrous Oxide Emissions in Switchgrass: Nonlinear Response Reveals Emission Reduction at Low Phosphorus Rates Augustine Kwame Osei, Nadia Gabbanelli, Maren Oelbermann This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5328479/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Aims Phosphorus (P) fertilization can play a critical role in increasing switchgrass biomass yields for bioenergy production. However, applying mineral P to switchgrass can stimulate nitrous oxide (N 2 O) emissions, offsetting its climate mitigation benefits. The effect of P fertilization on N 2 O emissions is not well understood, with previous studies producing conflicting results. Moreover, studies evaluating the effect of P fertilizer on nitrogen (N) dynamics and its contribution to N 2 O emissions under switchgrass are lacking. Methods A 43-day study was conducted in a controlled-environment to evaluate the effect of different P fertilizer rates on N 2 O emissions in N-fertilized switchgrass. Four different fertilizer rates [(i) 60 kg N ha –1 (60N0P); (ii) 60 kg N and 20 kg P ha –1 (60N20P); (iii) 60 kg N and 40 kg P ha –1 (60N40P); (iv) 60 kg N and 60kg P ha –1 (60N60P)] and a treatment with no fertilizer (0N0P) were evaluated. Results While switchgrass biomass yield was less responsive to N and P fertilization, P fertilization had a nonlinear effect on N 2 O emissions. Applying P at 20 kg ha –1 resulted in lower emissions compared to N-only treatment. In contrast, increasing the P rate to 40 kg ha –1 led to the highest N 2 O emissions, surpassing those of N-only treatment. At 60 kg P ha –1 , emissions were comparable to the N-only treatment. Conclusions These findings suggest that while moderate P application can reduce N 2 O emissions, higher P rates may increase emissions, offsetting the climate benefits of switchgrass as a bioenergy crop. Switchgrass biomass yield Nitrogen and phosphorus fertilizer Biomass N and P uptake N2O emissions Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Bioenergy plays a critical role in decarbonizing our energy systems as well as limiting global warming (Lemus and Lal 2005 ). While current bioenergy feedstocks from food crops such as corn ( Zea mays L.), soybeans ( Glycine max L.), canola ( Brassica napus L.), etc., have come under scrutiny due to the myriad of environmental and climate challenges associated with their production (Pool 2013 ; Searchinger and Heimlich 2015 ), the use of lignocellulosic crops such as herbaceous and short rotation woody crops have attracted great interests (Agostini et al. 2015 ; Perea-Moreno et al. 2019 ; Osei et al. 2024 ). Key among the lignocellulosic crops recommended for bioenergy production is switchgrass ( Panicum virgatum L.) – a perennial warm season grass native to North America (Samson et al. 2016 ). The interest in switchgrass as bioenergy feedstock stems from its low maintenance cost and ability to adapt and produce high amounts of biomass under marginal conditions (Samson et al. 2016 ). Additionally, switchgrass can provide climate mitigation benefits through increased soil carbon (C) sequestration and reduced greenhouse gas (GHG) emissions (Blanco-Canqui 2010 ; Coleman et al. 2018 ; Bazrgar et al. 2020 ). Although, switchgrass can produce high yields without fertilizer inputs (Ruan et al. 2016 ; Wang et al. 2020 ), optimizing yields may require fertilizer additions (Lee et al. 2007 ; Kering et al. 2012 ). For instance, the Ontario Biomass Producers Co-operative in Canada recommend an annual application of 60–70 kg N ha –1 for improved switchgrass biomass yields (Samson et al. 2016 ). Schmer et al ( 2012 ) measured 6.8 Mg ha –1 aboveground dry matter yield for switchgrass receiving 67 kg N ha –1 compared to 3.2 Mg ha –1 without nitrogen (N) fertilizer. Nikièma et al ( 2011 ) also observed 1.5- and 2.5-fold increase in switchgrass biomass with 56 kg ha –1 and 112 kg ha –1 N fertilization, respectively. While these results point to higher potential biomass yields from N fertilization in switchgrass, the resultant nitrous oxide (N 2 O) emissions from N fertilizer may offset any climate benefits derived from the increased yield of switchgrass for bioenergy production. McGowan et al ( 2018 ) studied the impact of different N rates in switchgrass and observed that total N 2 O emissions increased from 0.2 to 3.0 kg N 2 O-N ha − 1 as N rates increased from 0 to 150 kg N ha − 1 . Wile et al ( 2014 ) observed that fertilization of switchgrass with 120 kg N ha –1 resulted in a 73% increase in N 2 O emissions over 2 years compared to the unfertilized crop. Jin et al ( 2019 ) argued that N fertilization of switchgrass can result in increased N 2 O emissions offsetting the GHG balance of switchgrass as a bioenergy crop. Whereas these and previous studies ( e.g ., Nikièma et al. 2011 ; Pannu et al. 2019 ; Rau et al. 2019 ) examined N fertilization on N 2 O emissions in switchgrass, studies measuring the response of phosphorus (P) fertilization on N 2 O emissions in switchgrass have seldom been conducted. This is despite the contradictory findings on the impact of P fertilization on N cycling and N 2 O emissions. For instance, Ullah et al ( 2016 ) and Zhang et al ( 2019 ) found increased N 2 O emissions from P fertilization due to the increased stimulation of nitrifying and/or denitrifying bacteria activities. Mori et al ( 2010 ) also suggested that P addition enhanced N cycling and caused accumulation of available N for nitrification and denitrification which resulted in increased N 2 O emission. Contrary to these findings, other studies (Baral et al. 2014 ; Mori et al. 2013 , 2014 ) observed reduced N 2 O emissions with P addition to soils due to increased N uptake in plant biomass. Zhang et al ( 2019 ), on the other hand found no impact of P addition on N 2 O emissions. These contrasting results of N 2 O emissions with P addition indicate that the effect of P on N 2 O emissions may differ with crop type, soil nutrient content, and environmental conditions. From an agronomic perspective, N and P are two major limiting macronutrients whose application positively influences switchgrass yield (Nikièma et al. 2011 ; Kering et al. 2012 ; Sawyer et al. 2019 ). Studies on N and P fertilization in switchgrass have focused on economic benefits, biomass and ethanol yields, and soil nutrients removal (Haque et al. 2012 ; Jungers et al. 2015 ; McGowan et al. 2018 ; Ashworth et al. 2019 ; Tang et al. 2020 ), with less emphasis on N 2 O emissions. However, considering the significance of P in N cycling and N 2 O emissions (Mori et al. 2010 ; Ullah et al. 2016 ), it is important to understand how P fertilization can influence N 2 O emissions in N-fertilized switchgrass and what that may mean for the climate mitigation benefit of switchgrass as a bioenergy feedstock. An experiment was conducted under controlled-environment to assess the impact of different P application rates in N-fertilized switchgrass to enhance our understanding of how P fertilization impacts N 2 O emissions in switchgrass. We hypothesized that P application at high rates would enhance below and aboveground biomass growth resulting in increased N uptake and lower soil available N, with a consequent reduction in N 2 O emissions. Materials and methods Experimental design A 43-day experiment was conducted in a controlled-environment chamber (Conviron PGR-15, Controlled Environments Inc., Winnipeg, MB) at the University of Waterloo, Ontario – Canada. Equal amount (equivalent of 500 g air-dry soil) of commercial potting mix soil (Vigoro® natural garden soil) was weighed into white polyvinyl chloride (PVC) tubes of 10 cm diameter and 25 cm height. Soil in each tube were packed at a bulk density of 0.64 g cm –3 occupying the bottom 10 cm depth of the tubes leaving a 15 cm headspace. A fiberglass mesh screen was attached to the bottom of each tube to allow for free drainage. The soil in each PVC tube was then saturated with deionized water and allowed to drain overnight to attain soil moisture at field capacity (Evett et al. 2019 ). The soil moisture at field capacity for each of the tubes was measured with a WET-2 sensor (Delta-T Devices, Cambridge, UK) and recorded. The tubes were then seeded with cave-in-rock switchgrass variety at a seeding rate of 10 kg ha –1 (Samson et al. 2016 ). At the time of seeding, the following fertilizer rates, representing different treatments were supplied to each tube; (i) 60 kg N ha –1 (60N0P); (ii) 60kg N and 20 kg P ha –1 (60N20P); (iii) 60 kg N and 40 kg P ha –1 (60N40P); (iv) 60 kg N and 60 kg P ha –1 (60N60P); and (v) 0 kg N and 0 kg P ha –1 (0N0P). The 60 kg N ha –1 , 20 kg P ha –1 , 40 kg P ha –1 , and 60 kg P ha –1 fertilizer rates were based on actual N and P additions of 566 mg N kg –1 , 188 mg P kg –1 , 376 mg P kg –1 , and 566 mg P kg –1 , respectively. Urea [(NH 2 ) 2 CO] and potassium dihydrogen phosphate (KH 2 PO 4 ) were used as N and P sources, respectively. Each treatment (fertilizer rate) was replicated three times. The tubes were randomly placed inside the growth chamber (Fig. 1 ) and were systematically rotated every 3 days to account for any potential spatial variations inside the growth chamber. The growth chamber was set up in 14-hour light and 10-hour dark photoperiod cycles. Carbon dioxide (CO 2 ), temperature, relative humidity, and light intensity during the 14-hour light photoperiod cycle were set at 420 ppm, 25 o C, 65%, and 450 µmol m –2 s − 1 , respectively, whereas their corresponding settings for the 10-hour dark photoperiod cycle were 420 ppm, 15 o C, 80%, and 0 µmol m − 2 s − 1 . These climate data are typical of the average peak growing season local conditions in the Waterloo Region in southern Canada (Environment Canada). Gas sampling and nitrous oxide measurement The concentration of N 2 O in the headspace of each tube was taken daily for the first 7 days and at 3-day intervals after day 7 of the experiment, starting at t = 0 days and terminated at t = 43 days. The experiment was terminated on day 43 as N 2 O fluxes had stabilized, and the switchgrass biomass had grown to a size that made it challenging to manage beyond this point. At the time of gas sampling, the tubes were capped with a lid containing a central sampling port and a 10-cm-long (3 mm inner diameter) ventilation duct to account for pressure differences during sampling (Lutes et al. 2019 ) (Fig. 1 ). A 10 ml gas was drawn from the headspace of each tube using a syringe and needle into previously evacuated 3 ml Labco Exetainer vials with grey butyl rubber septa (Exetainer, Labco Ltd., Buckinghamsire, UK) at 0, 15, and 30 minutes after the tubes were caped (Lutes et al. 2016 ). During lid closure for gas sampling in tubes with bigger plants, plants were carefully folded to prevent breaking and soil disturbance. The gas samples were analysed for N 2 O using a Gas Chromatograph (GC) 6890 equipped with a micro-electron capture detector (ECD) (Agilent Technologies Inc., Santa Clara, USA) (Lutes et al. 2019 ). Gas fluxes were calculated with the HMR model in R using the chamber volume, surface area of the chamber base, and rate of change in chamber gas concentration (Pedersen et al. 2010 ). Following each gas sampling event, soil moisture content in each tube was measured with the WET-2 sensor and where required, moisture content was adjusted with deionized water to field capacity based on the recorded soil moisture at field capacity for each tube that was measured at the time of seeding. Soil sampling and analysis for chemical properties After 43 days, when the experiment terminated, ~ 100 g of soil was removed from the top, middle, and bottom in each tube. The collected soil from each treatment replicate was thoroughly mixed, air dried and sieved (2 mm), and analyzed for pH, EC, SOC, total N, available N, and available P. All soil analyses were performed on soil before the experiment (herein referred to as control) and after the experiment. Total N and SOC was determined using an elemental analyzer (Costech 4010, Cernusco, CA, USA) whereas the Olsen P method was used for available P analysis. Prior to soil analyses for C and total N, soil carbonates were removed according to Dyer et al ( 2012 ). Soil extract for orthophosphate was prepared by extracting 2.5 g of 2 mm sieved soil with 50 ml of 0.5 M NaHCO 3 (pH 8.5) and analyzed using a UV-Vis Spectrophotometer (Kuo 1996). Soil pH (1:1 soil: ultrapure water suspension) (Miller and Kissel 2010 ) was measured using a pH meter (Fisherbrand, Accumet AB 15) and the EC with the WET-2 sensor. For NH 4 + and NO 3 – analyses, soil extracts were prepared using 2 M KCl solution and the filtered extracts were analyzed for NH 4 + and NO 3 – on a Shimadzu 1800 UV-Vis Spectrophotometer (Shimadzu Corp., Kyoto, Japan) (Doane and Horwáth 2003 ). Biomass yield of and determination nitrogen and phosphorus concentrations in biomass Beginning day 8 of the experiment, plant height for each treatment replicate was measured and recorded weekly until termination of the study on day 43. The data were used to determine switchgrass growth rate. Following termination of the experiment, aboveground biomass was harvested by cutting plants in each tube 0.5 cm above soil surface. After collecting soil from each tube for analysis, the remaining soil containing switchgrass roots was placed on top of a 2 mm sieve and the soil washed off to obtain root biomass for each tube. Both shoot and root biomass for each tube was weighed and the fresh weights recorded. The biomass was then oven-dried at 40 o C until no moisture loss was detected. Dry biomass weight for shoot and roots were recorded. After oven drying, root and shoot biomasses were ground with a Kinematica Polymix Grinding Mill (Kinematica AG, Switzerland) and sieved through a 0.5 mm sieve. Subsamples of the 0.5 mm sieved root and shoot biomass for each replicate were analyzed for total N and P. Total N of the ground and 0.5 mm sieved biomass was analyzed on the elemental analyzer and total P on the spectrophotometer. Before total P analysis on the spectrophotometer, plant samples were digested in concentrated HCl (12 M) and filtered according to a method adapted from Flindt and Lillebø ( 2005 ). Briefly, the ground and 0.5 mm sieved biomass samples were combusted in a muffle furnace at 500 o C for 4 hours. After biomass combustion, 5 mg of the combusted ash were weighed into a 200 mL Erlenmeyer flask and mixed with 1 mL concentrated HCl and 25 mL deionized water. The mixture was placed on a heating plate at 120 o C until the solution turned yellow and transparent after the water had evaporated. Volume of the transparent yellowish solution was then adjusted to 100 ml using deionized water and filtered through a Whatman 42 filter paper (Whatman 42, 2.5 µm). The filtrate was analyzed for total P using the ascorbic acid method (Murphy and Riley 1962 ) with the spectrophotometer. Biomass N and P removals were calculated by multiplying N and P concentrations with dry matter biomass yields. Statistical analysis Data processing and analysis were carried out using SPSS (IBM Corp.). A one-way analysis of variance (ANOVA) was used to compare the effect of different fertilizer rate on yield, cumulative N 2 O emissions, biomass N and P concentrations, and soil properties before (control) and after the experiment. Prior to the ANOVA, data was assessed for homogeneity of variance (Levene's) and normality (Shapiro-Wilk Test) (Steel et al. 1980). The shoot biomass P and root biomass N concentrations were found to have violated the assumption of normality, hence, were log-transformed. The Fisher least significant difference (LSD) was used to detect significant differences between individual treatments. Type I error rate for all statistical analyses was set at p ≤ 0.05 . Results Soil characteristics The initial pH, EC, SOC, total N, soil C/N ratio, and available P contents of the commercial potting mix are reported in Table 1 . Growing switchgrass for 43 days significantly increased ( p ≤ 0.015 ) soil total N in the 60N0P, 60N40P and 60N60P fertilizer rates and unfertilized treatment compared to the total N in the control. Although total N was higher in the 60N20P treatment compared to the control, this difference was not statistically significant ( p = 0.123 ). Compared to the control, soil available P was significantly lower ( p ≤ 0.005 ) in the unfertilized and 60N0P treatments after the 43 days. Soil available P did not differ significantly ( p ≥ 0.066 ) between any of the P-fertilized treatments compared to the control. Other than the significantly higher ( p ≤ 0.003 ) soil NH 4 + content in the 60N60P compared to all the other treatments, NH 4 + content did not differ among any of the other treatments. Meanwhile, significantly higher ( p ≤ 0.047 ) soil NO 3 – contents were observed in the control and 60N0P compared to the 60N60P and unfertilized treatment after the 43 days (Table 1 ). Table 1 Soil chemical properties of the commercial potting mix soil before (control) and after the 43-day experiment in switchgrass with different fertilizer rates grown in a controlled-environmental chamber. Soil Properties Before experiment (control) After experiment 0N0P 60N0P 60N20P 60N40P 60N60P pH 6.99 ± 0.01 b 7.47 ± 0.04 a 7.41 ± 0.04 ab 7.45 ± 0.04 ab 7.46 ± 0.02 a 7.55 ± 0.06 a EC (mS m –1 ) 183.33 ± 3.48 a 174.00 ± 6.66 a 178.33 ± 12.45 a 185.00 ± 6.11 a 187.67 ± 11.20 a 180.33 ± 6.69 a SOC (g kg –1 ) 301.13 ± 0.35 ab 308.47 ± 1.27 a 306.83 ± 3.60 ab 285.60 ± 12.61 b 306.86 ± 8.18 a 303.11 ± 7.71 ab Total N (g kg –1 ) 3.38 ± 0.22 b 14.13 ± 3.27 a 20.29 ± 1.23 a 10.30 ± 5.35 ab 18.36 ± 1.91 a 18.82 ± 3.06 a Soil C/N Ratio 89.92 ± 5.65 a 25.10 ± 7.18 ab 15.31 ± 0.74 b 38.66 ± 22.02 ab 17.18 ± 2.23 b 16.96 ± 2.64 b NH 4 + (mg kg –1 ) 5.03 ± 0.09 b 4.94 ± 0.11 b 5.15 ± 0.27 b 5.18 ± 0.10 b 4.98 ± 0.09 b 6.22 ± 0.34 a NO 3 – (mg kg –1 ) 19.64 ± 0.06 a 11.20 ± 1.70 b 19.85 ± 0.02 a 16.29 ± 1.54 ab 16.93 ± 1.59 ab 9.09 ± 0.32 b Olsen P (mg kg –1 ) 27.22 ± 0.16 a 23.65 ± 0.23 b 22.70 ± 1.17 b 24.67 ± 0.39 ab 24.93 ± 0.84 ab 25.52 ± 0.38 ab Means of soil properties with same lowercase letters, comparing differences among treatments, are not significantly different ( p > 0.05 ) according to Fisher's least significant difference (LSD). Biomass growth rate and yield The daily switchgrass growth rates over each 7-day period, measured as the change in plant height over time, ranged from 0.03 cm day –1 in the 60N40P fertilizer rate to 2.23 cm day –1 in the unfertilized crop (Fig. 2 ). The highest switchgrass growth rates for all treatments occurred on Day 36, where daily growth rates in the unfertilized, 60N20P, 60N60P, 60N40P, and the 60N0P were 2.23 cm day –1 , 2.16 cm day –1 , 2.02 cm day –1 , 1.98 cm day –1 , and 1.83 cm day –1 , respectively. Throughout the 43 days, the 60N0P treatment had the lowest biomass growth rate (Fig. 2 ). Total dry matter yield of the switchgrass ranged from 52.20 to 198.25 g m –2 (Table 2 ). Contrary to our assumption that increasing P application rate would increase switchgrass yield, P fertilization at any rate had no significant effect on dry matter yields compared to the unfertilized treatment. Although, total dry matter yield was highest in the 60N60P treatment and was 21% higher than the unfertilized treatment, this difference in total dry matter yield was not significant ( p = 0.352 ). Surprisingly, application of 60N0P decreased total dry matter yields by 68% compared to the unfertilized crop (Table 2 ). Root biomass constituted ~ 13 to 15% of the total dry matter yield for all the treatments (Table 2 ). Similar in trend to the total dry matter yield, root and shoot biomasses, despite being highest in the 60N60P treatment were not significantly higher ( p ≥ 0.320 ) than the unfertilized treatment. Rather, the application of 60N0P without any P addition resulted in significantly lower ( p ≤ 0.015 ) root and shoot biomass yields compared to the unfertilized and 60N60P treatments. Table 2 The effect of N and P fertilizer on yield (dry matter basis), biomass concentration, and removal rates of N and P in switchgrass harvested after 43 days in a controlled-environmental chamber. Fertilizer Rate (kg ha –1 ) Dry Matter Yield (g m –2 ) Nitrogen Concentration (%) Nitrogen Removal (g m –2 ) Phosphorus Concentration (%) Phosphorus Removal (g m –2 ) Shoot Root Total Shoot Root Shoot Root Total Shoot Root Shoot Root Total 0N0P 138.99 ± 20.12 ab 24.92 ± 4.63 a 163.91 ± 24.33 a 5.48 ± 0.81 a 6.41 ± 0.61 a 7.92 ± 2.20 ab 1.54 ± 0.16 a 9.46 ± 2.35 ab 0.33 ± 0.01 b 0.59 ± 0.00 b 0.46 ± 0.07 b 0.15 ± 0.03 a 0.61 ± 0.10 b 60N 45.58 ± 1.65 c 6.62 ± 0.43 b 52.20 ± 2.08 b 3.44 ± 0.01 b 1.74 ± 0.01 bc 1.57 ± 0.05 c 0.11 ± 0.01 c 1.68 ± 0.06 c 0.41 ± 0.06 b 0.54 ± 0.01 c 0.18 ± 0.05 c 0.04 ± 0.00 c 0.22 ± 0.05 c 60N20P 118.72 ± 15.19 ab 18.34 ± 3.95 ab 137.06 ± 19.03 a 3.85 ± 0.03 ab 1.70 ± 0.02 bc 4.57 ± 0.56 bc 0.31 ± 0.07 b 4.88 ± 0.63 bc 1.63 ± 0.01 a 0.39 ± 0.01 d 1.94 ± 0.25 a 0.07 ± 0.02 bc 2.01 ± 0.27 a 60N40P 101.77 ± 25.07 bc 17.21 ± 4.28 ab 118.98 ± 29.25 ab 3.85 ± 0.01 ab 1.69 ± 0.01 c 3.92 ± 0.96 bc 0.29 ± 0.07 b 4.21 ± 1.03 c 1.28 ± 0.20 ab 0.70 ± 0.02 a 1.39 ± 0.54 a 0.12 ± 0.01 ab 1.51 ± 0.57 ab 60N60P 169.92 ± 17.27 a 28.33 ± 1.50 a 198.25 ± 18.77 a 5.88 ± 0.73 a 4.63 ± 1.73 ab 11.03 ± 2.62 a 0.90 ± 0.44 a 11.93 ± 3.06 a 1.24 ± 0.27 ab 0.60 ± 0.01 b 1.90 ± 0.82 a 0.17 ± 0.01 a 2.07 ± 0.83 a Means with same lowercase letters, comparing differences among treatments for dry matter yield, nitrogen concentration, nitrogen removal, phosphorus concentration, and phosphorus removal, are not significantly different ( p > 0.05 ) according to Fisher's least significant difference (LSD). Biomass nitrogen and phosphorus concentrations and removal Nitrogen and P fertilization of switchgrass influenced the distribution of N concentration between shoot and root biomass. In the fertilized crops, the N concentration in the shoots was higher compared to the roots, while in the unfertilized treatment (0N0P), the reverse was observed, with a higher N concentration in the roots than in the shoots (Table 2 ). Phosphorus fertilization at rates lower than 60 kg ha –1 had no significant effect on shoot N concentration. Only at the highest P rate (60 kg ha –1 ) was the shoot N concentration significantly higher ( p = 0.003 ) compared to the 60N0P treatment. A similar trend was observed for root N concentration, with the highest P rate yielding greater root N concentrations compared to the 0, 20, and 40 kg P ha –1 rates. The overall total switchgrass biomass N removal was highest (12 g m –2 ) in the 60N60P and lowest (2 g m –2 ) in the 60N0P-fertilized crop. Except for the unfertilized treatment, total biomass N removal was significantly higher ( p ≤ 0.023 ) in the 60N60P than all the other treatments. While P fertilization resulted in higher P concentration in shoots than roots, higher P concentrations in roots than shoots were observed in the non-P-fertilized (unfertilized and 60N0P) crops. Shoot P concentration was significantly higher ( p ≤ 0.006 ) in the 60N20P than the unfertilized and 60N0P but did not differ significantly ( p ≥ 0.201 ) in the 60N40P and 60N60P than the unfertilized and 60N0P treatments. The higher shoot P concentrations with P fertilization resulted in an overall increased biomass P removal compared to the non-P-fertilized treatments (Table 2 ). Nitrogen fertilization without P addition (60N0P) significantly reduced ( p = 0.030 ) total biomass P removal compared to the unfertilized crop. Nitrous oxide emission Mean N 2 O fluxes from all treatments ranged from − 0.026 to 0.953 µg N 2 O–N m – 2 hr – 1 (Fig. 3 ) and were greatest in the first 6 days of the study with peak fluxes occurring on day 4 (Fig. 3 ). Within the first 6 days of the experiment, ~ 46%, 72%, 73%, 75%, and 78% of the cumulative N 2 O emissions occurred in the unfertilized, 60N20P, 60N40P, 60N60P, and 60N0P-fertilized crops, respectively. After this period, N 2 O fluxes decreased and were near zero for all the treatments. The 43-day cumulative N 2 O emissions ranged from 25 to 103 µg N 2 O–N m –2 and was highest in the 60N40P and lowest in the unfertilized treatment (Fig. 4 ). Cumulative N 2 O emissions were 2– 4 times greater in the fertilized than the unfertilized crop. Among the fertilized crops, the switchgrass which received the 60N20P fertilizer rate had the lowest cumulative N 2 O emissions (Fig. 4 ). Discussions Effect of phosphorus addition on soil nitrogen and phosphorus contents Contrarily to our hypothesis that increasing P application rates would induce higher root and shoot biomass and N uptake, thereby depleting soil available N, we did not observe lower soil NH 4 + and NO 3 – contents at high P rates. Instead, soil analyses conducted before and after the experiment indicated an increase in soil total N following switchgrass cultivation. The increase in soil total N following N and P fertilization in our study may be due to enhanced plant and microbial immobilization of the applied N. Our findings are consistent with Wang et al ( 2022 ), who reported increased soil total N levels following fertilization, which they attributed to enhanced plant and microbial immobilization of available N in the soil. Fertilizing high organic C soils with mineral N can promote N immobilization, resulting in higher soil total N (Wang et al. 2022 ). Given that the potting mix soil used in this study had a very high SOC content, it is possible that a significant proportion of the applied N was immobilized into organic forms, contributing to the observed increase in soil total N. While N immobilization by the SOC-rich potting mix soil may have contributed to increased soil total N following fertilization in the switchgrass treatments, the higher soil total N observed in the unfertilized crop, despite receiving no N fertilization compared to the control, suggests the involvement of additional biological processes. One possible explanation is the biological N-fixation capability of switchgrass through its association with free-living diazotrophs in the soil (Mao et al. 2011 ). These free-living N-fixers can contribute 2–3 kg N ha − 1 yr –1 (Son 2001 ). Agronomic practices such as fertilization can reduce diazotrophs, with unfertilized systems enhancing the abundance and diversity of these N-fixing bacteria (Mao et al. 2011 ). Hence, in the unfertilized crop, the potential increased activities of N-fixing bacteria associated with the switchgrass may have contributed to the total N pool resulting in higher soil total N in the unfertilized treatment compared to the total N measured in the control. Additionally, the lack of any significant increase in the soil available P in the P-fertilized crops may be due to increased biomass and microbial removal of soil available P which may have immobilized the P into unavailable pools. This could be evidenced by the fact that biomass P removal was higher in the P-fertilized compared to 60N0P-fertilized crop. Switchgrass biomass growth rate and yield response to nitrogen and phosphorus fertilization The significant reduction in biomass yield observed with the 60N0P treatment, although not entirely clear, may be attributed to a potential N-induced nutrient imbalance caused by the application of N without P addition. Khan et al ( 2023 ) noted that such imbalances can cause inefficient N use by plants, leading to suboptimal growth and yield. Guo et al (2016) found that the combined application of multiple fertilizers had a more pronounced effect on the physiological metabolism of plants compared to the use of a single type of fertilizer. Similarly, Anas et al ( 2020 ) reported that the combined application of N and P produced better positive interactions, enhancing N use efficiency compared to applying N alone. Therefore, unlike in the unfertilized and P-fertilized crops, the sole application of N in the 60N0P treatment may have disrupted the balance of other essential nutrients needed to promote biomass productivity, consequently, resulting in a drastic reduction in biomass yield. Another potential explanation for the reduced biomass yield observed with the application of N fertilizer, compared to the unfertilized crop, could be attributed to a decrease in arbuscular mycorrhizal fungi (AMF) colonization of switchgrass roots. Previous studies have shown that increased levels of inorganic N application can lower AMF biomass, species richness, and diversity (Liu et al. 2012 ; Albizua et al. 2015 ). For instance, Williams et al ( 2017 ) reported that unfertilized agricultural soils in Sweden were associated with a more distinct AMF community, while the application of 50 kg ha –1 yr –1 N was sufficient to reduce AMF biomass in these soils. We suggest that the application of N fertilizer may have reduced AMF biomass compared to the unfertilized crop, which likely maintained enhanced AMF colonization. The higher AMF biomass in the unfertilized crop could have supported improved nutrient uptake and balanced nutrition, contributing to the higher biomass yield compared to the N-only fertilized treatment. This reduction in AMF colonization in response to N fertilization could also explain the lack of yield response to P fertilization. Brejda ( 2000 ) determined that the combined application of high P rates with low N rates (as low as 56 kg N ha –1 ) in native warm-season grasses, such as switchgrass, would likely result in little to no yield response. This is because AMF colonization of switchgrass roots can facilitate P uptake even at low soil P levels, reducing the impact of P fertilization on switchgrass biomass yield (Brejda 2000 ). Consistent with this, Muir et al ( 2001 ) found no yield response in switchgrass when P was applied at rates of 0, 10, 20, 30, or 39 kg P ha –1 to low-P soils (4–11 mg P kg⁻¹) in Texas, USA. Similarly, Jung et al ( 1988 ) observed no significant yield response with 20 and 40 kg ha –1 P fertilizer application to switchgrass on a strongly acidic, low-P soil in western Pennsylvania due to the presence and activities of AMF. Thus, it is possible that the association of AMF with switchgrass roots in our study may have enhanced P uptake, obscuring any potential yield benefits from P fertilization and contributing to the overall lack of yield response to P application. Also, the yield response of warm-season grasses to P fertilization is largely influenced by the soil P status, with positive yield responses more likely to occur in low-P compared to high-P soils (Kering et al. 2012 ; Siri-Prieto et al. 2020 ). Ros et al ( 2020 ) observed that P fertilization in grasses grown on soils with ≤ 5 mg kg − 1 available P increased yields by 110%, whereas yield increases were limited to only 7–25% in soils with > 5 mg kg − 1 available P. Anderson and Shapiro ( 1990 ) categorized soil available P levels based on Olsen-P soil tests as very low (0–3 mg kg –1 ), low (4–10 mg kg –1 ), medium (10–17 mg kg –1 ), and high (> 17 mg kg –1 ). This categorization indicates that the potting mix soil used in our experiment, with an Olsen-P level of 27 mg kg –1 , had a high P status. Therefore, P may not have been in limited supply to the switchgrass, reducing the likelihood of a positive yield response to P fertilization. Brejda ( 2000 ) also noted that warm-season grasses, such as switchgrass, typically have a lower P requirement compared to cool-season grasses. This lower P requirement, combined with the high Olsen-P status of the potting mix soil, may explain the lack of a significant yield response to P fertilization in our study as the sufficient soil P levels likely met the switchgrass nutrient needs without additional P supplementation, resulting in no observable yield benefit from fertilizer P application. It is equally possible that the short duration of our study may have also contributed to the lack of yield response to P fertilization. Switchgrass typically does not reach its full potential yield until its third year of growth, as it allocates much of its energy during the first two years to establishing an extensive root network (Siri-Prieto et al. 2020 ). This initial focus on root development over shoot biomass production can minimize the above-ground yield response to P fertilization in early growth stages. Therefore, it is essential to replicate this study over longer time periods to fully capture the effects of P fertilization on switchgrass yields. Long-term experiments would allow for a comprehensive understanding of how different P application rates influence yield as the crop transitions from root establishment to full productivity. Such studies are critical for determining the long-term nutrient requirements of switchgrass and optimizing P management strategies for sustainable biomass production. Nitrogen and phosphorus concentrations and removal in biomass The amount of nutrients removed from soil is a function of yield and the concentration of that nutrient in the plant sample (Massey 2020 ). It was expected that increased yield from high P rates would increase yield with resultant increase in biomass N and P concentrations and removal from soil. However, lower yield, particularly, in the N-only fertilized crop led to significantly lower N and P concentrations and removal in both shoot and root biomasses compared to the unfertilized crop. While lower N concentrations and removal in the 60N0P and 60N40P fertilized compared to unfertilized crop could be attributed to lower yield, higher P concentrations and removal in the P-fertilized crops may be due to the higher P contents from the P fertilization compared to the treatments which did not receive any P application. Our observation of higher P concentrations and removals in the switchgrass biomass at higher P rates is consistent with findings from other studies (Kering et al. 2012 ; Ashworth et al. 2019 ; Sawyer et al. 2019 ). Ashworth et al ( 2019 ) observed highest P uptake in switchgrass biomass occurred with higher P fertilizer rates. Kering et al ( 2012 ) also determined 11% higher P concentrations with the application of 45 kg P ha –1 in switchgrass compared to the unfertilized crop across two sites in Oklahoma, USA, whereas Sawyer et al ( 2019 ) determined a linear increase in switchgrass biomass P concentration and removal with increased P application rate, with P removal increasing by 0.027 kg for every kg ha − 1 of applied P. Although, other studies observed increased yield with higher P application rates, the lack of significant impact of increased P application rate on yield in our study implies that increasing P application rates in P-sufficient soils could result in increased P removal with no beneficial impact on yield, reducing the P-use efficiency in switchgrass. At the onset of switchgrass growth, nutrients are translocated from roots to shoots (Massey 2020 ). However, as switchgrass matures and move towards flowering and senescence, nutrients is translocated from shoots back to roots (Massey 2020 ). Whereas translocation of N and P from shoot to roots will increase N and P concentrations in roots relative to shoots, the opposite would be observed with translocation from roots to shoots. Hence, the higher concentrations of N and P in shoots than roots in the N- and P-fertilized crops in our study may mean that the increased availability of N and P in soil from fertilization made it possible for increased translocation of these nutrients from roots to shoots. This is especially so when it is considered that biomass N and P concentrations were determined only 43 days after seeding. Makaju et al ( 2013 ) measured highest N and P concentrations in switchgrass biomass one month (May) after the beginning (April) of switchgrass growth. This period of increased biomass accumulation of N and P coincides with the 43 days after seeding switchgrass in our study. Therefore, we allude to the higher N and P concentrations in the shoots of our N- and P-fertilized switchgrass than roots to N and P translocation from roots (belowground) to shoots (aboveground). While the higher N and P concentrations in the shoots than roots of the N- and P-fertilized crops may have been enhanced by higher N and P availability from fertilization, lower N and P concentrations in shoots than roots of the unfertilized switchgrass may have been due to relatively lower soil N and P availability from lack of fertilization. Changes in resource supply such as soil nutrient concentrations can impact how plants allocate soil nutrient resources (Wang et al. 2023 ). It is, therefore, possible that at the relatively lower available N and P concentrations, the unfertilized switchgrass may have invested much of the available N and P in roots before shoots. This strategy of N and P allocation could potentially result in higher concentrations of N and P in roots compared to shoots at the early stages of biomass growth. Further investigations are required to understand how switchgrass may allocate soil nutrient resources when in limited amounts and how that could potentially impact biomass productivity and the provisioning of other ecological services such as soil C sequestration and reduced GHG emissions. Effect of phosphorus fertilization on nitrous oxide emissions The nonlinear response of N 2 O emissions to P fertilization observed in our study, where the lowest P rate reduced N₂O emissions but increased with higher P rates did not support our hypothesis that increasing P rates would reduce N 2 O emissions. This observation suggests a potentially more complex interaction between P application and soil N₂O dynamics at the initial stage of switchgrass cultivation, and that while moderate P application might help mitigate N 2 O emissions, excessive P inputs could potentially promote conditions that enhance N 2 O emissions. We suspect that increasing the rate of P application may have stimulated N mineralization, increasing the availability of soil inorganic N (NH 4 + and NO 3 – ) at a time when N uptake by switchgrass was lowest. At higher P rate of 2000 mg P kg –1 soil, Mori et al ( 2010 ) observed increased N 2 O emissions in laboratory incubated soil from a 1-year-old Acacia mangium plantation in Indonesia due to increased stimulation of N cycling and losses as N 2 O. Wang et al ( 2022 ) also determined that P application accelerated some of the N cycling processes in the soil such as gross N mineralization, gross nitrification, and denitrification, with the effect sizes increasing with P addition rates. Evidence from these studies indicate that increased P application rates can result in increased N cycling and losses as N 2 O as observed in this study. This potential increase in soil N availability, combined with reduced N uptake by switchgrass during its early growth stage, may have led to the lost of the available N as N 2 O. The implication of this is that the timing of P application relative to plant N demand is critical in minimizing N 2 O emissions. Aside the potential increased stimulation of N cycling in the soil which may have caused higher N losses as N 2 O under higher P rates, the ability of N and P fertilization to stimulate nitrifying and denitrifying bacteria (Mori et al. 2010 ; Thompson et al. 2018 ) could have equally resulted in higher N 2 O emissions with higher P fertilization rates and under the 60N0P fertilization compared to the unfertilized crop. Mori et al ( 2013 ) demonstrated the significant positive impact P fertilization have on stimulating the activities of denitrifying bacteria in the soil. He and Dijkstra ( 2015 ) also explained that the simultaneous application of inorganic N and P fertilizers stimulate nitrifiers and denitrifiers, increasing N 2 O emissions. Thompson et al ( 2018 ) measured increased denitrifying bacteria genes ( nirS and nosZ ) in N-fertilized switchgrass plots compared to the unfertilized plots. Hence, compared to the unfertilized crop where majority of the soil residual N and P may have possibly been used for biomass production, the increased availability of soil N and P content from fertilization may have stimulated the soil nitrifier and denitrifier community which resulted in increased N 2 O emissions compared to the unfertilized crop. The increased N 2 O emissions within the first 6 days of our study could be explained by the absence of roots prior to the germination of the switchgrass seeds. The first germination in our study occurred on the 5th day of the experiment. Mori et al ( 2013 ) explained that in the absence of roots, N 2 O emission is elevated due to reduced N uptake in plants. Therefore, our findings support the argument that N-fertilization of switchgrass at the initial establishment phase should be avoided (Samson et al. 2016 ). This could ensure that N 2 O emissions from N application is curtailed while inhibiting early weed growth. Conclusions Contrary to our hypothesis that higher P application would enhance soil N uptake in switchgrass biomass, thereby reducing N 2 O emissions, our study showed an increase in total soil N. This increase was likely due to enhanced microbial and plant immobilization of applied N, as well as the contribution of N from biological N-fixation in the unfertilized switchgrass. Despite the increase in soil total N, the combined application of N and P did not significantly impact switchgrass biomass yield. In contrast, N-only application significantly reduced biomass yields compared to the unfertilized crop. The lack of yield response from combined N and P application was attributed to high baseline soil P levels and a possible reduction in AMF colonization whereas the yield reduction from N-only application was likely due to N-induced nutrient imbalances that affected soil nutrient uptake and utilization. Although P fertilization resulted in the highest nutrient removal in biomass, its use efficiency was reduced as yield did not increase proportionately with P application rate. The 43-day cumulative N 2 O emissions ranged from 25 to 103 µg N 2 O-N m –2 , with the highest emissions observed at the 60N40P rate and the lowest in the unfertilized crop. P fertilization exhibited a nonlinear effect on N 2 O emissions. While P at 20 kg ha –1 reduced emissions compared to N-only treatment, increasing P to 40 kg ha –1 resulted in the highest emissions, surpassing those of N-only treatments. Emissions at 60 kg ha –1 were similar to N-only treatment. These results indicate that while moderate P application may reduce N 2 O emissions, higher P rates may enhance emissions due to changes in microbial activity and N cycling in the soil. Additionally, N 2 O emissions were greatest during the first week of our study prior to switchgrass root development, supporting the argument that fertilization of switchgrass at the initial establishment phase should be avoided to mitigate N 2 O emissions. Even though this study increases our knowledge of P-fertilization on N 2 O emissions in switchgrass and may serve as basis for long-term replication on the field or in the lab using field soils, further research is needed to identify optimal timing and combined N and P application strategies to maximize biomass yield and minimize N 2 O emissions under field conditions. Declarations Acknowledgement Funding for this study was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC) (Grant # RGPIN-2018-04346) and the Canadian Foundation for Innovation (CFI) (Grant # 12896). This work was supported by the University of Waterloo by providing research infrastructure and funding to Augustine K. Osei. Global Affairs Canada’s Emerging Leaders in the Americas Program also provided funding to support Nadia Gabbanelli’s internship at the University of Waterloo. We are also grateful to the editors and reviewers that helped improve this manuscript. Competing Interests The authors have no relevant financial or non-financial interests to disclose. Author Contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Augustine K. Osei and Nadia Gabbanelli. The first draft of the manuscript was written by Augustine K. Osei and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Agostini, F., Gregory, A. S., & Richter, G. M. (2015). 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Assessing soil nitrous oxide emission as affected by phosphorus and nitrogen addition under two moisture levels. Journal of Integrative Agriculture , 15 (12), 2865-2872. https://doi.org/10.1016/S2095-3119(16)61353-9 Wang, L., Chen, X., Yan, X., Wang, C., Guan, P., & Tang, Z. (2023). A response of biomass and nutrient allocation to the combined effects of soil nutrient, arbuscular mycorrhizal, and root-knot nematode in cherry tomato. Frontiers in Ecology and Evolution , 11 , 1106122. https://doi.org/10.3389/fevo.2023.1106122 Wang, R., Bicharanloo, B., Hou, E., Jiang, Y., & Dijkstra, F. A. (2022). Phosphorus supply increases nitrogen transformation rates and retention in soil: a global meta‐analysis. Earth's Future , 10 (3), e2021EF002479. https://doi.org/10.1029/2021EF002479 Wang, S., Sanford, G. R., Robertson, G. P., Jackson, R. D., & Thelen, K. D. (2020). Perennial bioenergy crop yield and quality response to nitrogen fertilization. BioEnergy Research , 13 , 157-166. https://doi.org/10.1007/s12155-019-10072-z Wile, A., Burton, D. L., Sharifi, M., Lynch, D., Main, M., & Papadopoulos, Y. A. (2014). Effect of nitrogen fertilizer application rate on yield, methane and nitrous oxide emissions from switchgrass (Panicum virgatum L.) and reed canarygrass (Phalaris arundinacea L.). Canadian Journal of Soil Science , 94 (2), 129–137. https://doi.org/10.4141/CJSS2013-058 Williams, A., Manoharan, L., Rosenstock, N. P., Olsson, P. A., & Hedlund, K. (2017). Long‐term agricultural fertilization alters arbuscular mycorrhizal fungal community composition and barley (H ordeum vulgare) mycorrhizal carbon and phosphorus exchange. New Phytologist , 213 (2), 874-885. https://doi.org/10.1111/nph.14196 Zhang, Y., Wang, C., & Li, Y. (2019). Contrasting effects of nitrogen and phosphorus additions on soil nitrous oxide fluxes and enzyme activities in an alpine wetland of the Tibetan Plateau. Plos One , 14 (5), e0216244. https://doi.org/10.1371/journal.pone.0216244 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5328479","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":376934892,"identity":"8161617c-16c8-45cd-b61a-cfcce2113cb5","order_by":0,"name":"Augustine Kwame Osei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYLCCBAMJOQYGHhiXjRgtBTbGJGph+JCW2EC0Fv729ocPHhgcTt9w/OzBBx8Y7OQYJNIS8GqROHPG2CDB4HDuhjN5yYYzGJKNgVoO4LfmRg6bBFjLgRwzaR6GA4kNEukNeHXI30h//gOoJd3g/BsitRjcSDADBnJagsENuC0EHGYI9AvQYTaGM2+8MTacYZBszMbzLAGvFrnj7Q8//vgjIc93PsfwwYcKOzl+9jQDvFrgQAHsGgMiIxIM5BuIVjoKRsEoGAUjDQAATIhGRUSCf40AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-3797-5701","institution":"University of Waterloo Faculty of Environment","correspondingAuthor":true,"prefix":"","firstName":"Augustine","middleName":"Kwame","lastName":"Osei","suffix":""},{"id":376934893,"identity":"b52cb219-4762-46ad-be48-f99151619a1a","order_by":1,"name":"Nadia Gabbanelli","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Nadia","middleName":"","lastName":"Gabbanelli","suffix":""},{"id":376934894,"identity":"304ee925-2e12-4ab9-90dd-67ff60ad2122","order_by":2,"name":"Maren Oelbermann","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Maren","middleName":"","lastName":"Oelbermann","suffix":""}],"badges":[],"createdAt":"2024-10-24 23:03:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5328479/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5328479/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":70177206,"identity":"32732935-48b5-429e-9ca7-ee964cb894cb","added_by":"auto","created_at":"2024-11-29 07:40:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":367973,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental design using PVC tubes with (A) young plants, (B) matured plants, and (C) tube covers with gas sampling ports inside the Conviron PGR-15 controlled environmental chamber.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5328479/v1/bde96c5f1ae6770c253245b6.png"},{"id":70177204,"identity":"c77218ed-3399-4b46-a2dc-9c5190947877","added_by":"auto","created_at":"2024-11-29 07:40:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":57693,"visible":true,"origin":"","legend":"\u003cp\u003eAverage weekly plant height (cm) represented by bar graph and growth rate (cm day\u003csup\u003e–1\u003c/sup\u003e) by line graph of switchgrass with different fertilizer rates during a 43-day period.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5328479/v1/135d3cd835bfedecf9bea373.png"},{"id":70177207,"identity":"fef97cf0-67ca-4c32-ac7f-1de2dffc48b0","added_by":"auto","created_at":"2024-11-29 07:40:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":42553,"visible":true,"origin":"","legend":"\u003cp\u003eMean N\u003csub\u003e2\u003c/sub\u003eO-N fluxes (µg m\u003csup\u003e–2\u003c/sup\u003e hr\u003csup\u003e–1\u003c/sup\u003e) from switchgrass with different fertilizer rates during a 43-day period.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5328479/v1/963e74cabc5554a2f13573e8.png"},{"id":70177381,"identity":"18102f34-83ff-449f-8ea9-91abf7ebdc16","added_by":"auto","created_at":"2024-11-29 07:48:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":18295,"visible":true,"origin":"","legend":"\u003cp\u003e43-day Cumulative N\u003csub\u003e2\u003c/sub\u003eO-N emissions (µg m\u003csup\u003e–2\u003c/sup\u003e) from switchgrass with different fertilizer rates in a controlled-environmental chamber. Bars represent standard error of cumulative means (n = 3). Error bars with same uppercase letters show cumulative means are not significantly different (\u003cem\u003ep\u0026gt;0.05\u003c/em\u003e) according to Fisher's least significant difference (LSD).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5328479/v1/bce5506a56a50c5e0490ccca.png"},{"id":74311735,"identity":"d488c995-430a-4116-b5d1-66fde181d980","added_by":"auto","created_at":"2025-01-21 02:15:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1673569,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5328479/v1/bcd2d76a-1539-44a8-ab2c-7505a6730e7e.pdf"}],"financialInterests":"","formattedTitle":"Impact of Phosphorus Fertilization Rates on Nitrous Oxide Emissions in Switchgrass: Nonlinear Response Reveals Emission Reduction at Low Phosphorus Rates","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBioenergy plays a critical role in decarbonizing our energy systems as well as limiting global warming (Lemus and Lal \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). While current bioenergy feedstocks from food crops such as corn (\u003cem\u003eZea mays\u003c/em\u003e L.), soybeans (\u003cem\u003eGlycine max\u003c/em\u003e L.), canola (\u003cem\u003eBrassica napus\u003c/em\u003e L.), etc., have come under scrutiny due to the myriad of environmental and climate challenges associated with their production (Pool \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Searchinger and Heimlich \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), the use of lignocellulosic crops such as herbaceous and short rotation woody crops have attracted great interests (Agostini et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Perea-Moreno et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Osei et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Key among the lignocellulosic crops recommended for bioenergy production is switchgrass (\u003cem\u003ePanicum virgatum\u003c/em\u003e L.) \u0026ndash; a perennial warm season grass native to North America (Samson et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The interest in switchgrass as bioenergy feedstock stems from its low maintenance cost and ability to adapt and produce high amounts of biomass under marginal conditions (Samson et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Additionally, switchgrass can provide climate mitigation benefits through increased soil carbon (C) sequestration and reduced greenhouse gas (GHG) emissions (Blanco-Canqui \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Coleman et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Bazrgar et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAlthough, switchgrass can produce high yields without fertilizer inputs (Ruan et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Wang et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), optimizing yields may require fertilizer additions (Lee et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Kering et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). For instance, the Ontario Biomass Producers Co-operative in Canada recommend an annual application of 60\u0026ndash;70 kg N ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e for improved switchgrass biomass yields (Samson et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Schmer et al (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) measured 6.8 Mg ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e aboveground dry matter yield for switchgrass receiving 67 kg N ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e compared to 3.2 Mg ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e without nitrogen (N) fertilizer. Niki\u0026egrave;ma et al (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) also observed 1.5- and 2.5-fold increase in switchgrass biomass with 56 kg ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e and 112 kg ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e N fertilization, respectively. While these results point to higher potential biomass yields from N fertilization in switchgrass, the resultant nitrous oxide (N\u003csub\u003e2\u003c/sub\u003eO) emissions from N fertilizer may offset any climate benefits derived from the increased yield of switchgrass for bioenergy production.\u003c/p\u003e \u003cp\u003eMcGowan et al (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) studied the impact of different N rates in switchgrass and observed that total N\u003csub\u003e2\u003c/sub\u003eO emissions increased from 0.2 to 3.0 kg N\u003csub\u003e2\u003c/sub\u003eO-N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e as N rates increased from 0 to 150 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Wile et al (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) observed that fertilization of switchgrass with 120 kg N ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e resulted in a 73% increase in N\u003csub\u003e2\u003c/sub\u003eO emissions over 2 years compared to the unfertilized crop. Jin et al (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) argued that N fertilization of switchgrass can result in increased N\u003csub\u003e2\u003c/sub\u003eO emissions offsetting the GHG balance of switchgrass as a bioenergy crop. Whereas these and previous studies (\u003cem\u003ee.g\u003c/em\u003e., Niki\u0026egrave;ma et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Pannu et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Rau et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) examined N fertilization on N\u003csub\u003e2\u003c/sub\u003eO emissions in switchgrass, studies measuring the response of phosphorus (P) fertilization on N\u003csub\u003e2\u003c/sub\u003eO emissions in switchgrass have seldom been conducted. This is despite the contradictory findings on the impact of P fertilization on N cycling and N\u003csub\u003e2\u003c/sub\u003eO emissions. For instance, Ullah et al (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and Zhang et al (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) found increased N\u003csub\u003e2\u003c/sub\u003eO emissions from P fertilization due to the increased stimulation of nitrifying and/or denitrifying bacteria activities. Mori et al (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) also suggested that P addition enhanced N cycling and caused accumulation of available N for nitrification and denitrification which resulted in increased N\u003csub\u003e2\u003c/sub\u003eO emission. Contrary to these findings, other studies (Baral et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Mori et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) observed reduced N\u003csub\u003e2\u003c/sub\u003eO emissions with P addition to soils due to increased N uptake in plant biomass. Zhang et al (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), on the other hand found no impact of P addition on N\u003csub\u003e2\u003c/sub\u003eO emissions. These contrasting results of N\u003csub\u003e2\u003c/sub\u003eO emissions with P addition indicate that the effect of P on N\u003csub\u003e2\u003c/sub\u003eO emissions may differ with crop type, soil nutrient content, and environmental conditions.\u003c/p\u003e \u003cp\u003eFrom an agronomic perspective, N and P are two major limiting macronutrients whose application positively influences switchgrass yield (Niki\u0026egrave;ma et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Kering et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Sawyer et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Studies on N and P fertilization in switchgrass have focused on economic benefits, biomass and ethanol yields, and soil nutrients removal (Haque et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Jungers et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; McGowan et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Ashworth et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Tang et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), with less emphasis on N\u003csub\u003e2\u003c/sub\u003eO emissions. However, considering the significance of P in N cycling and N\u003csub\u003e2\u003c/sub\u003eO emissions (Mori et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Ullah et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), it is important to understand how P fertilization can influence N\u003csub\u003e2\u003c/sub\u003eO emissions in N-fertilized switchgrass and what that may mean for the climate mitigation benefit of switchgrass as a bioenergy feedstock. An experiment was conducted under controlled-environment to assess the impact of different P application rates in N-fertilized switchgrass to enhance our understanding of how P fertilization impacts N\u003csub\u003e2\u003c/sub\u003eO emissions in switchgrass. We hypothesized that P application at high rates would enhance below and aboveground biomass growth resulting in increased N uptake and lower soil available N, with a consequent reduction in N\u003csub\u003e2\u003c/sub\u003eO emissions.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design\u003c/h2\u003e \u003cp\u003eA 43-day experiment was conducted in a controlled-environment chamber (Conviron PGR-15, Controlled Environments Inc., Winnipeg, MB) at the University of Waterloo, Ontario \u0026ndash; Canada. Equal amount (equivalent of 500 g air-dry soil) of commercial potting mix soil (Vigoro\u0026reg; natural garden soil) was weighed into white polyvinyl chloride (PVC) tubes of 10 cm diameter and 25 cm height. Soil in each tube were packed at a bulk density of 0.64 g cm\u003csup\u003e\u0026ndash;3\u003c/sup\u003e occupying the bottom 10 cm depth of the tubes leaving a 15 cm headspace. A fiberglass mesh screen was attached to the bottom of each tube to allow for free drainage. The soil in each PVC tube was then saturated with deionized water and allowed to drain overnight to attain soil moisture at field capacity (Evett et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The soil moisture at field capacity for each of the tubes was measured with a WET-2 sensor (Delta-T Devices, Cambridge, UK) and recorded. The tubes were then seeded with cave-in-rock switchgrass variety at a seeding rate of 10 kg ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e (Samson et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). At the time of seeding, the following fertilizer rates, representing different treatments were supplied to each tube; (i) 60 kg N ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e (60N0P); (ii) 60kg N and 20 kg P ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e (60N20P); (iii) 60 kg N and 40 kg P ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e (60N40P); (iv) 60 kg N and 60 kg P ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e (60N60P); and (v) 0 kg N and 0 kg P ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e (0N0P). The 60 kg N ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, 20 kg P ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, 40 kg P ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, and 60 kg P ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e fertilizer rates were based on actual N and P additions of 566 mg N kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, 188 mg P kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, 376 mg P kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, and 566 mg P kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, respectively. Urea [(NH\u003csub\u003e2\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eCO] and potassium dihydrogen phosphate (KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e) were used as N and P sources, respectively. Each treatment (fertilizer rate) was replicated three times. The tubes were randomly placed inside the growth chamber (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and were systematically rotated every 3 days to account for any potential spatial variations inside the growth chamber. The growth chamber was set up in 14-hour light and 10-hour dark photoperiod cycles. Carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e), temperature, relative humidity, and light intensity during the 14-hour light photoperiod cycle were set at 420 ppm, 25\u003csup\u003eo\u003c/sup\u003eC, 65%, and 450 \u0026micro;mol m\u003csup\u003e\u0026ndash;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively, whereas their corresponding settings for the 10-hour dark photoperiod cycle were 420 ppm, 15\u003csup\u003eo\u003c/sup\u003eC, 80%, and 0 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. These climate data are typical of the average peak growing season local conditions in the Waterloo Region in southern Canada (Environment Canada).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGas sampling and nitrous oxide measurement\u003c/h3\u003e\n\u003cp\u003eThe concentration of N\u003csub\u003e2\u003c/sub\u003eO in the headspace of each tube was taken daily for the first 7 days and at 3-day intervals after day 7 of the experiment, starting at t\u0026thinsp;=\u0026thinsp;0 days and terminated at t\u0026thinsp;=\u0026thinsp;43 days. The experiment was terminated on day 43 as N\u003csub\u003e2\u003c/sub\u003eO fluxes had stabilized, and the switchgrass biomass had grown to a size that made it challenging to manage beyond this point. At the time of gas sampling, the tubes were capped with a lid containing a central sampling port and a 10-cm-long (3 mm inner diameter) ventilation duct to account for pressure differences during sampling (Lutes et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). A 10 ml gas was drawn from the headspace of each tube using a syringe and needle into previously evacuated 3 ml Labco Exetainer vials with grey butyl rubber septa (Exetainer, Labco Ltd., Buckinghamsire, UK) at 0, 15, and 30 minutes after the tubes were caped (Lutes et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). During lid closure for gas sampling in tubes with bigger plants, plants were carefully folded to prevent breaking and soil disturbance. The gas samples were analysed for N\u003csub\u003e2\u003c/sub\u003eO using a Gas Chromatograph (GC) 6890 equipped with a micro-electron capture detector (ECD) (Agilent Technologies Inc., Santa Clara, USA) (Lutes et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Gas fluxes were calculated with the HMR model in R using the chamber volume, surface area of the chamber base, and rate of change in chamber gas concentration (Pedersen et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Following each gas sampling event, soil moisture content in each tube was measured with the WET-2 sensor and where required, moisture content was adjusted with deionized water to field capacity based on the recorded soil moisture at field capacity for each tube that was measured at the time of seeding.\u003c/p\u003e\n\u003ch3\u003eSoil sampling and analysis for chemical properties\u003c/h3\u003e\n\u003cp\u003eAfter 43 days, when the experiment terminated, ~\u0026thinsp;100 g of soil was removed from the top, middle, and bottom in each tube. The collected soil from each treatment replicate was thoroughly mixed, air dried and sieved (2 mm), and analyzed for pH, EC, SOC, total N, available N, and available P. All soil analyses were performed on soil before the experiment (herein referred to as control) and after the experiment. Total N and SOC was determined using an elemental analyzer (Costech 4010, Cernusco, CA, USA) whereas the Olsen P method was used for available P analysis. Prior to soil analyses for C and total N, soil carbonates were removed according to Dyer et al (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSoil extract for orthophosphate was prepared by extracting 2.5 g of 2 mm sieved soil with 50 ml of 0.5 M NaHCO\u003csub\u003e3\u003c/sub\u003e (pH 8.5) and analyzed using a UV-Vis Spectrophotometer (Kuo 1996). Soil pH (1:1 soil: ultrapure water suspension) (Miller and Kissel \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) was measured using a pH meter (Fisherbrand, Accumet AB 15) and the EC with the WET-2 sensor. For NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e analyses, soil extracts were prepared using 2 M KCl solution and the filtered extracts were analyzed for NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e on a Shimadzu 1800 UV-Vis Spectrophotometer (Shimadzu Corp., Kyoto, Japan) (Doane and Horw\u0026aacute;th \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eBiomass yield of and determination nitrogen and phosphorus concentrations in biomass\u003c/h3\u003e\n\u003cp\u003eBeginning day 8 of the experiment, plant height for each treatment replicate was measured and recorded weekly until termination of the study on day 43. The data were used to determine switchgrass growth rate. Following termination of the experiment, aboveground biomass was harvested by cutting plants in each tube 0.5 cm above soil surface. After collecting soil from each tube for analysis, the remaining soil containing switchgrass roots was placed on top of a 2 mm sieve and the soil washed off to obtain root biomass for each tube. Both shoot and root biomass for each tube was weighed and the fresh weights recorded. The biomass was then oven-dried at 40\u003csup\u003eo\u003c/sup\u003eC until no moisture loss was detected. Dry biomass weight for shoot and roots were recorded. After oven drying, root and shoot biomasses were ground with a Kinematica Polymix Grinding Mill (Kinematica AG, Switzerland) and sieved through a 0.5 mm sieve. Subsamples of the 0.5 mm sieved root and shoot biomass for each replicate were analyzed for total N and P.\u003c/p\u003e \u003cp\u003eTotal N of the ground and 0.5 mm sieved biomass was analyzed on the elemental analyzer and total P on the spectrophotometer. Before total P analysis on the spectrophotometer, plant samples were digested in concentrated HCl (12 M) and filtered according to a method adapted from Flindt and Lilleb\u0026oslash; (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Briefly, the ground and 0.5 mm sieved biomass samples were combusted in a muffle furnace at 500 \u003csup\u003eo\u003c/sup\u003eC for 4 hours. After biomass combustion, 5 mg of the combusted ash were weighed into a 200 mL Erlenmeyer flask and mixed with 1 mL concentrated HCl and 25 mL deionized water. The mixture was placed on a heating plate at 120\u003csup\u003eo\u003c/sup\u003eC until the solution turned yellow and transparent after the water had evaporated. Volume of the transparent yellowish solution was then adjusted to 100 ml using deionized water and filtered through a Whatman 42 filter paper (Whatman 42, 2.5 \u0026micro;m). The filtrate was analyzed for total P using the ascorbic acid method (Murphy and Riley \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1962\u003c/span\u003e) with the spectrophotometer. Biomass N and P removals were calculated by multiplying N and P concentrations with dry matter biomass yields.\u003c/p\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData processing and analysis were carried out using SPSS (IBM Corp.). A one-way analysis of variance (ANOVA) was used to compare the effect of different fertilizer rate on yield, cumulative N\u003csub\u003e2\u003c/sub\u003eO emissions, biomass N and P concentrations, and soil properties before (control) and after the experiment. Prior to the ANOVA, data was assessed for homogeneity of variance (Levene's) and normality (Shapiro-Wilk Test) (Steel et al. 1980). The shoot biomass P and root biomass N concentrations were found to have violated the assumption of normality, hence, were log-transformed. The Fisher least significant difference (LSD) was used to detect significant differences between individual treatments. Type I error rate for all statistical analyses was set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003e\u0026le;\u003c/span\u003e\u0026thinsp;\u003cem\u003e0.05\u003c/em\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eSoil characteristics\u003c/h2\u003e \u003cp\u003eThe initial pH, EC, SOC, total N, soil C/N ratio, and available P contents of the commercial potting mix are reported in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Growing switchgrass for 43 days significantly increased (\u003cem\u003ep\u0026thinsp;\u0026le;\u0026thinsp;0.015\u003c/em\u003e) soil total N in the 60N0P, 60N40P and 60N60P fertilizer rates and unfertilized treatment compared to the total N in the control. Although total N was higher in the 60N20P treatment compared to the control, this difference was not statistically significant (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.123\u003c/em\u003e). Compared to the control, soil available P was significantly lower (\u003cem\u003ep\u0026thinsp;\u0026le;\u0026thinsp;0.005\u003c/em\u003e) in the unfertilized and 60N0P treatments after the 43 days. Soil available P did not differ significantly (\u003cem\u003ep\u0026thinsp;\u0026ge;\u0026thinsp;0.066\u003c/em\u003e) between any of the P-fertilized treatments compared to the control. Other than the significantly higher (\u003cem\u003ep\u0026thinsp;\u0026le;\u0026thinsp;0.003\u003c/em\u003e) soil NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e content in the 60N60P compared to all the other treatments, NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e content did not differ among any of the other treatments. Meanwhile, significantly higher (\u003cem\u003ep\u0026thinsp;\u0026le;\u0026thinsp;0.047\u003c/em\u003e) soil NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e contents were observed in the control and 60N0P compared to the 60N60P and unfertilized treatment after the 43 days (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSoil chemical properties of the commercial potting mix soil before (control) and after the 43-day experiment in switchgrass with different fertilizer rates grown in a controlled-environmental chamber.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoil Properties\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBefore experiment\u003c/p\u003e \u003cp\u003e(control)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c7\" namest=\"c3\"\u003e \u003cp\u003eAfter experiment\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0N0P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60N0P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e60N20P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e60N40P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e60N60P\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e7.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEC (mS m\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e183.33\u0026thinsp;\u0026plusmn;\u0026thinsp;3.48\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e174.00\u0026thinsp;\u0026plusmn;\u0026thinsp;6.66\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e178.33\u0026thinsp;\u0026plusmn;\u0026thinsp;12.45\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e185.00\u0026thinsp;\u0026plusmn;\u0026thinsp;6.11\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e187.67\u0026thinsp;\u0026plusmn;\u0026thinsp;11.20\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e180.33\u0026thinsp;\u0026plusmn;\u0026thinsp;6.69\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSOC (g kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e301.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e308.47\u0026thinsp;\u0026plusmn;\u0026thinsp;1.27\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e306.83\u0026thinsp;\u0026plusmn;\u0026thinsp;3.60\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e285.60\u0026thinsp;\u0026plusmn;\u0026thinsp;12.61\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e306.86\u0026thinsp;\u0026plusmn;\u0026thinsp;8.18\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e303.11\u0026thinsp;\u0026plusmn;\u0026thinsp;7.71\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal N (g kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14.13\u0026thinsp;\u0026plusmn;\u0026thinsp;3.27\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.29\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.30\u0026thinsp;\u0026plusmn;\u0026thinsp;5.35\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e18.36\u0026thinsp;\u0026plusmn;\u0026thinsp;1.91\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e18.82\u0026thinsp;\u0026plusmn;\u0026thinsp;3.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSoil C/N Ratio\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e89.92\u0026thinsp;\u0026plusmn;\u0026thinsp;5.65\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25.10\u0026thinsp;\u0026plusmn;\u0026thinsp;7.18\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e38.66\u0026thinsp;\u0026plusmn;\u0026thinsp;22.02\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e17.18\u0026thinsp;\u0026plusmn;\u0026thinsp;2.23\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e16.96\u0026thinsp;\u0026plusmn;\u0026thinsp;2.64\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e (mg kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e6.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e (mg kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.29\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16.93\u0026thinsp;\u0026plusmn;\u0026thinsp;1.59\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOlsen P (mg kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e24.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e24.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e25.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMeans of soil properties with same lowercase letters, comparing differences among treatments, are not significantly different (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) according to Fisher's least significant difference (LSD).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eBiomass growth rate and yield\u003c/h3\u003e\n\u003cp\u003eThe daily switchgrass growth rates over each 7-day period, measured as the change in plant height over time, ranged from 0.03 cm day\u003csup\u003e\u0026ndash;1\u003c/sup\u003e in the 60N40P fertilizer rate to 2.23 cm day\u003csup\u003e\u0026ndash;1\u003c/sup\u003e in the unfertilized crop (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The highest switchgrass growth rates for all treatments occurred on Day 36, where daily growth rates in the unfertilized, 60N20P, 60N60P, 60N40P, and the 60N0P were 2.23 cm day\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, 2.16 cm day\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, 2.02 cm day\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, 1.98 cm day\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, and 1.83 cm day\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, respectively. Throughout the 43 days, the 60N0P treatment had the lowest biomass growth rate (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTotal dry matter yield of the switchgrass ranged from 52.20 to 198.25 g m\u003csup\u003e\u0026ndash;2\u003c/sup\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Contrary to our assumption that increasing P application rate would increase switchgrass yield, P fertilization at any rate had no significant effect on dry matter yields compared to the unfertilized treatment. Although, total dry matter yield was highest in the 60N60P treatment and was 21% higher than the unfertilized treatment, this difference in total dry matter yield was not significant (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.352\u003c/em\u003e). Surprisingly, application of 60N0P decreased total dry matter yields by 68% compared to the unfertilized crop (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Root biomass constituted\u0026thinsp;~\u0026thinsp;13 to 15% of the total dry matter yield for all the treatments (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Similar in trend to the total dry matter yield, root and shoot biomasses, despite being highest in the 60N60P treatment were not significantly higher (\u003cem\u003ep\u0026thinsp;\u0026ge;\u0026thinsp;0.320\u003c/em\u003e) than the unfertilized treatment. Rather, the application of 60N0P without any P addition resulted in significantly lower (\u003cem\u003ep\u0026thinsp;\u0026le;\u0026thinsp;0.015\u003c/em\u003e) root and shoot biomass yields compared to the unfertilized and 60N60P treatments.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe effect of N and P fertilizer on yield (dry matter basis), biomass concentration, and removal rates of N and P in switchgrass harvested after 43 days in a controlled-environmental chamber.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"14\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c14\" colnum=\"14\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFertilizer Rate\u003c/p\u003e \u003cp\u003e(kg ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eDry Matter Yield (g m\u003csup\u003e\u0026ndash;2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eNitrogen Concentration (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e \u003cp\u003eNitrogen Removal \u003c/p\u003e \u003cp\u003e(g m\u003csup\u003e\u0026ndash;2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e \u003cp\u003ePhosphorus Concentration (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c14\" namest=\"c12\"\u003e \u003cp\u003ePhosphorus Removal \u003c/p\u003e \u003cp\u003e(g m\u003csup\u003e\u0026ndash;2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShoot\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRoot\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eShoot\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRoot\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eShoot\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eRoot\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eShoot\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eRoot\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003eShoot\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003eRoot\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0N0P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e138.99\u0026thinsp;\u0026plusmn;\u0026thinsp;20.12\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.92 \u0026plusmn;\u003c/p\u003e \u003cp\u003e4.63\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e163.91 \u0026plusmn; 24.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.92\u0026thinsp;\u0026plusmn;\u0026thinsp;2.20\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e9.46\u0026thinsp;\u0026plusmn;\u0026thinsp;2.35\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.46\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60N\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e45.58\u0026thinsp;\u0026plusmn;\u0026thinsp;1.65\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e52.20\u0026thinsp;\u0026plusmn;\u0026thinsp;2.08\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e1.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60N20P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e118.72\u0026thinsp;\u0026plusmn;\u0026thinsp;15.19\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e18.34 \u0026plusmn;\u003c/p\u003e \u003cp\u003e3.95\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e137.06\u0026thinsp;\u0026plusmn;\u0026thinsp;19.03\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e1.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e2.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60N40P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e101.77\u0026thinsp;\u0026plusmn;\u0026thinsp;25.07\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e17.21 \u0026plusmn;\u003c/p\u003e \u003cp\u003e4.28\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e118.98\u0026thinsp;\u0026plusmn;\u0026thinsp;29.25\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.92\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e4.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e1.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.54\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e1.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e60N60P\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e169.92\u0026thinsp;\u0026plusmn;\u0026thinsp;17.27\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28.33 \u0026plusmn;\u003c/p\u003e \u003cp\u003e1.50\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e198.25\u0026thinsp;\u0026plusmn;\u0026thinsp;18.77\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e5.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.63\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e11.03\u0026thinsp;\u0026plusmn;\u0026thinsp;2.62\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e11.93\u0026thinsp;\u0026plusmn;\u0026thinsp;3.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e1.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e0.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e1.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.82\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c14\"\u003e \u003cp\u003e2.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eMeans with same lowercase letters, comparing differences among treatments for dry matter yield, nitrogen concentration, nitrogen removal, phosphorus concentration, and phosphorus removal, are not significantly different (\u003cem\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e) according to Fisher's least significant difference (LSD).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBiomass nitrogen and phosphorus concentrations and removal\u003c/h2\u003e \u003cp\u003eNitrogen and P fertilization of switchgrass influenced the distribution of N concentration between shoot and root biomass. In the fertilized crops, the N concentration in the shoots was higher compared to the roots, while in the unfertilized treatment (0N0P), the reverse was observed, with a higher N concentration in the roots than in the shoots (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Phosphorus fertilization at rates lower than 60 kg ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e had no significant effect on shoot N concentration. Only at the highest P rate (60 kg ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e) was the shoot N concentration significantly higher (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.003\u003c/em\u003e) compared to the 60N0P treatment. A similar trend was observed for root N concentration, with the highest P rate yielding greater root N concentrations compared to the 0, 20, and 40 kg P ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e rates. The overall total switchgrass biomass N removal was highest (12 g m\u003csup\u003e\u0026ndash;2\u003c/sup\u003e) in the 60N60P and lowest (2 g m\u003csup\u003e\u0026ndash;2\u003c/sup\u003e) in the 60N0P-fertilized crop. Except for the unfertilized treatment, total biomass N removal was significantly higher (\u003cem\u003ep\u0026thinsp;\u0026le;\u0026thinsp;0.023\u003c/em\u003e) in the 60N60P than all the other treatments.\u003c/p\u003e \u003cp\u003eWhile P fertilization resulted in higher P concentration in shoots than roots, higher P concentrations in roots than shoots were observed in the non-P-fertilized (unfertilized and 60N0P) crops. Shoot P concentration was significantly higher (\u003cem\u003ep\u0026thinsp;\u0026le;\u0026thinsp;0.006\u003c/em\u003e) in the 60N20P than the unfertilized and 60N0P but did not differ significantly (\u003cem\u003ep\u0026thinsp;\u0026ge;\u0026thinsp;0.201\u003c/em\u003e) in the 60N40P and 60N60P than the unfertilized and 60N0P treatments. The higher shoot P concentrations with P fertilization resulted in an overall increased biomass P removal compared to the non-P-fertilized treatments (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Nitrogen fertilization without P addition (60N0P) significantly reduced (\u003cem\u003ep\u0026thinsp;=\u0026thinsp;0.030\u003c/em\u003e) total biomass P removal compared to the unfertilized crop.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eNitrous oxide emission\u003c/h2\u003e \u003cp\u003eMean N\u003csub\u003e2\u003c/sub\u003eO fluxes from all treatments ranged from \u0026minus;\u0026thinsp;0.026 to 0.953 \u0026micro;g N\u003csub\u003e2\u003c/sub\u003eO\u0026ndash;N m\u003csup\u003e\u0026ndash; 2\u003c/sup\u003e hr\u003csup\u003e\u0026ndash; 1\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) and were greatest in the first 6 days of the study with peak fluxes occurring on day 4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Within the first 6 days of the experiment, ~ 46%, 72%, 73%, 75%, and 78% of the cumulative N\u003csub\u003e2\u003c/sub\u003eO emissions occurred in the unfertilized, 60N20P, 60N40P, 60N60P, and 60N0P-fertilized crops, respectively. After this period, N\u003csub\u003e2\u003c/sub\u003eO fluxes decreased and were near zero for all the treatments. The 43-day cumulative N\u003csub\u003e2\u003c/sub\u003eO emissions ranged from 25 to 103 \u0026micro;g N\u003csub\u003e2\u003c/sub\u003eO\u0026ndash;N m\u003csup\u003e\u0026ndash;2\u003c/sup\u003e and was highest in the 60N40P and lowest in the unfertilized treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Cumulative N\u003csub\u003e2\u003c/sub\u003eO emissions were 2\u0026ndash; 4 times greater in the fertilized than the unfertilized crop. Among the fertilized crops, the switchgrass which received the 60N20P fertilizer rate had the lowest cumulative N\u003csub\u003e2\u003c/sub\u003eO emissions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussions","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEffect of phosphorus addition on soil nitrogen and phosphorus contents\u003c/h2\u003e \u003cp\u003eContrarily to our hypothesis that increasing P application rates would induce higher root and shoot biomass and N uptake, thereby depleting soil available N, we did not observe lower soil NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e contents at high P rates. Instead, soil analyses conducted before and after the experiment indicated an increase in soil total N following switchgrass cultivation. The increase in soil total N following N and P fertilization in our study may be due to enhanced plant and microbial immobilization of the applied N. Our findings are consistent with Wang et al (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), who reported increased soil total N levels following fertilization, which they attributed to enhanced plant and microbial immobilization of available N in the soil. Fertilizing high organic C soils with mineral N can promote N immobilization, resulting in higher soil total N (Wang et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Given that the potting mix soil used in this study had a very high SOC content, it is possible that a significant proportion of the applied N was immobilized into organic forms, contributing to the observed increase in soil total N.\u003c/p\u003e \u003cp\u003eWhile N immobilization by the SOC-rich potting mix soil may have contributed to increased soil total N following fertilization in the switchgrass treatments, the higher soil total N observed in the unfertilized crop, despite receiving no N fertilization compared to the control, suggests the involvement of additional biological processes. One possible explanation is the biological N-fixation capability of switchgrass through its association with free-living diazotrophs in the soil (Mao et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). These free-living N-fixers can contribute 2\u0026ndash;3 kg N ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e yr\u003csup\u003e\u0026ndash;1\u003c/sup\u003e (Son \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Agronomic practices such as fertilization can reduce diazotrophs, with unfertilized systems enhancing the abundance and diversity of these N-fixing bacteria (Mao et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Hence, in the unfertilized crop, the potential increased activities of N-fixing bacteria associated with the switchgrass may have contributed to the total N pool resulting in higher soil total N in the unfertilized treatment compared to the total N measured in the control. Additionally, the lack of any significant increase in the soil available P in the P-fertilized crops may be due to increased biomass and microbial removal of soil available P which may have immobilized the P into unavailable pools. This could be evidenced by the fact that biomass P removal was higher in the P-fertilized compared to 60N0P-fertilized crop.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSwitchgrass biomass growth rate and yield response to nitrogen and phosphorus fertilization\u003c/h2\u003e \u003cp\u003eThe significant reduction in biomass yield observed with the 60N0P treatment, although not entirely clear, may be attributed to a potential N-induced nutrient imbalance caused by the application of N without P addition. Khan et al (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) noted that such imbalances can cause inefficient N use by plants, leading to suboptimal growth and yield. Guo et al (2016) found that the combined application of multiple fertilizers had a more pronounced effect on the physiological metabolism of plants compared to the use of a single type of fertilizer. Similarly, Anas et al (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) reported that the combined application of N and P produced better positive interactions, enhancing N use efficiency compared to applying N alone. Therefore, unlike in the unfertilized and P-fertilized crops, the sole application of N in the 60N0P treatment may have disrupted the balance of other essential nutrients needed to promote biomass productivity, consequently, resulting in a drastic reduction in biomass yield.\u003c/p\u003e \u003cp\u003eAnother potential explanation for the reduced biomass yield observed with the application of N fertilizer, compared to the unfertilized crop, could be attributed to a decrease in arbuscular mycorrhizal fungi (AMF) colonization of switchgrass roots. Previous studies have shown that increased levels of inorganic N application can lower AMF biomass, species richness, and diversity (Liu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Albizua et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). For instance, Williams et al (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) reported that unfertilized agricultural soils in Sweden were associated with a more distinct AMF community, while the application of 50 kg ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003eyr\u003csup\u003e\u0026ndash;1\u003c/sup\u003e N was sufficient to reduce AMF biomass in these soils. We suggest that the application of N fertilizer may have reduced AMF biomass compared to the unfertilized crop, which likely maintained enhanced AMF colonization. The higher AMF biomass in the unfertilized crop could have supported improved nutrient uptake and balanced nutrition, contributing to the higher biomass yield compared to the N-only fertilized treatment. This reduction in AMF colonization in response to N fertilization could also explain the lack of yield response to P fertilization.\u003c/p\u003e \u003cp\u003eBrejda (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) determined that the combined application of high P rates with low N rates (as low as 56 kg N ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e) in native warm-season grasses, such as switchgrass, would likely result in little to no yield response. This is because AMF colonization of switchgrass roots can facilitate P uptake even at low soil P levels, reducing the impact of P fertilization on switchgrass biomass yield (Brejda \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Consistent with this, Muir et al (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2001\u003c/span\u003e) found no yield response in switchgrass when P was applied at rates of 0, 10, 20, 30, or 39 kg P ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e to low-P soils (4\u0026ndash;11 mg P kg⁻\u0026sup1;) in Texas, USA. Similarly, Jung et al (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1988\u003c/span\u003e) observed no significant yield response with 20 and 40 kg ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e P fertilizer application to switchgrass on a strongly acidic, low-P soil in western Pennsylvania due to the presence and activities of AMF. Thus, it is possible that the association of AMF with switchgrass roots in our study may have enhanced P uptake, obscuring any potential yield benefits from P fertilization and contributing to the overall lack of yield response to P application.\u003c/p\u003e \u003cp\u003eAlso, the yield response of warm-season grasses to P fertilization is largely influenced by the soil P status, with positive yield responses more likely to occur in low-P compared to high-P soils (Kering et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Siri-Prieto et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Ros et al (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) observed that P fertilization in grasses grown on soils with \u0026le;\u0026thinsp;5 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e available P increased yields by 110%, whereas yield increases were limited to only 7\u0026ndash;25% in soils with \u0026gt;\u0026thinsp;5 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e available P. Anderson and Shapiro (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1990\u003c/span\u003e) categorized soil available P levels based on Olsen-P soil tests as very low (0\u0026ndash;3 mg kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e), low (4\u0026ndash;10 mg kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e), medium (10\u0026ndash;17 mg kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e), and high (\u0026gt;\u0026thinsp;17 mg kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e). This categorization indicates that the potting mix soil used in our experiment, with an Olsen-P level of 27 mg kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, had a high P status. Therefore, P may not have been in limited supply to the switchgrass, reducing the likelihood of a positive yield response to P fertilization. Brejda (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) also noted that warm-season grasses, such as switchgrass, typically have a lower P requirement compared to cool-season grasses. This lower P requirement, combined with the high Olsen-P status of the potting mix soil, may explain the lack of a significant yield response to P fertilization in our study as the sufficient soil P levels likely met the switchgrass nutrient needs without additional P supplementation, resulting in no observable yield benefit from fertilizer P application.\u003c/p\u003e \u003cp\u003eIt is equally possible that the short duration of our study may have also contributed to the lack of yield response to P fertilization. Switchgrass typically does not reach its full potential yield until its third year of growth, as it allocates much of its energy during the first two years to establishing an extensive root network (Siri-Prieto et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This initial focus on root development over shoot biomass production can minimize the above-ground yield response to P fertilization in early growth stages. Therefore, it is essential to replicate this study over longer time periods to fully capture the effects of P fertilization on switchgrass yields. Long-term experiments would allow for a comprehensive understanding of how different P application rates influence yield as the crop transitions from root establishment to full productivity. Such studies are critical for determining the long-term nutrient requirements of switchgrass and optimizing P management strategies for sustainable biomass production.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eNitrogen and phosphorus concentrations and removal in biomass\u003c/h2\u003e \u003cp\u003eThe amount of nutrients removed from soil is a function of yield and the concentration of that nutrient in the plant sample (Massey \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). It was expected that increased yield from high P rates would increase yield with resultant increase in biomass N and P concentrations and removal from soil. However, lower yield, particularly, in the N-only fertilized crop led to significantly lower N and P concentrations and removal in both shoot and root biomasses compared to the unfertilized crop. While lower N concentrations and removal in the 60N0P and 60N40P fertilized compared to unfertilized crop could be attributed to lower yield, higher P concentrations and removal in the P-fertilized crops may be due to the higher P contents from the P fertilization compared to the treatments which did not receive any P application. Our observation of higher P concentrations and removals in the switchgrass biomass at higher P rates is consistent with findings from other studies (Kering et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Ashworth et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sawyer et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Ashworth et al (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) observed highest P uptake in switchgrass biomass occurred with higher P fertilizer rates. Kering et al (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) also determined 11% higher P concentrations with the application of 45 kg P ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e in switchgrass compared to the unfertilized crop across two sites in Oklahoma, USA, whereas Sawyer et al (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) determined a linear increase in switchgrass biomass P concentration and removal with increased P application rate, with P removal increasing by 0.027 kg for every kg ha\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of applied P. Although, other studies observed increased yield with higher P application rates, the lack of significant impact of increased P application rate on yield in our study implies that increasing P application rates in P-sufficient soils could result in increased P removal with no beneficial impact on yield, reducing the P-use efficiency in switchgrass.\u003c/p\u003e \u003cp\u003eAt the onset of switchgrass growth, nutrients are translocated from roots to shoots (Massey \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). However, as switchgrass matures and move towards flowering and senescence, nutrients is translocated from shoots back to roots (Massey \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Whereas translocation of N and P from shoot to roots will increase N and P concentrations in roots relative to shoots, the opposite would be observed with translocation from roots to shoots. Hence, the higher concentrations of N and P in shoots than roots in the N- and P-fertilized crops in our study may mean that the increased availability of N and P in soil from fertilization made it possible for increased translocation of these nutrients from roots to shoots. This is especially so when it is considered that biomass N and P concentrations were determined only 43 days after seeding. Makaju et al (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) measured highest N and P concentrations in switchgrass biomass one month (May) after the beginning (April) of switchgrass growth. This period of increased biomass accumulation of N and P coincides with the 43 days after seeding switchgrass in our study. Therefore, we allude to the higher N and P concentrations in the shoots of our N- and P-fertilized switchgrass than roots to N and P translocation from roots (belowground) to shoots (aboveground). While the higher N and P concentrations in the shoots than roots of the N- and P-fertilized crops may have been enhanced by higher N and P availability from fertilization, lower N and P concentrations in shoots than roots of the unfertilized switchgrass may have been due to relatively lower soil N and P availability from lack of fertilization. Changes in resource supply such as soil nutrient concentrations can impact how plants allocate soil nutrient resources (Wang et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). It is, therefore, possible that at the relatively lower available N and P concentrations, the unfertilized switchgrass may have invested much of the available N and P in roots before shoots. This strategy of N and P allocation could potentially result in higher concentrations of N and P in roots compared to shoots at the early stages of biomass growth. Further investigations are required to understand how switchgrass may allocate soil nutrient resources when in limited amounts and how that could potentially impact biomass productivity and the provisioning of other ecological services such as soil C sequestration and reduced GHG emissions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEffect of phosphorus fertilization on nitrous oxide emissions\u003c/h2\u003e \u003cp\u003eThe nonlinear response of N\u003csub\u003e2\u003c/sub\u003eO emissions to P fertilization observed in our study, where the lowest P rate reduced N₂O emissions but increased with higher P rates did not support our hypothesis that increasing P rates would reduce N\u003csub\u003e2\u003c/sub\u003eO emissions. This observation suggests a potentially more complex interaction between P application and soil N₂O dynamics at the initial stage of switchgrass cultivation, and that while moderate P application might help mitigate N\u003csub\u003e2\u003c/sub\u003eO emissions, excessive P inputs could potentially promote conditions that enhance N\u003csub\u003e2\u003c/sub\u003eO emissions. We suspect that increasing the rate of P application may have stimulated N mineralization, increasing the availability of soil inorganic N (NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e and NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026ndash;\u003c/sup\u003e) at a time when N uptake by switchgrass was lowest. At higher P rate of 2000 mg P kg\u003csup\u003e\u0026ndash;1\u003c/sup\u003e soil, Mori et al (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) observed increased N\u003csub\u003e2\u003c/sub\u003eO emissions in laboratory incubated soil from a 1-year-old \u003cem\u003eAcacia mangium\u003c/em\u003e plantation in Indonesia due to increased stimulation of N cycling and losses as N\u003csub\u003e2\u003c/sub\u003eO. Wang et al (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) also determined that P application accelerated some of the N cycling processes in the soil such as gross N mineralization, gross nitrification, and denitrification, with the effect sizes increasing with P addition rates. Evidence from these studies indicate that increased P application rates can result in increased N cycling and losses as N\u003csub\u003e2\u003c/sub\u003eO as observed in this study. This potential increase in soil N availability, combined with reduced N uptake by switchgrass during its early growth stage, may have led to the lost of the available N as N\u003csub\u003e2\u003c/sub\u003eO. The implication of this is that the timing of P application relative to plant N demand is critical in minimizing N\u003csub\u003e2\u003c/sub\u003eO emissions.\u003c/p\u003e \u003cp\u003eAside the potential increased stimulation of N cycling in the soil which may have caused higher N losses as N\u003csub\u003e2\u003c/sub\u003eO under higher P rates, the ability of N and P fertilization to stimulate nitrifying and denitrifying bacteria (Mori et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Thompson et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) could have equally resulted in higher N\u003csub\u003e2\u003c/sub\u003eO emissions with higher P fertilization rates and under the 60N0P fertilization compared to the unfertilized crop. Mori et al (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) demonstrated the significant positive impact P fertilization have on stimulating the activities of denitrifying bacteria in the soil. He and Dijkstra (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) also explained that the simultaneous application of inorganic N and P fertilizers stimulate nitrifiers and denitrifiers, increasing N\u003csub\u003e2\u003c/sub\u003eO emissions. Thompson et al (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) measured increased denitrifying bacteria genes (\u003cem\u003enirS\u003c/em\u003e and \u003cem\u003enosZ\u003c/em\u003e) in N-fertilized switchgrass plots compared to the unfertilized plots. Hence, compared to the unfertilized crop where majority of the soil residual N and P may have possibly been used for biomass production, the increased availability of soil N and P content from fertilization may have stimulated the soil nitrifier and denitrifier community which resulted in increased N\u003csub\u003e2\u003c/sub\u003eO emissions compared to the unfertilized crop.\u003c/p\u003e \u003cp\u003eThe increased N\u003csub\u003e2\u003c/sub\u003eO emissions within the first 6 days of our study could be explained by the absence of roots prior to the germination of the switchgrass seeds. The first germination in our study occurred on the 5th day of the experiment. Mori et al (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) explained that in the absence of roots, N\u003csub\u003e2\u003c/sub\u003eO emission is elevated due to reduced N uptake in plants. Therefore, our findings support the argument that N-fertilization of switchgrass at the initial establishment phase should be avoided (Samson et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). This could ensure that N\u003csub\u003e2\u003c/sub\u003eO emissions from N application is curtailed while inhibiting early weed growth.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eContrary to our hypothesis that higher P application would enhance soil N uptake in switchgrass biomass, thereby reducing N\u003csub\u003e2\u003c/sub\u003eO emissions, our study showed an increase in total soil N. This increase was likely due to enhanced microbial and plant immobilization of applied N, as well as the contribution of N from biological N-fixation in the unfertilized switchgrass. Despite the increase in soil total N, the combined application of N and P did not significantly impact switchgrass biomass yield. In contrast, N-only application significantly reduced biomass yields compared to the unfertilized crop. The lack of yield response from combined N and P application was attributed to high baseline soil P levels and a possible reduction in AMF colonization whereas the yield reduction from N-only application was likely due to N-induced nutrient imbalances that affected soil nutrient uptake and utilization. Although P fertilization resulted in the highest nutrient removal in biomass, its use efficiency was reduced as yield did not increase proportionately with P application rate. The 43-day cumulative N\u003csub\u003e2\u003c/sub\u003eO emissions ranged from 25 to 103 \u0026micro;g N\u003csub\u003e2\u003c/sub\u003eO-N m\u003csup\u003e\u0026ndash;2\u003c/sup\u003e, with the highest emissions observed at the 60N40P rate and the lowest in the unfertilized crop. P fertilization exhibited a nonlinear effect on N\u003csub\u003e2\u003c/sub\u003eO emissions. While P at 20 kg ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e reduced emissions compared to N-only treatment, increasing P to 40 kg ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e resulted in the highest emissions, surpassing those of N-only treatments. Emissions at 60 kg ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e were similar to N-only treatment. These results indicate that while moderate P application may reduce N\u003csub\u003e2\u003c/sub\u003eO emissions, higher P rates may enhance emissions due to changes in microbial activity and N cycling in the soil. Additionally, N\u003csub\u003e2\u003c/sub\u003eO emissions were greatest during the first week of our study prior to switchgrass root development, supporting the argument that fertilization of switchgrass at the initial establishment phase should be avoided to mitigate N\u003csub\u003e2\u003c/sub\u003eO emissions. Even though this study increases our knowledge of P-fertilization on N\u003csub\u003e2\u003c/sub\u003eO emissions in switchgrass and may serve as basis for long-term replication on the field or in the lab using field soils, further research is needed to identify optimal timing and combined N and P application strategies to maximize biomass yield and minimize N\u003csub\u003e2\u003c/sub\u003eO emissions under field conditions.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding for this study was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC)\u0026nbsp;(Grant # RGPIN-2018-04346) and the Canadian Foundation for Innovation (CFI)\u0026nbsp;(Grant # 12896). This work was supported by the University of Waterloo by providing research infrastructure and funding to Augustine K. Osei. Global Affairs Canada\u0026rsquo;s Emerging Leaders in the Americas Program also provided funding to support Nadia Gabbanelli\u0026rsquo;s internship at the University of Waterloo. We are also grateful to the editors and reviewers that helped improve this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Augustine K. Osei and Nadia Gabbanelli. The first draft of the manuscript was written by Augustine K. Osei and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAgostini, F., Gregory, A. S., \u0026amp; Richter, G. M. (2015). Carbon Sequestration by Perennial Energy Crops: Is the Jury Still Out? \u003cem\u003eBioenergy Research\u003c/em\u003e 8 (3), 1057\u0026ndash;1080. https://doi.org/10.\u003cbr\u003e 1007/s12155-014-9571-0\u003c/li\u003e\n\u003cli\u003eAlbizua, A., Williams, A., Hedlund, K., \u0026amp; Pascual, U. (2015). 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Phosphorus supply increases nitrogen transformation rates and retention in soil: a global meta‐analysis. \u003cem\u003eEarth\u0026apos;s Future\u003c/em\u003e, \u003cem\u003e10\u003c/em\u003e(3), e2021EF002479. https://doi.org/10.1029/2021EF002479\u003c/li\u003e\n\u003cli\u003eWang, S., Sanford, G. R., Robertson, G. P., Jackson, R. D., \u0026amp; Thelen, K. D. (2020). Perennial bioenergy crop yield and quality response to nitrogen fertilization. \u003cem\u003eBioEnergy Research\u003c/em\u003e, \u003cem\u003e13\u003c/em\u003e, 157-166. https://doi.org/10.1007/s12155-019-10072-z\u003c/li\u003e\n\u003cli\u003eWile, A., Burton, D. L., Sharifi, M., Lynch, D., Main, M., \u0026amp; Papadopoulos, Y. A. (2014). Effect of nitrogen fertilizer application rate on yield, methane and nitrous oxide emissions from switchgrass (Panicum virgatum L.) and reed canarygrass (Phalaris arundinacea L.). \u003cem\u003eCanadian Journal of Soil Science\u003c/em\u003e, \u003cem\u003e94\u003c/em\u003e(2), 129\u0026ndash;137. https://doi.org/10.4141/CJSS2013-058\u003c/li\u003e\n\u003cli\u003eWilliams, A., Manoharan, L., Rosenstock, N. P., Olsson, P. A., \u0026amp; Hedlund, K. (2017). Long‐term agricultural fertilization alters arbuscular mycorrhizal fungal community composition and barley (H ordeum vulgare) mycorrhizal carbon and phosphorus exchange. \u003cem\u003eNew Phytologist\u003c/em\u003e, \u003cem\u003e213\u003c/em\u003e(2), 874-885. https://doi.org/10.1111/nph.14196\u003c/li\u003e\n\u003cli\u003eZhang, Y., Wang, C., \u0026amp; Li, Y. (2019). Contrasting effects of nitrogen and phosphorus additions on soil nitrous oxide fluxes and enzyme activities in an alpine wetland of the Tibetan Plateau. \u003cem\u003ePlos One\u003c/em\u003e, \u003cem\u003e14\u003c/em\u003e(5), e0216244. https://doi.org/10.1371/journal.pone.0216244\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Switchgrass biomass yield, Nitrogen and phosphorus fertilizer, Biomass N and P uptake, N2O emissions","lastPublishedDoi":"10.21203/rs.3.rs-5328479/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5328479/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eAims\u003c/h2\u003e \u003cp\u003ePhosphorus (P) fertilization can play a critical role in increasing switchgrass biomass yields for bioenergy production. However, applying mineral P to switchgrass can stimulate nitrous oxide (N\u003csub\u003e2\u003c/sub\u003eO) emissions, offsetting its climate mitigation benefits. The effect of P fertilization on N\u003csub\u003e2\u003c/sub\u003eO emissions is not well understood, with previous studies producing conflicting results. Moreover, studies evaluating the effect of P fertilizer on nitrogen (N) dynamics and its contribution to N\u003csub\u003e2\u003c/sub\u003eO emissions under switchgrass are lacking.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eA 43-day study was conducted in a controlled-environment to evaluate the effect of different P fertilizer rates on N\u003csub\u003e2\u003c/sub\u003eO emissions in N-fertilized switchgrass. Four different fertilizer rates [(i) 60 kg N ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e (60N0P); (ii) 60 kg N and 20 kg P ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e (60N20P); (iii) 60 kg N and 40 kg P ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e (60N40P); (iv) 60 kg N and 60kg P ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e (60N60P)] and a treatment with no fertilizer (0N0P) were evaluated.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWhile switchgrass biomass yield was less responsive to N and P fertilization, P fertilization had a nonlinear effect on N\u003csub\u003e2\u003c/sub\u003eO emissions. Applying P at 20 kg ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e resulted in lower emissions compared to N-only treatment. In contrast, increasing the P rate to 40 kg ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e led to the highest N\u003csub\u003e2\u003c/sub\u003eO emissions, surpassing those of N-only treatment. At 60 kg P ha\u003csup\u003e\u0026ndash;1\u003c/sup\u003e, emissions were comparable to the N-only treatment.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings suggest that while moderate P application can reduce N\u003csub\u003e2\u003c/sub\u003eO emissions, higher P rates may increase emissions, offsetting the climate benefits of switchgrass as a bioenergy crop.\u003c/p\u003e","manuscriptTitle":"Impact of Phosphorus Fertilization Rates on Nitrous Oxide Emissions in Switchgrass: Nonlinear Response Reveals Emission Reduction at Low Phosphorus Rates","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-29 07:40:39","doi":"10.21203/rs.3.rs-5328479/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"340b7e5c-5fd2-47f8-8e48-d34dd477a77f","owner":[],"postedDate":"November 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-21T02:07:38+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-29 07:40:39","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5328479","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5328479","identity":"rs-5328479","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}