Soil Nitrogen Cycling in a Pine Forest Exposed to 5 Years of Elevated Carbon Dioxide

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1 Ecosystems (2003) 6: DOI: /s ECOSYSTEMS 2003 Springer-Verlag Soil Nitrogen Cycling in a Pine Forest Exposed to 5 Years of Elevated Carbon Dioxide Adrien C. Finzi 1 * and William H. Schlesinger 2 1 Department of Biology, Boston University, Boston, Massachusetts 02215, USA; 2 Nicholas School of the Environment and Earth Sciences, Duke University, Durham, North Carolina 27708, USA ABSTRACT Empirical and modeling studies have shown that the magnitude and duration of the primary production response to elevated carbon dioxide (CO 2 ) can be constrained by limiting supplies of soil nitrogen (N). We have studied the response of a southern US pine forest to elevated CO 2 for 5 years ( ). Net primary production has increased significantly under elevated CO 2. We hypothesized that the increase in carbon (C) fluxes to the microbial community under elevated CO 2 would increase the rate of N immobilization over mineralization. We tested this hypothesis by quantifying the pool sizes and fluxes of inorganic and organic N in the forest floor and top 30 cm of mineral soil during the first 5 years of CO 2 fumigation. We observed no statistically significant change in the gross or net rate of inorganic N mineralization and immobilization in any soil horizon under elevated CO 2. Similarly, elevated CO 2 had no statistically significant effect on the concentration or flux of organic N, including amino acids. Microbial biomass N was not significantly different between CO 2 treatments. Thus, we reject our hypothesis that elevated CO 2 increases the rate of N immobilization. The quantity and chemistry of the litter inputs to the forest floor and mineral soil horizons can explain the limited range of microbially mediated soil N cycling responses observed in this ecosystem. Nevertheless a comparative analysis of ecosystem development at this site and other loblolly pine forests suggests that rapid stand development and C sequestration under elevated CO 2 may be possible only in the early stages of stand development, prior to the onset of acute N limitation. Key words: elevated carbon dioxide; nitrogen cycling; forest productivity; organic nitrogen; amino acids. INTRODUCTION Experimental increases in the concentration of atmospheric carbon dioxide (CO 2 ) increase the rate of plant growth, net primary production (NPP), and net ecosystem production (NEP) in many different terrestrial ecosystems (for example, see Curtis and Wang 1998; Cheng and others 2000; Hamilton and others 2002). As atmospheric concentrations of CO 2 rise due to human activities, studies suggest Received 1 July 2002; accepted 9 October 2002; published online May 30, *Corresponding author; afinzi@bu.edu that the productivity of most terrestrial ecosystems will increase. Currently, the largest estimated sink for atmospheric CO 2 is in midlatitude ecosystems (Schimel and others 2001) ecosystems that are dominated by temperate forests and grasslands. The productivity of these ecosystems is often limited by the availability of soil nitrogen (N) (Vitousek and Howarth 1991). Empirical and modeling studies have shown that the magnitude and duration of the primary production response to elevated CO 2 can be constrained by limiting supplies of soil N (Rastetter and others 1997; Luo and Reynolds 1999; Thornley and Cannell 2000; Medlyn and others 2000; Oren and others 2001). Consequently, the 444

2 Soil N Cycling Responses to Elevated CO modulation of soil N availability by primary producers may ultimately control the magnitude of the carbon (C) sink in midlatitude ecosystems. There is no consensus on the effects of elevated CO 2 on soil N cycling. Various researchers have reported either increases, decreases, or no change in the rate of N mineralization under elevated CO 2 (see, for example, Hungate and others 1997; Zak and others 2000c; Williams and others 2001; Gill and others 2002). This range of responses is surprising given that carbon (C) and N cycles in soils are tightly coupled (Schlesinger 1997), that microbial function is C-limited (Burke 1989; Zak and others 1994), and that elevated CO 2 increases substrate availability to the decomposer community (Zak and others 2000c). In theory, the larger inputs of organic matter under elevated CO 2 should initially create a large biosynthetic demand for N by the microbial community as the C-limited microbial community increases in biomass. Assuming that the abundance of mesofaunal grazers does not increase (for example, see Zak and others 1993), the rate of N immobilization should exceed the rate of mineralization during the first growing season to several years under elevated CO 2. Over time, however, enhanced inputs of C to the microbial community under elevated CO 2 should increase gross and net fluxes of N through the microbial community as these communities reach a new equilibrium biomass (see Reich and others 1997; Thornley and Cannell 2000). The source of the additional N that is cycled through the microbial community could include inputs via fixation and atmospheric deposition or the mobilization of N from soil organic matter (Richter and others 2000; Vitousek and Field 1999; Martin-Olmedo and others 2002). Zak and others (2000c) hypothesized that the diversity of the short-term soil N cycling responses to elevated CO 2 could be explained not only by the quantity but also by the chemistry of the plantderived substrates available to microbes. They suggested that a large influx of labile substrates (for example, simple carbohydrates and organic acids) could stimulate microbial growth, creating a biosynthetic demand for N. This in turn would increase N immobilization within the microbial biomass and decrease the net rate of N mineralization to the plant-available pool. In contrast, they suggested that an influx of substrates that provide little energy for microbial metabolism (for example, lignins, tannins, and polyphenols) would create little biosynthetic demand for N and have a negligible effect on N immobilization. This hypothesis is not well tested because few studies link variation in the quantity and chemistry of C and N inputs under elevated CO 2 to the rate of N release from soil organic matter. We studied the response of a southern US pine forest to free-air CO 2 enrichment for 5 years. NPP and NEP in this ecosystem increased significantly under elevated CO 2 (Finzi and others 2002; Hamilton and others 2002). The increase in forest productivity under elevated CO 2 has increased the quantity and altered the chemistry of the organic matter reaching the forest floor (Table 1) (Finzi and others 2001, 2002; Finzi and Schlesinger 2002) and the mineral soil horizons (Matamala and Schlesinger 2000; Pritchard and others 2001; Schlesinger and Lichter 2001). It has also increased the rate of heterotrophic respiration by 165%, implying greater substrate availability to the microbial community (Hamilton and others 2002). The objectives of this study were (a) to quantify the pools and fluxes of organic and inorganic N under ambient and elevated CO 2, and (b) to relate potential changes in soil N cycling under elevated CO 2 with the quantity and chemistry of fresh litter inputs and soil organic matter. We tested the hypothesis that increases in substrate availability to the decomposer community under elevated CO 2 increase the rate of N immobilization relative to mineralization. We present data collected during the first 5 years of CO 2 fumigation. METHODS Site Description The free-air CO 2 enrichment (FACE) experiment in the Duke Forest (Orange County, North Carolina, USA) is composed of six 30-m diameter plots. Three experimental plots are fumigated with CO 2 to maintain the atmospheric CO 2 concentration at 200 l L 1 above ambient (that is, 565 l L 1 ). Three control plots are fumigated with ambient air only (365 l L 1 ). The experiment began on 27 August 1996 and is ongoing on a continuous basis (24 h d 1,365dy 1 ), with the exception of periods when the air temperature is below 4 C. Additional details on the FACE operation can be found in Hendrey and others (1999). The forest is derived from 3-year-old loblolly pine (Pinus taeda) seedlings that were planted in 1983 in a m spacing. In 1996, the 13-year-old pine trees were approximately 14 m tall and accounted for 98% of the basal area of the stand. Since the planting, a deciduous understory layer has been recruited from nearby hardwood forests and stump sprouts. The most abundant understory tree species is sweet gum (Liquidambar styraciflua), with admix-

3 446 A. C. Finzi and W. H. Schlesinger Table 1. The Flux (g m 2 y 1 ) and Ratio of Different Carbon (C) Fractions and Nitrogen (N) in Leaf Litterfall under Ambient (A) and Elevated (E) CO 2 during the First 4 Years of Fumigation Year Carbon TNC Lignin Nitrogen C:N Lignin:N A E A E A E A E A E A E (5) 176 (5) 38.0 (3.3) 44.7 * (3.3) 85.7 (12) 88.9 (1.2) 2.65 (0.06) 2.74 (0.06) 64 (2) 64 (2) 32 (1) 32 (1) (7) 243 * (7) 57.8 (2.5) 71.0 * (2.5) 116 (5) 130 (5) 2.94 (0.03) 3.36 ** (0.03) 70 (1) 72 (1) 39 (1) 39 (1) (8) 289 * (8) 61.6 (4.6) 71.6 (4.6) 108 (7) 138 * (7) 3.44 (0.15) 3.84 (0.15) 68 (1) 75 * (1) 31 (1) 39 **** (1) (1) 284 *** (1) 73.8 (3.2) 78.1 (3.2) 96.3 (2.2) 114 * (2.2) 3.11 (0.07) 3.32 (0.07) 80 (2) 86 (2) 31 (1) 35 ** (1) Cumulative 864 (8) 992 ** (8) 231 (20.6) 265 (4.2) 407 (8) 470 ** (8) 12.1 (0.3) 13.3 **** (0.3) 71 (1) 74 (1) 33 (1) 37 ** (1) TNC, total nonstructural carbohydrates Each value is the mean ( 1 SE) of leaf litterfall in loblolly pine and the deciduous species. Modified from Finzi and others (2002) and Finzi and Schlesinger (2003) * P 0.05 ** P 0.01 *** P x.xx **** P 0.10 The cumulative value in the C:N and lignin:n ratio columns is the ratio of the cumulative C, lignin, and N fluxes. tures of red maple (Aæer rubrum), red bud (Cercis canadensis), and dogwood (Cornus florida). The 32-ha site contains an elevation gradient of 15 m between the highest and lowest points, but topographic relief is less than 1 throughout. Soils are from the Enon Series (fine, mixed, active, thermic Ultic Hapludalfs). Enon soils are derived from mafic bedrock; they are slightly acidic (0.1 M CaCl 2 ph 5.75), and they have well-developed soil horizons with mixed clay mineralogy. Additional site details can be found in Schlesinger and Lichter (2001) and Finzi and others (2001). Gross and Net N Transformations We measured the net rate of potential N mineralization in the top 15 cm of mineral soil from 1997 through In 2000 and 2001, we also measured the rate of net mineralization in the forest floor and the cm soil horizon. Samples were collected from the FACE site in April, June, August, and October of each year, with the exception of 1997, when they were taken in September only, and 2001, when they were taken in June and October only. The mineral soil cores were 5 cm in diameter and 15 cm in depth. The forest floor was sampled by excavating a cm block of the surface organic horizon to the top of the A horizon. Four replicate soil cores and forest floor samples were collected from each of the FACE plots. These samples were combined into a single composite sample in the lab, and each soil horizon was kept separate from the others. From each of the composite samples, we measured net and gross rates of N transformations. We measured the rate of potential net N mineralization in two ( ) or three ( ) replicate 20-g samples of soil from each soil horizon (forest floor, 0 15 cm mineral soil, cm mineral soil). Soils were incubated in 250-ml plastic bottles in the lab. The same number of replicates was used for both the initial and the incubated samples. We estimated the rate of net N mineralization by measuring the concentration of NO 3 and NH 4 in 2M KCl extracts using an autoanalyzer (Lachat QuickChem FIA 8000 Series; Zellweger Analytics, Milwaukee, WI, USA). These extracts were obtained before and after a 30-day aerobic laboratory incubation at 28 C (Binkley and Hart 1989). We used 15 N isotope dilution to determine gross rates of microbial N mineralization and N immobilization in the 0 15-cm mineral soil horizon in June and August of 1999 and 2000 (Davidson and others 1992; Hart and others 1994). Four 20-g subsamples were taken from each of the composited

4 Soil N Cycling Responses to Elevated CO soils in each FACE plot and placed in 250-ml plastic bottles. All subsamples were initially labeled with 1 mlofa30mgl 1 99-atom% enriched ( 15 NH 4 ) 2 SO 4 solution prepared in nanopure water. The 1 ml of 15 N solution was added drop by drop across the surface of the soil in the plastic bottle. Two of the four samples from each FACE plot were extracted in 2M KCl after 15 mins. The two remaining samples were incubated for 40 h at 22 C, after which time they were extracted in 2M KCl. Gross rates of NH 4 mineralization and immobilization were calculated using the equations presented in Hart and others (1994). We determined the initial and final pool size of NH 4 by measuring the concentration of NH 4 of each sample on an autoanalyzer (Lachat QuickChem FIA 8000 Series, Zellweger Analytics). We determined the atom percent 15 N excess of the initial and incubated samples by diffusing the N in each sample onto an acidified cellulose disc, followed by analysis on a mass spectrometer (Finnegan Delta S; Thermo Finnigan, San Jose, CA, USA). In brief, 1 ml of the 15 N-labeled sample was added to a specimen cup containing 40 l of2m KCl and 6.43 mol 14 N (as [ 14 NH 4 ] 2 SO 4 ). The addition of 14 N brought the concentration and isotope ratio of N into the optimal detection range for our mass spectrometer (R. Michener personal communication). A cellulose disc (Whatman no. 3; Whatman, Clifton, NJ, USA) was acidified with 10 l) of 2.5 mol/l KHSO 4 and suspended on a metal wire at the top of each specimen cup. To each specimen cup we added 270 mg MgO to volatilize the NH 4 (to NH 3 ), which was then trapped on the acidified cellulose disc. The diffusion lasted for 7 days, after which time the acidified filter discs were air-dried and stored in a dessicator with a sulfuric acid trap until analysis on the mass spectrometer. Extractable Organic N and Amino Acid Analysis In 2001, we quantified the concentrations and fluxes of organic N and amino acids in all three soil horizons. The concentration of extractable organic N was determined by persulfate digestion of a subsample of the 2M KCl extracts used in the analysis of the rate of potential net N mineralization (Shepherd and others 2001). In 25-ml culture tubes, 2.5-ml samples of each 2M KCl extract were added to 10 ml of a concentrated potassium persulfate solution containing boric acid (Cabrera and Beare 1993). The samples were capped immediately and autoclaved for 1hat121 C. The samples were then diluted to 25 ml with nanopure water and analyzed for NO 3 on an autoanalyzer using the hydrazine reduction method (Lachat QuickChem FIA 8000 Series; Zellweger Analytics). The concentration of extractable organic N ( g g 1 ) in a given sample was calculated as the difference in the concentration of N released from the persulfate digestion and the concentration of inorganic N in that sample (Shepherd and others 2001). The initial samples provided information on the standing pool of extractable organic N in each sample. The net production or consumption of extractable organic N was calculated as the difference between the concentration of the persulfate-reactive N in the incubated and initial samples after correction for their inorganic N concentration (Shepherd and others 2001). We also quantified the concentration and flux of amino acids in a second subsample of the 2M KCl extract used for the analysis of net N mineralization. Each subsample was reacted with an acetatecyanide buffer and 3% ninhydrin (Rosen 1957; Amato and Ladd 1988). In brief, we placed 1 ml of the sample in a 15-ml test tube. To each test tube we added 0.5 ml of acetate-cyanide buffer and 0.5 ml of a 3% ninhydrin solution. The samples were transferred to an 80 C water bath and incubated for 10 min. After it was heated, we added 5 ml of 95% ethanol. We measured the concentration of amino acids in the 2M KCl extracts at 570 nm on a spectrophotometer (Hitachi U-2000; Hitachi Instruments, San Diego, CA, USA). We used a sevenpoint standard curve with leucine as our standard and concentrations ranging from 0 to 0.5 mm (r ). Ninhydrin also reacts with NH 4 in solution (Rosen 1957). In an independent analysis, we determined that approximately 28% of the available NH 4 reacts with ninhydrin (Reacted NH [NH 4 ]) in a linear manner up to 10 mg L 1 NH 4 (the highest measured concentration of NH 4 in our samples). We decreased all of our initial amino acid concentrations by the amount of ninhydrin-reactive NH 4 present in each sample. The initial samples provided information on the standing pool of amino acids in each sample. We calculated the net production or consumption of amino acids as the difference in the amino acid concentration in the incubated and initial samples, respectively. Microbial Biomass N We quantified microbial biomass N using the chloroform fumigation extraction procedure (Brookes and others 1985; Gallardo and Schlesinger 1990). Two ( ) or three ( ) replicate 10-g samples of soil from each soil horizon and three replicate 5-g samples from the forest floor were

5 448 A. C. Finzi and W. H. Schlesinger Table 2. Repeated-measures Analyses of Variance for Inorganic Nitrogen (N) Concentrations, Rate of Net N Mineralization, Microbial Biomass N, Gross NH 4 Mineralization, and Gross NH 4 Immobilization in the Top 15 cm of Mineral Soil under Ambient and Elevated CO 2 Source of Variation df Inorganic N Concentration Net Mineralization Microbial Biomass N Gross NH 4 Mineralization Gross NH 4 Immobilization MS F MS F MS F MS F MS F Between subjects CO Error Within subjects Year * ** *** Year CO Error df, degrees of freedom; Mean Square,...; Fratlo,... Gross N dynamics were only measured in 1999 and Thus, there was one degree of freedom for the CO 2, Year, and Year CO 2 interaction terms. There were four degrees of freedom for the error term both within and between subjects. * P 0.05 ** P 0.01 *** P placed into 50-ml centrifuge tubes (with the same number of replicates for the initial and incubated samples). The initial samples were immediately extracted with 0.5M K 2 SO 4. The incubated samples were placed under C 2 H 5 OH-free CHCl 3 for 7 days. After they were fumigated, we extracted the incubated samples with 0.5M K 2 SO 4. Both the initial and incubated samples underwent persulfate digestion to oxidize all N species to NO 3. Microbial biomass N was estimated as the difference in the flush of N following CHCl 3 fumigation and that extracted in the initial sample divided K EN 0.54 (Jorgensen and Mueller 1996). Data Presentation and Statistical Analysis We used repeated-measures analysis of variance (ANOVA) to test for the effect of elevated CO 2 (370 and 570 L 1 ), soil horizon (forest floor, 0 15-cm mineral soil, cm mineral soil), and sample year on the rate of soil N cycling. For analyses in which the year CO 2 or year soil horizon interaction term was statistically significant, we used one-way ANOVA to test for significant differences between CO 2 treatments or among soil horizons on a year-by-year basis. We log-transformed the data when variances were heterogeneous or nonnormally distributed. We used Tukey s test for post hoc comparisons among means. In cases where a log transformation did not satisfy model assumptions, we used nonparametric, distribution-free statistics (Wilcoxon rank sums, Kruskal-Wallis). RESULTS N Cycling in the 0 15-cm Mineral Soil Horizon Elevated CO 2 did not affect the pool size of inorganic N in the top 15 cm of mineral soil; however, the pool of inorganic N varied significantly among years (Table 2). Pool sizes of inorganic N were largest in 1998; intermediate in 1997, 1999, and 2000; and lowest in 2001 (Figure 1A). There was no interaction between CO 2 treatment and the year of study. Elevated CO 2 did not affect the rate of potential net mineralization in the top 15 cm of mineral soil (Table 2). The rate of net N mineralization varied significantly among years. Potential net N mineralization was highest in 1998, intermediate in 1997 and 2000, and lowest in 1999 and 2001 (Figure 1B). The CO 2 year interaction was not statistically significant (Table 2). Elevated CO 2 did not alter the quantity of microbial biomass N (Table 2). Microbial biomass N was significantly higher in 1997, 1998, and 1999 than 2000 (Figure 1C). There was no interaction between CO 2 treatment and year with respect to the quantity of N in microbial biomass. Gross rates of NH 4 mineralization and immobilization were not significantly different between CO 2 treatments or between sample years (Table 2, and Figure 2). The CO 2 year interaction term was not statistically significant. Rates of NH 4 immobi-

6 Soil N Cycling Responses to Elevated CO Figure 2. The gross rate of 15 NH 4 mineralization and immobilization in the top 15 cm of mineral soil measured during the 3rd (1999) and 4th (2000) growing seasons after the initiation of carbon dioxide (CO 2 ) treatment. The open bars indicate the average (n 3) for the plots under ambient CO 2 ; the filled bars are the average (n 3) for plots under elevated CO 2. Figure 1. A Concentration of inorganic nitrogen (N), B rate of potential net N mineralization, and C microbial biomass N in the top 15 cm of mineral soil during the first 5 treatment years of forest growth under ambient and elevated carbon dioxide (CO 2 ). The open bars indicate the average (n 3) for the plots under ambient CO 2 ; the filled bars are the average (n 3) for plots under elevated CO 2. lization exceeded rates of mineralization during the 40-h incubation period (Figure 2). Depth Variation in Soil N Cycling Elevated CO 2 did not affect the pool size or rate of net N mineralization across soil horizons, within a given soil horizon, or differentially between years (Table 3). In contrast, there was significant variation in the pool size and rate of net N mineralization among soil horizons and between years. Concentrations of inorganic N and the rate of potential net N mineralization were highest in the forest floor, intermediate in the 0 15-cm soil horizon, and lowest in the cm soil horizon (Figure 3). Pools and fluxes of inorganic N were significantly higher in 2000 than in There was a large pool of extractable organic N (ON) in all soil horizons (Table 4). In general, the pool of extractable ON was an order of magnitude larger than the pool of inorganic N (Figure 3). There was no effect of elevated CO 2 on the pool size or the net flux of extractable ON during the 28-day aerobic incubation. There was a statistically significant difference in the pool size but not in the net flux of extractable ON among soil horizons (Table 4). The pool of inorganic N was significantly higher in the forest floor than in the 0 15-cm or the cm layer of mineral soil. The CO 2 soil horizon interaction was not statistically significant for the pool size of ON. The initial pool size of extractable ON was significantly correlated (r ) with the rate of net N mineralization (Figure 4). A small fraction (about 10%) of the extractable ON pool was in the form of amino acids (Table 4). The pool of amino acids was two- to threefold higher than that of the inorganic N within a given soil horizon (Figure 3). Elevated CO 2 did not affect the concentration or net flux of amino acids during incubation. The concentration and net flux of amino acids varied significantly among soil horizons. The concentration and net consumption of amino acids was significantly higher in the forest floor than in the and cm soil horizons (Table 4). The CO 2 soil horizon interaction was not statistically significant.

7 450 A. C. Finzi and W. H. Schlesinger Table 3. Repeated-measures Analyses of Variance for Inorganic Nitrogen (N) Concentrations and Rate of Net N Mineralization under Ambient and Elevated CO 2 and across Three Different Soil Horizons Source of Variation df Inorganic N Concentration Net Mineralization MS F MS F Between subjects CO Horizon * * CO 2 Horizon Error Within subjects Year * ** Year CO * Year Horizon Year Horizon CO Error df, degrees of freedom; Mean Square,...; Fratlo,... Forest floor, 0 15-cm mineral soil, and cm mineral soil * P 0.05 ** P 0.01 DISCUSSION Elevated CO 2 and Soil N Cycling Increases in the availability of organic C to soil microbial communities can alter rates of N cycling (Diaz and others 1993; Zak and others 1993). In this forest ecosystem, 4 years of growth under elevated CO 2 significantly increased the input of C and N to the forest floor and the mineral soil via above- and belowground turnover (Finzi and others 2002; Matamala and Schlesinger 2000). Increased inputs of C and N under elevated CO 2 are highly correlated with increases in the surface efflux of CO 2 and the rate of heterotrophic respiration (Hamilton and others 2002; Hungate and others 1997; Zak and others 2000c; Andrews and Schlesinger 2001). In this ecosystem, the rate of heterotrophic respiration has more than doubled under elevated CO 2 (Hamilton and others 2002), indicating an increase in substrate availability to the microbial community under elevated CO 2 (see Zak and others 2000c). Despite the increase in substrate availability to the decomposer community, there was no statistically significant change in the cycling rate of N derived from soil organic matter under elevated CO 2. Neither the rate of net N mineralization (Figure 1) nor gross 15 NH 4 dynamics (Figure 2) were significantly altered by elevated CO 2. There was no statistically significant difference in the concentration or net flux of organic and inorganic N in the Figure 3. Depth variation in the concentration ( g g 1 ) of inorganic nitrogen (N) and the rate of net mineralization ( g g 1 28d 1 ) in 2000 and Each observation is the mean ( 1 SE) of samples (n 3) under ambient (open bars) and elevated (filled bars) carbon dioxide (CO 2 ). Superscript letters within a row indicate statistically significant differences among soil horizons. forest floor and top 30-cm of mineral soil after 5 years of CO 2 fumigation (Figure 3 and Table 4). Microbial biomass was not a larger sink for N (Fig-

8 Soil N Cycling Responses to Elevated CO Table 4. Initial Concentrations ( g g 1 ) and Net Fluxes ( g g 1 28 d 1 ) of 2M KCl Extractable Organic Nitrogen (ON) and Amino Acids (AA) in Soils under Ambient (A) and Elevated (E) CO 2 Interaction Means Forest Floor 0 15 cm cm P Values CO 2 Horizon A E A E A E CO 2 Horizon ON Concentration a (10.80) a (12.00) 7.10 b (0.58) 6.64 b (0.62) 2.48 b (0.31) 2.67 b (0.46) ON Flux (15.80) (11.05) 3.34 (1.59) 3.57 (1.59) 2.23 (0.82) 2.07 (0.98) AA Concentration a (4.49) a (6.48) 4.03 b (2.09) 3.80 b (1.49) 1.17 b (2.01) 1.23 b (2.01) a (1.73) 1.16 a (1.20) a (2.06) 0.43 a (1.20) AA Flux 9.27 b (3.33) b (5.74) 1 Non parametric analysis ure 1). Based on these results, we reject the hypothesis that elevated CO 2 significantly increases the rate of N immobilization by the microbial community. Past studies have shown that the rate of inorganic N cycling can increase, decrease, or remain unchanged in ecosystems exposed to elevated CO 2. Zak and others (2000c) proposed that the input of organic matter via primary production relative to the size of the native soil organic matter (SOM) pool could, in part, explain the range of soil N cycling responses observed under elevated CO 2.In ecosystems where SOM pools are small and the input of organic matter via plant production is high, the rates of soil N cycling should be strongly modulated by plant processes under elevated CO 2.In contrast, they argued that in ecosystems where SOM pools are large, additional inputs of organic matter under elevated CO 2 would not alter the influence of native SOM on soil N transformations. Support for the conceptual model of Zak and others (2000c) is limited by the small number of studies that have complete C and N budgets for ecosystems exposed to elevated CO 2. For example, Hungate and others (1997a), (1997b) measured a significant increase in belowground plant C allocation that increased the size of microbial biomass and the rate of gross NH 4 mineralization relative to immobilization in California grasslands dominated by annual species exposed to elevated CO 2. In this system, SOM contained only fold more C than was added to the soil each year through belowground turnover (Zak and others 2000c). In contrast, Pregitzer and others (2000) and Zak and others (2000a, 2000b) measured significant increases in belowground C and N inputs in Populus clones under elevated CO 2 but found no change in the size of the microbial biomass or gross and net rates of N mineralization. Background SOM pools in these Populus ecosystems were 1000 times larger than litter inputs (Zak and others 2000c). In the Duke Forest, inputs of C and N in aboveground litterfall are more than 10-fold higher than those in fine-root turnover (Table 5), suggesting that aboveground inputs control soil N cycling in this ecosystem. In particular, N cycling in the forest floor should be very sensitive to the increase in litter inputs under elevated CO 2. The mean residence time of C in the forest floor is 3.5 years (Schlesinger and Lichter 2001), suggesting that 28% of the organic matter in this horizon is consumed by the microbial community or exported as dissolved organic C within 1 year. In 1999, forest floor C content was 837 and 1106 g m 2 in the plots under ambient and elevated CO 2, respectively (Ta-

9 452 A. C. Finzi and W. H. Schlesinger Figure 4. The relationship between the rate of potential net nitrogen (N) mineralization and the initial concentration of 2M KCl extractable organic N. The regression line is statistically significant (P 0.01) with r The open symbols indicate samples from the plots under ambient carbon dioxide (CO 2 ) the filled symbols indicate samples from the plots under elevated CO 2. This plot includes data from both sample dates in 2001 and all horizons. ble 5). If we conservatively assume that only 10% of this mass is in the form of labile C substrates, there is an additional 27 gcm 2 a 32% increase over ambient CO 2 that could be available for microbial biosynthesis under elevated CO 2. We estimate that microbial biomass C in the forest floor under ambient and elevated CO 2 is 5.4 and 4.9 g C m 2, respectively (microbial biomass N is 0.45 and 0.41 g N m 2 under ambient and elevated CO 2 [Finzi and others 2002], and we assumed that the microbial community was dominated by fungi with a C:N ratio of 12:1 [see Paul and Clark 1986]). The additional input of labile C to the microbial community under elevated CO 2 is more than five times the size of the microbial biomass C pool, and native pools of C are only three times larger than the additional input of C under elevated CO 2 (that is, 10%of837gCm 2 under ambient CO 2 divided by the additional 27 g C m 2 under elevated CO 2 ). Consistent with increased rates of microbial N immobilization, the concentration of inorganic and organic N and the specific rate of net N mineralization were lower in the plots under elevated CO 2 (Figure 3). However, the differences were not statistically significant (Table 3). The chemistry of aboveground inputs appears to be as important as the quantity of inputs in explaining the N cycling responses in the forest floor horizon. During the first 4 years of CO 2 fumigation, nearly 50% of the cumulative flux of C in litterfall was accounted for by lignin (Table 1). The C in lignin occurs as complex ring structures that are recalcitrant to the decomposition process, making lignin a low energy-yielding substrate for microbial biosynthesis. Thus, the nonsignificant soil N cycling responses may reflect the large proportion of refractory C in aboveground litterfall. Only a fourth of the litter inputs was in the form of nonstructural carbohydrates labile substrates that are rapidly consumed by soil microbes and that yield considerable energy for microbial biosynthesis. Had elevated CO 2 consistently increased the fraction of nonstructural carbohydrates in leaves throughout the first 4 years of fumigation (Table 1), the increase in the rate of N immobilization we observed might have been statistically significant (Figure 3). Mineral soil N cycling responses to elevated CO 2 are correlated with the low rates of fine-root turnover in this system. The rate of fine-root turnover in the top 30 cm of mineral soil was 14 and 21gCm 2 y 1 under ambient and elevated CO 2, respectively, in 1998 (Matamala and Schlesinger 2000) and nearly identical in 1999 (Pritchard and others 2001). Ninety percent of the biomass of fine roots is concentrated in the top 15 cm of mineral soil (Matamala and Schlesinger 2000). These rates of fine-root turnover are very low in comparison to other temperate forest ecosystems (Pritchard and others 2001). Moreover, inputs of C via fine-root turnover are 1/100th of the standing pool of C in the mineral soil (Table 5). Thus, inputs of C via fine-root production may not have substantially increased C availability to the microbial community above what was already available from the degradation of the larger pool of soil organic matter. Consistent with the hypothesis of Zak and others (2000c), there was no statistically significant modulation of soil N cycling under elevated CO 2 (Figure 3). The rate of net N mineralization in laboratory incubations can differ from field-based incubations because of variations in temperature or moisture and because soils in laboratory incubations are often sieved prior to incubation, whereas intact cores that are collected and incubated in the field maintain soil structure (Binkley and Hart 1989). We measured the annual rate of net N mineralization in the field from June 1997 through May 1998 (Finzi and others 2002). The annual rate of net N mineralization varies greatly among plots, ranging from 12 to 43 kg N ha 1 y 1 in the top 15 cm of mineral soil. NPP and foliar N concentrations in this stand are highly correlated with the annual rate of net mineralization, indicating that NPP in this ecosys-

10 Soil N Cycling Responses to Elevated CO Table 5. Inputs and Pool of Carbon (C) and Nitrogen (N) in the Duke Forest under Ambient and Elevated CO 2 in 1999 Carbon Nitrogen Ambient Elevated Ambient Elevated Inputs (g m 2 yr 1 ) Leaf Litterfall a Root Turnover * Total Input * Pools (g m 2 ) 2 Forest Floor ** * Soil 0 15-cm depth Soil cm depth Total 33, Notes:Data sources: study 1, Matamala and Schlesinger , Schlesinger and Lichler 2001 (pools). tem is N-limited under ambient and elevated CO 2 (Finzi and others 2002). The average rate of potential net N mineralization measured in the lab in 1997 and 1998 is highly correlated with, and has the same rank order as, the annual rate of net N mineralization measured in the field (Spearman s 0.90, P 0.05, n 6). Thus, laboratory incubations of soils collected from this FACE site are representative of the between-plot variation measured in the field and should provide a good index of the effects of elevated CO 2 on the rate of net mineralization. Organic N Cycling Organic N (and amino acids in particular) is increasingly recognized as an important component of the terrestrial N cycle (for example, see Perakis and Hedin 2002; Lipson and Nasholm 2001; Jones and Kielland 2002). The bulk of the research on amino acid production and uptake by plants has been conducted in nutrient-poor ecosystems, where inorganic N production appears to lag behind the annual demand for N by vegetation (Kielland 1997; Raab and others 1996; Nasholm and others 1998). Our data show that organic N is an important component of the N cycle in this warm temperate ecosystem. Concentrations of extractable ON are on average an order of magnitude larger than concentrations of inorganic N (Table 4). Concentrations of amino acids are nearly double the concentration of inorganic N (Table 4 and Figure 3). Extractable ON also appears to be an important source of the N that is mineralized to inorganic forms by the microbial community (Figure 4). There is a clear spatial component to the production and consumption of ON and amino acids in this ecosystem (Table 4). The highest concentrations of ON and amino acids are in the forest floor; they are five to 10 times higher than the concentrations in the mineral soil horizons. During the 28-day aerobic incubations, net immobilization of amino acids was significantly higher in the forest floor than in the mineral soil. Our data suggest a very rapid rate of ON and amino acid production and consumption in the forest floor horizon. This presumably reflects the more labile constituents of the organic matter in the forest floor than in mineral soil horizons (Schlesinger 1997). Leaf litter contains significant quantities of proteins, amino acids, and nucleic acids, all of which are precursors to the release of amino acids. In contrast, much of the organic matter in soils is in the form of humic and fulvic acids, which are bound to soil particles. This organic matter is much harder to decompose or it is in forms that do not result in the generation of amino acids as a byproduct of microbial degradation. A variety of mechanisms can account for free amino acids in soils. These include the release of amino acids during the lysis of microbial cells, exudation by plant roots, and the hydrolysis of proteins and peptides by extracellular enzymes (Lipson and Nasholm 2001). The quantity of microbial biomass per unit of soil mass is much higher in the forest floor than in the mineral soil horizons (data not shown). This is consistent with a larger pool of labile C in the forest floor horizon than the mineral soil horizons. Thus we postulate that the greater turnover of microbial cells and the larger production of extracellular, proteolytic enzymes explain the higher concentration of amino acids in the forest floor than in the mineral soil horizon.

11 454 A. C. Finzi and W. H. Schlesinger Summary and Implications On a variable-by-variable basis, there is very little statistical support for significant differences in the pools or fluxes of soil N under ambient and elevated CO 2. Nevertheless, there appears to be a series of consistent responses that enable us to hypothesize that N limitation will ultimately decrease the NPP response to elevated CO 2 in this forest. During the course of stand development, southeastern US pine hardwood forests grow to a state of acute nutrient deficiency that can only be reversed with fertilization (Valentine and Allen 1990; Piatek and Allen 2000; Oren and others 2001). The immobilization of N in tree biomass and the forest floor appears to explain this result (Richter and others 2000; Piatek and Allen 2001). Richter and others (2000) found that 40 years of forest growth accumulated 36.6 and 74.0 gnm 2 in tree biomass and the forest floor, respectively, while mineral soil N pools declined by 82.3 gnm 2 (the difference between accumulation and loss was accounted for by atmospheric N deposition). They also found significant decreases in mineralizable N during the course of stand development. Data from the Duke Forest FACE experiment suggest a similar pathway for stand development and N cycling. Based on a recently completed N budget for this ecosystem (Finzi and others 2002), the quantity of N immobilized in plant biomass and the forest floor is increasing under elevated CO 2.By the end of the 4th year of this experiment, the quantity of N in plant biomass was 38.6 and 40.2 g Nm 2 under ambient and elevated CO 2, respectively (Finzi and others 2002). By October of the 3rd year of this experiment, the quantity of N in the forest floor was 16.6 and 20.6 gnm 2 under ambient and elevated CO 2, respectively (Table 5). Atmospheric N deposition contributes 0.7 gnm 2 y 1 to this forest ecosystem (Finzi and others 2002). The difference in the quantity of N accrued in biomass and the forest floor by the end of the 4th year of CO 2 fumigation (conservatively estimated at 5.6gNm 2 ) is more than double the quantity of N inputs via atmospheric deposition during the same time period (2.8 gnm 2 ). This implies that N is being translocated from mineral soil to vegetation more rapidly under elevated CO 2 ; the difference in the standing pool of N in the top 30 cm of mineral soil appears to reflect pretreatment differences among experimental units (Table 5) (Schlesinger and Licther 2001). There are at least three mechanisms that could accelerate the decrease in the quantity of mineralizable N under elevated CO 2. First, the immobilization of N in perennial tissues should decrease the quantity of N that is cycling through relatively labile soil pools (see Richter and others 2000). Second, the increase in forest floor mass and C:N ratio under elevated CO 2 should increase the demand for N by the microbial community leading to greater N immobilization (see Piatek and Allen 2001). Third, the significant increase in the flux of lignin to the forest floor (Table 1) should increase N immobilization because the decomposition of lignin and its subsequent reaction with N forms compounds that resist further decay (see Berg 1986; Fogg 1988). Consistent with these mechanisms, the rate of net mineralization was systematically lower in the forest floor horizon in the 4th and 5th growing seasons under elevated CO 2 (Figure 3). Net mineralization was lower in the 4th growing season but nearly identical in the 5th growing season under elevated CO 2 (Figure 3). Consistent with decreases in the rate of N mineralization and greater uptake of N by plants under elevated CO 2, the concentration of inorganic N was systematically lower in the forest floor and top 15 cm of mineral soil in the 4th and 5th growing seasons (Figure 3). The decrease in the concentration of inorganic N is a necessary, although insufficient, condition to document greater N limitation to forest production under elevated CO 2 through time. None of these differences in soil N cycling in response to elevated CO 2 can be substantiated statistically. However, there are important similarities in the pattern of stand development and N cycling between the Duke Forest and other southeastern pine forests (Richter and others 2000; Piatek and Allen 2001). Our current results suggest that more rapid stand development under elevated CO 2 will accelerate the rate at which this forest reaches a state of severe nutrient limitation. Thus, we hypothesize that elevated CO 2 will only increase the productivity of this forest during the initial stages of stand development, with N limitation constraining additional C sequestration under elevated CO 2 well before this stand reaches its equilibrium biomass. ACKNOWLEDGMENTS George Hendrey, John Nagy, and Keith Lewin were instrumental in the construction and maintenance of the FACE facilities. We thank Heather Hemric, Anthony Mace, and Jeffrey Pippen for their assistance in the field and Ariana Sutton, Damon Bradbury, and Meredith Zaccherio for their assistance in the lab. We also thank Don Zak and Bill Holmes for their technical assistance with the 15 N pool dilution experiments. This study was supported by the US Department of Energy, with additional support from the National Science Foundation (DEB 98-

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