Microbial Denitrification Dominates Nitrate Losses from Forest Ecosystems

Microbial denitrification dominates nitrate losses from forest ecosystems

Yunting Fanga,b, Keisuke Kobab,1, Akiko Makabeb, Chieko Takahashib, Weixing Zhuc, Takahiro Hayashib, Azusa A. Hokarib, Rieko Urakawad, Edith Baia, Benjamin Z. Houltone, Dan Xia, Shasha Zhanga, Kayo Matsushitab, Ying Tua, Dongwei Liua, Feifei Zhua, Zhenyu Wanga, Guoyi Zhouf, Dexiang Cheng, Tomoko Makitab, Hiroto Todab, Xueyan Liub, Quansheng Chena,h, Deqiang Zhangf, Yide Lig, and Muneoki Yohb

aState Key Laboratory of Forest and Soil Ecology, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110164, China; bInstitute of Agriculture, Tokyo University of Agriculture and Technology, Tokyo 1838509, Japan; cDepartment of Biological Sciences, Binghamton University, The State University of New York, Binghamton, NY 13902; dGraduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 1138657, Japan; eDepartment of Land, Air, and Water Resources, University of California, Davis, CA 95616; fKey Laboratory of Vegetation Restoration and Management of Degraded Ecosystems, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China; gResearch Institute of Tropical Forestry, Chinese Academy of Forestry, Guangzhou 510520, China; and hState Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China

Edited by Sarah E. Hobbie, University of Minnesota, St. Paul, Saint Paul, MN, and approved December 10, 2014 (received for review September 1, 2014) Denitrification removes fixed nitrogen (N) from the biosphere, watershed mass balance techniques are nonintrusive and can pro- thereby restricting the availability of this key limiting nutrient for vide larger scale insights into gaseous N losses, this approach relies terrestrial plant productivity. This microbially driven process has on difficult-to-measure N input fluxes and assumptions therein, been exceedingly difficult to measure, however, given the large and is geographically constrained to environments that lack ground background of nitrogen gas (N2) in the atmosphere and vexing water seepage (7). scaling issues associated with heterogeneous soil systems. Here, As an alternative, natural composition of N and oxygen (O) we use natural abundance of N and oxygen isotopes in nitrate − − isotopes in NO3 provides nonintrusive, quantitative, and in- (NO3 ) to examine dentrification rates across six forest sites in tegrative constraints on denitrification across a myriad of space- southern China and central Japan, which span temperate to tropical time scales (8, 9). This approach takes advantage of the bi- climates, as well as various stand ages and N deposition regimes. ological imprint of soil denitrification on the N cycle and the Our multiple stable isotope approach across soil to watershed tendency for kinetic isotope effects to elevate 15N/14Nand18O/16O scales shows that traditional techniques underestimate terrestrial − of NO3 systematically in forests (8). Here, we extend on this ap- denitrification fluxes by up to 98%, with annual losses of 5.6– − proach by developing a new way to estimate NO3 supply rates to 30.1 kg of N per hectare via this gaseous pathway. These N export 17 15 18 − denitrification, which involves a combined Δ O, δ N, and δ O fluxes are up to sixfold higher than NO3 leaching, pointing to − analysis. We hypothesize that denitrification rates examined via our widespread dominance of denitrification in removing NO3 from forest ecosystems across a range of conditions. Further, we report multiple-isotope approach will be larger than denitrification rates − that the loss of NO3 to denitrification decreased in comparison to based on conventional soil techniques because they are generally leaching pathways in sites with the highest rates of anthropo- applicable to the upper part of soil [e.g., the upper 50 cm (9)] and genic N deposition. are challenged by spatial and temporal complexities in the soil denitrification process. We examine this hypothesis across an array of denitrification | nitrate isotopes | nitrogen cycling | forested watersheds sites spanning tropical (southern China) to temperate (central Japan) regions, as well as various stand ages and N input levels, including itrogen (N) is an essential, although ecologically limiting, Nnutrient in many terrestrial ecosystems (1). Anthropogenic Significance emissions of reactive forms of N due to fossil fuel combustion and modern agriculture practices have drastically increased N de- Nitrogen (N) losses from terrestrial ecosystems can occur as inert − position inputs to forests globally (2). Atmospherically deposited N forms or heat-trapping greenhouse gases, and via nitrate (NO3 ) represents a new N input to terrestrial ecosystems and may enhance leaching to drainage waters, which can contribute to eutrophi- carbon dioxide sequestration, potentially reducing some global cation and anoxia in downstream ecosystems. Here, we use nat- climate impacts (3). On the other hand, long-term N deposition ural isotopes to demonstrate that microbial gaseous N production − − could result in N saturation and increased nitrate (NO3 )losses via denitrification is the dominant pathway of NO3 removal from to leaching and denitrification (4, 5), pathways that have dif- forest ecosystems, with gaseous N losses that are up to ∼60-fold ferent consequences for the Earth system, including con- higher than those based on traditional techniques. Denitrification − sequences on climate, biodiversity, and water and air quality becomes less efficient compared with NO3 leaching in more N- for human health (6). Thus, in the context of global climate polluted ecosystems, which has important implications for as- and other biogeochemical changes, it is critically important to sessing the connections between terrestrial soils and down- understand terrestrial N balances and their responses to anthro- stream ecosystems under rising anthropogenic N deposition. pogenic N inputs. Denitrification is considered the most poorly resolved pathway Author contributions: Y.F. and K.K. designed research; Y.F., K.K., A.M., C.T., T.H., A.A.H., of N removal from the soil, owing to difficulties in quantifying R.U., D.X., S.Z., K.M., Y.T., D.L., F.Z., Z.W., G.Z., D.C., T.M., H.T., X.L., Q.C., D.Z., Y.L., and M.Y. performed research; Y.F., K.K., A.M., C.T., W.Z., E.B., and B.Z.H. contributed new denitrification rates using standard methods (7). Conventional reagents/analytic tools; Y.F., K.K., A.M., C.T., W.Z., T.H., A.A.H., R.U., E.B., B.Z.H., D.X., S.Z., approaches used to estimate denitrification rates are largely in- K.M., Y.T., D.L., F.Z., Z.W., G.Z., D.C., T.M., H.T., X.L., Q.C., D.Z., Y.L., and M.Y. analyzed trusive and challenged by issues of pattern and scale. Acetylene data; and Y.F., K.K., W.Z., E.B., and B.Z.H. wrote the paper. 15 block, N tracer applications, and direct nitrogen gas (N2) quan- The authors declare no conflict of interest. tification are examples of approaches that have deepened our This article is a PNAS Direct Submission. understanding of denitrification; however, they are inherently 1To whom correspondence should be addressed. Email: [email protected]. disruptive and cannot be applied at scales larger than individual This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. soil cores without a high degree of extrapolation (7). Whereas 1073/pnas.1416776112/-/DCSupplemental.

1470–1474 | PNAS | February 3, 2015 | vol. 112 | no. 5 www.pnas.org/cgi/doi/10.1073/pnas.1416776112 Downloaded by guest on September 23, 2021 − Table 1. Site information and NO3 budget for the six study forests Jianfengling Jianfengling Dinghushan FM Ohyasan FM Ohyasan FM Tamakyuryo (primary, JFL-P) (secondary, JFL-S) (DHS) (old-aged, OYS-O) (middle-aged, OYS-M) (TM)

Climate Tropical Tropical Tropical Temperate Temperate Temperate Latitude and 18°43′47′′N, 18°44′17′′N, 23°10′18′′N, 36°33′44′′N, 36°33′42′′N, 35°38′18′′N, longitude 108°53′23′′E 108°52′14′′E 112°32′23′′E 139°21′13′′E 139°21′07′′E 139°22′35′′E Forest type Broad-leaved Broad-leaved Broad-leaved Coniferous Coniferous Broad-leaved Age, y Primary ∼50 >400 107 38 ∼60 MAP, mm 2,449 2,449 1,997 1,743 1,743 1,780 MAT, °C 19.8 19.8 21.0 9.7 9.7 14.0 DIN deposition* 6.1 6.1 33.5 11.3 11.3 14.8 + NH4 deposition* 3.0 3.0 20.3 6.1 6.1 8.7 − NO3 deposition* 3.1 3.1 13.2 6.2 6.2 6.1 Gross nitrification† 48 43 116 88 119 118 − NO3 leaching* 2.5 2.4 18.4 13.0 12.4 13.3 + NH4 leaching* 0.2 0.2 1.2 0.4 0.3 0.2 Denitrification‡ 15.4 (3.6) 5.6 (2.7) 30.1 (8.3) 12.1 (2.7) 19.7 (9.4) 22.3 (5.9) Denitrification by 3.0 6.0 9.4 0.2 0.2 1.8 § N2O/(N2O + N2) ratio Gaseous N loss by soil 2.0 2.1 9.8 8.5 7.7 7.4 15N enrichment§

DIN, dissolved inorganic nitrogen; FM, field museum; MAP, mean annual precipitation; MAT, mean annual temperature. − − *Measured in this study in kg of N ha 1·y 1, except for TM, where data were cited from a previous study (SI Text). † − − Calculated in kg of N ha 1·y 1 based on Eq. 4 as described in the main text. ‡ − − Mean and SD (in parentheses) calculated in kg of N ha 1·y 1 based on Eq. 7. §Calculated in kg of N ha−1·y−1 (Methods).

−1 −1 −1 −1 − moderate (6.1 kg of N ha ·y )tohigh(33.5kgofNha ·y ; transferred from ozone to NO3 in the atmosphere during pho- Table 1) rates of N deposition from anthropogenic activities. tochemical reactions. In contrast, O atoms incorporated into soil − NO3 during nitrification are derived from atmospheric O2 and − 17 Principles of NO3 Isotope Approach soil H2O molecules, which have Δ O values of −0.34‰ and 0, − We conceptualized the soil NO3 pool as an open, continuous flow- respectively (12). Because the nitrification process is a normal, 17 − through system with competing plant-soil-microbe sinks. There mass-dependent kinetic process, the Δ O value of NO3 pro- − − are two NO3 input sources, atmospheric NO3 deposition and duced from nitrification can be taken as 0 (14, 15). Furthermore, − soil nitrification, and three NO sinks, organismal uptake, deni- other terrestrial processes, including denitrification and micro- 3 Δ17 − trification, and leaching to streams, in the forest N cycle. The bial and plant assimilation, do not affect the O of soil NO3 − because these processes impart mass-dependent effects on δ17O turnover of NO3 is rapid, with a residence time as short as 2 d in and δ18O (14, 15). As a result, the Δ17O signature can be used most forest soils (10). Further, past work in some of our forest sites − − to distinguish NO3 that originates from the atmosphere (i.e., reveals little accumulation of NO3 in the soil across a range of N − 17 Δ OA > 0) relative to that produced via soil nitrification reac- addition treatments (Fig. S1), reflecting the high mobility of NO3 . − Δ17 = We therefore assume no net NO accumulation on an annual tions (i.e., ON 0). Hence, according to a two-source mixing 3 model, we derive Eq. 3: basis (SI Text) and derive the following mass balance equation: À Á 17 17 17 FA × Δ OA + FN × Δ ON ðFA + FNÞ = Δ OsoilNO3 ; [3] FA + FN = FU + FD + FL; [1] -

− 17 17 17 17 where Δ OA, Δ ON, and Δ OsoilNO3− are the averaged Δ O where FA,FN,FU,FD, and FL represent annual NO3 fluxes of − atmospheric deposition, soil gross nitrification, plant or micro- values of NO3 derived from atmospheric deposition, nitrifica-

tion, and their fractional mixtures, respectively. SCIENCES bial gross uptake, denitrification, and leaching, respectively. 17 The seasonal change in Δ OsoilNO3− is integrated into the FA and FL can be monitored with a fair degree of precision (11). ENVIRONMENTAL Δ17 − Δ17 However, estimating gross nitrification rates at scales larger than seasonal change in O of stream NO3 leaching ( OL). Given Δ17 − individual soil columns has remained challenging. Traditionally, that microbial and plant sinks do not alter the OofsoilNO3 , 15 17 17 gross nitrification rates have been determined by using the Npool Δ OsoilNO3− is approximately equivalent to Δ OL. Hence, taking 17 dilution method in individual soil cores (10); however, spatial Δ ON = 0, we combine Eqs. 1 and 3 to calculate FN: and temporal heterogeneities in nitrification call the applica- À Á 17 17 17 tion of this technique into question at larger scales. Here, we FN = FA × Δ OA − Δ OL Δ OL: [4] apply a method to estimate gross nitrification rates for forested catchments using Δ17O (defined by Eq. 2, in per million) (12) of This method has been used to evaluate gross nitrification rates in − atmospheric NO3 as a naturally conserved tracer: a lake system (16) and recently in soils (17), and it requires de- 17 − Â À Á À ÁÃ termination of Δ OofNO3 in atmospheric deposition and 17 17 18 − Δ O = ln 1 + δ O 1;000 − 0:5247 × ln 1 + δ O 1;000 stream water, as well as NO3 deposition mass fluxes. [2] − 17 In addition to NO3 source partitioning via Δ O, we partitioned × 1;000: − NO3 losses to streams (FL) and denitrification (FD), and gross − δ15 Past work has shown that atmospheric NO3 exhibits systematically biological uptake by microbes and plants (FU)using Nand positive Δ17O (13, 14). This positive Δ17O occurs because the corresponding isotope effects of these processes on 15N/14N. excess 17O (compared with mass-dependent expectations) is According to isotope balance, we thereby derive Eqs. 5 and 6:

Fang et al. PNAS | February 3, 2015 | vol. 112 | no. 5 | 1471 Downloaded by guest on September 23, 2021 À Á À Á δ15 = f × δ15 − e + f × δ15 − e NNO3-input D NsoilNO3- D U NsoilNO3- U + f × δ15 L NsoilNO3- [5]

fD + fU + fL = ðFD + FU + FLÞð= FA + FNÞ = 1; [6]

15 15 − where δ NsoilNO3− refers to the δ NofNO3 in the whole soil 15 pool and δ NNO3−input includes both nitrification and atmo- − spheric NO3 deposition. The abbreviations eD and eU denote isotope effects (e = 14k/ 15k − 1, reported in ‰, with k being the − rate constant) by denitrification and biological NO3 uptake. The abbreviations fD, fU, and fL are proportionality constants − of the various NO3 sinks. Hence, combining and reorganizing Eqs. 5 and 6 yields: Â À Á = ð + Þ × δ15 − δ15 − e FD FA FN NsoilNO3- NNO3-input U Fig. 1. Concentrations (A and C)andδ15N(B and D) of bulk soil and Ã [7] − extracted NO along soil profiles (mean ± 1SE,n = 2–6). con., concentration. + FL × eU ðeD − eUÞ: 3

− Finally, FD is determined by NO3 inputs (FA and FN) and − 15 − This finding points to stratified NO3 dynamics in our sites, with leaching losses (FL), the δ N of inputs and soil NO3 , and iso- − NO3 production via nitrification in near-surface soils and micro- tope effects associated with denitrification and biological uptake. − bial NO3 uptake and denitrification in deeper soil zones. − Results and Discussion We quantified rates of NO3 production (i.e., nitrification) by parameterizing our Δ17Oisotopemodel(Eq.4) with measured We used natural N and O isotope approach (described above) − 17 − annual NO3 fluxes in precipitation (Table 1) and Δ Oforat- to quantify patterns and rates of gross NO production and − 3 mospheric and stream water NO (Table S2). The mean Δ17Ofor dentrification across an array of forest ecosystems. The sites 3 atmospheric deposition ranged from 13 to 24‰, whereas the mean we examined covered a broad spectrum of atmospheric N de- − Δ17O in stream NO was low, varying from 0.8 to 1.7‰ (Table position rates, soil N contents, and stand development ages, and 3 S2) across our sites. The mixing model suggests that atmospheric they included temperate vs. tropical forest biomes (Table 1). − − − NO3 deposition accounts for 5–10% of the soil NO3 pool, similar NO3 leaching to streams also showed a large range among these − − forest sites (2.4–18.4 kg of N ha 1·y 1, Table 1), thus providing us to the results from synoptic or event sampling in other studies (3–10%; Table S2). Estimated gross nitrification rates using our Δ17 with the opportunity to examine a broad range of influences on 4 −1· −1 forest N balances and denitrification fluxes. Oapproach(Eq. ) varied from 43 to 119 kg of N ha y (Table − – We found isotopic evidence for NO consumption via de- 1), being 40 200% higher than their corresponding net nitrification 3 rates quantified previously by field incubation methods (Table S1 nitrification across all of our forest sites, particularly from shallow − − 15 and SI Text). Gross NO3 production rates are usually much higher to deeper soils. The δ N of soil-extracted NO3 was generally − − negative, particularly in surface soils, being much lower (by 6 to than net NO3 production rates due to substantial microbial NO3 15 immobilization, particularly in N-limited coniferous forests, where 13.4‰) than the δ N of soil organic N, which ranged from 0 to − − 7.5‰ (Fig. 1 and Table S1). The δ15N and δ18OofNO in soil soil NO3 concentrations are generally low (10). 3 7 SI extracts and soil water samples increased with increasing depth Parameterizing our model with the field data (i.e., Eq. , − Text,andFig. S7), we estimated that denitrification rates varied and in phase with the declines in NO3 concentrations observed − − − between5.6and30.1kgofNha 1·y 1 in our forest sites (Table 1). in profile (Figs. 1 and 2 and Figs. S2 and S3). The NO3 concentration-weighted δ15N integrated to 1 m beneath the soil A model sensitivity analysis conducted by changing the model ± surface varied from −4.2 to −1.0‰ across our sites (Fig. 3). These input parameters by 10% (21) indicates a confidence interval of 15 − 0.5–11.4% in our denitrification estimates (Fig. S8). Our approach averages were lower (by 3.9 to 6.6‰) than the δ NofNO3 in stream water, which was mostly positive (Fig. 3); lower (by 0.9 to is especially sensitive to the isotope effects of denitrification and − 15 ‰ microbial uptake (Fig. S7), NO3 supply estimates, and the δ N 6.1 ) than the average values in soil water (Figs. S2 and S3); and − 15 − much lower (by 6 to 13‰) than the δ NofsurfacesoilNO3 of soil NO3 input used in the model (Fig. S8). Nevertheless, there extractions (Table S1). These results, in combination with the is reason to believe that our estimates of denitrification are con- − δ15 changes in NO3 concentration observed (Figs. 1C and 2), dem- servative. In particular, we used the concentration-weighted N − 15 14 integrated to a depth of 1 m to estimate the δ15N of the initial onstrate that NO3 was both consumed and elevated in N/ Nand − 18 16 − 15 NO3 available for denitrification (i.e., δ NNO3−input; SI Text). O/ O as it moved from environments of NO3 production in − surface soils to deeper soils and streams. However, our data suggest that much of this NO3 was already These changes and the correlations between N and O isotopes consumed by denitrification (22) and biological uptake (Fig. 1C − δ15 − and NO3 concentrations point to a balance between kinetic and Fig. S3). When the average NofNO3 in the uppermost − isotope effects during microbial NO3 reduction (18) and other soil samples (Table S1) is used instead, we estimate even higher − − − organismal uptake processes, which consume NO without a denitrification rates (22.0–48.9 kg of N ha 1·y 1). Further, in this 3 − substantial isotope effect in natural sites (19, 20) (SI Text and Fig. study, we used bulk collectors to estimate NO3 deposition inputs. − S4). The δ15Nandδ18OofNO were significantly correlated in all This approach has been shown to underestimate or miss altogether 3 − but the mature Ohyasan forest site (OYS-O), with slopes ranging dry NO3 deposition and forest canopy scavenging effects (23). A − from 0.3 to 1.5 (Fig. S5). These slopes correspond to microbial 10% increase in NO3 deposition would correspond to roughly − 15 NO3 consumption in the soil, with past work revealing δ Nand a 6% increase in denitrification rates in our approach (Fig. S8). δ18O slopes between 0.5 and 1 for denitrification (18) and between Our results suggest a dominant role for soil denitrification in − 1 and 2 for microbial NO3 assimilation (19) (SI Text and Fig. S4). removingplantavailableNacrossawidevarietyofforestsitesand 15 18 − – In contrast, such correlations between δ Nandδ OinNO3 conditions (Table 1). Denitrification accounts for 48 86%(65%on − − were not apparent in the uppermost surface soil samples (Fig. S6). average) of total NO3 losses (denitrification plus NO3 leaching)

1472 | www.pnas.org/cgi/doi/10.1073/pnas.1416776112 Fang et al. Downloaded by guest on September 23, 2021 − Fig. 2. Concentrations of NO3 in soil water and stream water from the study forests. Box plots show median values, 25th and 75th percentiles, and outliers. Different letters indicate significance within the same forest, analyzed by one-way ANOVA. The numbers below boxes are replicates.

from our forest ecosystems. This result is consistent with the high against the composition of bulk soil 15N/14N (9, 21). This approach denitrification fractions (∼25% to >50%) vs. total N losses (de- similarly yields lower denitrification fluxes than our method, which − −1 −1 nitrification plus total dissolved N leaching, including dissolved isbasedonNO3 isotope composition (i.e., 2.0–9.8 kg of N ha ·y ; organic N) that have been estimated for five Hawaiian tropical Table 1). This difference is consistent with the idea that bulk soil forests by N isotope balance (8). These high denitrification frac- δ15N data yield a lower bound for denitrification rates from the land biosphere (9), because this approach misses zones of complete tions are much greater than implied by traditional soil-based − approaches (Table 1). NO3 consumption and isotope effects of denitrification that can be At both local and global scales, denitrification losses are often dilutedatlargerscales(8,9). estimated by nitrous oxide (N2O)/(N2 + N2O) ratios and the N2O The isotope-based evidence we advance for high denitrification emission rates, with an average N Ofractionof∼0.5 observed for fluxes is supported by two cases studies using the mass balance ap- 2 −1· −1 soils under natural vegetation (24). Applying this fraction to data proach in a temperate forest (35 kg of N ha y ) (25) and a trop- −1· −1 collected across our study sites yields denitrification fluxes that are ical forest (49 kg of N ha y ) (26). High denitrification rates but substantially lower than our isotope-based approach (i.e., 0.2– low N2O loss rates for our study forests suggest that N2 and perhaps − − 9.4 kg of N ha 1·y 1; Table 1). Another method has taken advantage nitrogen oxide are important byproducts of denitrification. This as- sertion is consistent with reports based on gas flow-soil core tech- of total ecosystem N isotope balances and isotope fractionations 15 niques (27), local N-N2 flux measures (28), and previous natural isotope approaches in wet tropical forests (8), which show that N2 is the dominant product of denitrification, with N2/N2Oratiosex- ceeding 48 (28) to 85 (29) in local measures. Additional research on denitrification and gaseous N2 production will help advance our understanding of the missing N sink in the terrestrial biosphere (9). Our results have implications for climate and other global environmental changes. Denitrification is a critically important SCIENCES

process that removes bioavailable N from the Earth system, in ENVIRONMENTAL which N2 is the terminal product and nitric oxide (NO) and N2O are intermediates. NO is a free radical and is rapidly oxidized in air to nitrogen dioxide. At Dinghushan (DHS), NO and N2O account for 20–23% and 16% of denitrification, respectively, and at Jianfengling (JFL), N2O accounts for 10% and 54% in the primary and secondary forests, respectively (Table 1 and Table S1). In contrast, N2O accounts for 1–4% of denitrification in our temperate sites [OYS and Tamakyuryo (TM)] (Table 1 and Table S1). These results agree with the idea that tropical forests are a large natural source of atmospheric N2O (21, 30). − 15 − Further, NO3 leaching and denitrification losses significantly Fig. 3. δ NofNO3 in stream water (○, individual samples) and extracted − increased with increasing N deposition, but at different rates NO3 at 0–100 cm of soil depth (ranges of concentration-weighted means are − across the six forests we examined (Table 1). The proportion of shown in gray) in the study forests. NO3 flux-weighted means (thick lines) and − − 15 – NO3 concentration-weighted means (thin lines) ± 1SEareshownforδ N N lost to denitrification vs. total NO3 losses decreased from 70 − − of stream water NO and the extracted soil NO (atthedepthof0–100 cm), 86% in the lowest N deposition sites (JFL) to 48–63% in the four 3 3 − respectively. Replicates are shown in parentheses. other forests, where N deposition and NO3 leaching were

Fang et al. PNAS | February 3, 2015 | vol. 112 | no. 5 | 1473 Downloaded by guest on September 23, 2021 15 14 higher (Table 1). This result is consistent with the reports for gaseous N loss rate by soil N/ N enrichment (9, 21). Soil N2O emission rates – −1· −1 + streams (31) and coastal waters (32), showing that the efficiency were 0.1 4.7 kg of N ha y (Table S1), and an average N2O/(N2O N2) − ratio of 0.5 (24) was used. We constrained gaseous N losses through the soil of biological NO3 removal via denitrification decreases with increasing N loading. With the global trend of increasing N de- 15N/14N approach using Eqs. 8 and 9 (9, 21): − position, an increased proportion of NO3 leaching might be δ15 = δ15 + × e + × e expected, posing additional risks of acidification and eutrophi- NNsoil NNinput fG G fL L [8] cation on downstream ecosystems. fG + fL = 1, [9]

15 15 15 15 Methods where δ NNsoil and δ NNinput represent the δ N of bulk soil and the δ NofN Study Site, Sampling, and Laboratory Analysis. We applied our natural isotope inputs derived from N fixation and atmospheric deposition (different from the

approach to six intensively studied forest sites (Table 1). Bulk precipitation, Eq. 5), respectively. eG and eL are the isotope effects associated with gaseous N

soil water, stream water, and soil were collected from each site. Water loss (denitrification) and N leaching to streams, and fG and fL denote their + − samples and soil extracts were analyzed for ammonium (NH4 ) and NO3 proportion of gaseous N loss and total dissolved nitrogen leaching loss, re- − concentrations, as well as for N and O isotopes of NO3 . The denitrifier spectively. The details on parameterization of Eqs. 8 and 9 can found in SI Text. 15 18 − method (33, 34) was used to determine δ Nandδ OofNO3 in all samples, excluding stream waters collected from JFL forests in 2012 and soil extracts ACKNOWLEDGMENTS. We thank X. M. Fang, M. Kuroiwa, and S. Y. Fan for from JFL and DHS forest soil profiles collected in 2013, which were determined field sampling or help during laboratory analysis. This work was financially Δ17 − by the chemical conversion method (35). The OofNO3 of selected pre- supported by the Japan Society for the Promotion of Science [JSPS; Funding cipitation and stream water samples was determined by combining bacterial Program for Next Generation World-Leading Researchers (GS008) and reduction (i.e., denitrifier method) and the thermal decomposition method Grants-in-Aid for Scientific Research (26252020)]; the National Natural (36). Subsamples of soil were measured for concentrations of carbon (C), N, Science Foundation of China (Grant 31370464); the State Key Laboratory and δ15N. N inputs and N leaching losses were determined by multiplying N of Forest and Soil Ecology, Institute of Applied Ecology, Chinese Academy of Sciences (Grant Y1SRC211S1); and a grant-in-aid for Scientific Research from concentrations by water fluxes. Additional details can be found in SI Text. the JSPS (Grant 21310008). B.Z.H. was supported by National Science Foundation Grant DEB-1150246. Y.F. and X.L. were also supported by the Comparison with Conventional Methods. For the purpose of comparison, we JSPS, with a Postdoctoral Fellowship for Foreign Researchers and a Grant-in- estimated denitrification rates using the N2O/(N2O + N2) ratio (24) and total Aid for JSPS Fellows (Grants 20-08421 and 21-09318, respectively).

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