Increased plant biomass accumulation in response to elevated atmospheric CO2 (eCO2) is largely constrained by limited soil nitrogen (N) availability (1–7), so increasing the supply of N is expected to mitigate CO2 emissions and associated climate warming by sustaining the CO2 enhancement of carbon (C) storage in terrestrial ecosystems (1–7). Ecosystem inputs of human-made N compounds, mostly in the form of ammonium (NH4+)–based fertilizers, are enormous, but the distribution between natural and managed/agricultural systems is unbalanced (8–12). The majority of NH4+ fertilizers have been applied to agroecosystems (8–14), driven by a growing global demand for food. In particular, the global application of NH4+ fertilizers to rice ecosystems, which supply the staple food to more than half of the world’s population, was estimated at 19 Tg N in 2015, ~20% of N fertilizer use in all crops (8), and this value is projected to increase under future eCO2 (8–11). Increasing the use of NH4+ over nitrate (NO3−) fertilizers may enhance the growth of rice plants under eCO2, in part because plants under eCO2 prefer NH4+ over NO3− (5, 6). However, whether high NH4+ inputs sustain the response of rice ecosystems to eCO2 hinges, in part, on the extent to which eCO2 influences the stability of NH4+ in paddy soils, especially under anaerobic conditions.
Effects of eCO2 and N supply on plant N uptake
Here, we present results from a long-term free-air CO2 enrichment (FACE) study in which three ambient (aCO2) and three elevated (ambient + 200 μmol mol−1 CO2) atmospheric CO2 plots have been maintained to investigate the response of a rice ecosystem to eCO2 (fig. S1) (15, 16). We first examined the effect of eCO2 on rice growth at seven levels of NH4+ fertilization: 0, 8, 12.5, 15, 22.5, 25, and 35 g urea-N m−2 (see Materials and Methods). At each N level, eCO2 significantly increased aboveground biomass of rice plants (P < 0.01; fig. S2A). Theory predicts (1, 3, 4) that the net CO2effect on plant N uptake should be higher with high N supply than with low N supply, regardless of whether it is within a N-limited or a N-rich system (fig. S2B). However, eCO2 did not increase plant N uptake as much as anticipated at high N supply compared to low N supply both across the four rice growth stages in an early experimental year (fig. S2C) and for the entire experimental period (fig. S2D). The CO2-induced aboveground alterations such as photosynthetic acclimation (17) and tissue N concentrations (fig. S2A) could not adequately explain the lower than expected net CO2effect on plant N uptake, especially at high compared to low levels of NH4+-N supply (fig. S2, C and D). We, therefore, reason that alterations in NH4+-N transformation in the soil may contribute partly to the CO2-induced negative feedback on plant N uptake in the rice paddy system.
N loss via AOA under eCO2
We explored the possible microbial mechanisms by which eCO2 transforms NH4+-N in the paddy soil. We first used metagenomic sequencing to analyze microbial functional genes associated with N cycling in the field (see Materials and Methods). Compared to aCO2, eCO2 increased the abundance of many functional genes, especially those involved in anaerobic N transformations (fig. S3). For much of the time rice is growing, anaerobic conditions prevail in paddy soils. In previous work, we demonstrated that eCO2 promoted soil anaerobic conditions by stimulating microbial activity and decreasing redox potential in the rice paddy system (15). In this research, we further investigated the CO2 effect on the NH4+-N transformations under anaerobic conditions by conducting three separate but complementary experiments (see Materials and Methods). Experiment 1 included two parallel microcosm experiments carried out in 2014 and 2015 (Fig. 1A). Experiment 2 was an in situ field study conducted in 2016 (Fig. 1B). Experiment 3 consisted of two parallel stable isotope probing (SIP) microcosm experiments performed in 2017 (Fig. 1C). We determined whether NH4+-N could be converted to an oxidized form using 15N-NH4+ in experiments 1 and 2. We measured the rate of NH4+ consumption in experiment 3.
Fig. 1 Losses of ammonium (NH4+)–nitrogen from a rice ecosystem exposed to long-term FACE.
(A to C) Experimental scheme: experiment 1 (A), two parallel microcosms in which soils, taken from ambient (aCO2) and elevated (eCO2) CO2 plots at the heading stage of rice plants in 2014 and 2015, were incubated with the addition of 15NH4+ under strictly anaerobic conditions; experiment 2 (B), a field work with an anaerobic bioreactor conducted in the 2016 rice growing season; experiment 3 (C), two parallel SIP microcosms in which soils, taken from aCO2 and eCO2plots in the 2017 rice growing season, were anaerobically incubated with the addition of 13C-labeled either 13CH313COO− + NH4+ (SIP 1) or 13CO2 + NH4+ (SIP 2). (D) Influence of eCO2 on 15N-N2 production (total amount of 29N2 and 30N2; light blue bars) in experiments 1 (P < 0.05) and 2 (P < 0.01), and NH4+ consumption (dark blue bar) in experiment 3 (P < 0.05). The net CO2 effect was calculated: (value at eCO2 − value at aCO2)/(value at aCO2). Values are means (n = 3) ± SEM.
We found that eCO2 led to considerable losses of N from the anaerobic paddy soil when NH4+ was added alone (hereafter +NH4+) in experiments 1 to 3. In experiment 1 in which soils were amended with 15NH4+ (Fig. 1A), headspace 15N-labeled N2 (30N2 + 29N2) production averaged across two experimental years was significantly higher in eCO2 (3.84 ± 0.08 μg N g−1 day−1) than in aCO2 soils (3.45 ± 0.13 μg N g−1 day−1; P < 0.05; Fig. 1D). The magnitude of the CO2 effect on 15N-N2production following 15NH4+ addition was even larger in the field (Fig. 1B); in situ 15N-N2production under eCO2 (44.37 ± 2.06 mg N m−2 day−1) was 88% higher than that under aCO2 (P < 0.01; Fig. 1D). In experiment 3 (Fig. 1C), similarly, the average rate of NH4+ consumption increased significantly with eCO2 (3.61 ± 0.42 μg N g−1 day−1) compared to aCO2 (2.61 ± 0.43 μg N g−1 day−1) across the two SIP microcosms (P < 0.05; Fig. 1D). These results suggest that the eCO2-associated NH4+-N loss was a consequence of anaerobic oxidation of ammonium (AOA) in the paddy soil.
AOA coupled to IR under eCO2
Soils contain a variety of compounds capable of catalyzing oxidation reactions under anaerobic conditions, and iron oxides are among the most abundant (see Discussions I and II in the Supplementary Materials) (15, 18, 19). In the rice-FACE system, we previously demonstrated that eCO2 significantly stimulated root and microbial respiration, decreased soil redox potential, and facilitated ferric iron reduction (IR) (15). To test whether eCO2-induced changes in IR were coupled to AOA, we investigated the CO2 effect on ferrous iron (Fe2+) production in soils with the addition of either amorphous ferric oxyhydroxide alone [hereafter +Fe(III)] or NH4+ and Fe(III) together [hereafter +NH4++Fe(III)] in experiment 1 (fig. S4A). Both the +NH4+ + Fe(III) and +Fe(III) treatments enhanced Fe2+ production, with the average rate of the former being 1.7-fold faster than that of the latter across two experimental years (P < 0.01; fig. S5, A and B). The stimulatory effect of +NH4+ on Fe2+ production [+NH4+ + Fe(III) versus +Fe(III)] was further reinforced by eCO2 (64%) compared to aCO2 (41%; Fig. 2B and fig. S5, A and B).
Fig. 2 AOA coupled to IR under elevated CO2.
Elevated CO2 increased the production of 15N-N2 (total amount of 29N2and 30N2) (A and D) and Fe2+ (B and E) following the addition of 15NH4++ Fe(III) (A and B) or 15NH4+ + Fe(III) + C2H2 together (D and E) in experiment 1 (see fig. S4A for the experimental scheme). (A and D) AOA (NH4+ → N2); CO2 effect: P < 0.01; C2H2 effect: P < 0.05. (B and E) IR [Fe(III) → Fe2+]; CO2 effect: P < 0.05; C2H2 effect: P = 0.61. Open bars, ambient CO2 (aCO2); filled bars, elevated CO2 (eCO2). Data represent means (n = 3) ± SEM. (C and F) The stoichiometry of AOA-IR reactions and the overall molar ratio (R) of Fe2+ to 15N-N2, following the addition of either +15NH4+ + Fe(III) (C) or +15NH4+ + Fe(III) + C2H2 (F), were estimated using a general linear model.
We then compared the CO2 effect on 15N-N2 production when soils were given either +15NH4+ + Fe(III) or +15NH4+ in experiment 1. The +15NH4+ + Fe(III) treatment produced 15N-N2 2.4-fold faster than did the +15NH4+ treatment across two experimental years (P < 0.01; fig. S6, A and B). Corresponding to the CO2 stimulation of N2 production following +15NH4+ (fig. S6, A and B), 15N-N2production following +15NH4+ + Fe(III) addition was increased, on average, by 40% in eCO2compared to aCO2 soils (P < 0.01; Fig. 2A and fig. S6, A and B). These results indicate that the CO2enhancement of N2 production was a result of AOA coupled to IR [AOA-IR; also referred to as Feammox (19)] in the paddy soil.
We also examined whether AOA-IR occurred in experiment 2 (fig. S4B). Again, relative to the +NH4+treatment, the +NH4+ + Fe(III) treatment increased the production of Fe2+ (P < 0.05; fig. S5C) and 15N-N2 (P < 0.05; fig. S6C), confirming the occurrence of AOA-IR in the field. The CO2 stimulation of AOA-IR was evidenced by the CO2 enhancement of Fe2+ (P < 0.05; fig. S5C) and 15N-N2 (P < 0.05; fig. S6C) production in the +NH4+ + Fe(III) treatment. By mass balance (see Materials and Methods), we estimated that N2 production throughout the rice growing season via AOA-IR following +NH4+ + Fe(III) accounted for 17 and 25% of the applied NH4+-N, respectively, under aCO2and eCO2 plots.
(3)where ΔG°′ is the energy yield of the redox reaction that was calculated at pH 6.5, as the pH value of most paddy soils under waterlogging is approximately neutral (20). Reaction 1 produces N2, while reactions 2 and 3 yield NO2− and NO3−. Both NO2− and NO3− could be subsequently converted to N2 through the reduction of NO3−/NO2− to N2 (denitrification) (9) and/or AOA coupled to nitrite reduction (NR; AOA-NR, also referred to as anammox) (21, 22). Two lines of evidence, however, suggest that AOA-NR contributes little to N2 production in the rice-FACE system. First, 29N2 production did not differ between the +15NH4+ and +15NH4+ + 14NO3− treatment in AOA-NR tests accompanying with experiments 1 (P = 0.44; figs. S4A and S7A) and 2 (P = 0.76; figs. S4B and S7B). Second, the expression of hzsA, a gene encoding the subunit of hydrazine (N2H4) synthase in anammox bacteria (22), was not detectable in experiment 1 (table S1). If reactions 2 and 3 generated N2 through denitrification, the addition of acetylene (C2H2), an inhibitor of the reduction of N2O to N2 during denitrification (23), would lower N2 production. As expected, the presence of C2H2 reduced N2 production, on average, by 34% (P < 0.05; Fig. 2, A and D, and fig. S6, A and B), whereas it did not alter Fe2+ production (P = 0.61; Fig. 2, B and E) following +NH4+ + Fe(III)addition across two experimental years.
To assess the relative contributions of the three reactions to N2 production, we analyzed the stoichiometry of AOA-IR reactions in experiment 1. Because eCO2 did not alter the Fe2+:N2 ratio either with (P = 0.18) or without (P = 0.90) C2H2, we used a general linear model to calculate the overall Fe2+:N2 ratio across two CO2 treatments (see Materials and Methods). The Fe2+:N2 ratio was estimated to be 9.8:1 (Fig. 2C), suggesting that reaction 1 co-occurred with reaction 2 and/or reaction 3, as their theoretical mole ratios are 6:1, 12:1, and 16:1, respectively. The presence of C2H2 impeded denitrification and, as a result, increased the Fe2+:N2 ratio (Fig. 2F). Although we could not distinguish N2 production from reactions 2 and 3, the proportion of N2 produced from reaction 1 was estimated to account for 76% of the total N2 production in experiment 1 (see Materials and Methods).
Microbial mediation of AOA-IR
To examine whether N2 production via AOA-IR was mediated by soil microorganisms, we set up a microcosm study in which soils in half the replicates were sterilized to eliminate microbes, and the soils in all replicates were incubated following either +NH4+ + Fe(III) or +Fe(III) additions (see Materials and Methods). Under nonsterilized conditions, Fe2+ production was elevated in both +NH4+ + Fe(III) (P < 0.01) and +Fe(III) (P < 0.05) compared to the soil control (Fig. 3A), in line with results from experiments 1 and 2 (Fig. 2B and fig. S5). When soils were sterilized, however, neither +NH4+ + Fe(III) nor +Fe(III) significantly altered Fe2+ production, as compared to the soil control (Fig. 3A), suggesting a key role for soil microbes in coupling IR to AOA.
Fig. 3 Microbial mediation of the coupling of AOA and IR.
(A) Fe2+ production following the addition of Fe(III) or NH4+ + Fe(III) after 2-week incubation using sterilized or nonsterilized soils. (B) 13CO2consumption following the addition of NH4+, Fe(III), or NH4++Fe(III)(experiment 3). Different letters above bars denote statistically significant differences (P < 0.05). (C) Nonmetric multidimensional analysis (NMDS) of community dissimilarities based on 16S rRNA gene profiling in experiment 3 (see fig. S4C for the experimental scheme). Squares (I and II), 12C-DNA; triangles (III and IV), 13C-DNA; green symbols (I and III), +NH4+; red symbols (II and IV), +NH4+ + Fe(III). (D) Images of live members of β-Proteobacteria using FISH during incubation (experiment 3). DAPI, 4′,6-diamidino-2-phenylindole (blue). The BET 42a probe (red) was used to target β-Proteobacteria. (E) Unclassified OTU dominated in both 12C- and 13C-labeled microbiota (experiment 3). Data are the relative abundance of taxa.
To determine whether autotrophic or heterotrophic microbes were involved in the AOA-IR process, we designed two SIP microcosm incubations by feeding soil microbial communities with 13C-labeled acetate (13CH313COO−, SIP 1), a major organic substrate for anaerobes in paddy soils (24), and 13CO2 (SIP 2), separately (fig. S4C). Upon the measurement of Fe2+ production and NH4+consumption (fig. S8, A to D), we verified the occurrence of AOA-IR in two SIP microcosms. We then monitored the changes of headspace 13CO2 to determine whether microbes involved in AOA-IR required C from organic (CH3COO−) and/or inorganic (CO2) sources. When soils were amended with 13CH313COO−, 13CO2 production remained unchanged in the +NH4+, +Fe(III), and +NH4+ + Fe(III)treatments (fig. S8E). By contrast, with the addition of 13CO2, headspace 13CO2 consumption by microbial communities increased with time (P < 0.05; fig. S8F), with a higher CO2 consumption following +NH4+ + Fe(III) compared to +NH4+, +Fe(III), or the soil control treatment (P < 0.01; Fig. 3B). These results imply that microbes involved in AOA-IR were largely autotrophic, rather than heterotrophic.
To identify key microbial community members associated with AOA-IR, we sequenced 16Sribosomal RNA (rRNA) gene amplicons for genomic DNA prepared from 13CO2-fed soil samples in SIP 2. We separated 13C- from 12C-labeled DNA through ultracentrifugation to identify active autotrophs (see Materials and Methods). A total of 16,366 bacterial and archaeal operational taxonomic units (OTUs) were detected in both 12C- and 13C-labeled microbiota. Microbial communities given +NH4+ + Fe(III) were distinctive from those given +NH4+ as revealed by the ordination analysis of 16S rRNA gene profiles (Fig. 3C) and three nonparametric multivariate statistical tests (table S2). Within both 12C- and 13C-labeled microbiota, the most dominant taxon was the β-Proteobacteria, its relative abundance being higher in the +NH4+ + Fe(III) than that of the +NH4+ treatment (fig. S9, A and B). β-Proteobacteria were difficult to culture, but the presence of the most cultures of live β-Proteobacteria in the +NH4+ + Fe(III) treatment was confirmed using fluorescence in situ hybridization (FISH) with the BET42a probe (Fig. 3D). Assessment at lower taxonomic levels further points to the prominent role of an unclassified genus within the family Alcaligenaceae, order Burkholderiales (fig. S9, C to F). Within this unclassified genus, a hitherto unclassified OTU accounted for 47% of all detected OTUs at +NH4+ + Fe(III) within the 13C-labeled community (Fig. 3E). These results suggest that a previously unknown autotrophic member of β-Proteobacteria is responsible for mediating AOA-IR, although disentangling its complete set of functions will require further metagenomic analyses of enriched cultures.
Ecosystem N balance under eCO2
This set of investigations reveals a previously unidentified mechanism of CO2-induced N losses from the rice ecosystems in which rising atmospheric CO2 may significantly shift the functions of rice paddy soils by favoring an unusual group of autotrophic anaerobes responsible for N2volatilization due to ammonium oxidation linked to IR (Fig. 4; also see Discussion III in the Supplementary Materials). Two results emerge. First, CO2-induced N losses via AOA-IR will cause a decline in soil N retention under eCO2, as shown by 15N remaining in soil in the in situ field experiment (fig. S10A). The decrease in soil N retention under eCO2 was coincident with CO2-induced changes in the size of soil N pool [soil-extractable NH4+ (fig. S10B) and soil total N (fig. S10C)] across the experimental years of 2014–2018, likely mitigating the net CO2 effect on plant N uptake over time (figs. S2B and S10D; also see Discussions I and IV in the Supplementary Materials). Second, our results imply that N2 production associated with AOA-IR may be substantial (Fig. 2A, fig. S6, and table S3). On the basis of the current projection of NH4+-N fertilization in rice ecosystems (8–10), N losses via AOA-IR from paddy rice systems would be estimated approximately at 4.8 Tg N year−1 by the middle of this century (see Materials and Methods), substantially altering terrestrial N cycling under future eCO2.
Fig. 4 A conceptual framework of microbial mediation of AOA coupled to IR in a rice ecosystem in response to long-term FACE.
CO2 stimulation of root and microbial activities increases soil CO2production, generating a novel niche for certain autotrophic anaerobes that take advantage of CO2 as their C source; meanwhile, they conserve energy by catalyzing AOA-IR. Left inset: Reactions 1 to 3 of AOA. Right inset: IR. Solid and dashed arrows represent positive and negative CO2 effects, respectively.
The discovery that eCO2 stimulates anaerobic microbial mediation of AOA-IR might be extrapolated into systems other than rice paddies such as in wetlands and humid tropical forests where anaerobic soil conditions are prevalent (18, 19). Given the widespread observations that eCO2 enhances anaerobic microbial activity (1, 15, 25), understanding the CO2 effect on AOA coupled to reduction of iron and other oxidizers (see Discussion II in the Supplementary Materials) in terrestrial soils should be an active area of research. Although accurate projections of global N losses via AOA-IR from terrestrial ecosystems under future eCO2 would require further experimental (see Discussion IV in the Supplementary Materials) and modeling efforts, our results do provide insights into the linkage between terrestrial Fe and N cycles. Last, our findings raise important questions about the efficacy of applying excessive NH4+-N fertilizers to sustain crop production under rising atmospheric CO2 concentrations. We suggest, as have others (12, 14, 26), that careful matching of crop N demand to N supply in terms of timing and quantity would effectively reduce N fertilizer use and unnecessary loss from agroecosystems.