Xue-Yan Liu

Microbial denitrification dominates nitrate losses from forest ecosystems

Significance Nitrogen (N) losses from terrestrial ecosystems can occur as inert forms or heat-trapping greenhouse gases, and via nitrate (NO3−) leaching to drainage waters, which can contribute to eutrophication and anoxia in downstream ecosystems. Here, we use natural isotopes to demonstrate that microbial gaseous N production via denitrification is the dominant pathway of NO3− removal from forest ecosystems, with gaseous N losses that are up to ∼60-fold higher than those based on traditional techniques. Denitrification becomes less efficient compared with NO3− leaching in more N-polluted ecosystems, which has important implications for assessing the connections between terrestrial soils and downstream ecosystems under rising anthropogenic N deposition. Denitrification removes fixed nitrogen (N) from the biosphere, thereby restricting the availability of this key limiting nutrient for terrestrial plant productivity. This microbially driven process has been exceedingly difficult to measure, however, given the large background of nitrogen gas (N2) in the atmosphere and vexing scaling issues associated with heterogeneous soil systems. Here, we use natural abundance of N and oxygen isotopes in nitrate (NO3−) to examine dentrification rates across six forest sites in southern China and central Japan, which span temperate to tropical climates, as well as various stand ages and N deposition regimes. Our multiple stable isotope approach across soil to watershed scales shows that traditional techniques underestimate terrestrial denitrification fluxes by up to 98%, with annual losses of 5.6–30.1 kg of N per hectare via this gaseous pathway. These N export fluxes are up to sixfold higher than NO3− leaching, pointing to widespread dominance of denitrification in removing NO3− from forest ecosystems across a range of conditions. Further, we report that the loss of NO3− to denitrification decreased in comparison to leaching pathways in sites with the highest rates of anthropogenic N deposition.

Low δ 18 O Values of Nitrate Produced from Nitrification in Temperate Forest Soils

Analyses of δ(18)O of nitrate (NO(3)(-)) have been widely used in partitioning NO(3)(-) sources. However the δ(18)O value of NO(3)(-) produced from nitrification (microbial NO(3)(-)) is commonly estimated using the δ(18)O of environmental water and molecular oxygen in a 2:1 ratio. Here our laboratory incubation of nine temperate forest soils across a 1500 m elevation gradient demonstrates that microbial NO(3)(-) has lower δ(18)O values than the predicted using the 2:1 ratio (by 5.2-9.5‰ at low elevation sites), in contrast to previous reports showing higher δ(18)O values (up to +15‰) than their predicted values. Elevated δ(18)O values of microbial NO(3)(-) were observed at high elevation sites where soil was more acidic, perhaps due to accelerated O-exchange between nitrite, an intermediate product of nitrification, and water. Lower δ(18)O of microbial NO(3)(-) than the predicted and from previous observations suggests that the contribution of anthropogenic N inputs, such as fertilizer and atmospheric deposition, to a given ecosystem and the progress of denitrification in nitrogen removal are greater than we know. More than half of the δ(18)O of stream NO(3)(-) lower than the predicted value along the elevation gradient also indicate the impropriety using the 2:1 ratio for differentiating NO(3)(-) sources.

Ammonium first: natural mosses prefer atmospheric ammonium but vary utilization of dissolved organic nitrogen depending on habitat and nitrogen deposition

Mosses, among all types of terrestrial vegetation, are excellent scavengers of anthropogenic nitrogen (N), but their utilization of dissolved organic N (DON) and their reliance on atmospheric N remain uncharacterized in natural environments, which obscures their roles in N cycles. Natural (15) N abundance of N sources (nitrate (NO(3)(-)), ammonium (NH(4)(+)) and DON in deposition and soil) for epilithic and terricolous mosses was analyzed at sites with different N depositions at Guiyang, China. Moss NO(3)(-) assimilation was inhibited substantially by the high supply of NH(4)(+) and DON. Therefore, contributions of NH(4)(+) and DON to moss N were partitioned using isotopic mass-balance methods. The N contributions averaged 56% and 46% from atmospheric NH(4)(+), and 44% and 17% from atmospheric DON in epilithic and terricolous mosses, respectively. In terricolous mosses, soil NH(4)(+) and soil DON accounted for 16% and 21% of bulk N, which are higher than current estimations obtained using (15) N-labeling methods. Moreover, anthropogenic NH(4)(+) deposition suppressed utilization of DON and soil N because of the preference of moss for NH(4)(+) under elevated NH(4)(+) deposition. These results underscore the dominance of, and preference for, atmospheric NH(4)(+) in moss N utilization, and highlight the importance of considering DON and soil N sources when estimating moss N sequestration and the impacts of N deposition on mosses.

Atmospheric transport of urban-derived NHx: Evidence from nitrogen concentration and δ15N in epilithic mosses at Guiyang, SW China

Nitrogen concentration and delta15N in 175 epilithic moss samples were investigated along four directions from urban to rural sites in Guiyang, SW China. The spatial variations of moss N concentration and delta15N revealed that atmospheric N deposition is dominated by NHx-N from two major sources (urban sewage NH3 and agricultural NH3), the deposition of urban-derived NHx followed a point source pattern characterized by an exponential decline with distance from the urban center, while the agricultural-derived NHx was shown to be a non-point source. The relationship between moss N concentration and distance (y=1.5e(-0.13x)+1.26) indicated that the maximum transporting distance of urban-derived NHx averaged 41 km from the urban center, and it could be determined from the relationship between moss delta(15)N and distance [y=2.54ln(x)-12.227] that urban-derived NHx was proportionally lower than agricultural-derived NHx in N deposition at sites beyond 17.2 km from the urban center. Consequently, the variation of urban-derived NHx with distance from the urban center could be modeled as y=56.272e(-0.116x)-0.481 in the Guiyang area.

Pitfalls and New Mechanisms in Moss Isotope Biomonitoring of Atmospheric Nitrogen Deposition

Moss N isotope (δ(15)N(bulk)) has been used to monitor N deposition, but it remains questionable whether inhibition of nitrate reductase activity (NRA) by reduced dissolved N (RDN) engenders overestimation of RDN in deposition when using moss δ(15)N(bulk). We tested this question by investigation of δ(15)N(bulk) and δ(15)NO(3)(-) in mosses under the dominance of RDN in N depositions of Guiyang, SW China. The δ(15)N(bulk) of mosses on bare rock (-7.9‰) was unable to integrate total dissolved N (TDN) (δ(15)N = -6.3‰), but it reflected δ(15)N-RDN (-7.5‰) exactly. Moreover, δ(15)N-NO(3)(-) in mosses (-1.7‰) resembled that of wet deposition (-1.9‰). These isotopic approximations, together with low isotopic enrichment with moss [NO(3)(-)] variations, suggest the inhibition of moss NRA by RDN. Moreover, isotopic mixing modeling indicated a negligible contribution from NO(3)(-) to moss δ(15)N(bulk) when the RDN/NO(3)(-) reaches 3.8, at which maximum overestimation (21%) of RDN in N deposition can be generated using moss δ(15)N(bulk) as δ(15)N-TDN. Moss δ(15)N-NO(3)(-) can indicate atmospheric NO(3)(-) under distinctly high RDN/NO(3)(-) in deposition, although moss δ(15)N(bulk) can reflect only the RDN therein. These results reveal pitfalls and new mechanisms associated with moss isotope monitoring of N deposition and underscore the importance of biotic N dynamics in biomonitoring studies.

Dual N and O isotopes of nitrate in natural plants: first insights into individual variability and organ-specific patterns

Nitrate (NO3−) is an important form of nitrogen (N) available to plants. The measurements of NO3− concentration [NO3−] and isotopes (δ15N and δ18O) in plants provide unique insights into ecosystem NO3− availability and plant NO3− dynamics. This work investigated the variability of these parameters in individuals of a broadleaved (Aucuba japonica) plant and a coniferous (Platycladus orientalis) plant, and explored the applicability of tissue NO3− isotopes for deciphering plant NO3− utilization mechanisms. The NO3− in washed leaves showed concentration and isotopic ratios that were much lower than that in unwashed leaves, indicating a low contribution of atmospheric NO3− to NO3− in leaves. Current leaves showed higher [NO3−] and isotopic ratios than mature leaves. Moreover, higher leaf [NO3−] and isotopic enrichments (relative to soil NO3−) were found under higher soil NO3− availability for A. japonica. In contrast, leaves of P. orientalis showed low [NO3−] and negligible isotopic enrichments despite high soil NO3−. Higher [NO3−] was found in both fine and coarse roots of the P. orientalis plant, but significant isotopic enrichment was found only in coarse roots. These results reflect that the NO3− accumulation and isotopic effects decreased with leaf age, but increased with soil NO3− supply. Leaves are therefore identified as a location of NO3− reduction for A. japonica, while P. orientalis did not assimilate NO3− in leaves but in coarse roots. This work provided the first organ-specific information on NO3− isotopes in plant individuals, which will stimulate further studies of NO3− dynamics in a broader spectrum of plant ecosystems.

Response of stable carbon isotope in epilithic mosses to atmospheric nitrogen deposition.

Epilithic mosses are characterized by insulation from substratum N and hence meet their N demand only by deposited N. This study investigated tissue C, total Chl and delta13C of epilithic mosses along 2 transects across Guiyang urban (SW China), aiming at testing their responses to N deposition. Tissue C and total Chl decreased from the urban to rural, but delta13C(moss) became less negative. With measurements of atmospheric CO2 and delta13CO2, elevated N deposition was inferred as a primary factor for changes in moss C and isotopic signatures. Correlations between total Chl, tissue C and N signals indicated a nutritional effect on C fixation of epilithic mosses, but the response of delta13C(moss) to N deposition could not be clearly differentiated from effects of other factors. Collective evidences suggest that C signals of epilithic mosses are useful proxies for N deposition but further works on physiological mechanisms are still needed.

Patterns of foliar δ15N and their control in Eastern Asian forests

Foliar δ15N has been used increasingly in research on ecosystem nitrogen (N) cycling, because it can serve as an integrator of ecosystem N cycling and thus has a potential to reveal temporal and spatial patterns of N cycling as well as how the N cycle is altered by disturbances. However, the current understanding on controls of foliar δ15N is based principally on studies from America, Europe, Australia and Africa. Here we compiled data from 65 forests at 33 sites across East Asia to explore regional patterns and what controls foliar δ15N by linking it to climate, species composition, soil depth, slope position, N deposition, and soil N availability. In East Asia, foliar δ15N ranged from −7.1 to +2.7‰. Mean foliar δ15N values for tropical, subtropical and temperate forests were all −3.1‰, which was unexpected. The patterns of foliar δ15N with precipitation, temperature and altitude were not clear. The variation in foliar δ15N among species and between different slope positions appeared to be small within a given forest. The δ15N for both bulk soil N and extractable inorganic N generally increased with soil depth as expected, strengthening the idea that deep-rooted trees may have access to 15N-enriched N. Different from the positive correlations reported across America and Europe, in East Asia we found that foliar δ15N decreased with increasing N deposition and did not relate to soil N availability. These discrepancies deserve more research to elucidate the mechanisms by which foliar δ15N is affected by ecosystem N availability at a regional scale.

Tissue S/N ratios and stable isotopes (δ34S and δ15N) of epilithic mosses (Haplocladium microphyllum) for showing air pollution in urban cities in Southern China

In urban cities in Southern China, the tissue S/N ratios of epilithic mosses (Haplocladium microphyllum), varied widely from 0.11 to 0.19, are strongly related to some atmospheric chemical parameters (e.g. rainwater SO(4)(2-)/NH(4)(+) ratios, each people SO(2) emission). If tissue S/N ratios in the healthy moss species tend to maintain a constant ratio of 0.15 in unpolluted area, our study cities can be divided into two classes: class I (S/N > 0.15, S excess) and class II (S/N < 0.15, N excess), possibly indicative of stronger industrial activity and higher density of population, respectively. Mosses in all these cities obtained S and N from rainwater at a similar ratio. Sulphur and N isotope ratios in mosses are found significantly linearly correlated with local coal delta(34)S and NH(4)(+)-N wet deposition, respectively, indicating that local coal and animal NH(3) are the major atmospheric S and N sources.

Assessment of atmospheric sulfur with the epilithic moss Haplocladium microphyllum: Evidences from tissue sulfur and δ34S analysis

The application of geochemical signals in mosses is more and more popular to investigate the deposition of atmospheric pollutants, but it is unclear whether records of atmospheric sulfur in mosses differ between their diverse habitats. This study aimed to investigate the influence of growing condition on tissue sulfur and delta34S of Haplocladium microphyllum. Epilithic and terricolous mosses in open fields, mosses under different canopy conditions were considered. We found that tissue sulfur and delta34S of mosses under different habitats were not consistent and could not be compared for atmospheric sulfur research with each other even collected at the same site, moss sulfur and delta34S records would be distorted by subsoil and upper canopies in different degrees, which possibly mislead the interpretation of atmospheric sulfur level and sources. Consequently, mosses on open rocks can be used reliably to assess atmospheric-derived sulfur in view of their identical sulfur and delta34S evidences.