Muneoki Yoh

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.

The natural abundance of 15N in plant and soil‐available N indicates a shift of main plant N resources to NO 3− from NH 4+ along the N leaching gradient

To investigate which of ammonium (NH(4)(+)) or nitrate (NO(3)(-)) is used by plants at gradient sites with different nitrogen (N) availability, we measured the natural abundance of (15)N in foliage and soil extractable N. Hinoki cypress (Chamaecyparis obtusa Endlicher) planted broadly in Japan was selected for use in this study. We estimated the source proportion of foliar N (NH(4)(+) vs. NO(3)(-)) quantitatively using mass balance equations. The results showed that C. obtusa used mainly NH(4)(+) in N-limited forests, although the dependence of C. obtusa on NO(3)(-) was greater in other NO(3)(-)-rich forests. We regarded dissolved organic N (DON) as a potential N source because a previous study demonstrated that C. obtusa can take up glycine. Thus we added DON to our mass balance equations and calculated the source proportion using an isotope-mixing model (IsoSource model). The results still showed a positive correlation between the calculated plant N proportion of NO(3)(-) and the NO(3)(-) pool size in the soil, indicating that high NO(3)(-) availability increases the reliance of C. obtusa on NO(3)(-). Our data suggest the shift of the N source for C. obtusa from NH(4)(+) to NO(3)(-) according to the relative availability of NO(3)(-). They also show the potential of the foliar delta(15)N of C. obtusa as an indicator of the N status in forest ecosystems with the help of the delta(15)N values of soil inorganic and organic N.

N2O and NO emissions from grassland soils after the application of cattle and swine excreta

N2O and NO fluxes from grassland soil after the application of cattle and swine excreta were measured by a closed chamber method in the autumn and winter of 1994 to 1995. Fresh excrement and urine were spread on the grassland experimental plots and these gas fluxes were measured one or two times a week. In the autumn experiment, N2O and NO fluxes began to increase several days after the application, the NO flux reaching a maximum after 16 days. In the winter experiment, N2O and NO fluxes began to increase 45 days after the application and reached a maximum after 80 days. Nitrous oxide flux was influenced by soil water content, high water content leading to high N2O flux. The ratio of NO-N/N2O-N in the flux was in the range of 1.1 to 13.7, and negatively correlated to the soil water content. In the winter experiment, the total emission rate of NO was 0.48% and 0.45% of total nitrogen in the applied cattle and swine excreta, respectively. The total emission rate of N2O was 0.085% and 0.098% in the applied cattle and swine excreta, respectively.

Diffusion analysis of N2O cycling in a fertilized soil

The behavior of nitrous oxide (N2O) in fertilized soil was studied in terms of soil fluxes, the production rates at various depths and the turnover in soil. The diffusive losses of N2O to the atmosphere calculated from soil N2O profile compared favorably with the flux directly determined with a closed chamber technique. The estimate of N2O production rates at several depths demonstrated that the sites of N2O production was only near the soil surface. The calculated residence time of N2O in the entire soil column studied was only 1.4 hour during active emission period and less than 1 day even in the later period having trace N2O emission. The prolonged N2O emission observed after the active phase was due likely to a lasting N2O production rather than a supply from the soil N2O reservoir. The results suggested that most N2O in soil was emitted quite promptly to the atmosphere after its production. A minor role of soil as an N2O reservoir is emphasized from the viewpoint of the origin of groundwater N2O.

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.

Regional Distribution of Natural Stream Nitrate in Central Japan

Over 60 natural streams with forested catchments were surveyed for nitrate (NO3−) concentration in central Japan. Stream NO3− concentrations showed a clear zonal gradient with a range nearly as large as 100-fold, where their small seasonal variations (av. CV = 27%) justified respective statuses. Elevated NO3− concentrations (more than 70 µM) described a distribution of N saturation along a course of polluted air advection in the Kanto Plain. The extent of NO3− leaching from forests was not proportional to atmospheric N deposition probably because of ecosystem N retention. A dependency of NO3− level on the watershed altitude and quite constant δ15N of stream NO3− over a wide NO3− range were observed, which could be also related to this cause.

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.

The 15N natural abundance of the N lost from an N‐saturated subtropical forest in southern China

The 15N-enrichment of plants and soils is believed to indicate characteristics of the open nitrogen (N) cycle in terrestrial ecosystems because N lost from an ecosystem is presumably 15N-depleted through isotopic fractionation. However, because of a lack of an appropriate analytical methodology to confirm that supposition, the δ15N value for total dissolved nitrogen (TDN, the sum of ammonium, nitrate, and dissolved organic N) in stream water from forests has been measured only rarely. This report describes the δ15N values for TDN, ammonium, and nitrate in precipitation and stream water, together with those for soil-emitted nitrous oxide (N2O; measured once) in an N-saturated subtropical forest in southern China. Concentration-weighted δ15N values of TDN were −0.7‰ in precipitation and +1.2‰ in stream water. The difference in δ15N between soil (+3.9‰) and TDN in the stream water was 2.7‰. In contrast, soil-emitted N2O was strongly 15N-depleted (−14.3‰): 18‰ lower than that of the soil. Our results demonstrate that the discharged N loss is 15N-depleted only slightly compared with soil N, and gaseous N losses can be a strong driver for raising the terrestrial ecosystem δ15N. Our findings suggest that the relation between ecosystem δ15N and the open N cycle can be interpreted better by considering the net discrimination against 15N determined by the balance between gaseous and discharge N losses. Steady state 15N budget calculations proposed by Houlton and Bai (2009) can provide important information about the gaseous N fluxes, which are difficult to measure directly. The steady state calculation for the relationships among gaseous N loss, apparent isotopic fractionation during gaseous N loss, and isotopic signature of N inputs suggests that precise measurements of unmeasured components (e.g., dry deposition, NO and N2 emission) are quite important for better estimation of gaseous N losses from the ecosystem.

Nutrient Limitation in Three Lowland Tropical Forests in Southern China Receiving High Nitrogen Deposition: Insights from Fine Root Responses to Nutrient Additions

Elevated nitrogen (N) deposition to tropical forests may accelerate ecosystem phosphorus (P) limitation. This study examined responses of fine root biomass, nutrient concentrations, and acid phosphatase activity (APA) of bulk soil to five years of N and P additions in one old-growth and two younger lowland tropical forests in southern China. The old-growth forest had higher N capital than the two younger forests from long-term N accumulation. From February 2007 to July 2012, four experimental treatments were established at the following levels: Control, N-addition (150 kg N ha–1 yr–1), P-addition (150 kg P ha–1 yr–1) and N+P-addition (150 kg N ha–1 yr–1 plus 150 kg P ha–1 yr–1). We hypothesized that fine root growth in the N-rich old-growth forest would be limited by P availability, and in the two younger forests would primarily respond to N additions due to large plant N demand. Results showed that five years of N addition significantly decreased live fine root biomass only in the old-growth forest (by 31%), but significantly elevated dead fine root biomass in all the three forests (by 64% to 101%), causing decreased live fine root proportion in the old-growth and the pine forests. P addition significantly increased live fine root biomass in all three forests (by 20% to 76%). The combined N and P treatment significantly increased live fine root biomass in the two younger forests but not in the old-growth forest. These results suggest that fine root growth in all three study forests appeared to be P-limited. This was further confirmed by current status of fine root N:P ratios, APA in bulk soil, and their responses to N and P treatments. Moreover, N addition significantly increased APA only in the old-growth forest, consistent with the conclusion that the old-growth forest was more P-limited than the younger forests.

δ15N of soil N and plants in a N‐saturated, subtropical forest of southern China

We investigated the delta(15)N profile of N (extractable NH(4)(+), NO(3)(-), and organic N (EON)) in the soil of a N-saturated subtropical forest. The order of delta(15)N in the soil was EON > NH(4)(+) > NO(3)(-). Although the delta(15)N of EON had been expected to be similar to that of bulk soil N, it was higher than that of bulk soil N by 5 per thousand. The difference in delta(15)N between bulk soil N and EON (Delta(15)N(bulk-EON)) was correlated significantly with the soil C/N ratio. This correlation implies that carbon availability, which determines the balance between N assimilation and dissimilation of soil microbes, is responsible for the high delta(15)N of EON, as in the case of soil microbial biomass delta(15)N. A thorough delta(15)N survey of available N (NH(4)(+), NO(3)(-), and EON) in the soil profiles from the organic layer to 100 cm depth revealed that the delta(15)N of the available N forms did not fully overlap with the delta(15)N of plants. This mismatch in delta(15)N between that of available N and that of plants reflects apparent isotopic fractionation during N uptake by plants, emphasizing the high N availability in this N-saturated forest.