Nitrogen and Oxygen Isotope Signatures of Nitrogen Compounds during Anammox in the Laboratory and a Wastewater Treatment Plant.

Isotopic fractionation factors against 15N and 18O during anammox (anaerobic ammonia oxidization by nitrite) are critical for evaluating the importance of this process in natural environments. We performed batch incubation experiments with an anammox-dominated biomass to investigate nitrogen (N) and oxygen (O) isotopic fractionation factors during anammox and also examined apparent isotope fractionation factors during anammox in an actual wastewater treatment plant. We conducted one incubation experiment with high δ18O of water to investigate the effects of water δ18O. The N isotopic fractionation factors estimated from incubation experiments and the wastewater treatment plant were similar to previous values. We also found that the N isotopic effect (15εNXR of -77.8 to -65.9‰ and 15ΔNXR of -31.3 to -30.4‰) and possibly O isotopic effect (18εNXR of -20.6‰) for anaerobic nitrite oxidation to nitrate were inverse. We applied the estimated isotopic fractionation factors to the ordinary differential equation model to clarify whether anammox induces deviations in the δ18O vs δ15N of nitrate from a linear trajectory of 1, similar to heterotrophic denitrification. Although this deviation has been attributed to nitrite oxidation, the O isotopic fractionation factor for anammox is crucial for obtaining a more detailed understanding of the mechanisms controlling this deviation. In our model, anammox induced the trajectory of the δ18O vs δ15N of nitrate during denitrification to less than one, which strongly indicates that this deviation is evidence of nitrite oxidation by anammox under denitrifying conditions.

Control of the Nitrogen Isotope Composition of the Fungal Biomass: Evidence of Microbial Nitrogen Use Efficiency.

Changes in 15N/14N in the soil microbial biomass during nitrogen (N) mineralization have been hypothesized to influence 15N/14N in soil organic matter among ecosystem sites. However, a direct experimental test of this mechanism has not yet been performed. To evaluate the potential control of microbial N mineralization on the natural N isotope composition, we cultured fungi (Aspergillus oryzae) in five types of media of varying C:N ratios of 5, 10, 30, 50, and 100 for 4 d, and tracked changes in δ15N in the microbial biomass, NH4+, and dissolved organic N (DON: glycine) over the course of the experiment. High rates of NH4+ excretion from A. oryzae were accompanied by an increase in δ15N in the microbial biomass in low C:N media (i.e., C/N<30). In contrast, NH4+ was strongly retained in higher C/N treatments with only minor (i.e., <1 ‰) changes being detected in δ15N in the microbial biomass. Differences in δ15N in the microbial biomass were attributed to the loss of low-δ15N NH4+ in low, but not high C/N substrates. We also detected a negative linear correlation between microbial nitrogen use efficiency (NUE) and Δ15N (δ15N-biomass-δ15N-glycine). These results suggest an isotope effect during NH4+ excretion in relatively N-repleted environments in which microbial NUE is low, which may explain the vertical patterns of organic matter δ15N in soil profiles.

Evaluation of N environmental risks on Andosols from an intensive dairy farming watershed using DNDC.

Manure nitrogen (N) in the livestock sector has become a key driver of environmental change. The denitrification-decomposition (DNDC) model was used to evaluate N pollution strengths on Andosols with intensive dairy manure application in Upper Naka River Watershed, Japan. The calibrated model was capable of predicting Andosol N flows because the simulated soil mineral N content, soil nitrogen oxide (N2O) fluxes, denitrification rate, and crop N uptake matched the patterns and magnitudes of the field observations from a wide range of soil textures, as well as manure management and cropping systems. The simulations showed that current intensive manure application systems caused low crop N use efficiency and a large amount of NO3(-)-N leaching and N2O emission. The crop N use efficiency was 27%-42% and 37%-55% of input N for uplands and paddy rice, respectively. The uplands showed much more serious N environmental pollution risks with N leaching 123-362 kg N ha(-1) yr(-1) and N2O emissions 6.53-11.8 kg N ha(-1) yr(-1) than that in the lowland paddy rice with N leaching 17.4-103 kg N ha(-1) yr(-1) and N2O emissions 0.59-2.77 kg N ha(-1) yr(-1). Forage rice/barley crop systems have high N cleaning capability due to the greater crop N uptake which reached to 304 kg N ha(-1) yr(-1). High precipitation stimulated more NO3(-)-N leaching. Sandy soil also showed higher N leaching and was unsuitable for paddy rice. Slurry application stimulated more N2O emission than compost manure. To mitigate the current high N pollution, the critical N application rate was recommended to be approximately 380, 470, 640, and 390 kg N ha(-1) yr(-1) for loam sand planted with maize/grass, loam soil with maize/grass, forage rice/barley, and rice/fallow with winter manure application, respectively.

Microbial denitrification dominates nitrate losses from forest ecosystems.

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.

Variations in nitrogen-15 natural abundance of plant and soil systems in four remote tropical rainforests, southern China.

The foliar stable N isotope ratio (δ(15)N) can provide integrated information on ecosystem N cycling. Here we present the δ(15)N of plant and soil in four remote typical tropical rainforests (one primary and three secondary) of southern China. We aimed to examine if (1) foliar δ(15)N in the study forests is negative, as observed in other tropical and subtropical sites in eastern Asia; (2) variation in δ(15)N among different species is smaller compared to that in many N-limited temperate and boreal ecosystems; and (3) the primary forest is more N rich than the younger secondary forests and therefore is more (15)N enriched. Our results show that foliar δ(15)N ranged from -5.1 to 1.3‰ for 39 collected plant species with different growth strategies and mycorrhizal types, and that for 35 species it was negative. Soil NO3 (-) had low δ(15)N (-11.4 to -3.2‰) and plant NO3 (-) uptake could not explain the negative foliar δ(15)N values (NH4 (+) was dominant in the soil inorganic-N fraction). We suggest that negative values might be caused by isotope fractionation during soil NH4 (+) uptake and mycorrhizal N transfer, and by direct uptake of atmospheric NH3/NH4 (+). The variation in foliar δ(15)N among species (by about 6‰) was smaller than in many N-limited ecosystems, which is typically about or over 10‰. The primary forest had a larger N capital in plants than the secondary forests. Foliar δ(15)N and the enrichment factor (foliar δ(15)N minus soil δ(15)N) were higher in the primary forest than in the secondary forests, albeit differences were small, while there was no consistent pattern in soil δ(15)N between primary and secondary forests.

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.

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.

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.

Low δ18O 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.

High abundance of ammonia-oxidizing archaea in acidified subtropical forest soils in southern China after long-term N deposition.

Nitrification has been believed to be performed only by autotrophic ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) until the recent discovery of ammonia-oxidizing archaea (AOA). Meanwhile, it has been questioned whether AOB are significantly responsible for NH(3) oxidation in acidic forest soils. Here, we investigated nitrifying communities and their activity in highly acidified soils of three subtropical forests in southern China that had received chronic high atmospheric N deposition. Nitrifying communities were analyzed using PCR- and culture (most probable number)-based approaches. Nitrification activity was analyzed by measuring gross soil nitrification rates using a (15) N isotope dilution technique. AOB were not detected in the three forest soils: neither via PCR of 16S rRNA and ammonia monooxygenase (amoA) genes nor via culture-based approaches. In contrast, an extraordinary abundance of the putative archaeal amoA was detected (3.2 × 10(8) -1.2 × 10(9)  g soil(-1) ). Moreover, this abundance was correlated with gross soil nitrification rates. This indicates that amoA-possessing archaea rather than bacteria were predominantly responsible for nitrification of the soils. Furthermore, sequences of the genus Nitrospira, a dominant group of soil NOB, were detected. Thus, nitrification of acidified subtropical forest soils in southern China could be performed by a combination of AOA and NOB.

Preliminary insights into δ15N and δ18O of nitrate in natural mosses: a new application of the denitrifier method.

Natural mosses have been employed as reactive and accumulative indicators of atmospheric pollutants. Using the denitrifier method, the concentration, δ(15)N and δ(18)O of moss nitrate (NO(3)(-)) were measured to elucidate the sources of NO(3)(-) trapped in natural mosses. Oven drying at 55-70 °C, not lyophilization, was recommended to dry mosses for NO(3)(-) analyses. An investigation from urban to mountain sites in western Tokyo suggested that moss [NO(3)(-)] can respond to NO(3)(-) availability in different habitats. NO(3)(-) in terricolous mosses showed isotopic ratios as close to those of soil NO(3)(-), reflecting the utilization of soil NO(3)(-). Isotopic signatures of NO(3)(-) in corticolous and epilithic mosses elucidated atmospheric NO(3)(-) sources and strength from the urban (vehicle NO(x) emission) to mountain area (wet-deposition NO(3)(-)). However, mechanisms and isotopic effects of moss NO(3)(-) utilization must be further verified to enable the application of moss NO(3)(-) isotopes for source identification.

Nitrogen and oxygen isotope effects of tissue nitrate associated with nitrate acquisition and utilisation in the moss Hypnum plumaeforme.

Mosses are effective accumulators and indicators of N deposition, but the mechanisms of moss N utilisation remain unclear. This study monitored nitrate concentrations ([NO3-]) in solutions supplied to Hypnum plumaeforme Wils. to characterise NO3- uptake from rain events. Concentrations and isotopic ratios (δ15N and δ18O) of residual NO3- in moss tissues were measured to interpret induced NO3- reduction. Noninduced NO3- reduction was inferred from endogenous [NO3-] and isotopic variations that occurred during 65 days of N deprivation. H. plumaeforme scavenges NO3- effectively from supplied solutions. The uptake rate increased with substrate [NO3-] (0.4-3.9mgNL-1) and generally obeyed saturation (Michaelis-Menten) kinetics. The uptake rate was maximised within 60min after receiving NO3-, irrespective of the initial substrate [NO3-]. Lower tissue [NO3-] and greater isotopic enrichment verified the inducibility of nitrate reductase activity (NRA) by NO3- availability, but short-term darkness did not markedly influence moss NO3- uptake or reduction. Significant reduction and isotopic enrichment were detected in moss NO3- reserves during N deprivation, showing 15ε of 12.1‰ and 18ε of 14.4‰. The Δδ15N:Δδ18O ratios of ~1: 1 implied that NRA is the single process driving 15N and 18O fractionations. These results provide new isotopic insights into the nitrate reductase dynamics of the moss.

Analytical techniques for quantifying (15)N/(14)N of nitrate, nitrite, total dissolved nitrogen and ammonium in environmental samples using a gas chromatograph equipped with a quadrupole mass spectrometer.

The measurement of (15)N concentrations in environmental samples requires sophisticated pretreatment devices and expensive isotope-ratio mass spectrometry (IRMS). This report describes the use of a gas chromatograph equipped with a quadrupole-type mass spectrometer (GC/MS) to measure (15)N concentrations of ammonium, nitrate, nitrite, and total dissolved nitrogen (TDN) in distilled water, a 2 M KCl solution and a 0.5 M K(2)SO(4) solution. The system measures nitrous oxide (N(2)O) that is ultimately converted from the target N compound, requiring no special apparatus such as a purge-and-trap pretreatment device. It uses a denitrifier lacking N(2)O reductase, which produces N(2)O from nitrate. Persulfate oxidation was applied to convert TDN to nitrate, while additional pretreatment with ammonia diffusion was required for ammonium prior to the persulfate oxidation. Up to 100 samples can be measured daily using the system. We can generally run (15)N measurements with only 1-10 mL of sample for each chemical species of N, a volume 1/10-1/100 times smaller than the amount necessary for conventional methods. Our method is useful for measuring (15)N with GC/MS, offering greater convenience than IRMS.

Temporal variability of iron concentrations and fractions in wetland waters in Sanjiang Plain, northeast China.

Chemical forms, reactivities and transformation of iron fractions in marshy waters were investigated with cross-flow filtration technique to study the iron environmental behavior. Iron fractions were divided into four parts: acid-labile iron (pre-acidification of unfiltered marshy water samples, > 0.7 microm), high-molecular-weight iron (0.7-0.05 microm), medium-molecular-weight iron (0.05-0.01 microm), and low-molecular-weight iron (< 0.01 microm). The cross-flow filtration suggested that iron primarily exist in both the > 0.7 microm and < 0.01 microm size fractions in marshy waters. Rainfall is the key for rain-fed wetland to determine fate of iron by changing the aquatic biochemical conditions. By monitoring the variation of iron concentrations and fractions over three years, it was found that dissolved iron and acid-labile iron concentrations exhibit a large variation extent under different annual rainfalls from 2006 to 2008. The seasonal variation for iron species proved that the surface temperature could control some conversion reactions of iron in marshy waters. Low-molecular-weight iron would convert to acid-labile iron gradually with temperature decreasing. The photochemical reactions of iron fractions, especially low-molecular-weight iron had occurred under solar irradiation. The relative proportion of low-molecular-weight in total dissolved iron ranging from 28.3% to 43.2% were found during the day time, which proved that the observed decreasing concentration of acid lability iron was caused by its degradation to low molecular weight iron.

The natural abundance of 15N in plant and soil-available N indicates a shift of main plant N resources to NO3(-) from NH4(+) 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.