Revisiting the involvement of ammonia oxidizers and denitrifiers in nitrous oxide emission from cropland soils.

Nitrous oxide (N2O), an ozone-depleting greenhouse gas, is generally produced by soil microbes, particularly NH3 oxidizers and denitrifiers, and emitted in large quantities after N fertilizer application in croplands. N2O can be produced via multiple processes, and reduced, with the involvement of more diverse microbes with different physiological constraints than previously thought; therefore, there is a lack of consensus on the production processes and microbes involved under different agricultural practices. In this study, multiple approaches were applied, including N2O isotopocule analyses, microbial gene transcript measurements, and selective inhibition assays, to revisit the involvement of NH3 oxidizers and denitrifiers, including the previously-overlooked taxa, in N2O emission from a cropland, and address the biological and environmental factors controlling the N2O production processes. Then, we synthesized the results from those approaches and revealed that the overlooked denitrifying bacteria and fungi were more involved in N2O production than the long-studied ones. We also demonstrated that the N2O production processes and soil microbes involved were different based on fertilization practices (plowing or surface application) and fertilization types (manure or urea). In particular, we identified the following intensified activities: (1) N2O production by overlooked denitrifying fungi after manure fertilization onto soil surface; (2) N2O production by overlooked denitrifying bacteria and N2O reduction by long-studied N2O-reducing bacteria after manure fertilization into the plowed layer; and (3) N2O production by NH3-oxidizing bacteria and overlooked denitrifying bacteria and fungi when urea fertilization was applied into the plowed layer. We finally propose the conceptual scheme of N flow after fertilization based on distinct physiological constraints among the diverse NH3 oxidizers and denitrifiers, which will help us understand the environmental context-dependent N2O emission processes.

Influence of δ18 O of water on measurements of δ18 O of nitrite and nitrate.

RATIONALE: Oxygen isotope ratio measurements of NO2 - and NO3 - by the azide method and denitrifier method are sensitive to the δ18 O value of the sample water. However, the influence of δ18 OH2O on those measurements has not been quantitatively evaluated and documented so far. Therefore, we investigated the influence of δ18 OH2O of a sample on the δ18 O analysis of NO2 - and NO3 - . METHODS: We prepared NO2 - and NO3 - standards (with known δ18 ONO2- and δ18 ONO3- values) dissolved in waters having different δ18 OH2O values (δ18 OH2O = -12.6, 25.9, 56.7, and 110.1‰). Nitrite and nitrate were converted into N2 O using the azide method and the denitrifier method, respectively. The isotope ratios of the generated N2 O were measured with a Sercon purge-and-trap gas chromatography/isotope ratio mass spectrometry (PT-GC/IRMS) system. The measured δ18 O values of the produced N2 O were plotted against known δ18 ONO2- and δ18 ONO3- values to evaluate the influence of exchange of an oxygen atom with H2 O during the conversion of NO2 - into N2 O and NO3 - into N2 O, respectively. RESULTS: The degree of oxygen isotope exchange was 10.8 ± 0.3% in the azide method and 5.5 ± 1.0% in the denitrifier method, indicating that the azide method is more susceptible to artifacts arising from differences in the δ18 OH2O value of water than the denitrifier method. Thus, the intercept of the standard calibration curve must be corrected to account for differences in δ18 OH2O . Abiotic NO2 -H2 O equilibrium isotope effect experiments yielded a rate constant of (1.13 ± 007) × 10-2 (h-1 ) and an equilibrium isotope effect of 11.9 ± 0.1‰ under the condition of pH = 7.5, 30°C, and 2.5% salinity. CONCLUSIONS: Oxygen isotope ratio measurements of NO2 - by the azide method are highly sensitive to δ18 OH2O as a result of significant oxygen isotope exchange between NO2 - and H2 O. Therefore, to obtain the most accurate measurements water with the same δ18 OH2O value as that of the sample must be used to make the NO2 - and NO3 - standards.

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.

Dual nitrogen and oxygen isotope fractionation during anaerobic ammonium oxidation by anammox bacteria.

Natural abundance of stable nitrogen (N) and oxygen (O) isotopes are invaluable biogeochemical tracers for assessing the N transformations in the environment. To fully exploit these tracers, the N and O isotope effects (15ε and 18ε) associated with the respective nitrogen transformation processes must be known. However, the N and O isotope effects of anaerobic ammonium oxidation (anammox), one of the major fixed N sinks and NO3- producers, are not well known. Here, we report the dual N and O isotope effects associated with anammox by three different anammox bacteria including "Ca. Scalindua japonica", a putative marine species, which were measured in continuous enrichment culture experiments. All three anammox species yielded similar N isotope effects of NH4+ oxidation to N2 (15εNH4→N2) ranging from 30.9‰ to 32.7‰ and inverse kinetic isotope effects of NO2- oxidation to NO3- (15εNO2→NO3 = -45.3‰ to -30.1‰). In contrast, 15εNO2→N2 (NO2- reduction to N2) were significantly different among three species, which is probably because individual anammox bacteria species might possess different types of nitrite reductase. We also report the combined O isotope effects for NO2- oxidation (18ENO2→NO3) by anammox bacteria. These obtained dual N and O isotopic effects could provide significant insights into the contribution of anammox bacteria to the fixed N loss and NO2- reoxidation (N recycling) in various natural environments.

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.

Nitrate is an important nitrogen source for Arctic tundra plants.

Plant nitrogen (N) use is a key component of the N cycle in terrestrial ecosystems. The supply of N to plants affects community species composition and ecosystem processes such as photosynthesis and carbon (C) accumulation. However, the availabilities and relative importance of different N forms to plants are not well understood. While nitrate (NO3-) is a major N form used by plants worldwide, it is discounted as a N source for Arctic tundra plants because of extremely low NO3- concentrations in Arctic tundra soils, undetectable soil nitrification, and plant-tissue NO3- that is typically below detection limits. Here we reexamine NO3- use by tundra plants using a sensitive denitrifier method to analyze plant-tissue NO3- Soil-derived NO3- was detected in tundra plant tissues, and tundra plants took up soil NO3- at comparable rates to plants from relatively NO3--rich ecosystems in other biomes. Nitrate assimilation determined by 15N enrichments of leaf NO3- relative to soil NO3- accounted for 4 to 52% (as estimated by a Bayesian isotope-mixing model) of species-specific total leaf N of Alaskan tundra plants. Our finding that in situ soil NO3- availability for tundra plants is high has important implications for Arctic ecosystems, not only in determining species compositions, but also in determining the loss of N from soils via leaching and denitrification. Plant N uptake and soil N losses can strongly influence C uptake and accumulation in tundra soils. Accordingly, this evidence of NO3- availability in tundra soils is crucial for predicting C storage in tundra.

Angiopoietin-2 may contribute to multiple organ dysfunction and death in sepsis*.

OBJECTIVE: : In sepsis, quiescent blood vessels become leaky and inflamed by mechanisms that are incompletely understood. We hypothesized that angiopoietin-2, a partial antagonist of the endothelium-stabilizing receptor Tie-2 secreted by endothelium, contributes to adverse outcomes in this disease. DESIGN: : Laboratory and animal research. SETTINGS: : Research laboratories and Emergency Department of Beth Israel Deaconess Medical Center, Boston, MA. SUBJECTS: : Angiopoietin-2 heterozygous mice, emergency department patients. MEASUREMENTS AND MAIN RESULTS: : Mice with one functional angiopoietin-2 allele developed milder kidney and lung injury, less tissue inflammation, and less vascular leakage compared to wild-type counterparts. Heterozygotes experienced >40% absolute survival advantage following two different models of sepsis (p = .004 and .018). In human subjects presenting to our emergency department with suspected infection (n = 270 combined), circulating angiopoietin-2 was markedly elevated within the first hour of clinical care. First-hour angiopoietin-2 concentrations were proportional to current disease severity (p < .0001), rose further over time in eventual nonsurvivors (p < .0001), and predicted the future occurrence of shock (p < .0001) or death (p < .0001) in the original cohort and an independent validation group. Finally, septic human serum disrupted the barrier function of microvascular endothelial cells, an effect fully neutralized by an angiopoietin-2 monoclonal antibody. CONCLUSIONS: : We conclude that angiopoietin-2 induction precedes and contributes to the adverse outcomes in sepsis, opening a new avenue for therapeutic investigation.

Adiponectin diminishes organ-specific microvascular endothelial cell activation associated with sepsis.

Experimental sepsis was induced in male C57BL/6j, adiponectin-deficient mice (ADPNKO), and wild-type littermates by i.p. injection of 16 mg/kg lipopolysaccharide or cecal ligation and puncture. Blood and tissue samples were harvested 24 h after model induction. Circulating adiponectin is reduced in mice with endotoxemic challenge and after cecal ligation and puncture compared with healthy control mice. Quantitative reverse transcriptase-polymerase chain reaction for adiponectin reveals a pattern of response that is both model- and organ-specific. When challenged with sepsis, adiponectin deficiency results in increased expression of endothelial adhesion and coagulation molecules in the lung, liver, and kidney as quantified by reverse transcriptase-polymerase chain reaction, increased macrophage and neutrophil infiltration by immunohistochemistry, and vascular leakage in the liver and kidney. Adiponectin-deficient mice have reduced survival following cecal ligation and puncture and increased blood levels of interleukin 6, soluble vascular endothelial growth factor receptor 1, and soluble endothelial adhesion molecules E-selectin and intercellular adhesion molecule 1. Finally, ADPNKO promoted end-organ injury in the liver and kidney, whereas the lungs were not affected. These data suggest a protective role of adiponectin in diminishing microvascular organ-specific endothelial cell activation during sepsis.

Leptin exacerbates sepsis-mediated morbidity and mortality.

The adipose-derived hormone leptin is well known for its contribution to energy metabolism and satiety signaling in the hypothalamus. Previous studies suggested that obesity is an independent risk factor for sepsis morbidity and mortality, and it is associated with elevated baseline levels of circulating leptin in normal, nonseptic patients. In mouse endotoxemia and cecal ligation puncture models of sepsis, we observed elevated levels of leptin and soluble leptin receptor (sLR). Exogenously administered leptin increased mortality in endotoxemia and cecal ligation puncture models and was associated with increased expression of adhesion and coagulation molecules, macrophage infiltration into the liver and kidney, and endothelial barrier dysfunction. Conversely, longform leptin receptor-deficient mice were protected from sepsis morbidity and mortality and had less endothelial dysfunction. Furthermore, an in vitro study revealed that leptin-induced endothelial dysfunction is likely mediated, at least in part, by monocytes. Moreover, administration of an sLR conferred a survival benefit. Human septic patients have increased circulating sLR concentrations, which were correlated with disease severity indices. Together, these data support a pathogenic role for leptin signaling during sepsis.