Sakae Toyoda

Constraining the atmospheric N2O budget from intramolecular site preference in N2O isotopomers

Nitrous oxide (N2O) is an important trace gas in the atmosphere. It is an active greenhouse gas in the troposphere and it also controls ozone concentration in the stratosphere through nitric oxide production. One way to trace the geochemical cycle of N2O is by measuring the natural abundance of stable isotopes, namely 15N and 18O (refs 2,3,4,5,6,7,8,9,10,11, 12,13,14,15). Here we report the intramolecular distribution of 15N within the linear NNO molecule, determined by measuring molecular and fragment ions of N2O on a modified mass spectrometer. This revealed a preference for 15N at the central N position, or α-site, within N2O isotopomers (isotope-containing molecules). Moreover, this preference varied significantly throughout the atmosphere. In the troposphere, low α-site preference indicates local emission of N2O from soils and fossil-fuel combustion, each with distinct isotopomer signatures, which then mixes with background N2O. In the stratosphere, on the other hand, loss of N2O is observed as enhanced α-site preference for 15N, due to fractionation during ultraviolet photolysis of N2O. We have constructed an atmospheric mass balance of N 2O, incorporating isotopomer abundance, which shows that the intramolecular distribution of 15N is a parameter that has the potential to increase significantly the resolution with which sources and sinks of N 2O can be identified and quantified in the atmosphere.

Source of nitrous oxide emissions during the cow manure composting process as revealed by isotopomer analysis of and amoA abundance in betaproteobacterial ammonia-oxidizing bacteria.

ABSTRACT A molecular analysis of betaproteobacterial ammonia oxidizers and a N2O isotopomer analysis were conducted to study the sources of N2O emissions during the cow manure composting process. Much NO2−-N and NO3−-N and the Nitrosomonas europaea-like amoA gene were detected at the surface, especially at the top of the composting pile, suggesting that these ammonia-oxidizing bacteria (AOB) significantly contribute to the nitrification which occurs at the surface layer of compost piles. However, the 15N site preference within the asymmetric N2O molecule (SP = δ15Nα − δ15Nβ, where 15Nα and 15Nβ represent the 15N/14N ratios at the center and end sites of the nitrogen atoms, respectively) indicated that the source of N2O emissions just after the compost was turned originated mainly from the denitrification process. Based on these results, the reduction of accumulated NO2−-N or NO3−-N after turning was identified as the main source of N2O emissions. The site preference and bulk δ15N results also indicate that the rate of N2O reduction was relatively low, and an increased value for the site preference indicates that the nitrification which occurred mainly in the surface layer of the pile partially contributed to N2O emissions between the turnings.

Microbiology of nitrogen cycle in animal manure compost.

Composting is the major technology in the treatment of animal manure and is a source of nitrous oxide, a greenhouse gas. Although the microbiological processes of both nitrification and denitrification are involved in composting, the key players in these pathways have not been well identified. Recent molecular microbiological methodologies have revealed the presence of dominant Bacillus species in the degradation of organic material or betaproteobacterial ammonia‐oxidizing bacteria on nitrification on the surface, and have also revealed the mechanism of nitrous oxide emission in this complicated process to some extent. Some bacteria, archaea or fungi still would be considered potential key players, and the contribution of some pathways, such as nitrifier denitrification or heterotrophic nitrification, might be involved in composting. This review article discusses these potential microbial players in nitrification–denitrification within the composting pile and highlights the relevant unknowns through recent activities that focus on the nitrogen cycle within the animal manure composting process.

Fractionation of N2O isotopomers in the stratosphere

The vertical distribution of isotopomers of N2O (14N15N16O, 15N14N16O, and 14N14N18O) in the lower and middle stratosphere was observed over Japan (39°N, 142°E) in 1999 using a balloon-borne cryogenic sampler and ground-based mass spectrometry. The abundance of the heavier isotopomers relative to 14N14N16O increased with altitude, while the mixing ratio of N2O decreased due to photochemical depletion. Maximum fractionation was observed at the highest altitude, 34.5 km, where δ15Nαair (isotopomer ratio of center nitrogen), δ15Nβair (end nitrogen), and δ18OSMOW were 144.l‰, 42.4‰, and 119.0‰, respectively. The observed distribution is mostly accounted for by isotopic fractionation during consumption processes, which is in accordance with reported simulation experiments and theoretical prediction for photolytic fractionation of N2O isotopomers. However, the apparent fractionation factors in the lower (<∼24 km) and higher regions are different, which suggests that (1) the fractionation factor for stratospheric photolysis may depend on altitude or latitude, (2) transport and mixing processes in the stratosphere can affect the vertical profile, and (3) the relative contribution of photolysis and photo-oxidation to total N2O sink is possibly dependent on altitude.

N2O production, a widespread trait in fungi.

N2O is a powerful greenhouse gas contributing both to global warming and ozone depletion. While fungi have been identified as a putative source of N2O, little is known about their production of this greenhouse gas. Here we investigated the N2O-producing ability of a collection of 207 fungal isolates. Seventy strains producing N2O in pure culture were identified. They were mostly species from the order Hypocreales order—particularly Fusarium oxysporum and Trichoderma spp.—and to a lesser extent species from the orders Eurotiales, Sordariales, and Chaetosphaeriales. The N2O 15N site preference (SP) values of the fungal strains ranged from 15.8‰ to 36.7‰, and we observed a significant taxa effect, with Penicillium strains displaying lower SP values than the other fungal genera. Inoculation of 15 N2O-producing strains into pre-sterilized arable, forest and grassland soils confirmed the ability of the strains to produce N2O in soil with a significant strain-by-soil effect. The copper-containing nitrite reductase gene (nirK) was amplified from 45 N2O-producing strains, and its genetic variability showed a strong congruence with the ITS phylogeny, indicating vertical inheritance of this trait. Taken together, this comprehensive set of findings should enhance our knowledge of fungi as a source of N2O in the environment.

Isotopomer analysis of production and consumption mechanisms of N2O and CH4 in an advanced wastewater treatment system.

Wastewater treatment processes are believed to be anthropogenic sources of nitrous oxide (N(2)O) and methane (CH(4)). However, few studies have examined the mechanisms and controlling factors in production of these greenhouse gases in complex bacterial systems. To elucidate production and consumption mechanisms of N(2)O and CH(4) in microbial consortia during wastewater treatment and to characterize human waste sources, we measured their concentrations and isotopomer ratios (elemental isotope ratios and site-specific N isotope ratios in asymmetric molecules of NNO) in water and gas samples collected by an advanced treatment system in Tokyo. Although the estimated emissions of N(2)O and CH(4) from the system were found to be lower than those from the typical treatment systems reported before, water in biological reaction tanks was supersaturated with both gases. The concentration of N(2)O, produced mainly by nitrifier-denitrification as indicated by isotopomer ratios, was highest in the oxic tank (ca. 4000% saturation). The dissolved CH(4) concentration was highest in in-flow water (ca. 3000% saturation). It decreased gradually during treatment. Its carbon isotope ratio indicated that the decrease resulted from bacterial CH(4) oxidation and that microbial CH(4) production can occur in anaerobic and settling tanks.

Isotopomeric analysis of N2O dissolved in a river in the Tokyo metropolitan area.

River water has been suggested as a potential source of nitrous oxide (N2O), which is a greenhouse gas that is accumulating rapidly in the troposphere and which is a precursor to stratospheric NOx that depletes ozone. Previous studies on freshwater N2O sources have specifically examined estuaries where sedimentary N2O production might be important and a few points near anthropogenic nitrogen sources such as agricultural or municipal wastewater areas. Here we present the first observation of a temporal and horizontal distribution of N2O and its isotopomers between the midstream and estuary of an urban river. Surface water was supersaturated (100-6800%) with N2O at all stations during the study period. The average or maximum saturation value was greater than described in most previous reports. High N2O concentrations were observed near sewage plants and the unique signature of isotopomer ratios implied direct N2O addition from the plants. The isotopomer ratios also suggested N2O production/consumption at the sediment-water interface. Fluxes and isotopomer ratios of N2O, from the river to the atmosphere, estimated from our observations, indicated that the urban river is indeed a source of atmospheric N2O and that its production could be distinguished from other natural or anthropogenic sources using isotopomer ratios.

Interlaboratory assessment of nitrous oxide isotopomer analysis by isotope ratio mass spectrometry and laser spectroscopy: current status and perspectives.

RATIONALE In recent years, research and applications of the N2O site-specific nitrogen isotope composition have advanced, reflecting awareness of the contribution of N2O to the anthropogenic greenhouse effect, and leading to significant progress in instrument development. Further dissemination of N2O isotopomer analysis, however, is hampered by a lack of internationally agreed gaseous N2O reference materials and an uncertain compatibility of different laboratories and analytical techniques. METHODS In a first comparison approach, eleven laboratories were each provided with N2O at tropospheric mole fractions (target gas T) and two reference gases (REF1 and REF2). The laboratories analysed all gases, applying their specific analytical routines. Compatibility of laboratories was assessed based on N2O isotopocule data for T, REF1 and REF2. Results for T were then standardised using REF1 and REF2 to evaluate the potential of N2O reference materials for improving compatibility between laboratories. RESULTS Compatibility between laboratories depended on the analytical technique: isotope ratio mass spectrometry (IRMS) results showed better compatibility for δ(15)N values, while the performance of laser spectroscopy was superior with respect to N2O site preference. This comparison, however, is restricted by the small number of participating laboratories applying laser spectroscopy. Offset and two-point calibration correction of the N2O isotopomer data significantly improved the consistency of position-dependent nitrogen isotope data while the effect on δ(15)N values was only minor. CONCLUSIONS The study reveals that for future research on N2O isotopocules, standardisation against N2O reference material is essential to improve interlaboratory compatibility. For atmospheric monitoring activities, we suggest N2O in whole air as a unifying scale anchor.

Source identification of nitrous oxide on autotrophic partial nitrification in a granular sludge reactor

Emission of nitrous oxide (N2O) during biological wastewater treatment is of growing concern since N2O is a major stratospheric ozone-depleting substance and an important greenhouse gas. The emission of N2O from a lab-scale granular sequencing batch reactor (SBR) for partial nitrification (PN) treating synthetic wastewater without organic carbon was therefore determined in this study, because PN process is known to produce more N2O than conventional nitrification processes. The average N2O emission rate from the SBR was 0.32 ± 0.17 mg-N L(-1) h(-1), corresponding to the average emission of N2O of 0.8 ± 0.4% of the incoming nitrogen load (1.5 ± 0.8% of the converted NH4(+)). Analysis of dynamic concentration profiles during one cycle of the SBR operation demonstrated that N2O concentration in off-gas was the highest just after starting aeration whereas N2O concentration in effluent was gradually increased in the initial 40 min of the aeration period and was decreased thereafter. Isotopomer analysis was conducted to identify the main N2O production pathway in the reactor during one cycle. The hydroxylamine (NH2OH) oxidation pathway accounted for 65% of the total N2O production in the initial phase during one cycle, whereas contribution of the NO2(-) reduction pathway to N2O production was comparable with that of the NH2OH oxidation pathway in the latter phase. In addition, spatial distributions of bacteria and their activities in single microbial granules taken from the reactor were determined with microsensors and by in situ hybridization. Partial nitrification occurred mainly in the oxic surface layer of the granules and ammonia-oxidizing bacteria were abundant in this layer. N2O production was also found mainly in the oxic surface layer. Based on these results, although N2O was produced mainly via NH2OH oxidation pathway in the autotrophic partial nitrification reactor, N2O production mechanisms were complex and could involve multiple N2O production pathways.

Isotopocule analysis of biologically produced nitrous oxide in various environments

Natural abundance ratios of isotopocules, molecules that have the same chemical constitution and configuration, but that only differ in isotope substitution, retain a record of a compounds origin and reactions. A method to measure isotopocule ratios of nitrous oxide (N2 O) has been established by using mass analysis of molecular ions and fragment ions. The method has been applied widely to environmental samples from the atmosphere, ocean, fresh water, soils, and laboratory-simulation experiments. Results show that isotopocule ratios, particularly the 15 N-site preference (difference between isotopocule ratios 14 N15 N16 O/14 N14 N16 O and 15 N14 N16 O/14 N14 N16 O), have a wide range that depends on their production and consumption processes. Observational and laboratory studies of N2 O related to biological processes are reviewed and discussed to elucidate complex material cycles of this trace gas, which causes global warming and stratospheric ozone depletion.