B11C-01 08:00h
The Chemistry and Biochemistry of Biological Denitrification
The pathway by which NO2- is reduced to N2O in denitrifying bacteria was controversial for many years, but evidence from several laboratories has now established that NO is an obligatory intermediate in most, if not all, bacteria. Reports of the X-ray structures of examples of both copper- and heme cd1-containing nitrite reductases (NiRs) and the unique copper enzyme nitrous oxide reductase (NoS), along with spectroscopic and genetic studies of nitric oxide reductase (NoR), provide new insight into questions such as how NO2- is reduced to NO by the nitrite reductases, why two redox centers are needed to effect this one-electron transformation, why the copper NiRs are able to produce N2O under certain conditions, how NoRs reduce NO to N2O, and why the unique tetranuclear copper cluster in NoS is essential for the reduction of N2O. This talk will present an overview of recent work from a number of laboratories, focussing on the systems that produce and consume N2O and on the chemical and biochemical factors that may affect these processes.
B11C-02 INVITED 08:25h
Methods and Some Applications of Isotopomer Analysis to Identify Microbial Sources and Sinks of N$_{2}$O
Nitrous oxide (N$_{2}$O) is an important trace gas in the atmosphere since it is radiatively active in the troposphere and also a precursor of nitric oxide which catalytically destroys ozone in the stratosphere. Natural isotope abundance in N$_{2}$O has been studied to understand its complex geochemical cycle. We first developed a high-precision analytical technique for determining intramolecular $^{15}$N-site preference in asymmetric molecule of NNO and observed the site preference for $\alpha$(center)-site over $\beta$(end)-site in atmospheric N$_{2}$O. Thus, isotopomer (isotopic isomer) analysis can give us a new information on sources and sinks of N$_{2}$O. Data from laboratory simulation experiments and field observations are now being accumulated with respect to natural and anthropogenic sources and sinks. However, microbial processes, which are responsible for most of the terrestrial and oceanic sources, have not been well characterized on isotopomer basis in order to close N$_{2}$O budget or to identify dominant pathways (nitrification or denitrification) in various environment. In this paper, we show analytical techniques for measuring isotopomer ratios in N$_{2}$O in several type of samples and some results from microbial incubation experiments and observations on gases emitted from agricultural soils. Theoretical consideration on isotopomeric fractionation in microbial processes and outstanding problems are also discussed.
B11C-03 08:40h
Variable Nitrogen Isotope Effects Associated With N$_{2}$O Isotopologue Production: Towards an Understanding of Denitrification Mechanism
There is much current interest in the use of the isotopic composition of N$_{2}$O, including site-specific $\delta^{15}$N (i.e., the $^{15}$N/$^{14}$N ratios of the central or terminal positions, expressed as $\delta^{15}$N$^{\alpha}$ and $\delta^{15}$N$^{\beta}$ respectively) and $\delta^{15}$N$^{bulk}$ (the integrated N isotope value) to understand biological sources of this important greenhouse gas. However, mechanisms driving the variability of $\delta^{15}$N$^{bulk}$, $\delta^{15}$N$^{\alpha}$, and $\delta^{15}$N$^{\beta}$ values of biologically produced N$_{2}$O need to be better understood for the effective interpretation of field observations. In denitrification, a major source of N$_{2}$O, the formation of the N=N bond is generally understood to occur via NO + NO. However evidence suggests that enzyme-bound NO$^{+}$ + NO$_{2}^{-}$ may also form N$_{2}$O (Ye et al. 1994), generating potentially significant differences in relative values of $\delta^{15}$N$^{bulk}$, $\delta^{15}$N$^{\alpha}$, and $\delta^{15}$N$^{\beta}$ depending on production pathways. A useful way to explore reaction mechanism is to determine whether an isotope effect is dependent on substrate concentration, in this case nitrate. Nitrate concentrations are also highly variable in terrestrial and aquatic environments where denitrification occurs. We investigated the $\delta^{15}$N values of N$_{2}$O produced by the denitrifier ${\it P. aureofaciens}$ at nitrate concentrations ranging from 0.03 mg/L to 2000 mg/L at which the total conversion was $<$ 15 $%$ of the initial nitrate. The bulk $^{15}$N enrichment factor ($\epsilon$) increased with production rate when [NO$_{3}^{-}$]$_{init}$ varied between 25 mg/L to 844 mg/L, but was constant below this [NO$_{3}^{-}$]$_{init}$ range (here referred to as low nitrate). The maximum production rate and observed enrichment factors were reached at [NO$_{3}^{-}$]$_{init}$ = $\sim$1000 mg/L and remained constant up to 2000 mg/L (here referred to as high nitrate). Results were $\epsilon$= -22.7 $\pm$ 2.6 $\permil$ (n=18) at low nitrate, $\epsilon$= -33.2 $\pm$ 3.3 $\permil$ (n=4) at [NO$_{3}^{-}$]$_{init}$ = 250 mg/L, and constant at $\epsilon$= -42.3 $\pm$ 0.3 $\permil$ (n=10) at high nitrate. $\delta^{15}$N$^{\alpha}$ and $\delta^{15}$N$^{\beta}$ values relative to tropospheric N$_{2}$O were respectively -36.6 $\pm$ 2.8 $\permil$ (n=15) and -11.8 $\pm$ 5.0 $\permil$ (n=15) at low nitrate, -50.9 $\pm$ 3.2 $\permil$ (n=4) and -23.2 $\pm$ 3.6 $\permil$ (n=4) at [NO$_{3}^{-}$]$_{init}$ = 250 mg/L, and -60.0 $\pm$ 0.3 $\permil$ (n=6) and -32.6 $\pm$ 1.5 $\permil$ (n=6) at high nitrate. We found no significant variability of the difference between $\delta^{15}$N$^{\alpha}$ and $\delta^{15}$N$^{\beta}$ with nitrate availability, despite large differences in $\epsilon$. Implications of these results with respect to the mechanism of N$_{2}$O production will be discussed. Ye R.W., Averill B.A., and Tiedje J.M (1994) Denitrification: Production and Consumption of Nitric Oxide, Appl. Environ. Microbiol. 60: 1053-1058.
B11C-04 08:55h
Use of Stable Isotope Techniques to Differentiate Between Processes Contributing to N2O Emissions From Soils
N2O is produced biologically in soils during denitrification, nitrification, nitrifier denitrification and dissimilatory reduction of nitrate to ammonium (DNRA). These processes may occur simultaneously in different microsites of the same soil but there is often uncertainty associated with which process is predominantly contributing to measured emissions. Recent advances in stable isotope techniques facilitating direct measurement of 15N-N2O and natural abundance 15N218O allows determination of the source of N2O and an accurate quantification (15N enrichment) or estimation in natural systems (natural abundance) of emissions from each source. Here we will introduce the techniques we have developed and present selected results from studies where they have been applied. We are able to directly determine the respective contributions of nitrification and denitrification in soil by measurement of 15N-N2O after application of (a) 15NH415NO3 and (b) 14NH415NO3 (10 atom % excess 15N) to different replicates, where 15N-N2O measured from (b) replicates are attributed to denitrification and 15N-N2O measured from (a) replicates minus 15N-N2O from (b) replicates are attributed to nitrification, provided that dissimilatory 15NO3- reduction or immobilisation and remineralisation of 15NO3- are negligible. Addition of C2H2 (0.01 % v/v) to inhibit autotrophic ammonia oxidation enables us to differentiate between autotrophic and heterotrophic nitrified N2O. We have shown heterotrophic nitrification to be contributing to N2O emissions from a clay loam soil at 50 % water-filled pore space (WFPS) and evidence for aerobic denitrification at 20 % WFPS after application of 200 kg N ha-1. We have further developed this technique to determine the contribution of nitrifier denitrification by addition of 100 kPa O2 to inhibit anaerobic processes, and derivation of the nitrifier denitrification emission on a difference basis. In culture studies we have quantified 15N-N2O production during nitrifier denitrification by beta-proteobacterial ammonia oxidising bacteria strains representative of phylogenetic clusters 0, 2, 3 and 4 in the Nitrosospira lineage and the Nitrosomonas europaea/ `Nitrosococcus mobilis' lineage after application of 15NO2-. Based on knowledge from our 15N enrichment experiments of soil conditions where each process predominates, we are currently examining natural abundance 15N218O signatures for differentiating N2O produced during different processes. Natural abundance studies are more appropriate for low input systems, but are currently hindered by uncertainty of the isotopic signature of N2O during nitrification, nitrifier denitrification or DNRA. Different N and O sources and isotopic discrimination will result in different natural abundance 15N218O signatures and fractionation factors, enabling differentiation of processes producing N2O.
B11C-05 INVITED 09:10h
Separating Terrestrial, Oceanic and Stratospheric Signals in Atmospheric N$_{2}$O: Seasonal Cycles and Isotopic Signatures
Seasonal cycles in atmospheric N$_{2}$O provide potentially important information about surface source distributions. Previous attempts to reproduce observed N$_{2}$O seasonal cycles in atmospheric transport models (ATMs) were largely unsuccessful, for reasons that may include the following: 1) The observed cycles are very small. 2) The influence of the backflux of N$_{2}$O-depleted air from the stratosphere was neglected. Here, an interpretation of the observed atmospheric N$_{2}$O seasonal cycle at Cape Grim, Tasmania is presented and successfully compared to the results of an ATM run with prescribed surface sources. The exercise suggests that the observed N$_{2}$O seasonal cycle can be partitioned into distinct oceanic and stratospheric components, and offers a model for future exercises at northern hemisphere monitoring stations, where terrestrial sources are also likely to influence observed seasonal cycles. Like seasonal cycles, the observed isotopic signature of tropospheric N$_{2}$O represents a combination of terrestrial, oceanic, and stratospheric influences, all of which have distinct isotopic characteristics. A simple box model is used to predict the effect of seasonality on the isotopic signature of tropospheric N$_{2}$O and to examine how isotopic data might complement mixing ratio measurements. Some speculations on the oceanic influence on the isotopic signature of tropospheric N$_{2}$O will also be presented.
B11C-06 INVITED 09:30h
Partition of the relative contribution of nitrification and denitrification from Amazon forest soils using a model based on bulk $^{15}$N of N$_{2}$O natural abundance determinations.
Most of the available methods for the determination of the relative contribution of nitrification and denitrification to the soil emitted N$_{2}$O are invasive. Therefore, they could produce biased results due to the change in soil structure, alteration to the microbial community and substrates. However, the soil community bacterial activity has intrinsic properties such as isotopic fractionation factors that are relative constant through different sets of soil conditions. We took advantage of these bacterial properties and devised a mass balance method for partitioning the relative contribution of each process by using: (1) The $^{15}$N enrichment factors for N$_{2}$O production via nitrification and denitrification for soils (determined previously by acetylene addition soil incubation methods) and (2) the $\delta$$^{15}$N$-$N$_{2}$O soil emission values from the selected studied soils. We selected soils from a forest soil texture gradient from the Tapajos National Forest (TNF), in the Amazon Basin, Par State, Brazil and Nova Vida Farm (NV), Rondonia State, Brazil where we had determined the $^{15}$N enrichment factors for each microbial process and collected N$_{2}$O soil emissions for bulk stable isotope analysis during the rainy season of 2002. The soils selected were Oxisol (clay) and Ultison (sandy) at TNF and Latosol (sandy loam) at NV. We found that for all studied soils, the relative contribution of nitrification was smaller than 40 %. This corroborates the assumption that the N$_{2}$O emitted from Amazon forest is mostly denitrification-derived. The advantage of this method is that is non invasive. However, the uncertainties associated with the method increase when $\delta$$^{15}$N$-$N$_{2}$O values of emitted N2O are smaller than -25 per mil.
B11C-07 09:45h
Measurement and Modeling of Isotopomer Signatures of Nitrous Oxide from Soils and Groundwater
18O and average 15N signatures of N2O have contributed to a better understanding of global N2O fluxes and of N2O source processes in aquatic and terrestric environments. Recently, new methods have been developed to analyze site-specific 15N abundance in order to further improve isotopic characterization of N2O. The aim of our study is to evaluate isotopomer signatures of N2O (i) as a tool to identify N2O production processes in soils and groundwater and (ii) to constrain the isotopic fingerprint of soil-derived N2O. A microcosm study was conducted with arable loess soil incubated at varying oxygen and moisture levels in order establish different levels of process rates. Analysis of soil extracts and emitted gases was conducted in order to quantify gross rates and N2O production of nitrification and denitrification, respectively, N2O reduction of denitrification and the isotopic fingerprint of emitted N2O. N2O production and reduction during denitrification and the isotopic fingerprint of groundwater N2O was investigated in sandy aquifers using in situ and laboratory techniques and simulations. 18O and site specific 15N-signatures of N2O produced in the soil and in the groundwater study were analyzed by isotope ratio mass spectrometry after cryo-focussing and GC-separation of the gas phase. The average delta 15N was related to soil moisture and denitrification in the soil microcosm study and to the reduction of groundwater dissolved nitrate by denitrification. The difference in delta 15N between the central and terminal N-position within the N2O molecule ("site preference") as well as delta 18O increased with soil moisture and with increasing nitrate reduction of groundwater nitrate. For the groundwater study, a simulated time course of isotopic signatures could be fitted to the measurements. The isotopic fingerprints of N2O derived from wet and dry soil and from the groundwater were clearly distinct. It is concluded that isotopomer signatures of N2O from soils and groundwater are suitable to characterize source processes.