B43A-0888 INVITED
An absolute calibration of the site preference of 15N isotopic fractionation in N2O using FTIR spectroscopy
The bulk isotopic fractionation of 15N in nitrous oxide (N2O) provides a valuable diagnostic for the pathways by which N2O is formed and processed in the natural environment. As the two N atoms in N2O are chemically inequivalent and do not exchange, 15N fractionation can be different at the two different sites. The preference for 15N fractionation between the two sites provides a further valuable diagnostic for N2O processing, but its exploitation has been limited in part due to a lack of agreement in defining an absolute calibration of the site preference. Here we present an independent method for an absolute calibration of the 15N site preference based on high resolution FTIR spectroscopy of N2O. IR spectroscopy naturally distinguishes the two isotopomers as separate species, provides absolute calibrations for both isotopomers independently, and is completely independent of mass spectrometric measurements.
B43A-0889
Laboratory incubation studies to investigate the control of isotopologue signatures of soil- emitted N2O
Isotopologue signatures of N2O such as δ18O, average δ15N (δ15Nbulk) and 15N site preference (SP = difference in δ15N between the central and peripheral N positions of the asymmetric N2O molecule) can be used to constrain the atmospheric N2O budget and to characterize N2O turnover processes. However, the use of this approach to study N2O dynamics in soils requires knowledge of isotopologue fractionation factors (ε) for the various partial processes involved, e.g. N2O production by nitrification or denitrification, and N2O reduction by denitrification. The aim of our study is to investigate the control of isotopologue signatures of soil-emitted N2O. Two soils were incubated in the laboratory under varying conditions in order to manipulate the partial processes of N2O turnover. ε of δ18O, δ15Nbulk and SP was determined in experiments, where only one of the partial processes was governing the isotopic signature of N2O in the incubation system of the respective treatment. ε of nitrification was determined by favoring N2O fluxes originating from NH4+ oxidation using high NH4+ levels and low soil moisture (30 to 50 % water-filled pore space). ε of N2O production by denitrification was obtained by anaerobic incubation of NO3- amended soils when N2O reduction was inhibited by 10 kPa acetylene. ε of N2O reduction was derived by (i) comparing treatments with and without inhibition of N2O reduction or (ii) by monitoring the time course of isotopic signatures of N2O applied to the headspace of NO3-- depleted anaerobic soil. To investigate the combined effect of various simultaneous processes, isotopologue signatures of N2O emitted from soils incubated under varying N-fertilizer level and moisture were measured while gross rates of the partial processes were determined independently. Results of fractionation factors and isotopologue signatures will be presented and discussed in view of previous studies and theoretical considerations.
B43A-0890
Isotopomer Analysis of N2O Produced During Waste Water Treatment
Nitrous oxide (N2O) 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. Isotopomer ratios (elemental N and O isotope ratios and site-specific N isotope ratios in asymmetric molecule of NNO) have been studied to understand its complex geochemical cycle. Microbial processes such as nitrification and denitrification are the largest N2O sources, and pure culture incubation studies showed that intramolecular 15N-site preference (SP) in N2O can differentiate the two N2O producing processes, hydroxylamine oxidation and nitrite reduction. However, there have been still few studies on N2O isotopomer ratios in complex bacterial systems. In this paper, we investigated the isotopomer ratios in N2O produced in waste water treatment system in order to evaluate characteristics of N2O emitted from human sewage and to understand N2O dynamics in microbial consortia (activated sludge). Water and gas samples were collected step by step in two different treatment systems in a sewage plant in Tokyo. High dissolved N2O concentration (up to 7600%\ saturation) was observed in biological reaction tanks and isotopomer ratios confirmed active N2O production by microbes. Moreover, isotopomer ratios showed large variations throughout the whole treatment system and suggested that N2O is produced in settling and chlorination steps as well as biological reaction steps.
B43A-0891
In-Situ Quantification of Methanotrophic Activity in a Landfill Cover Soil Using Gas Push-Pull Tests
Landfills are both a major anthropogenic source and a sink for the greenhouse gas CH4. Methanogenic bacteria produce CH4 during the anaerobic digestion of landfill waste, whereas, methanotrophic bacteria consume CH4 as it is transported through a landfill cover soil. Methanotrophs are thought to be ubiquitous in soils, but typically exist in large numbers at oxic/anoxic interfaces, close to anaerobic methane sources but exposed to oxygen required for metabolism. Accurate in-situ quantification of the sink strength of methanotrophs in landfill cover soils is needed for global carbon balances and for local emissions mitigation strategies. We measured in-situ CH4 concentrations at 30, 60, and 100 cm depth at 18 evenly spaced locations across a landfill cover soil. Furthermore, we performed Gas Push-Pull Tests (GPPTs) to estimate in-situ rates of methanotrophic activity in the cover soil. The GPPT is a gas-tracer test in which a gas mixture containing CH4, O2, and non-reactive tracer gases is injected (pushed) into the soil followed by extraction (pull) from the same location. Quantification of CH4 oxidation rates is based upon comparison of the breakthrough curves of CH4 and tracer gases. We present the results of a series of GPPTs conducted at two locations in the cover soil to assess the feasibility and reproducibility of this technique to quantify methanotrophic activity. Additional GPPTs were performed with a methanotrophic inhibitor in the injection gas mixture to confirm the appropriate choice of tracers to quantify CH4 oxidation. Estimated CH4 oxidation rate constants indicate that the cover soil contains a highly active methanotrophic community.
B43A-0892
Isotopomers as a method for differentiating between bacterial and fungal production of nitrous oxide
In order to study the importance of fungi to nitrous oxide (N2O) production in the environment it is critical to have a non-intrusive method for differentiating between fungal and bacterial N2O production. Site preference (SP), the difference in d15N between the central and outer N atoms in N2O, has been used to differentiate between bacterial nitrification and denitrification. In this study we compare the SP, d15N and d18O of N2O produced by the two best-studied fungal denitrifiers, Fusarium oxysporum and Cylindrocarpon tonkinense, to data from our previous bacterial studies. Both d18O and SP values remained fairly constant during the course of nitrite reduction which likely reflects isotopic exchange with water in the case of d18O and conservative behavior in SP that has been observed previously (Sutka et al., 2006). We observed a wide range of fractionation factors for fungal denitrification, -74.7 to -6.6 ‰, and non-linear behavior indicating that fractionation was controlled by more than one step. We interpret the small degree of fractionation as reflecting fractionation during diffusion and the more negative values as being controlled by enzymatic fractionation. Data from this and our previous study of bacterial production (Sutka et al., 2006) reveals that N2O produced via nitrification by fungi can be differentiated from N2O produced by bacterial denitrification primarily on the basis of d18O. The site preference of N2O produced by F. oxysporum and C. tonkinense was 37.1 ± 2.5 ‰ and 36.9 ± 2.8 ‰, respectively. These results indicate that isotopomers can be used as a basis for differentiating bacterial and fungal denitrification. Our work further reveals the role that fungal and bacterial nitric oxide reductases have in determining site preference during N2O production.
B43A-0893
Isotopic and Reporter Techniques to Verify Links Between Plant C Flow and Denitrification
The availability of organic C is considered paramount for the production and reduction of the greenhouse gas nitrous oxide (N2O) during denitrification in the rhizosphere. Despite this, the role of organic C in the regulation of N2O- and N2-genic enzymes is poorly understood. Stable isotopes are fundamental in resolving this. Here we will present selected results from experiments in which we have applied isotopic and reporter techniques to verify the effect of plant C in driving denitrification, and the potential feedbacks of this on climate change. Changes in C input to soil, such as under elevated atmospheric CO2, is significant for N2O production and reduction. Following application of 15N-labelled fertiliser to Lolium perenne swards we showed increased denitrifier-N2O and N2 production under elevated pCO2 (60 Pa) in the Swiss FACE experiment. This was attributed to greater below ground C allocation providing the energy for denitrification, and emissions were strongly positively correlated with TOC. By converting all rhizosphere soil to Redox conditions conducive to denitrification, rhizosphere C flow was quantified via N2O flux, and estimates agreed with measurements using 13C and 14C approaches. Little is known about the effect of different C substrates in regulating N2O and N2 production nor their effects on community structure, activity or species selection of denitrifying bacteria in the rhizosphere. We provide the first evidence for differences in N2O and N2 production with different C compounds typically present in root exudate, which suggest differences in regulation of the NO and N2O reductases, or preference for different C compounds in the rhizosphere denitrifier community. Such differences in gaseous N production are being related to the function and activity of the denitrifier community associated with this root C flow, with the link between C flow and denitrifier activity being verified by stable isotope probing and NanoSIMS imaging. Further ecophysiological evidence is presented of reporter gene expression by denitrifiers in response to different substrate classes of rhizosphere C.
B43A-0894
Recent Advances in Stable Isotope Techniques for N2O Source Partitioning in Soils
The use of 13C, 15N and 18O enables us to overcome uncertainties associated with soil C and N processes and to assess the links between species diversity and ecosystem function. Recent advances in stable isotope techniques enable determination of process rates, and are fundamental for examining interactions between C and N cycles. Here we will introduce the 15N-, 18O- and 13C-enrichment techniques we have developed to distinguish between different N2O-producing processes in situ in soils, presenting selected results, and will critically assess their potential, alone and in combination with molecular techniques, to help address key research questions for soil biogeochemistry and microbial ecology. We have developed 15N- 18O-enrichment techniques to distinguish between, and to quantify, N2O production during ammonia oxidation, nitrifier denitrification and denitrification. This provides a great advantage over natural abundance approaches as it enables quantification of N2O from each microbial source, which can be coupled with quantification of N2 production, and used to examine interactions between different processes and cycles. These approaches have also provided new insights into the N cycle and how it interacts with the C cycle. For example, we now know that ammonia oxidising bacteria significantly contribute to N2O emissions from soils, both via the traditionally accepted ammonia oxidation pathway, and also via denitrification (nitrifier denitrification) which can proceed even under aerobic conditions. We are also linking emissions from each source to diversity and activity of relevant microbial functional groups, for example through the development and application of a specific nirK primer for the nitrite reductase in ammonia oxidising bacteria. Recently, isotopomers have been proposed as an alternative for source partitioning N2O at natural abundance levels, and offers the potential to investigate N2O production from nitrate ammonification, and overcomes the need to apply 18O-H2O to determine nitrifier denitrification. However, this only provides an estimated, not a quantified, contribution, and further developments are required for quantification using isotope enrichment. Despite some limitations, such techniques become even more powerful when linked with other recent developments, such as nanoSIMS, gene expression and 13C-stable isotope probing of microbial RNA, and when linked to other disciplines. These may help address remaining questions such as: which microbes are producing N2O in soil, what is the influence of plants and mycorrhizal fungi on rhizosphere processes, and where is denitrification occurring in soil?
B43A-0895
Rapid Land-Use Changes for Large-Scale Industrialized Agriculture and Associated Estimates of Greenhouse Gas Emissions in the Brazilian Amazon
Rapid changes in land use in the southwestern Amazon, particularly conversion of native vegetation to mechanized row-crop agriculture, have many ecological consequences, including changes in biogeochemical cycles. Quantifying the patterns and magnitude of changes in row-crop agriculture, and subsequent intensification, are important boundary conditions for estimating regional-scale environmental impacts. We used five years of MODIS data to identify annual changes in row-crop agriculture, focusing on expansion and intensification of croplands in Mato Grosso and Rondônia. Our approach involved a wavelet-smoothing methodology for processing and analyzing MODIS time series data. We validated our results against crop histories from farms in both states. From annual statistics of phenology, we distinguished areas of cropland from other land covers. Results show increases in area of row-crop agriculture of 1% in Rondônia and 6% in Mato Grosso between 2001-2005. Within row-crop agriculture, we calculated the timing and number of crops per year (single or double cropping). Between 2001 and 2005, farmers in Mato Grosso have intensified from single to double cropping on more than 11,000 km2, while in Rondônia, farmers have transitioned to double cropping on over 20 km2. These remote sensing products are now being used as boundary conditions for our biogeochemical model the Terrestrial Ecosystems Model (TEM). We are using TEM to estimate greenhouse-gas emissions, particularly nitrous oxide, associated with the rapid conversion of native ecosystems and managed pastures to heavily industrialized agriculture.
B43A-0896
The 14CH4 Measurement Record, Geologic Sources of Methane, and a new Reconciliation of the Global Methane Budget
An enduring consensus of the global methane budget, as expressed in recent IPCC Assessment Reports, is that about 20%\ of emissions have geologic origin (ie, are free of 14CH4). Based on atmospheric 14CH4 measurements and confounded by ill-quantified 14CH4 emissions from the nuclear- power industry, this "fossil fraction" estimate is quite uncertain, typically 18± 9%. A very recent re-determination of 30± 5%\ (± 2σ) for the fossil fraction which does not require that the nuclear-power emission be specified may still be compatible with the earlier assessment despite an upward revision by 50%. Anthropogenic emissions via identified pathways of fossil-fuel exploitation are assessed in the Fourth IPCC Assessment Report at 90± 16 Tg~yr-1. Natural emissions extrapolated conservatively from new and widespread measurements of terrestrial seeps are 50± 10 Tg~yr-1, at least three times larger than previously accepted. The aggregate fossil emission of 140± 19 Tg~yr-1 is conservative yet still accounts for 24± 5%\ of the global source of 582± 87 Tg~yr-1, a value that is compatible with both 14CH4-based estimates of the fossil fraction. Nevertheless, while the fossil contribution to the methane source inventory remains incompletely determined the currently accepted estimate of ~20%\ for the fossil fraction may need to be revised upward, with substantial consequences for pre-industrial and contemporary source inventories.
B43A-0897
Isotopologue fractionation during nitrous oxide reduction in soil
Reduction of N2O is a challenge to studies using isotope values to resolve global budgets and microbial sources of this critical greenhouse gas. Prior research has demonstrated that the difference in δ15N between the central (α) and outer (β) N atoms in the N2O can be used to distinguish N2O derived from nitrification and denitrification (Sutka et al., 2003; 2006; Toyoda et al., 2005). If the intramolecular distribution of 15N, however, is altered during reduction, apportionments of N2O to nitrification and denitrification will be inaccurate. Isotopologue analyses of N2O within soil mesocosm experiments were used to investigate fractionation during N2O reduction at four different levels of water filled pores space (WFPS) 60, 80, 100 and 110%. Uncultivated successional soils were obtained from the Kellogg Biological Station Long Term Ecological Research Site located in Michigan (KBS LTER). Isotopic enrichment factors (ε) for δ15N, δ18O, δ15Nα and δ15Nβ ranged from -4.2 to -9.0, -12.5 to -23.6, -6.4 to -10.0 and -2.0 to -7.9, respectively. With the exception of SP, lower fractionation factors were observed at higher WFPS demonstrating the importance of diffusion in limiting the expression of enzymatic fractionation. Isotopic discrimination in SP during N2O reduction was small and the ε values varied between -4.5 and 0 ‰. Strong correlations were evident between δ18O and δ15N during reduction and segregation against 18O was 2.7 times greater than 15N. Similarly, 18O was discriminated against approximately 2.0 times more than 15Nα. These relationships (1) provide a definitive means for establishing that isotope effects during reduction are present and (2) may provide a means to determine the source signatures even when reduction occurs.
B43A-0898
Environmental controls on methanogen viability in the hydrothermal waters of the El Tatio geyser field, Chile.
At the El Tatio geyser field, a unique hydrothermal site located in the Andes Mountains in Chile, methanogenic archaea were found in only two of the hundreds of hydrothermal features. Reported here is an investigation into the environmental and geochemical controls on the distribution of methanogenic archaea. Located in the hyper- arid Atacama Desert, El Tatio waters are characterized by high salinity (95-175mM), Na-Cl type waters and circum-neutral pH (6.5-7), with very low inorganic carbon (0.1-0.5 mM TIC), but very high concentrations of As and Sb (300-700 uM As, 10-30uM Sb). Extensive bacterial mats thrive in most of the shallow run-off streams originating from hydrothermal features. In order to determine geochemical controls on methanogen populations, major and trace elements, including As and Sb speciation and concentrations, were determined using IC and HPLC-ICP-MS methods. The structure of microbial communities was analyzed using MPN enumeration of methanogens, culturing, and phylogenetic analysis using molecular techniques. Here, as in many hydrothermal regions, temperature and geochemical gradients influence the microbial ecology. Results from MPN enumeration indicate methanogen populations are dominated by H2-utilizing (carbonate reducing) archaea at both of the sites, with some acetate-oxidizing archaea present. These sites contain comparatively high DIC concentrations; however, it is unclear whether this is a control or a product of methanogenic archaea. Water quality analyses also show a strong correlation between antimony concentrations and the presence of methanogens; methanogenic archaea being present only at sites with 17 uM Sb concentrations or less.