Dominika Lewicka-Szczebak

Dual isotope and isotopomer signatures of nitrous oxide from fungal denitrification – a pure culture study

RATIONALE The contribution of fungal denitrification to the emission of the greenhouse gas nitrous oxide (N2O) from soil has not yet been sufficiently investigated. The intramolecular (15)N site preference (SP) of N2O could provide a tool to distinguish between N2O produced by bacteria or fungi, since in previous studies fungi exhibited much higher SP values than bacteria. METHODS To further constrain isotopic evidence of fungal denitrification, we incubated six soil fungal strains under denitrifying conditions, with either NO3(-) or NO2(-) as the electron acceptor, and measured the isotopic signature (δ(18)O, δ(15)Nbulk and SP values) of the N2O produced. The nitrogen isotopic fractionation was calculated and the oxygen isotope exchange associated with particular fungal enzymes was estimated. RESULTS Five fungi of the order Hypocreales produced N2O with a SP of 35.1 ± 1.7 ‰ after 7 days of anaerobic incubation independent of the electron acceptor, whereas one Sordariales species produced N2O from NO2(-) only, with a SP value of 21.9 ± 1.4 ‰. Smaller isotope effects of (15)Nbulk were associated with larger N2O production. The δ(18)O values were influenced by oxygen exchange between water and denitrification intermediates, which occurred primarily at the nitrite reduction step. CONCLUSIONS Our results confirm that SP of N2O is a promising tool to differentiate between fungal and bacterial N2O from denitrification. Modelling of oxygen isotope fractionation processes indicated that the contribution of the NO2(-) and NO reduction steps to the total oxygen exchange differed among the various fungal species studied. However, more information is needed about different biological orders of fungi as they may differ in denitrification enzymes and consequently in the SP and δ(18)O values of the N2O produced.

Soil denitrification potential and its influence on N2O reduction and N2O isotopomer ratios

RATIONALE N2O isotopomer ratios may provide a useful tool for studying N2O source processes in soils and may also help estimating N2O reduction to N2. However, remaining uncertainties about different processes and their characteristic isotope effects still hamper its application. We conducted two laboratory incubation experiments (i) to compare the denitrification potential and N2O/(N2O+N2) product ratio of denitrification of various soil types from Northern Germany, and (ii) to investigate the effect of N2O reduction on the intramolecular (15)N distribution of emitted N2O. METHODS Three contrasting soils (clay, loamy, and sandy soil) were amended with nitrate solution and incubated under N2 -free He atmosphere in a fully automated incubation system over 9 or 28 days in two experiments. N2O, N2, and CO2 release was quantified by online gas chromatography. In addition, the N2O isotopomer ratios were determined by isotope-ratio mass spectrometry (IRMS) and the net enrichment factors of the (15)N site preference (SP) of the N2O-to-N2 reduction step (η(SP)) were estimated using a Rayleigh model. RESULTS The total denitrification rate was highest in clay soil and lowest in sandy soil. Surprisingly, the N2O/(N2O+N2) product ratio in clay and loam soil was identical; however, it was significantly lower in sandy soil. The IRMS measurements revealed highest N2O SP values in clay soil and lowest SP values in sandy soil. The η(SP) values of N2O reduction were between -8.2 and -6.1‰, and a significant relationship between δ(18)O and SP values was found. CONCLUSIONS Both experiments showed that the N2O/(N2O+N2) product ratio of denitrification is not solely controlled by the available carbon content of the soil or by the denitrification rate. Differences in N2O SP values could not be explained by variations in N2O reduction between soils, but rather originate from other processes involved in denitrification. The linear δ(18)O vs SP relationship may be indicative for N2O reduction; however, it deviates significantly from the findings of previous studies.

Carbon isotope signature of dissolved inorganic carbon (DIC) in precipitation and atmospheric CO2

This paper describes results of chemical and isotopic analysis of inorganic carbon species in the atmosphere and precipitation for the calendar year 2008 in Wrocław (SW Poland). Atmospheric air samples (collected weekly) and rainwater samples (collected after rain episodes) were analysed for CO2 and dissolved inorganic carbon (DIC) concentrations and for δ13C composition. The values obtained varied in the ranges: atmospheric CO2: 337-448 ppm; δ13CCO2 from -14.4 to -8.4‰; DIC in precipitation: 0.6-5.5 mg dm(-3); δ13CDIC from -22.2 to +0.2‰. No statistical correlation was observed between the concentration and δ13C value of atmospheric CO2 and DIC in precipitation. These observations contradict the commonly held assumption that atmospheric CO2 controls the DIC in precipitation. We infer that DIC is generated in ambient air temperatures, but from other sources than the measured atmospheric CO2. The calculated isotopic composition of a hypothetical CO2 source for DIC forming ranges from -31.4 to -11.0‰, showing significant seasonal variations accordingly to changing anthropogenic impact and atmospheric mixing processes.

Isotope fractionation factors controlling isotopocule signatures of soil-emitted N₂O produced by denitrification processes of various rates.

RATIONALE This study aimed (i) to determine the isotopic fractionation factors associated with N2O production and reduction during soil denitrification and (ii) to help specify the factors controlling the magnitude of the isotope effects. For the first time the isotope effects of denitrification were determined in an experiment under oxic atmosphere and using a novel approach where N2O production and reduction occurred simultaneously. METHODS Soil incubations were performed under a He/O2 atmosphere and the denitrification product ratio [N2O/(N2 + N2O)] was determined by direct measurement of N2 and N2O fluxes. N2O isotopocules were analyzed by mass spectrometry to determine δ(18)O, δ(15)N and (15)N site preference within the linear N2O molecule (SP). An isotopic model was applied for the simultaneous determination of net isotope effects (η) of both N2O production and reduction, taking into account emissions from two distinct soil pools. RESULTS A clear relationship was observed between (15)N and (18)O isotope effects during N2O production and denitrification rates. For N2O reduction, diverse isotope effects were observed for the two distinct soil pools characterized by different product ratios. For moderate product ratios (from 0.1 to 1.0) the range of isotope effects given by previous studies was confirmed and refined, whereas for very low product ratios (below 0.1) the net isotope effects were much smaller. CONCLUSIONS The fractionation factors associated with denitrification, determined under oxic incubation, are similar to the factors previously determined under anoxic conditions, hence potentially applicable for field studies. However, it was shown that the η(18)O/η(15)N ratios, previously accepted as typical for N2O reduction processes (i.e., higher than 2), are not valid for all conditions.

An enhanced technique for automated determination of 15N signatures of N2, (N2 +N2O) and N2O in gas samples.

RATIONALE An enhanced analytical approach for analyzing gaseous products from (15)N-enriched pools has been developed. This technique can be used to quantify nitrous oxide (N2O) and dinitrogen (N2) fluxes from denitrification. It can also help in distinguishing different N2- and N2O-forming processes, such as denitrification, nitrification, anaerobic ammonium oxidation or co-denitrification. METHODS The measurement instrumentation was based on a commercially available automatic preparation system allowing collection and separation of gaseous samples. The sample transfer paths, valves, liquid nitrogen traps, gas chromatography column and open split of the original system were modified. A reduction oven (Cu) was added in order to eliminate oxygen and measure N2O-N as N2. Gases leaving the separation system entered an isotope ratio mass spectrometer where masses (28)N2, (29)N2 and (30)N2 were measured. RESULTS The enhanced technique enabled rapid simultaneous measurement of stable isotope ratios (29)N2/(28)N2 and (30)N2/(28)N2 originating from dinitrogen alone (N2) and from the sum of the denitrification products (N2 +N2O) as well as the determination of (15)N enrichment in N2O. The (15)N fraction in the N pool undergoing N2 and N2O production ((15)X(N)) and the contribution of N2 and N2O originating from this pool (d) were determined with satisfactory accuracy of better than 3.3% and 2.9%, respectively. CONCLUSIONS The precision and accuracy of this method were comparable with or better than previously reported for similar measurements. The proposed method allows for the analysis of all quantities within one run, thus reducing the measurement and sample preparation time as well as increasing the reliability of the results.

Carbon and nitrogen isotope analyses coupled with palynological data of PM10 in Wrocław city (SW Poland) – assessment of anthropogenic impact

We have applied both palynological and carbon and nitrogen isotopic analyses of PM10 (particulate matter with a diameter of 10 μm or less) to trace its origin and to assess the anthropogenic impact for the area under study. The PM10 samples were collected in Wrocław (SW Poland) by the Regional Inspectorate for Environment Protection during the year 2007. The usefulness of the palynological observations in the case of PM10 is much lower than that for total suspended particles due to the resolution of absorbed particles, but is still helpful for distinguishing C3/C4 plants that indicate long-distance transport of pollutants. The δ13C(PM10) values varied seasonally from−26.9 to−25.1‰. The δ15N(PM10) values showed chaotic fluctuations and varied from 5.0 to 13.7‰. Our results indicated that during the heating period, the PM10 particles in Wrocław are derived mainly from local home heaters, whereas in the growing period, PM10 particles are derived from local transport and are partially generated by the industrial application of coal combustion outside the city of Wrocław.

N2O source partitioning in soils using 15N site preference values corrected for the N2O reduction effect

RATIONALE The aim of this study was to determine the impact of isotope fractionation associated with N2O reduction during soil denitrification on N2O site preference (SP) values and hence quantify the potential bias on SP-based N2O source partitioning. METHODS The N2O SP values (n = 431) were derived from six soil incubation studies in N2-free atmosphere, and determined by isotope ratio mass spectrometry (IRMS). The N2 and N2O concentrations were measured directly by gas chromatography. Net isotope effects (NIE) during N2O reduction to N2 were compensated for using three different approaches: a closed-system model, an open-system model and a dynamic apparent NIE function. The resulting SP values were used for N2O source partitioning based on a two end-member isotopic mass balance. RESULTS The average SP0 value, i.e. the average SP values of N2O prior to N2O reduction, was recalculated with the closed-system model, resulting in -2.6 ‰ (±9.5), while the open-system model and the dynamic apparent NIE model gave average SP0 values of 2.9 ‰ (±6.3) and 1.7 ‰ (±6.3), respectively. The average source contribution of N2O from nitrification/fungal denitrification was 18.7% (±21.0) according to the closed-system model, while the open-system model and the dynamic apparent NIE function resulted in values of 31.0% (±14.0) and 28.3% (±14.0), respectively. CONCLUSIONS Using a closed-system model with a fixed SP isotope effect may significantly overestimate the N2O reduction effect on SP values, especially when N2O reduction rates are high. This is probably due to soil inhomogeneity and can be compensated for by the application of a dynamic apparent NIE function, which takes the variable reduction rates in soil micropores into account.

Diurnal variations in the photosynthesis-respiration activity of a cyanobacterial bloom in a freshwater dam reservoir: an isotopic study†

The stable isotopic analyses of molecular oxygen dissolved in water (δ18O(DO)) and dissolved inorganic carbon (δ13C(DIC)), supplemented with basic chemical measurements, have been carried out on a diurnal basis to better understand the dynamics of photosynthesis and respiration in freshwater systems. Our observations have been carried out in a lowland dam reservoir, the Sulejów Lake (central Poland), during the summer cyanobacterial bloom. All data obtained, isotopic, hydrochemical, and biological, show a high mutual consistency. Namely, the lowest δ18O(DO) values, obtained at 10:00 and 14:00 (16.0 and 15.5‰, respectively), correspond to the highest amount of cyanobacterial cells observed (66 and 63 mg dm−3, respectively), whereas the minimum δ13C(DIC) (−10.6‰) obtained at 22:00 corresponds to the maximum content of organic matter (110 mg dm−3). This evidence suggests that isotopic assays of δ18O(DO) and δ13C(DIC) are a reliable tool for the quantitative study of biochemical processes in freshwater systems. † Revised version of a paper presented at the 9th. Symposium of the European Society for Isotope Research (ESIR), 23 to 28 June 2007, Cluj-Napoca, Romania.

Comparison of methods to determine triple oxygen isotope composition of N2O

RATIONALE The oxygen isotope anomaly, Δ(17) O, of N2 O and nitrate is useful to elucidate nitrogen oxide dynamics. A comparison of different methods for Δ(17) O measurement was performed. METHODS For Δ(17) O measurements, N2 O was converted into O2 and N2 using microwave-induced plasma in a quartz or corundum tube reactor, respectively, or conversion was carried out in a gold wire oven. In each case, isotope ratios were measured by isotope ratio mass spectrometry. RESULTS All the tested methods showed acceptable precision (coefficient of variation <2.4 % at 160 nmol N2 O) with high sample size but the sample size dependence was lowest when using microwave-induced plasma in a corundum tube reactor. CONCLUSIONS The use of microwave-induced plasma in a corundum tube yields best results for Δ(17) O measurement on N2 O gas samples.

Tracing and quantifying lake water and groundwater fluxes in the area under mining dewatering pressure using coupled O and H stable isotope approach

Oxygen and hydrogen stable isotopic compositions of precipitation, lake water and groundwater were used to quantitatively asses the water budget related to water inflow and water loss in natural lakes, and mixing between lake water and aquifer groundwater in a mining area of the Lignite Mine Konin, central Poland. While the isotopic composition of precipitation showed large seasonal variations (δ2H from−140 to+13 ‰ and δ18O from−19.3 to+7.6 ‰), the lake waters were variously affected by evaporation (δ2H from−44 to−21 ‰ and δ18O from−5.2 to−1.7 ‰) and the groundwater showed varying contribution from mixing with surface water (δ2H from−75 to−39 ‰ and δ18O from−10.4 to−4.8 ‰). The lake water budget was estimated using a Craig–Gordon model and isotopic mass balance constraint, which enabled us to identify various water sources and to quantify inflow and outflow for each lake. Moreover, we documented that a variable recharge of lake water into the Tertiary aquifer was dependent on mining drainage intensity. A comparison of coupled δ2H–δ18O data with hydrogeological results indicated better precision of the δ2H-based calculations.