The mechanism of oxygen isotope fractionation during N2O production by denitrification

The isotopic composition of soil-derived N2O can help differentiate between N2O production pathways and estimate the fraction of N2O reduced to N2. Until now, δ18O of N2O has been rarely used in the interpretation of N2O isotopic signatures because of the rather complex oxygen isotope fractionations during N2O production by denitrification. The latter process involves nitrate reduction mediated through the following three enzymes: nitrate reductase (NAR), nitrite reductase (NIR) and nitric oxide reductase (NOR). Each step removes one oxygen atom as water (H2O), which gives rise to a branching isotope effect. Moreover, denitrification intermediates may partially or fully exchange oxygen isotopes with ambient water, which is associated with an exchange isotope effect. The main objective of this study was to decipher the mechanism of oxygen isotope fractionation during N2O production by denitrification and, in particular, to investigate the relationship between the extent of oxygen isotope exchange with soil water and the δ18O values of the produced N2O. We performed several soil incubation experiments. For the first time, ∆17 O isotope tracing was applied to simultaneously determine the extent of oxygen isotope exchange and any associated oxygen isotope effect. We found bacterial denitrification to be typically associated with almost complete oxygen isotope exchange and a stable difference in δ18O between soil water and the produced N2O of δ18O(N2O / H2O) = (17.5±1.2) ‰. However, some experimental setups yielded oxygen isotope exchange as low as 56 % and a higher δ18O(N2O / H2O) of up to 37‰. The extent of isotope exchange and δ18O(N2O / H2O) showed a very significant correlation (R2 = 0.70, p < 0.00001). We hypothesise that this observation was due to the contribution of N2O from another production process, most probably fungal denitrification. An oxygen isotope fractionation model was used to test various scenarios with different magnitudes of branching isotope effects at different steps in the reduction process. The results suggest that during denitrification the isotope exchange occurs prior to the isotope branching and that the mechanism of this exchange is mostly associated with the enzymatic nitrite reduction mediated by NIR. For bacterial denitrification, the branching isotope effect can be surprisingly low, about (0.0±0.9) ‰; in contrast to fungal denitrification where higher values of up to 30‰ have been reported previously. This suggests that δ18O might be used as a tracer for differentiation between bacte- 5 rial and fungal denitrification, due to their different magnitudes of branching isotope effect

Oxygen isotope fractionation during N2O production by soil denitrification

The isotopic composition of soil-derived N₂O can help differentiate between N₂O production pathways and estimate the fraction of N₂O reduced to N₂. Until now, <i>δ</i>¹⁸O of N₂O has been rarely used in the interpretation of N₂O isotopic signatures because of the rather complex oxygen isotope fractionations during N₂O production by denitrification. The latter process involves nitrate reduction mediated through the following three enzymes: nitrate reductase (NAR), nitrite reductase (NIR) and nitric oxide reductase (NOR). Each step removes one oxygen atom as water (H₂O), which gives rise to a branching isotope effect. Moreover, denitrification intermediates may partially or fully exchange oxygen isotopes with ambient water, which is associated with an exchange isotope effect. The main objective of this study was to decipher the mechanism of oxygen isotope fractionation during N₂O production by soil denitrification and, in particular, to investigate the relationship between the extent of oxygen isotope exchange with soil water and the <i>δ</i>¹⁸O values of the produced N₂O. <br><br> In our soil incubation experiments Δ¹⁷O isotope tracing was applied for the first time to simultaneously determine the extent of oxygen isotope exchange and any associated oxygen isotope effect. We found that N₂O formation in static anoxic incubation experiments was typically associated with oxygen isotope exchange close to 100 % and a stable difference between the ¹⁸O ∕ ¹⁶O ratio of soil water and the N₂O product of <i>δ</i>¹⁸O(N₂O ∕ H₂O)  =  (17.5 ± 1.2) ‰. However, flow-through experiments gave lower oxygen isotope exchange down to 56 % and a higher <i>δ</i>¹⁸O(N₂O ∕ H₂O) of up to 37 ‰. The extent of isotope exchange and <i>δ</i>¹⁸O(N₂O ∕ H₂O) showed a significant correlation (<i>R</i>² = 0.70, <i>p</i> &lt;  0.00001). We hypothesize that this observation was due to the contribution of N₂O from another production process, most probably fungal denitrification. <br><br> An oxygen isotope fractionation model was used to test various scenarios with different magnitudes of branching isotope effects at different steps in the reduction process. The results suggest that during denitrification, isotope exchange occurs prior to isotope branching and that this exchange is mostly associated with the enzymatic nitrite reduction mediated by NIR. For bacterial denitrification, the branching isotope effect can be surprisingly low, about (0.0 ± 0.9) ‰, in contrast to fungal denitrification where higher values of up to 30 ‰ have been reported previously. This suggests that <i>δ</i>¹⁸O might be used as a tracer for differentiation between bacterial and fungal denitrification, due to their different magnitudes of branching isotope effects

Investigations on the use of alcohols in modern gasoline engines

Motiviert durch die aktuellen Trends der Motorenentwicklung und die sich abzeichnenden Kraftstoffszenarien der nahen Zukunft wird im Rahmen dieser Arbeit der Einsatz von Alkoholen als Benzinsubstitut bewertet. Unter Berücksichtigung der Randbedingungen eines modernen Downsizingkonzepts wird so der Beitrag der alternativen Kraftstoffe zu einer wirkungsvollen Reduktion der CO2-Emissionen untersucht. Betrachtet wird die gesamte Prozesskette von der Gemischbildung über die Verbrennung bis zu den Emissionen. Zunächst erfolgt eine Bewertung der fünf einfachsten Alkohole anhand ihrer für den Verbrennungsrozess relevanten Stoffeigenschaften. Untersuchungen zum Dosierverhalten etablierter Injektorkonzepte zeigen den Einfluss der im Vergleich zu Benzin veränderten Stoffeigenschaften. Mittels speziell angepasster optischer Messtechnik werden u.a. die geometrischen Spraygrößen und die Zerstäubungsgüte bewertet. Die Ergebnisse aus dem Stationärversuch werden anschließend auf den Motorbetrieb unter instationären Bedingungen übertragen. Neben grundlegenden Untersuchungen zur Verbrennung in verschiedenen Lastzuständen wird auch der Einfluss einer Anhebung des Verdichtungsverhältnisses betrachtet. Verschiedene Ansätze zur Wirkungsgradsteigerung werden miteinander verglichen und bewertet. Die Arbeit schließt mit der Untersuchung eines Systemansatzes, bei dem ein zusätzlicher klopffester Kraftstoff bedarfsgerecht zudosiert wird. Ziel hierbei ist, die Klopfgrenze eines Downsizingmotors anzuheben und so den Wirkungsgrad zu verbessern.Motivated by the current trends in engine technology development and by the upcoming fuel scenarios of the near future the use of alcohols as a substitute of gasoline is evaluated. Considering the boundary conditions of a modern downsizing concept the alternative fuel's contribution to an efficient reduction of CO2 emissions is investigated. The engine's entire process chain of mixture formation, combustion, and emissions is examined. First of all five different alcohols are assessed by their physical and chemical properties relevant to the combustion process. Investigations on the dosing behavior of established injector concepts reveal different reactions to the changed fuel properties. Specially adapted optical measurement techniques are used to examine the geometric properties of the fuel sprays and the quality of spray atomization. The results of the stationary investigations are transferred to the transient conditions of the engine operation. Besides fundamental research on combustion at different operating points the influence of increasing the compression ratio is analyzed. Different scenarios to improve the efficiency are compared and evaluated. Finally a system concept is investigated injecting a knock-resistant fuel appropriately to increase efficiency by advancing the knock limit of a downsized engine

SPIN-MIMS simplifying the SPIN-MAS instrumentation for online measurement of 15N-abundances of ammonium, nitrite and nitrate in aqueous solutions

Common methods for measuring selectively the 15N abundances in individual N-species such as NH4+, NO2- and NO3- in samples with multiple N-species are laborious and time consuming. The SPIN-MAS technique (Stange et al. 2007) offers an automated, rapid and selective determination of 15N abundances in NH4+, NO2- and NO3- in aqueous samples. During a SPIN-MAS measurement one of three different reaction solutions is mixed with the aqueous sample in a Sample Preparation unit for Inorganic N-species (SPIN). The reaction solution is chosen in dependence on the N-species of interest. The gaseous reaction products (N2 or NO) are then conducted to a quadrupole mass spectrometer (MAS) in a helium stream. This measurement technique is not commonly used due to its complex instrumentation. The instrumentation can be significantly simplified by the use of a membrane inlet mass spectrometer (MIMS). The presented SPIN-MIMS approach relies on the use of a reaction capillary in which the sample containing the N-species of interest is mixed with the corresponding reaction solution. The mixture of reaction solution and sample is pumped from the reaction capillary directly to the membrane inlet of the mass spectrometer. The reaction products (N2 or NO) formed during the reaction of NH4+, NO2- and NO3- with the reaction solutions are passed through the gas-permeable membrane of the inlet directly into the ion source of the mass spectrometer. 15N standards with different at% 15N (NH4+, NO2- and NO3- respectively in dist. Water) were used to assess the performance of the system. Overall, SPIN-MIMS measurements showed a good agreement between measured and expected 15N abundances (range 0.36 – 10 at% 15N deviations: &lt;0.5 at% 15N for NH4+-, &lt;0.23 for NO2-- and &lt;0.15 at% 15N for NO3-- standards)

Turnover of soil monosaccharides: Recycling versus Stabilization

Soil organic matter (SOM) represents a mixture of differently degradable compounds. Each of these compounds are characterised by different dynamics due to different chemical recalcitrance, transformation or stabilisation processes in soil. Carbohydrates represent one of these compounds and contribute up to 25 % to the soil organic matter. Vascular plants are the main source of pentose sugars (Arabinose and Xylose), whereas hexoses (Galactose and Mannose) are primarily produced by microorganisms. Several studies suggest that the mean turnover times of the carbon in soil sugars are similar to the turnover dynamics of the bulk carbon in soil. The aim of the study is to characterise the influence of stabilisation and turnover of soil carbohydrates. Soil samples are collected from (i)a continuous maize cropping experiment (“Höhere Landbauschule” Rotthalmünster, Bavaria) established 1979 on a Stagnic Luvisol and (ii) from a continuous wheat cropping, established 1969, as reference site. The effect of stabilisation is estimated by the comparison of turnover times of microbial and plant derived soil carbohydrates. As the dynamics of plant derived carbohydrate are solely influenced by stabilisation processes, whereas the dynamics of microbial derived carbohydrates are affected by recycling of organic carbon compounds derived from C3 plant substrate as well as stabilisation processes. The compound specific isotopic analysis (CSIA) of soil carbohydrates was performed using a HPLC/o/IRMS system. The chromatographic and mass spectrometric subunits were coupled with a LC–Isolink interface. Soil sugars were extracted after mild hydrolysis using 4 M trifluoroacetic acid (TFA)

Is tagging with visual implant elastomer a reliable technique for marking earthworms?

Visual implant elastomer (VIE) has recently been employed to investigate different aspects of earthworm ecology. However, a number of fundamental questions relating to the detection and positioning of the tag, its persistence and potential effects on earthworms remain unknown. Seven earthworm species belonging to three ecological groupings, with different pigmentation and burrowing behaviour, were tagged using different coloured VIE. External inspection after two days, one week and 1, 10 and 27 months were followed by preservation, dissection and internal inspection. Tags could be seen in living specimens to 27 months, and dissection revealed that in most cases they were lodged in the coelomic cavity, held in place by septa. However, over longer time periods (more than two years), the chlorogogenous tissue tended to bind to the tags and made external observation increasingly difficult. Migration of the VIE material towards the posterior of the earthworm and potential loss of the tag were only observed on rare occasions, and a recovery rate in excess of 98% was recorded. By introducing a reasonable amount of VIE into segments, just after the clitellum, this technique can become a valuable tool in earthworm ecology and life history studies, particularly in short-medium term laboratory and field experiments

Stable isotope analysis (δ¹³C and δ¹⁵N) of soil nematodes from four feeding groups

Soil nematode feeding groups are a long-established trophic categorisation largely based on morphology and are used in ecological indices to monitor and analyse the biological state of soils. Stable isotope ratio analysis (13C/12C and 15N/14N, expressed as δ13C and δ15N) has provided verification of, and novel insights into, the feeding ecology of soil animals such as earthworms and mites. However, isotopic studies of soil nematodes have been limited to date as conventional stable isotope ratio analysis needs impractically large numbers of nematodes (up to 1,000) to achieve required minimum sample weights (typically >100 µg C and N). Here, micro-sample near-conventional elemental analysis–isotopic ratio mass spectrometry (μEA–IRMS) of C and N using microgram samples (typically 20 µg dry weight), was employed to compare the trophic position of selected soil nematode taxa from four feeding groups: predators (Anatonchus and Mononchus), bacterial feeders (Plectus and Rhabditis), omnivores (Aporcelaimidae and Qudsianematidae) and plant feeder (Rotylenchus). Free-living nematodes were collected from conventionally and organically managed arable soils. As few as 15 nematodes, for omnivores and predators, were sufficient to reach the 20 µg dry weight target. There was no significant difference in δ15N (p = 0.290) or δ13C (p = 0.706) between conventional and organic agronomic treatments but, within treatments, there was a significant difference in N and C stable isotope ratios between the plant feeder, Rotylenchus (δ15N = 1.08 to 3.22 mUr‰, δ13C = –29.58 to –27.87 mUr) and all other groups. There was an average difference of 9.62 mUr in δ15N between the plant feeder and the predator group (δ15N = 9.89 to 12.79 mUr, δ13C = –27.04 to –25.51 mUr). Isotopic niche widths were calculated as Bayesian derived standard ellipse areas and were smallest for the plant feeder (1.37 mUr2) and the predators (1.73 mUr2), but largest for omnivores (3.83 mUr2). These data may reflect more preferential feeding by the plant feeder and predators, as assumed by classical morphology-based feeding groups, and indicate that omnivory may be more widespread across detritivore groups i.e. bacterial feeders (3.81 mUr2). Trophic information for soil nematodes derived from stable isotope analysis, scaled as finely as species level in some cases, will complement existing indices for soil biological assessment and monitoring, and can potentially be used to identify new trophic interactions in soils. The isotopic technique used here, to compare nematode feeding group members largely confirm their trophic relations based on morphological studies

Die unterschätzte Rolle des Recyclings intakter Metabolite für die Umsatzraten der organischen Bodensubstanz

Obwohl Sorption an Mineraloberflächen als dominierender Prozess zur Erklärung des langsamen Umsatzes mineralassoziierter organischer Bodensubstanz (OBS) dient, widerspricht diese Idee einer zunehmenden Zahl an Inkubationsstudien, die zeigen, dass für niedermolekulare Substanzen nicht nur die mikrobielle Aufnahme kompetitivier als die Sorption ist, sondern auch sorbierte Substanzen in hohem Maße desorbiert und mikrobiell verwertet werden können. Dabei wurde gezeigt, dass sich die Verstoffwechselung desorbierter Substanzen zugunsten eines erhöhten Recyclings verschiebt. Dies wirft die Frage auf, ob Recycling von intakten Metaboliten, d.h. unter Erhalt des Kohlenstoffgerüstes, generell ein bisher stark unterschätzter Prozess ist, der die relativ hohen 14C Alter der OBS teilweise erklären kann. Nach Applikation hoher Toxindosen konnte nachgewiesen werden, dass die nachfolgende Reetablierung der mikrobiellen Gemeinschaft zu großem Anteil auf Recycling der Nekromasskomponenten der Vorgeneration basiert. Intaktes Metabolitrecycling ist jedoch unter steady-state Bedingungen nur äußerst schwierig von der direkten Stabiliserung zu unterscheiden. Um diesen Prozess im Fließgleichgewicht nachzuweisen muss 1) ein Biomolekül untersucht werden, dessen Biosyntheseweg so aufwändig ist, dass Recycling einen deutlichen Vorteil für die Zelle im Vergleich zur Neusynthese darstellt, 2) dieses Biomolkül in lebenden Zellen in einer anderen Form gebunden sein, als die zu recycelnde Einheit in der Bodenlösung, so dass beide Zustandsformen unterschieden werden können und 3) dieses Biomolekül positionsspezifisch isotopenmarkiert zugegeben werden, so dass über einen identischen Einbau der Positionen die Intaktheit des Kohlenstoffgerüstes nachgewiesen werden kann. Am Beispiel der Alkylketten von Fettsäuren, die in mikrobiellen Zellen primär als Phospholipide in den Membranen gebunden sind, soll dieses Prinzip veranschaulicht werden. Eine erste Abschätzung des intakten Recyclings von Alkylketten durch Mikroorganismen in Böden zeigt, dass von den 0.03% der basierend auf Alkyl-Kohlenstoff neugebildeten PLFA mehr als 75% aus intaktem Recycling dieser Ketten hervor gingen. Obwohl der Beitrag des Recyclings intakter Metabolite zur Umsatzzeit der gesamten OBS aufgrund der geringen Anzahl bisher untersuchter Metabolite noch nicht final quantifiziert werden kann, untermauern die hier vorgestellten Ergebnisse jedoch die hohe Relevanz dieses Prozesses für die Dynamik der OBS