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

Compound-specific isotope analysis of amino acids as a new tool to uncover trophic chains in soil food webs

Food webs in soil differ fundamentally from those aboveground; they are based on inputs from both living plants via root exudates, and from detritus, which is a complex mixture of fungi, bacteria, and dead plant remains. Trophic relationships are difficult to disentangle due to the cryptic lifestyle of soil animals and inevitable microbial contributions to their diet. Compound‐specific isotope analysis of amino acids (AAs) is increasingly used to explore complex food webs. The combined use of AA δ^(13)C and δ^(15)N values is a promising new approach to disentangle trophic relationships since it provides independent but complementary information on basal resources, as well as the trophic position of consumers. We conducted a controlled feeding study in which we reconstructed trophic chains from main basal resources (bacteria, fungi, plants) to primary consumers (springtails, oribatid mites) and predators (gamasid mites, spiders). We analyzed dual compound‐specific isotope AA values of both resources and consumers. By applying an approach termed “stable isotope (^(13)C) fingerprinting” we identified basal resources, and concomitantly calculated trophic positions using ^(15)N values of trophic and source AAs in consumers. In the ^(13)C fingerprinting analysis, consumers in general grouped close to their basal resources. However, higher than usual offsets in AA δ^(13)C between diet and consumers suggest either gut microbial supplementation or the utilization of specific resource fractions. Identification of trophic position crucially depends on correct estimates of the trophic discrimination factor (TDF_(Glu‐Phe)), which was close to the commonly applied value of 7.6‰ in primary consumers feeding on microbial resources, but considerably lower in arachnid predators (~2.4‰), presumably due to higher diet quality, excretion of guanine, and fluid feeding. While our feeding study demonstrates that dual compound‐specific AA analyses hold great promise in delineating trophic linkages among soil‐dwelling consumers and their resources, it also highlights that a “one‐size‐fits‐all” approach to TDF_(Glu‐Phe) does not apply to soil food webs

Determination of fungal activity in modified wood by means of micro-calorimetry and determination of total esterase activity

Beech and pine wood blocks were treated with 1,3-dimethylol-4,5-dihydroxyethylen urea (DMDHEU) to increasing weight percent gains (WPG). The resistance of the treated specimens against Trametes versicolor and Coniophora puteana, determined as mass loss, increased with increasing WPG of DMDHEU. Metabolic activity of the fungi in the wood blocks was assessed as total esterase activity (TEA) based on the hydrolysis of fluorescein diacetate and as heat or energy production determined by isothermal micro-calorimetry. Both methods revealed that the fungal activity was related with the WPG and the mass loss caused by the fungi. Still, fungal activity was detected even in wood blocks of the highest WPG and showed that the treatment was not toxic to the fungi. Energy production showed a higher consistency with the mass loss after decay than TEA; higher mass loss was more stringently reflected by higher heat production rate. Heat production did not proceed linearly, possibly due to the inhibition of fungal activity by an excess of carbon dioxide