Comparing modified substrate-induced respiration with selective inhibition (SIRIN) and N<sub>2</sub>O isotope approaches to estimate fungal contribution to denitrification in three arable soils under anoxic conditions

Abstract

Abstract. The coexistence of many N2O production pathways in soil hampers differentiation of microbial pathways. The question of whether fungi are significant contributors to soil emissions of the greenhouse gas nitrous oxide (N2O) from denitrification has not yet been resolved. Here, three approaches to independently investigate the fungal fraction contributing to N2O from denitrification were used simultaneously for, as far as we know, the first time (modified substrate-induced respiration with selective inhibition (SIRIN) approach and two isotopic approaches, i.e. end-member mixing approach (IEM) using the 15N site preference of N2O produced (SPN2O) and the SP/δ18O mapping approach (SP/δ18O Map)). This enabled a comparison of methods and a quantification of the importance of fungal denitrification in soil. Three soils were incubated in four treatments of the SIRIN approach under anaerobic conditions to promote denitrification. While one treatment without microbial inhibition served as a control, the other three treatments were amended with inhibitors to selectively inhibit bacterial, fungal, or bacterial and fungal growth. These treatments were performed in three variants. In one variant, the 15N tracer technique was used to estimate the effect of N2O reduction on the N2O produced, while two other variants were performed under natural isotopic conditions with and without acetylene. All three approaches revealed a small contribution of fungal denitrification to N2O fluxes (fFD) under anaerobic conditions in the soils tested. Quantifying the fungal fraction with modified SIRIN was not successful due to large amounts of uninhibited N2O production. In only one soil could fFD be estimated using modified SIRIN, and this resulted in 28 ± 9 %, which was possibly an overestimation, since results obtained by IEM and SP/δ18O Map for this soil resulted in fFD of below 15 % and 20 %, respectively. As a consequence of the unsuccessful SIRIN approach, estimation of fungal SPN2O values was impossible. While all successful methods consistently suggested a small or missing fungal contribution, further studies with stimulated fungal N2O fluxes by adding fungal C substrates and an improved modified SIRIN approach, including alternative inhibitors, are needed to better cross-validate the methods.

Increased Nitrogen Loading Facilitates Nitrous Oxide Production through Fungal and Chemodenitrification in Estuarine and Coastal Sediments

“…Fungal contribution to N 2 O production was 54–66% under high NO 3 – concentration treatments, which was in line with the result from isotope model (48–63%), suggesting that fungal denitrification dominated N 2 O production in the sediments with high NO 3 – concentration . Therefore, the contribution of N 2 O production pathways is highly dependent on environmental conditions and NO 3 – concentrations. − Fungal contribution to N 2 O production from inhibition approaches can be overestimated (28% with inhibition approach vs ≤15% with isotope model) due to incomplete inhibition . Our isotope approaches demonstrated 2.2–4.8% of bacterial N 2 O production in the STP group and 5.3–7.8% of fungal N 2 O production in the CYH group (Table S5), suggesting that the inhibition may be not complete.…”

“…57−59 Fungal contribution to N 2 O production from inhibition approaches can be overestimated (28% with inhibition approach vs ≤15% with isotope model) due to incomplete inhibition. 58 Our isotope approaches demonstrated 2.2−4.8% of bacterial N 2 O production in the STP group and 5.3−7.8% of fungal N 2 O production in the CYH group (Table S5), suggesting that the inhibition may be not complete. The reason may be that the inhibitors only deactivate microorganisms in the growing phase, whereas minor N 2 O can be produced from the processes that have been theoretically inhibited.…”

“…Our isotope approaches demonstrated 2.2–4.8% of bacterial N 2 O production in the STP group and 5.3–7.8% of fungal N 2 O production in the CYH group (Table S5), suggesting that the inhibition may be not complete. The reason may be that the inhibitors only deactivate microorganisms in the growing phase, whereas minor N 2 O can be produced from the processes that have been theoretically inhibited . N 2 O production in the CK group may be not equal to the sum of STP + CYH + (STP+CYH) groups as we could not determine how much N 2 O was reduced (simultaneous occurrence of N 2 O production and consumption) and how the microbial inhibitors affect N 2 O reductase .…”

“…The reason may be that the inhibitors only deactivate microorganisms in the growing phase, whereas minor N 2 O can be produced from the processes that have been theoretically inhibited . N 2 O production in the CK group may be not equal to the sum of STP + CYH + (STP+CYH) groups as we could not determine how much N 2 O was reduced (simultaneous occurrence of N 2 O production and consumption) and how the microbial inhibitors affect N 2 O reductase . Therefore, improved inhibitor approaches or molecular-based methods are required to cross-validate N 2 O production pathways and associated relative contribution in further studies.…”

“…Technol. XXXX, XXX, XXX−XXX 2 O production pathways is highly dependent on environmental conditions and NO 3 − concentrations.57−59 Fungal contribution to N 2 O production from inhibition approaches can be overestimated (28% with inhibition approach vs ≤15% with isotope model) due to incomplete inhibition 58. Our isotope approaches demonstrated 2.2−4.8% of bacterial N 2 O production in the STP group and 5.3−7.8% of fungal N 2 O production in the CYH group (Table…”