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

Abstract. Pure culture studies provide evidence of the ability of soil fungi to produce nitrous oxide (N2O) during denitrification. Soil studies with selective inhibition indicated a possible dominance of fungal compared to bacterial N2O production in soil, which drew more attention to fungal denitrification. Analyzing the isotopic composition of N2O, especially the 15N site preference of N2O produced (SPN2O), showed that N2O of pure bacterial or fungal cultures differed in SPN2O values, which might enable the quantification of fungal N2O based on the isotopic endmember signatures of N2O produced by fungi and bacteria. This study aimed to identify the fungal contribution to N2O emissions under anaerobic conditions in incubated repacked soil samples by using different approaches to disentangle sources of N2O. Three soils were incubated under anaerobic conditions to promote denitrification with four treatments of a modified substrate induced respiration with selective inhibition (SIRIN) approach. 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 varieties. In one variety the 15N tracer technique was used to estimate the effect of N2O reduction on N2O produced, while two other varieties were performed under natural isotopic conditions but with and without acetylene. Three approaches were established to estimate the N2O production by a fungal community in soil: i) A modification of the SIRIN approach was used to calculate N2O evolved from selected organism groups, and ii) SPN2O values from the acetylated treatment were used in the isotope endmember mixing approach (IEM), and iii) the SP/δ18O mapping approach (SP/δ18O Map) was used to estimate the fungal contribution to N2O production and N2O reduction under anaerobic conditions from the non-acetylated treatment. The three approaches tested revealed a small fungal contribution to N2O fluxes under anaerobic conditions in the soils tested. Quantifying the fungal fraction with modified SIRIN was only possible in one soil and totaled 0.28 ± 0.09. This was higher than the results obtained by IEM and SP/δ18O Map, which accounted zero to 0.20 of N2O produced to the fungal community. To our knowledge, this study was the first attempt to quantify the fungal contribution to anaerobic N2O production by simultaneous application of three approaches, i.e. modified SIRIN, IEM and SP/δ18O Map. While all methods coincided by suggesting a small or missing fungal contribution, further studies under conditions ensuring larger fungal N2O fluxes and including alternative inhibitors are needed to better cross-validate the methods.