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.

中文翻译：

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比较改良基质诱导呼吸与选择性抑制（SIRIN）和N ₂ O同位素方法，以评估缺氧条件下三种耕作土壤中真菌对反硝化的贡献

摘要。纯培养研究提供了土壤真菌在反硝化过程中产生一氧化二氮（N ₂ O）的能力的证据。具有选择性抑制作用的土壤研究表明，与土壤中细菌N ₂ O的产生相比，真菌可能占主导地位，这引起了真菌反硝化的更多关注。分析N ₂ O的同位素组成，特别是产生的N ₂ O（SPN ₂ O）的¹⁵ N位点偏爱，表明纯细菌或真菌培养物的N ₂ O SPN ₂ O值不同，这可能使基于N的同位素末端成员特征的真菌N ₂ O₂ Ó通过真菌和细菌中产生。这项研究的目的是通过使用不同的方法分解N ₂ O的来源来确定在厌氧条件下培养的重装土壤样品中真菌对N ₂ O排放的贡献。在三种厌氧条件下对三种土壤进行了培养，以四种改良的基质处理来促进反硝化作用选择性抑制（SIRIN）诱导呼吸。尽管没有微生物抑制作用的一种治疗方法作为对照，但其他三种治疗方法都经过了抑制剂的改良，以选择性抑制细菌，真菌或细菌和真菌的生长。这些处理分为三个品种。在一个品种中，使用¹⁵ N示踪技术来估算N的影响₂上N 2 O还原₂ O产生，而另外两个品种的天然同位素的条件下，但使用和不使用乙炔进行。建立了三种方法来估计土壤中真菌群落产生的N ₂ O：i）使用SIRIN方法的一种改进方法来计算从选定生物体中释放出来的N2O，并且ii）使用乙酰化处理中的SPN ₂ O值在同位素端元混合方法（IEM），以及iii）SP /δ ¹⁸ O映射方法（SP /δ ¹⁸ O映射）来估计到N真菌贡献₂ ö生产和N ₂非乙酰化处理在厌氧条件下降低了O含量。测试的三种方法表明，在厌氧条件下，测试土壤中的真菌对N ₂ O通量的贡献很小。仅可在一种土壤中用改良的SIRIN定量真菌级分，总计0.28±0.09。这比通过IEM和SP /δ中获得的结果高¹⁸ O映射，占零的N 0.20 ₂产生的真菌社区O操作。据我们所知，这项研究是量化厌氧n中的真菌贡献的第一次尝试₂通过三种方法同时应用Ø生产，即修改丝琳，IEM和SP /δ ¹⁸O地图。尽管所有方法都暗示了真菌的贡献很小或缺失，但需要在确保更大的真菌N ₂ O通量并包括替代抑制剂的条件下进行进一步研究，以更好地交叉验证方法。