Gas kinetics and stoichiometry from four fungi incubated under conditions favouring denitrification
Authors: Rohe Lena , Well Reinhard , Nadeem Shahid , Dörsch Peter 
Published: March 2022

&lt;p&gt;Even though the ability of fungi to produce the greenhouse gas nitrous oxide (N&lt;sub&gt;2&lt;/sub&gt;O) during denitrification has been demonstrated, the proportion N&lt;sub&gt;2&lt;/sub&gt;O emissions from fungal denitrification in soils cannot yet be determined or predicted. In order to develop methods for estimating the fungal proportion, N&lt;sub&gt;2&lt;/sub&gt;O must be partitioned to bacterial and fungal denitrification. The denitrification regulatory phenotype (DRP) is well described for a number of bacterial strains (Bergaust et al. 2010, Bergaust et al. 2011), but to our knowledge there are only few data relating to the fungal DRP in terms of oxygen (O&lt;sub&gt;2&lt;/sub&gt;) tension in fully stirred cultures at which they start producing N&lt;sub&gt;2&lt;/sub&gt;O. The aim of this study was to analyse the kinetics of fungal denitrification combined with analysis of the isotopic composition of N&lt;sub&gt;2&lt;/sub&gt;O. In particular, the &lt;sup&gt;15&lt;/sup&gt;N site preference of N&lt;sub&gt;2&lt;/sub&gt;O (SP-N&lt;sub&gt;2&lt;/sub&gt;O) is known to be a promising tool to differentiate between N&lt;sub&gt;2&lt;/sub&gt;O produced during bacterial and fungal denitrification.&lt;/p&gt;&lt;p&gt;Four fungal species (&lt;em&gt;Fusarium oxysporum&lt;/em&gt;, &lt;em&gt;Fusarium decemcellulare&lt;/em&gt;, &lt;em&gt;Fusarium solani&lt;/em&gt; fsp. &lt;em&gt;pisi &lt;/em&gt;and &lt;em&gt;Chaetomium funicola&lt;/em&gt;) were incubated as batch cultures in a robotized incubation system (Molstad et al. 2007) for 165h. Batch cultures were incubated in 120 ml flasks containing 50 ml of growth medium amended with ample amounts of carbon and nitrate in a He atmosphere with 2 vol%O&lt;sub&gt;2&lt;/sub&gt;. To test for pH effects, a complex medium (Shoun et al. 1992) with pH values adjusted to 6.9 and 7.4 as well a minimal medium (Dox 1910) with a pH value of about 7.9 were used. O&lt;sub&gt;2&lt;/sub&gt; consumption and production of nitric oxide (NO), N&lt;sub&gt;2&lt;/sub&gt;O, dinitrogen (N&lt;sub&gt;2&lt;/sub&gt;) and carbon dioxide (CO&lt;sub&gt;2&lt;/sub&gt;) were monitored at high temporal resolution while isotopic composition of N&lt;sub&gt;2&lt;/sub&gt;O was analysed in samples taken manually at selected time points.&lt;/p&gt;&lt;p&gt;All four fungal cultures quickly consumed O&lt;sub&gt;2&lt;/sub&gt;. NO production increased strongly before O&lt;sub&gt;2&lt;/sub&gt; was completely consumed and was followed by immediate N&lt;sub&gt;2&lt;/sub&gt;O production. The kinetics of N&lt;sub&gt;2&lt;/sub&gt;O production differed to published kinetics of denitrifying prokaryotes by showing a lower sensitivity to O&lt;sub&gt;2&lt;/sub&gt;. This could result in a larger share of fungal denitrification under microaerobic conditions in soil.&lt;/p&gt;&lt;p&gt;Isotopic analysis of N&lt;sub&gt;2&lt;/sub&gt;O confirmed previous results of specifically high SP-N&lt;sub&gt;2&lt;/sub&gt;O values of fungal produced N&lt;sub&gt;2&lt;/sub&gt;O. We further showed that SP-N&lt;sub&gt;2&lt;/sub&gt;O values of fungal N&lt;sub&gt;2&lt;/sub&gt;O are quite stable and do not depend on denitrification kinetics. Likewise, incubation conditions such as pH of the medium had little impact on SP-N&lt;sub&gt;2&lt;/sub&gt;O values. These findings support the usage of SP-N&lt;sub&gt;2&lt;/sub&gt;O values for partitioning N&lt;sub&gt;2&lt;/sub&gt;O soil fluxes and provide a tool to study the biology of fungal denitrification under field conditions, which is needed to develop mitigation strategies of N&lt;sub&gt;2&lt;/sub&gt;O from fungal denitrification.&lt;/p&gt;&lt;p&gt;References:&lt;/p&gt;&lt;ul&gt;&lt;li&gt;Bergaust, Y. Mao, L. R. Bakken, &amp;#197;. Frosteg&amp;#229;rd, Appl Environ Microbiol 2010, 76.&lt;/li&gt;
&lt;li&gt;Bergaust, L. R. Bakken, &amp;#197;. Frosteg&amp;#229;rd, Biochem Soc Trans 2011, 39.&lt;/li&gt;
&lt;li&gt;Molstad, P. D&amp;#246;rsch, L. R. Bakken, J Microbiol Methods 2007, 71, 202.&lt;/li&gt;
&lt;li&gt;Shoun, D.-H. Kim, H. Uchiyama, J. Sugiyama, FEMS Microbiol. Lett. 1992, 94, 277.&lt;/li&gt;
&lt;li&gt;W. Dox, U.S. Dept. of Agriculture, Bureau of Animal Industry, Washington, D.C., 1910, 70 p.&lt;/li&gt;
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