Man-Young Jung^1,2, Joo-Han Gwak¹, Lena Rohe³, Anette Giesemann⁴, Jong-Geol Kim¹, Reinhard Well⁴, Eugene L. Madsen⁵, Craig W. Herbold^2,6, Michael Wagner^2,6,7, Sung-Keun Rhee^1,*

¹ Department of Microbiology, Chungbuk National University, 1 Chungdae-ro, Seowon-Gu, Cheongju 28644, South Korea
² University of Vienna, Centre for Microbiology and Environmental Systems Science, Division of Microbial Ecology, Althanstrasse 14, A-1090 Vienna, Austria
³ Helmholtz Centre for Environmental Research-UFZ, Department of Soil System Sciences, Theodor-Lieser-Strasse 4, D-06120, Halle (Saale), Germany
⁴ Thünen Institute of Climate-Smart Agriculture, Bundesallee 50, D-38116 Braunschweig, Germany
⁵ Department of Microbiology, Cornell University, Ithaca, NY14853-8101, USA
⁶ The Comammox Research Platform, University of Vienna, Althanstrasse 14, A-1090 Vienna, Austria
⁷ Department of Biotechnology, Chemistry and Bioscience, Aalborg University, Fredrik Bajers Vej 7H, 9220 Aalborg, Denmark

^*Correspondence to Sung-Keun Rhee

Nitrous oxide (N₂O) is a key climate change gas and nitrifying microbes living in terrestrial ecosystems contribute significantly to its formation. Many soils are acidic and global change will cause acidification of aquatic and terrestrial ecosystems, but the effect of decreasing pH on N₂O formation by nitrifiers is poorly understood. Here, we used isotope-ratio mass spectrometry to investigate the effect of acidification on production of N₂O by pure cultures of two ammonia-oxidizing archaea (AOA; Nitrosocosmicus oleophilus and Nitrosotenuis chungbukensis) and an ammonia-oxidizing bacterium (AOB; Nitrosomonas europaea). For all three strains acidification led to increased emission of N₂O. However, changes of 15N site preference (SP) values within the N₂O molecule (as indicators of pathways for N₂O formation), caused by decreasing pH, were highly different between the tested AOA and AOB. While acidification decreased the SP value in the AOB strain, SP values increased to a maximum value of 29‰ in N. oleophilus. In addition, 15N-nitrite tracer experiments showed that acidification boosted nitrite transformation into N₂O in all strains, but the incorporation rate was different for each ammonia oxidizer. Unexpectedly, for N. oleophilus more than 50% of the N₂O produced at pH 5.5 had both nitrogen atoms from nitrite and we demonstrated that under these conditions expression of a putative cytochrome P450 NO reductase is strongly upregulated. Collectively, our results indicate that N. oleophilus might be able to enzymatically denitrify nitrite to N₂O at low pH.