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.
N2O is an important ozone-depleting substance with a high global warming potential [1,2,3]. The processes of biological N2O production include partial dissimilatory nitrate (NO3−) or nitrite (NO2−) reduction (denitrification), nitrifier denitrification , ammonia [hydroxylamine (NH2OH)] oxidation, and NOx detoxification (also known as the “nitrosative stress” pathway) . AOB are recognized as a major source of N2O production from terrestrial environments  and AOA are also considered to be important contributors to N2O production in various environments, based on their high abundance in many ecosystems and the documented formation of N2O during AOA-mediated ammonia oxidation [5,6,7,8]. Despite the presence of a nitrite reductase gene (nirK) in almost all AOA genomes, canonical nitric oxide reductase (NOR) genes have not been detected in these organisms. Instead, isotope labeling experiments suggested hybrid N2O formation in the AOA Nitrososphaera viennensis from nitrite and an intermediate of ammonia oxidation, and was attributed to either an enzymatically catalyzed (codenitrification) or abiotic N-nitrosation reaction . Under low-oxygen conditions, abiotic formation of N2O from hydroxylamine or NO was observed in experiments with killed N. viennensis biomass in AOA media . Furthermore, it was recently demonstrated that N2O is formed abiotically under aerobic conditions from hydroxylamine and nitrite produced by aerobic ammonia-oxidizing microbes and that this hybrid pathway can account for a large proportion of the aerobically produced N2O . Interestingly, we observed in a previous study for selected AOA strains variable N2O isotopomer SP (enrichment of 15N at the alpha, or beta site of N2O) values (ca. 20–30‰) and a very variable contribution of both N atoms in N2O from nitrite (8.4–53% of the N2O produced by the AOA strains had both N from nitrite) at high initial nitrite concentrations. These results are consistent with the hypothesis that nitrifier denitrification might contribute to N2O production by some AOA in the presence of excess nitrite .
Terrestrial and ocean environments experience acidification due to natural or human activities [11, 12] and a large part (ca. 30%) of the world’s natural and arable soils are acidic (pH < 5.5) . However, most of the studies of N2O production by AOA and AOB have been performed in circumneutral pH ranges only, although various biological and chemical reactions involved in N2O production are pH-dependent . Thus, responses of N2O production by AOA and AOB to acidification need to be better understood to estimate the present and future contribution of soil N2O production to the global N2O budget. The present study was designed to reveal the impact of acidification upon N2O production by two selected AOA strains and by a model strain of AOB. We analyzed N2O production and SP of the N2O molecule under varying pH conditions. In addition, pH-dependent changes of the source of nitrogen of N2O were investigated using 15N-nitrite tracer experiments in order to obtain insights into potential differences of the N2O production pathways between the analyzed strains.
In order to assess the effect of acidification on N2O production, axenic AOA (Nitrosotenuis chungbukensis MY2 of thaumarchaeal group I.1a; Nitrosocosmicus oleophilus MY3 of thaumarchaeal group I.1b) and AOB (Nitrosomonas europaea ATCC 19718) strains were incubated in growth media spanning a pH range of 5.5–8.5 (at intervals of 0.5 pH units). More details on cultivation methods, media, and incubation conditions are described in the Supplementary Materials and Methods. The lowest pH used in these assays was selected after screening the three cultures for retention of ∼20% of the growth rate found at optimal pH. Thus, pHs of 5.5, 6.0, and 6.5 were used as most acidic incubation conditions for N. oleophilus MY3, N. chungbukensis MY2, and for N. europaea ATCC 19718, respectively. For all tested ammonia oxidizers the N2O yield (N2O-N/oxidized NH4+-N) increased as pH decreased (Supplementary Fig. S1). The N2O yield of N. chungbukensis MY2 at low pH was much higher than that of N. oleophilus MY3. The increased N2O production at acidic conditions may be caused by increased production of enzymes involved in N2O production , an acidic pH optimum of N2O-producing enzymes [15, 16], or acceleration of abiotic hybrid N2O formation [8, 17]. In coculture experiments of the AOA N. oleophilus MY3 with the nitrite-oxidizing bacterium (NOB), Nitrobacter winogradskyi Nb-255 nitrite accumulation was not observed during nitrification at pH 7.5 and pH 5.5 (Supplementary Fig. S2). This Nitrobacter strain has no known enzymatic repertoire to produce or oxidize N2O, but encodes a reversible nitrite oxidoreductase that is able to catalyze the oxidation of nitrite to nitrate . Interestingly, for both AOA and AOB at the lowest pH tested, the presence of the NOB in the cultures caused a significant decrease of N2O yields (28% and 48%, respectively) (P < 0.001), suggesting that the accumulation of nitrite in the experiments without addition of NOB contributed to the increased N2O production under these conditions (Supplementary Fig. S1). Thus, it is tempting to speculate that the higher N2O production at lower pH may possibly be connected to an upregulation of nitrite detoxification due to increased formation of the reactive compound nitrous acid (HNO2) from nitrite (NO2−, pKa = 3.39) at lower pH.
Many isotopic studies have documented a positive SP value (around 30‰) that is the characteristic for N2O produced by ammonia oxidizers via the formation of NH2OH , with similar values reported for fungal denitrification  or chemical formation of N2O from hydroxylamine . In contrast, SP values for N2O produced by bacterial heterotrophic denitrification or nitrifier denitrification are both near or below zero . The SP values of N2O produced by the strains in the present study (N. oleophilus MY3, N. chungbukensis MY2, and N. europaea ATCC 19718) at pH 7.5 were 26‰, 29‰, and 28‰, respectively, resembling the signatures of N2O produced mostly via NH2OH formation (Fig. 1a). The SP values of N2O produced by the AOB strain ATCC 19718 decreased dramatically from ~30‰ at pH 8.5 to ~0.5‰ (P < 0.001) at pH 6.5 (Fig. 1a). This shift is consistent with prior observations that increased nitrifier denitrification activity of AOB at decreasing pH  may be associated with removal of HNO2, the toxic form of NO2-. In contrast, for N. chungbukensis MY2 only a slight decrease of SP values of N2O (from 29 to 27‰; P = 0.05) was observed when the pH dropped from pH 7.5 to 6.0 (Fig. 1a). Intriguingly, for N. oleophilus MY3 the SP values of N2O even increased from 26 to 29‰ (P < 0.05), when the pH decreased from 7.5 to 5.5 suggesting no involvement of canonical nitrifier denitrification. Meanwhile, continuous nitrite removal from the N. oleophilus MY3 culture by cocultivation with a NOB had no significant effect on the SP values of N2O at pH 5.5 (Fig. 1a) indicating independence of N2O production mechanisms on the external nitrite concentration at low pH. Our observations of opposing and/or variable trends in SP values of N2O in the different strains as a function of pH indicates that, although N2O yields increased under acidic conditions in the tested AOA and AOB cultures (see Supplementary Fig. S1), the underlying mechanisms responding to acidification likely to differ.
The SP value of the N2O produced by the tested AOA does not exclude production of N2O by a chemical reaction of NO2− with NH2OH during ammonia oxidation , as the SP of N2O abiotically produced from NH2OH and NO2−(or HNO2) at acidic conditions was reported to be ∼34‰ . However, in our abiotic control experiments at pH 5.5 and 7.5, N2O production was much lower in the presence of 500 µM of NO2−with or without addition of 10 and 50 µM of NH2OH compared to corresponding experiments with active AOA and AOB cultures (see Supplementary Table S1 and 2). The NH2OH concentrations in the abiotic controls were selected based on the recently published data that showed maximum concentrations of extracellular NH2OH of <10 µM in AOA, AOB, and comammox strains during oxidation of 2 mM ammonia . In this context it should be noted that previously higher N2O production in abiotic control incubations that we report here has been detected [9, 10]. This difference likely reflects differences in media composition (for example, we used a ~ 10 × lower trace metal concentration than Kozlowski et al.  and Liu et al.  for our biotic and abiotic experiments) and/or incubation conditions (e.g., our abiotic experiments were performed fully aerobic in contrast to Kozlowski et al. ) between studies.
The increase of the N2O yield by both tested AOA (and the increase of the SP value of N2O for N. oleophilus MY3) at lower pH cannot be explained by conventional nitrifier denitrification for HNO2 detoxification as this would be expected to strongly lower the SP value (as observed for N. europaea). To further investigate the underlying processes of N2O production, nitrogen incorporation into N2O produced by the three nitrifiers was traced by using 15N-labeled nitrite. With this setup, most of m/e 44 (14,14N2O) and all of m/e 46 (15,15N2O) is produced by conversion of unlabeled ammonia and 15N-labeled nitrite, respectively (see Fig. 1b, c). Interestingly, the relative contribution of labeled nitrite to N2O production (m/e 45 – one labeled N atom + m/e 46) was increased by acidification in all strains (Fig. 1b).
For N. europaea ATCC 19718, the increased N2O yield and decreased SP at low pH suggests production of N2O via nitrifier denitrification by the combined action of its nitrite reductase and nitric oxide reductase enzymes. Dissolved O2 concentrations in our cultures of AOA and AOB were >95% saturation and those enzymes are also known to be expressed under aerobic conditions [14, 22]. Unexpectedly however, in the 15N-nitrite labeling experiment double-labeled N2O (m/e 46) was not much increased at low pH and instead more hybrid-N2O formation (m/e 45) was observed (Fig. 1b). It is important to keep in mind that N. europaea ATCC 19718 produces much more NO than AOA at circumneutral pH , and the expression of nitrite reductase is further induced by low pH and high nitrite , which will lead to increased NO production from nitrite under these conditions. Furthermore, recent biochemical experiments demonstrated that the hydroxylamine dehydrogenase (HAO) of N. europaea produces NO and not nitrite during ammonia oxidation . Consequently, in our labeling experiment at acidic conditions (unlabeled) 14NO formed from ammonia oxidation by the HAO activity will mix with (labeled) 15NO formed from nitrite via nitrite reductase. Reduction of this partially labeled NO by NorB (or NorSY , or cytochrome c554 ; with possibly different contributions of the different NO reductases with varying pH) will result in hybrid N2O formation (m/e 45). This hypothesis might also explain the results from recent stable isotope labeling experiments with a natural AOB community in a lake that also indicated increased hybrid N2O formation with decreasing pH .
For N. chungbukensis MY2, the nitrite-labeling experiment also suggested an increased contribution of hybrid-N2O formation (m/e 45) at pH 6.5 (Fig. 1b). Lowering the pH might have increased the N2O yield in N. chungbukensis MY2 by increasing chemical hybrid N2O formation from nitrite and hydroxylamine as previously described [10,11,12]. However, this is inconsistent with the data from our abiotic control experiments with nitrite and hydroxylamine that showed a much lower N2O production at pH 6.5 than measured in the corresponding biotic experiment. Thus, our data indicate that in N. chungbukensis MY2 N2O formation at low pH might be catalyzed by an unknown NO-reducing enzyme.
Surprisingly, for N. oleophilus MY3 up to 56.5% of the N2O produced in our labeling experiment had both N from nitrite (15,15N2O) implicating a substantial involvement of an unusual nitrifier denitrification process in N2O formation (Fig. 1b). In this context it is interesting to note that the SP values for N2O produced from fungal denitrification show values of up to 35‰ [19, 20], similar to those observed by us for N. oleophilus MY3 at low pH. The most characteristic feature of the fungal-denitrifying system is the involvement of cytochrome P450, as NOR (P450nor) [20, 26]. The proposed overall mechanisms for the reduction of NO by the enzyme P450nor is [2NO + NAD(P)H + H+ → N2O + H2O + NAD(P)+] and the enzyme accept two electrons directly from NAD(P)H, in contrast to other P450 enzymes (non-NOR type) where the electrons are donated one at a time via redox partners involving flavins and iron–sulfur centers . The fungal denitrifying system seems to lack N2O reductase (NOS) and therefore evolves N2O as the final product . Interestingly, putative cytochrome P450-encoding genes were detected by us in the genomes of N. oleophilus MY3 and some other thaumarchaeal group I.1b members, as well as in some nitrifying bacterial strains (AOB, NOB, and comammox) (Supplementary Fig. S3). The cytochrome P450 of Nitrosocosmicus spp., like the fungal P450nor clade (CYP55_NOR), is related to bacterial members of this enzyme superfamily. However, the fungal P450nor clade and the respective enzyme superfamily members of nitrifying bacteria, Nitrososphaera spp. and Nitrosocosmicus spp. are polyphyletic, and without biochemical data no direct proof of their specific enzymatic activity can be obtained. However, at pH 5.5 (compared to pH 7.5), we observed significantly increased transcription of two cytochrome P450-like genes (MY3_00641 and MY3_01637) of strain N. oleophilus MY3 compared to transcription of housekeeping genes (see Supplementary Table S4 for information on qPCR primers) such as those encoding the 16S rRNA, AmoA, and enzymes required for CO2 fixation (Fig. 2 and Supplementary Fig. S4). N2O yields are significantly increased due to nitrifier denitrification in AOB under low-oxygen conditions [8, 27, 28], which can be higher than those from nitrifier denitrification in N. oleophilus MY3 under aerobic conditions at acidic conditions (Supplementary Table S1). Altogether, this suggests that in N. oleophilus MY3 cytochrome P450 might be possibly involved in the production of N2O via nitrifier denitrification acting as NOR under aerobic conditions at a lower pH, a hypothesis that warrants further experimental investigation. If confirmed, cytochrome P450-catalyzed N2O production in AOA and possibly some other nitrifiers would significantly expand our perception of the metabolic repertoire of these important N-cycle microorganisms and their contribution to global change.
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We would like to thank Kerstin Gilke for assistance with GC measurements, Martina Heuer for assistance in IRMS analysis, and Shurong Liu and Kerim Dimitri Kits for helpful discussions. This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Ministry of Science, ICT & Future Planning (NRF-2015R1A4A1041869, NRF-2015M3D3A1A01064881, and NRF-2018R1A2B6008861), and the National Institute of Biological Resources (NIBR) grant, funded by the Ministry of Environment (MOE) of the Republic of Korea (NIBR201701107). CH and MW were supported by the Comammox Research Platform of the University of Vienna.
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Jung, MY., Gwak, JH., Rohe, L. et al. Indications for enzymatic denitrification to N2O at low pH in an ammonia-oxidizing archaeon. ISME J 13, 2633–2638 (2019). https://doi.org/10.1038/s41396-019-0460-6
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