No abstract

Stable isotope analysis of oxygen (O) is increasingly used to determine the origin of nitrate (NO(3)-) and nitrous oxide (N(2)O) in the environment. The assumption underlying these studies is that the (18)O signature of NO(3)- and N(2)O provides information on the different O sources (O(2) and H(2)O) during the production of these compounds by various biochemical pathways. However, exchange of O atoms between H(2)O and intermediates of the (de)nitrification pathways may change the isotopic signal and thereby bias its interpretation for source determination. Chemical exchange of O between H(2)O and various nitrogenous oxides has been reported, but the probability and extent of its occurrence in terrestrial ecosystems remain unclear. Biochemical O exchange between H(2)O and nitrogenous oxides, NO(2)- in particular, has been reported for monocultures of many nitrifiers and denitrifiers that are abundant in nature, with exchange rates of up to 100%. Therefore, biochemical O exchange is likely to be important in most soil ecosystems, and should be taken into account in source determination studies. Failing to do so might lead to (i) an overestimation of nitrification as NO(3)- source, and (ii) an overestimation of nitrifier denitrification and nitrification-coupled denitrification as N(2)O production pathways. A method to quantify the rate and controls of biochemical O exchange in ecosystems is needed, and we argue this can only be done reliably with artificially enriched (18)O compounds. We conclude that in N source determination studies, the O isotopic signature of especially N(2)O should only be used with extreme caution.

To effectively mitigate emissions of the greenhouse gas nitrous oxide (N(2)O) it is essential to understand the biochemical pathways by which it is produced. The (18)O signature of N(2)O is increasingly used to characterize these processes. However, assumptions on the origin of the O atom and resultant isotopic composition of N(2)O that are based on reaction stoichiometry may be questioned. In particular, our deficient knowledge on O exchange between H(2)O and nitrogen oxides during N(2)O production complicates the interpretation of the (18)O signature of N(2)O.Here we studied O exchange during N(2)O formation in soil, using a novel combination of (18)O and (15)N tracing. Twelve soils were studied, covering soil and land-use variability across Europe. All soils demonstrated the significant presence of O exchange, as incorporation of O from (18)O-enriched H(2)O into N(2)O exceeded their maxima achievable through reaction stoichiometry. Based on the retention of the enrichment ratio of (18)O and (15)N of NO(3)(-) into N(2)O, we quantified O exchange during denitrification. Up to 97% (median 85%) of the N(2)O-O originated from H(2)O instead of from the denitrification substrate NO(3)(-).We conclude that in soil, the main source of atmospheric N(2)O, the (18)O signature of N(2)O is mainly determined by H(2)O due to O exchange between nitrogen oxides and H(2)O. This also challenges the assumption that the O of N(2)O originates from O(2) and NO(3)(-), in ratios reflecting reaction stoichiometry.

Methane is an important greenhouse gas and the most abundant hydrocarbon in the Earth's atmosphere. Methanotrophic microorganisms can use methane as their sole energy source and play a crucial role in the mitigation of methane emissions in the environment. "Candidatus Methylomirabilis oxyfera" is a recently described intra-aerobic methanotroph that is assumed to use nitric oxide to generate internal oxygen to oxidize methane via the conventional aerobic pathway, including the monooxygenase reaction. Previous genome analysis has suggested that, like the verrucomicrobial methanotrophs, "Ca. Methylomirabilis oxyfera" encodes and transcribes genes for the Calvin-Benson-Bassham (CBB) cycle for carbon assimilation. Here we provide multiple independent lines of evidence for autotrophic carbon dioxide fixation by "Ca. Methylomirabilis oxyfera" via the CBB cycle. The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO), a key enzyme of the CBB cycle, in cell extracts from an "Ca. Methylomirabilis oxyfera" enrichment culture was shown to account for up to 10% of the total methane oxidation activity. Labeling studies with whole cells in batch incubations supplied with either 13 CH 4 or [ 13 C]bicarbonate revealed that "Ca. Methylomirabilis oxyfera" biomass and lipids became significantly more enriched in 13 C after incubation with 13 C-labeled bicarbonate (and unlabeled methane) than after incubation with 13 C-labeled methane (and unlabeled bicarbonate), providing evidence for autotrophic carbon dioxide fixation. Besides this experimental approach, detailed genomic and transcriptomic analysis demonstrated an operational CBB cycle in "Ca. Methylomirabilis oxyfera." Altogether, these results show that the CBB cycle is active and plays a major role in carbon assimilation by "Ca. Methylomirabilis oxyfera" bacteria. Our results suggest that autotrophy might be more widespread among methanotrophs than was previously assumed and implies that a methanotrophic community in the environment is not necessarily revealed by 13 C-depleted lipids.

Nitrifier denitrification (i.e. nitrite reduction by ammonia oxidizers) is one of the biochemical pathways of nitrous oxide (N 2 O) production. It is increasingly suggested that this pathway may contribute substantially to N 2 O production in soil, the major source of this greenhouse gas. However, although monoculture studies recognize its potential, methodological drawbacks prohibit conclusive proof that nitrifier denitrification occurs in actual soils. Here we suggest and apply a new isotopic approach to identify its presence in soil. In incubation experiments with 12 soils, N 2 O production was studied using oxygen (O) and nitrogen (N) isotope tracing, accounting for O exchange. Microbial biomass C and N and phospholipid fatty acid (PLFA) patterns were analysed to explain potential differences in N 2 O production pathways. We found that in at least five of the soils nitrifier denitrification must have contributed to N 2 O production. Moreover, it may even have been responsible for all NH 4 + -derived N 2 O in most soils. In contrast, N 2 O as a by-product of ammonia oxidation contributed very little to total production. Microbial biomass C and N and PLFA-distinguished microbial community composition were not indicative of differences in N 2 O production pathways. Overall, we show that combined O and N isotope tracing may still provide a powerful tool to understand N 2 O production pathways, provided that O exchange is accounted for. We conclude that nitrifier denitrification can indeed occur in soils, and may in fact be responsible for the greater proportion of total nitrifier-induced N 2 O production.