Habitats favouring denitrification are those with fluctuating O2 concentrations. In these settings, carbon sources may be limited but still available, and NO3− is generated by nitrification during oxygenated periods or supplied through fertilization. The prevailing understanding is that the ability to denitrify provides a fitness advantage by securing survival and growth when confronted with anoxic conditions [33]. It therefore lies close at hand to infer that microbial communities adapted to regular shifts between O2-rich and deprived conditions will develop an ‘efficient’ denitrification process with minimal transient production of intermediates. The present study does not support this assumption. Instead, and contrary to our hypothesis, it was the Ox treatment that showed the most efficient denitrification process during the final anoxic incubation, seen through its faster denitrification rate and lower accumulation of NO2−, NO and N2O compared to both SA and LA (Fig. 3, Table 1, and Supplementary Fig. 3b). Our experiment is based on one agricultural soil, cautioning against drawing extensive conclusions. Nonetheless, the results underscore that our current understanding of the fitness value of denitrification is incomplete. Importantly, our findings are supported by a study of complex denitrifying communities in seawater [34], which showed that samples collected from the oxic layer consumed N2O under anoxic conditions at a faster rate than samples collected from anoxic depths at the same station.
The rapid denitrification in Ox compared to LA and SA could theoretically be due to a greater biomass of denitrifiers, but this was apparently not the case. The total microbial biomass reached similar levels across all three legacy treatments (Fig. 4) and the denitrification gene abundances were also similar (Fig. 5a). This may appear counterintuitive since the growth yield (per mol C) is greater in aerobic than in anaerobic respiration [9]. However, the soils in SA were kept primarily under oxic conditions throughout the O2 legacy establishment phase, while soils in LA were kept oxic for over 50% of this time (Fig. 1). This indicates that most growth occurred via aerobic respiration in all three legacy treatments during the establishment phase.
The metatranscriptomic analysis also failed to provide a clearcut explanation for the ‘efficient’ denitrification in the Ox treatment, as the denitrification transcripts were generally more abundant in LA than in the other treatments (Fig. 5c, Supplementary Table 3). The higher abundance of NAR genes (particularly narG) and transcripts relative to NIR in both LA and SA during the final anoxic incubation (Fig. 5) may have caused the initially greater accumulation of NO2− (Supplementary Fig. 3) and subsequently a higher transient net production of NO and N2O (Fig. 3) compared to Ox. It has been suggested that NO2− accumulation impairs N2O reduction both in soils and wastewater treatments [35, 36], yet the exact mechanism has not been identified.
A notable difference between the treatments was that Ox had the greatest level of nosZ II transcription (Fig. 5c; 6b) and the lowest IN2O (Table 1). This may be attributed to a selection process during the establishment phase that favoured bacteria that thrived on clover, used O2 as an electron acceptor, and had a general propensity towards the nosZ II gene type. Studies have demonstrated that a pre-existing history of oxic conditions increased nosZ II transcription at the onset of anoxia [37, 38], and others have suggested that nosZ II has an enhanced affinity for N2O compared to nosZ I [39]. Collectively, these characteristics may explain the rapid N2O consumption in Ox during the final anoxic incubation.
The nosZ clade II gene is found in a wide range of taxonomically diverse organisms, many of which have truncated denitrification pathways or are classified as ‘non-denitrifier’ N2O reducers [40]. Conversely, the nosZ clade I gene is more commonly found in canonical denitrifiers, many of which belong to the Proteobacteria and carry other denitrification genes such as nirS [6, 40, 41]. In our study, the differences between the nosZ clades align with the slightly greater diversity and increased activity of Bacteroidetes in Ox and the increased transcription of nirS and activity of Proteobacteria in LA (Fig. 5, 7, and Supplementary Fig. 5). This implies that LA favoured active canonical denitrifiers, while Ox favoured active truncated denitrifiers that performed denitrification in a modular fashion with each reductase providing selectable benefits independent of the others [6, 7]. These findings highlight the uncertainty surrounding any selective advantage of being a complete denitrifier in a complex denitrifying community and suggest that the ‘the sharing of work’ (partnering) between organisms may result in a more efficient biogeochemical process, emphasizing the need for more research into this understudied field of microbial ecology.
A comparison of LA and SA during the final anoxic incubation shows, in agreement with our hypothesis, that antecedent soil conditions of long anoxic pulses led to a lower accumulation of NO and N2O (Table 1) and an earlier completion of denitrification (Fig. 3) compared to antecedent conditions of short anoxic pulses. These functional differences in denitrification progression were not due to differences in the abundance of denitrification genes (Fig. 5a) or in initial VNIR, VNOR, and VNOS (Table 1). Transcription of the denitrification genes was, however, generally higher in LA than in SA during the first 2 h of the final anoxic incubation (Fig. 5c, Supplementary Table 3), indicating that antecedent soil conditions characterized by brief anoxic pulses favoured organisms employing distinct transcriptional control mechanisms. This could be due to low transcription rates of all cells or some sort of bet-hedging, where only a fraction of the cells in SA transcribed the denitrification genes as a strategy to conserve energy if O2 were to return [16, 18]. If so, the initial denitrification rates in SA being equal to those in LA (Table 1) may be explained by the activity of denitrification reductases that were produced during earlier anoxic events. In contrast, LA favoured organisms which synthesized a full denitrification proteome at the onset of anoxia. The metabolic cost was rewarded in terms of a more rapid denitrification progression and a lower transient production of intermediates. This points to the succession of a fluctuation-adapted denitrifier community in LA that was strongly dependent on the length of the oxic-anoxic cycles, similar to what was suggested by Pett-Ridge and Firestone [42].
Interestingly, despite greater transcription of NIR genes in LA compared to Ox during the final anoxic incubation (Fig. 5c, Supplementary Table 3), the initial VNIR was significantly lower in LA (Table 1). One tentative explanation for this inconsistency is the potential O2-induced damage to NIR during the establishment phase in LA, possibly in particular to nirS, which has been shown to be irreversibly damaged by O2 [25]. As the final anoxic phase commenced, the relative rate of NO2- reduction would be low in LA due to O2 damage, resulting in NO2- accumulation. In contrast, Ox did not produce NIR during the establishment phase, thus avoiding oxidative damage and utilizing only ‘freshly’ synthesized NIR during the final anoxic phase. This would explain the lower NO2- accumulation in Ox compared to LA and SA during the final incubation (Supplementary Fig. 3b).
During the O2 legacy establishment phase, LA exhibited a progressively more efficient denitrification phenotype through repeated exposure to denitrifying conditions, as evidenced by an increasing denitrification rate and the concurrent decrease in N2O index and maximum NO concentration with each successive oxic-anoxic cycle (Fig. 2; Supplementary Fig. 1c, d). One reason could be that frequent switches between oxic and anoxic conditions enhanced denitrification at high O2 concentrations as a result of ‘aerobic denitrification’ [43]. While we have reservations about this term, we acknowledge the potential for detectable denitrification in the presence of O2 in two scenarios: (1) denitrification that occurs in anoxic microsites of a seemingly oxic system [44]; or (2) the co-respiration of O2 and N-oxides within the same cell when electron flow to terminal oxidases is restricted by low O2 concentrations [26], provided there is no O2-induced damage to the denitrification reductases. However, it is uncertain whether any denitrification took place in LA during the oxic periods of the establishment phase. If reductases were active in both anoxic and oxic states, our results suggest that this would likely occur during shorter periods of oxygenation amidst longer cycles of anoxia, as this cyclic pattern would minimize exposure of reductases to O2-related damage.
In the LA treatment during the final anoxic incubation, the gas kinetics showed a slowed denitrification phenotype and increased accumulation of intermediates compared to Cycle 11 of the establishment phase (Fig. 3 and Supplementary Fig. 1). This was unexpected, considering the gradual shift in denitrification rates and N2O indices with each successive anoxic-oxic cycle (Fig. 2). One possibility could be that at the start of the final anoxic incubation, the soil was amended with double the amount of NO3− (4 mM vs. 2 mM), and of note, the community struggled to control NO concentrations (Supplementary Fig. 1c, d). A similar disturbance causing accumulation of NO and N2O in soils with an intact denitrification proteome was observed by Highton et al. [45], in that case by adding NO2−. This suggests that the control of denitrification is easily influenced by sudden rises in NO3− levels, making it susceptible to disruptions.
The specific type of active denitrifiers favoured by SA remains unclear. The transcriptional behavior during the final anoxic incubation was similar to that of Ox, as evidenced by the close clustering of their denitrification MT profiles (Fig. 5d). Yet, SA was genotypically and phenotypically more similar to LA, subsequently sharing close clustering of denitrification MG profiles (Fig. 5b) and exhibiting slowed denitrification kinetics during the final anoxic spell (Fig. 3). A recent study pointed to the role of ROS in the inhibition of N2O reduction in soils and sediments during the transition from anoxic to oxic conditions [46]; however, the absence of a clear legacy treatment effect on the abundance of ROS-scavenging genes and transcripts across treatments (Supplementary Fig. 8) suggests that oxidative stress did not play a significant role in shaping the denitrifying community in the SA treatment. Future approaches should prioritize metaproteomic analysis to provide a more holistic understanding of functional changes in response to fluctuating O2 conditions.
Our study supports the hypothesis that antecedent conditions of long anoxic pulses resulted in a faster denitrification phenotype at the onset of anoxia compared to a history of short anoxic pulses. However, this adaptation was easily disturbed by sudden rises in NO3−. Unexpectedly, a history of constant oxic soil conditions gave rise to the fastest denitrification phenotype at the onset of anoxia and favoured a denitrifier community dominated by nosZ clade II-bearing partial or non-denitrifiers, suggesting efficient partnering of the reduction steps among organisms. Overall, our study underscores the necessity for further investigations into the interactions among organisms involved in denitrification and highlights that knowing the O2 legacy of a complex environment is crucial for accurately predicting N2O emissions arising from denitrification.