Dose-dependent effect of soluble ammonium on methane uptake rates
Methane was rapidly consumed within 2–3 days after NH4Cl addition in all the soil slurry incubations (T1–T6) when compared to the reference incubation (T0), where headspace methane decreased by < 50%v/v even after 7 days, indicating that supplemented NH4Cl in AMS stimulated methane uptake rates (Fig. 1a, c). On the other hand, NH4Cl-induced stimulation of methane uptake was dose-dependent in the soil enrichment (Fig. 1b, d), where the stimulatory effect was significant at < 2.50 gL−1 NH4Cl (T4). Although methane uptake was still detected in the 3.25 and 4.75 gL−1 NH4Cl-supplemented incubations (T5 and T6), the uptake rates were comparable to the reference (T0) incubation. Therefore, optimum methane uptake occurred at < 2.50 gL−1 NH4Cl in the soil enrichment incubation, whereas the NH4Cl-induced stimulatory effect in the soil slurry incubation seemingly occurred at all NH4Cl-supplemented concentrations.
Methane uptake could be related particularly to the soluble ammonium concentration in both soil slurry and enrichment incubations. Soluble ammonium was appreciably lower than the total ammonium concentration in all soil slurry incubations after 5 days, indicating biological ammonium uptake and/or adsorption of a large fraction of the ammonium to the soil (Fig. 2). Considering the relatively little change in the initial total ammonium concentration after the incubation (i.e., total ammonium uptake; Fig. 2a), the appreciably lower soluble ammonium concentration detected is more likely caused by a higher adsorption to the soil than microbially mediated ammonium consumption. To additionally assess the contribution of biological ammonium consumption, future studies could employ isotopically labeled ammonium to track ammonium-derived nitrate (i.e., microbially mediated nitrification) after saturating the soil (and hence, adsorption sites) with unlabeled ammonium. It appears that the supplemented ammonium levels did not reflect on the amounts accessible to the microorganisms, where approximately 36–63% of supplemented ammonium (i.e., difference in the total and soluble ammonium fraction relative to the initial supplemented concentration) was adsorbed to the soil (Fig. 2).
In contrast, soluble ammonium concentrations commensurate with the NH4Cl-supplemented amount in the soil enrichment incubations (Fig. 2b). Here, at < 3.25 gL−1 NH4Cl (T5), the mean soluble ammonium concentrations after the incubation were lower or comparable to the initial values and were proportionate to the supplemented concentrations (Fig. 2b). In the soil enrichment supplemented with 4.75 gL−1 NH4Cl (T6), however, ammonium concentration was higher after the incubation, possibly released by lysed cells of organisms adversely affected by the inhibitory ammonium levels. The inhibitory effect was corroborated by the significantly lower methane uptake and methanotrophic abundance when compared to incubations supplemented with lower NH4Cl concentrations (Figs. 1 and 3). Therefore, methane uptake taken together with the methanotrophic abundance and ammonium concentrations after the soil enrichment incubation indicates that supplemented NH4Cl was largely accessible to the methanotrophs and could explain the dose-dependent effect on methanotrophic activity (i.e., stimulation at < 2.5 gL−1 NH4Cl and inhibition at 4.75 gL−1 NH4Cl). Since soluble ammonium concentration was appreciably lower (< 1.6 gL−1 NH4Cl) in all soil slurry incubations, methane uptake was seemingly stimulated, regardless of the supplemented NH4Cl concentrations (Figs. 1 and 2). Hence, ammonium-induced effects can be influenced by the ion exchange capacity (adsorption/desorption) of the mineral form of ammonium in different soils (King and Schnell 1998). In agreement with our hypothesis, the soluble ammonium, rather than the total ammonium fraction, is relevant to determine ammonium-induced response in methane uptake.
The effects of NH4Cl addition on the methanotrophic activity may be confounded by other parameters (e.g., AMS-derived copper, salt stress, nitrate, and pH) (DiSpirito et al. 2016; Ho et al. 2013b; 2018; Figs. S2 and S3) during the incubation. Although we cannot completely exclude the effects of these confounding factors, the response in methane uptake appears to be largely induced by the supplemented NH4Cl in AMS, extended discussion given in the Supplementary Information.
Response of the methanotrophic abundance and community composition to NH4Cl-AMS amendments
The pmoA gene abundance, which determines the abundance of the methanotrophs, was significantly higher after incubation in T4 to T6 in the soil slurry incubations relative to the reference and starting (after pre-incubation) values (Fig. 3). Although the mean pmoA gene abundance gradually increased in the soil slurry from T1 to T3, the values were not significantly different. On the other hand, the significant increase (p < 0.05) in the pmoA gene abundance (T1 to T4) in the soil enrichment is not consistent with the decreasing methane uptake rates (Figs. 1 and 3). Although statistically significant, the mean pmoA gene abundances in T1 to T4 were within a relatively narrow range (3.5 × 106 to 2.0 × 107 pmoA copy no. ml−1). Admittedly, the DNA-based qPCR analysis may not be as sufficiently sensitive as other biological indicators (e.g., transcript-based analyses) to capture relatively subtle differences, as detected in the activity measurements, likely being obscured by persistent relic DNA in the soil matrix (Carini et al. 2017; Schloter et al. 2018). Nevertheless, the qPCR was effective at capturing more pronounced differences (Ho et al. 2015; Reumer et al. 2018); the severe inhibition in methane uptake rate in T6 is clearly reflected in the appreciably lower mean pmoA gene abundance (1–2 orders of magnitude) when compared to the other treatments in the soil enrichment (Figs. 1d and 3).
To account for the discrepancy in the initial pmoA gene abundances in the soil slurry and enrichment incubations (soil slurry, 5.0 × 106 pmoA copy no. g soil−1; soil enrichment, 1.6 × 105 pmoA copy no. ml−1; Fig. 3), we determined the magnitude increase in the pmoA gene abundance for the NH4Cl-supplemented incubations that were significantly higher relative to the initial pmoA gene abundance (i.e., soil slurry, T4-T6; soil enrichment, T1-T5; Fig. 3). During the soil slurry incubation, the mean pmoA gene abundance increased by 6.7-fold, 10.7-fold, and 13.9-fold, respectively, in T4, T5, and T6, whereas a 22.5-fold, 41.0-fold, 47.0-fold, 134.0-fold, and 48.3-fold increase were detected in the T1, T2, T3, T4, and T5 soil enrichment incubations, respectively. The difference in the pmoA gene abundance (initial and after incubation) indicates methanotrophic growth.
The response of the methanotrophic community composition to the added NH4Cl was visualized as a principal component analysis (PCA), based on the pmoA gene sequences (Figs. 3 and S4). Notably, introducing two successive pre-incubation steps (Fig. S1) in the soil enrichment altered the composition of the methanotrophs; compared to the soil slurry incubation, methanotrophs belonging to the rice paddy cluster (RPC) decreased in relative abundance in the soil enrichment (Fig. S4). Nevertheless, the predominant methanotrophs in both the soil slurry and enrichment consistently comprised of Methylosarcina (type Ia) and other members of gammaproteobacterial methanotrophs (RPC and other uncultured type Ib methanotrophs), as well as alphaproteobacterial methanotrophs belonging to Methylocystis (type II) (Figs. 3 and S4). These taxa collectively represent > 90% of the total methanotrophic population in all incubations. A compositional shift in the methanotrophic community was detected in the soil slurry incubations, whereby Methylosarcina became dominant with increasing NH4Cl concentrations, diverging from the community in the reference incubation (Fig. 3). Methylosarcina also predominate the population at < 3.25 gL−1 NH4Cl (T5) in the soil enrichment, but community composition in T6 and reference incubations were more similar (Fig. 3).
Methylosarcina was favored in both the soil slurry and enrichment with increasing supplemented NH4Cl after incubation, despite of the discrepancy in the composition of the methanotrophic community prior to NH4Cl addition (Figs. 3 and S4). This indicates that NH4Cl strongly influenced the community composition during the incubation. However, the relative abundance of Methylosarcina decreased in the soil enrichment supplemented with 4.75 gL−1 NH4Cl (T6), corresponding to significantly lower methanotrophic activity, suggesting that this methanotroph contributed to methane oxidation under moderate NH4Cl levels (up to 3.25 gL−1, T5). This is not surprising given that gammaproteobacterial methanotrophs of subgroup type Ia are thought to be rapid responders to substrate availability (Ho et al. 2013a; 2017) and would benefit from the sudden availability of extraneous nitrogen sources. Indeed, besides Methylosarcina, other type Ia methanotrophs (e.g., Methylobacter and Methylomicrobium) have been consistently enriched after ammonium amendments in short-term incubations and showed higher N assimilation (Hu and Lu 2015; Nold et al. 1999; Noll et al. 2008; Yang et al. 2020). In line with previous studies (e.g., Alam and Jia 2012; Bodelier et al. 2000; Noll et al. 2008; Qiu et al. 2008), the apparent stimulation of type Ia methanotrophs can be attributable to a relief of N limitation, as indicated by the significant NH4Cl-induced stimulation (exception, 4.75 gL−1 NH4Cl in the soil enrichment) of methanotrophic activity.
Contrastingly, an alphaproteobacterial methanotroph, Methylocystis, was enriched in incubations of the same soil where NH4Cl concentration was increased step-wise from 0.5 to 4.75 gL−1 at 0.25 gL−1 increments (Ho et al. 2020). Because of the prolonged incubation over approximately 3 months with a constantly increasing selection pressure (elevated NH4Cl concentrations), the predominant ammonium-tolerant methanotroph that emerged is presumably able to detoxify products of ammonium oxidation (i.e., hydroxylamine, nitrite, nitrate (Ho et al. 2020; López et al. 2019; Versantvoort et al. 2020). Although further oxidation of hydroxylamine and nitrite is not confined to specific methanotroph subgroups (Hoefman et al. 2014; Nyerges and Stein 2009; Poret-Peterson et al. 2008), some Methylocystis appear to be more effective at detoxifying products of ammonium oxidation and hence were relatively more tolerant to ammonium inhibition (Nyerges and Stein 2009). Recently, a hydroxylamine oxidoreductase (responsible for the oxidation of hydroxylamine to nitric oxide) was purified from Methylacidiphilum fumariolicum SolV, a thermophilic verrucomicrobial methanotroph, but is also likely to occur in other aerobic methanotrophs (Versantvoort et al. 2020). Therefore, different mechanisms may underlie the selection of the gammaproteobacterial (including Methylosarcina) and alphaproteobacterial (including Methylocystis) methanotrophs at high ammonium levels. While a relief of N limitation may favor members of gammaproteobacterial methanotrophs in the short-term as in this study, metabolic capacity to detoxify inhibitory compounds of ammonium oxidation may become relevant over time, determining the fitness and dominance of specific methanotrophs.