Recovery of Methanotrophic Activity Is Not Reflected in the Methane-Driven Interaction Network after Peat Mining

Aerobic methanotrophs are crucial in ombrotrophic peatlands, driving the methane and nitrogen cycles. Peat mining adversely affects methanotrophs, but activity and community composition/abundances may recover after restoration. Considering that the methanotrophic activity and growth are significantly stimulated in the presence of other microorganisms, the methane-driven interaction network, which encompasses methanotrophs and nonmethanotrophs (i.e., the methanotrophic interactome), may also be relevant in conferring community resilience. Yet, little is known of the methanotrophic interactome's response to and recovery from disturbances. Here, we determined the recovery of the methanotrophic interactome as inferred by a co-occurrence network analysis comparing pristine and restored peatlands. We coupled a DNA-based stable isotope probing (SIP) approach using [13C]CH4 to a co-occurrence network analysis derived from the 13C-enriched 16S rRNA gene sequences to relate the response in methanotrophic activity to the structuring of the interaction network. Methanotrophic activity and abundances recovered after peat restoration since 2000. “Methylomonaceae” taxa were the predominantly active methanotrophs in both peatlands, but the peatlands differed in the relative abundances of Methylacidiphilaceae and Methylocystis. However, bacterial community compositions were distinct in both peatlands. Likewise, the methanotrophic interactome was profoundly altered in the restored peatland. Structuring of the interaction network after peat mining resulted in the loss of complexity and modularity, indicating a less connected and efficient network, which may have consequences in the event of recurring/future disturbances. Therefore, determining the response of the methane-driven interaction network, in addition to relating methanotrophic activity to community composition/abundances, provided a more comprehensive understanding of the resilience of the methanotrophs

The methane-driven interaction network in terrestrial methane hotspots

Background: Biological interaction affects diverse facets of microbial life by modulating the activity, diversity, abundance, and composition of microbial communities. Aerobic methane oxidation is a community function, with emergent community traits arising from the interaction of the methane-oxidizers (methanotrophs) and non-methanotrophs. Yet little is known of the spatial and temporal organization of these interaction networks in naturally-occurring complex communities. We hypothesized that the assembled bacterial community of the interaction network in methane hotspots would converge, driven by high substrate availability that favors specific methanotrophs, and in turn influences the recruitment of non-methanotrophs. These environments would also share more co-occurring than site-specific taxa. Results: We applied stable isotope probing (SIP) using 13C-CH4 coupled to a co-occurrence network analysis to probe trophic interactions in widespread methane-emitting environments, and over time. Network analysis revealed predominantly unique co-occurring taxa from different environments, indicating distinctly co-evolved communities more strongly influenced by other parameters than high methane availability. Also, results showed a narrower network topology range over time than between environments. Co-occurrence pattern points to Chthoniobacter as a relevant yet-unrecognized interacting partner particularly of the gammaproteobacterial methanotrophs, deserving future attention. In almost all instances, the networks derived from the 13C-CH4 incubation exhibited a less connected and complex topology than the networks derived from the unlabelledC-CH4 incubations, likely attributable to the exclusion of the inactive microbial population and spurious connections; DNA-based networks (without SIP) may thus overestimate the methane-dependent network complexity. Conclusion: We demonstrated that site-specific environmental parameters more strongly shaped the co-occurrence of bacterial taxa than substrate availability. Given that members of the interactome without the capacity to oxidize methane can exert interaction-induced effects on community function, understanding the co-occurrence pattern of the methane-driven interaction network is key to elucidating community function, which goes beyond relating activity to community composition, abundances, and diversity. More generally, we provide a methodological strategy that substantiates the ecological linkages between potentially interacting microorganisms with broad applications to elucidate the role of microbial interaction in community function. © 2022, The Author(s)

Disentangling abiotic and biotic controls of aerobic methane oxidation during re-colonization

Aerobic methane oxidation is driven by both abiotic and biotic factors, which are often confounded in the soil environment. Using a laboratory-scale reciprocal inoculation experiment with two native soils (paddy and upland agricultural soils) and the gamma-irradiated fraction of these soils, we aim to disentangle and determine the relative contribution of abiotic (i.e., soil edaphic properties) and biotic (i.e., initial methanotrophic community composition) controls of methane oxidation during re-colonization. Methane uptake was appreciably higher in incubations containing gamma-irradiated paddy than upland soil despite the initial difference in the methanotrophic community composition. This suggested an overriding effect of the soil edaphic properties, which positively regulated methane oxidation. Community composition was similar in incubations with the same starting inoculum, based on quantitative and qualitative pmoA gene analyses. Thus, results suggested that the initial community composition affects the trajectory of community succession to an extent, but not at the expense of the methanotrophic activity under high methane availability. Still, methane oxidation was affected more by soil edaphic properties than by the initial composition of the methanotrophic community

When the going gets tough: Emergence of a complex methane-driven interaction network during recovery from desiccation-rewetting

Microorganisms interact in complex communities, affecting microbially-mediated processes in the environment. Particularly, aerobic methanotrophs showed significantly stimulated growth and activity in the presence of accompanying microorganisms in an interaction network (interactome). Yet, little is known of how the interactome responds to disturbances, and how community functioning is affected by the disturbance-induced structuring of the interaction network. Here, we employed a time-series stable isotope probing (SIP) approach using 13C–CH4 coupled to a co-occurrence network analysis after Illumina MiSeq sequencing of the 13C-enriched 16S rRNA gene to directly relate the response in methanotrophic activity to the network structure of the interactome after desiccation-rewetting of a paddy soil. Methane uptake rate decreased immediately (<5 days) after short-term desiccation-rewetting. Although the methanotroph subgroups differentially responded to desiccation-rewetting, the metabolically active bacterial community composition, including the methanotrophs, recovered after the disturbance. However, the interaction network was profoundly altered, becoming more complex but, less modular after desiccation-rewetting, despite the recovery in the methanotrophic activity and community composition/abundances. This suggests that the legacy of the disturbance persists in the interaction network. The change in the network structure may have consequences for community functioning with recurring desiccation-rewetting

Discrepancy in exchangeable and soluble ammonium-induced effects on aerobic methane oxidation: a microcosm study of a paddy soil

Ammonium-induced stimulatory, inhibitory, and/or neutral effects on soil methane oxidation have been attributable to the ammonium concentration and mineral forms, confounded by other edaphic properties (e.g., pH, salinity), as well as the site-specific composition of the methanotrophic community. We hypothesize that this inconsistency may stem from the discrepancy in the cation adsorption capacity of the soil. We postulate that the effects of ammonium on the methanotrophic activity in soil are more accurately portrayed by relating methane uptake rates to the soluble ammonium (bioavailable), rather than the exchangeable (total) ammonium. To reduce adsorption (exchangeable) sites for ammonium in a paddy soil, two successive pre-incubation steps were introduced resulting in a 1000-fold soil dilution (soil enrichment), to be compared to a soil slurry (tenfold dilution) incubation. Ammonium was supplemented as NH4Cl at 0.5–4.75gL−1 after pre-incubation. While NH4Cl significantly stimulated the methanotrophic activity at all concentrations in the soil slurry incubation, methane uptake showed a dose-dependent effect in the soil enrichment. The trend in methane uptake could be explained by the soluble ammonium concentration, which was proportionate to the supplemented ammonium in the soil enrichment. In the soil slurry incubation, a fraction (36–63%) of the supplemented ammonium was determined to be adsorbed to the soil. Accordingly, Methylosarcina was found to predominate the methanotrophic community after the incubation, suggesting the relevance of this methanotroph at elevated ammonium levels (< 3.25gL−1 NH4Cl). Collectively, our results showed that the soluble, rather than the exchangeable ammonium concentration, is relevant when determining the effects of ammonium on methane oxidation, but this does not exclude other (a)biotic factors concurrently influencing methanotrophic activity

Discrepancy in exchangeable and soluble ammonium-induced effects on aerobic methane oxidation: a microcosm study of a paddy soil

Ammonium-induced stimulatory, inhibitory, and/or neutral effects on soil methane oxidation have been attributable to the ammonium concentration and mineral forms, confounded by other edaphic properties (e.g., pH, salinity), as well as the site-specific composition of the methanotrophic community. We hypothesize that this inconsistency may stem from the discrepancy in the cation adsorption capacity of the soil. We postulate that the effects of ammonium on the methanotrophic activity in soil are more accurately portrayed by relating methane uptake rates to the soluble ammonium (bioavailable), rather than the exchangeable (total) ammonium. To reduce adsorption (exchangeable) sites for ammonium in a paddy soil, two successive pre-incubation steps were introduced resulting in a 1000-fold soil dilution (soil enrichment), to be compared to a soil slurry (tenfold dilution) incubation. Ammonium was supplemented as NH4Cl at 0.5–4.75gL−1 after pre-incubation. While NH4Cl significantly stimulated the methanotrophic activity at all concentrations in the soil slurry incubation, methane uptake showed a dose-dependent effect in the soil enrichment. The trend in methane uptake could be explained by the soluble ammonium concentration, which was proportionate to the supplemented ammonium in the soil enrichment. In the soil slurry incubation, a fraction (36–63%) of the supplemented ammonium was determined to be adsorbed to the soil. Accordingly, Methylosarcina was found to predominate the methanotrophic community after the incubation, suggesting the relevance of this methanotroph at elevated ammonium levels (< 3.25gL−1 NH4Cl). Collectively, our results showed that the soluble, rather than the exchangeable ammonium concentration, is relevant when determining the effects of ammonium on methane oxidation, but this does not exclude other (a)biotic factors concurrently influencing methanotrophic activity

Interkingdom interaction: the soil isopod Porcellio scaber stimulates the methane-driven bacterial and fungal interaction

Porcellio scaber (woodlice) are (sub-)surface-dwelling isopods, widely recognized as “soil bioengineers”, modifying the edaphic properties of their habitat, and affecting carbon and nitrogen mineralization that leads to greenhouse gas emissions. Yet, the impact of soil isopods on methane-cycling processes remains unknown. Using P. scaber as a model macroinvertebrate in a microcosm study, we determined how the isopod influences methane uptake and the associated interaction network in an agricultural soil. Stable isotope probing (SIP) with 13C-methane was combined to a co-occurrence network analysis to directly link activity to the methane-oxidizing community (bacteria and fungus) involved in the trophic interaction. Compared to microcosms without the isopod, P. scaber significantly induced methane uptake, associated to a more complex bacteria-bacteria and bacteria-fungi interaction, and modified the soil nutritional status. Interestingly, 13C was transferred via the methanotrophs into the fungi, concomitant to significantly higher fungal abundance in the P. scaber-impacted soil, indicating that the fungal community utilized methane-derived substrates in the food web along with bacteria. Taken together, results showed the relevance of P. scaber in modulating methanotrophic activity with implications for bacteria-fungus interaction

Response of a methane-driven interaction network to stressor intensification

Microorganisms may reciprocally select for specific interacting partners, forming a network with interdependent relationships. The methanotrophic interaction network, comprising methanotrophs and non-methanotrophs, is thought to modulate methane oxidation and give rise to emergent properties beneficial for the methanotrophs. Therefore, microbial interaction may become relevant for community functioning under stress. However, empirical validation of the role and stressor-induced response of the interaction network remains scarce. Here, we determined the response of a complex methane-driven interaction network to a stepwise increase in NH4Cl-induced stress (0.5–4.75 g L−1, in 0.25–0.5 g L−1 increments) using enrichment of a naturally occurring complex community derived from a paddy soil in laboratory-scale incubations. Although ammonium and intermediates of ammonium oxidation are known to inhibit methane oxidation, methanotrophic activity was unexpectedly detected even in incubations with high ammonium levels, albeit rates were significantly reduced. Sequencing analysis of the 16S rRNA and pmoA genes consistently revealed divergent communities in the reference and stressed incubations. The 16S rRNA-based co-occurrence network analysis revealed that NH4Cl-induced stress intensification resulted in a less complex and modular network, likely driven by less stable interaction. Interestingly, the non-methanotrophs formed the key nodes, and appear to be relevant members of the community. Overall, stressor intensification unravels the interaction network, with adverse consequences for community functioning