Methane-oxidizing bacteria; “the gatekeepers of methane” : Insights into their niche differentiation in freshwater systems

Methane (CH₄) is a potent greenhouse gas whose atmospheric concentration continues to rise, strongly influencing Earth’s climate. As the end product of archaeal metabolism, methane is mainly produced under anoxic conditions, making freshwater systems an important natural source of biogenic CH₄. Lakes, ponds, rivers, and agricultural drainage systems are now recognized as significant contributors to global methane emissions. Because these ecosystems are highly vulnerable to anthropogenic pressures such as eutrophication, their methane emissions are expected to increase in the future.A major natural process counteracting methane emissions is performed by methane-oxidizing bacteria (MOB). These bacteria use methane as a source of carbon and energy and can prevent up to 80% of sediment-produced methane from reaching the atmosphere. Despite their importance, the ecology of freshwater MOB has received limited attention, largely because the contribution of freshwater systems to the global methane budget has only recently been acknowledged. Understanding the abiotic and biotic factors shaping MOB diversity, distribution, and activity is therefore essential for predicting methane emissions from freshwater ecosystems.In this thesis, I investigated freshwater MOB using a combination of field surveys and experimental approaches. The overall aim was to describe MOB abundance, activity, and diversity, and to identify the abiotic and biotic drivers underlying MOB niche differentiation in freshwater environments.Chapter 2 examined MOB niche differentiation in the upper sediment layer (~1 cm) of 50 agricultural drainage ditches across 10 regions in the Netherlands. MOB Type Ib dominated most sites, and nitrogen-related variables emerged as the main drivers of MOB distribution. Clear niche differentiation among closely related species suggested trait divergence at the species level. MOB interactions were strongly context dependent, potentially shifting from facilitation to competition under different environmental conditions. MOB accounted for 3–7% of the total bacterial community and showed strong associations with other microbial taxa.Chapter 3 explored whether methane-derived carbon provided by MOB could support cyanobacterial carbon demands. Highly negative delta¹³C signatures in phytoplankton indicated the use of methane-derived carbon. Co-culturing experiments confirmed syntrophic interactions between MOB and cyanobacteria, and activity assays revealed active MOB on cyanobacterial filaments in stratified lakes, pointing to bidirectional interactions affecting methane emissions.Chapter 4 investigated interactions between methane-cycling microorganisms and cyanobacteria as a potential methane mitigation strategy in rice paddies. Microcosm experiments using two indigenous cyanobacterial isolates showed methane emissions decreased up to 20-fold, depending on cyanobacterial identity and soil depth.Chapter 5 assessed the effects of fish-mediated bioturbation on methane dynamics in shallow systems. Fish activity increased methane ebullition but reduced CO₂ emissions. Although planktonic MOB abundance increased due to trophic cascading effects, methane oxidation rates remained unchanged.Overall, this thesis demonstrates that MOB niche differentiation in freshwater systems is shaped by a complex interplay of abiotic and biotic drivers. These interactions link MOB to multiple trophic levels and strongly influence methane cycling, emphasizing the need for a polyphasic approach to studying freshwater methane dynamics.