Methane (CH4) is markedly more potent than carbon dioxide (CO2) in retaining heat within the atmosphere, having a global warming potential 28 times greater over a 100-year timescale1. Numerous sources of fugitive CH4 emissions exist in the natural environment, such as landfills, wetlands, and paddy fields. Concurrently, anthropogenic influences significantly shape the levels of CH4 emissions to varying degrees within these areas2,3. One of the most effective methods for removing fugitive CH4 is through its biological oxidation of CH4 by methane-oxidizing bacteria (MOB), which exhibit long-term persistence in CH4-rich environments. MOB can capture fugitive CH4 and metabolize it into water and CO2 in the presence of sufficient O2 through aerobic CH4 oxidation. The efficiency of MOB communities is influenced by various environmental factors, such as CH4 concentration, ambient temperature, soil moisture levels, and nutrient content4–6.
Extensive research efforts have been focused on identifying the key indicators that promote the growth of MOB and enhance the oxidation of fugitive CH4. However, the influence of nitrogen on CH4 oxidation remains highly debated. In addition to their influence on the carbon cycle through CH4 oxidation, various nitrogen forms are directly involved in the complex nitrogen cycle as substrates7,8. Microorganisms primarily utilize two forms of nitrogen: NH4+ and NO3−. Some studies suggest that NO3− may slightly inhibit CH4 oxidation9. However, this inhibition becomes evident only at concentrations exceeding 10 Mm10. Additionally, numerous studies have demonstrated that the introduction of NH4+ in soils can suppress CH4 oxidation due to competitive inhibition of methane monooxygenase enzymes (MMOs) by NH4 + 11. This may be because the molecular size and structure between CH4 and NH4+ share similarities, and the MMOs and ammonia monooxygenase (AMO) from the common ancestor share high sequence identity in gene encoding12,13. In addition, intermediates of ammonia oxidation, i.e., hydroxylamine (NH2OH) and NO2−, are known to be toxic to MOB, thereby inhibiting CH4 oxidation14,15. However, some research indicates that NH4+ could promote CH4 oxidation by stimulating MOB growth. Several studies have demonstrated that adding a small amount of NH4+ can benefit CH4 oxidation16,17. For instance, the introduction of 100 mg N kg− 1 dry soil has been shown to double the peak CH4 oxidation value18. The mechanisms underlying the stimulatory effects of NH4+ and NO3− on CH4 oxidation remain to be elucidated.
Various researchers have attempted to clarify the coupling mechanism between denitrification and CH4 oxidation. Byproducts of CH4 oxidation, such as methanol, can serve as electron donors in denitrification, and recent research has shown that some microorganisms can perform both processes simultaneously19,20. However, the impact of nitrification on CH4 oxidation remains contentious. Traditional ecological perspectives posit a robust competitive dynamic between nitrification and CH4 oxidation. For example, a recent study using DNA-stable isotope probing has revealed a negative correlation between the relative abundance of primary active MOB and nitrifying bacteria (NOB) in the soil, supporting the competitive interaction hypothesis21. Nevertheless, contemporary research has unveiled a heterotrophic influence of CH4 oxidation on nitrification. Studies have shown that the introduction of 20%-25% CH4 into wastewater treatment systems promotes the rapid removal of NH4 + 22. These observations lend credence to the concept of a coupled CH4 oxidation and nitrification-denitrification process. However, the metabolic mechanisms between CH4 oxidation and nitrification process remain to be clarified. Nitrification is a unidirectional and almost irreversible process, including ammonia oxidation by ammonia-oxidizing bacteria (AOB) and nitrification by NOB. However, recent findings have revealed that specific bacteria (Nitrospira spp.) can perform complete nitrification in a single step, directly transforming NH4+ into NO3−, thereby circumventing the requirement for a rate-limiting step in nitrification, i.e., ammonia oxidation by AOB23,24. Notably, Nitrospirota phylum is a distinctive bacteria coexisting in a significant population of MOB that can utilize NH4+ as a nitrogen source25. This nitrification pathway is likely to result in a different impact of nitrogen on CH4 oxidation compared with what has been previously reported.
The influence of alterations in microbial metabolism is most intuitively reflected in the composition and concentration of extracellular polymers (EPS). MOB produce a considerable amount of EPS, consisting mainly of extracellular polysaccharides (EPSs), extracellular proteins (ECPs), and nucleic acids. EPS exhibit a layered structure and can be divided into soluble EPS (S-EPS), loosely bound EPS (LB-EPS), and tightly bound EPS (TB-EPS)26. NH4+ can be significantly adsorbed in both LB-EPS and TB-EPS, with a portion of it persisting within the TB-EPS fraction27. Moreover, EPS are pivotal mediators in the facilitation of extracellular electron transfer (EET). Microorganisms primarily simulate EET through redox-active proteins as electron transfer chains that span their cell membranes28. In addition, different species of exoelectrogens develop distinct EET pathways during their growth and metabolic activities. Extensively studied exoelectrogens include Gram-negative bacteria, such as Geobacter and Shewanella29. Most Gram-positive electroactive strains belong to the Bacillus and Clostridium classes of the Firmicutes phylum. Recent studies have identified a flavin-based EET mechanism in diverse Gram-positive bacteria30. A specific type of NADH dehydrogenase separates electron transfer from aerobic respiration by directing electrons to a specific group of quinones in the cell membrane. Other proteins help build an abundant extracellular flavoprotein with free molecule flavin shuttles to mediate electron transfer to extracellular acceptors29,30. NH4+ serves as an electron donor in the EET mechanisms of anammox bacteria, which couple the oxidation of NH4+ to the transfer of electrons onto insoluble extracellular electron acceptors. In this process, NH4+ is oxidized to NH2OH as an intermediate, ultimately producing NO3 − 31.
Despite evidence that nitrogen forms can modulate CH4 oxidation, the specific microbial and metabolic mechanisms underlying this modulation remain to be fully elucidated. To address this limitation, in this study, NH4+ and NO3− absorbed in zeolites were introduced into MOB-rich soil to investigate the metabolic activity of different nitrogen forms and the influence of nitrification on CH4 oxidation, while maintaining stable pH and water content. The results are expected to provide a comprehensive understanding of the alterations in specific metabolites and metabolic pathways and clarify the interplay between nitrification and CH4 oxidation processes.