Community composition and ultrastructure of a nitrate-dependent anaerobic methane-oxidizing enrichment culture

Methane is a very potent greenhouse gas and can be oxidized aerobically or anaerobically through microbial-mediated processes, thus decreasing methane emissions to the atmosphere. Using a complementary array of methods including phylogenetic analysis, physiological experiments, and light and electron microscopy techniques (including electron tomography), we investigated the community composition and ultrastructure of a continuous bioreactor enrichment culture, in which anaerobic methane oxidation (AOM) was coupled to nitrate reduction. A membrane bioreactor was seeded with AOM biomass and continuously fed with excess methane. After 150 days the bioreactor reached a daily consumption of 10 mmol nitrate L-1 d-1. The biomass consisted of aggregates that were dominated by nitrate-dependent anaerobic methane-oxidizing Methanoperedens-like archaea (40%) and nitrite-dependent anaerobic methane-oxidizing Methylomirabilis-like bacteria (50%). The Methanoperedens sp. were identified by fluorescence in situ hybridization and immunogold localization of the Methyl-Coenzyme M Reductase (Mcr) enzyme, which was located in the cytoplasm. The Methanoperedens sp. aggregates consisted of slightly irregular coccoid cells (∼1.5 μm diameter) which produced extruding tubular structures and putative cell-to-cell contacts among each other. Methylomirabilis sp. bacteria exhibited the polygonal cell shape typical of this genus. In AOM archaea and bacteria cytochrome c proteins were localized in the cytoplasm and periplasm respectively by cytochrome staining. Our results indicate that AOM bacteria and archaea might work closely together in the process of anaerobic methane oxidation as the bacteria depend on the archaea for nitrite. Future studies will be aimed at elucidating the function of the cell-to-cell interactions in nitrate-dependent AOM.IMPORTANCE Microorganisms performing nitrate- and nitrite-dependent anaerobic methane oxidation are important in both natural and man-made ecosystems such as wastewater treatment plants. In both systems, complex microbial interactions take place that are largely unknown. Revealing these microbial interactions would enable us to understand how the oxidation of the important greenhouse gas methane occurs in nature, and pave the way for an application of these microbes in wastewater treatment plants. Here, we elucidated the microbial composition, ultrastructure, and physiology of a nitrate-dependent AOM community of archaea and bacteria and describe the cell plan of Methanoperedens-like methanotrophic archaea