Detection of Metabolic Key Enzymes of Methane Turnover Processes in Cold Seep Microbial Biofilms

Abstract

In anoxic environments, methane oxidation is conducted in a syntrophic process between methanotrophic archaea (ANME) and sulfate reducing bacteria (SRB). Microbial mats consisting of ANME, SRB and other microorganisms form methane seep-related carbonate buildups in the anoxic bottom waters of the Black Sea Crimean shelf. To shed light on the localization of the biochemical processes at the level of single cells in the Black Sea microbial mats, we applied antibody-based markers for key enzymes of the relevant metabolic pathways. The dissimilatory adenosine-5′-phosphosulfate (APS) reductase, methyl-coenzyme M reductase (MCR) and methanol dehydrogenase (MDH) were selected to localize sulfate respiration, reverse methanogenesis and aerobic methane oxidation, respectively. The key enzymes could be localized by double immunofluorescence and immunocytochemistry at light- and electron microscopic levels. In this study we show that sulfate reduction is conducted synchronized and in direct proximity to reverse methanogenesis of ANME archaea. Microcolonies in interspaces between ANME/SRB express methanol dehydrogenase, which is indicative for oxidation of C₁ compounds by methylotrophic or methanotrophic bacteria. Thus, in addition to syntrophic AOM, oxygen-dependent processes are also conducted by a small proportion of the microbial population.

Keywords:

• anaerobic oxidation of methane
• microbial mat
• sulfate reduction
• methylotrophy
• immunoelectron microscopy
• immunofluorescence

Acknowledgments

We thank the crew of the R/V “Professor Logachev”, the Hamburg research group of. W. Michaelis, and the Jago-Submersible Team (J. Schauer & K. Hissmann) for collaboration during the sampling cruise for deep sea sediments. We also thank J. Peckmann (Univ. Vienna) for helpful discussions in an initial phase of this project. We thank C. Wichmann for critically reading this manuscript. Part of this study received financial support by the Bundesministerium für Bildung und Forschung (BMBF-GEOTECHNOLOGIEN-Program GHOSTDABS 03G0559A) and the Deutsche Forschungsgemeinschaft (DFG-Research Unit 571 - Geobiology of Organo- and Biofilms, DFG-Research Projects Re 665/31-1, Ho 1830/2-1). This is Courant Research Centre Geobiology Scientific Contribution n. 109.

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