Characterization of the Redox-Active Extracellular Polymeric Substances in an Anaerobic Methanotrophic Consortium

Anaerobic oxidation of methane (AOM) is a microbial process of importance in the global carbon cycle. AOM is predominantly mediated by anaerobic methanotrophic archaea (ANME), the physiology of which is still poorly understood. Here we present a new addition to the current physiological understanding of ANME by examining, for the first time, the biochemical and redox-active properties of the extracellular polymeric substances (EPS) of an ANME enrichment culture. Using a ‘Candidatus Methanoperedens nitroreducens’-dominated methanotrophic consortium as the representative, we found it can produce an EPS matrix featuring a high protein-to-polysaccharide ratio of ∼8. Characterization of EPS using FTIR revealed the dominance of protein-associated amide I and amide II bands in the EPS. XPS characterization revealed the functional group of C-(O/N) from proteins accounted for 63.7% of total carbon. Heme-reactive staining and spectroscopic characterization confirmed the distribution of c-type cytochromes in this protein-dominated EPS, which potentially enabled its electroactive characteristic. Redox-active c-type cytochromes in EPS mediated the EET of ‘Ca. M. nitroreducens’ for the reduction of Ag+ to metallic Ag, which was confirmed by both ex-situ experiments with extracted soluble EPS and in-situ experiments with pristine EPS matrix surrounding cells. The formation of nanoparticles in the EPS matrix during in-situ extracellular Ag + reduction resulted in a relatively lower intracellular Ag distribution fraction, beneficial for alleviating the Ag toxicity to cells. The results of this study provide the first biochemical information on EPS of anaerobic methanotrophic consortia and a new insight into its physiological role in AOM process.