Extracellular electron uptake in Methanosarcinales is independent of multiheme c-type cytochromes

The co-occurrence of Geobacter and Methanosarcinales is often used as a proxy for the manifestation of direct interspecies electron transfer (DIET) in man-made and natural aquatic environments. We previously reported that not all Geobacter are capable of DIET with Methanosarcina. Here we tested 15 new artificial co-culture combinations with methanogens and electrogenic bacteria, including an electrogen outside of the Geobacter clade – Rhodoferax ferrireducens.Consistently, highly effective electrogenic bacteria (G. metallireducens, G. hydrogenophilus and R. ferrireducens) formed successful associations with Methanosarcinales. Highly effective electrogens could not sustain the growth of H2-utilizing methanogens of the genera Methanococcus, Methanobacterium, Methanospirillum, Methanolacinia or Methanoculleus.Methanosarcinales, including strict non-hydrogenotrophic methanogens of the genus Methanothrix (Mtx. harundinacea and Mtx. shoeghenii) and Methanosarcina horonobensis, conserved their ability to interact with electrogens. Methanosarcinales were classified as the only methanogens containing c-type cytochromes, unlike strict hydrogenotrophic methanogens. It was then hypothesized that multiheme c-type cytochromes give Methanosarcinales their ability to retrieve extracellular electrons. However, multiheme c-type cytochromes are neither unique to this group of methanogens nor universal. Only two of the seven Methanosarcinales tested had multiheme c-type cytochromes (MCH). In one of these two species - M. mazei a deletion mutant for its MCH was readily available. Here we tested if the absence of this MHC impacts extracellular electron uptake. Deletion of the MHC in M. mazei did not impact the ability of this methanogens to retrieve extracellular electrons from G. metallireducens or a poised cathode. Since Methanosarcina did not require multiheme c-type cytochromes for direct electron uptake we proposed an alternative strategy for extracellular electron uptake.