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 the environment. Here we tested eleven new co-culture combinations between methanogens and electrogens. Previously, only the most electrogenic Geobacter paired by DIET with Methanosarcinales methanogens, namely G. metallireducens and G. hydrogenophilus. Here we provide additional support, and show that five additional Methanosarcinales paired with G. metallireducens, while a strict hydrogenotroph could not. We also show that G. hydrogenophilus, which is incapable to grow with a strict hydrogenotrophic methanogen, could pair with a strict non-hydrogenotrophic Methanosarcinales. Likewise, an electrogen outside the Geobacter cluster (Rhodoferrax ferrireducens) paired with Methanosarcinales but not with strict hydrogenotrophic methanogens. The ability to interact with electrogens appears to be conserved among Methanosarcinales, the only methanogens with c-type cytochromes, including multihemes (MHC). Nonetheless, MHC, which are often linked to extracellular electron transfer, were neither unique nor universal to Methanosarcinales and only two of seven Methanosarcinales tested had MHC. Of these two, one strain had an MHC-deletion knockout available, which we hereby show is still capable to retrieve extracellular electrons from G. metallireducens or an electrode suggesting an MHC-independent strategy for extracellular electron uptake.

There are indications for direct interspecies electron transfer occuring in methane producing environments such as anaerobic digesters 15 , rice paddy soils 16 , and aquatic sediments 17,18 , as well as in methane consuming environments such as hydrothermal vents 19,20 . In these environments, DIET is typically inferred either because conductive materials stimulate the syntrophic metabolism but also by the co-presence of DNA and/or RNA of phylotypes related to DIET-microorganisms. DIET-pairing of Geobacter with methanogens was only described in two Geobacter species (G. metallireducens, G. hydrogenophilus) 5,10-12 . On the other hand, a series of six Geobacter species were unable to interact syntrophically with Ms. barkeri 800. All these Geobacter were modest anode respiring bacteria and did not produce high current densities at the anode 5 . Species outside of the Geobacter-clade have never been shown to do DIET with methanogens.
In this study, we expand the list of syntrophic-DIET pairs and investigated whether electrogenic bacteria other than Geobacter, could interact syntrophically with methanogens. We determined whether DIET was widespread among methanogens or was a specific trait of Methanosarcinales because of their high c-type cytochrome content 21 . Multiheme c-type cytochromes (MHC) were previously implicated in EET in bacteria 22 and an MHC of Methanosarcina acetivorans was required for anthraquinone-2, 6-disulfonate (AQDS) respiration 23 . Here we asked whether MHC in Methanosarcina are required for DIET and electron uptake from electrodes.

Materials and Methods
Microorganisms and cultivation conditions. Cultures were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) and grown on the media advised by the collection until pre-adaption to co-cultivation media.
Electrogens. We used the following strains of bacteria: Rhodoferax ferrireducens (DSM 15236), Pelobacter carbinolicus (DSM 2380), Geobacter hydrogenophilus (DSM 13691) and Geobacter metallireducens GS-15 (DSM 7210) available at the University of Massachusetts. For experiments with G. metallireducens strain GS15 at the University of Southern Denmark GS-15 (DSM 7210) was purchased from the DSMZ culture collection. G. metallireducens, G. hydrogenophilus and R. ferrireducens were maintained on the typical co-culture freshwater media 10 with 55 mM ferric citrate as electron acceptor. The two Geobacter species were maintained with 10-20 mM ethanol as electron donor 11,12 whereas Rhodoferax was maintained with 5 mM glucose. P. carbinolicus which was used as H 2 -donating strain (DIET-negative control), was pre-cultivated under fermentative conditions on 10 mM acetoin as previously described 2 .
Methanogens. The following strains of methanogens were tested: Methanoculleus marisnigri JR1 (DSM 1498) Methanothrix soehengii, Methanosaeta harundinacea, Methanospirilum hungatei and Methanoculleus marisnigri were pre-grown on the typical co-culture freshwater media however without ethanol 10  For the multiheme cytochrome mutant of M. mazei (633 k.o.) an antibiotic mix (ampicillin 100 µg/ml and puromycin 5 µg/ml) was added for selection and safe maintanance. This antibiotic mix was omitted in the transfer prior to co-culture inoculation and in the co-culture media.
Co-cultures with electrically conductive granular activated carbon (GAC). For experiments with conductive materials, we added 25 g/L GAC (charcoal activated, Merck kGaA, Darmstadt, Germany) to the co-culture media and controls were run in parallel. No-food controls were set up to monitor for background product formation from carry-over substrates or the possible use of GAC as a food source. Single-species culture controls with and without the addition of GAC to assess the probability of ethanol oxidation using GAC as electron acceptor by a single species.
All co-cultures were incubated at 37 °C at a final volume of 10 ml and all co-cultures were set up in duplicates or more replicates.
Analytical measurements. Samples were withdrawn anaerobically using N 2 :CO 2 (80:20) flushed hypodermic needles to verify the levels of methane, hydrogen, acetate, ethanol, and glucose. Determination of methane (CH 4 ), hydrogen gas (H 2 ), ethanol and volatile fatty acids (e.g. acetate, formate) was carried out as described before 11,12 . Briefly, for experiments done at the University of Southern Denmark, gases were measured with a Trace 1300 gas chromatograph (GC) (Thermo-Scientific) equiped with a TracePLOT ™ TG-BOND Msieve 5A column and a thermal conductivity detector (TCD). The carrier gas was argon at a flow rate of 25 mL/min with the temperatures set for the injector, oven and detector at 150 °C, 70 °C and 200 °C, respectively. To monitor ethanol, the same GC system was used with a different column, TRACE ™ TR-Wax and a flame ionization detector (FID). The filter-sterilised liquid sample (0.5 mL) was first heated to 60 °C for 5 mins in an air-tight exetainer after which the vaporised sample was collected for measurement. The carrier gas was nitrogen flowing at 1 mL/min, with the temperatures of injector, oven and detector set at 220 °C, 40 °C and 230 °C respectively. Acetate was analysed with a Dionex ™ ICS-1500 Ion Chromatography system, using a Dionex ™ IonPac ™ AS15 IC Column. The eluent was a mixture of 1.4 mM NaHCO 3 and 4.5 mM Na 2 CO 3 detected on an electron capture detector (ECD) at 30 mA. For experiments done at the University of Massachusetts, gases were measured by a Shimadzu 8 A gas chromatograph with a 80/100 Hayasep Q column connected to an FID. The temperatures of injector, oven and detector were set at 200 °C, 120 °C and 200 °C respectively. Ethanol was measured on a Perkin Elmer GC equiped with an Elite 5 column with helium as the carrier gas following a gradient separation protocol as follows: 50 °C for 1 min, a increase of 12 °C per minute to reach 200 °C, and a final 1.5 min at 200 °C. The temperatures for injector and detector were set at 200 °C and 300 °C, respectively.
Acetate was measured on a high-pressure liquid chromatography (HPLC) via an Aminex NPX-87H column using 8 mM H 2 SO 4 as the eluent with a UV detector set at 210 nm. Glucose was determined at the end of the incubation, as previously described 25 by separating on an HPLC with an Aminex Ion-Exclusion organic acid analysis column HPX-87H (Bio-Rad) and detected with a SP8430 refractive-index detector (Spectra-Physics). electrochemical reactor setup. Incubations using a cathode as sole electron donor, were carried out as previously described 11 , with slight modifications. Briefly, a two-chambered H cell reactor (Adams and Chittenden, USA) with a total volume of 650 ml in each chamber was separated via a Nafion ™ N117 proton exchange membrane (Ion power). The working and counter electrodes were made of graphite rods with dimensions of 2.5 × 7.5 × 1.2 cm and were connected to titanium rods. A leak-free Ag/AgCl reference electrode (3.4 M KCl) (CMA Microdialysis, Sweden). To ensure low carry over of substrates, cells were harvested in an anaerobic chamber at 4000 rpm for 10 mins, resuspended in fresh media (modified 120c -see above) prior to inoculation of the cell suspension at a final concentration of 20% in the reactor. In this set-up, the working and counter electrodes were connected via a resistor (250 Ω) and a potentiostat, however the resistor was unecessary since the cathodic potential was fully controlled by the potentiostat. The potential of working electrode was controlled with the MultiEmstat potentiostat (Palmsens, The Netherlands) set at −400 mV (vs. the standard hydrogen electrode).

Results and Discussion
electrogens establish Diet syntrophy with Methanosarcinales methanogens. To examine the ability for direct interspecies electron uptake in new strains of methanogens, G. metallireducens was used as the default DIET-partner for co-culture experiments due to its high electrogenic capability 5 , correlated to a syntrophic ability 10,12 , which was independent of H 2 -transfer, as this Geobacter was incapable to produce H 2 9,26 . We selected seven methanogenic strains as representatives of two groups: strict hydrogenotrophic methanogens (H 2 -consuming) and Methanosarcinales-methanogens, including H 2 -utilizing and strict non-hydrogenotrophic strains.
G. metallireducens paired syntrophically with five new Methanosarcinales, independent of the methanogen's ability to consume H 2 . The electrogen Geobacter metallireducens was previously shown to establish DIET interactions with three species of the order Methanosarcinales of which two were non-hydrogenotrophic methanogens (Methanosarcina horonobensis and Methanosaeta harundinacea) and one was a H 2 -utilizing methanogen (Methanosarcina barkeri strain MS) [10][11][12] . Nevertheless, we recently reported that of two Methanosarcina species which are capable of DIET with G. metallireducens only one could use a cathode at −400 mV (vs. SHE) 11 as sole electron donor. This variability in the Methanosarcinales capability to carry out direct electron uptake prompted us to verify whether DIET is conserved in other Methanosarcinales.
In this study, we evaluated five aditional strains of Methanosarcina, two of M. barkeri (M. barkeri 227 and M. barkeri strain Fusaro/804) two of M. mazei (Go1 and 633 k.o.) and one of the strict non-hydrogenotrophic methanogen -Methanothrix soehngenii.
All co-cultures were provided with ethanol as electron donor in the absence of any electron acceptor other than carbon dioxide. If successful, co-cultures were anticipated to reach mid exponential growth after circa two months, in agreement to preliminary tests and previous reports on DIET-consortia [10][11][12] . After 75 days, all co-cultures of G. metallireducens with the five new Methanosarcinales effectively converted their substrate (ethanol) to products (methane) (Fig. 1). The respiratory metabolism of G. metallireducens (see reaction 1) resulted in extracellular transfer of electrons and transient formation of acetate (Fig. 2). The products of ethanol oxidation were then converted into methane (reaction 2 & 3; Fig. 2). Alone, none of the five Methanosarcinales converted ethanol to methane (Fig. 2).
Previously, it was shown that interactions between G. metallireducens and M. barkeri strain MS (DSM 800) could be initiated much faster and continue at a faster pace, when amended with electrically conductive materials such as granular activated carbon (GAC) 12,27 whereas non-conductive materials such as cotton cloth had no effect 28 . GAC was shown to promote the respiratory metabolism of G. metallireducens, which oxidizes ethanol and releases electrons extracellularly onto this conductor until it reaches charge-saturation 29,30 . Afterwards, the presence of a methanogen reclaiming electrons keeps the process from coming to a halt 11 .
In this study, we subjected three additional co-cultures of G. metallireducens and Methanosarcina (two strains of M. mazei and one M. barkeri 227) to 25 g/L GAC to verify whether GAC can accelerate their growth. All co-cultures amended with this electrically conductive material exhibited shorter lag-phases, reached mid-exponential growth much quicker, and at a minimum tripled their methanogenesis rates (Fig. 2). On the other hand, the addition of electrically conductive particles to the three Methanosarcina-strains in pure culture had no impact on methane production or ethanol utilization (Fig. 2).
We have now expanded the repertoire of strains of methanogens capable of DIET with G. metallireducens by an additional five strains adding to the original three strains described in the past. Of these eight strains, three were not hydrogenotrophic. We could conclude that the capacity to carry out DIET with G. metallireducens was conserved among all tested Methanosarcinales, independent of their ability to use H 2 or not. www.nature.com/scientificreports www.nature.com/scientificreports/ G. metallireducens was unsuccessful to pair syntrophically with a strict hydrogenotroph -Methanoculleus marisnigri. Although G. metallireducens does not have the genetic possibility to form H 2 26 , and could not evolve H 2 when grown in pure culture on its own substrate 9 , we nevertheless previously tested whether H 2 -utilizing methanogens may have discovered a strategy to interact with this electrogenic Geobacter. In these former investigations we have shown that G. metallireducens was unsuccessful in developing ethanol-utilizing consortia in co-culture with two strict hydrogen-utilizing methanogens Methanospirillum hungatei (order Methanomicrobiales) and Methanobacterium formicicum (order Methanobacteriales) 10 , even in the presence of electrically conductive materials 11 .
In this study, we evaluated DIET between G. metallireducens with a third strict hydrogenotrophic species: Methanoculleus marisnigri (order Methanomicrobiales). Order Methanomicrobiales comprises the families Methanomicrobiaceae and Methanospirillaceae. Members of the family Methanomicrobiaceae (includins Methanoculleus marisnigri), were high in transcript abundance (13%) in rice paddies co-dominated by transcripts of Geobacter 16 , whereas Methanospirillaceae were not. This was hinting at the possibility of an interaction between Geobacter and Methanomicrobiaceae. We incubated G. metallireducens with Mcl. marisnigri for ca. 5 months, but did not observe methane production from ethanol during this timeframe (Fig. 1).
DIET evaluation by co-cultivation with G. metallireducens has now been carried out for three strict hydrogenotrophs from two major orders: Methanomicrobiales (M. hungatei, M. marisnigri) and Methanobacteriales (M. formicicum), showcasing the inability of strict hydrogenotrophic methanogens to pair with G. metallireducens (Fig. 1).
G. hydrogenophilus paired syntrophically with a second Methanosarcinales. Previously, we demonstrated that a second Geobacter species (G. hydrogenophilus) paired syntrophically with M. barkeri strain MS (DSM800). This was the only other Geobacter out of seven strains tested which exhibited the highest current density on the anode, similar to G. metallireducens 5 . Unlike G. metallireducens, G. hydrogenophilus does produce H 2 during respiration 9 , but could not pair with the strict H 2 -utilizing methanogen -Methanospirillum hungatei 5 .
G. hydrogenophilus was only tested with strains that did have the possibility to use H 2 and it was never tested whether it can pair syntrophically with strict non-hydrogenotrophic methanogens. In this study, we tested whether G. hydrogenophilus can pair syntrophically with the non-hydrogenotrophic methanogen Methanosaeta harundinacea (order Methanosarcinales). Methane production and acetate accumulation from ethanol were used as a proxy for the efficiency of the interaction. Previously, we have shown that acetate and methane can only accumulate in successfully paired co-cultures. For example, control tests with Methanosaeta could not sustain methane production and acetate accumulation from ethanol in the absence of a functional ethanol-oxidizing Geobacter 10 . In this study, we show that over the course of 117 days, co-cultures of G. hydrogenophilus and www.nature.com/scientificreports www.nature.com/scientificreports/ Methanosaeta harundinacea accumulated 5-times more methane and 4-times more acetate than a control culture of Methanosaeta on ethanol run in paralel (Fig. 3). However, a small amount of methane (ca. 1 mM) was produced by Methanosaeta in the control culture likely due to cells being transferred together with traces of acetate (an effective substrate for this methanogen). This was consistent with previous observations 10 .
Moreover, the co-culture of G. hydrogenophilus and Methanosaeta harundinacea produced 9-times more methane than co-cultures with Methanospirillum run in parallel (Fig. 1, p = 0.03). Methanospirrilum was previously shown not to pair successfully with Geobacter hydrogenophilus 5 . Here we have shown additionally that that G. hydrogenophilus favors pairing with a strict non-hydrogenotrophic methanogen (Methanosaeta harundinacea) over a strict hydrogenotroph (Methanospirillum hungatei). This supports the notion that while G. hydrogenophilus can produce H 2 9 it is an ineffective H 2 donor, unlike the H 2 -donating syntroph -P. carbinolicus 12 (Fig. 1).

Rhodoferrax ferrireducens paired syntrophically with Methanosarcinales but not with strict hydrogenotrophic methanogens.
With only two out of seven Geobacter species showing aptitude for DIET with Methanosarcinales, we verified whether other highly effective electrogens outside the Geobacter clade could pair syntrophically via DIET. We tested this possibility for an effective anode respiring Betaproteobacteria -Rhodoferax ferrireducens 25 . Interestingly, Rhodoferax was predicted to outcompete Geobacter in a subsurface environment with low substrate flux and relatively high ammonia 31 , where non-hydrogenotrophic Methanosarcina species co-exists 32 . The co-existence of Rhodoferax, Geobacter and Methanosarcina was also noted in coastal Baltic Sea sediments 32 . It is possible Methanosarcina species could receive DIET-electrons from Rhodoferax as well as Geobacter. Here we examined whether R. ferrireducens could establish interspecies electron transfer in co-cultures with 2 DIET methanogens (M. harundinacea, M. barkeri) or with 2 strict hydrogenotrophic methanogens (M. hungatei and M. formicicum). We expected that this efficient anode-respiring bacterium 25 would prefer DIET syntrophic partners to H 2 -utilizing partners. R. ferrireducens cannot utilize ethanol, therefore these co-cultures were provided with glucose (5 mM) as sole electron donor 33 . On glucose, Rhodoferax acts a respiratory organism and could not oxidize this substrate in the absence of an electron acceptor 34 . In these co-cultures, methane was used as a proxy for syntrophic metabolism. In order to estimate electron recovery from glucose, volatile fatty acid accumulation was determined during stationary phase. All co-cultures consumed the 5 mM glucose added (<4 µM detected after 270 days). Product recoveries varied significantly in Rhodoferax co-cultures with DIET-methanogens versus co-cultures with hydrogenotrophic methanogens (Fig. 4 and 4-inset). By comparing methane production in co-cultures with Rhodoferax, it was evident that M. harundinacea was the most effective at accumulating methane followed by Ms. barkeri and then strict hydrogenotrophs (Fig. 4). Rhodoferax in co-culture with Methanothrix had the highest total electron recovery (45%) with all electrons being recovered as methane, and none as acetate (Fig. 4-inset). The electrons not accounted for in products, are likely assimilated into biomass, typical of methanogenic metabolisms 35 . The Rhodoferax co-cultured with Methanosarcina was 3-fold less effective at recovering electrons into products (14%; Fig. 4-inset), yet the majority of the electrons were recovered as methane (12%) and only traces as acetate (2%). On the other hand, both co-cultures of Rhodoferax with strict hydrogenotrophs resulted in low electron recoveries as methane (ca. 5%) and more as acetate (18%) indicating hydrogenotrophic methanogens do not thrive in partnership with Rhodoferax. These results indicate that Rhodoferax favors interactions with DIET-methanogens rather than strict hydrogenotrophic methanogens. However, we do not know how Rhodoferax releases electrons to DIET methanogenic partners, although hints about its EET metabolism have been projected from genome screening 34,36 . The genome of R. ferrireducens contains 45 putative c-type cytochromes 34 and the entire Mtr-pathway suggesting R. ferrireducens may be doing EET similar to Shewanella 36,37 . It remains to be tested whether this pathway is also used for DIET syntrophy with methanogens.
Multiheme c-type cytochromes were not required for Diet or cathodic eet. Many studies have shown that multi-heme c-type cytochromes (MHC) are important for extracellular electron transfer 38 including EET during DIET 1,3,12,19,39 . However, MHCs are neither ubiquitous nor restricted to DIET-methanogens (Fig. 5).
Of the hydrogenotrophic methanogens tested, M. hungatei and M. marisnigri contained potential multiheme cytochrome proteins (Fig. 5), apparently localized in the cytoplasm (Fig. 5). Of the DIET-methanogens, 5 out of 7 species did not contain MHCs including all of the Methanosarcina barkeri strains and both Methanosaetaceae species (M. harundinacea and M. soehngenii). M. horonobensis and M. mazei were the only 2 species with MHC apparently localized on the membrane or secreted (Fig. 5). M. mazei's predicted MHC was detected within the surface/membrane-bound fraction by biochemical testing and predicted to be secreted extracellularly through leaderless secretion 40 where it is suggested to join a membrane-bound complex containing flavoproteins and iron-sulfur flavoproteins 41 . Our hypothesis was that if M. mazei required its MHC for extracellular electron transfer, cells without it would be unable to interact with a DIET syntroph or with a poised electrode. This approach was previously used to determine Geobacter's necessity for cell surface MHCs during EET to electrodes 42 , iron-oxide minerals 22 and DIET-partners 1,3,12 .
To test this hypothesis, we used a knock-out mutant of M. mazei (633k.o.) in which the gene (MM_0633) encoding for the putative multiheme c-type cytochrome was deleted 24 . The deletion mutant showed no phenotypic variability to the wild type when growing on its typical substrates (methanol and acetate) 24 . To determine whether this MHC was required to receive DIET electrons from Geobacter, M. mazei 633k.o. was incubated with G. metallireducens in syntrophic media with ethanol (Fig. 6). Methane production and ethanol oxidation progressed similar to wild type control incubations (Fig. 6) demonstrating that this MHC is not required for DIET.
Recently, it was reported that M. mazei was not electroactive and incapable to retrieve electrons from a poised cathode at −700 mV (vs. SHE) 43 . However, these experiments were carried out under conditional typically associated with electrochemical H 2 -generation 44 , and for a time frame of only 3 days, which is too short to test for the formation of electrical contacts for direct electromethanogenesis 11,45 . In fact, the authors observed only the growth of strict hydrogenotrophic methanogens on cathodes poised at −700 mV 43 . Strict hydrogenotrophic methanogens have low H 2 -thresholds (ca. 6 nM) 46 , unlike M. mazei, which grows poorly on H 2 , and has a high H 2 -threshold (ca. 300 nM) like all other Methanosarcinales 46 . Unlike the study by Meyer et al. 43 , our experiements were run at −400 mV vs. SHE, condition that does not allow for abiotic electrochemical H 2 accumulation even after several months 11 . In a recent study, we showed that a cathode at −400 mV could not be used as sole electron donor by H 2 -utilizing methanogens like Methanobacterium formicicum, but it was used by a strain capable of direct electron uptake (via DIET) -Methanosarcina barkeri strain MS 11 .
Here we tested whether another Methanosarcina, M. mazei could carry out direct electron uptake from a cathode at −400 mV. We compared a wild type M. mazei and an MHC deletion mutant of M. mazei (633k.o.) to www.nature.com/scientificreports www.nature.com/scientificreports/ see whether the absence of its one and only MHC impacts EET from a cathode. Both M. mazei strains with and without the multiheme cytochrome were incubated with a cathode poised at a voltage of −400 mV (vs. SHE), unfavorable for the H 2 -evolution reaction 11 . Control experiments were run alongside, without applying a voltage at the cathode, to verify whether methanogenesis can be induced by carry-over substrates. Only in experiments Figure 5. Predicted c-type cytochromes in methanogens tested for DIET. c-type cytochrome heme biding sites in 10 species of methanogens as predicted by CxxCH motif and/or annotated, this includes multiheme cytochromes; the predicted localization of the multiheme c-type cytochromes according to various bioinformatics tools. PSORTb -subcellular localization prediction tool 58 ; TMHMM -prediction of transmembrane helices in proteins 59 ; SOSUI-classification and secondary structure prediction system for membrane proteins 60 ; SignalP -location of signal peptide cleavage sites, which is Archaea specific 61 ; Pred-Signal -prediction of signal peptides in Archaea 62 ; TatP -prediction of Twin-arginine signal peptide cleavage sites 63 ; SecretomeP -prediction of non-classical protein secretion 64 . www.nature.com/scientificreports www.nature.com/scientificreports/ with a poised cathode, methane production proceeded effectively for 633k.o. and wild type M. mazei (Fig. 7), showing that the MHC is not required for electron uptake from a cathode. Previously, we observed that M. horonobensis which contains the highest number of MHCs among DIET-methanogens was also unable to use a cathode as electron donor 11 . Combined, these results disprove the hypothesis that Methanosarcina species require a multiheme c-type cytochrome for extracellular electron uptake.
For Methanosarcinales involved in EET/DIET, the first barrier for electrons to enter a cell is the cell envelope. Thus, for DIET to take place, the cell surface of Methanosarcinales is anticipated to harbor charge transferring molecules. Methanogens exhibit very different cell envelopes and among the methanogens examined in this paper, as many as five types of distinct cellular surface composition have been observed (Fig. 8).
DIET-associations have now been demonstrated strictly with methanogens of the order Methanosarcinales including members of the families Methanosarcinaceae (Methanosarcina-genus) and Methanosaetaceae (Methanothrix and Methanosaeta). What distinguishes the Methanosarcina genus from all other methanogens is a thick methanochondroitin sulfate (MS) layer (Fig. 8), which is steadily represented on cell surfaces grown at low osmolarity 47 . Until now, DIET has been only demonstrated under freshwater conditions when Methanosarcina cells would be coated by methanochodroitin sulfate (MCS). MCS is an exopolysacharide resembling chondroitin sulfate in eukaryotes 48 where it confers conduction via axonal length 49 . It is typical of exopolysaccharides like MCS to absorb metals 50 or even trap redox cofactors and c-type cytochromes 51 , thus the embedded redox centers within the surface matrix may confer very different electric properties. What distinguishes the cell surface of Methanosaeta/Methanothrix genus from other methanogens is that only a protein sheet is delineating the cell surface from the environment 52 (Fig. 8). The protein sheet was recently described in Methanosaeta thermophila to be composed of amyloid proteins 53 . Amyloid proteins are known to cluster together, while binding peptides 54 , and concentrating metal ions 55 . For example, the protein sheet of Methanothrix shoeghenii was described to concentrate metal ions like iron, copper, nickel and zinc 56 .
The surface structures of DIET methanogens of the order Methanosarcinales, although different in structure, have a shared attribute in binding/traping metal-ions to the cell surface, which we hypothesize to play a role in extracellular electron uptake by these Archaea.
Of the strict H 2 -utilizing methanogens, we observed that members of Methanomicrobiales and one Methanobacteriales were incapable to interact by DIET with electrogens. Typically, the membrane of these two groups are delineated from the environment by one single layer made either of pseudomurein (Methanobacteriales) or glycosylated S-layer proteins (Methanomicrobiales) 57 . Methanospirillum which is coated by an S-layer protein sheath was often compared to the protein sheet of Methanothrix (Fig. 8). However, the S-layer protein sheath of Methanospirillum traps less metal ions (2-5 fold less) than that of Methanothrix 56 .
These differences in surface biology may provide DIET-methanogens with a specific advantage to retrieve electrons from the extracellular environment and consequently an ecological niche where they outcompete H 2 -utilizers (e.g. mineral rich environments).

conclusion
The incidence of Geobacter in methanogenic environments is often used as signature for direct interspecies electron transfer. However, only two (G. metallireducens, G. hydrogenophilus), which were the most electroactive species out of seven Geobacter species tested were previously shown to pair succesfully with Methanosarcinales. On the other hand these two Geobacter were previously shown to be unable to pair with methanogens of the orders Methanobacteriales (Methanobacterium formicicum) and a Methanomicrobiales (Methanospirillum hungatei). Here, we have expanded the list of DIET partnerships between Geobacter and Methanosarcinales by six additional combinations; and confirm using another Methanomicrobiales (Methanoculleus marisnigri) that these electrogenic Geobacter cannot pair with a strict hydrogenotrophic methanogens. Additionally, we show that DIET may favor another effective electrogen outside the Geobacter-cluster -Rhodoferrax ferrireducens. We observed that