Extracellular electron uptake by two Methanosarcina species

Direct electron uptake by prokaryotes is a recently described mechanism with a potential application for energy and CO2 storage into value added chemicals. Members of Methanosarcinales, an environmentally and biotechnologically relevant group of methanogens, were previously shown to retrieve electrons from an extracellular electrogenic partner performing Direct Interspecies Electron Transfer (DIET) and were therefore proposed to be electroactive. However, their intrinsic electroactivity has never been examined. In this study, we tested two methanogens belonging to Methanosarcina, M. barkeri and M. horonobensis, regarding their ability to accept electrons directly from insoluble electron donors like other cells, conductive particles and electrodes. Both methanogens were able to retrieve electrons from Geobacter metallireducens via DIET. Furthermore, DIET was also stimulated upon addition of electrically conductive granular activated carbon (GAC) when each was co-cultured with G. metallireducens. However, when provided with a cathode poised at – 400 mV (vs SHE), only M. barkeri could perform electromethanogenesis. In contrast, the strict hydrogenotrophic methanogen, Methanobacterium formicicum, did not produce methane regardless of the type of insoluble electron donor provided (Geobacter cells, GAC or electrodes). A comparison of functional gene categories between Methanobacterium and the two Methanosarcinas revealed a higher abundance of genes associated with extracellular electron transfer in Methanosarcina species. Between the two Methanosarcina we observed differences regarding energy metabolism, which could explain dissimilarities concerning electromethanogenesis at fixed potentials. We suggest that these dissimilarities are minimized in the presence of an electrogenic DIET partner (i.e. Geobacter), which can modulate its surface redox potentials by adjusting the expression of electroactive surface proteins.

(Thermo-Scientific) with a TracePLOT™ TG-BOND Msieve 5A column and a thermal conductivity 147 detector (TCD). The carrier gas was argon at a flow rate of 25 mL/min. The injector, oven and detector 148 temperatures were 150 o C, 70 o C and 200 o C respectively. The detection limit for CH 4 and H 2 was ca. 149 500 ppm for both. The concentration unit was converted to molarity by using the ideal gas law (pV =  168 horonobensis and M. formicicum respectively. For searching the cytochrome motif (CxxCH), the 3of5 pattern matching application (Seiler et al., 2006) in addition to manual search was used to scan through 170 all the genomes.

172
It was previously shown that two Methanosarcinales, M. barkeri and M. harundinacea grew via DIET 173 whereas strict hydrogenotrophs did not (Rotaru et al., 2014a(Rotaru et al., , 2014b. This indicated that the 174 Methanosarcinales members were likely capable of extracellular electron uptake. Here we show that 175 indeed M. barkeri could retrive electrons not only from an exoelectrogen but also from an electrode 176 poised at -400 mV (non-hydrogen generating conditions) to carry electromethanogenesis. As expected, 177 a hydrogenotrophic methanogen M. formicicum did not carry electromethanogenesis under this 178 condition. We tested a non-hydrogenotrophic Methanosarcina, M. horonobensis for extracellular 179 electron uptake from cells and electrodes, and we observed it could only retrieve electrons from 180 exoelectrogenic Geobacter and from granular activated carbon but not from electrodes.

181
A strict hydrogenotroph, Methanobacterium formicicum, was unable to produce methane using 182 extracellular electron uptake from cells or solid electron donors 183 M. formicicum was chosen as the representative hydrogenotrophic methanogen due to its low hydrogen 184 uptake threshold (approximately 6 nM (Lovley, 1985)). Previously it was demonstrated that the 185 electrogen G. metallireducens could not establish DIET co-cultures with the hydrogenotroph M. 186 formicicum even after 6 months of incubation (Rotaru et al., 2014b). This result was anticipated 187 because G. metallireducens is a respiratory organism without the genetic potential to produce H 2 188 (Aklujkar et al., 2009;Shrestha et al., 2013), which was also demonstrated in early physiological 189 experiments (Cord-Ruwisch et al., 1998) Fig. 2A). Still, since the inoculum was a mid-exponential active culture, there was a possibility that 209 microbial enzymes attach onto the electrode to produce hydrogen or formate (Deutzmann et al., 2015).

210
M. formicicum is particularly well-suited for such a test because, the H 2 threshold of such a strict 211 hydrogenotroph is lower than that of Methanosarcina-methanogens (Thauer et al., 2008(Thauer et al., , 2010. For instance, the dissolved H 2 threshold of the hydrogenotrophic M. formicicum (~ 6 nM) is ~60 times 213 lower than that of M. barkeri (296 nM -376 nM) (Kral et al., 1998;Lovley, 1985). In electrochemical 214 reactors with M. formicicum, no hydrogen or methane was detected and no substantial current draw 215 was observed at -400 mV (Fig. 2B). Thus, M. formicicum could not carry electromethanogenesis 216 neither via electrochemical nor enzyme-mediated H 2 in our bioelectrochemical set-up.

217
The non-hydrogenotrophic Methanosarcina horonobensis produced methane using extracellular 218 electron uptake from Geobacter directly or via conductive particles 219 The non-hydrogenotrophic, Methanosarcina horonobensis paired syntrophically with G. 220 metallireducens with or without conductive particles as electric conduit (Fig 3A & B). This is the  M. barkeri encoded for more 86% more N 2 -fixation proteins and 13% more heme-biosynthesis 284 proteins.

285
Beside these differences, we also observed significant differences regarding energy metabolism (Table   286 3). M. barkeri utilizes hydrogen-cycling for its energy metabolism employing the energy-converting