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Favorable interspecies associations prevail in natural microbial assemblages. Some of these favorable associations are co-metabolic dependent partnerships in which extracellular electrons are exchanged between species. For such electron exchange to occur, the cells must exhibit electroactive interfaces and get involved in direct cell-to-cell contact (Direct Interspecies Electron Transfer/DIET) or use available conductive mineral grains from their environment (Conductive-particle-mediated Interspecies Electron Transfer/CIET). This review will highlight recent discoveries and knowledge gaps regarding DIET and CIET interspecies associations in artificial co-cultures and consortia from natural and man-made environments and emphasize approaches to validate DIET and CIET. Additionally, we acknowledge the initiation of a movement towards applying electric syntrophies in biotechnology, bioremediation and geoengineering for natural attenuation of toxic compounds. Next, we have highlighted the urgent research needs that must be met to develop such technologies.

Electromicrobiology is an emerging field investigating and exploiting the interaction of microorganisms with insoluble electron donors or acceptors. Some of the most recently categorized electroactive microorganisms became of interest to sustainable bioengineering practices. However, laboratories worldwide typically maintain electroactive microorganisms on soluble substrates, which often leads to a decrease or loss of the ability to effectively exchange electrons with solid electrode surfaces. In order to develop future sustainable technologies, we cannot rely solely on existing lab-isolates. Therefore, we must develop isolation strategies for environmental strains with electroactive properties superior to strains in culture collections. In this article, we provide an overview of the studies that isolated or enriched electroactive microorganisms from the environment using an anode as the sole electron acceptor (electricity-generating microorganisms) or a cathode as the sole electron donor (electricity-consuming microorganisms). Next, we recommend a selective strategy for the isolation of electroactive microorganisms. Furthermore, we provide a practical guide for setting up electrochemical reactors and highlight crucial electrochemical techniques to determine electroactivity and the mode of electron transfer in novel organisms.

Electromicrobiology is an emerging field investigating and exploiting the interaction of microorganisms with insoluble electron donors or acceptors. Some of the most recently categorized electroactive microorganisms became of interest to sustainable bioengineering practices. However, laboratories worldwide typically maintain electroactive microorganisms on soluble substrates, which often leads to a decrease or loss of the ability to effectively exchange electrons with solid electrode surfaces. In order to develop future sustainable technologies, we cannot rely solely on existing lab-isolates. Therefore, we must develop isolation strategies for environmental strains with electroactive properties superior to strains in culture collections. In this article, we provide an overview of the studies that isolated or enriched electroactive microorganisms from the environment using an anode as the sole electron acceptor (electricity-generating microorganisms) or a cathode as the sole electron donor (electricity-consuming microorganisms). Next, we recommend a selective strategy for the isolation of electroactive microorganisms. Furthermore, we provide a practical guide for setting up electrochemical reactors and highlight crucial electrochemical techniques to determine electroactivity and the mode of electron transfer in novel organisms.

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.

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 H 2 -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.

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 H 2 -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.

Direct electron uptake by prokaryotes is a recently described mechanism with a potential application for energy and CO 2 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 the genus 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 the two Methanosarcina showed 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 (e.g. Geobacter ), which can modulate its surface redox potentials by adjusting the expression of electroactive surface proteins.

Previous studies of acetate-promoted bioremediation of uranium-contaminated aquifers focused on Geobacter because no other microorganisms that can couple the oxidation of acetate with U(VI) reduction had been detected in situ. Monitoring the levels of methyl CoM reductase subunit A (mcrA) transcripts during an acetate-injection field experiment demonstrated that acetoclastic methanogens from the genus Methanosarcina were enriched after 40 days of acetate amendment. The increased abundance of Methanosarcina corresponded with an accumulation of methane in the groundwater. In order to determine whether Methanosarcina species could be participating in U(VI) reduction in the subsurface, cell suspensions of Methanosarcina barkeri were incubated in the presence of U(VI) with acetate provided as the electron donor. U(VI) was reduced by metabolically active M. barkeri cells; however, no U(VI) reduction was observed in inactive controls. These results demonstrate that Methanosarcina species could play an important role in the long-term bioremediation of uranium-contaminated aquifers after depletion of Fe(III) oxides limits the growth of Geobacter species. The results also suggest that Methanosarcina have the potential to influence uranium geochemistry in a diversity of anaerobic sedimentary environments.

Direct electron uptake by prokaryotes is a recently described mechanism with a potential application for energy and CO 2 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 the genus 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 the two Methanosarcina showed 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 (e.g. Geobacter ), which can modulate its surface redox potentials by adjusting the expression of electroactive surface proteins.

This document is a research article published in the journal Frontiers in Microbiology. It acknowledges the contributions of a researcher named Barny to the field of methanogen research and the broader microbial field. The article highlights Barny's work in clarifying the taxonomy of methanogens and recognizes his wider contributions as an editor. The article also includes information about the funding sources for the research and acknowledges the authors and reviewers who contributed to the research topic. It concludes with a statement about potential conflicts of interest and a note from the publisher. [Extracted from the article]

Improving methane production through electrical current application to anaerobic digesters has garnered interest in optimizing such microbial electrochemical technologies, with claims suggesting direct interspecies electron transfer (DIET) at the cathode enhances methane yield. However, previous studies with mixed microbial communities only reported interspecies interactions based on species co‐occurrence at the cathode, lacking insight into how a poised cathode influences well‐defined DIET‐based partnerships. To address this, we investigated the impact of continuous and discontinuous exposure to a poised cathode (−0.7 V vs. standard hydrogen electrode) on a defined consortium of Geobacter metallireducens and Methanosarcina barkeri, known for their DIET capabilities. The physiology of DIET consortia exposed to electrical current was compared to that of unexposed consortia. In current‐exposed incubations, overall metabolic activity and cell numbers for both partners declined. The consortium, receiving electrons from the poised cathode, accumulated acetate and hydrogen, with only 32% of the recovered electrons allocated to methane production. Discontinuous exposure intensified these detrimental effects. Conversely, unexposed control reactors efficiently converted ethanol to methane, transiently accumulating acetate and recovering 88% of electrons in methane. Our results demonstrate the overall detrimental effect of electrochemical stimulation on a DIET consortium. Besides, the data indicate that the presence of an alternative electron donor (cathode) hinders efficient electron retrieval by the methanogen from Geobacter, and induces catabolic repression of oxidative metabolism in Geobacter. This study emphasizes understanding specific DIET‐based interactions to enhance methane production during electrical stimulation, providing insights for optimizing tailored interspecies partnerships in microbial electrochemical technologies. [ABSTRACT FROM AUTHOR]

Diverse physiological groups congregate into environmental corrosive biofilms, yet the interspecies interactions between these corrosive physiological groups are seldom examined. We, therefore, explored Fe0-dependent cross-group interactions between acetogens and methanogens from lake sediments. On Fe0, acetogens were more corrosive and metabolically active when decoupled from methanogens, whereas methanogens were more metabolically active when coupled with acetogens. This suggests an opportunistic (win–loss) interaction on Fe0 between acetogens (loss) and methanogens (win). Clostridia and Methanobacterium were the major candidates doing acetogenesis and methanogenesis after four transfers (metagenome sequencing) and the only groups detected after 11 transfers (amplicon sequencing) on Fe0. Since abiotic H2 failed to explain the high metabolic rates on Fe0, we examined whether cell exudates (spent media filtrate) promoted the H2-evolving reaction on Fe0 above abiotic controls. Undeniably, spent media filtrate generated three- to four-fold more H2 than abiotic controls, which could be partly explained by thermolabile enzymes and partly by non-thermolabile constituents released by cells. Next, we examined the metagenome for candidate enzymes/shuttles that could catalyze H2 evolution from Fe0 and found candidate H2-evolving hydrogenases and an almost complete pathway for flavin biosynthesis in Clostridium. Clostridial ferredoxin-dependent [FeFe]-hydrogenases may be catalyzing the H2-evolving reaction on Fe0, explaining the significant H2 evolved by spent media exposed to Fe0. It is typical of Clostridia to secrete enzymes and other small molecules for lytic purposes. Here, they may secrete such molecules to enhance their own electron uptake from extracellular electron donors but indirectly make their H2-consuming neighbors— Methanobacterium —fare five times better in their presence. The particular enzymes and constituents promoting H2 evolution from Fe0 remain to be determined. However, we postulate that in a static environment like corrosive crust biofilms in lake sediments, less corrosive methanogens like Methanobacterium could extend corrosion long after acetogenesis ceased, by exploiting the constituents secreted by acetogens. [ABSTRACT FROM AUTHOR]

Direct interspecies electron transfer (DIET) via electrically conductive minerals can play a role in the anaerobic oxidation of petroleum hydrocarbons in contaminated sites and can be exploited for the development of new, more effective bioremediation approaches. The first observation of a DIET association was between two I Geobacter i species, I Geobacter metallireducens i and I Geobacter sulfurreducens, i grown on a medium provided with an electron donor (ethanol) for the first species, and an electron acceptor (fumarate) for the other species. Similarly, the electron transfer mechanisms actually involved in the biodegradation processes have been rarely experimentally verified, primarily due to intrinsic methodological difficulties in tracking the involved electron carriers (i.e. H SB 2 sb , formate, acetate) transiently accumulating only at extremely low concentration levels and the lack of alternative investigation techniques. (2010) may have a role in recycling electron acceptors such as sulphate in hydrocarbon contaminated anoxic environments (Müller I et al i ., 2016) and thus sustaining anaerobic hydrocarbon degradation. [Extracted from the article]