Interspecies Electron Exchange Research Articles

Unnatural Direct Interspecies Electron Transfer Enabled by Living Cell-Cell Click Chemistry.

Direct interspecies electron transfer (DIET) is essential for maintaining the function and stability of anaerobic microbial consortia. However, only limited natural DIET modes have been identified and DIET engineering remains highly challenging. In this study, an unnatural DIET between Shewanella oneidensis MR-1 (SO, electron donating partner) and Rhodopseudomonas palustris (RP, electron accepting partner) was artificially established by a facile living cell-cell click chemistry strategy. By introducing alkyne- or azide-modified monosaccharides onto the cell outer surface of the target species, precise covalent connections between different species in high proximity were realized through a fast click chemistry reaction. Remarkably, upon covalent connection, outer cell surface C-type cytochromes mediated DIET between SO and RP was achieved and identified, although this was never realized naturally. Moreover, this connection directly shifted the natural H2 mediated interspecies electron transfer (MIET) to DIET between SO and RP, which delivered superior interspecies electron exchange efficiency. Therefore, this work demonstrated a naturally unachievable DIET and an unprecedented MIET shift to DIET accomplished by cell-cell distance engineering, offering an efficient and versatile solution for DIET engineering, which extends our understanding of DIET and opens up new avenues for DIET exploration and applications.

Unnatural Direct Interspecies Electron Transfer Enabled by Living Cell‐Cell Click Chemistry

Direct interspecies electron transfer (DIET) is essential for maintaining the function and stability of anaerobic microbial consortia. However, only limited natural DIET modes have been identified and DIET engineering remains highly challenging. Here, an unnatural DIET between Shewanella oneidensis MR‐1 (SO, electron donating partner) and Rhodopseudomonas palustris (RP, electron accepting partner) was artificially established by a facile living cell‐cell click chemistry strategy. By introducing alkyne‐ or azide‐modified monosaccharides onto the cell outer surface of the target species, precise covalent connections between different species in high proximity were realized via a fast click chemistry reaction. Remarkably, upon covalent connection, outer cell surface C‐type cytochromes mediated DIET between SO and RP was achieved and identified, although this was never realized naturally. Moreover, this connection directly shifted the natural H2 mediated interspecies electron transfer (MIET) to DIET between SO and RP, which delivered superior interspecies electron exchange efficiency. Therefore, this work demonstrated a naturally unachievable DIET and an unprecedented MIET shift to DIET accomplished by cell‐cell distance engineering, offering an efficient and versatile solution for DIET engineering, which would extend our understanding of DIET and open up new avenue for DIET exploration and applications.

The survival strategy of direct interspecies electron transfer-capable coculture under electron donor-limited environments

Direct interspecies electron transfer (DIET) has been considered as an effective mechanism for interspecies electron exchange in microbial syntrophy. Understanding DIET-capable syntrophic associations under energy-limited environments is important because these conditions more closely approximate those found in natural subsurface environments than in the batch cultures in the laboratory. This study, investigated the metabolic dynamics and electron transfer mechanisms in DIET-capable syntrophic coculture of Geobacter metallireducens and Geobacter sulfurreducens under electron donor-limited condition. The wild-coculture and the mutant-coculture with a citrate synthase-deficient G. sulfurreducens exhibited similar rates of syntrophic metabolism under ethanol-limited and ethanol-replete conditions. Transcriptomic analyses revealed that, in the mutant-coculture in which interspecies electron exchange was the sole electron source for G. sulfurreducens, the transcription of genes associated with uptake hydrogenase in G. sulfurreducens were significantly repressed and thus DIET tended to be the preferred mode of interspecies electron exchange under electron donor-limited condition. To overcome electron donor limitation, c-type cytochromes in the coculture actively moved from outer membrane to extracellular environment, potentially via increased secretion of outer-membrane vesicles. These results suggested a preferred electron transfer mechanism for DIET-capable syntrophic communities' survival in the electron donor-limited environments, providing valuable insights into the biogeochemical processes mediated by DIET in natural and engineered environments.

Genomic Insights into Syntrophic Lifestyle of 'Candidatus Contubernalis alkaliaceticus' Based on the Reversed Wood-Ljungdahl Pathway and Mechanism of Direct Electron Transfer.

The anaerobic oxidation of fatty acids and alcohols occurs near the thermodynamic limit of life. This process is driven by syntrophic bacteria that oxidize fatty acids and/or alcohols, their syntrophic partners that consume the products of this oxidation, and the pathways for interspecies electron exchange via these products or direct interspecies electron transfer (DIET). Due to the interdependence of syntrophic microorganisms on each other's metabolic activity, their isolation in pure cultures is almost impossible. Thus, little is known about their physiology, and the only available way to fill in the knowledge gap on these organisms is genomic and metabolic analysis of syntrophic cultures. Here we report the results of genome sequencing and analysis of an obligately syntrophic alkaliphilic bacterium 'Candidatus Contubernalis alkaliaceticus'. The genomic data suggest that acetate oxidation is carried out by the Wood-Ljungdahl pathway, while a bimodular respiratory system involving an Rnf complex and a Na+-dependent ATP synthase is used for energy conservation. The predicted genomic ability of 'Ca. C. alkaliaceticus' to outperform interspecies electron transfer both indirectly, via H2 or formate, and directly, via pili-like appendages of its syntrophic partner or conductive mineral particles, was experimentally demonstrated. This is the first indication of DIET in the class Dethiobacteria.

Electro-polarization of the sludge with dynamic magnetic field enhanced the interspecies electron transfer in ZVI-added anaerobic digesters

Zero-valent iron (ZVI) has been widely reported to strengthen anaerobic digestion; however, ZVI passivation prevents its continuous function. In this study, a dynamic magnetic field (DMF) was imposed on a ZVI-added anaerobic digester treating food waste to promote microbial interspecies electron transfer to enhance methane production. Results showed that methane production with DMF was 15.3% higher than that without DMF. Meanwhile, the Fe(II) concentration from ZVI increased by 39.46% with DMF, but the electron equivalent was far less than the increased methane production even if all electrons from the ZVI were used for hydrogenotrophic methanogenesis. In agreement, the relative abundances of hydrogenotrophic methanogens were less changed after DMF. Notably, an induced electric current ranging from −0.017–0.021 μA was detected with DMF. With the DMF-induced current as electrical stimulation, the sludge conductivity and capacitance increased by 242% and 18%, respectively. In-situ electrochemical tests on the functional groups of the sludge showed that the protein-like substances were polarized by the induced electromotive force to enhance the proton-coupled electron transfer in the sludge. Together with the enriched electrotrophic methanogens, the enhanced microbial interspecies electron exchange was the main reason for the improved performance of anaerobic digestion with DMF.

Coupling direct voltage and granular activated carbon modified nanoscale zero valent iron for enhancing anaerobic methane production

Anaerobic digestion technology has been widely used because it has a unique advantage of producing biogas as a renewable energy source. Therefore, several methods were studied to facilitate anaerobic methane production process. Coupling direct voltage and single conductive particles was an effective method to improve anaerobic wastewater treatment efficiency and methane production. However, the enhancement method was limited in this process due to the current of direct voltage or the toxicity of nanoparticles. Therefore, the granular activated carbon loaded with nanoscale zero valent iron (GAC-NZVI) particles prepared by co-precipitation method were added to the anaerobic synthetic wastewater system with direct voltage (0.10 V) to improve the treatment efficiency in this study. GAC-NZVI particles were added into anaerobic system with 0.10 V direct voltage to enhance CH4 production process. The COD removal and total CH4 production were enhanced by 4.22 % and 10.83 % with GAC-NZVI particles. The measurement results of EPS and Fe concentration showed that GAC-NZVI particles promoted the secretion of EPS by microorganisms, which could improve the floc strength of granular sludge. The measurements of conductivity and cyclic voltammetry (CV) showed that particles accelerated the metabolism of microorganism and promoted the electron transfer process. The increasing of Methanothrix and Methanobacterium could strengthen the methanogenesis. The abundances of bacteria and archaea using indirect interspecies electron exchange form (such as H2 or formate transfer microorganisms) were decreased after adding the particles. The results indicated that anaerobic treatment efficiency could be enhanced under the combined action of direct voltage and particles.

Sparking Anaerobic Digestion: Promoting Direct Interspecies Electron Transfer to Enhance Methane Production.

Anaerobic digestion was one of the first bioenergy strategies developed, yet the interactions of the microbial community that is responsible for the production of methane are still poorly understood. For example, it has only recently been recognized that the bacteria that oxidize organic waste components can forge electrical connections with methane-producing microbes through biologically produced, protein-based, conductive circuits. This direct interspecies electron transfer (DIET) is faster than interspecies electron exchange via diffusive electron carriers, such as H2. DIET is also more resilient to perturbations such as increases in organic load inputs or toxic compounds. However, with current digester practices DIET rarely predominates. Improvements in anaerobic digestion associated with the addition of electrically conductive materials have been attributed to increased DIET, but experimental verification has been lacking. This deficiency may soon be overcome with improved understanding of the diversity of microbes capable of DIET, which is leading to molecular tools for determining the extent of DIET. Here we review the microbiology of DIET, suggest molecular strategies for monitoring DIET in anaerobic digesters, and propose approaches for re-engineering digester design and practices to encourage DIET.

Probing the Role of Metal Coordination and pH in Assembly and Function of Cytochrome Nanowires

Geobacter sulfurreducens produces protein nanowires for extracellular electron transfer during respiration and interspecies electron exchange to survive in environments that lack soluble electron acceptors. It was previously thought that nanowires were Type IV pili comprised by PilA monomers, but we showed via electron cryo-microscopy (cryo-EM) that nanowires instead constitute a helical polymer of the six-heme outer membrane cytochrome OmcS, yielding a periodic arrangement of hemes over the micrometer scale of the filaments (Cell, 2019, 177, 361). This 3.7 Å-resolution structure revealed several features which appear to stabilize this polymer, including close stacking of covalently bound heme ligands within ∼ 3.5-6 Å of each other, and axial coordination of a heme metal center in each subunit by a histidine residue in the adjacent subunit. These OmcS filaments represent to our knowledge the first micrometer-scale cytochrome polymer naturally evolved for electron transfer, but the mechanism of this polymerization remains unknown. Furthermore, lowering the pH of nanowire samples enhances their conductivity and improves heme stacking, yet the structural changes in OmcS underlying this conformation change are unclear. Here, we develop a method to obtain OmcS nanowires and monomers in high purity. We then combine cryo-EM with spectroscopic methods such as UV-vis and circular dichroism to examine the changes in OmcS polymers and their inter-subunit interactions as a result of changes to pH, further enabling tuning of the conductivity of purified microbial nanowires. Furthermore, we explore the spectroscopic features and biochemical properties of OmcS monomers after reversing their polymerization, to obtain a mechanistic understanding of nanowire assembly that will aid engineering of synthetic protein nanowires for bioelectronics. This research was supported by Career Award from Burroughs Welcome Fund, NIH New Innovator award NSF CAREER award and DARPA (to N.S.M.).

Syntrophus conductive pili demonstrate that common hydrogen-donating syntrophs can have a direct electron transfer option.

Syntrophic interspecies electron exchange is essential for the stable functioning of diverse anaerobic microbial communities. Hydrogen/formate interspecies electron transfer (HFIT), in which H2 and/or formate function as diffusible electron carriers, has been considered to be the primary mechanism for electron transfer because most common syntrophs were thought to lack biochemical components, such as electrically conductive pili (e-pili), necessary for direct interspecies electron transfer (DIET). Here we report that Syntrophus aciditrophicus, one of the most intensively studied microbial models for HFIT, produces e-pili and can grow via DIET. Heterologous expression of the putative S. aciditrophicus type IV pilin gene in Geobacter sulfurreducens yielded conductive pili of the same diameter (4 nm) and conductance of the native S. aciditrophicus pili and enabled long-range electron transport in G. sulfurreducens. S. aciditrophicus lacked abundant c-type cytochromes often associated with DIET. Pilin genes likely to yield e-pili were found in other genera of hydrogen/formate-producing syntrophs. The finding that DIET is a likely option for diverse syntrophs that are abundant in many anaerobic environments necessitates a reexamination of the paradigm that HFIT is the predominant mechanism for syntrophic electron exchange within anaerobic microbial communities of biogeochemical and practical significance.

Recyclable magnetite-enhanced electromethanogenesis for biomethane production from wastewater

Improving the yield and methane content of biogas is of great concern for wastewater treatment by anaerobic digestion. Herein we developed a nanomagnetite-enhanced electromethanogenesis (EMnano) process for the first time, the sustainable utilization of which improved the biomethane production rate from dairy wastewater. The maximum CH4 production rate in the EMnano process is 2.3 ± 0.3-fold higher than it is in the conventional methanogenesis (CM) process, and it is accompanied by an almost delay-free start-up. The technical-economic evaluation revealed that an 82.1 ± 5.0% greater net benefit was obtained in the third generation of the EMnano process compared with the CM process. The improved methanogenesis was attributed to the formation of dense planktonic cell co-aggregates that are triggered by nanomagnetite, which facilitated the interspecies electron exchange during acetoclastic methanogenesis. Simultaneously, a cathode biofilm with high viability and catalytic activity was also formed in the EMnano process that decreased the biofilm resistance and facilitated the electron transfer during electromethanogenesis. This study is a worthwhile attempt to combine recyclable conductive materials with an electromethanogenesis process for wastewater treatment, and it effectively achieves energy recovery with high stability and cost-effectiveness.

Application of ethanol-type fermentation in establishment of direct interspecies electron transfer: A practical engineering case study

A promising strategy for establishing direct interspecies electron transfer (DIET)-based syntrophic metabolism during anaerobic digestion (AD) is to stimulate the methanogenic communities with ethanol to rapidly produce biological electrical connections. In this study the strategy was further explored in an engineering-scale up-flow anaerobic sludge blanket (UASB) reactor treating bagasse wastes. The results demonstrated that, under the conditions employed, the ethanol-abundant acidogenic products from ethanol-type fermentation could stimulate the UASB reactor to form the aggregates with a higher conductivity (22.6 ± 0.6 uS/cm) than that in the laboratory-scale UASB reactor treating brewery wastes (8.1 ± 1.7 uS/cm), in which DIET was the primary working mode of interspecies electron exchange. Furthermore, these aggregates also presented a positive response with granular active carbon (GAC) to methanogenesis, suggesting that the DIET-based methanogenic communities were established. As a result, the engineering-scale UASB reactor was capable of maintaining stability in response to the environmental disturbance. Microbial community analysis revealed that specific and substantial enrichments of Petrimonas (21.8% of abundance) and Methanothrix species (64.8%) were detected in the aggregates. These results, and the known ability of Petrimonas species to transfer electrons to elemental sulfur, suggested that Petrimonas species might participate in DIET with Methanothrix species.

Geobacter Strains Expressing Poorly Conductive Pili Reveal Constraints on Direct Interspecies Electron Transfer Mechanisms

ABSTRACTCytochrome-to-cytochrome electron transfer and electron transfer along conduits of multiple extracellular magnetite grains are often proposed as strategies for direct interspecies electron transfer (DIET) that do not require electrically conductive pili (e-pili). However, physical evidence for these proposed DIET mechanisms has been lacking. To investigate these possibilities further, we constructed Geobacter metallireducens strain Aro-5, in which the wild-type pilin gene was replaced with the aro-5 pilin gene that was previously shown to yield poorly conductive pili in Geobacter sulfurreducens strain Aro-5. G. metallireducens strain Aro-5 did not reduce Fe(III) oxide and produced only low current densities, phenotypes consistent with expression of poorly conductive pili. Like G. sulfurreducens strain Aro-5, G. metallireducens strain Aro-5 displayed abundant outer surface cytochromes. Cocultures initiated with wild-type G. metallireducens as the electron-donating strain and G. sulfurreducens strain Aro-5 as the electron-accepting strain grew via DIET. However, G. metallireducens Aro-5/G. sulfurreducens wild-type cocultures did not. Cocultures initiated with the Aro-5 strains of both species grew only when amended with granular activated carbon (GAC), a conductive material known to be a conduit for DIET. Magnetite could not substitute for GAC. The inability of the two Aro-5 strains to adapt for DIET in the absence of GAC suggests that there are physical constraints on establishing DIET solely through cytochrome-to-cytochrome electron transfer or along chains of magnetite. The finding that DIET is possible with electron-accepting partners that lack highly conductive pili greatly expands the range of potential electron-accepting partners that might participate in DIET.

Gene expression and ultrastructure of meso- and thermophilic methanotrophic consortia.

SummaryThe sulfate‐dependent, anaerobic oxidation of methane (AOM) is an important sink for methane in marine environments. It is carried out between anaerobic methanotrophic archaea (ANME) and sulfate‐reducing bacteria (SRB) living in syntrophic partnership. In this study, we compared the genomes, gene expression patterns and ultrastructures of three phylogenetically different microbial consortia found in hydrocarbon‐rich environments under different temperature regimes: ANME‐1a/HotSeep‐1 (60°C), ANME‐1a/Seep‐SRB2 (37°C) and ANME‐2c/Seep‐SRB2 (20°C). All three ANME encode a reverse methanogenesis pathway: ANME‐2c encodes all enzymes, while ANME‐1a lacks the gene for N5,N10‐methylene tetrahydromethanopterin reductase (mer) and encodes a methylenetetrahydrofolate reductase (Met). The bacterial partners contain the genes encoding the canonical dissimilatory sulfate reduction pathway. During AOM, all three consortia types highly expressed genes encoding for the formation of flagella or type IV pili and/or c‐type cytochromes, some predicted to be extracellular. ANME‐2c expressed potentially extracellular cytochromes with up to 32 hemes, whereas ANME‐1a and SRB expressed less complex cytochromes (≤ 8 and ≤ 12 heme respectively). The intercellular space of all consortia showed nanowire‐like structures and heme‐rich areas. These features are proposed to enable interspecies electron exchange, hence suggesting that direct electron transfer is a common mechanism to sulfate‐dependent AOM, and that both partners synthesize molecules to enable it.

Syntrophy Goes Electric: Direct Interspecies Electron Transfer.

Direct interspecies electron transfer (DIET) has biogeochemical significance, and practical applications that rely on DIET or DIET-based aspects of microbial physiology are growing. Mechanisms for DIET have primarily been studied in defined cocultures in which Geobacter species are one of the DIET partners. Electrically conductive pili (e-pili) can be an important electrical conduit for DIET. However, there may be instances in which electrical contacts are made between electron transport proteins associated with the outer membranes of the partners. Alternatively, DIET partners can plug into conductive carbon materials, such as granular activated carbon, carbon cloth, and biochar, for long-range electron exchange without the need for e-pili. Magnetite promotes DIET, possibly by acting as a substitute for outer-surface c-type cytochromes. DIET is the primary mode of interspecies electron exchange in some anaerobic digesters converting wastes to methane. Promoting DIET with conductive materials shows promise for stabilizing and accelerating methane production in digesters, permitting higher organic loading rates. Various lines of evidence suggest that DIET is important in terrestrial wetlands, which are an important source of atmospheric methane. DIET may also have a role in anaerobic methane oxidation coupled to sulfate reduction, an important control on methane releases. The finding that DIET can serve as the source of electrons for anaerobic photosynthesis further broadens its potential environmental significance. Microorganisms capable of DIET are good catalysts for several bioelectrochemical technologies and e-pili are a promising renewable source of electronic materials. The study of DIET is in its early stages, and additional investigation is required to better understand the diversity of microorganisms that are capable of DIET, the importance of DIET to carbon and electron flow in anaerobic environments, and the biochemistry and physiology of DIET.

Stimulation of the anaerobic digestion of the dry organic fraction of municipal solid waste (OFMSW) with carbon-based conductive materials

Growth of bacterial and archaeal species capable of interspecies electron exchange was stimulated by addition of conductive materials (carbon cloth or granular activated carbon (GAC)) to anaerobic digesters treating dog food (a substitute for the dry-organic fraction of municipal solid waste (OFMSW)). Methane production (772–1428mmol vs <80mmol), volatile solids removal (78%–81% vs 54%–64%) and COD removal efficiencies (∼80% vs 20%–30%) were all significantly higher in reactors amended with GAC or carbon cloth than controls. OFMSW degradation was also significantly accelerated and VFA concentrations were substantially lower in reactors amended with conductive materials. These results suggest that both conductive materials (carbon cloth and GAC) can promote conversion of OFMSW to methane even in the presence of extremely high VFA concentrations (∼500mM).

Potentially shifting from interspecies hydrogen transfer to direct interspecies electron transfer for syntrophic metabolism to resist acidic impact with conductive carbon cloth

Anaerobic digesters usually become sour during the operation and inhibit methanogenesis. This effect occurs due to the interspecies electron exchange for the syntrophic metabolism of alcohols and volatile fatty acids (VFAs) via interspecies H2 transfer (IHT). Conductive materials can promote direct interspecies electron transfer (DIET) between the syntrophs and methanogens, providing an alternative to IHT. However, the cooperative mechanism of these two working modes during syntrophic metabolism is unknown, especially during acidic impacts. The results of this study demonstrated that anaerobic digesters supplemented with conductive carbon cloth had a higher capacity to resist the acidic impacts. Conversely, the digesters supplemented with non-conductive cotton cloth (control) were nearly stagnant when the H2 partial pressure of the anaerobic systems increased. Further experiments at high H2 partial pressure to inhibit IHT exhibited almost no effect on the syntrophic metabolism in the carbon cloth-supplemented group, in which the methane production approached the stoichiometric production (350mLCH4/gCODremoval), while the methane production in the control group ceased. The microbial community analysis revealed that the surface sludge attached to the carbon cloth had the highest abundance of Geobacter and Methanosaeata species known to participate in DIET. These results suggested that the predominant working mode for the interspecies electron exchange might have shifted from IHT to DIET in the presence of the conductive carbon cloth during acidic impacts. DIET primarily occurred on the surface sludge of the carbon cloth and replaced IHT to proceed the syntrophic metabolism and maintained stable anaerobic methanogenesis.

Plugging in or going wireless: strategies for interspecies electron transfer.

Interspecies exchange of electrons enables a diversity of microbial communities to gain energy from reactions that no one microbe can catalyze. The first recognized strategies for interspecies electron transfer were those that relied on chemical intermediates that are recycled through oxidized and reduced forms. Well-studied examples are interspecies H2 transfer and the cycling of sulfur intermediates in anaerobic photosynthetic communities. Direct interspecies electron transfer (DIET) in which two species establish electrical contact is an alternative. Electrical contacts documented to date include electrically conductive pili, as well as conductive iron minerals and conductive carbon moieties such as activated carbon and biochar. Interspecies electron transfer is central to the functioning of methane-producing microbial communities. The importance of interspecies H2 transfer in many methanogenic communities is clear, but under some circumstances DIET predominates. It is expected that further mechanistic studies and broadening investigations to a wider range of environments will help elucidate the factors that favor specific forms of interspecies electron exchange under different environmental conditions.

Long-range electron transport to Fe(III) oxide via pili with metallic-like conductivity

The mechanisms for Fe(III) oxide reduction by Geobacter species are of interest because Geobacter species have been shown to play an important role in Fe(III) oxide reduction in a diversity of environments in which Fe(III) reduction is a geochemically significant process. Geobacter species specifically express pili during growth on Fe(III) oxide compared with growth on soluble chelated Fe(III), and mutants that cannot produce pili are unable to effectively reduce Fe(III) oxide. The pili of Geobacter sulfurreducens are electrically conductive along their length under physiologically relevant conditions and exhibit a metallic-like conductivity similar to that observed previously in synthetic organic metals. Metallic-like conductivity in a biological protein filament is a previously unrecognized mechanism for electron transport that differs significantly from the more well-known biological strategy of electron hopping/tunnelling between closely spaced redox-active proteins. The multihaem c-type cytochrome OmcS is specifically associated with pili and is necessary for Fe(III) oxide reduction. However, multiple lines of evidence, including the metallic-like conductivity of the pili and the fact that OmcS molecules are spaced too far apart for electron hopping/tunnelling, indicate that OmcS is not responsible for long-range electron conduction along the pili. The role of OmcS may be to facilitate electron transfer from the pili to Fe(III) oxide. Long-range electron transport via pili with metallic-like conductivity is a paradigm shift that has important implications not only for Fe(III) oxide reduction, but also for interspecies electron exchange in syntrophic microbial communities as well as microbe-electrode interactions and the emerging field of bioelectronics.

Interspecies Electron Transfer via Hydrogen and Formate Rather than Direct Electrical Connections in Cocultures of Pelobacter carbinolicus and Geobacter sulfurreducens

Direct interspecies electron transfer (DIET) is an alternative to interspecies H(2)/formate transfer as a mechanism for microbial species to cooperatively exchange electrons during syntrophic metabolism. To understand what specific properties contribute to DIET, studies were conducted with Pelobacter carbinolicus, a close relative of Geobacter metallireducens, which is capable of DIET. P. carbinolicus grew in coculture with Geobacter sulfurreducens with ethanol as the electron donor and fumarate as the electron acceptor, conditions under which G. sulfurreducens formed direct electrical connections with G. metallireducens. In contrast to the cell aggregation associated with DIET, P. carbinolicus and G. sulfurreducens did not aggregate. Attempts to initiate cocultures with a genetically modified strain of G. sulfurreducens incapable of both H(2) and formate utilization were unsuccessful, whereas cocultures readily grew with mutant strains capable of formate but not H(2) uptake or vice versa. The hydrogenase mutant of G. sulfurreducens compensated, in cocultures, with significantly increased formate dehydrogenase gene expression. In contrast, the transcript abundance of a hydrogenase gene was comparable in cocultures with that for the formate dehydrogenase mutant of G. sulfurreducens or the wild type, suggesting that H(2) was the primary electron carrier in the wild-type cocultures. Cocultures were also initiated with strains of G. sulfurreducens that could not produce pili or OmcS, two essential components for DIET. The finding that P. carbinolicus exchanged electrons with G. sulfurreducens via interspecies transfer of H(2)/formate rather than DIET demonstrates that not all microorganisms that can grow syntrophically are capable of DIET and that closely related microorganisms may use significantly different strategies for interspecies electron exchange.