Relevance of extracellular electron uptake mechanisms for electromethanogenesis applications.

Electromethanogenesis has emerged as a biological branch of Power-to-X technologies that implements methanogenic microorganisms, as an alternative to chemical Power-to-X, to convert electrical power from renewable sources, and CO2 into methane. Unlike biomethanation processes where CO2 is converted via exogenously added hydrogen, electromethanogenesis occurs in a bioelectrochemical set-up that combines electrodes and microorganisms. Thereby, mixed, or pure methanogenic cultures catalyze the reduction of CO2 to methane via reducing equivalents supplied by a cathode. Recent advances in electromethanogenesis have been driven by interdisciplinary research at the intersection of microbiology, electrochemistry, and engineering. Integrating the knowledge acquired from these areas is essential to address the specific challenges presented by this relatively young biotechnology, which include electron transfer limitations, low energy and product efficiencies, and reactor design to enable upscaling. This review approaches electromethanogenesis from a multidisciplinary perspective, putting emphasis on the extracellular electron uptake mechanisms that methanogens use to obtain energy from cathodes, since understanding these mechanisms is key to optimize the electrochemical conditions for the development of these systems. This work summarizes the direct and indirect extracellular electron uptake mechanisms that have been elucidated to date in methanogens, along with the ones that remain unsolved. As the study of microbial corrosion, a similar bioelectrochemical process with Fe0 as electron source, has contributed to elucidate different mechanisms on how methanogens use solid electron donors, insights from both fields, biocorrosion and electromethanogenesis, are combined. Based on the repertoire of mechanisms and their potential to convert CO2 to methane, we conclude that for future applications, electromethanogenesis should focus on the indirect mechanism with H2 as intermediary. By summarizing and linking the general aspects and challenges of this process, we hope that this review serves as a guide for researchers working on electromethanogenesis in different areas of expertise to overcome the current limitations and continue with the optimization of this promising interdisciplinary technology.

Considerations on the use of microsensors to profile dissolved H2 concentrations in microbial electrochemical reactors.

Measuring the distribution and dynamics of H2 in microbial electrochemical reactors is valuable to gain insights into the processes behind novel bioelectrochemical technologies, such as microbial electrosynthesis. Here, a microsensor method to measure and profile dissolved H2 concentrations in standard H-cell reactors is described. Graphite cathodes were oriented horizontally to enable the use of a motorized microprofiling system and a stereomicroscope was used to place the H2 microsensor precisely on the cathode surface. Profiling was performed towards the gas-liquid interface, while preserving the electric connections and flushing the headspace (to maintain anoxic conditions) and under strict temperature control (to overcome the temperature sensitivity of the microsensors). This method was tested by profiling six reactors, with and without inoculation of the acetogen Sporomusa ovata, at three different time points. H2 accumulated over time in the abiotic controls, while S. ovata maintained low H2 concentrations throughout the liquid phase (< 4 µM) during the whole experimental period. These results demonstrate that this setup generated insightful H2 profiles. However, various limitations of this microsensor method were identified, as headspace flushing lowered the dissolved H2 concentrations over time. Moreover, microsensors can likely not accurately measure H2 in the immediate vicinity of the solid cathode, because the solids cathode surface obstructs H2 diffusion into the microsensor. Finally, the reactors had to be discarded after microsensor profiling. Interested users should bear these considerations in mind when applying microsensors to characterize microbial electrochemical reactors.

An Acetobacterium strain isolated with metallic iron as electron donor enhances iron corrosion by a similar mechanism as Sporomusa sphaeroides.

Sporomusa sphaeroides related strains are to date the only homoacetogens known to increase metallic iron corrosion. The goal of this work was to isolate additional homoacetogenic bacteria capable of using Fe(0) as electron donor and to explore their extracellular electron transfer mechanism. Enrichments were started from anoxic corrosion products and yielded Acetobacterium as main homoacetogenic genus. Isolations were performed with a new procedure using plates with a Fe(0) powder top layer. An Acetobacterium strain, closely related to A. malicum and A. wieringae, was isolated, in addition to a S. sphaeroides strain. The Acetobacterium isolate significantly increased Fe(0) corrosion ((1.44 ± 0.16)-fold) compared to abiotic controls. The increase of corrosion by type strains ranged from (1.28 ± 0.13)-fold for A. woodii to (2.03 ± 0.22)-fold for S. sphaeroides. Hydrogen mediated the electron uptake from Fe(0) by the acetogenic isolates and tested type strains. Exchange of the medium and SEM imaging suggested that cells were attached to Fe(0). The corrosion enhancement mechanism is for all tested strains likely related to free extracellular components catalyzing hydrogen formation on the Fe(0) surface, or to the maintenance of low hydrogen concentrations on the Fe(0) surface by attached cells thereby thermodynamically favoring hydrogen formation.

Extracellular Electron Uptake by Acetogenic Bacteria: Does H2 Consumption Favor the H2 Evolution Reaction on a Cathode or Metallic Iron?

Some acetogenic bacteria are capable of using solid electron donors, such as a cathode or metallic iron [Fe(0)]. Acetogens using a cathode as electron donor are of interest for novel applications such as microbial electrosynthesis, while microorganisms using Fe(0) as electron donor cause detrimental microbial induced corrosion. The capacity to use solid electron donors strongly differs between acetogenic strains, which likely relates to their extracellular electron transfer (EET) mechanism. Different EET mechanisms have been proposed for acetogenic bacteria, including a direct mechanism and a H2 dependent indirect mechanism combined with extracellular hydrogenases catalyzing the H2 evolution reaction on the cathode or Fe(0) surface. Interestingly, low H2 partial pressures often prevail during acetogenesis with solid electron donors. Hence, an additional mechanism is here proposed: the maintenance of low H2 partial pressures by microbial H2 consumption, which thermodynamically favors the H2 evolution reaction on the cathode or Fe(0) surface. This work elaborates how the H2 partial pressure affects the H2 evolution onset potential and the H2 evolution rate on a cathode, as well as the free energy change of the anoxic corrosion reaction. In addition, the H2 consumption characteristics, i.e., H2 threshold (thermodynamic limit for H2 consumption) and H2 consumption kinetic parameters, of acetogenic bacteria are reviewed and evidence is discussed for strongly different H2 consumption characteristics. Different acetogenic strains are thus expected to maintain different H2 partial pressures on a cathode or Fe(0) surface, while those that maintain lower H2 partial pressures (lower H2 threshold, higher H2 affinity) more strongly increase the H2 evolution reaction. Consequently, I hypothesize that the different capacities of acetogenic bacteria to use solid electron donors are related to differences in their H2 consumption characteristics. The focus of this work is on acetogenic bacteria, but similar considerations are likely also relevant for other hydrogenotrophic microorganisms.

A Novel Shewanella Isolate Enhances Corrosion by Using Metallic Iron as the Electron Donor with Fumarate as the Electron Acceptor.

The involvement of Shewanella spp. in biocorrosion is often attributed to their Fe(III)-reducing properties, but they could also affect corrosion by using metallic iron as an electron donor. Previously, we isolated Shewanella strain 4t3-1-2LB from an acetogenic community enriched with Fe(0) as the sole electron donor. Here, we investigated its use of Fe(0) as an electron donor with fumarate as an electron acceptor and explored its corrosion-enhancing mechanism. Without Fe(0), strain 4t3-1-2LB fermented fumarate to succinate and CO2, as was shown by the reaction stoichiometry and pH. With Fe(0), strain 4t3-1-2LB completely reduced fumarate to succinate and increased the Fe(0) corrosion rate (7.0 ± 0.6)-fold in comparison to that of abiotic controls (based on the succinate-versus-abiotic hydrogen formation rate). Fumarate reduction by strain 4t3-1-2LB was, at least in part, supported by chemical hydrogen formation on Fe(0). Filter-sterilized spent medium increased the hydrogen generation rate only 1.5-fold, and thus extracellular hydrogenase enzymes appear to be insufficient to explain the enhanced corrosion rate. Electrochemical measurements suggested that strain 4t3-1-2LB did not excrete dissolved redox mediators. Exchanging the medium and scanning electron microscopy (SEM) imaging indicated that cells were attached to Fe(0). It is possible that strain 4t3-1-2LB used a direct mechanism to withdraw electrons from Fe(0) or favored chemical hydrogen formation on Fe(0) through maintaining low hydrogen concentrations. In coculture with an Acetobacterium strain, strain 4t3-1-2LB did not enhance acetogenesis from Fe(0). This work describes a strong corrosion enhancement by a Shewanella strain through its use of Fe(0) as an electron donor and provides insights into its corrosion-enhancing mechanism.IMPORTANCEShewanella spp. are frequently found on corroded metal structures. Their role in microbial influenced corrosion has been attributed mainly to their Fe(III)-reducing properties and, therefore, has been studied with the addition of an electron donor (lactate). Shewanella spp., however, can also use solid electron donors, such as cathodes and potentially Fe(0). In this work, we show that the electron acceptor fumarate supported the use of Fe(0) as the electron donor by Shewanella strain 4t3-1-2LB, which caused a (7.0 ± 0.6)-fold increase of the corrosion rate. The corrosion-enhancing mechanism likely involved cell surface-associated components in direct contact with the Fe(0) surface or maintenance of low hydrogen levels by attached cells, thereby favoring chemical hydrogen formation by Fe(0). This work sheds new light on the role of Shewanella spp. in biocorrosion, while the insights into the corrosion-enhancing mechanism contribute to the understanding of extracellular electron uptake processes.

Biofilm Formation by Clostridium ljungdahlii Is Induced by Sodium Chloride Stress: Experimental Evaluation and Transcriptome Analysis.

The acetogen Clostridium ljungdahlii is capable of syngas fermentation and microbial electrosynthesis. Biofilm formation could benefit both these applications, but was not yet reported for C. ljungdahlii. Biofilm formation does not occur under standard growth conditions, but attachment or aggregation could be induced by different stresses. The strongest biofilm formation was observed with the addition of sodium chloride. After 3 days of incubation, the biomass volume attached to a plastic surface was 20 times higher with than without the addition of 200 mM NaCl to the medium. The addition of NaCl also resulted in biofilm formation on glass, graphite and glassy carbon, the latter two being often used electrode materials for microbial electrosynthesis. Biofilms were composed of extracellular proteins, polysaccharides, as well as DNA, while pilus-like appendages were observed with, but not without, the addition of NaCl. A transcriptome analysis comparing planktonic (no NaCl) and biofilm (NaCl addition) cells showed that C. ljungdahlii coped with the salt stress by the upregulation of the general stress response, Na+ export and osmoprotectant accumulation. A potential role for poly-N-acetylglucosamines and D-alanine in biofilm formation was found. Flagellar motility was downregulated, while putative type IV pili biosynthesis genes were not expressed. Moreover, the gene expression analysis suggested the involvement of the transcriptional regulators LexA, Spo0A and CcpA in stress response and biofilm formation. This study showed that NaCl addition might be a valuable strategy to induce biofilm formation by C. ljungdahlii, which can improve the efficacy of syngas fermentation and microbial electrosynthesis applications.

Motile Geobacter dechlorinators migrate into a model source zone of trichloroethene dense non-aqueous phase liquid: experimental evaluation and modeling.

Microbial migration towards a trichloroethene (TCE) dense non-aqueous phase liquid (DNAPL) could facilitate the bioaugmentation of TCE DNAPL source zones. This study characterized the motility of the Geobacter dechlorinators in a TCE to cis-dichloroethene dechlorinating KB-1(™) subculture. No chemotaxis towards or away from TCE was found using an agarose in-plug bridge method. A second experiment placed an inoculated aqueous layer on top of a sterile sand layer and showed that Geobacter migrated several centimeters in the sand layer in just 7days. A random motility coefficient for Geobacter in water of 0.24±0.02cm(2)·day(-1) was fitted. A third experiment used a diffusion-cell setup with a 5.5cm central sand layer separating a DNAPL from an aqueous top layer as a model source zone to examine the effect of random motility on TCE DNAPL dissolution. With top layer inoculation, Geobacter quickly colonized the sand layer, thereby enhancing the initial TCE DNAPL dissolution flux. After 19days, the DNAPL dissolution enhancement was only 24% lower than with an homogenous inoculation of the sand layer. A diffusion-motility model was developed to describe dechlorination and migration in the diffusion-cells. This model suggested that the fast colonization of the sand layer by Geobacter was due to the combination of random motility and growth on TCE.

Carbon cloth stimulates direct interspecies electron transfer in syntrophic co-cultures.

This study investigated the possibility that the electrical conductivity of carbon cloth accelerates direct interspecies electron transfer (DIET) in co-cultures. Carbon cloth accelerated metabolism of DIET co-cultures (Geobacter metallireducens-Geobacter sulfurreducens and G.metallireducens-Methanosarcina barkeri) but did not promote metabolism of co-cultures performing interspecies H2 transfer (Desulfovibrio vulgaris-G.sulfurreducens). On the other hand, DIET co-cultures were not stimulated by poorly conductive cotton cloth. Mutant strains lacking electrically conductive pili, or pili-associated cytochromes participated in DIET only in the presence of carbon cloth. In co-cultures promoted by carbon cloth, cells were primarily associated with the cloth although the syntrophic partners were too far apart for cell-to-cell biological electrical connections to be feasible. Carbon cloth seemingly mediated interspecies electron transfer between the distant syntrophic partners. These results suggest that the ability of carbon cloth to accelerate DIET should be considered in anaerobic digester designs that incorporate carbon cloth.

Inhibition of microbial trichloroethylene dechlorination [corrected] by Fe (III) reduction depends on Fe mineralogy: a batch study using the bioaugmentation culture KB-1.

Microbial reductive dechlorination of trichloroethylene (TCE) in groundwater can be stimulated by adding of electron donors. However, side reactions such as Fe (III) reduction competes with this reaction. This study was set-up to relate the inhibition of microbial TCE dechlorination to the quantity and quality (mineralogy) of Fe (III) in the substrate and to calibrate a substrate extraction procedure for testing bioavailable Fe (III) in sediments. Batch experiments were set-up with identical inoculum (KB-1 culture) and liquid medium composition, and adding either 1) variable amounts of ferrihydrite or 2) 14 different Fe (III) minerals coated onto or mixed in with quartz sand (at constant total Fe) at a stoichiometric excess Fe (III) over electron donor. Increasing amounts of ferrihydrite significantly increased the time for complete TCE degradation from 8 days (control sand) to 28 days (excess Fe). Acid extractable Fe (II) increased and magnetite formed during incubation, confirming Fe (III) reduction. At constant total Fe in the sand, TCE dechlorination time varied with Fe mineralogy between 8 days (no Fe added) to >120 days (Fe-containing bentonite). In general, poorly crystalline Fe (III) minerals inhibited TCE dechlorination whereas crystalline Fe (III) minerals such as goethite or hematite had no effect. The TCE inhibition time was positively correlated to the Fe (II) determined after 122 days and to the surface area of the Fe (III) minerals. Only a fraction of total Fe (III) is reduced, likely because of solubility constraints and/or coating of Fe (III) minerals by Fe (II) minerals. Iron extraction tests based on Fe (III) reduction using NH2OH(.)HCl predict the competitive inhibition of TCE degradation in these model systems. This study shows that Fe mineralogy rather that total Fe content determines the competitive inhibition of TCE dechlorination.

Acidification due to microbial dechlorination near a trichloroethene DNAPL is overcome with pH buffer or formate as electron donor: experimental demonstration in diffusion-cells.

Acidification due to microbial dechlorination of trichloroethene (TCE) can limit the bio-enhanced dissolution of TCE dense non-aqueous phase liquid (DNAPL). This study related the dissolution enhancement of a TCE DNAPL to the pH buffer capacity of the medium and the type of electron donor used. In batch systems, dechlorination was optimal at pH7.1-7.5, but was completely inhibited below pH6.2. In addition, dechlorination in batch systems led to a smaller pH decrease at an increasing pH buffer capacity or with the use of formate instead of lactate as electron donor. Subsequently, bio-enhanced TCE DNAPL dissolution was quantified in diffusion-cells with a 5.5 cm central sand layer, separating a TCE DNAPL layer from an aqueous top layer. Three different pH buffer capacities (2.9 mM-17.9 mM MOPS) and lactate or formate as electron donor were applied. In the lactate fed diffusion-cells, the DNAPL dissolution enhancement factor increased from 1.5 to 2.2 with an increase of the pH buffer capacity. In contrast, in the formate fed diffusion-cells, the DNAPL dissolution enhancement factor (2.4±0.3) was unaffected by the pH buffer capacity. Measurement of the pore water pH confirmed that the pH decreased less with an increased pH buffer capacity or with formate instead of lactate as electron donor. These results suggest that the significant impact of acidification on bio-enhanced DNAPL dissolution can be overcome by the amendment of a pH buffer or by applying a non acidifying electron donor like formate.

Inhibition of Geobacter dechlorinators at elevated trichloroethene concentrations is explained by a reduced activity rather than by an enhanced cell decay.

Microbial dechlorination of trichloroethene (TCE) is inhibited at elevated TCE concentrations. A batch experiment and modeling analysis were performed to examine whether this self-inhibition is related to an enhanced cell decay or a reduced dechlorination activity at increasing TCE concentrations. The batch experiment combined four different initial TCE concentrations (1.4-3.0 mM) and three different inoculation densities (4.0 × 10(5) to 4.0 × 10(7)Geobacter cells·mL(-1)). Chlorinated ethene concentrations and Geobacter 16S rRNA gene copy numbers were measured. The time required for complete conversion of TCE to cis-DCE increased with increasing initial TCE concentration and decreasing inoculation density. Both an enhanced decay and a reduced activity model fitted the experimental results well, although the reduced activity model better described the lag phase and microbial decay in some treatments. In addition, the reduced activity model succeeded in predicting the reactivation of the dechlorination reaction in treatments in which the inhibiting TCE concentration was lowered after 80 days. In contrast, the enhanced decay model predicted a Geobacter cell density that was too low to allow recovery for these treatments. Conclusively, our results suggest that TCE self-inhibition is related to a reduced dechlorination activity rather than to an enhanced cell decay at elevated TCE concentrations.

Electron donor limitations reduce microbial enhanced trichloroethene DNAPL dissolution: a flux-based analysis using diffusion-cells.

Electron donor limitations likely reduce microbial enhanced trichloroethene (TCE) dense non-aqueous phase liquid (DNAPL) dissolution. This study quantitatively examined the relation between the DNAPL dissolution enhancement and the electron donor supply rate. An experiment used diffusion-cells with a 5.5 cm central sand layer, separating a DNAPL layer from an aqueous top layer. Top layers were amended with different concentrations of formate (0-16 mM). The TCE DNAPL dissolution rate increased from no enhancement compared to abiotic dissolution without formate, to a 2.4 times dissolution enhancement with 16 mM formate amended to the top layer. With 2, 4 and 8 mM formate amended the top layer, the TCE diffusion flux out of the DNAPL layer equaled the formate diffusion flux out of the top layer, which illustrates their stoichiometric interdependence under electron donor limiting conditions. In contrast, with 16 mM formate amended to the top layer, the TCE diffusion flux was lower than the formate diffusion flux, demonstrating that the dechlorination kinetics limited the DNAPL dissolution enhancement. The DNAPL dissolution flux under electron donor limiting conditions was readily predicted from the electron donor concentration in the top layer.

Distribution of a dechlorinating community in relation to the distance from a trichloroethene dense nonaqueous phase liquid in a model aquifer.

The toxicity of trichloroethene (TCE) likely restricts microbial activity in close vicinity of a TCE dense nonaqueous phase liquid (DNAPL). This study examined the distribution of a dechlorinating community in relation to the distance from a TCE DNAPL using a diffusion-cell set-up. Subcultures of the KB-1(™) culture dechlorinating TCE to cis-dichloroethene and grown with either formate or lactate as electron donor were used to inoculate the diffusion-cells. 16S rRNA gene clone library analysis showed that both inocula consisted of dechlorinating bacteria similar to Geobacter lovleyi SZ and fermentative microorganisms related to Clostridium and Clostridiales. qPCR and RFLP analyses of pore water and sand samples showed a stratified microbial community composition in the diffusion-cells. Geobacter dominated where TCE was present, that is, in the lower 3 cm of the 5.5-cm-thick sand layer. Even at 0.5 cm distance from the DNAPL layer, Geobacter densities were two orders of magnitude higher than at inoculation, despite the expected TCE toxicity. In the upper 2.5 cm of the sand layer, where TCE was depleted, apparently fermenting populations prevailed, corresponding to Clostridium in some diffusion-cells. This analysis demonstrates that the microbial community composition in a source zone is related to the distance from the DNAPL.

A three-layer diffusion-cell to examine bio-enhanced dissolution of chloroethene dense non-aqueous phase liquid.

Microbial reductive dechlorination of trichloroethene (TCE) and perchloroethene (PCE) in the vicinity of their dense non-aqueous phase liquid (DNAPL) has been shown to accelerate DNAPL dissolution. A three-layer diffusion-cell was developed to quantify this bio-enhanced dissolution and to measure the conditions near the DNAPL interface. The 12 cm long diffusion-cell setup consists of a 5.5 cm central porous layer (sand), a lower 3.5 cm DNAPL layer and a top 3 cm water layer. The water layer is frequently refreshed to remove chloroethenes at the upper boundary of the porous layer, while the DNAPL layer maintains the saturated chloroethene concentration at the lower boundary. Two abiotic and two biotic diffusion-cells with TCE DNAPL were tested. In the abiotic diffusion-cells, a linear steady state TCE concentration profile between the DNAPL and the water layer developed beyond 21 d. In the biotic diffusion-cells, TCE was completely converted into cis-dichloroethene (cis-DCE) at 2.5 cm distance of the DNAPL. Dechlorination was likely inhibited up to a distance of 1.5 cm from the DNAPL, as in this part the TCE concentration exceeded the culture's maximum tolerable concentration (2.5mM). The DNAPL dissolution fluxes were calculated from the TCE concentration gradient, measured at the interface of the DNAPL layer and the porous layer. Biotic fluxes were a factor 2.4 (standard deviation 0.2) larger than abiotic dissolution fluxes. This diffusion-cell setup can be used to study the factors affecting the bio-enhanced dissolution of DNAPL and to assess bioaugmentation, pH buffer addition and donor delivery strategies for source zones.