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The anaerobic oxidation of methane (AOM) carried out by anaerobic methanotrophic archaea (ANME) plays an important role in mitigating methane emissions from aqueous environments and has applications in bioremediation and wastewater treatment. Previous studies showed that AOM could be coupled to chromate reduction. However, the specific responsible microorganisms and the biochemical mechanisms are unclear. Herein, we showed that a consortium dominated by ANME "Candidatus Methanoperedens" was able to couple AOM to the reduction of Cr(VI) to Cr(III) at a stoichiometry close to the theoretical ratio. Quantitative distribution analysis of Cr(III) products suggested Cr(VI) was predominantly reduced via the extracellular respiratory pathways. Further Cr(III)-targeted fluorescent visualization combined with single-cell electron microscopic imaging suggested that Cr(VI) was reduced by "Ca. Methanoperedens" independently. Biochemical mechanism investigation via proteomic analysis showed proteins for nitrate reduction under nitrate-reducing conditions were significantly downregulated in Cr(VI)-reducing incubation. Instead, many multiheme cytochrome c (MHCs) were among the most upregulated proteins during the Cr(VI) reduction process, suggesting MHC-governed pathways for extracellular Cr(VI) reduction. The significant upregulation of a formate-dependent nitrite reductase during Cr(VI) reduction indicated its potential contribution to the small proportion of Cr(VI) reduction inside cells., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Cathodic Electro-Fermentation (CEF) is an innovative approach to manage the spectrum of products deriving from anaerobic fermentation. Herein, mixed microbial culture fermentation using a ternary mixture containing labelled 13 C glucose and non-labelled acetate and ethanol was studied to identify the role of polarization on the metabolic pathways of glucose fermentation. CEF at an applied potential of -700 mV (vs. SHE, Standard Hydrogen Electrode) enhanced the production yield of acetate, propionate, and butyrate (0.90±0.10, 0.22±0.03, and 0.34±0.05 mol/mol; respectively) compared to control tests performed at open circuit potential (OCP) (0.54±0.09, 0.15±0.04, and 0.20±0.001 mol/mol, respectively). Results indicate that CEF affected the 13 C labelled fermented product levels and their fractional 13 C enrichments, allowing to establish metabolic pathway models. This work demonstrates that, under cathodic polarization, the abundance of both fully 13 C labelled propionate and butyrate isotopomers increased compared to control tests. The effect of CEF is mainly due to intermediates initially produced from the glucose metabolic transformation in the presence of non-labelled acetate and ethanol as external substrates. These findings represent a significant advancement in current knowledge of CEF, which offers a promising tool to control mixed cultures bioprocesses., (© 2024 The Authors. ChemSusChem published by Wiley-VCH GmbH.)

Anaerobic oxidation of methane (AOM) is a microbial process of importance in the global carbon cycle. AOM is predominantly mediated by anaerobic methanotrophic archaea (ANME), the physiology of which is still poorly understood. Here we present a new addition to the current physiological understanding of ANME by examining, for the first time, the biochemical and redox-active properties of the extracellular polymeric substances (EPS) of an ANME enrichment culture. Using a 'Candidatus Methanoperedens nitroreducens'-dominated methanotrophic consortium as the representative, we found it can produce an EPS matrix featuring a high protein-to-polysaccharide ratio of ∼8. Characterization of EPS using FTIR revealed the dominance of protein-associated amide I and amide II bands in the EPS. XPS characterization revealed the functional group of C-(O/N) from proteins accounted for 63.7% of total carbon. Heme-reactive staining and spectroscopic characterization confirmed the distribution of c-type cytochromes in this protein-dominated EPS, which potentially enabled its electroactive characteristic. Redox-active c-type cytochromes in EPS mediated the EET of 'Ca. M. nitroreducens' for the reduction of Ag + to metallic Ag, which was confirmed by both ex-situ experiments with extracted soluble EPS and in-situ experiments with pristine EPS matrix surrounding cells. The formation of nanoparticles in the EPS matrix during in-situ extracellular Ag  +  reduction resulted in a relatively lower intracellular Ag distribution fraction, beneficial for alleviating the Ag toxicity to cells. The results of this study provide the first biochemical information on EPS of anaerobic methanotrophic consortia and a new insight into its physiological role in AOM process., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Motivated by the ambition to establish an enzyme-driven bioleaching pathway for copper extraction, properties of the Type-1 copper protein rusticyanin from Acidithiobacillus ferrooxidans (AfR) were compared with those from an ancestral form of this enzyme (N0) and an archaeal enzyme identified in Ferroplasma acidiphilum (FaR). While both N0 and FaR show redox potentials similar to that of AfR their electron transport rates were significantly slower. The lack of a correlation between the redox potentials and electron transfer rates indicates that AfR and its associated electron transfer chain evolved to specifically facilitate the efficient conversion of the energy of iron oxidation to ATP formation. In F. acidiphilum this pathway is not as efficient unless it is up-regulated by an as of yet unknown mechanism. In addition, while the electrochemical properties of AfR were consistent with previous data, previously unreported behavior was found leading to a form that is associated with a partially unfolded form of the protein. The cyclic voltammetry (CV) response of AfR immobilized onto an electrode showed limited stability, which may be connected to the presence of the partially unfolded state of this protein. Insights gained in this study may thus inform the engineering of optimized rusticyanin variants for bioleaching processes as well as enzyme-catalyzed solubilization of copper-containing ores such as chalcopyrite., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

Anaerobic methanotrophic archaea (ANME) belonging to the family Methanoperedenaceae are crucial for the global carbon cycle and different biogeochemical processes, owing to their metabolic versatility to couple anaerobic oxidation of methane (AOM) with different electron acceptors. A universal feature of Methanoperedenaceae is the abundant genes encoded in their genomes associated with extracellular electron transfer (EET) pathways. Candidatus. 'Methanoperedens manganicus', an archaeon belonging to the family Methanoperedenaceae, was recently enriched in a bioreactor performing AOM coupled with Mn (IV) reduction. Using this EET-capable ANME, we tested the hypothesis in this study that ANME can catalyse the humic-dependent AOM for growth. A two-year incubation showed that AOM activity can be sustained by Ca. 'M. manganicus' consortium in a bioreactor fed only with humic acids and methane. An isotopic mass balance batch test confirmed that the observed AOM was coupled to the reduction of humic acids. The increase of relative abundance of Ca. 'M. manganicus', and the total archaea population in the microbial community suggested that Ca. 'M. manganicus' can grow on methane and humic acids. The observation of humic-dependent AOM led to a subsequent hypothesis that humic acids could be used as the electron shuttle to mediate the EET in dissimilatory Mn (IV) reduction by Ca. 'M. manganicus'. We tested this hypothesis by adding humic acids to a Ca. 'M. manganicus' dominated-culture, which showed that the AOM rate was doubled by the addition of humic acids. X-ray photoelectron spectroscopy (XPS) showed that quinone moieties were consumed when humic acids worked as electron acceptors while remaining stable when functioning as a shuttle for electron transfer. The results of our study suggest that humic acids may serve as electron shuttles to allow ANME to access more electron acceptors through long-range EET., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Pyrogenic carbon (PC) can mediate electron transfer and thus catalyze biogeochemical processes to impact greenhouse gas (GHG) emissions. Here, we demonstrate that PC can contribute to mitigating GHG emissions by promoting the Fe(III)-dependent anaerobic oxidation of methane (AOM). It was found that the amendment PCs in microcosms dominated by Methanoperedenaceae performing Fe(III)-dependent AOM simultaneously promoted the rate of AOM and Fe(III) reduction with a consistent ratio close to the theoretical stoichiometry of 1:8. Further correlation analysis showed that the AOM rate was linearly correlated with the electron exchange capacity, but not the conductivity, of added PC materials, indicating the redox-cycling electron transfer mechanism to promote the Fe(III)-dependent AOM. The mass content of the C═O moiety from differentially treated PCs was well correlated with the AOM rate, suggesting that surface redox-active quinone groups on PCs contribute to facilitating Fe(III)-dependent AOM. Further microbial analyses indicate that PC likely shuttles direct electron transfer from Methanoperedenaceae to Fe(III) reduction. This study provides new insight into the climate-cooling impact of PCs, and our evaluation indicates that the PC-facilitated Fe(III)-dependent AOM could have a significant contribution to suppressing methane emissions from the world's reservoirs.

Anaerobic methanotrophic archaea (ANME) carry out anaerobic oxidation of methane, thus playing a crucial role in the methane cycle. Previous genomic evidence indicates that multi-heme c-type cytochromes (MHCs) may facilitate the extracellular electron transfer (EET) from ANME to different electron sinks. Here, we provide experimental evidence supporting cytochrome-mediated EET for the reduction of metals and electrodes by 'Candidatus Methanoperedens nitroreducens', an ANME acclimated to nitrate reduction. Ferrous iron-targeted fluorescent assays, metatranscriptomics, and single-cell imaging suggest that 'Ca. M. nitroreducens' uses surface-localized redox-active cytochromes for metal reduction. Electrochemical and Raman spectroscopic analyses also support the involvement of c-type cytochrome-mediated EET for electrode reduction. Furthermore, several genes encoding menaquinone cytochrome type-c oxidoreductases and extracellular MHCs are differentially expressed when different electron acceptors are used., (© 2023. Springer Nature Limited.)

Formate is a promising energy carrier that could be used to transport renewable electricity. Some acetogenic bacteria, such as Eubacterium limosum, have the native ability to utilise formate as a sole substrate for growth, which has sparked interest in the biotechnology industry. However, formatotrophic metabolism in E. limosum is poorly understood, and a system-level characterisation in continuous cultures is yet to be reported. Here, we present the first steady-state dataset for E. limosum formatotrophic growth. At a defined dilution rate of 0.4 d -1 , there was a high specific uptake rate of formate (280 ± 56 mmol/gDCW/d; gDCW = gramme dry cell weight); however, most carbon went to CO 2 (150 ± 11 mmol/gDCW/d). Compared to methylotrophic growth, protein differential expression data and intracellular metabolomics revealed several key features of formate metabolism. Upregulation of phosphotransacetylase (Pta) appears to be a futile attempt of cells to produce acetate as the major product. Instead, a cellular energy limitation resulted in the accumulation of intracellular pyruvate and upregulation of pyruvate formate ligase (Pfl) to convert formate to pyruvate. Therefore, metabolism is controlled, at least partially, at the protein expression level, an unusual feature for an acetogen. We anticipate that formate could be an important one-carbon substrate for acetogens to produce chemicals rich in pyruvate, a metabolite generally in low abundance during syngas growth. KEY POINTS: First Eubacterium limosum steady-state formatotrophic growth omics dataset High formate specific uptake rate, however carbon dioxide was the major product Formate may be the cause of intracellular stress and biofilm formation., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The market of biobased products obtainable via fermentation processes has steadily increased over the past few years, driven by the need to create a decarbonized economy. To date, industrial fermentation (IF) employs either pure or mixed microbial cultures (MMC), whereby the type of the microbial catalysts and the used feedstock affect metabolic pathways and, in turn, the type of product(s) generated. In many cases, especially when dealing with MMC, the economic viability of IF is still hindered by factors such as the low attained product titer and selectivity, which ultimately challenge the downstream recovery and purification steps. In this context, electro-fermentation (EF) represents an innovative approach, based on the use of a polarized electrode interface to trigger changes in the rate, yield, titer or product distribution deriving from traditional fermentation processes. In principle, the electrode in EF can act as an electron acceptor (i.e., anodic electro-fermentation, AEF) or donor (i.e., cathodic electro-fermentation, CEF), or simply as a means to control the oxidation-reduction potential of the fermentation broth. However, the molecular and biochemical basis underlying EF are still largely unknown. This review provides a comprehensive overview of recent literature studies including both AEF and CEF examples using pure or mixed microbial cultures. A critical analysis of biochemical, microbiological, and engineering aspects which presently hamper the transition of the EF technology from the laboratory to the market is also presented., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Anaerobic oxidation of methane (AOM) is an important microbial process mitigating methane (CH 4 ) emission from natural sediments. Anaerobic methanotrophic archaea (ANME) have been shown to mediate AOM coupled to the reduction of several compounds, either directly (i.e. nitrate, metal oxides) or in consortia with syntrophic bacterial partners (i.e. sulfate). However, the mechanisms underlying extracellular electron transfer (EET) between ANME and their bacterial partners or external electron acceptors are poorly understood. In this study, we investigated electron and carbon flow for an anaerobic methanotrophic consortium dominated by 'Candidatus Methanoperedens nitroreducens' in a CH 4 -fed microbial electrolysis cell (MEC). Acetate was identified as a likely intermediate for the methanotrophic consortium, which stimulated the growth of the known electroactive genus Geobacter. Electrochemical characterization, stoichiometric calculations of the system, along with stable isotope-based assays, revealed that acetate was not produced from CH 4 directly. In the absence of CH 4 , current was still generated and the microbial community remained largely unchanged. A substantial portion of the generated current in the absence of CH 4 was linked to the oxidation of the intracellular polyhydroxybutyrate (PHB) and the breakdown of extracellular polymeric substances (EPSs). The ability of 'Ca. M. nitroreducens' to use stored PHB as a carbon and energy source, and its ability to donate acetate as a diffusible electron carrier expands the known metabolic diversity of this lineage that likely underpins its success in natural systems., (Copyright © 2022. Published by Elsevier Ltd.)

Unlike gaseous C 1 feedstocks for acetogenic bacteria, there has been less attention on liquid C 1 feedstocks, despite benefits in terms of energy efficiency, mass transfer and integration within existing fermentation infrastructure. Here, we present growth of Eubacterium limosum ATCC8486 using methanol and formate as substrates, finding evidence for the first time of native butanol production. We varied ratios of methanol-to-formate in batch serum bottle fermentations, showing butyrate is the major product (maximum specific rate 220 ± 23 mmol-C gDCW -1 day -1 ). Increasing this ratio showed methanol is the key feedstock driving the product spectrum towards more reduced products, such as butanol (maximum titre 2.0 ± 1.1 mM-C). However, both substrates are required for a high growth rate (maximum 0.19 ± 0.011 h -1 ) and cell density (maximum 1.2 ± 0.043 gDCW l -1 ), with formate being the preferred substrate. In fact, formate and methanol are consumed in two distinct growth phases - growth phase 1, on predominately formate and growth phase 2 on methanol, which must balance. Because the second growth varied according to the first growth on formate, this suggests butanol production is due to overflow metabolism, similar to 2,3-butanediol production in other acetogens. However, further research is required to confirm the butanol production pathway in E. limosum, particularly given, unlike other substrates, methanol likely results in mostly NADH generation, not reduced ferredoxin., (© 2021 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

A circular carbon bioeconomy has potential to halt atmospheric accumulation of greenhouse gases causing climate change and sustainably produce chemical, agricultural and fuel products. Here, we report application of a simplified technoeconomic assessment to critically review two approaches in this space - microbial electrosynthesis and gas fermentation. For microbial electrosynthesis, decoupling of surface-dependant abiotic process for electron delivery from volume-dependant biotic carbon fixation, is shown as the only economically viable strategy to scale-up due to comparatively low biofilm electron consumption rate. This is effectively an electrolyser-assisted gas fermentation system. Targeting high-value products, such as protein for human food consumption is one of the few pathways forward for electrolyser-assisted gas fermentation. Alternatively, gas fermentation of reformed biogas presents an interesting and potentially more sustainable implementation pathway to improve economic viability of chemicals. This critical review suggests linking water treatment resource recovery with gas fermentation is attractive for bioplastics and butanol in terms of competitiveness and market demand., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Mixed-culture fermentation (MCF) enables carbon recycling from complex organic waste streams into valuable feedstock chemicals. Using complex microbial consortia, MCF systems can be tuned to produce a range of biochemicals to meet market demand. However, the metabolic mechanisms and community interactions which drive biochemical production changes under differing conditions are currently poorly understood. These mechanisms are critical to useful MCF production models. Furthermore, predictable product transitions are currently limited to pH-driven changes between butyrate and ethanol, and chain-elongation (fed by lactate, acetate, and ethanol) to butyrate, valerate, and hexanoate. Lactate, a high-value biopolymer feedstock chemical, has been observed in transition states, but sustained production has not been described. In this study, steady state lactate production was achieved by increasing the organic loading rate of a butyrate-producing system from limiting to nonlimiting conditions at pH 5.5. Crucially, butyrate production resumed upon return to substrate-limited conditions. 16S ribosomal DNA community profiling combined with metaproteomics demonstrated that the butyrate-producing lineage Megasphaera redirected carbon flow through the methylglyoxal bypass when substrate was nonlimiting, which altered the community structure and metabolic expression toward lactate production. This metabolic mechanism can be included in future MCF models to describe the changes in product generation in substrate nonlimiting conditions., (© 2021 Wiley Periodicals LLC.)

Carboxylic acids obtained via the microbial electrochemical conversion of waste gases containing carbon dioxide ( i.e. , microbial electrosynthesis) can be used in lieu of nonrenewable building-block chemicals in the manufacture of a variety of products. When targeting valuable medium-chain carboxylic acids such as caproic acid, electricity-driven fermentations can be limited by the accumulation of fermentation products in the culturing media, often resulting in low volumetric productivities and titers due to direct toxicity or inhibition of the biocatalyst. In this study, we tested the effectiveness of a simple electrodialysis system in upconcentrating carboxylic acids from a model solution mimicking the effluent of a microbial electrochemical system producing short- and medium-chain carboxylic acids. Under batch extraction conditions, the electrodialysis scheme enabled the recovery of 60% (mol mol -1 ) of the total carboxylic acids present in the model fermentation broth. The particular arrangement of conventional monopolar ion exchange membranes and hydraulic recirculation loops allowed the progressive acidification of the extraction solution, enabling phase separation of caproic acid as an immiscible oil with 76% purity., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Background: Bioelectrochemical methane oxidation catalysed by anaerobic methanotrophic archaea (ANME) is constrained by limited methane bioavailability as well as by slow kinetics of extracellular electron transfer (EET) of ANME. In this study, we tested a combination of two strategies to improve the performance of methane-driven bioelectrochemical systems that includes (1) the use of hollow fibre membranes (HFMs) for efficient methane delivery to the ANME organisms and (2) the amendment of ferricyanide, an effective soluble redox mediator, to the liquid medium to enable electrochemical bridging between the ANME organisms and the anode, as well as to promote EET kinetics of ANME., Results: The combined use of HFMs and the soluble mediator increased the performance of ANME-based bioelectrochemical methane oxidation, enabling the delivery of up to 196 mA m -2 , thereby outperforming the control system by 244 times when HFMs were pressurized at 1.6 bar., Conclusions: Improving methane delivery and EET are critical to enhance the performance of bioelectrochemical methane oxidation. This work demonstrates that by process engineering optimization, energy recovery from methane through its direct oxidation at relevant rates is feasible., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

In this review, the use of MESe are evaluated in the monitoring of volatile fatty acids (VFAs) during the anaerobic digestion of high strength wastewater, with a focus on slaughterhouse wastewater. VFAs are identified as a key intermediary in anaerobic digestion, hence their accumulation could be used to infer possible process instability of anaerobic digesters. Current sample measurement for VFAs through off-line laboratory analysis can be costly, time consuming, and require specialist skills. Consequently, microbial electrochemical sensors (MESe) are currently being investigated as a low-cost alternative method for in-line VFA measurement. In this paper, the fundamental operation of MESe is summarised, including the exploration of several factors that would impact the operation of MESe in real wastewater applications. It is found that, in the context of wastewater sensing, MESe technology has been unable to bridge the gap between the laboratory and real-world anaerobic digesters effectively. Important issues surrounding biofouling, sensitivity, and detection range are explored and prioritised in this review, and an overview of potential research pathways is provided. These include the potential to further explore alternate electrode materials, use of ion exchange membranes, and development of other sensor components, as further described in the review., (Copyright © 2020 Elsevier B.V. All rights reserved.)

A microbial electrosynthesis cell comprising two biological cathode chambers sharing the same anode compartment is used to promote the production of C2-C4 carboxylic acids and alcohols from carbon dioxide. Each cathode chamber provides ideal pH conditions to favor acetogenesis/carbon chain elongation (pH = 6.9), and solventogenesis (pH = 4.9), respectively, without the requirement of external acid/base dosing.

Microbial electrochemical technologies (MET) are promising to drive metabolic processes for the production of chemicals of interest. They provide microorganisms with an electrode as an electron sink or an electron source to stabilize their redox and/or energy state. Here, we applied an anode as additional electron sink to enhance the anoxic metabolism of the industrial bacterium Corynebacterium glutamicum through an anodic electro-fermentation. In using ferricyanide as extracellular electron carrier, anaerobic growth was enabled and the feedback-deregulated mutant Corynebacterium glutamicum lysC further accumulated L-lysine. Under such oxidizing conditions we achieved L-lysine titers of 2.9 mM at rates of 0.2 mmol/L/hr. That titer is comparable to recently reported L-lysine concentrations achieved by anaerobic production under reductive conditions (cathodic electro-fermentation). However unlike other studies, our oxidative conditions allowed anaerobic cell growth, indicating an improved cellular energy supply during anodic electro-fermentation. In that light, we propose anodic electro-fermentation as the right choice to support C. glutamicum stabilizing its redox and energy state and empower a stable anaerobic production of L-lysine., (© 2018 Wiley Periodicals, Inc.)

Maintaining low concentrations of nitrogen compounds (ammonium, nitrate and nitrite) in recirculating aquaculture waters is extremely important for a larger and healthier fish production, as well as for water discharge purposes. Although ammonium removal from aquaculture streams is usually done within a nitrifying step, nitrate removal via denitrification is still partially limited by the low organic matter availability. Therefore, an easy-to-operate autotrophic denitrifying bioelectrochemical system is herein proposed for the treatment of seawater aquaculture streams. The nitrate-containing synthetic stream flows sequentially through a biological denitrifying cathode (placed at the lower portion of a tubular reactor) and an abiotic anode (generating electrons and oxygen from water splitting, at the upper portion). Experimental results with synthetic seawater showed that the system reached denitrification rates of 0.13 ± 0.01 kg N m -3 day -1 , operating with minimum ammonium and nitrite accumulation, as well as minimum chlorine formation in the abiotic anode, despite the high chloride concentration. There results support the technical potential for simultaneous bioelectrochemical denitrification and partial re-oxygenation of aquaculture waters either for recirculation or discharge purposes., Competing Interests: The authors declare no competing financial interest.

Electron transfer is central to cellular life, from photosynthesis to respiration. In the case of anaerobic respiration, some microbes have extracellular appendages that can be utilised to transport electrons over great distances. Two model organisms heavily studied in this arena are Shewanella oneidensis and Geobacter sulfurreducens. There is some debate over how, in particular, the Geobacter sulfurreducens nanowires (formed from pilin nanofilaments) are capable of achieving the impressive feats of natural conductivity that they display. In this article, we outline the mechanisms of electron transfer through delocalised electron transport, quantum tunnelling, and hopping as they pertain to biomaterials. These are described along with existing examples of the different types of conductivity observed in natural systems such as DNA and proteins in order to provide context for understanding the complexities involved in studying the electron transport properties of these unique nanowires. We then introduce some synthetic analogues, made using peptides, which may assist in resolving this debate. Microbial nanowires and the synthetic analogues thereof are of particular interest, not just for biogeochemistry, but also for the exciting potential bioelectronic and clinical applications as covered in the final section of the review., Statement of Significance: Some microbes have extracellular appendages that transport electrons over vast distances in order to respire, such as the dissimilatory metal-reducing bacteria Geobacter sulfurreducens. There is significant debate over how G. sulfurreducens nanowires are capable of achieving the impressive feats of natural conductivity that they display: This mechanism is a fundamental scientific challenge, with important environmental and technological implications. Through outlining the techniques and outcomes of investigations into the mechanisms of such protein-based nanofibrils, we provide a platform for the general study of the electronic properties of biomaterials. The implications are broad-reaching, with fundamental investigations into electron transfer processes in natural and biomimetic materials underway. From these studies, applications in the medical, energy, and IT industries can be developed utilising bioelectronics., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Shewanella species respire using iron and manganese oxides as well as electrodes as solid terminal electron acceptors. Shewanella oneidenis MR-1 exploits mediated as well as direct extracellular electron transfer (EET) modes to transfer electrons at different formal potentials. These different EET modes at different potentials may utilise alternate electron transfer pathways. Therefore, we investigated how different anode potentials, providing different maximum microbial energy gains impacted S. oneidensis microbial physiology. Using quantitative proteomics, comparative analysis of the cellular variations to different anode potentials was performed. A label-free proteomic mass spectrometric analysis method, SWATH-MS, was used to gather quantitative information to determine physiological changes of Shewanella oneidensis MR-1 grown at different anodic potentials. S. oneidensis was cultured and grown in electrochemical cells at the set anode potentials of +0.71V, +0.21V & -0.19V versus SHE reference electrode, while the current production was monitored. At maximum current, electrodes were removed and whole-cell proteins extracted. Subsequent SWATH-MS analysis revealed information on 740 identified proteins across the three electrode potentials. For the first time, we show the abundance of S. oneidensis electron transfer proteins differs with electrode potential., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Three bioelectrochemical systems were operated with set anode potentials of +300 mV, +550 mV and +800 mV vs. Standard Hydrogen Electrode (SHE) to test the hypothesis that anode potential influences microbial diversity and is positively associated with microbial biomass and activity. Bacterial and archaeal diversity was characterized using 16 S rRNA gene amplicon sequencing, and biofilm thickness was measured as a proxy for biomass. Current production and substrate utilization patterns were used as measures of microbial activity and the mid-point potentials of putative terminal oxidases were assessed using cyclic voltammetry. All measurements were performed after 4, 16, 23, 30 and 38 days. Microbial biomass and activity differed significantly between anode potentials and were lower at the highest potential. Anodic electrode and electrolyte associated community composition was also significantly influenced by anode potential. While biofilms at +800 mV were thinner, transferred less charge and oxidized less substrate than those at lower potentials, they were also associated with putative terminal oxidases with higher mid-point potentials and generated more biomass per unit charge. This indicates that microbes at +800 mV were unable to capitalize on the potential for additional energy gain due to a lack of adaptive traits to high potential solid electron acceptors and/or sensitivity to oxidative stress.

Background: Microbial electrosynthesis is a novel approach that aims at shifting the cellular metabolism towards electron-dense target products by extracellular electron supply. Many organisms including several acetogenic bacteria have been shown to be able to consume electrical current. However, suitable hosts for relevant industrial processes are yet to be discovered, and major knowledge gaps about the underlying fundamental processes still remain., Results: In this paper, we present the first report of electron uptake by the Gram-positive, ethanol-producing acetogen, Clostridium   autoethanogenum . Under heterotrophic conditions, extracellular electron supply induced a significant metabolic shift away from acetate. In electrically enhanced fermentations on fructose, acetate production was cut by more than half, while production of lactate and 2,3-butanediol increased by 35-fold and threefold, respectively. The use of mediators with different redox potential revealed a direct dependency of the metabolic effect on the redox potential at which electrons are supplied. Only electrons delivered at a redox potential low enough to reduce ferredoxin caused the reported effect., Conclusions: Production in acetogenic organisms is usually challenged by cellular energy limitations if the target product does not lead to a net energy gain as in the case of acetate. The presented results demonstrate a significant shift of carbon fluxes away from acetate towards the products, lactate and 2,3-butanediol, induced by small electricity input (~0.09 mol of electrons per mol of substrate). This presents a simple and attractive method to optimize acetogenic fermentations for production of chemicals and fuels using electrochemical techniques. The relationship between metabolic shift and redox potential of electron feed gives an indication of possible electron-transfer mechanisms and helps to prioritize further research efforts.

Interspecies electron transfer (IET) is important for many anaerobic processes, but is critically dependent on mode of transfer. In particular, direct IET (DIET) has been recently proposed as a metabolically advantageous mode compared with mediated IET (MIET) via hydrogen or formate. We analyse relative feasibility of these IET modes by modelling external limitations using a reaction-diffusion-electrochemical approach in a three-dimensional domain. For otherwise identical conditions, external electron transfer rates per cell pair (cp) are considerably higher for formate-MIET (317 × 10(3) e(-) cp(-1) s(-1)) compared with DIET (44.9 × 10(3) e(-) cp(-1) s(-1)) or hydrogen-MIET (5.24 × 10(3) e(-) cp(-1) s(-1)). MIET is limited by the mediator concentration gradient at which reactions are still thermodynamically feasible, whereas DIET is limited through redox cofactor (for example, cytochromes) activation losses. Model outcomes are sensitive to key parameters for external electron transfer including cofactor electron transfer rate constant and redox cofactor area, concentration or count per cell, but formate-MIET is generally more favourable for reasonable parameter ranges. Extending the analysis to multiple cells shows that the size of the network does not strongly influence relative or absolute favourability of IET modes. Similar electron transfer rates for formate-MIET and DIET can be achieved in our case with a slight (0.7 kJ mol(-1)) thermodynamic advantage for DIET. This indicates that close to thermodynamic feasibility, external limitations can be compensated for by improved metabolic efficiency when using direct electron transfer.

Background: Pseudomonas putida is a promising host for the bioproduction of chemicals, but its industrial applications are significantly limited by its obligate aerobic character. The aim of this paper is to empower the anoxic metabolism of wild-type Pseudomonas putida to enable bioproduction anaerobically, with the redox power from a bioelectrochemical system (BES)., Results: The obligate aerobe Pseudomonas putida F1 was able to survive and produce almost exclusively 2-Keto-gluconate from glucose under anoxic conditions due to redox balancing with electron mediators in a BES. 2-Keto-gluconate, a precursor for industrial anti-oxidant production, was produced at an overall carbon yield of over 90 % based on glucose. Seven different mediator compounds were tested, and only those with redox potential above 0.207 V (vs standard hydrogen electrode) showed interaction with the cells. The productivity increased with the increasing redox potential of the mediator, indicating this was a key factor affecting the anoxic production process. P. putida cells survived under anaerobic conditions, and limited biofilm formation could be observed on the anode's surface. Analysis of the intracellular pools of ATP, ADP and AMP showed that cells had an increased adenylate energy charge suggesting that cells were able to generate energy using the anode as terminal electron acceptor. The analysis of NAD(H) and NADP(H) showed that in the presence of specific extracellular electron acceptors, the NADP(H) pool was more oxidised, while the NAD(H) pool was unchanged. This implies a growth limitation under anaerobic conditions due to a shortage of NADPH and provides a way to limit biomass formation, while allowing cell maintenance and catalysis at high purity and yield., Conclusions: For the first time, this study proved the principle that a BES-driven bioconversion of glucose can be achieved for a wild-type obligate aerobe. This non-growth bioconversion was in high yields, high purity and also could deliver the necessary metabolic energy for cell maintenance. By combining this approach with metabolic engineering strategies, this could prove to be a powerful new way to produce bio-chemicals and fuels from renewables in both high yield and high purity.

Anaerobic propionic acid degradation relies on interspecies electron transfer (IET) between propionate oxidisers and electron acceptor microorganisms, via either molecular hydrogen, formate or direct transfers. We evaluated the possibility of stimulating direct IET, hence enhancing propionate oxidation, by increasing availability of proton carriers to decrease solution resistance and reduce pH gradients. Phosphate was used as a proton carrying anion, and chloride as control ion together with potassium as counter ion. Propionic acid consumption in anaerobic granules was assessed in a square factorial design with ratios (1:0, 2:1, 1:1, 1:2 and 0:1) of total phosphate (TP) to Cl(-), at 1X, 10X, and 30X native conductivity (1.5 mS.cm(-1)). Maximum specific uptake rate, half saturation, and time delay were estimated using model-based analysis. Community profiles were analysed by fluorescent in situ hybridisation and 16S rRNA gene pyrosequencing. The strongest performance was at balanced (1:1) ratios at 10X conductivity where presumptive propionate oxidisers namely Syntrophobacter and Candidatus Cloacamonas were more abundant. There was a shift from Methanobacteriales at high phosphate, to Methanosaeta at low TP:Cl ratios and low conductivity. A lack of response to TP, and low percentage of presumptive electroactive organisms suggested that DIET was not favoured under the current experimental conditions.

Quantitative proteomics from low biomass, biofilm samples is not well documented. In this study we show successful use of SWATH-MS for quantitative proteomic analysis of a microbial electrochemically active biofilm. Shewanella oneidensis MR-1 was grown on carbon cloth electrodes under continuous anodic electrochemical polarizations in a bioelectrochemical system (BES). Using lactate as the electron donor, anodes serving as terminal microbial electron acceptors were operated at three different electrode potentials (+0.71 V, +0.21 V & -0.19 V vs. SHE) and the development of catalytic activity was monitored by measuring the current traces over time. Once maximum current was reached (usually within 21-29 h) the electrochemical systems were shut off and biofilm proteins were extracted from the electrodes for proteomic assessment. SWATH-MS analysis identified 704 proteins, and quantitative comparison was made of those associated with tricarboxcylic acid (TCA) cycle. Metabolic differences detected between the biofilms suggested a branching of the S. oneidensis TCA cycle when grown at the different electrode potentials. In addition, the higher abundance of enzymes involved in the TCA cycle at higher potential indicates an increase in metabolic activity, which is expected given the assumed higher energy gains. This study demonstrates high numbers of identifications on BES biofilm samples can be achieved in comparison to what is currently reported. This is most likely due to the minimal preparation steps required for SWATH-MS., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Mixed-culture fermentation is a key central process to enable next generation biofuels and biocommodity production due to economic and process advantages over application of pure cultures. However, a key limitation to the application of mixed-culture fermentation is predicting culture product response, related to metabolic regulation mechanisms. This is also a limitation in pure culture bacterial fermentation. This review evaluates recent literature in both pure and mixed culture studies with a focus on understanding how regulation and signaling mechanisms interact with metabolic routes and activity. In particular, we focus on how microorganisms balance electron sinking while maximizing catabolic energy generation. Analysis of these mechanisms and their effect on metabolism dynamics is absent in current models of mixed-culture fermentation. This limits process prediction and control, which in turn limits industrial application of mixed-culture fermentation. A key mechanism appears to be the role of internal electron mediating cofactors, and related regulatory signaling. This may determine direction of electrons towards either hydrogen or reduced organics as end-products and may form the basis for future mechanistic models., (© 2014 Wiley Periodicals, Inc.)

Anaerobic digestion is the default process for biological conversion of residue organics to renewable energy and biofuel in the form of methane. However, its scope of application is expanding, due to availability of new technologies, and the emerging drivers of energy and nutrient conservation and recovery. Here, we outline two of these new application areas, namely wastewater nutrient and energy recovery, and generation of value added chemicals through mixed culture biotechnology. There exist two options for nutrient and energy recovery from domestic wastewater: low energy mainline and partition-release-recovery. Both are heavily dependent on anaerobic digestion as an energy generating and nutrient release step, and have been enabled by new technologies such as low emission anaerobic membrane processes. The area of mixed culture biotechnology has been previously identified as a key industrial opportunity, but is now moving closer to application due application of existing and new technologies. As well as acting as a core technology option in bioproduction, anaerobic digestion has a key role in residual waste valorization and generation of energy for downstream processing. These new application areas and technologies are emerging simultaneously with substantial advances in knowledge of underlying mechanisms such as electron transfer, understanding of which is critical to development of the new application areas., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

An individual-based, mass-spring modeling framework has been developed to investigate the effect of cell properties on the structure of biofilms and microbial aggregates through Lagrangian modeling. Key features that distinguish this model are variable cell morphology described by a collection of particles connected by springs and a mechanical representation of deformable intracellular, intercellular, and cell-substratum links. A first case study describes the colony formation of a rod-shaped species on a planar substratum. This case shows the importance of mechanical interactions in a community of growing and dividing rod-shaped cells (i.e., bacilli). Cell-substratum links promote formation of mounds as opposed to single-layer biofilms, whereas filial links affect the roundness of the biofilm. A second case study describes the formation of flocs and development of external filaments in a mixed-culture activated sludge community. It is shown by modeling that distinct cell-cell links, microbial morphology, and growth kinetics can lead to excessive filamentous proliferation and interfloc bridging, possible causes for detrimental sludge bulking. This methodology has been extended to more advanced microbial morphologies such as filament branching and proves to be a very powerful tool in determining how fundamental controlling mechanisms determine diverse microbial colony architectures., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Confocal Resonance Raman Microscopy (CRRM) was used to probe variations of redox state of c-type cytochromes embedded in living mixed-culture electroactive biofilms exposed to different electrode polarizations, under potentiostatic and potentiodynamic conditions. In the absence of the metabolic substrate acetate, the redox state of cytochromes followed the application of reducing and oxidizing electrode potentials. Real-time monitoring of the redox state of cytochromes during cyclic voltammetry (CV) in a potential window where cytochromes reduction occurs, evidenced a measurable time delay between the oxidation of redox cofactors probed by CV at the electrode interface, and oxidation of distal cytochromes probed by CRRM. This delay was used to tentatively estimate the diffusivity of electrons through the biofilm. In the presence of acetate, the resonance Raman spectra of young (10 days, j = 208 ± 49 µA cm(-2)) and mature (57 days, j = 267 ± 73 µA cm(-2)) biofilms show that cytochromes remained oxidized homogeneously even at layers as far as 70 µm from the electrode, implying the existence of slow metabolic kinetics that do not result in the formation of a redox gradient inside the biofilm during anode respiration. However, old biofilms (80 days, j = 190 ± 37 µA cm(-2)) with thickness above 100 µm were characterized by reduced catalytic activity compared to the previous developing stages. The cytochromes in these biofilm were mainly in the reduced redox state, showing that only aged mixed-culture biofilms accumulate electrons during anode respiration. These results differ substantially from recent observations in pure Geobacter sulfurreducens electroactive biofilms, in which accumulation of reduced cytochromes is already observed in thinner biofilms, thus suggesting different bottlenecks in current production for mixed-culture and G. sulfurreducens biofilms.

Anthropogenic nitrous oxide (N(2)O) emissions represent up to 40% of the global N(2)O emission and are constantly increasing. Mitigation of these emissions is warranted since N(2)O is a strong greenhouse gas and important ozone-depleting compound. Until now, only physicochemical technologies have been applied to mitigate point sources of N(2)O, and no biological treatment technology has been developed so far. In this study, a bioelectrochemical system (BES) with an autotrophic denitrifying biocathode was considered for the removal of N(2)O. The high N(2)O removal rates obtained ranged between 0.76 and 1.83 kg N m(-3) net cathodic compartment (NCC) d(-1) and were proportional to the current production, resulting in cathodic coulombic efficiencies near 100%. Furthermore, our experiments suggested the active involvement of microorganisms as the catalyst for the reduction of N(2)O to N(2), and the optimal cathode potential ranged from -200 to 0 mV vs standard hydrogen electrode (SHE) in order to obtain high conversion rates. Successful operation of the system for more than 115 days with N(2)O as the sole cathodic electron acceptor strongly indicated that N(2)O respiration yielded enough energy to maintain the biological process. To our knowledge, this study provides for the first time proof of concept of biocathodic N(2)O removal at long-term without the need for high temperatures and expensive catalysts.

The aeration of the cathode compartment of bioelectrochemical systems (BESs) was recently shown to promote simultaneous nitrification and denitrification (SND). This study investigates the cathodic metabolism under different operating conditions as well as the structural organization of the cathodic biofilm during SND. Results show that a maximal nitrogen removal efficiency of 86.9 ± 0.5%, and a removal rate of 3.39 ± 0.08 mg NL(-1)h(-1) could be achieved at a dissolved oxygen (DO) level of 5.73 ± 0.03 mg L(-1) in the catholyte. The DO levels used in this study are higher than the thresholds previously reported as detrimental for denitrification. Analysis of the cathodic half-cell potential during batch tests suggested the existence of an oxygen gradient within the biofilm while performing SND. FISH analysis corroborated this finding revealing that the structure of the biofilm included an outer layer occupied by putative nitrifying organisms, and an inner layer where putative denitrifying organisms were most dominant. To our best knowledge this is the first time that nitrifying and denitrifying microorganisms are simultaneously observed in a cathodic biofilm., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Microbial fuel cells (MFCs) have applications other than electricity production, including the capacity to power desirable reactions in the cathode chamber. However, current knowledge of the microbial ecology and physiology of biocathodes is minimal, and as a result more research dedicated to understanding the microbial communities active in cathode biofilms is required. Here we characterize the microbiology of denitrifying bacterial communities stimulated by reducing equivalents generated from the anodic oxidation of acetate. We analyzed biofilms isolated from two types of cathodic denitrification systems: (1) a loop format where the effluent from the carbon oxidation step in the anode is subjected to a nitrifying reactor which is fed to the cathode chamber and (2) an alternative non-loop format where anodic and cathodic feed streams are separated. The results of our study indicate the superior performance of the loop reactor in terms of enhanced current production and nitrate removal rates. We hypothesized that phylogenetic or structural features of the microbial communities could explain the increased performance of the loop reactor. We used PhyloChip with 16S rRNA (cDNA) and fluorescent in situ hybridization to characterize the active bacterial communities. Our study results reveal a greater richness, as well as an increased phylogenetic diversity, active in denitrifying biofilms than was previously identified in cathodic systems. Specifically, we identified Proteobacteria, Firmicutes and Chloroflexi members that were dominant in denitrifying cathodes. In addition, our study results indicate that it is the structural component, in terms of bacterial richness and evenness, rather than the phylogenetic affiliation of dominant bacteria, that best corresponds to cathode performance.