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Microbial electrochemical technologies (METs) are promising for sustainable applications. Recently, electron storage during intermittent operation of electroactive biofilms (EABs) has been shown to play an important role in power output and electron efficiencies. Insights into electron storage mechanisms, and the conditions under which these occur, are essential to improve microbial electrochemical conversions and to optimize biotechnological processes. Here, we discuss the two main mechanisms for electron storage in EABs: storage in the form of reduced redox active components in the electron transport chain and in the form of polymers. We review electron storage in EABs and in other microorganisms and will discuss how the mechanisms of electron storage can be influenced. Intermittent operation of bioanodes influences electron storage in the electroactive biofilm.Longer-term electron storage in the carry over of polymers is an unexplored mechanism.Microbial electrochemical technologies are unique because electrodes provide a new way to control biological conversions.Understanding electron storage may provide new ways to influence the selectivity of biotechnological processes.

The possibility of the developing a biochemical oxygen demand (BOD) biosensor based on electroactive biofilms of activated sludge grown on the surface of a graphite-paste electrode modified with carbon nanotubes was studied. A complex of microscopic methods controlled biofilm formation: optical microscopy with phase contrast, scanning electron microscopy, and laser confocal microscopy. The features of charge transfer in the obtained electroactive biofilms were studied using the methods of cyclic voltammetry and electrochemical impedance spectroscopy. The rate constant of the interaction of microorganisms with the extracellular electron carrier (0.79 ± 0.03 dm3(g s)−1) and the heterogeneous rate constant of electron transfer (0.34 ± 0.02 cm s−1) were determined using the cyclic voltammetry method. These results revealed that the modification of the carbon nanotubes’ (CNT) electrode surface makes it possible to create electroactive biofilms. An analysis of the metrological and analytical characteristics of the created biosensors showed that the lower limit of the biosensor based on an electroactive biofilm of activated sludge is 0.41 mgO2/dm3, which makes it possible to analyze almost any water sample. Analysis of 12 surface water samples showed a high correlation (R2 = 0.99) with the results of the standard method for determining biochemical oxygen demand.

Conductive nanowires are thought to contribute to long-range electron transfer (LET) in Geobacter sulfurreducens anode biofilms. Three types of nanowires have been identified: pili, OmcS, and OmcZ. Previous studies highlighted their conductive function in anode biofilms, yet a structural function also has to be considered. We present here a comprehensive analysis of the function of nanowires in LET by inhibiting the expression of each nanowire. Meanwhile, flagella with poor conductivity were expressed to recover the structural function but not the conductive function of nanowires in the corresponding nanowire mutant strain. The results demonstrated that pili played a structural but not a conductive function in supporting biofilm formation. In contrast, the OmcS nanowire played a conductive but not a structural function in facilitating electron transfer in the biofilm. The OmcZ nanowire played both a structural and a conductive function to contribute to current generation. Expression of the poorly conductive flagellum was shown to enhance biofilm formation, subsequently increasing current generation. These data support a model in which multiheme cytochromes facilitate long-distance electron transfer in G. sulfurreducens biofilms. Our findings also suggest that the formation of a thicker biofilm, which contributed to a higher current generation by G. sulfurreducens, was confined by the biofilm formation deficiency, and this has applications in microbial electrochemical systems. The low power generation of microbial fuel cells limits their utility. Many factors can affect power generation, including inefficient electron transfer in the anode biofilm. Thus, understanding the mechanism(s) of electron transfer provides a pathway for increasing the power density of microbial fuel cells. Geobacter sulfurreducens was shown to form a thick biofilm on the anode. Cells far away from the anode reduce the anode through long-range electron transfer. Based on their conductive properties, three types of nanowires have been hypothesized to directly facilitate long-range electron transfer: pili, OmcS, and OmcZ nanowires. However, their structural contributions to electron transfer in anode biofilm have not been elucidated. Based on studies of mutants lacking one or more of these facilitators, our results support a cytochrome-mediated electron transfer process in biofilms and highlight the structural contribution of nanowires in anode biofilm formation, which contributes to biofilm formation and current generation, thereby providing a strategy to increase current generation.

Bioelectrochemical systems (BESs) exploit electroactive biofilms (EABs) for promising applications in biosensing, wastewater treatment, energy production, and chemical biosynthesis. However, during the operation of BESs, EABs inevitably decay. Seeking approaches to rejuvenate decayed EABs is critical for the sustainability and practical application of BESs. Prophage induction has been recognized as the primary reason for EAB decay. Herein, we report that introducing a competitive species of Geobacter uraniireducens suspended prophage induction in Geobacter sulfurreducens and thereby rejuvenated the decayed G. sulfurreducens EAB. The transcriptomic profile of G. sulfurreducens demonstrated that the addition of G. uraniireducens significantly affected the expression of metabolism- and stress response system-related genes and in particular suppressed the induction of phage-related genes. Mechanistic analyses revealed that interspecies ecological competition exerted by G. uraniireducens suppressed prophage induction. Our findings not only reveal a novel strategy to rejuvenate decayed EABs, which is significant for the sustainability of BESs, but also provide new knowledge for understanding phage–host interactions from an ecological perspective, with implications for developing therapies to defend against phage attack. Graphical Abstract Graphical Abstract

Electroactive biofilms (EABs) have aroused wide concern in waste treatment due to their unique capability of extracellular electron transfer with solid materials. The combined effect of different operating conditions on the formation, microbial architecture, composition, and metabolic activity of EABs is still unknown. In this study, the impact of three different factors (anode electrode, substrate concentration, and resistance) on the acclimation and performance of EABs was investigated. The results showed that the shortest start-up time of 127.3 h and highest power density of 0.84 W m −2 were obtained with carbon brush as electrode, low concentration of substrate (1.0 g L −1 ), and 1000 Ω external resistance (denoted as N1). The EABs under N1 condition also represented strongest redox capacity, lowest internal resistance, and close arrangement of bacteria. Moreover, the EABs cultured under different conditions both showed similar results, with direct electron transfer (DET) dominated from EABs to anode. Microbial community compositions indicated that EABs under N1 condition have lowest diversity and highest abundance of electroactive bacteria (46.68%). Higher substrate concentration (3.0 g L −1 ) promoted the proliferation of some other bacteria without electroactivity, which was adverse to EABs. The metabolic analysis showed the difference of genes related to electron transfer (cytochrome C and pili) and biofilm formation (xap) of EABs under different conditions, which further demonstrated the higher electroactivity of EABs under N1. These results provided a comprehensive understanding of the effect of different operating conditions on EABs including biofilm formation and electrochemical activity.

Microbial fuel cells (MFCs) have been demonstrated as a renewable energy strategy to efficiently recover chemical energy stored in wastewater into clean electricity, yet the limited power density limits their practical application. Here, Fe-doped carbon and nitrogen (Fe@CN) nanoparticles were synthesized by a direct pyrolysis process, which was further decorated to fabricate Fe@CN carbon paper anode. The modified Fe@CN anode with a higher electrochemically active surface area was not only benefit for the adhesion of electrochemically active microorganisms (EAMs) and extracellular electron transfer (EET) between the anode and EAMs but also selectively enriched Geobacter , a typical EAMs species. Accordingly, the MFCs with Fe@CN anode successfully achieved a highest voltage output of 792.76 mV and a prolonged stable voltage output of 300 h based on the mixed culture feeding with acetate. Most importantly, the electroactive biofilms on Fe@CN anode achieved more content ratio of proteins to polysaccharides (1.40) in extracellular polymeric substances for the balance between EET and cell protection under a harsh environment. This work demonstrated the feasibility of development on anode catalysts for the elaboration of the catalytic principle about interface modification, which may contribute to the practical application of MFC in energy generation and wastewater treatment.

Microbial mediator biosensors for surface water toxicity determination make it possible to carry out an early assessment of the environmental object’s quality without time-consuming standard procedures based on standard test-organisms, and provide broad opportunities for receptor element modifying depending on the required operational parameters analyzer. Four microorganisms with broad substrate specificity and nine electron acceptors were used to form a receptor system for toxicity assessment. Ferrocene was the most effective mediator according to its high rate constant of interaction with the microorganisms (0.33 ± 0.01 dm3/(g × s) for yeast Saccharomyces cerevisiae). Biosensors were tested on samples containing four heavy metal ions (Cu2+, Zn2+, Pb2+, Cd2+), two phenols (phenol and p-nitrophenol), and three natural water samples. The «ferrocene- Escherichia coli» and «ferrocene-Paracoccus yeei, E. coli association» systems showed good operational stability with a relative standard deviation of 6.9 and 7.3% (14 measurements) and a reproducibility of 7 and 5.2% using copper (II) ions as a reference toxicant. Biosensor analysis with these systems was shown to highly correlate with the results of the standard method using Chlorella algae as a test object. Developed biosensors allow for a valuation of the polluted natural water’s impact on the ecosystem via an assessment of the influence on bacteria and yeast in the receptor system. The systems could be used in toxicological monitoring of natural waters.

Electroactive biofilms (EABs) are essential components of both natural and artificial bio‐electrochemical systems (BESs). However, the inevitable decay of EABs during prolonged operation can diminish their performance. In this contribution, an effective and noninvasive strategy for rejuvenating aging biofilms by the elastic deformation of anode material is approved. The synthesized wood tracheid‐like structures anode material showed excellent compressibility and fatigue resistance in a wet state. The findings indicate that after the elastic deformation of the anode, aged biofilm exhibited a 37.5% increase in metabolic activity, and multi‐SIM images confirmed the removal of dead cells. Analysis of the extruded substance revealed a significant removal of loosely bound extracellular polymeric substance which doesn't contribute directly to electron transfer. Community analysis demonstrated the rejuvenation process suppressed the ecological competition from non‐exoelectrogens. Overall, there is a notable 25.97% increase in power density following the elastic deformation of the anode. Additionally, ion diffusion, specific capacitance, and catalytic response current all improved. To the knowledge, this is the first report employing an anode deformation strategy to restore decayed mix‐cultured electroactive biofilm, which is vital for the practical long‐term application of BESs. This work also offers new insights into the mechanical influence of anode materials on microorganisms. A new strategy for revitalizing aged biofilms using the elastic deformation of biomass‐based flexible carbon aerogel is proposed. The elastic deformation removes the nonelectrogenic bacteria and the dead electrogenic bacteria, as well as a lot of loosely bound extracellular polymer materials. As a result, after elastic deformation, electroactive biofilms restore metabolic activity for efficient extracellular electron transfer (EET).

Electrical stimulation is increasingly explored as a strategy to accelerate the development of electroactive biofilms in microbial fuel cells (MFCs), yet its integration with photosynthetic MFCs (pMFCs) remains insufficiently understood. This study evaluated how short-term anodic stimulation (0.5–5 V, 4 days) affects biofilm formation and COD removal, and how subsequent operation with photosynthetic cathodes—Chlorella sp., Arthrospira platensis and Tetraselmis subcordiformis—modulates anodic microbial communities and functional potential. Stimulation at 1 V yielded the best activation effect, resulting in the highest voltage output, power density and fastest COD removal kinetics, whereas 5 V inhibited biofilm development. During pMFC operation, Chlorella produced the highest voltage (0.393 ± 0.064 V), current density (0.14 ± 0.02 mA·cm−2) and Coulombic efficiency (~19%). Arthrospira showed moderate performance, while Tetraselmis generated no current despite efficient COD removal. 16S rRNA sequencing revealed distinct cathode-driven community shifts: Chlorella enriched facultative electroactive taxa, Arthrospira promoted sulfur-cycling bacteria and Actinobacteria, and Tetraselmis induced strong methanogenic dominance. Functional prediction and qPCR confirmed these trends, with Chlorella showing increased pilA abundance and Tetraselmis displaying enriched methanogenic pathways. Overall, the combined use of optimal anodic stimulation and photosynthetic cathodes demonstrates that cathodic microalgae strongly influence anodic redox ecology and energy recovery, with Chlorella-based pMFCs offering the highest electrochemical performance.

This study explores the feasibility of producing biohydrogen from winery wastewater using a dual-chamber microbial electrolysis cell (MEC). A mixed microbial consortium pre-adapted to heavy-metal environments and enriched with Geobacter sulfurreducens was anaerobically cultivated from diverse waste streams. Over 5000 h of development, the MEC system was progressively adapted to winery wastewater, enabling long-term electrochemical stability and high organic matter degradation. Upon winery wastewater addition (5% v/v), the system achieved a sustained hydrogen production rate of (0.7 ± 0.3) L H2 L−1 d−1, with an average current density of (60 ± 4) A m−3, and COD removal efficiency exceeding 55%, highlighting the system’s resilience despite the presence of inhibitory compounds. Coulombic efficiency and cathodic hydrogen recovery reached (75 ± 4)% and (87 ± 5)%, respectively. Electrochemical impedance spectroscopy provided mechanistic insight into charge transfer and biofilm development, correlating resistive parameters with biological adaptation. These findings demonstrate the potential of MECs to simultaneously treat agro-industrial wastewaters and recover energy in the form of hydrogen, supporting circular resource management strategies.