Status Update on Bioelectrochemical Systems: Prospects for Carbon Electrode Design and Scale-Up

Bioelectrochemical systems (BES) employ enzymes, subcellular structures or whole electroactive microorganisms as biocatalysts for energy conversion purposes, such as the electrosynthesis of value-added chemicals and power generation in biofuel cells. From a bioelectrode engineering viewpoint, customizable nanostructured carbonaceous matrices have recently received considerable scientific attention as promising electrode supports due to their unique properties attractive to bioelectronics devices. This review demonstrates the latest advances in the application of nanoand micro-structured carbon electrode assemblies in BES. Specifically, in view of the gradual increase in the commercial applicability of these systems, we aim to address the stability and scalability of different BES designs and to highlight their potential roles in a circular bioeconomy.


Current Developments
Bioelectrochemical systems (BES) are made of biocatalytic assemblies capable of converting electrical into chemical energy or vice-versa. The advantages of biocatalysts in electrochemical conversion are numerous and include the possibility to work under mild operating conditions with high substrate selectivity accompanied with resilience to the presence of impurities, and the ability to seamlessly integrate decentralized energy conversion concepts [1][2][3][4][5][6][7][8][9]. The substitution or complementation of chemically catalyzed processes with biocatalysis are envisaged to make a multidimensional impact in the bioeconomy [10][11][12][13], where the latter has been defined by the European Commission as "the production of renewable biological resources and the conversion of these resources and waste streams into value added products, such as food, feed, bio-based products and bioenergy" [14]. From a general standpoint, BES may play a major role in a circular bioeconomy: (i) as biodegradable tools, (ii) consequential to their assembly from renewable or recycled resources, and (iii) by their conjugation to energy reco99very processes.
In regard to the first contribution (i), single-use and short-lifetime BES devices demonstrate clear opportunities in plastic/electronic waste reduction, which may be applied to biodegradable medical implants, environmental sensors, and wearable devices [15]. These include, for example, cellulose-fiber-based sensors and fuel cell components [16,17], or degradable fuel cell stacks [18].
In terms of the second role suggested for the BES (ii), intensive efforts are currently invested in developing practical approaches reducing the environmental impact of BES components by using renewable or recycled materials and energy resources. For example, Hernandez-Flores et al. introduced an agar-containing membrane for microbial electrosynthesis cells, which was discovered to be a low-cost and sustainable replacement for conventional membranes, and also resulted in a safer pretreatment procedure [14]. In another interesting recycling approach reported, high-performance microbial fuel cell electrodes were produced from waste tires [19]. In the process of designing a BES, one may select a biocatalyst from a constantly expanding pool of options [27]. Depending on the targeted substrate, product, and process specifications, broad possibilities to select from exist, and these include, for example: purified redox enzymes such as oxidoreductases, unpurified "crude" extracts of microbial cells, organelles such as chloroplasts or mitochondria, or whole cells such as electroactive bacteria, archaea or eukaryotes in mono-or co-cultures, given they can be immobilized in a way that allows them to effectively exchange electrons with an electrode surface in a receiving (cathodic) or ejecting (anodic) manner [1,27,28]. These are typically integrated into assemblies representing the four main BES categories, which consist of enzymatic fuel cells, microbial fuel cells, enzymatic electrosynthesis cells, and microbial electrosynthesis cells. While microbial systems are generally considered more stable and reliable in terms of energy or VAC recovery from organic wastes and wastewater streams, enzymatic systems benefit from high efficiencies due to the lack of membranes and metabolic processes that increase the inherent overpotentials and limited efficiencies associated with whole- Figure 1. Illustration of the two main bioelectrochemical systems used for the production of power and value-added chemicals and their basic operation principles: (A) In the biofuel cell assembly the biocatalyzed oxidation of substrates on the anode occurring simultaneously with the cathodic reduction of an oxidizer accounts for the generation of an electric power; (B) the bioelectrosynthesis cell converts the power it is fed from renewable sources to value-added chemicals via biocatalyzed cathodic transformations.
In the process of designing a BES, one may select a biocatalyst from a constantly expanding pool of options [27]. Depending on the targeted substrate, product, and process specifications, broad possibilities to select from exist, and these include, for example: purified redox enzymes such as oxidoreductases, unpurified "crude" extracts of microbial cells, organelles such as chloroplasts or mitochondria, or whole cells such as electroactive bacteria, archaea or eukaryotes in mono-or co-cultures, given they can be immobilized in a way that allows them to effectively exchange electrons with an electrode surface in a receiving (cathodic) or ejecting (anodic) manner [1,27,28]. These are typically integrated into assemblies representing the four main BES categories, which consist of enzymatic fuel cells, microbial fuel cells, enzymatic electrosynthesis cells, and microbial electrosynthesis cells. While microbial systems are generally considered more stable and reliable in terms of energy or VAC recovery from organic wastes and wastewater streams, enzymatic systems benefit from high efficiencies due to the lack of membranes and metabolic processes that increase the inherent overpotentials and limited efficiencies associated with whole-cell microbial systems [29][30][31]. Outside of these main categories, biomimetic systems are further promising objectives for optimization [1,13,27], and specific target systems such as microbial desalination and solar cells can be indicated as rapidly emerging technologies [32][33][34]. Along with the fundamental biocatalyst-electrode support interactions and electron transfer routes becoming better understood by the scientific community, the progress of the lab-scale research in this field requires an assessment of scalability and commercial applicability. Whereas substantial efforts are currently being invested in improving the performance of biocatalysts, other factors, such as the composition and the surface functionalization of the electrode show manifold possibilities for improvement. In this regard, the efficiency (i.e., rate) of the electron transfer between the catalysts and the electrodes defines the resulting space-time-yield/power density performance of the systems [1].

Recent Highlights in Carbon Bioelectrodes
In search of appropriate materials that (i) provide a stable immobilization matrix for biocatalysts, (ii) efficiently transfer electrons to/from their active centers, and (iii) are economically beneficial, carbon-based materials and additives offer a broad topographical diversity and opportunities for surface chemistry functionalization [35][36][37][38]. Compared to most metallic electrodes, carbonaceous structures naturally offer much higher specific surface areas and adsorption characteristics for biocatalyst immobilization [1,[39][40][41][42].
Furthermore, whereas non-precious metals are prone to corrosion and passivation, precious metals are hard and expensive to obtain, with these efforts often leaving negative social and environmental impacts. In comparison, a great deal of scientific efforts are invested into the sustainable production of carbon nanomaterials for electrodes that can be customized in size and shape as needed [43][44][45][46][47][48][49][50].
Additionally, current advances in material science and nanofabrication allow the synthesis-dependent scaling and tuning of carbon electrode materials suitable to match specific biological component dimensions, thus potentially increasing the catalytic performance of such biocatalysts in integrated assemblies [46].

Enzymatic Electrode Designs
One of the major challenges in enzyme-based bioelectronics is the electrical connection, known as "wiring", between the redox enzyme's active center, which is typically buried in the electrically insulating protein, and the electrode surface. Since the beginning of bioelectrochemical research in the field, numerous studies have highlighted the promising merits of carbonaceous materials to serve as efficient matrices for enzyme immobilization and wiring. A clear distinction is commonly accepted between direct electron transfer (DET) and mediated electron transfer (MET) routes. While most redox enzymes can only communicate with the electrode supports via mediating electroactive units, certain protein structures such as bilirubin oxidase (BOD) and fructose dehydrogenase (FDH) are known to facilitate direct electron exchange in engineered electrode configurations [51,52]. Such direct configurations obviate the need to use potentially leaking mediators and reduce toxicity. They tend to support more integrated assemblies, requiring fewer components to operate. A recent review by Xia et al. discusses current wiring and surface modification techniques used in optimizing enzyme-electrode interface supporting DET [53]. Here, we set the focus on mesoporous carbon, which has been repeatedly demonstrated as a highsurface-area, conductive, biocompatible, and cheap material showing a tunable structural versatility for both MET and DET wiring strategies [54][55][56][57][58].
One of the advantages of the mesoporous carbon lies in the dimensions of its pores (2-50 nm). As the pores fall in the size range of most redox enzymes (~5-100 nm), unique features for enzymatic biosensors, fuel cells, and electrolysis cells are expected from the carbon/enzyme hybrids. The optimal size range of the pores was further emphasized in other studies, showing that matrices with micropores (d < 2 nm) increased diffusion limitations within the electrolyte, while macropores (d > 50 nm) supported a lower surface loading of the enzyme biocatalyst [59][60][61]. Such drawbacks were found to be absent in the presence of the mesoporous materials, thus making them attractive for all scales of BES [62][63][64][65]. Further recent studies in the related field of enzymatic biosensors indicated that mesoporous carbon can be successfully implemented as a host matrix in amperometric devices [66][67][68][69][70], immunosensors [71], and electrochemiluminescent sensors [72]. These were subsequently used for environmental applications such as pollution detection [72][73][74][75], compost bioremediation [76], and in the health and food industries [77][78][79].
In 2010, Kwon et al. reported the improved stability and electron transfer characteristics observed for functionalized bioelectrodes, which were obtained by immobilizing glucose oxidase (GOx) in the pores of a mesoporous carbon matrix with a narrowing "bottleneck" pore structure [80]. This inspired the development of different "entrapment" techniques that were based on matching the pore sizes of the host matrix to the dimensions of different enzymes such as bilirubin oxidase (BOD) [81] or hemoglobin [82]. Using this approach, the immobilized enzymes showed superior stability and electroactivity when compared to other carbonaceous hosts or non-matching pore size assemblies [83]. The increased stability was attributed to the "biocompatible microenvironment" confinement leading to decreased biofouling effects [84], while the increase in the bioelectrocatalytic responses was attributed to the catalytically favorable unfolding of the protein's tertiary structure, allowing a favorable electron transfer between the cofactors of the enzymes and the pores [80,85].
A novel methodology to electrically wire redox enzymes on mesoporous conductive carbon supports was shown in 2013 [86]. In the first step, a contact adsorption of mesoporous carbon nanoparticles (mpCNPs) with an average pore size of 6 nm was carried out on top of a glassy carbon support. The matrix was then interacted with redox-relay molecules, such as ferrocene methanol (FcMeOH), methylene blue (MB + ), or 2,2 -azinobis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS 2− ), which are known to mediate the enzymes GOx, horseradish peroxidase (HRP), and BOD, respectively. The subsequent adsorption of these enzymes on the matrix, followed by a chemical crosslinking and immobilization of the protein units using Nafion TM , led to stable assemblies in which the enzymes capped the relay molecule-filled nanopores. As an example, the GOx-capped FcMeOH-containing mpCNP assembly is illustrated in Figure 2A. Following the observation that only a minor leakage of mediator molecules occurs from the matrix, this assembly was tested as a bioelectrocatalytic anode, as shown in Figure 2B. Evidently, the glucose-responsive anodic currents, mediated by the FcMeOH species trapped in the pores, showed a linear increase before their saturation at 60 mM glucose, as shown in Figure 2C. From the saturation current and the enzymatic loading of GOx on the surface, a turnover rate of k et~1 000 s −1 was calculated for the assembly. This value, nearly 50% larger than the transfer rate between GOx and its native O 2 acceptor, was rationalized by the efficient electron exchange between the mpCNPs matrix and the FAD cofactor of the enzyme through the nanopore-confined diffusion of the mediator units. The generic wiring paradigm was further implemented to other enzymes, such as BOD, in the construction of an ABTS 2-/BOD-mpCNPs cathode assembly. The latter was coupled to the GOx-based anode to provide a fully functional membrane-less biofuel cell operated by glucose and O 2 , and supporting a power output of nearly 100 µW cm −2 . The enzyme-capped mpCNPimmobilization approach was further implemented in architectures employing multiple enzymes [87,88]. Electrodes modified with mixed compositions of redox enzymes were demonstrated to effectively conduct multiple anodic and/or cathodic bioelectrocatalytic transformations. In one system employing two redox relay units, FcMeOH and MB + , trapped in the nanopores by a mixture of GOx and HRP, electrocatalytic currents were observed both for the oxidation of glucose and the reduction of H 2 O 2 . The presence of FcMeOH was further shown to simultaneously mediate the two FAD-containing enzymes GOx and lactate oxidase, resulting in oxidation currents in the presence of one or both of the sugar fuels glucose and lactate. Notably, these electrically contacted bienzymefunctionalized mpCNP electrodes show promise to be applied in applications such as a dual amperometric biosensors, as well as in a multifuel-driven biofuel cells, as depicted in Figure 2D [87]. The cathodes employed in these multi-sugar fuel cells are based on ABTS 2--loaded mpCNPs matrices capped by the BOD and catalase (CAT) enzymes. For them, two cathodic pathways were associated: (a) aerobic, at which the 4ereduction of O 2 (as an internal oxygen source) were present in the cell, the power output depended on the composition of the fuel introduced into the chamber, as shown in Figure 2E. Typical power discharge curves were obtained in the presence of either glucose or lactate, with a maximal current response recorded upon the introduction of both fuels. The power outputs correlated with the relative bioelectrocatalytic responses measured for the different fuels on the anode assembly. The effect of the oxidizer composition in the cell on its maximal power output was also demonstrated, as measured in the presence, separately and combined, of the fuels in Figure 2F. Interestingly, when H 2 O 2 was solely present in the electrolyte, the power generation reached peak values that were correlated to the localized formation of O 2 near the BOD, avoiding its interference to the buildup of the catalytic current at the anode surface. The co-immobilization techniques were highlighted as promising for harnessing electrical power from mixed biomass environments containing multiple fuel resources.
amperometric biosensors, as well as in a multifuel-driven biofuel cells, as depicted in Figure 2D [87]. The cathodes employed in these multi-sugar fuel cells are based on ABTS 2-loaded mpCNPs matrices capped by the BOD and catalase (CAT) enzymes. For them, two cathodic pathways were associated: (a) aerobic, at which the 4e -reduction of O2 to H2O was catalyzed by BOD through a pore-confined mediation by the ABTS 2-species, and (b) anaerobic, at which introduction of H2O2 to the electrolyte in the absence of O2 led to its catalytic disproportionation to O2 and H2O by the CAT, with the generated O2 triggering the electrocatalytic transformations at the BOD. Whenever both O2 and H2O2 (as an internal oxygen source) were present in the cell, the power output depended on the composition of the fuel introduced into the chamber, as shown in Figure 2E. Typical power discharge curves were obtained in the presence of either glucose or lactate, with a maximal current response recorded upon the introduction of both fuels. The power outputs correlated with the relative bioelectrocatalytic responses measured for the different fuels on the anode assembly. The effect of the oxidizer composition in the cell on its maximal power output was also demonstrated, as measured in the presence, separately and combined, of the fuels in Figure 2F. Interestingly, when H2O2 was solely present in the electrolyte, the power generation reached peak values that were correlated to the localized formation of O2 near the BOD, avoiding its interference to the buildup of the catalytic current at the anode surface. The co-immobilization techniques were highlighted as promising for harnessing electrical power from mixed biomass environments containing multiple fuel resources. In a later study, the pore-entrapment methodology was found to be of a further benefit in the efforts to immobilize communicating enzymatic cascades on electrodes In a later study, the pore-entrapment methodology was found to be of a further benefit in the efforts to immobilize communicating enzymatic cascades on electrodes [30,41,89,90]. A mpCNP-entrapped enzymatic cascade-functionalized assembly was designed consisting of invertase (INV), mutarotase, GOx, and FDH [88]. When triggered in the presence of a sugar mixture (glucose, fructose, sucrose) and hydrogen peroxide, the biofuel cell allowed several sequential conversion pathways between the enzymatic catalysts, which resulted in A further unique aspect of mesoporous carbon-based BES relates to the mpCNPs' versatility in serving as a matrix for the tailored electrosynthesis or biosynthesis of poreconfined metallic nanoparticles (NPs). This aspect is particularly interesting, as several studies showed that an enhanced catalysis is facilitated by mpCNP structures with embedded Ag [91], Au [92][93][94][95][96], Pt [97][98][99], or Pd NPs [99]. In one example, Au NP-ordered mesoporous carbon hybrid electrodes were designed by Korani et al., where the respective modification of the cathodic and anodic assemblies with a BOD and glucose dehydrogenase biocatalysts yielded a biofuel cell with a maximum power density of 33 µW cm −2 [93].
Recently, carbonaceous mesoporous materials are being increasingly explored as matrices assisting DET in bioelectrocatalytic processes [53,82,100]. In one of the first examples, metal nanoparticle-mpCNP hybrid structures were tested in an assembly in which the enzymes served as caps, trapping metal ion molecules inside the mesopores [96]. Figure 3A illustrates the methodology used in constructing Pt or Au nanocluster-entrapped mpCNPs. The pores were initially loaded with platinum or gold salt solutions and capped by enzyme units. The latter were cross-linked to ensure a good mechanical stability of the assembly. In the next stage, a negative potential was applied, reducing the metal ions to their respective atoms, thus creating metallic nanoclusters (NCs) whose growth was confined to the nanopores, as shown for Pt in Figure 3B. The use of the protease proteinase K to remove the enzyme caps yielded an electro-active surface which was then demonstrated, e.g., with Pt NCs, to electrocatalytically reduce both O 2 and H 2 O 2 .
The study further showed that by recapping the pore-entrapped metal NCs with specific redox enzymes, an electron transport from the enzymatic cofactors through the NCs to the conductive carbonaceous matrix was facilitated and allowed an effective DET pathway for electrocatalysis. The resulting assemblies showed enhanced bioelectrocatalytic functions, either in the absence or the presence of the capping enzymes (i.e., both by the metallic NPs themselves, as well as them transferring the charge to/from the enzymes). Among the systems described in this study were GOx-capped Pt NCs/mpCNPs assemblies allowing the direct oxidation of glucose, and HRP-capped Au NCs/mpCNP assemblies facilitating the direct reduction of H 2 O 2 .
In search for means to exploit the mpCNP matrix in enhancing the efficiency of the DET towards even more facile bioelectrocatalysis, a strategy to self-wire GOx to its carbon-supported surface matrix was recently reported [51]. The method was inspired by previous successful attempts to enzymatically reduce metal ions at the vicinity of GOx's activated FADH 2 cofactor [101]. Following the loading of glassy carbon-adsorbed mpCNPs with platinum ions and glucose, the nanopores were capped with GOx. Under N 2 -saturated anaerobic conditions, the GOx oxidized the pore-entrapped glucose, with the concurrent reduction of its oxidized FAD cofactor to FADH 2 . Under these conditions, the cofactor acted as a strong reducing agent for the pore-entrapped Pt ions, thus allowing the formation of a Pt NC in its vicinity. The extension of the cluster from the cofactor towards the walls of the nanopore defined the "inside-out" growth direction associated with the enzymatic synthesis paradigm, as shown in Figure 3C, Panel I. As the clusters reached the carbon walls of the matrix, an electrical contact was established, allowing a bioelectrocatalytic DET path for GOx to oxidize its glucose substrate. The resulting electrode was investigated and compared to an "outside-in"-grown assembly obtained by the direct electrochemical deposition of mpCNP-entrapped Pt ions in the presence of GOx caps, as shown in Figure 3C, Panel II. The Pt NC/GOx-modified mpCNP assembly achieved by this methodology was shown to possess a larger nano-gap bridging the FAD cofactor of the GOx and the metal cluster, as compared to the enzymatically synthesized "inside-out" electrode. The larger gap increased the overpotential required to regenerate the bioelectrocatalytic reaction and led to a total decrease in the performance of the electrode towards oxidizing glucose. The differences between the two approaches were further reflected by comparative discharges of biofuel cells, employing a DET biocathode based on BOD-adsorbed mpCNPs, and the respective anodes, as shown in Figure 3D. As can be seen both from the polarization curves in Panel I, and the power outputs in Panel II, upon introduction of an equivalent concentration of glucose to the cells, the enzymatically "inside-out"-based assembly supported favorable discharge voltage and power generation. These were even higher than the values measured for a mediated assembly employing a GOx-capped FcMeOH-entrapped mpCNP anode. The results highlight a novel approach to self-wire redox enzymes immobilized to confining mesoporous carbon structures, thus allowing a maximal proximity between their redox centers and relaying NCs to support enhanced bioelectrocatalysis. direct electrochemical deposition of mpCNP-entrapped Pt ions in the presence of GOx caps, as shown in Figure 3C, Panel II. The Pt NC/GOx-modified mpCNP assembly achieved by this methodology was shown to possess a larger nano-gap bridging the FAD cofactor of the GOx and the metal cluster, as compared to the enzymatically synthesized "inside-out" electrode. The larger gap increased the overpotential required to regenerate the bioelectrocatalytic reaction and led to a total decrease in the performance of the electrode towards oxidizing glucose. The differences between the two approaches were further reflected by comparative discharges of biofuel cells, employing a DET biocathode based on BOD-adsorbed mpCNPs, and the respective anodes, as shown in Figure 3D. As can be seen both from the polarization curves in Panel I, and the power outputs in Panel II, upon introduction of an equivalent concentration of glucose to the cells, the enzymatically "inside-out"-based assembly supported favorable discharge voltage and power generation. These were even higher than the values measured for a mediated assembly employing a GOx-capped FcMeOH-entrapped mpCNP anode. The results highlight a novel approach to self-wire redox enzymes immobilized to confining mesoporous carbon structures, thus allowing a maximal proximity between their redox centers and relaying NCs to support enhanced bioelectrocatalysis.

Microbial Electrode Designs
Compared to their enzymatic counterparts, fewer studies have been reported on base supports associated with defined nanostructure features for microbial electrode assemblies, until recently [102][103][104][105][106][107][108]. Indeed, most studies combining microbial bioelectronics with carbonaceous supports report the use of carbon macrostructures such as glassy carbon, graphite plates, carbon fiber brushes, foams, and felts [109][110][111][112]. These are typically preferred over the use of a stainless steel, hence benefitting from the biocompatibility, low cost, conductivity, and high surface area of the carbon [113][114][115][116][117][118]. Among these materials, porous carbon electrodes with varying pore sizes were successfully implemented [119][120][121][122][123]. A recent increase in research activity directed towards implementing 3D-nanostructure architectures for microbial electrodes indicates that the nanotopographical modification of the electrode surfaces enhance the adhesion and growth of electroactive organisms, leading to beneficial biofilm formations and superior electron transfer properties [124][125][126][127][128][129][130]. In one study, Chen et al. showed a biofuel cell anode employing Escherichia coli adsorbed on a custom macroporous carbon paste with a defined pore size of 400 nm. Notably, the pore size in the assembly, constructed on a carbon felt matrix, was slightly larger than the microorganism. The controlled pore size of the porous paste was achieved by using silica spheres as templates and sucrose as a carbon precursor. Evidently, a four times higher power output (160 µW cm −2 ) was measured in this configuration compared to plain carbon felt matrices. The results were explained by the size matching between the bacteria and the pore, facilitating the formation of a bacterial biofilm with superior electron transfer between the microorganisms and the surface [119]. A similar strategy was pursued by Jeon et al. in 2018, who fabricated porous carbon scaffolds with tunable pore sizes and achieved a 40-fold higher loading density of E. coli confinement compared to conventional 3D porous reticulated vitreous carbon scaffolds [131]. This technique might be interesting for microbial BES electrode fabrication, as the pore sizes can be tuned by the controlled growth of polystyrene spheres by dispersion polymerization, which lead to a self-assembled arraytemplate for the carbonization of a resorcinol formaldehyde gel. Accordingly, the resulting 3D porous carbon scaffolds can be customized to the size of the targeted microorganism. In a different study, a microbial fuel cell anode employing anaerobic sludge as a biocatalytic source showed a twofold increase in its power output (ca. 280 µW cm -2 ) compared to an unmodified carbon cloth anode, by employing a 3D N 2 -doped porous carbon material fabricated using a one-step pyrolysis route [132]. In regard to BES in a circular bioeconomy, promising power outputs were recently published for coffee waste-based activated carbon matrices employing E. coli biocatalysts [133]. The power density of the assembled fuel cell exceeded the one of commercial activated carbon (ca. 390 µW cm −2 as opposed to ca. 100 µW cm −2 ) with a pore size adaptable to a certain degree by the amount of added KOH during the carbonization process. Another sustainable and cost-effective alternative for microbial BES supports with a mixture of micro-, meso-and macropores was tested with the use of activated carbon sourced from silver grass and demonstrated a power output of ca. 100 µW cm −2 [134]. Such approaches also hold a promise for microbial electrosynthesis processes. Notably, scalable microbial electrosynthesis cells are particularly interesting for power-to-x applications, where decentrally generated excess renewable energy is stored chemically in the form of fuel molecules such as methane, known as electromethanogenesis [135,136]. This microbial electrosynthesis technology employing methanogenic archaea as biocatalysts that metabolize CO 2 and hydrogen is quickly growing. The produced methane can be stored, injected into the existing gas grid, or used as a transportation fuel [136]. For these reasons, the methodology was recently named "bioelectrochemical power-to-gas" [11], and it is rapidly aligning with other powerto-x methodologies that are currently being investigated for electricity-enhanced biofuel production and fossil fuel substitution [137][138][139]. With first field tests implying a high resilience of biocatalysts under standby conditions and contaminated CO 2 feeds, which are essential for a seamless operation accompanied by fluctuating solar/wind surplus electricity recovery and real syngas streams, this methodology is attractive and holds a promise for the future [140,141].

Stability
To date, the operating lifetime expectancy in all enzyme-based systems remains a major obstacle to commercialize enzyme-based BES applications, a predicament that does not apply to their microbial counterparts as the living organisms provide optimized conditions for the membrane encapsulated enzymes [30,31]. Under full load, enzymatic fuel cells charged with a single batch of active biomaterial rarely exceed a 24 h discharge cycle. Despite the challenge, a few attempts showing novel approaches were carried out to prolong the discharge lifetimes of the BES. In one study [142], a stack of sealed chambers containing fresh glucose dehydrogenase deposited on multi-array electrodes facilitated a continuous operation through the sequential opening of chambers. Another study [143] demonstrated the possibility to continuously supply fresh biocatalysts into the system and to maintain an active air-breathing laccase cathode over 33 days using a laccase-producing trametes versicolor fungus present in the supernatant.
A different approach that was recently presented highlights the immobilization of redox enzymes on magnetic carbon structures [144,145]. In this study, the protein degradation limitation was bypassed by magnetically assembling and exchanging the active biocatalyst layers on stationary electrodes, as shown in Figure 4A [52]. In this design, the biocatalysts FDH and BOD were immobilized on carbon-coated magnetic nanoparticles (ccMNPs), thus generating the active components of the anode and the cathode, respectively. Directed by magnetic field gradients, these nanoparticles were deposited on planar electrode surfaces, released upon degradation of the catalytic activity, and reloaded as a fresh batch to facilitate the renewal of the active biocatalysts on the surface. The method was demonstrated for a fructose/oxygen biofuel cell. The dramatic extension in the operation lifetime of the fuel cell provides a promising approach that leaves the need for exchanging the entire systems, including chambers and electrodes, redundant. Shown in Figure 4B is another ccMNP-based methodology to extend the operational lifetime of enzymatically modified electrodes. In this study, ccMNPs were used as a removable protection layer covering a BOD/carbon nanotubes-deposited electrode surface. Upon the magnetic attraction of the protective layer to the surface, a stable, mediatorless oxygen reduction was obtained, and even remained while testing the biocatalysts in a harmful proteolytic environment [146]. Under these conditions, a 30% loss in the bioelectrocatalytic currents over 30 days was observed, as compared to a 100% loss in the case of the unprotected BOD electrode. Evidently, these reports imply that the immobilization of the biocatalysts on magnetic nanoparticles offers various merits to increase the applicability of BES.

Substrate Flexibility
A further aspect of BES applicability lies in the flexibility of the system to adapt to a changing substrate supply. In this regard, the high affinity and selectivity of the biocatalysts towards their substrates can be of a disadvantage when the homogenity of feed solutions change over time, a case often observed in waste water treatment plants, side-product accumulations in fermentation processes, or biomass-derived syngas feeds [147][148][149]. To adress this issue, a recent study suggested a methodology based on a combination of different enzyme-modified carbonaceous nanoparticles [150]. With the base-electrode modified with relay-loaded, redox enzyme-capped mpCNPs, a second layer containing predesigned enzyme-functionalized ccMNPs was introduced through an external magnetic field gradient, as shown in Figure 4C. The close proximity between the enzymes then allowed the facile conversion of the substrate, resulting in the activation of the cascade and generation of bioelectrocatalytic currents. A further demonstration of an in-situ generation of power along changing the substrate feed composition was then exemplified. Following the methodology, a multi-substrate biofuel cell consisting of a cathodic CAT/BOD cascade and an anodic INV/FDH was constructed. The cell was successfully operated on fructose and sucrose and their combination. Other advantages of this cell included its ability to operate reliably in anaerobic and aerobic conditions, its exclusive operation (both cathode and anode) by DET reactions, and that the power output could be magnetically controlled. The highlighted results exemplify the promising possibilities of magnetically responsive BES to be implemented in multi-biomass environments and for applications requiring bioelectrocatalytic functions under the control of non-invasive external triggers.
layer containing predesigned enzyme-functionalized ccMNPs was introduced through an external magnetic field gradient, as shown in Figure 4C. The close proximity between the enzymes then allowed the facile conversion of the substrate, resulting in the activation of the cascade and generation of bioelectrocatalytic currents. A further demonstration of an in-situ generation of power along changing the substrate feed composition was then exemplified. Following the methodology, a multi-substrate biofuel cell consisting of a cathodic CAT/BOD cascade and an anodic INV/FDH was constructed. The cell was successfully operated on fructose and sucrose and their combination. Other advantages of this cell included its ability to operate reliably in anaerobic and aerobic conditions, its exclusive operation (both cathode and anode) by DET reactions, and that the power output could be magnetically controlled. The highlighted results exemplify the promising possibilities of magnetically responsive BES to be implemented in multi-biomass environments and for applications requiring bioelectrocatalytic functions under the control of non-invasive external triggers.

Future Synergies
Nanostructured carbon electrodes hold an opportunity to reduce the investment costs of manufacturing BES. This, however, might be a consequence of further research and development efforts building on the recent advancements and achievements. It can be expected, for example, that by tailoring the size of carbonaceous 3D architectures to the biocatalysts, the power outputs and conversion efficiencies can be further increased.
Furthermore, the use of carbon-metallic NC hybrid structures and magnetically responsive nanostructures may contribute to biofuel cell stability and operational versatility in different environments and conditions. The encouraging findings from fuel cell assemblies might also be translated to bioelectrosynthesis. Specifically, this knowledge transfer can be beneficial to the relatively new field of enzymatic electrosynthesis, where an increasing number of VACs are already synthesized via enzymatic routes. Among these are VACs synthesized, for example, from CO 2 using single-or multi-enzymatic electrode configurations [24,25,[151][152][153]. One may envisage that such reactions will be optimized by a tailored functionalization of nanostructured carbon electrodes, thus overcoming challenges associated with high ohmic losses, and will increase the observed low yields. A better adsorption of CO 2 on nano-and microstructured support materials may also influence the transformation rates, as was recently shown with silica microspheres and their use as carrier units for immobilized carbonic anhydrase [154]. Furthermore, customized high-surface-area carbon matrices offer new possibilities for enzymatic N 2 valorization [26]. Apart from classic enzymatic electrosynthesis cells, transferable electrode designs can furthermore promote the direct enzymatic conversion of sunlight [155], as well as the development of hybrid systems, which incorporate a biofuel/bioelectrosynthesis cell combination for an internal electron supply for the electrosynthesis reaction [156]. Such systems may find usage in specialized applications such as the decarbonylation of fatty aldehydes [24], or a D-sorbitol conversion [157]. For symbiotic processes, purely enzyme-based self-powered systems as well as the combination of microbial and enzymatic electrosynthesis/fuel cells are forthcoming for decentral VAC, fuel, or pharmaceutical production [24,152,155].
Interestingly, the knowledge transfer from the enzymatic systems regarding wiring and stabilization techniques may be highly valuable for the field of microbial electrosynthesis. In microbial reactor design, the unique quality of micro-/nanostructured features of the carbonaceous immobilization matrix can be successfully matched to serve a distinct purpose in the electron transfer routes [38,158]. Of the systems described, microbial electrosynthesis shows the highest promise for up-scaling, due to lower investment costs, self-replication and functional biofilm formation and resilience in changing operation conditions [117,159].
From the current point of knowledge, the assessment of techno-economical evaluations and possible impacts on circular bioprocesses for distributed energy systems remains difficult because of limited available data [138,160,161]. Reliable data from scalable processes and reactor designs, in addition to regulatory developments in carbon taxing, availability of precious electrode materials, and enforcements of bioeconomic strategies, will ultimately determine the commercial competitiveness of carbon electrode-based BES technologies.