Microbial electrosynthesis: opportunities for microbial pure cultures

Highlights

• The time is right for targeted engineering of electroactive microbes for electrobioproduction.

• Current mixed culture microbial electrosynthesis (MES) and microbial electromethanogenesis are limited to a few low-value products.

• The product spectrum of MES will greatly benefit from engineered microbial pure cultures, especially with regard to high-value products.

• To quickly advance MES, standardized, off-the-shelf electrobioreactors are urgently needed.

Abstract

Microbial electrosynthesis (MES) is an emerging technology that couples renewable electricity to microbial production processes. Although advances in MES performance have been driven largely by microbial mixed cultures, we see a great limitation in the diversity, and hence value, of products that can be achieved in undefined mixed cultures. By contrast, metabolic control of pure cultures and genetic engineering could greatly expand the scope of MES, and even of broader electrobiotechnology, to include targeted high-value products. To leverage this potential, we advocate for more efforts and activities to develop engineered electroactive microbes for synthesis, and we highlight the need for a standardized electrobioreactor infrastructure that allows the establishment and engineering of electrobioprocesses with these novel biocatalysts.

Microbial electrosynthesis (MES) is an emerging technology that couples renewable electricity to microbial production processes. Although advances in MES performance have been driven largely by microbial mixed cultures, we see a great limitation in the diversity, and hence value, of products that can be achieved in undefined mixed cultures. By contrast, metabolic control of pure cultures and genetic engineering could greatly expand the scope of MES, and even of broader electrobiotechnology, to include targeted high-value products. To leverage this potential, we advocate for more efforts and activities to develop engineered electroactive microbes for synthesis, and we highlight the need for a standardized electrobioreactor infrastructure that allows the establishment and engineering of electrobioprocesses with these novel biocatalysts.

Achievements and limitations of MES with mixed cultures

It has been long known that microbes can interact with electrodes [1,2], and it was already suggested in the 1970s that electrochemistry can be used to steer fermentations [3]. Soon after the first isolation of solid metal oxide-reducing bacteria in around 1990 [4., 5., 6.], interest was sparked in producing electrical energy through microbial transformations in microbial fuel cells [7., 8., 9.]. However, it took time and two key publications before the first concepts emerged of microbial production fueled by electrical energy. First, in 2009, the concept of microbial electromethanogenesis (MEM, see Glossary) was presented, where cathode-derived (cathodic) electrons fed the microbial electrochemical production of methane [10]. Then, in 2010, the overarching concept of MES was introduced with the example of acetogens consuming cathodic electrons to drive the reduction of CO₂, leading to the formation of acetate as the main product [11].

Since then the scientific activities in electrobiotechnology for synthesis have exploded, and the promise of coupling renewable electrical energy resources to biotechnological production has attracted tremendous interest. Developments have proceeded in two directions: defined microbial physiology research to understand and manipulate microbial electrode interactions (Box 1), and bioprocess development research for the technical realization of MES. The latter has mostly employed undefined anaerobic mixed cultures, mainly because of difficulties in handling the obligate anaerobic pure cultures and limited understanding of their biology. A recent review from Hengsbach and colleagues [12] comprehensively compiled progress in MES and highlighted the current superiority of mixed culture approaches over pure cultures. The average space-time yield of all MEM studies examined was 330 mol CH₄ m^–3.day^–1 for undefined mixed cultures (20 studies) versus 22 mol CH₄ m^–3.d^–1 for pure cultures (five studies), although there was a wide spread in the data. Similarly, the space-time yields for MES from CO₂ to acetate were much higher for mixed cultures; these generated 1900 g acetate.m^–3.d^–1 (14 studies) versus 87 g acetate.m^–3.d^–1 for pure cultures (16 studies). However, this divide in overall process performance between mixed and pure cultures is to be expected. Mixed culture research has treated the biological component mostly as a black box, and instead focused on optimizing electrode materials and reactor designs to boost process performance. By contrast, much pure culture work was intended for fundamental research into the specific biocatalysts or biochemical reactions. This was often carried out in reactors that were suitable for this type of research, but inadequate from a (process) engineering perspective (i.e., H-type reactors), and therefore did not allow leveraging of their full potential for synthesis. In addition, anaerobic pure cultures require aseptic conditions and are prone to damage from potential oxygen leakage (from the anodic reaction or intrusion via reactor ports), whereas mixed cultures are robust in performance and oxygen-tolerant through the resilience of the community. Thus, mixed microbial cultures provide several benefits for microbial electroproduction. Jourdin and Burdyny recently tried to predict a path forward for MES and suggested that the most promising developments are in engineering more efficient biofilm–electrode composites and in integrated process optimization by tuning temperature, salinity, and pressure [13].

Box 1. Microbial extracellular electron transfer.

The biochemical mechanism by which electroactive microorganisms are able to exchange electrons with solid substrates is known as microbial extracellular electron transfer. Evolutionarily, these mechanisms are crucial to maintain redox homeostasis under conditions in which the metabolic electron donor or acceptor is a reduced or an oxidized solid compound, for example, a metal or a metal oxide, respectively. The most widespread case is that of iron minerals, which, like an electrode, can release electrons when they are oxidized, or receive electrons when they are reduced.

In pure cultures, two main types of microbial extracellular electron transfer are known: direct and mediated electron transfer (Figure I). Direct electron transfer involves close physical contact between the cells and the solid substrate, and requires a dedicated biomolecular machinery that is capable of conducting electrons from the outside to the inside of the cells, or vice versa. The well-known stacked-heme nanowires of Geobacter sulfurreducens [75], and the cytochrome-based Mtr pathway of S. oneidensis [76], are the most studied examples of direct electron transfer (Figure IA,B). Mediated electron transfer requires the action of a diffusible electron shuttle molecule that transports the electrons and donates or accepts them, and flavins [77], quinones [78], H₂ [79], and formate [31] are most widespread electron mediators (Figure IC). These natural electron mediators, as well as their synthetic equivalents, can also be externally added to enable extracellular electron transfer in microorganisms that lack intrinsic mechanisms.

The microbial growth phenotype in a bioelectrochemical system (BES) is closely related to the extracellular electron transfer mechanism. Strains that use direct electron transfer are prone to generate biofilms to ensure their proximity to the electrode, while biofilm formation is not a requirement for those that rely on the use of suspended mediators.

Figure I.

Illustration of the different modes of extracellular electron transfer to an anode (unbroken blue bar, pink arrows) or from a cathode (shaded blue bar, blue arrows).

(A) Direct electron transfer via specialized redox proteins in the cell wall, for example, cytochromes. (B) Direct electron transfer via conductive nanowires. (C) Electron transfer via diffusible redox mediators.

Alt-text: Box 1

However, there is one significant limitation to all mixed culture approaches (Figure 1): despite the amazing metabolic capacities of the biocatalysts, because of thermodynamic constraints current microbial electroproduction with mixed cultures can only deliver a few products – namely methane, acetate, sometimes ethanol, and in rare examples longer-chain carboxylates (up to hexanoate). Given this drawback, we see a new trend arising: the development of highly specific microbial pure culture electrocatalysts to open the treasure box of biochemical pathways for MES.

Figure 1.

Metabolic driving forces in microbial electrosynthesis.

(A) With undefined mixed cultures, thermodynamic constraints drive the overall metabolic balance of cathodic reactions (shaded left electrode) to stable fermentation products such as methane or acetate. (B) A defined microbial pure culture catalyst enables the tailored production of a high-value target product at the cathode.

The opportunities of pure cultures

Physiological studies and targeted engineering of non-model microorganisms are often slow and tedious endeavors. However, they allow a stepwise approach to a more rational and knowledge-based understanding of the limitations of microbial reactions, and lay the foundation for the targeted design of new pathways. Multiomic comparisons of electrochemically influenced microbial reactions versus classical cultivations can elucidate important pathways and highlight bottlenecks. A more comprehensive understanding of metabolic driving forces would enable a rational re-engineering of microbial biocatalysts for better performance, higher efficiency, or the production of novel products. Although such approaches are well established in classical biotechnology, such as for the production of amino acids or secondary metabolites, they are still rarely discussed in relation to biocatalysts in electrobiotechnology. Instead, microbiological research on MES mostly stops at descriptive aspects of their performance. That is, deletions are generated to elucidate the function of particular genes such as those involved in electron transfer processes [14,15], and multiomic analyses provide insight into the physiological state of the biocatalysts [16., 17., 18.]. However, to date very few studies have sought to exploit this information to advance MES. Nevertheless, we see an enormous opportunity in these biology-driven developments to break through the current limitations of pure cultures in MES, and especially to overcome the narrow product spectrum of mixed culture MES. In this review we argue that it is now time to address electroactive microbial pure cultures as microbial hosts for a wide range of electrobioproductions (Figure 2). The genetic tools are generally available, the physiological knowledge base of electroactive microorganisms is rapidly expanding, and new biotechnological processes with in situ control of electron acceptor and donor availability meet the growing trend in bioprocess technology for online monitoring and control [19]. With this objective, we see two main tasks for the scientific and engineering community: (i) more lively attempts and activities in engineering electroactive microbes towards production, and (ii) rethinking of process and systems engineering to meet the specific needs of the electroactive biocatalysts while at the same time being compatible with classical bioprocess technology infrastructure. The latter approach will greatly lower the hurdle for advancing technology readiness levels once the biocatalysts are established. By contrast, if we continue to operate highly customized and individualized reactor infrastructure in every laboratory – as is currently the case – a transition to reliable and scalable electrobiotechnology will be cumbersome. In the following sections we first review the fairly limited, but promising, achievements in electrobiocatalyst engineering for better or new performance. We then present our suggestions and ideas for developing a future-ready and scalable electrobioprocess infrastructure.

Figure 2.

The promise of pure culture electrobioproduction.

Schematic of potential electrobioproduction with pure cultures for both anodic and cathodic reactions, and the expected advantages (blue) of physiological knowledge and synthetic biology approaches for strain development. The graphic also emphasizes the possible disadvantages (pink) of using pure cultures. Abbreviations: Fd_ox, oxidized ferredoxin; Fdred, reduced ferredoxin.

Advances in electrobiotechnology using pure cultures

State and prospects of pure culture electromethanogenesis

MEM is the reduction of CO₂ by cathode-derived electrons to generate methane by microorganisms. MEM is discussed as an energy storage technology to convert renewable electricity and waste CO₂ into methane (natural gas) for long-term storage. It was first described in mixed cultures [10,20], potentially because biomethanation processes often rely on mixed cultures. Indeed, biomass degradation to methane (i.e., anaerobic digestion for biogas production) necessitates a diverse community to break down biomass components efficiently. By contrast, the substrates in MEM are only CO₂ and electrons plus protons, respectively, hydrogen. Therefore, MEM in a pure methanogenic culture should be as efficient as in mixed cultures, provided that the pure culture can tolerate and thrive under the conditions of the MEM system. It is notable that commercial biomethanation based on gas fermentation of H₂ and CO₂ uses pure cultures of methanogens (e.g., Methanothermobacter) and can achieve exceptional microbial methanogenesis rates (Electrochaea GmbH, Germany, and Krajete GmbH, Austria). The current disparity between pure culture methane formation rates obtained via gas fermentation (up to 688 l.l^–1.d^–1 [21]) and the current record for MEM (12.5 l.l^–1.d^–1 [22]) indicates that the rate of electron delivery to the microbes is limiting during MEM, and not the microbial metabolic rate.

Pure culture studies have proved invaluable in identifying the limitations associated with MEM. Kracke and colleagues found that Methanococcus maripaludis is more active but also lyses at a higher rate during MEM than with H₂/CO₂ feeding [23]. However, proteomics did not identify unique stressors for MEM cultivation. Similarly, M. maripaludis was used to determine an inhibitory cathodic current density of 1–1.5 mA.cm^–2 [24] and to study the impact of fluctuating electricity supply on MEM [25]. The latter study found that M. maripaludis recovers activity quickly after substrate starvation, and utilizes electricity supplied according to a curtailed solar electricity pattern with a high efficiency of >95%.

Furthermore, pure cultures have been used to determine the electron uptake mechanisms of several methanogens. Several Methanosarcina species were shown to be capable of direct electron uptake from electrodes via heavily discussed mechanisms that can involve or be independent of multi-heme c-type cytochromes [26,27]. Methanobacterium strain IM1, an iron-corroding strain, has also been reported to take up electrons directly from electrodes and Fe(0) surfaces via an unknown mechanism [28,29]. Other methanogens such as M. maripaludis enhance cathodic currents on graphite electrodes by extracellular enzymes that facilitate hydrogen formation [30,31]. These studies highlight that even methanogens, a group with seemingly limited metabolic functionality and a simple core metabolism, have diverse mechanisms to take up electrons during MEM. In summary, these pure culture studies emphasize the importance of matching suitable microbial strains and system designs. Although specific MEM applications, such as biogas upgrading, certainly benefit from resilient mixed cultures, other MEM applications would benefit from pure cultures such as those used for commercial gas fermentation.

For future advances, in-depth knowledge about a strain will enable matching microbial needs and MEM system design – biofilm maintenance to enable direct electron transfer, and/or the use of hydrogen evolution electrocatalysts to improve the efficiency of MEM with hydrogenotrophic strains. One roadblock to efficient pure culture MEM systems is the need for pure culture isolates from high-performing MEM systems that often enrich for specific methanogens, such as Methanobacterium strains [32]. In-depth studies of such strains could lead to a deep understanding of the metabolic traits needed for efficient MEM. Although the specialized metabolism of methanogens likely impedes efforts to divert catabolic fluxes to produce other metabolic end-products, genetic engineering could improve adaptation to MEM systems, enhance production rates, and produce anabolism-derived compounds such as vitamins, lipids, coenzymes, and bioplastics, or aid nitrogen fixation [33,34].

State and prospect of MES with acetogens

Acetogens are well known for their ability to produce carboxylates with carbon chain lengths of one to four (C_1–4) as well as their corresponding alcohols in gas fermentation from syngas. Likewise, their portfolio in mixed culture MES is restricted to these products, and acetate is the most common. MES acetate production has so far benefited from mixed cultures, although recent advances in pure culture MES have shown significant improvements. The use of highly active catalytic cathodes proved to be successful in several independent studies and boosted the performance of an engineered Clostridium ljungdahlii strain [35], Sporomusa ovata [36], and Thermoanaerobacter kivui [37]. Abiotic syngas generation with highly porous cathodes covered by cobalt phthalocyanine allowed C. ljungdahlii to achieve the best acetate and ethanol production rates then reported in pure culture MES (2.00 g.l^–1.d^–1 and 0.47 g.l^–1d^–1, respectively) [38]. Even higher acetate titers were achieved by optimization of planktonic growth with C. ljungdahlii (up to 6.06 g.l^–1) [39]. S. ovata has also been shown to produce acetate in high amounts (up to 3.60 g.l^–1) without catalytic cathodes [40]. Despite these efforts, and even considering the best mixed culture MES, the underperformance of acetogens in MES compared to gas fermentation limits further technology development. The stressors and bottlenecks causing this underperformance in MES remain to be characterized.

In recent years the use of pure cultures has allowed us to determine the electron transfer mechanisms between the cathode and the electrobiocatalyst, which is of vital importance for leveraging the full potential of electrobioproduction. However, no direct electron transfer pathway has been elucidated in acetogens despite the presence of cytochromes in some species. Only a few studies have been able to empirically demonstrate the role of H₂-mediated electron transfer with C. ljungdahlii [39] and S. ovata [41]. Even when the acetogens are physiologically similar, the observed differences in H₂ uptake between different acetogen strains [42] highlight the importance of understanding the specific metabolic pathways involved.

Most importantly, pure culture MES has the potential to produce different and possibly more valuable compounds than gas fermentation or mixed culture MES. This can be achieved by the utilization of engineered strains, or by taking advantage of electrochemically created redox imbalances, to produce metabolites that would be consumed by the community in mixed cultures. The first example of the hidden potential of acetogens in pure culture MES was the report of trace amounts of 2-oxobutyrate with S. ovata [11], later also observed with other acetogens [43], as a consequence of a redox imbalance [44]. C. ljungdahlii was also shown to produce glycine and ethanolamine in the gram per liter range, which was predicted to be linked to an NADPH surplus [39]. A similar idea is also behind electrofermentation, in which electrodes can be used to control the redox potential of heterotrophic cultures (e.g., solventogenic Clostridium spp.) to modify their product profile (as extensively reviewed elsewhere [45]). Although scientific progress seems to be slow at the moment, the prospect of generating unique products by pure culture MES is intriguing, and more attention should be paid to the synthesis of unexpected or uncommon metabolites.

Biocatalysts and product groups beyond classical MES

The concept of coupling microbial synthesis to electron uptake (or discharge) in MES is not limited to CO₂ reduction to generate methane or acetate [46]. In fact, if we leave these target products behind and embrace the landscape of biochemical pathways, electrochemical tools become interesting aids in balancing many microbial redox processes. Examples include cathodic reductive reactions and anodic processes that benefit from electron discharge. Both approaches often are also called 'electrode-assisted fermentations' (in contrast to MES from CO₂). In this section we exclude the mere change of natural fermentation balances by electrochemical influences, which also falls within the term electrofermentation [45]. Instead, we focus on reactions that only become possible in a bioelectrochemical scenario and that rely on the stoichiometric supply or acceptance of electrons.

Remarkably, most targeted strain-engineering efforts for microbial electrobioproduction have in fact been reported for the diverse group of 'other microbial biocatalysts' rather than for the established – and most promoted – MES biocatalysts. The most advances and chances for wide-ranging future applications probably lie in this direction. However, the examples reported so far are mostly intended as 'proof-of-principle' reactions to show that microbial electroproduction is feasible. First, the model electroactive bacterium Shewanella oneidensis was engineered to allow balanced fermentation of glycerol to ethanol via the discharge of surplus electrons to the anode [47]. Additional studies with Shewanella showed anodic [48] or cathodic reaction balancing [49,50], mostly of acetoin-related pathways. Another study reported engineered S. oneidensis for isobutanol production that benefited from extracellular electron discharge [51], but the titers were still very low. However, beyond these reports, biotechnological studies on S. oneidensis or other well-known electroactive bacteria have remained surprisingly rare.

By contrast, Escherichia coli has been at the focus of creating a flexible host for future electrobioproduction. Intensive attempts to engineer synthetic direct extracellular electron transfer with outer-membrane cytochrome modules from S. oneidensis are only slowly progressing, and so far have not enabled sufficient electron discharge capacity to couple the synthetic electron pathway to a target reaction [52., 53., 54.]. However, some success has been reported with strains that couple electron transfer to soluble redox mediators which bridge electron transfer from the inner membrane of E. coli to the electrode to establish and improve the anodic production of acetoin [55,56] or the unbalanced fermentation of glycerol [57]. In addition, inwards electron transfer was utilized to balance and enhance the cathodic production of succinate [58] and mevalonate [59]. Although these reactions are coupled to the growth of the biocatalyst, resting (non-growing) cells have also been used for MES. Mayr and colleagues reported reduction of a chiral ketone to an alcohol using E. coli as the catalytic chassis and methyl viologen as the mediator [60].

Similarly to E. coli, the robust biocatalyst Pseudomonas putida has been explored for microbial electroproduction of 2-ketogluconate using synthetic mediators [61,62], or to produce rhamnolipids by using heterologously expressed natural phenazines as redox mediators [63,64]. Overall, however, these examples only scratch the surface of what is possible. One central obstacle to more widespread targeted engineering of strains for electroproduction of relevant (non-model) compounds is the lack of a reliable bioprocess infrastructure that would allow the full potential of these new biocatalysts to be evaluated and optimized.

Bioprocess technology for pure culture electrobiotechnology

Bioprocess technology and engineering cover a high diversity of microbial lifestyles that can be exploited for bioproduction, including heterotrophic fermenters and phototrophs. In these established fields, reactors and related peripherals across a wide range of scales (e.g., from single cells to liter-scale and cubic meter reactors) are available off-the-shelf or via experienced custom-engineering suppliers. However, this is not the case for MES. Reactors for MES need to serve both the requirements related to the lifestyle of the specific microbes as well as the need to interface electrochemical components, most importantly electrodes, with high efficiency. At the same time, commercial electrochemical reactors (e.g., H₂ electrolyzers) are not an option because these are not suitable for incorporating living microbes.

Thus, from the technical perspective, there needs to be a compromise between the 'optimal' conditions for microbial production (e.g., ambient temperature and pressure, close to neutral pH, etc.) and electrochemical operation (e.g., elevated temperature, high ionic conductivity, acidic or alkaline pH, etc.). Further, users in biotechnology generally focus on microbial physiology research and proof-of-concept studies, and have only limited interest in customizing reactors. Thus, there is a demand for off-the-shelf 'plug-and-play' electrobioreactors that can be operated without specialized electrochemical knowledge.

Ideally, the development of these off-the-shelf electrobioreactors will include well-established features of bioprocess technology such as online pH monitoring and control, controlled temperature, mixing and gas supply, and the option to install various other analytical probes (Figure 3). In addition, MES based on mixed versus pure cultures have different requirements for their reactors. Mixed culture-based MES is an open culture technology that does not require (energy-intensive and costly) sterilization of reactors and substrates; it is robust to changing reaction environments and operational variables. This is not the case for MES based on pure cultures that require a more defined 'technical habitat'. A main challenge in maintaining pure cultures is to assure sterility during reactor operation. This can be achieved by developing an adequate reactor infrastructure. In that regard, some researchers have 'tailored' existing, commercially available bioreactors/fermenters with electrochemical add-ons to build electrobioreactors for MES [60,65., 66., 67., 68., 69., 70., 71., 72., 73.].

Figure 3.

An envisaged off-the-shelf electrobioreactor in which the main components are labeled.

Based on a 2023 online survey among industrial and academic stakeholders regarding >100 physical, chemical, microbial, and other process parameters for bioelectrosynthesis, the mind-mapping list shows the most important desired design (upper panel) and process control (lower panel) features to guide off-the shelf electrobioreactor development. Please note that the listed design specifications are only exemplary and many are optional. One key design element is the flexible choice between a single-chamber electrochemical set-up with working and counter electrodes in the same electrolyte solution, and a two-chamber set-up where the working and counter electrodes are spatially separated by a membrane. The drawing proposes a single design solution to accommodate this task: the application of a counter electrode chamber with flexible installation options for a membrane.

Noteworthy, all of these systems are based on continuous stirred-tank reactors (CSTRs). Although some have been used for the cultivation of electroactive biofilms, they are (like all CSTRs) mainly suitable for MES using planktonic bioelectrocatalysts. In fact, for biofilm-based processes, namely those with mandatory direct extracellular electron transfer, specialized biofilm reactors might be a more appropriate starting point for standardization. Box 2 summarizes the main criteria for assessing the efficiency of cathodic MES. Whereas coulombic efficiency (CE) and final product concentration (titer) are usually reported, criteria such as selectivity are only suitable for some MES systems, for example, stereoselectivity when producing chiral compounds. The energy efficiency (EE) is strongly determined by both the reactor architecture and the ultimate economic criterion. Thus, reporting EE when using suboptimal reactor infrastructure does not allow a meaningful comparison. Standardized and off-the-shelf electrobioreactors would provide a clear advantage and thus allow targeted engineering of microbes.

Box 2. Assessing the efficiency of cathodic microbial electrosynthesis (MES).

Coulombic efficiency (CE)

For cathodic MES systems, CE is a measure of the ratio of electrons fed by the cathode and the electrons needed to form the product(s). Equation I exemplifies a batch process; it can be regarded as a combined measure of electrochemical and metabolic efficiency.

$${\mathit{CE}}_{\mathrm{product}}=\frac{n_{\mathrm{product}}\times z_{\mathrm{product}}\times F}{\int i\left(t\right)\mathrm{d}t}\times 100\%$$ [I]

where CE_product is the Coulombic efficiency for a desired product (percent), n_product is the amount of product (mol), z_product is the number of electrons per reaction for product formation, F is the Faraday constant (96.485 C.mol^–1), and i is the current (A).

Voltage efficiency (VE)

VE is a measure of the cell voltage (i.e., the difference in the formal potentials of the anode and cathode reactions) in an open circuit (i.e., zero current) and the cell voltage that is necessary to drive the reaction (Equation II). VE is determined mainly by reactor architecture (e.g., geometries and membranes and their fouling), electrode material, and the properties of the solution (e.g., conductivity, pH, as well as microbially secreted enzymes).

$$\mathit{VE}=\frac{E_{\mathrm{eq}}}{E_{\mathrm{cell}}}\times 100\%$$ [II]

where E_eq is the equilibrium cell voltage [i.e., the voltage (V) of the entire electrochemical reactor at zero current] and E_cell is the actual applied cell voltage during the MES production.

Energy efficiency (EE)

Evaluates the share of the invested (electric) energy that is stored in the product; EE is calculated by multiplying CE and VE (Equation III); it allows comparison of MES with other technologies that generate the same product.

$$\mathit{EE}=\mathit{VE}\times \mathit{CE}$$ [III]

Selectivity (S)

There are different types of selectivity; for example, the selective conversion of a carbon feedstock to the desired product, as well as chemical stereoselectivity when the product formed has different enantiomers. The selectivity of the formation of the desired product in relation to all product generated can be calculated with Equation IV.

$$S_{\mathrm{product}}=\frac{n_{\mathrm{product}}}{\sum n_{\mathrm{product}}}\times 100\%$$ [IV]

where S_product is the selectivity for the desired product (percent), n_product is the amount of the desired product obtained (mol), and $\sum n_{\mathit{products}}$ is the sum of amounts of all the products generated (mol) including the desired product as well as side products.

Concentration/titer

The concentration of product(s) per unit volume; for instance as given by $\frac{n_{\mathit{product}}}{V}$ in mol.l^–1 or g.l^–1.

Yield (Y)

The amount of product formed per consumed substrate/educt when considering the reaction stoichiometry. For example, Y for the formation of acetate from CO₂ can be calculated using Equation V.

$$Y_{\frac{\mathrm{acetate}}{{\mathrm{CO}}_2}}=\frac{2\times n_{\mathrm{acetate}}}{n_{{\mathrm{CO}}_2}}\times 100\%$$ [V]

where $Y_{\frac{\mathrm{acetate}}{{\mathrm{CO}}_2}}$ is the yield of acetate from CO₂ via MES, n_{acetate is} the amount of acetate formed (mol; note that it is multiplied by 2.0 for reasons of stoichiometry), and $n_{{\mathrm{CO}}_2}$ is the amount of CO₂ consumed (mol).

Current density (j)

The electric current is the number of electrons transferred per second per surface area or reactor volume. It is a measure of the electrode surface or volume normalized rate of the MES process.

Alt-text: Box 2

Finally, electrobioreactors cannot be considered to be stand-alone devices, and should be seen as 'modules' in a production line that includes other biological, chemical, and electrochemical production processes as well as pretreatments and downstream processing of different types. We consider that one main asset to be leveraged is to use both anode and cathode for the generation of value, as already exemplified by combining anodic electroorganic synthesis with gas fermentation using cathodic and anodic off-gases [74].

Concluding remarks and future perspectives

It is time to usher in a new era of electrobiotechnology – the use of flexible and tunable microbial catalysts for the tailored electrosynthesis of high-value compounds in an industrial environment. Opening this new chapter requires a more creative and flexible design of electrosynthetic pathways to generate high-value specialties and commodities with highest efficiency. To achieve this, the generation of highly specific production strains through genetic engineering will be required. Production strains can be based on pure culture isolates with native electroactivity (e.g., through high-throughput screening of promising habitats such as hydrothermal vents) or via engineered electroactivity of biotech workhorses such as E. coli. Simple-to-use and standardized off-the-shelf electrobioreactors will be crucial to enable widespread use and testing of pure cultures catalysts in application-relevant scales and environments, and will be key to transferring this effort to processes. These electrobioreactors need to be as convenient and intuitively operated as those used in other areas of biotechnology.

Going beyond these immediate tasks, strategies can be extended to optimize the overall microbial or process performance (see Outstanding questions). For example, some disadvantages of pure culture applications can be overcome by using defined cocultures. The benefits of collaborative biocatalysts extend from sharing metabolic tasks in the generation of a target product to alleviation of oxidative stress by consuming oxygen and producing reduced metabolites such as H₂S. New advances in spectroscopic online monitoring enable insight into the individual performance of coculture members and can unlock options for informed control of the biocatalysts to optimize MES performance. For process intensification, for instance, the combination of different microbial catalysts for both targeted anodic and cathodic reactions needs to be investigated more deeply. From an energetic and process-engineering perspective, creating value at both the anode and cathode of an electrochemical reactor is of utmost importance. This can be achieved, for instance by combining cathodic MES from CO₂ with anodic electrosynthesis of hydrocarbons from organic acids. The challenges are not trivial, but with more reliable and reproducible methodology and standardized platforms, as well as reporting of progress, groundbreaking scientific success is certain to follow.

Outstanding questions.

How can we go beyond proof-of-principle? Most current examples of engineered pure culture MES systems target model products without consideration of the economic driving forces. To go from academic exercise to biotechnological application, target products with high economic viability need to be defined and made accessible via MES.

The chicken versus egg problem: how can a good biocatalyst be evaluated without proper infrastructure? At this point, the lack of off-the-shelf electrobioreactors greatly limits the degrees of freedom for the larger academic as well as the early-stage industrial biotech field to explore MES. With standardized reactors, testing and benchmarking of new MES concepts will become feasible.

Application of redox mediators: can we afford to add external mediators? One great challenge for expanding MES concepts is the lack of electroactivity in established biotech hosts and the metabolic inflexibility of native electroactive microbes. As a solution, the application of externally added redox mediators has long been suggested. However, despite the additional costs of the addition and especially recovery of the mediator during downstream processing, this approach only makes sense if the reaction benefits from pure electron balancing (discharge or uptake). If the MES reaction needs to be wired into the metabolism of the host cell, the addition of external mediators alone will not be sufficient.

Can metabolic and technical hurdles be overcome by using defined cocultures? For many detailed aspects of MES function, the strategy of defined cocultivation might provide a valid option to overcome performance and compatibility challenges: combining an electroactive specialist that provides a simple fuel product with a bioproduction specialist; combining an oxygen-sensitive MES specialist with an oxygen scavenger; or combining a cathodic MES specialist with an anodic MES specialist for 200% cell MES (synthesis at both electrodes).

Alt-text: Outstanding questions

Glossary

Anode

the positive electrode of an electrochemical system that accepts electrons from the reaction process (i.e., oxidation).

Bioelectrochemical system (BES)

a generalized term for any electrochemical system with a biological redox reaction, all specialized systems are BES subcategories.

Cathode

the negative electrode of an electrochemical system that delivers electrons to the reaction process (i.e., reduction).

Electroactive microorganisms

microorganisms that are capable of extracellular electron transfer, namely the capacity to donate or uptake electrons from a solid electrode.

Electrobiotechnology

the application and integration of electrochemical processes with biotechnology.

Microbial electromethanogenesis (MEM)

cathodic MES by CO₂ reduction to methane.

Microbial electrosynthesis (MES)

in broad terms, this refers any microbial bioproduction that involves extracellular electron transfer with anode or cathode. In narrow terms, as mainly used in this article, MES refers to cathodic microbial CO₂ reduction to products.