Microbial electrosynthesis meets synthetic biology: Bioproduction from waste feedstocks

Abstract

Integrating electrochemistry and biology, microbial electrosynthesis (MES) enhances feedstock-to-product conversion by utilizing electroactive microorganisms to harness electrical energy for driving metabolic pathways. Advances in synthetic biology have improved microbial extracellular electron transfer and increased metabolic pathway efficiency, enabling optimized redox balance, expanded substrate versatility and enhanced bioproduction. Given the growing interest in sustainable chemical production and decarbonization, this mini-review highlights recent progress in MES enabled by synthetic biology, with a focus on engineering efficient microbial cell factories for electricity-mediated bioproduction through waste-derived feedstock utilization and carbon capture. We also highlight key challenges limiting MES scalability and propose future directions to enable industrial-scale deployment, unlocking its potential for sustainable, carbon-neutral production and driving transformative advances in biotechnology.

1. Introduction

The urgent need for sustainable solutions to meet the growing demands for chemicals and energy, while simultaneously addressing pollution and climate change issues arising from reliance on fossil resources, has driven the search for innovative technologies that align with circular bioeconomy principles. Among these, microbial electrosynthesis (MES) has emerged as a promising strategy. MES integrates electrochemistry and biology to enhance waste-derived feedstock transformation into high-value chemicals and fuels through the unique extracellular electron transfer (EET) capabilities of electroactive microorganisms (EAMs)1, 2, 3, 4, 5 by providing an electron sink or electron supply, ideally powered by green electrical sources like solar and wind energy. By leveraging these microorganisms’ ability to channel electrons between electrodes and their metabolic pathways, MES offers a powerful and sustainable platform to maximize the capabilities of microbial biotransformation, enabling redox-intensive processes that might be challenging to achieve efficiency in biological systems.

Advancements in synthetic biology have enhanced microbial metabolic pathways and broadened substrate versatility, enabling engineered strains to utilize diverse renewable feedstocks to produce valuable products.⁶ However, the efficiency of metabolic pathways is often constrained by an inadequate redox co-factor balance required to drive biochemical reactions. MES provides a solution by exploiting EAMs to provide the external electrical potential necessary to sustain the optimal redox conditions for these reactions, enhancing the biosynthesis pathways. Furthermore, understanding the EET mechanisms in EAMs enables synthetic biology to engineer non-electroactive microbes with the required machinery needed to participate in electron transfer, expanding MES's scope of applications.^7,8

As MES has gained traction, several recent reviews have been published on the subject. However, these reviews mainly focus on the application of MES to wild-type microbes and bioprocess design,9, 10, 11, 12, 13, 14 with few addressing the integration of synthetic biology with MES to enhance the performance of engineered microbial cell factories.^15,16 Given the increasing demand for sustainable chemical production and decarbonization, this mini-review delves into the current advancements and applications of MES enabled by synthetic biology, emphasizing and advocating electricity-assisted bioproduction through waste feedstock utilization and carbon capture using engineered microbes (Fig. 1). We also discuss the challenges for MES scale-up and highlight future directions critical for realizing its potential to enable sustainable, carbon-neutral production at an industrial scale.

Fig. 1.

Overview of MES.

MES integrates electrical and microbial systems to convert renewable feedstocks into high-value chemicals and fuels. Key processes include engineering electroactive microorganisms for EET, optimizing biosynthetic pathways, substrate utilization, and designing a sustainable MES environment for sustainable biochemical production.

2. Engineering microbes for enhanced electroactivity towards bioproduction

EAMs evolved electroactivity as an adaptive strategy to survive in environments where traditional energy generation and nutrient acquisition methods are constrained.¹⁷ This capability allows them to thrive by transferring electrons to or from extracellular substrates, enabling respiration and energy production under challenging conditions. There are two main EET mechanisms used by EAMs: direct electron transfer (DET) and indirect electron transfer (IET) (Fig. 2). In DET, EAMs facilitate direct electron flow from electron sources using specialized structures like conductive pili, nanowires and porin-cytochrome complexes; this mode of EET is used by prominent EAMs, such as Geobacter sulfurreducens and Shewanella oneidensis.^1,2 In IET, EAMs utilize redox-active small molecules (e.g., H₂, flavins, and phenazines) as electron shuttles to enable electron transfer (Fig. 2).3, 4, 5 Both EET mechanisms can be used cooperatively, as exemplified by interspecies electron transfer within a microbial consortium to support syntrophic growth.^18,19 In photosynthetic EAMs, IET has also been shown to couple with native photosystems to facilitate high-efficiency metabolism in light-rich environments using external electron sources.^20,21 By capitalizing on the various modes of microbial electroactivity, microbes can be engineered to serve as chassis for implementing MES.

Fig. 2.

Mechanisms of Electron Transfer in MES.

(a) Direct electron transfer (DET) occurs via (i) electron conduit transmembrane protein complex or (ii) pili/nanowires. Microbes with electron conduit transmembrane proteins can transfer electrons directly to and from electrodes through conductive porin-cytochrome protein complexes (such as MtrCAB), enabling efficient electron flow to drive redox reactions. Certain microbes utilize conductive pili or nanowires to transfer electrons directly to the electrode. This involves mechanisms such as electron transport through stacked aromatic residues or coherent/incoherent hopping through redox-active amino acid groups. (b) Indirect electron transfer (IET) uses electron carriers such as neutral red or hydrogen to shuttle electrons between microbial cells and the electrode, facilitating the reduction of NAD(P)⁺ to NAD(P)H and driving metabolic processes.

While native EAMs have inherent EET mechanisms, they can be engineered to further improve their EET efficiency, allowing them to serve as efficient chassis for MES to utilize waste-derived feedstocks and produce chemicals. For example, the exogenous phenazine-1-carboxylic acid (PCA) biosynthesis pathway genes from various microbes were screened and expressed in S. oneidensis to produce the PCA electron shuttle. By dynamically regulating PCA transport, EET was optimized and enhanced, leading to increased utilization of lactate,²² an organic substrate derived from dairy waste fermentation²³ that is the microbe's preferred carbon and energy source. To broaden the applicability of MES, EET machinery can be transferred to non-electroactive microbes to confer electron uptake abilities. Notably, the biotechnological workhorse Escherichia coli has been engineered with a complete electron transfer system from S. oneidensis to conduct extracellular electrons into the cell,²⁴ opening up opportunities to apply MES to the extensive bioproduction accomplishments already achieved in this well-established microbial host. Interestingly, a cyanobacterium has been engineered to uptake external electrons by deleting the photosynthetic microbe's native photosystem II (PSII), facilitating MES-mediated CO₂ assimilation.²⁵ This modification caused upregulation of genes, such as those associated with photosystem I (PSI), ferredoxin, and pili assembly, which were postulated to enable the transfer of extracellular electrons to the photoelectron transfer chain. Furthermore, non-electroactive microbes have been engineered to biosynthesize electron shuttle molecules for enhanced IET. For example, Pseudomonas putida, an emerging non-pathogenic microbial host capable of utilizing waste aromatic streams as carbon sources,²⁶ was engineered with the phenazine biosynthesis pathway genes from the pathogenic P. aeruginosa, enabling the production of redox-active phenazines to facilitate IET.²⁷ Taken together, these efforts in engineering microbial EET have enhanced the efficiency and versatility of using microbes as cellular factories for waste feedstock utilization and carbon capture via MES, particularly for pathways that are highly redox-intensive.

3. Applications of MES for enhanced chemical production from waste feedstocks

MES has shown promise in expanding the range of carbon substrates, aligning with the goal of sustainable biochemical production. It enables renewable feedstocks, such as organic waste materials and CO₂ from industrial emissions, to be efficiently utilized without competing with food resources.⁶ This section reviews recent efforts in sustainable chemical production from waste-derived feedstocks through the synergy between synthetic biology and MES.

3.1. Improved bioproduction from waste-derived organic substrates through MES

MES has gained attention in recent years for its ability to optimize microbes engineered for biochemical production, including the use of waste organic substrates (Table 1). By enhancing electron transfer, MES increases the efficiency of biosynthesis pathways for high-value compounds from waste-derived feedstocks. Native EAMs have been engineered with synthetic metabolic pathways to take advantage of their native DET mechanisms for improved biochemical production (Fig. 3). For example, S. oneidensis was engineered with a metabolic pathway to produce isobutanol from lactate.²⁸ Notably, this MES endeavour was achieved in conjunction with an electro-controlled system to modulate the redox balance and enhance biosynthesis efficiency, exemplifying the potential of MES when coupled with regulatory systems developed through synthetic biology to produce biofuel from a renewable feedstock. S. oneidensis has also been engineered for MES-driven (R)-acetoin production from glycerol,²⁹ an abundant by-product of the oleochemical industry that the microbe does not naturally assimilate, showcasing expansion of substrate range by synthetic biology for MES. Furthermore, an E. coli expressing S. oneidensis proteins involved in DET and enzymes for short branched-chain alcohol biosynthesis has been employed for MES-assisted anoxic utilization of glycerol to produce isobutanol and 3-methylbutanol.³⁰ This work highlights the impact of combining metabolic engineering and MES using a synthetic EAM to facilitate waste carbon stream assimilation for producing valuable compounds.

Table 1.

Application of microbial electrosynthesis for bioproduction.

Microorganism	EET Mode	Key engineering strategy	Substrate	Product	Key Findings	Ref
Shewanella oneidensis	DET	Two-stage electro-fermentation; electro-regulated CRISPRi for pathway suppression	Lactate	Isobutanol	Two-stage electro-fermentation process, using different voltages for growth and biosynthesis, enhanced isobutanol production. Directing reducing equivalents using NADH biosensor and suppressing byproduct pathways via CRISPRi led to an isobutanol titre of 1321.5 mg/L (94.9 % of theoretical yield).	28
Shewanella oneidensis	DET	Engineered glycerol utilization and (R)-acetoin biosynthesis pathway	Glycerol	(R)-Acetoin	Expanded substrate range of S. oneidensis to glycerol. (R)-acetoin production reached 313.61 mg/L.	29
Escherichia coli	DET	Expressed heterologous S. oneidensis electron transfer proteins and biosynthesis pathways for isobutanol and 3-methylbutanol	Glycerol	Isobutanol, 3-methylbutanol	Enhanced anoxic glycerol utilization. Total isobutanol and 3-methylbutanol production reached 232 mg/L, 25 % higher than using glucose	30
Escherichia coli	IET (Neutral red- and 2-hydroxy-1,4-naphthoquinone mediated)	Engineered glycerol reductive pathways to produce 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP)	Glycerol	1,3-PDO, 3-HP	Increased 1,3-PDO titre by 2.5-fold–15.5 mM with negative potential. Enable 3-HP production at 10.9 mM with positive potential.	33
Rhodopseudomonas palustris	DET	Engineered n-butanol biosynthesis pathway; deleted nitrogenases	CO2	n-Butanol	Produced 0.91 mg/L of n-butanol using CO2, electricity, and light. Demonstrated first solar panel-powered microbial electrosynthesis platform for n-butanol production.	39
Rhodopseudomonas palustris	DET	Overexpressed RuBisCO form I and II to increase CO2 fixation	CO2	Polyhydroxyalkanoate	Overexpression of RuBisCO increased polyhydroxyalkanoate production up to five-fold; engineered RuBisCO strains increased electron uptake under non-nitrogen-fixing conditions	40
Yarrowia lipolytica	IET (Neutral red-mediated)	Expressed AckA and Pta to increase acetyl-CoA; expressed pathway genes to produce fatty alcohols, lupeol and betulinic acid	Acetate, glucose	Fatty alcohols, lupeol, betulinic acid	Enhanced NADPH regeneration by MES drove acetate utilization, resulting in 6.17-fold increase in fatty alcohol production; demonstrated first MES application in Y. lipolytica	32
Synechocystis sp. PCC 6803	DET	Inactivated photosystem II; expressed heterologous ethylene-forming enzyme	CO2, HCO3−	Acetate, ethylene	External electrons and light enabled CO2 fixation at 9.3 % energy conversion efficiency; achieved acetate and ethylene production from CO2.	25
Cupriavidus necator	IET (H2-mediated)	Expressed heterologous mevalonate pathway and α-humulene synthase	H2, CO2	α-Humulene	Produced 10.8 mg/L α-humulene by MES; first example of electroautotrophic terpene production from CO2	37
Cupriavidus necator	IET (H2-mediated)	Expressed lycopene pathway	H2, CO2	Lycopene	Produced 1.73 mg/L lycopene from CO2 from power plant exhaust gas	38
Cupriavidus necator	DET and IET (flavin-mediated)	Expressed heterologous S. oneidensis MtrCAB electron conduit proteins and Gloeobacter violaceus rhodopsin; overexpressed native carbonic anhydrase	CO2	Biomass	Created an artificial photoelectrochemical microbial system that directs CO2 into the central metabolism	41

Fig. 3.

Examples on the application of MES for chemical production.

MES has been demonstrated to facilitate chemical production by providing electrons or electron sinks to microbes, ensuring an adequate supply of redox equivalents in the form of NAD(P)H and NAD(P)⁺, thus supporting redox-intensive biochemical reactions. Electrons have been transferred either directly through electron conduit complexes (ECC, such as MtrCAB) or indirectly through redox-active mediators (e.g. H₂, flavins, phenazines, neutral red and methyl viologen) that shuttle between their oxidized (MED_ox) and reduced (MED_red) forms. The transfer of electrons between electrodes and microbes has boosted the utilization of waste-derived feedstocks such as CO₂, lactate, glycerol and acetate. Moreover, both native pathways and metabolically engineered heterologous pathways have benefited from EET, receiving the necessary redox power for biochemical reactions and improving the production of a wide range of chemicals. By coupling EET-mediated utilization of renewable carbon sources with synthetic biology, MES using engineered microbes has demonstrated potential as a viable solution to meet the world's chemical needs while addressing environmental and energy issues. FAR, fatty acyl-CoA reductase; TS, terpene synthases; PHA, polyhydroxyalkanoate; 1,3-PDO, 1,3-propanediol; 3-HP, 3-hydroxypropionic acid.

Besides DET, IET has been shown to improve biochemical production via MES. Interestingly, although overproduction of natural redox mediators such as flavins and phenazines has been achieved to improve IET, these engineered strains have predominantly been demonstrated for electricity generation rather than MES.^27,31 Instead, synthetic electron mediators, such as neutral red (NR) and 2-hydroxy-1,4-naphthoquinone (HNQ), are added during MES to facilitate IET and improve EET efficiency,^32,33 possibly due to the ease of implementation alongside biosynthesis pathway engineering. Even in microbes lacking endogenous EET capabilities, these redox-active molecules can mediate EET and regenerate NAD(P)⁺ and NAD(P)H, thereby providing the necessary redox power. For example, an oleaginous yeast Yarrowia lipolytica was engineered for fatty alcohol production from acetate, a renewable carbon source generated from the anaerobic digestion of solid organic waste.³⁴ MES facilitated by NR improved NAD(P)H levels, driving acetate utilization by the NAD(P)H-dependent fatty acid and fatty alcohol biosynthesis pathway and increasing fatty alcohol production.³² In another study, the production of 1,3-propanediol and 3-hydroxypropionic acid from glycerol using E. coli was complemented with MES. NR (at the cathode) and HNQ (at the anode) were used to enhance EET, thereby increasing glycerol assimilation and production yield of the high-value plastic monomer molecules, as well as enabling tuning of product specificity by adjusting the electrical potential.³³ These examples collectively highlight the transformative potential of MES, particularly when integrated with synthetic biology strategies, to establish versatile and efficient platforms for the sustainable production of high-value biochemicals from waste carbon resources.

3.2. Enhanced carbon capture through MES for chemical production

In view of global warming and environmental concerns, much attention is placed on carbon capture and utilization for converting CO₂ into valuable products using engineered microbes. Hydrogen-mediated CO₂ fixation through the Wood–Ljungdahl and Calvin-Benson-Bassham (CBB) cycles is well-established in hydrogen-oxidizing microbes.^35,36 In these cycles, hydrogenase-mediated H₂ oxidation into H⁺ generates electrons needed for CO₂ assimilation into the central metabolism. By applying electrical potential in an MES system using Cupriavidus necator engineered with terpenoid pathways, H₂ generated in situ from H₂O in the culture and H⁺ from the hydrogenase-catalyzed reaction mediated IET to regenerate reducing equivalents and promoting CO₂ assimilation into the central metabolism. As a result, CO₂ was converted into valuable terpenoids, such as lycopene and α-humulene,^37,38 demonstrating a generalizable approach for MES-assisted CO₂ fixation with engineered hydrogen-oxidizing microbes for chemical production.

Anoxygenic phototrophic microorganisms, such as Rhodopseudomonas palustris and PSII-deleted cyanobacteria, have also been utilized for coupling EET to their PSI-driven electron transfer pathway for improved CO₂ fixation.^7,25 EET provides exogenous electrons to the photosynthetic electron transfer chain to increase the CO₂ fixation rate, while the absence of PSII prevents O₂ generation, eliminating O₂ fixation as a competing reaction to CO₂ fixation and thereby enhancing the efficiency of converting CO₂ into value-added molecules. These photosynthetic MES systems have successfully been coupled with metabolic engineering efforts, such as deletion of electron-consuming pathways³⁹ and overexpression of RuBisCO,⁴⁰ to improve light- and electricity-assisted CO₂ fixation. This has led to the production of a wide range of compounds, including n-butanol, amino acids, and polyhydroxyalkanoate (Fig. 3).^25,39,40

Building on foundational knowledge about various EET machineries, light-activated proton pumps, and CO₂ fixation, synthetic biology has recently enabled a combinatorial engineering approach to develop an artificial photosynthetic system in C. necator to direct CO₂ towards biomass.⁴¹ The MtrCAB electron conduit proteins of S. oneidensis were expressed in C. necator to enable DET and flavin-mediated IET. Concurrently, a cyanobacterial rhodopsin functions as a photo-driven proton pump to create a proton motive force that drives enzymatic reactions producing ATP, NADH, and NADPH, which are necessary for the native CBB cycle to fix CO₂. The outcome is a synthetic photoelectrochemical microbial system capable of assimilating CO₂ into its central metabolism, illustrating the extensiveness of microbial chassis engineering achievable with synthetic biology to advance MES for carbon capture.

4. Challenges and future directions

While synthetic biology has expanded and improved the utility of MES across a broader range of waste feedstocks to produce more complex molecules (Table S1), the production titer and volumetric productivity remain far below the thresholds typically considered to be commercially viable (100 g/L and 2.5 g/L/day, respectively).⁴² While MES holds potential as a platform for sustainable bioproduction, significant challenges remain in optimizing microbial performance and bioprocesses for complex biosynthetic pathways to achieve scalability of MES systems. Addressing these challenges will be critical for translating MES from laboratory benchtop to industrial applications and unlocking its full potential as a sustainable technology.

A key obstacle in MES lies in optimizing EET efficiency and expanding its use to more non-electroactive microbes. While effective in natural environments for native EAMs, natural EET machineries face scalability and pathway specificity limitations in engineered MES bioproduction systems. Despite progress, MES is implemented mostly with a few prokaryotes, such as E. coli, S. oneidensis, C. necator and Clostridium spp..¹⁶ One reason is the lack of tools for engineering EAMs. For example, Sporomusa ovata is an EAM capable of CO₂ fixation, making it an attractive microbe for MES-mediated carbon capture towards bioproduction. Although its application in MES has been demonstrated, no genetically engineered strain has been used so far.⁴³ Nevertheless, recent developments of genetic tools for S. ovata⁴⁴ may encourage its broader use in MES. Furthermore, toolkits have been created for precise electrochemical control of gene expression in EAMs,^28,45 demonstrating how synthetic biology can allow autonomous metabolic control for integration with MES to enhance bioproduction. Thus, synthetic biology shows potential in enabling the engineering of more diverse microbial hosts with more complex functionalities to expand MES applications, leveraging the distinct characteristics of different microbes for bioproduction from waste feedstocks.

Another critical challenge in MES-driven waste valorization is optimizing substrate utilization, as the EAMs may not have a sufficiently broad substrate range or high efficiency in assimilating carbon sources. By leveraging the advantages of both chemistry and biology, abiotic-biotic hybrid systems have shown promise in facilitating waste substrate utilization. For example, tunable syngas composition was achieved through electrocatalytic CO₂ conversion, which enhanced microbial syngas fermentation performance in medium-chain fatty acid production.⁴⁶ Electrocatalytic transformation of CO₂ to acetate and ethanol was also accomplished, enabling an engineered E. coli to utilize these CO₂-derived two-carbon substrates for producing l-tyrosine.⁴⁷ Moreover, electricity-assisted nitrogen recovery was realized, allowing concurrent MES-driven metabolism of waste carbon and nitrogen sources.¹¹ By coupling S. ovata with photo-excitable cadmium sulfide nanoparticles, an artificial photosynthetic system was created to enhance CO₂ assimilation via photoelectron-driven MES.⁴⁸ These examples exemplify the strength of integrating chemistry and biology to advance MES for sustainable chemical production. Furthermore, the application of microbial consortia for division of labor to improve biochemical production from renewable resources has gained traction.^6,49,50 This strategy has been employed in MES for concurrent valorization of food waste and CO₂ into volatile fatty acids,⁵¹ marking a breakthrough in the biotechnological application of microbial consortia while broadening the range of feedstocks compatible with MES. To further the use of microbial consortia in MES, future work could seek to understand the roles of individual microbes within consortia and optimize through synthetic biology approaches the cooperative interactions⁵² between electroactive and non-electroactive species for enhanced bioproduction performance from waste carbon streams.⁵³

In addition to microbial engineering, scaling MES systems for industrial applications requires bioprocess development, such as biofilm formation, reactor set-up and electrode design. The formation of stable microbial biofilms on electrode surfaces is crucial for sustained electron transfer and efficient bioproduction.⁵⁴ To reinforce biofilm stability, supplementation with quorum-sensing molecules, such as N-hexanoyl-l-homoserine lactone (C6HSL), has been used to increase product yields and ensure consistent EET in MES systems.^55,56 Instead of using expensive exogenous quorum-sensing molecules, synthetic biology approaches could be explored in the future for economical regulation of biofilm formation for MES applications.⁵⁷ Furthermore, the need for large-surface-area electrodes made of precious metals like platinum adds to the cost of MES reactors. This has motivated innovations in waste-derived biocathodes, such as those made from spent coffee grounds and tea leaves, which have demonstrated their potential to reduce costs while maintaining compatibility with microbial systems and enabling stable electron transfer.⁵⁸ Moreover, oxygen generated by electrolysis at the anode during MES negatively affects the cathodic reaction and viability of anaerobic EAMs. While this issue may be circumvented using strategies such as the use of specialized setups and membranes,⁵⁹ it remains uncertain whether these solutions are economical and effective at an industrial scale. Future development of electrodes made from sustainable resources, optimizing the ability of microbes to form biofilms on such surfaces and designing optimal MES reactor setups to ensure electron transfer efficiency will be crucial for the cost-effective scale-up of MES systems.

In conclusion, MES represents a transformative approach to sustainable chemical production and carbon capture, leveraging the unique capabilities of EAMs to integrate advancements in synthetic biology and electrochemistry. By building on interdisciplinary collaborations and fostering technological innovations, MES is poised to offer a viable solution to the pressing environmental and energy challenges of the twenty-first century.

CRediT authorship contribution statement

Dijin Zhang: Writing – review & editing, Writing – original draft, Conceptualization. Jee Loon Foo: Writing – review & editing, Writing – original draft, Conceptualization. Matthew Wook Chang: Writing – review & editing, Writing – original draft, Conceptualization.

Declaration of competing interest

The author Matthew Chang is an Editor-in-Chief for Biotechnology Notes and was not involved in the editorial review or the decision to publish this article.

The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article: