This is the accepted version of the journal article: Izadi, Paniz; Harnisch, Falk. «Microbial | electrochemical CO2 reduction : To integrate or not to integrate?». Joule, Vol. 6, Issue 5 (May 2022), p. 935-940. DOI 10.1016/j.joule.2022.04.005 This version is available at https://ddd.uab.cat/record/269099 under the terms of the license Microbial | Electrochemical CO2-reduction - to integrate or not to integrate?! Paniz Izadi and Falk Harnisch* Department of Environmental Microbiology, Helmholtz-Centre for Environmental Research - UFZ, Leipzig, 04318, Germany. falk.harnisch@ufz.de Calling for technology platforms to turn carbon dioxide into feedstock Carbon dioxide (CO2) has to turn from waste to feedstock for establishing a circular economy. Albeit there is consensus that chemicals and fuels for consumer goods will be based on the carbon that was recently CO2, one can only surmise the technology platform(s) to reach such an industrial transformation. Certainly, the current petro-based industries will not be simply replaced by one technology. Rather a broad portfolio of platforms comprising physical, chemical and biological technologies can be expected to allow development towards a true circularity of (bio)chemical synthesis. Among these are platforms based on interweaving microbial and electrochemical synthesis1. Microbial synthesis based on the immediate utilization of CO2 as a feedstock in gas fermentation, for instance, suffers from low solubility of gaseous compounds in liquid phase. It limits the accessibility of CO2 and/or H2 for microbial cells and therefore conversion efficiency2. Electrochemistry allows using electric and not chemical energy to exploit CO2 for synthesis. Yet, the electrochemical CO2-reduction reaction (CO2RR) is characterized by its low product portfolio that is commonly limited to C1- or C2- compounds3, but possesses high kinetics, particularly when compared to biological CO2-reduction4. Therefore, combining or integrating microbial conversions with CO2RR for synthesis seems very appealing. Integration may allow harnessing the best of both and hence using nothing but electric energy and CO2 for creating a broad portfolio of products. Microbial electrochemical synthesis from carbon dioxide Microbial electrochemical synthesis (MES) is based on interfacing of electrochemical and microbial transformations to allow the purposeful transformation of a feedstock into a desired product1. MES can be based on the activity of autotrophic microorganisms that reduce CO2 using cathodic electrons or hydrogen. Despite a first hype and significant research on MES from CO2, its development stagnates and seems to face insurmountable hurdles. The main obstacles are limited electron transfer rates and hence reaction rates, limited product portfolio, insufficient yields as well as long time required for developing a cathodic microbial electrocatalyst and hence production start-up5. Synthesis from CO2 can also be based on less immediate interfacing of microbial and electrochemical reactions. Through CO2RR, first CO2 is electrochemically reduced to yield formate (HCOOH) or other C1-compounds, which are already established feedstocks for microbial syntheses. Hence, one can foresee that in addition to using formatotrophs (formate consuming microorganisms) for synthesis, strain engineering will make formate an excellent feedstock for several production hosts, including yeasts6. Integration of CO2RR to formate with microbial synthesis from formate was first reported by Li et al7. Since then a significant development took place that concerned electrochemical and microbial engineering of components as well as process engineering. There is more than one way to integrate When integrating CO2RR and microbial synthesis, different approaches are feasible. The integration can occur in situ that is in one compartment (Figure 1a). CO2 is converted to formate by CO2RR proceeding at the cathode and linked to synthesis by microorganisms. Here the synchronisation of the CO2RR and microbial synthesis rates is a challenge. To avoid disturbing CO2RR at the electrode, microorganisms that form no biofilm thereon are favourable. The microorganisms submersed are also more appealing in terms of volumetric production rates and yields, especially when exploiting co- cultures of different microorganisms, which allows creating food webs for gaining complex products. It is noteworthy that often the main or only electrochemical side-product is H2 that is an ideal energy source for many microbial catalysts. Alternatively, CO2RR to formate proceeds spatially separated from the microbial synthesis (Figure 1b and 1c). The solution composition can be critical, as it needs to balance the requirements of both process steps and thus provide (optimal) conditions for CO2RR and microbial synthesis. A challenge can be inhibition of CO2RR by bio-products. Using two separate reactors (Figure 1b) including inline separation of the microbial products and reusing the solution for CO2RR can overcome this. Solution design can be especially challenging as some compounds vital for microbial growth or a pH required for microbial growth and activity can hamper CO2RR4. In this line the purity of the gaseous CO2-feed is also of relevance, for instance when assuming an industrial feed of flue gas containing impurities (e.g. traces of O2 or sulphur compounds). As well as chemical compounds, biomass can be detrimental for CO2RR when present at the electrode and be affected by the electrochemical reaction. Thus, biomass removal, e.g. filtering, is advantageous when integrating separate reactors. In addition, a small share of impurities (e.g. O2) in the feed gas can be tolerated by CO2RR. We are confident that engineering allows to balance the operational conditions for CO2RR and microbial synthesis as required, albeit controlling operational conditions such as pH for integrated processes remains challenging. Alternatively, the electrochemical and biological reactions can be fully separated (Figure 1c). This separation allows decoupling two process steps in space and time. As CO2RR and microbial synthesis are fully independent, they do not affect each other and the solutions can be optimized for the respective process step. Further, it allows concentration as well as amendment, for instance with trace elements, of the solution containing CO2RR products before supplying to a bioreactor. The rate of CO2RR and microbial synthesis can be disconnected in time when both are spatially fully separated (Figure 1c), whereas only a spatial separation requires synchronization to some extent (Figure 1b) and particularly full synchronization is required when operating in one compartment (Figure 1a). At the first glance, separated processes may be further advantageous by allowing precise control of the feed of formate to the bioreactor. This is in particular advantageous, as high formate concentration can be toxic as well as high concentration of the bio-products. The latter can be removed using downstream processing (DSP). Although the toxicity threshold of formate is different (ranging from <100 mM for weak (e.g. E.coli) to >100 mM for those with strong (e.g. yeasts) formate dehydrogenases), higher concentration than the threshold causes decline of biosynthesis6. However, when using locally separated process steps (Figure 1b, 1c), concentration gradients in the bioreactor do already limit the microbial synthesis. This requires improved mass transfer such as increased energy consuming agitation or implementation of baffles8. We argue that this limitation can be elegantly and economically overcome by using an appropriate electrode and reactor design for in situ integration, e.g. implementation of three-dimensional electrodes as discussed below. a) In situ integrating in one reactor  High kinetics of CO2RR to formate  High availability of formate for biosynthesis  Spatially well-provided formate (less concentration gradients), e.g. using GDE  Instantaneous electrochemical control of the microbial feed × Interference of electrochemical and biological reactions and challenge for synchronising the rates of the reactions × Requirement of the microorganisms to form no biofilms × Requirement of inline downstream processing due to the (very likely) toxicity of products for microbial cells × Requirement of a medium optimal for both electrochemical and biological reactions × Requirement of high-quality gas with high purity of CO2 b) Integrating of two reactors  High kinetics of CO2RR to formate  High to medium availability of formate for biosynthesis  Spatial separation of electrochemical and biological reactions  Microbial biofilms can be used  Small impurities in the CO2 gas feed can be tolerated × Requirement of inline downstream processing due to the toxicity of products for microbial cells and CO2RR × Requirement of a medium optimal for both electrochemical and biological reactions × Limited control over the final concentration of formate in the bioreactor × Mass transfer in the liquid may lead to gradients and limit accessibility of formate in the bioreactor c) Separated processes  High kinetics of CO2RR to formate  High availability of formate for microorganisms  Spatial and temporal separation of electrochemical and biological reactions and high control over synchronising the rates of the reactions  Microbial biofilms can be used  Small impurities in the CO2 gas feed can be tolerated  Different media optimal for each process step can be used  No requirement of inline downstream processing × Mass transfer in the liquid may lead to gradients limiting accessibility of formate in the bioreactor Figure 1. Schematic of the potential reactor designs for CO2-reduction reaction (CO2RR) and further microbial synthesis to value-added products a) integrated in one reactor (in situ) including product recovery, in which CO2RR and microbial synthesis occur in the cathodic compartment of an electrochemical reactor, b) integrated in two reactors including product recovery in which CO2RR and microbial synthesis occurring spatially separated in a cathodic compartment and a bioreactor, and c) separated processes, in which CO2RR and microbial synthesis occurring spatially as well as temporarily separated in a cathodic compartment and a bioreactor, linked with the concentrated formate stage. It seems a long way down, but worth it! We are confident that the above illustrated integration can become economically competitive, with their main assets being the kinetics of CO2RR leading to a low ratio of the needed active electrode surface area to reactor volume and the product diversity of microbial synthesis. The main competitor will be gas fermentation from syngas (from CO/H2O or CO2/H2). However, the solubility of gaseous feedstock is limited (e.g. 28mg L-1 CO, 1.6mg L-1 H2 and 1.7g L-1 CO2, 293K and 1atm8), and hence is setting boundaries to bio-production, especially in a large scale. Here we foresee a clear advantage for the in situ integration (Figure 1a) in case high surface three-dimensional electrodes are used. Among these three-dimensional electrodes, gas diffusion electrodes (GDEs) seem most promising to us due to their role in not only enhancing the CO2 mass transfer in aqueous media, but also decreasing the internal resistance by removing the bubbles covering the active surface areas of the electrodes when feeding CO2 directly in the aqueous media9. In GDE, CO2 feed is transported through the gas diffusion layer9 (Figure 2) allowing permanent availability of CO2 directly at the electrochemical reaction sites. The higher solubility of formate than CO2 in aqueous solution in turn assures the increased availability of feed for microbial synthesis. Further, the energetic efficiency of bioconversion using formate is higher compared to H2/CO2 (e.g. +80-90% compared to +60-80% in anaerobic acetogens)10. In addition to formate, production of other C1-compounds such as methanol from CO2RR can be achieved. Methanol is more energy-rich than formate and has been known as an established feedstock for microbial syntheses10. The selectivity of the products from CO2RR strongly depends on the electrode materials. For instance, while indium (In) or tin (Sn) (also Hg, Cd, etc.) are known for catalysing CO2RR to formate, oxidation state of copper (Cu2O) or addition of other metals to copper (e.g. Cu3Pt or Cu3Pd) can form methanol (although at different potentials). Figure 2. Schematic of CO2-reduction reaction to C1-compounds in an electrochemical reactor using a gas diffusion electrode (GDE) and different electrocatalysts. There are a number of external costs that needs to be considered when developing processes for a bio-based and circular economy from lab-bench to industrial reality. Product recovery through DSP such as extraction is required in any presented process design for turning CO2 and electric power to chemical products or fuels. Yet, it is of special importance for the designs with solution recirculation due to the potentially toxic effect of bio-products (Figure 1a and 1b). For instance, high concentration of microbial produced carboxylic acids and their effect on the pH can have damaging effects on the cell membrane resulting in growth inhibition. Production of longer chain carbon products than C1- compounds such as terpenes, drugs or even proteins not only increases the economic and energetic value but also makes the separation of the products more convenient. In addition to DSP, the quality of the gas feed is important particularly in the designs based on interfacing electrochemical and biological reactions (Fig. 1a and 1b). Even when the electrochemical reactor is separated from the bioreactor (Figure 1c), impurities such as high percentage of oxygen can affect the CO2RR4. The flue gas from cement industries, for instance, includes sulphur oxides (SOx) and nitrogen dioxides (NOx) in addition to dust and oxygen11 which could disturb the electrochemical and microbial reactions and hence decrease the overall efficiency. In this case, an extra gas-purification is required leading to increase in operational costs. CO2 purification and compression from flue gas of cement industry −1 through three different methods were calculated to require 256 − 466 𝑘𝐽 𝑘𝑔𝑐𝑙𝑖𝑛𝑘𝑒𝑟 energy11. As of today, ethanol and butanol produced by bio-based technologies are considered as a potential alternative to fossil-based transportation fuels. Syngas fermentation in an industrial scale is developed and pursued by some companies such as LanzaTech and its joint partner of Shougang group (Chinese Steel company), Genomatica, Kiverdi, IneosBio and Coskata 12,13, in particular for ethanol production (although IneosBio and Coskata stopped operation due to the operational and financial difficulties13). To make syngas fermentation even more economically and operationally feasible for industry, remarkable efforts were taken resulting in significant achievements over the past years. Different designs such as multi-stage fermentation or gas-liquid enhanced bioreactor for ethanol production have been patented13. Although COVID-19 pandemic decreased the ethanol production in 2020, ethanol biorefineries are remaining major drivers in clean ethanol production in U.S.A., Brazil, and certain parts in Asia and Europe14. Despite the pandemic, 2.7 million tons of CO2 were captured in 2021 for ethanol biorefineries which was used for further applications such as bottling and food processing. Production of other compounds than ethanol from syngas fermentation, such as acetate, isoprene or 3-hydroxypropionate using genetically modified microorganisms were also patented13. In spite of the growing progress observed in such biological syntheses, organic electrochemical syntheses on industrial scales seem not as mature. Thus far, electrochemical synthesis of valuable chemicals such as adiponitrile (a key-intermediate for production of nylon-6,6) is established since decades and used in industrial scales for instance in Japan (Asahi Chemical in Nobeoka)15. Electrochemical organic synthesis has the advantages of high product selectivity and purity, simple integration with renewable energies as well as with biosynthesis due to the ambient temperature and pressure required for its operation, low number of reaction steps and few polluting side-products. However, its limited application in industry especially without a known integration with developed biological synthesis in large scales can be seen as the consequence of several limits being in place up to now. These limits comprise missing suitable resources and manufacturing technologies, considered excessive costs for capital expenditures, especially for changing and implementing in existing production lines, lack of scientists and engineers being educated for integrating microbiology and electrochemistry, and lack of the collaboration between research and governmental sectors. To benefit from the electrochemical routes for production of diverse value-added organic compounds from CO2 using renewable energies, strong partnerships between universities and research institutes, as well as industry and government are required. We are confident these partnerships on the long run will create values for society as well as shareholders. ACKNOWLEDGEMENT The authors acknowledge the support of the VIVALDI project that has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement N°101000441. This work was supported by the Helmholtz Association in the frame of the Integration Platform "Tapping nature's potential for sustainable production and a healthy environment" at the UFZ. AUTHOR CONTRIBUTIONS The idea of this article and the manuscript were conceived and written by P.I. and F.H. 1. Harnisch, F., and Urban, C. (2018). Electrobiorefineries: unlocking the synergy of electrochemical and microbial conversions. 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Botte, G.G. (2014). Electrochemical manufacturing in the chemical industry. The Electrochemical Society Interface 23, 49. Biographies: Paniz Izadi is a lead scientist for the H2020-funded project VIVALDI at Helmholtz-Centre for Environmental Research – UFZ, Germany. Her current research focuses on developing methods for electrochemical CO2-reduction and its interfacing to microbiology for industrial applications, following her previous research on microbial CO2-reduction in bio-electrochemical systems. She received her PhD in chemical engineering from Newcastle University, UK in 2020. Falk Harnisch holds a Full Professorship at Leipzig University for Electrobiotechnology and is a group leader at the Helmholtz-Centre for Environmental Research – UFZ, Germany. He received his PhD from Greifswald University, Germany in 2009 as well as several fellowships and awards and is currently serving as a President of the International Society for Microbial Electrochemistry and Technology (ISMET). His research ranges from fundamentals of microbial electrochemistry via electroorganic synthesis to engineering of microbial electrochemical technologies.