Bioadaptive Ni single atoms unlock high rate microbial electrosynthesis of isopropanol from CO_2

Domain: chemistry
Published: 2026-01-15
Editorial outcome: major_revision

Abstract
Hybrid systems that integrate electrochemical CO2 reduction with microbial upgrading offer a viable route to high value organic compounds from CO2 at ambient conditions. However, electrocatalyst deactivation in microbial growth media remains a key barrier, limiting efficiency and increasing cost. Here we show that a bioadaptive single-atom nickel catalyst (NiSAC), coupled with genetically engineered Clostridium ljungdahlii, enables robust electrosynthesis of isopropanol (IPA) from CO2 via a CO-mediated pathway. Instead of relying on H2 as an electron carrier, the system applies high-rate CO formation in complex growth media, maintaining a tunable CO Faradaic efficiency up to 92%, which is 9.4 to 52.7 times greater than conventional Ag catalysts. This performance supports stable IPA production at current density of 10.8 A/m2 and production rate of 161.3 mg/L/day. In situ Raman and X-ray absorption spectroscopy, together with theoretical calculations, indicate that the NiSAC can resist competing organic adsorption and retain its coordination structure during CO2 reduction in bioelectrolytes, providing a mechanistic basis for the catalyst stability and integrated process performance.

Introduction
Isopropanol (IPA) is a critical platform chemical that is widely used in personal healthcare products and in industrial production of fuels and chemicals. Because IPA has a higher octane rating than conventional fuels, it is regarded as a promising fuel additive for enhancing combustion efficiency1. Its disinfectant properties also prompted a dramatic increase in global demand and prices during the COVID-19 pandemic. Moreover, IPA’s critical role in semiconductor cleaning has fueled overall IPA market expansion, as rising chip demand from AI technologies driven increasing market share within this segment2,3. The global IPA market was valued at $4.8 billion in 2024 and is projected to grow significantly over the next decade to more than $7 billion by 20334. Current industrial IPA production predominantly relies on energy-intensive hydration of propylene (two-thirds of supply) or hydrogenation of acetone. Both petro-based routes depend on fossil propylene or H2, as well as face challenging separations from IPA/H2O/diisopropylether azeotropes5. Renewable energy-driven electrochemical CO2 conversion offers a sustainable approach to both reduce carbon emissions and produce high-value chemicals6–9. However, prior research on C3 alcohol electrosynthesis has only achieved a maximum Faradic efficiency (FE) of 30-50% for n-propanol with pure CO2 supply10–13 and was only able to realize IPA generation under high-pressure CO2 conditions14. The synthesis of IPA via CO2 electrolysis at ambient pressure remains a major barrier.

Anaerobic microbial fermentation of syngas recently emerged as a powerful platform to convert C1 substrates into multi-carbon alcohols15,16. In particular, genetic engineering of Clostridium ljungdahlii has enabled the production of IPA from syngas, demonstrating the feasibility of tailoring microbial pathways toward target alcohols17. However, commercial syngas production still depends on coal gasification and natural gas refining, both of which are highly carbon-intensive18. To address this, a variety of high-selectivity, tunable CO2 electrolysis methods have been reported for on-demand syngas generation, offering a cleaner syngas source for bioconversion19. Integrated microbial electrochemical systems (iMES) based on gas diffusion electrodes have been shown to enhance kinetic mass transfer during microbial fermentation and potentially overcome limitations associated with separated abiotic electrolysis and fermentation20,21. Moreover, iMES systems enable microbials to convert simple electrode-derived intermediates (e.g., H2, CO, and formate) into a wide range of high-value products such as alcohols, carboxylic acids and esters through their native or engineered metabolic pathways22, which are nearly impossible on traditional inorganic catalyst surfaces. This material-microbe synergy can thus expand the accessible electrochemical product space beyond the limits of conventional electrocatalysis.

Considering the energy demand and carbon footprint, the iMES process may decouple IPA synthesis from fossil feedstocks by using renewable electricity to generate electron donors. This shifts energy and carbon inputs from fossil feedstocks to electrons and waste CO2, avoids steam reforming emissions, and is operationally attractive for distributed, point-source CO2 valorization. Notably, fermentation of syngas has already achieved carbon-negative IPA at pilot scale (−1.17 kg CO2 per kg IPA) when powered by low-carbon energy and waste-gas feeds23, which further highlights the decarbonization potential by interlinking the microbial conversion with renewable syngas supply.

Despite such promise, electrochemical syngas-mediated IPA synthesis via iMES has not yet been reported, primarily because traditional abiotic catalysts that were optimized for abiotic electrolyte lose significant activities when exposed to complex, nutrient-rich biological growth media24,25. The fundamental causes of catalyst deactivation in such mixed electrochemical-microbial environments remain poorly understood, impeding the development of an integrated, syngas-fed IPA production platform.

In this study, we designed a single-site Ni catalyst that can exhibit high tolerance to bioelectrolyte components (i.e., YT medium for microbial growth) and enable selective CO production, thereby significantly enhancing the CO supply rate in biological electrolytes. The synthesized Ni single-atom catalyst (NiSAC) demonstrated increased resistance to complex organic components in the electrolyte than conventional Ag catalyst, maintaining CO FE nearly identical to that in ideal electrolytes while allowing tunable syngas ratios. When we combined this catalyst with a genetically engineered IPA-producing C. Ljungdahlii biosynthetic platform, we achieved the CO-mediated CO2-to-IPA microbial electrochemical synthesis with an average rate of 161.3 mg/L/day. Additionally, we employed multiple in-situ characterization techniques to deeply probe the electrode-electrolyte interfacial properties in the bioelectrolyte and elucidated the deactivation mechanism of conventional electrocatalysts. We discovered that on continuous polycrystalline metal surfaces (e.g., Ag), extensive coverage of organics from the microbial growth medium disturbed the electrode-electrolyte interface and competitively occupied reactive sites, deactivating CO2 adsorption and conversion. We hope this study will provide different insights for future optimization of interfacial properties and the expansion of material–microbial hybrid systems.

Results
We employed a genetically tractable acetogenic bacterium, Clostridium Ljungdahlii, in the experiments due to its capacity to convert CO2/H2 or syngas into value-added biochemicals26. Through systematic thermodynamic optimization and theoretical modeling, we previously demonstrated that overexpression of acetoacetyl-CoA transferase (AACT) and acetoacetate decarboxylase (AADC) enabled robust conversion of CO2/CO/H2 mixtures into IPA (Fig. 1a), while synergistically enhancing ethanol biosynthesis17. In this study, our comparative fermentation analyses identified CO as a critical determinant for IPA synthesis. Under identical cultivation conditions, fermentation without CO feeding exhibited poor microbial growth (Supplementary Fig. S1) and yielded negligible IPA production (<0.1 mM). The maximum acetate and ethanol titers were constrained to ~42.9 mM (2531.8 mg/L) and ~8.9 mM (409.4 mg/L), respectively (Fig. 1b, c). In contrast, CO-supplemented fermentation produced ~11.4 mM IPA (681.3 mg/L), representing a 142-times enhancement, which was also accompanied by a 1.7 times (73.9 mM, 4359.3 mg/L) increase in acetate and a 3.3 times boost (29.3 mM, 1347.8 mg/L) in ethanol production (Fig. 1b, c). The cell viability results showed that the engineered C. Ljungdahlii strain cannot grow without CO/H2/CO2 feeding (Supplementary Fig. S2), but the introduction of CO/H2/CO2 apparently promoted viability during the logarithmic phase in the fermentation experiments, indicating that the organic components in YT medium alone should be insufficient to support IPA, acetate, or ethanol production. A drop in cell viability from day 3 to 5 was also observed, signifying that biological growth and metabolism had ceased and cells were entering apoptosis, which corresponds to the plateau observed in both metabolite concentrations and OD600. During this period, dead cells remained in suspension and continued to contribute to the optical density. As a result, the OD600 was distorted and could not serve as a reliable indicator of actual cell viability.

This CO-dependent metabolic shift arises from its dual role in precursor and energy metabolism (Fig. 1a). Beyond serving as a carbon source for acetyl-CoA formation, CO oxidation via CO dehydrogenase (CODH) generates reduced ferredoxin (Fdred). This electron carrier drives the Rnf complex to establish a transmembrane H+ gradient, thereby generating ATP to sustain growth and NADH to drive IPA biosynthesis26. H2 metabolism of C. ljungdahlii is less efficient, although it can also supply reducing equivalents via hydrogenases27. In H2/CO2 fermentation, H2 must simultaneously support both branches of the Wood-Ljungdahl pathway (WLP). In the methyl branch, CO2 is assimilated via the formate dehydrogenase (Fdh) and H2 is used by a bifurcating hydrogenase (in a complex with the Fdh) to generate Fdred for formate formation. While in the carbonyl branch, Fdred is required again for CO2-to-CO reduction by CODH. In addition, Fdred is required for ATP generation by the Rnf complex/ATPase, which is essential for autotrophic growth by these organisms. As a result, when C. ljungdahlii grows on H2/CO2, the available H2 pool is divided between carbon assimilation and energy conservation, leaving far fewer reducing equivalents available for product (e.g., IPA) formation. In contrast, CO oxidation directly provides Fdred via CODH, which can be efficiently utilized by the Rnf complex and other enzymes (e.g., Nfn) to generate NAD(P)H. Thus, in CO/CO2/H2 fermentation, the pool of Fdred is significantly larger, supporting both robust autotrophic growth and enhanced IPA production28. Consequently, microbial electrochemical systems targeting highly reduced compounds like IPA must prioritize sustained CO generation for C. ljungdahlii, ensuring both carbon flux and energy conservation requirements are met.

However, most microbial electrosynthesis studies supply H2 as the primary electron donor rather than generating CO from CO2 reduction. Although the thermodynamic standard potential for H2 evolution is only 0.1 V more positive than that for CO formation, media complexity (ones containing yeast extract and tryptone)29, low conductivity, and low alkali-cation concentration of bioelectrolyte make hydrogen evolution reaction (HER) kinetically more favorable and technically more convenient. Moreover, previous works have shown that CO2 reduction performance is severely impaired in microbial growth medium24,25. To address this challenge, we first evaluated Ag, a benchmark catalyst for CO2-to-CO electroconversion, in the C. ljungdahlii growth medium. Even at a deep reduction potential of −1.05 V vs. reversible hydrogen electrode (RHE), the FE for CO production remained below 25%, with a partial current density of only 2.7 mA/cm2 (Fig. 1d), representing a 1~2 order-of-magnitude decrease compared to previously reported Ag performances in abiotic electrolyte30–32. Resolving the deactivation of CO2 reduction catalysts in bioelectrolyte is therefore a critical step toward an integrated microbial electrochemical process for IPA production (Fig. 1a).

The CO dependency of engineered C. ljungdahlii necessitates a bioelectrolyte-compatible CO2 reduction catalyst capable of circumventing organic poisoning. Notably, recent advances in electrocatalysis revealed that atomically dispersed Ni-N-C configurations surpass conventional Ag catalysts in the abiotic medium33. In addition, the elevated potential of zero charge (PZC) of Ni single sites sustained increased surface cation charge density than silver, which stabilized the *CO2-intermediate and provided high cation insensitivity, enabling sustained CO FE even in cation-insufficient organic amine electrolytes34. While this frontier remained underexplored, we hypothesized that a structurally minimalist Ni single-atom structure could potentially exhibit high biocompatibility and overcome the limitations of conventional catalysts in biological medium. Hence, we engineered a N-coordinated Ni single-atom catalyst (NiSAC) on N-doped carbon using a modified pyrolysis approach (synthesis details and structure analysis in Methods and Materials). For conventional abiotic electrolysis, NiSAC achieved >95% of CO FE at industrial current densities (50–500 mA/cm2, Supplementary Fig. S3). In comparison to polycrystalline Ag NPs, Ni SAC exhibited a notable enhancement in CO selectivity and activity in the bioelectrolyte (Fig. 1e, Supplementary Fig. S4). Its CO FE was ~25.3 times that of Ag NPs at −0.85 V vs. RHE and can exceed 92% at higher overpotentials. Meanwhile, the maximum CO partial current density of Ni SAC in the concentrated YT medium surpassed 25.3 mA/cm2 at −1.05 V vs. RHE. In addition, the performance and controllable CO2/H2 ratio of NiSAC can also be extended to other commonly used microbial growth media (e.g. PETC medium, Supplementary Fig. S5), establishing it as a competitive candidate for integrated microbial electrochemical IPA production.

Adsorption-induced deactivation in bioelectrolyte
Unlike conventional abiotic electrolytes that facilitate CO2 reduction through optimized alkali cation effects35,36, the alkali ion-depleted biological medium (pH≈6) presents a fundamentally distinct interfacial environment. The insufficient ionic strength may change the interfacial properties and render both CO2 mass transport, adsorption, and subsequent intermediate stabilization thermodynamically unfavorable on the Ag surface37. Ionic chromatography (IC) analysis revealed that the bioelectrolyte contains approximately 93.3 mM Na+ and 13.3 mM K+ (Supplementary Table S1), and thus we used a 100 mM Na+ solution as an ideal electrolyte for comparative studies. Particularly, Ag NPs exhibited severe deactivation in the bioelectrolyte, with CO FE and partial current density reduced by >75% at identical potentials compared to the ideal electrolyte (Supplementary Fig. S6). In contrast, NiSAC maintained nearly identical CO generation activity, FE, and performance trends in both electrolytes (Supplementary Fig. S7). This phenomenon indicated that the complex medium may induce catalytic deactivation on Ag NPs via non-canonical mechanisms surpassing simple alkali cation depletion effects, while Ni SAC can inherently resist this. The bioelectrolyte contains dozens of organic compounds, including amino acids, vitamins, purines/pyrimidines and fragments of nucleotides/polypeptides, all of which may influence the catalyst interface properties and potentially interact with each other38. The complex composition of bioelectrolyte precludes deconvolution of individual organic species contributions.

To establish a mechanistic understanding of interfacial properties, we systematically characterized and compared the electrochemical behaviors of Ag NPs and Ni SAC. Electrochemical kinetics indicated that the rate-determining step for CO2RR on both Ag NPs and NiSAC was the first electron transfer during CO2 adsorption39, and Ni SAC always exhibited CO2RR kinetics better than Ag NPs (Supplementary Fig. S8). Cyclic voltammetry (CV) tests showed the double-layer capacitance (Cdl) of both catalysts decreased by ~30% in bioelectrolyte versus ideal conditions (Supplementary Fig. S9, 10), demonstrating the accumulation of hydrophilic organic macromolecules within the electrochemical double layer (EDL), inducing Helmholtz layer thickening, and thereby impeding mass transport of dissolving CO240,41. Conventionally, Cdl variations among electrodes in identical electrolytes were attributed to differences in electrochemically active surface area10,42,43. However, due to the significant changes in the electrolyte environment in this study, we directly normalized the partial current densities by Cdl. As shown in Fig. 2a, b, the normalized CO production rates of NiSAC in either ideal or bioelectrolyte were significantly higher than Ag NPs, indicating the greatly promoted CO2RR kinetics on each reactive site. More importantly, even excluding the effect of Cdl, Ag NPs exhibited a substantial intrinsic activity loss for CO production in the bioelectrolyte, whereas the performance of NiSACs remained nearly unchanged compared to that in the ideal electrolyte. This suggested that the presence or absence of adsorptive poisoning by organic species on the reactive sites, thereby affecting CO2 adsorption, may play a dominant role in altering CO2RR activity on Ag NPs and NiSACs.

Accordingly, we employed in-situ Raman to systematically investigate adsorption behavior and local environment on Ag NPs and Ni SAC reactive sites (Fig. 2c, d). During potential sweeps from −0.5 V to −0.9 V vs. RHE, the Ag electrode surface exhibited numerous complex organic adsorption signatures. Compared to previous studies using Ag colloids for yeast extract detection, the bioelectrolyte adsorbed on Ag nanoparticles exhibited changed and more complex Raman spectra44. For example, the adenine ring-breathing peak (~730 cm−1) was replaced by an in-phase ring stretching peak of guanine at 680 cm−1, revealing a shift in adsorption preference under negative potentials. At higher shifts (e.g., 1000–2000 cm−1), overlapping peaks from purines (e.g., various C–N vibrations of adenine or guanine from 1200–1500 cm−1)45 and amino acids (e.g., −COO− of alanine, ~1398 cm−1)46 complicated precise assignments. Despite this complexity, the spectra confirmed significant adsorption of organic compounds from bioelectrolyte on Ag NPs within the CO2RR potential window. Notably, all organic adsorption signals decreased with increasing overpotential, culminating in insignificant desorption beyond −0.8 V vs. RHE where Raman intensities diminish markedly. This potential-dependent desorption correlates well with the observed recovery of CO2RR activity at −0.85 V vs. RHE in our electrochemical measurements, suggesting that the competitive adsorption of organics from the bioelectrolyte and the resulting changes in interfacial properties would be the main reasons for the deactivation of the CO2RR on Ag NPs. Nevertheless, due to the rapid development of competing H2 evolution at higher overpotentials47,48, the higher energy and catalyst costs, as well as the increased risk of Ag+ leaching during long-time operation30, Ag is still a less suitable candidate than NiSAC for integration into iMES systems targeting IPA production.

In contrast, the Ni SAC surface did not exhibit any complex organic adsorption peaks like those on Ag, and only the D/G bands fingerprints of carbon were present49. It is noteworthy that although we enhanced the Raman response of NiSAC by using Au/SiO2 core-shell nanoparticles50, the signals from organic substances in the bioelectrolyte on Ni SAC should be much weaker than those on Ag particles with an inherent surface-enhancement effect51. Therefore, we cannot rule out the possibility of a small amount of organic adsorption on the NiSAC sites only based on in-situ Raman results. Nevertheless, compared with Ag NPs, the extent of organic poisoning on the NiSAC surface was significantly reduced or even absent, thus preserving a sufficient number of sites for CO2 adsorption and activation.

While distinguishing the individual roles of all organic species remains challenging, we employed Ag(111) and Ni–N4 as catalyst models and conducted DFT calculations to investigate differences in thermodynamic adsorption and interfacial properties (Supplementary Data 1). The enrichment of hydrophilic organic molecules within the double layer modified interfacial characteristics including ion concentration and electric field strength. CO2RR intermediates (*CO2, *COOH, *CO) responded differently to variations in electric field strength. The adsorption of CO2 with large dipole moment exhibited the strongest field dependence (Supplementary Fig. S11), and CO2 adsorption at Ni–N4 sites demonstrated greater electric field sensitivity compared to that on Ag(111), enabling effective CO2 activation under weaker fields. This suggested that Ni-N4 single-atom sites, which has discrete and narrow d state39, can maintain effective CO2 activation capability under demanding interfacial conditions. Guided by in-situ Raman results and the composition profile of microbial growth medium52, we selected adenine, guanine, alanine, and aspartic acid as representative organics to assess their adsorption energies differences. As shown in Fig. 2e, Supplementary Fig. S12, the adsorption of all the tested organics was feasible on Ag(111) but thermodynamically unfavorable on Ni–N4, and the adsorption free energy difference between these two sites all exceeded 0.3 eV, indicating that Ni–N4 sites are relatively repulsive toward these organic than Ag (Fig. 2f), aligning well with the proposed mechanism.

Atomic structure dynamics of NiSAC
Compared to Ag NPs, the promoted adaptability and activity of NiSAC in the bioelectrolyte were expected to be correlated with its distinctive atomic architecture. To elucidate the structure-performance correlation and monitor the dynamic structure evolution of NiSAC during electrolysis, comprehensive in-situ/ex-situ characterizations were conducted. Scanning transmission electron microscopy (STEM) images revealed that the carbon substrate in NiSAC exhibits an uneven, wrinkled morphology (Supplementary Fig. S13). Elemental mapping (Supplementary Fig. S14) and energy dispersive X-ray spectroscopy (EDS, Supplementary Fig. S15) confirmed the uniform distribution of Ni, N, C, and O elements with Ni loading amount of 0.33 at%, while X-ray diffraction (XRD, Supplementary Fig. S16) showed no characteristic peaks of Ni NPs. High-resolution high-angle annular dark-field STEM (HAADF-STEM, Fig. 3a) quantitatively verified the single atom dispersion with interatomic distances measuring 5.5 Å, which exceeds the theoretical Ni–Ni bond length (2.5 Å) and excludes cluster formation. The absence of a continuous metallic Ni surface may obstruct large organic molecules from achieving multidentate adsorption, thereby making the interaction between atomically dispersed Ni sites and organics more difficult53,54. Variations in the vertical height of these Ni atoms can be attributed to the unevenness of the carbon substrate. The locally uneven carbon framework surrounding the single-atom Ni sites can impose considerable steric hindrance, preventing large organic molecules, especially those with rigid and planar purine rings, from approaching the Ni center and forming strong bonding interactions, while these molecules can adjust their orientation to seek the most stable adsorption configuration on the broad Ag surface (Supplementary Fig. S12)54. High-resolution X-ray photoelectron spectroscopy (XPS) revealed a characteristic Ni 2p3/2 peak centered at 855.2 eV with a Ni atomic ratio of 0.34 at% (Fig. 3b, Supplementary Fig. S17), consistent with EDS results. Deconvolution of N 1s spectra identified catalytically active pyridinic (398.2 eV) and pyrrolic (400.8 eV) nitrogen as the dominant species, with a distinct Ni-N contribution (~399.5 eV) of ~1.93 at% (Supplementary Fig. S18). Comparative analysis with NiPc demonstrated a 0.7 eV negative shift in Ni binding energy (Fig. 3b), indicating the formation of an electron-rich Ni center with valence state lower than +255.

Synchrotron X-ray absorption spectroscopy (XAS) enables highly precise characterization of the valence state and coordination environment of single atoms, leading to a deeper understanding of the local environment of Ni single sites. Ex-situ X-ray absorption near edge structure (XANES) analysis revealed that the Ni K-edge absorption energy of the Ni SAC exhibited a shift to higher energy positions compared to Ni foil, resembling the XANES spectrum of NiPc (Fig. 3c). This suggested that the Ni SAC likely adopted a square planar-like structure with a valence state just below +255, supported by the first derivative analysis of the XANES spectrum (Supplementary Fig. S19) as well as XPS results. Fourier-transformed extended X-ray absorption fine structure (EXAFS, Fig. 3d) indicated exclusive Ni–N coordination at 1.87 Å (Supplementary Table S3), with no detectable Ni–Ni metallic bonds (2.49 Å) observed. Quantitative fitting yields an average coordination number of 3.5, implying a predominant planar Ni–N4 configuration with partial nitrogen loss during calcination. Previous studies on Ni–N structures have demonstrated that, compared to Ni–N3/4 structures, Ni–N3 configuration primarily reduced the energy barrier in the hydrogenation step (*CO2 → *COOH) of CO2RR, with minor impact on the CO2 adsorption energy56,57. Our DFT simulations that employed the Ni-N4 model as a representative configuration can therefore balance computational accuracy with experimental relevance.

The Ni K-edge XANES spectra reveal three primary molecular orbital transitions: 1s→3d (3d2 back-bonding/3dz2), 1s→4pz, and 1s→4pxy/continuum (Fig. 3c)58. While NiPc retained high D4h symmetry, the synthesized NiSAC sample exhibited structural distortion, facilitating hybridization between Ni 3d and 4p orbitals55. This manifests as intensified 1s→3d transition features and attenuated 1s→4pz transitions. Prior investigations of SCN−/NO3− adsorbate poisoning mechanisms suggested that, originating from the electron donation of the Ni 3dz2 orbital to the unoccupied orbitals of reactants, any stronger impurity adsorption than CO2 occupying Ni active sites could induce more pronounced splitting between 4pz and 4pxy/continuum orbitals58. Such interactions produce a red shift in the 1s→4pz transition and a concurrent blue shift in the 1s→4pxy/continuum transition within XANES profiles. To probe these effects, we performed in-situ XAS measurements on NiSACs under CO2RR conditions in both ideal and bio-electrolytes (Supplementary Fig. S20). Notably, the XANES spectra remained invariant under all tested conditions, irrespective of electrolyte composition (Fig. 3e). This indicates that the various complex organic molecules from the bioelectrolyte, unlike other reducible (i.e., NO3−/CO2) or poisoning (i.e., SCN−) species, cannot achieve stabilization on the Ni single sites via strong interactions with the Ni 3dz2 orbital58. In other words, their competitive adsorption with CO2 is likely negligible or, at least, cannot disrupt the orbital hybridization between CO2 and the Ni single sites under reducing potentials. Given the dynamic nature of electrocatalytic processes and controlled experimental parameters, enhanced EXAFS signal intensity would signify competitive adsorption of any transient poison at Ni sites, while no signal amplification of NiSAC was detected in the bioelectrolyte system (Fig. 3f). These observations further illustrate that organic constituents from the bioelectrolyte, including those not modeled in our DFT simulations, may accumulate in the electrochemical double layer without forming strong bonds to Ni active sites. Consequently, the adsorption competition between CO2 and organics from bioelectrolyte on Ni single sites is not significant or even absent, preserving the local coordination geometry and bond lengths of NiSAC, which aligns with in-situ Raman results and electrochemical performance.

Proof of an integrated electrobiosystem for IPA synthesis
The high activity and CO selectivity of NiSAC in the bioelectrolyte were validated through both experimental results and mechanistic insights. Building on this, we integrated NiSAC with the engineered C. ljungdahlii strain within a hybrid iMES system. As illustrated in Fig. 4a, Supplementary Fig. S21, to mitigate potential evaporation losses of EtOH/IPA during extended operation, the bioelectrolyte reservoir was connected to a cold trap to capture any volatilized alcohol products. To counteract pH fluctuations that may occur during long-term iMES operation, 100 mM phosphate-buffered saline (PBS) buffer was added to the bioelectrolyte. The introduction of PBS also enhanced the accessibility of alkali metal cations, thereby reducing the overpotential required to achieve similar levels of CO2RR activity and selectivity (Supplementary Fig. S22). The introduction of NiSAC electrode into the hybrid system will not have a significant effect on the cell viability of C. ljungdahlii strain (Supplementary Fig. 2). In addition, the NiSAC electrode exhibited stable current density and a consistent CO/H2 ratio for over 100 h in the bioelectrolyte containing 100 mM PBS (Supplementary Fig. S23). After 100 h of operation, post-characterizations (STEM, XPS and ICP, Supplementary Fig. S24–26) demonstrated that the overall structure of the NiSAC remained stable, and no apparent Ni2+ ion leaching was detected in the electrolyte, indicating the high biocompatibility and stability of NiSAC. Applying lower overpotentials may improve the FE of C2+ oxygenates but can also decrease the reducing equivalents supply rate and consequently diminish the overall production rate of C2+ oxygenates59. To ensure sufficient CO2 supply and a high CO/H2 ratio, the iMES system was operated continuously at −0.75 V vs. RHE (Supplementary Note S1).

Over four days of uninterrupted operation, the average current density remained stable at ~11.4 mA/cm2, and the optical density (OD600) of C. ljungdahlii steadily increased over the first three days, peaking at ~1.36 (Fig. 4b). The slightly decreased current density could be attributed to the loss of gas-diffusion layer (GDL) hydrophobicity over long-term operation60. Similar electrochemical performance observed before and after inoculating C. ljungdahlii further illustrated the stable operation and high bioadaptivity of the NiSAC in the flow cell (Fig. 4b, Supplementary Fig. S23). After accounting the evaporated amount, the average IPA titer reached ~10.8 mM over four days, with a total C2+ oxygenate (EtOH+IPA+Acetate) concentration of ~91.1 mM (Fig. 4c). The total electron recovery efficiency (ERE) for C2+ oxygenates reached 47.7 ± 6.9% (Fig. 4d). Specifically, the combined ERE for IPA and EtOH exceeded 26.4% (10.5% for IPA and 15.9% for EtOH). Reports on iMES for IPA production remain scarce, positioning this field as a largely unexplored area. Here, we present the demonstration of CO-mediated IPA iMES. Due to the sufficient CO supply and strong reducing power relative to H2, our system achieved a real production rate (3.23 mg/cm2/day) and volumetric production rates (161.3 mg/L/day) for IPA, which is competitive to prior studies (Fig. 5e, Supplementary Table S2)61–63. It is worth noting that a recent work by Liu et al. reported H2-mediated CO2 reduction using a different microbial strain and metabolic pathway64. The system achieved high rates of PHB generation (~116.7 mg/L/day) along with C4-5 alcohols coproduction, and a byproduct of IPA (~100 mg/L/day), highlighting a promising direction that combining alternative strains and metabolic pathways with bioadaptive catalysts to access higher-value products.

Methods
Catalyst synthesis
NiSAC was synthesized by a modified two-step pyrolysis method55,57. All the chemicals for Ni SAC synthesis, including nickel (II) nitrate hexahydrate (Ni(NO3)2·6H2O, 99.999%), polyvinylpyrrolidone (PVP, (C6H9NO)n, average Mw ~1300000), melamine (C3H6N6, >99%), cyanuric acid (CA, C3H3N3O3, >98%), and isopropanol (IPA, 99.9%), were purchased from Sigma-Aldrich and used without further purification. To synthesize Ni SAC, 6 g melamine, 3 g CA, 10 mg Ni(NO3)2·6H2O, and 1 g PVP were dispersed into 25 mL IPA and ultrasonicated for 1 h. The white dispersion was then dried by a freeze-dryer, and the solid mixture was further ground for 1 h. Thereafter, the precursor powder was carbonized in a tube furnace under Ar atmosphere by a two-stage calcination process: the calcination temperature was first raised from 25°C to 600°C with an increasing rate of 3°C/min and hold for 2 h; Then, the temperature was increased to 900°C with a rate of 2°C/min and hold for 1 h.

Electrode fabrication
Considering the long-term operation time of microbial electrosynthesis, a commercial gas diffusion carbon layer (Sigracet 39BB, Fuel Cell Store) was first modified by the previously reported method68 to improve hydrophobicity. 100 mg carbon black and 20 mg Teflon dispersion were dispersed into a 1:1 mixture of IPA/DI water. The mixture was sprayed onto the Sigracet 39BB electrode until a 10 wt% loading was achieved. Then, the modified electrode was placed in a 350°C furnace for 15 min and cooling to room temperature (20–25°C). Commercial Ag NPs (US Research Nanomaterials Inc) or synthesized Ni SAC, and 20% Nafion ionomer were first dispersed into a 1:1 mixture of IPA/DI water to make a 12.5 mg/mL catalyst ink. Then, the catalyst ink was sprayed onto the modified gas-diffusion carbon layer to fabricate the working electrode with a catalyst loading of 0.5 mg/cm2.

Electrochemical tests
All electrochemical measurements were performed by an electrochemical potentiostat (BioLogic, VMP3). All the electrochemical results were collected by a chronoamperometry mode at a temperature of 37°C. For the integrated iMES or abiotic CO2RR test, a purchased mixed metal oxide mesh (MMO, 2 cm * 2 cm, Standard Anode) was used as counter electrode and an Ag/AgCl electrode was used as reference electrode. All the potentials used in this work were converted to the RHE scale by the equation:
E(vs. RHE) = E Ag=AgCl + 0.21 V + 0.0591 V * pH (1)
Whenever calibration of the reference electrode was required, its potential was calibrated against RHE by measuring the open-circuit potential of a Pt electrode in a H2-saturated electrolyte. The stabilized open-circuit potential was taken as 0 V vs. RHE, and the resulting offset was used to convert all measured potentials to the RHE scale.

The active area of the working electrode was sealed to 1 cm2 by Kapton tape. Nafion 212 membrane (Fuel Cell Earth, 2.54*2.54 cm2) was pre-activated and used as CEM to separate the cathodic and anodic chambers. To activate the Nation membrane, the membrane was rinsed with DI water and then was immersed in 3 wt% H2O2 solution at 80°C for 1 h. Thereafter, the membrane was sequentially soaked in DI water and 0.5 M H2SO4 at 80°C for 1 h. Finally, the activated Nation membrane was repeatedly cleaned and stored in DI water. CO2 (99.999%, Airgas) was introduced to the electrolyzer at 5 sccm controlled by a mass flow controller. Acidic Na2SO4 solution (pH≈2) was used as an anolyte. Both catholyte and anolyte were flowed at 1 mL/min controlled by a peristaltic pump. Unless otherwise specified, bioelectrolyte refers to the YT medium, and the ideal electrolyte only contains 100 mM NaCl without any organics. All bioelectrolytes/ideal electrolytes (details as claimed below) were freshly prepared before testing. pH and conductivity of different electrolytes were tested by a pH meter (Fisher Scientific) and a conductivity meter (Fisher Scientific). All the electrochemical and microbial electrochemical tests were independently performed at least three times.

Products quantification
All gas products were tested by a gas chromatograph (GC, Agilent 8890). The liquid products were collected from the cathodic chamber or cold trap and detected by a high-performance liquid chromatograph (HPLC, Agilent 1260). To trap the evaporated liquid products as much as possible, a 15 mL glass tube containing 4 mL DI water at 0°C was connected to the bioelectrolyte reservoir outlet.

The FE or ERE of a specific product was calculated by the equation:
FE or ERE = n * electron transfer number * F * 100% / (Electrode area * I * t) (2)
Where n is the production amount (mol) of product X, determined by GC or HPLC; F is the Faradaic constant, 96485.33 C/mol; I is the total current density (mA/cm2). Although the equation is the same, we use FE and ERE to indicate direct electrochemical products (CO/H2) and indirect microbial products (IPA/EtOH/Acetate).

Bacterial strains and growth media
The IPA-producing C. ljungdahlii strain used in this study was created by Lo et al.17. Growth of C. ljungdahlii was in YTF-rich medium consisting of 10 g/L Bacto yeast extract, 16 g/L Bacto tryptone, 4 g/L NaCl, 0.5 g/L cysteine-HCl, with the addition of 5 g/L fructose when strains were grown from frozen stocks. Cell growth was monitored at 600 nm using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, USA).

Autotrophic growth of C. ljungdahlii for syngas vs CO2/H2 comparisons
Frozen stocks of the IPA-producing strain of C. ljungdahlii were inoculated into 10 mL of YTF containing fructose in a 15 mL Falcon tube. Growth overnight was at 37°C inside a COY (Grass Lake, MI) anaerobic growth chamber, flushed with 95% N2 and 5% H2 maintained anaerobic by palladium catalyst. Overnight cultures were added to 50 mL of YT (no fructose) in a 250-mL Duran pressure plus bottle (DWK Life Sciences, USA), pressurized to an additional 10 PSI above ambient with a syngas mix of 50%/20%/30% CO/H2/CO2 and left to grow shaking at 200 RPM at 37°C. Bottles were regularly pressurized back until the OD600 reached 1. Afterwards, C. ljungdahlii was transferred to fresh 50 mL YT medium in triplicate and grown either in a syngas mix of 50%/20%/30% CO/H2/CO2 or 20%/80% CO2/H2 pressurized to an additional 10 PSI above ambient again in 250-mL Duran pressure plus bottles. Bottles were routinely repressurized until growth ceased. For the cell viability assays, C. ljungdahlii was grown in 250-mL Duran pressure plus bottles in a syngas mix of 50%/20%/30% CO/H2/CO2 as previously described. Colony-forming units (CFU) per mL were assessed by taking 1 mL of culture from the bottles on selected days, serially diluting the culture to provide sufficient resolution to determine colonies and plating on YT + 5 g/L fructose medium. Colonies were counted after 5 days of growth on plates.

Growth of C. ljungdahlii in the biohybrid system
Before starting the iMES process, all the components (e.g., electrolyzer, bottles, connectors, and tubes) were autoclaved and sterilized. CO2 and electrolytes were flowed for at least 1 h to keep the anaerobic condition in the electrolyzer. A 0.22 μm filter was used between the gas flow controller and the electrolyzer to remove any possible contamination. Before adding the electrochemical potential (−0.75 V vs. RHE), seeds for the iMES system were prepared as above, and the initial OD600 was ~0.5. The operational temperature is 37°C. Liquid samples were collected from the bioelectrolyte reservoir once per day for each experiment. After finishing the long-term operation (4 days), all the liquids from the cold trap were collected for further detection. The calculations of volumetric production rate were based on the bioelectrolyte volume in the reservoir. All the results were averaged by at least three independent experiments.

Material characterizations
High-angle annular dark field scanning transmission electron microscopy (HAADF-STEM) images, energy-dispersive X-ray spectroscopy, and elemental mapping were obtained from Titan Cubed Themis 300 double Cs-corrected STEM operated at 300 kV and equipped with an extreme field emission gun source and a super-X energy-dispersive spectrometry system. X-ray photoelectron spectroscopy (XPS) data were collected by a Thermo Fisher K-alpha XPS/UPS with a monochromatic Al K-alpha x-ray source at 15 kV. All XPS results were corrected by the carbon 1s (assigned to 284.8 eV). X-ray diffraction (XRD) data were collected by a Bruker D8 Discover X-Ray Diffractometer. ICP-OES was conducted by an Agilent 5800 ICP-OES system.

In-situ Raman spectroscopy measurement
In-situ Raman spectroscopy measurement was carried out in a customized in-situ PTFE cell. A classy carbon electrode was used as the working electrode. The active area of the working electrode is ~0.2 cm2 (5 mm diameter). An Ag/AgCl electrode and a Pt wire were used as the reference electrode and counter electrode, respectively. The FAB-PK-130 membrane was used to separate the cathodic and anodic chambers. Au@SiO2 was used for enhancing the surface-enhanced Raman scattering of NiSAC50. 0.1 mg/cm2 of Au@SiO2 was first dropped onto the working electrode, and 0.5 mg/cm2 of NiSAC was then loaded. The in-situ Raman results were collected by a Horiba Aramis spectrometer (Imagin Analysis Center, Princeton University) with 785 nm excitation. The exposure time was set as 5 s, and all the Raman results were averaged at least 5 times of collection.

In-situ XAS spectroscopy measurement
Ni K-edge X-ray absorption spectroscopy (XAS) was measured at 8-ID at the National Synchrotron Light Source II (NSLS-II) at Brookhaven National Laboratory (BNL). For the ex-situ experiments, the samples were ground into a fine powder, pressed into a pellet, and sealed between Kapton tape before being scanned. The samples were scanned in their received condition at the Ni K edge. Two ion chambers with a Ni foil in between were placed behind the sample to calibrate the energy. For the in-situ XAS experiments, a 2 cm2 carbon paper loaded with 0.5 mg/cm2 NiSAC was used as the working electrode. An Ag/AgCl electrode was used as the reference electrode, and a graphite rod was used as the counter electrode. In-situ XAS was tested in a customized single-chamber XAS electrolyzer (Anhui Absorption Spectrum Equipment Co., LTD). Bioelectrolyte or ideal electrolyte was saturated by CO2 and flowed into the electrolyzer at a rate of 1 sccm. XAS data were fitted using the winXAS 3.1 software package (Supplementary Table 3). Least-squares fit in R-space of the k2-weighted Fourier transform data from 2.7 to 10.0 Å−1 and was used to obtain the EXAFS coordination parameters. The first shell was used to fit the EXAFS spectra. The amplitude reduction factor (S02) was determined by fitting a reference spectrum of the Ni foil (S02 = 0.8). Optimization of sigma-squared was obtained from fitting the isolated peaks in k-space.

DFT methods
All calculations in this work were performed using the Vienna Ab-Initio Simulation Package (VASP)69, where the RPBE functional was used70. The D3 correction method71 was employed to illustrate the long-range dispersion interactions between the adsorbates and surfaces. The core electrons were described with the projector augmented wave (PAW) method72. The convergence criteria for electronic and force minimization are set to 10−6 eV and 0.02 eV/Å for structural optimization. The cutoff energy for the kinetic energy of the plane-waves was 450 eV. The Gibbs free energy for gas-phase molecules and that for adsorbed species on the surface are obtained from the DFT energies with ZPE and entropy corrections determined from frequency calculations using the harmonic oscillator approximation. The Ag(111) surface was modeled by a 3-layer (5×4) supercell. The bottom two layers of the slab were constrained as the bulk region, and the top layer and adsorbates were allowed to relax as the interface region. The Brillouin zone was sampled using the 3×4×1 and 4×4×1 Gamma-centered k-point grids for the Ag(111) surface and the NiNC surface, respectively. The linearized Poisson-Boltzmann implicit solvation model implemented in VASPsol is used to represent the electrolyte region73. The dielectric constant of water, 78.4, and the Debye screening length corresponding to 1 M concentration of electrolytes, 3.0 Å, were used. As the exact catalytic material under operating conditions is difficult to model, the conclusions drawn from our DFT calculations should be treated with caution and regarded as qualitative. Our simplified, static models do not fully account for surface reconstruction, explicit solvent effects like H-bond interactions, or coverage-dependent phenomena; therefore, the DFT results are intended only as supportive guidance and plausible mechanistic scenarios, rather than definitive evidence.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript presents a highly innovative and impactful study integrating a poison-resistant Ni single-atom catalyst (Ni SAC) with engineered C. ljungdahlii for efficient CO2-to-isopropanol (IPA) conversion. The work addresses critical bottlenecks (catalyst deactivation in bioelectrolytes, low CO supply for microbial upgrading) and demonstrates record-breaking IPA production rates. The mechanistic investigation using in situ spectroscopy and DFT is rigorous. While the study is fundamentally sound and technologically significant, several key aspects require clarification or expansion to fully support the claims and ensure robustness.\nSpecific Comments:\n1.Faradaic Efficiency (FE) Calculation for C2+Oxygenates: Calculating FE for IPA and ethanol (C2+ oxygenates) based on the total current density (which generates CO/H2 via electrochemical CO2RR) is inappropriate. FE is an electrochemical metric defined for products directly generated at the electrode surface from electrons consumed. IPA and ethanol are microbial products derived from the consumption of the electrochemically generated CO/H2, not direct electrochemical products.\nSo it is suggested to Report FE only for the electrochemical products (CO, H2, possibly formate if detected). For microbial products (IPA, EtOH, acetate), report electron recovery efficiency (ERE) or carbon yield based on the electrons/carbon contained in the supplied CO/H2. Clearly state in the text and figure captions that the \"FE\" values for IPA/EtOH represent electron allocation efficiency from the electrochemical donor gases to the specific microbial product, not direct electrochemical FE.\n2.Assigning partial current densities to IPA and EtOH (e.g., \"partial current density for IPA\") with the fomula given is fundamentally incorrect for the same reason as point 1. Current density is a property of the electrochemical reaction step (CO2RR to CO/H2).\n3.Attributing the <50% FE (for liquids) solely to a mismatch between CO/H₂ generation and microbial uptake requires stronger evidence. Figure 4b-d shows a decreasing total current density alongside an increasing IPA \"FE\" and concentration over time. This complex trend needs deeper analysis beyond the simple mismatch hypothesis.\nIt is suggested to (1) Quantify CO/H2 concentrations in the headspace of the bioreactor/bioelectrolyte reservoir throughout the 4-day run. This is crucial to confirm accumulation and directly assess the mismatch; (2) Provide post-mortem characterization (XPS, STEM, ICP-MS for Ni leaching - see point 7) of the Ni SAC cathode after the 4-day operation to rule out catalyst degradation as a cause of current density drop; (3) Perform basic kinetic modeling of microbial CO/H2 consumption vs. electrochemical generation rates to substantiate the mismatch claim\n4.Could substrate inhibition (e.g., high CO/H₂/acetate) or pH shifts contribute to reduced uptake or current? Explain the apparent increase in IPA \"FE\" over time despite decreasing current density. Is this due to lag phase/adaptation, changing microbial metabolism, or accumulation making calculation artifacts?\n5.The observed current density decay suggests challenges for continuous operation. Discuss the implications explicitly. Acknowledge that the current system configuration appears more suited for batch or fed-batch operation, or propose strategies for enabling continuous operation (e.g., enhanced gas transfer/liquid mixing, cell recycling, product removal, optimized reactor design like membrane bioreactors).\n6.The stability of Ni SAC in the complex, potentially corrosive bioelectrolyte over long operation (4 days) is paramount but not adequately addressed. Ni leaching could poison microbes or alter catalyst performance. So (ICP-MS) analysis on the bioelectrolyte after the 4-day iMES experiment to quantify dissolved Ni ions is necessary.\n7.The justification for stopping at 4 days is unclear. While FE trends suggest potential improvement with longer runs, other factors (declining current, acetate accumulation, potential cell lysis/decline) might counteract this. Explain why the experiment was run for 4 days. Was microbial growth/productivity plateauing? Was acetate inhibition observed? Was current density becoming too low?\n8. Fig.1d&e, The description of bars vs. lines is ambiguous. Please explicitly state in the figure caption of Bar and Line.\n9.The term \"ideal electrolyte\" is used without clear definition in the context of each catalyst comparison. Define \"ideal electrolyte\" explicitly for each relevant experiment in the main text or figure captions.\n10.The in situ Raman data suggests organic desorption on Ag NPs at more negative potentials (> -0.8 V vs RHE), correlating with FE recovery. This implies Ag NPs can function in bioelectrolyte but require high overpotentials. Please Briefly discuss this nuance. While Ni SAC performs vastly better across the range, acknowledge that Ag NPs could be driven harder to overcome poisoning, albeit inefficiently.\n11.Line 112-117, Adding a schematic figure illustrating the electron flow from CO oxidation via CODH → Fd~red~ → Rnf complex → H+ gradient → ATP synthesis & reduced ferredoxin oxidation → NAD+ reduction → NADH for biosynthesis to enhance clarity. Indicate cellular localization, e.g. membrane for Rnf?\n12.Provide a brief stoichiometric comparison. Explain that using H2 as the sole energy source requires diverting more electrons through the energy-conserving Rnf complex solely for ATP generation via H+ gradient, leaving fewer electrons (as reduced Fd or NADH) directly available for the highly reducing IPA biosynthesis pathway compared to when CO, which directly generates Fd~red~ and provides carbon, is utilized.\n13.Line 140-142, cation insensitivity logic chain is problematic. Briefly restate the logic for clarity. linking PZC → field strength → field-dependent intermediate stabilization → reduced cation sensitivity.", "reviewer_2": "This is an interesting manuscript describing isopropanol production in an integrated CO2 electrolyzer – bioreactor system using a genetically modified strain of Clostridium ljungdahlii. Advantages of using single-atom nickel catalyst (Ni SAC) for CO2 reduction to CO and its compatibility with the microbial culture are clearly demonstrated. Overall, the reported IPA production rate and especially the current density is one of the highest reported for microbial electrosynthesis cells, although the integrated system used in this study is different from a typical MES cell, which typically uses microbial biofilm at the cathode for CO2 conversion and subsequent biotranformations.\nSeveral improvements to the manuscript could be suggested:\n1. Description of the experimental setup. To my understanding, the experimental setup in ref 20 is different from the one used in this work. In this reference, a gas diffusion cathode was used to facilitate CO2 transport to the cathode active layer and the biofilm. The experimental setup shown in figure 4A shows a CO2 electrolyzer and a bioreactor with liquid recirculation between the two units. A more detailed description of the setup and a better diagram, showing setup technical details rather than a general concept (e.g. a CO2-emitting plant) is needed. Perhaps other references should be verified for accuracy.\n2. Abbreviations: All abbreviations should be explained when used first time, e.g. L346 (PBS buffer, phosphate buffered saline, to my understanding); DFT (density functional theory); XANES (L290) and so on.\n3. Units: line 35 (and further in the text) refers to 161 mg/L/day, which is a volumetric production rate. It is not clear how this value was calculated, based on the fermenter volume or the combined electrolytic cell and fermenter volume? This should be specified in the text. Also, some performance values are given in mg/L and some concentrations in mM (e.g. L109).\n4. The duration of the experiments is not mentioned in the discussion and there are no graphs showing concentrations vs time. Perhaps such graphs can be added to the Supplementary Materials.\nL132. The term “partial current density” needs to be explained, it is not often used outside of electrochemistry. Also, what other products were formed, H2? Any liquid phase products?\nL148. Provide the range of current densities (50-500 mA/cm2, based on Figure S2)\nL149. Perhaps “dramatic” is not the best term.\nFigure 4a needs to be improved, as mentioned above\nL476: “anaerobic”\nL481: Specify the number of days and refer to a graph in Supplementary Materials.", "reviewer_3": "Reviewer #1 (Remarks to the Author):\nThis is a revised manuscript and the authors have carefully answered all the questions raised\nby reviewers, so this manuscript can be accepted for publication at present form.\nReviewer #2 (Remarks to the Author):\nThe manuscript can be accepted for publication at present form.\nReviewer #3 (Remarks to the Author):\nThe authors provided detailed responses but bypassed the critical experimental evidence of cell\nviability and performance in control groups, which are necessary for evaluating a material-\nmicrobe hybrid system for chemical synthesis or energy production1. The performance data in\nthe provided manuscript is only necessary but not sufficient to support that functionality of the\nproposed hybrid system; while the cell viability tests and all the control groups are crucial to\neliminate any potential false positive results. In details,\nreviewer indicated that previous studies and general understanding indicated that this is unlikely\nthe case. To make sure we don’t rule out any possibilities as the reviewer insisted, we performed\nadditional experiments of CO2 electrolysis in YT medium buffered with 100 mM PBS using a\nbare gas-diffusion electrode without any catalysts loading. The relevant data are shown below.\nIn the absence of the Ni SAC, the current densities at same potentials were remarkably lower\nthan those of the Ni SAC electrode. Due to the low conductivity of microbial growth medium,\nthe total current density was also lower than previous report (ACS Energy Letters 6.1 (2021):\n33-40). More importantly, no CO signals above the GC detection limit were detected at all\napplied potentials, indicating that the bare gas-diffusion electrode that we used in this work has\nno obvious CO2RR ability.\nFirst, as shown in the above figure, the bare electrode yielded a much lower current density,\nindicating fewer available electrons for microbial growth. For example, at -0.75 V vs. RHE the\ncurrent density is only 1.58 mA/cm2. Based on Supplementary N"}, "author_response": "Summary: We appreciate the thoughtful and constructive feedback from all the reviewers. The reviewers commented positively on the originality and significance of the work, and they raised several questions regarding the catalyst structure, performance analyses, and other aspects of the study. We have carefully studied the comments, and we revised the manuscript accordingly to improve the quality. We have performed all suggested essential experiments to the best of our ability, substantiated our findings with key references, and provided comprehensive analyses. We are confident that the concerns have been addressed, and we appreciate the reviewers’ consideration. The point-by-point responses can be found below. All the changes in the main manuscript and supplementary information have been highlighted.\nReviewer 1:\nThis manuscript presents a highly innovative and impactful study integrating a poison-resistant Ni single-atom catalyst (Ni SAC) with engineered C. ljungdahlii for efficient CO2-to-isopropanol (IPA) conversion. The work addresses critical bottlenecks (catalyst deactivation in bioelectrolytes, low CO supply for microbial upgrading) and demonstrates record-breaking IPA production rates. The mechanistic investigation using in situ spectroscopy and DFT is rigorous. While the study is fundamentally sound and technologically significant, several key aspects require clarification or expansion to fully support the claims and ensure robustness.\nResponse: We sincerely thank the reviewer for highlighting the novelty and impact of our work, and we are grateful for the constructive suggestions aimed at improving the clarity and quality of the work. We have provided point-by-point responses and added additional experiments and analytical discussions. Please refer to our detailed responses below.\nSpecific Comments:\n1. Faradaic Efficiency (FE) Calculation for C2+ Oxygenates: Calculating FE for IPA and ethanol (C2+ oxygenates) based on the total current density (which generates CO/H2 via electrochemical CO2RR) is inappropriate. FE is an electrochemical metric defined for products directly generated at the electrode surface from electrons consumed. IPA and ethanol are microbial products derived from the consumption of the electrochemically generated CO/H2, not direct electrochemical products.\nSo it is suggested to Report FE only for the electrochemical products (CO, H2, possibly formate if detected). For microbial products (IPA, EtOH, acetate), report electron recovery efficiency (ERE) or carbon yield based on the electrons/carbon contained in the supplied CO/H2. Clearly state in the text and figure captions that the \"FE\" values for IPA/EtOH represent electron allocation efficiency from the electrochemical donor gases to the specific microbial product, not direct electrochemical FE.\nResponse: We thank the reviewer for pointing out this potential confusion between Faradaic Efficiency (FE) and Electron Recovery Efficiency (ERE). This is a good question. While CO and H serve as the primary electron donors and dominate the energy flow from electrochemical system to C. ljungdahlii strain, some electrons could also be directly acquired from the electrode surface (Green Chemistry 25.11 (2023): 4375-4386.; Applied and environmental microbiology 79.4 (2013): 1102-1109.). Therefore, for microbial conversion process, it may not be accurate to attribute all electron input to CO/H , though we acknowledge and conducted research by our own group that the CO/H mediated pathways appear to be dominant. Under such circumstances, the calculation formulas for FE and ERE are similar (both representing the ratio of electrons associated with the product to the total electrons input into the electrochemical system). To make it clear, we have updated the descriptions in the Methods section as well as the relevant figures and analyses to clearly distinguish between the direct electrochemical products (CO/H ) and the indirect microbially synthesized products (IPA/EtOH/acetate), in order to avoid any misunderstanding:\n“Products quantification\nAll gas products were tested by a gas chromatograph (GC, Agilent 8890). The liquid products were collected from cathodic chamber or cold trap, and detected by a high-performance liquid chromatograph (HPLC, Agilent 1260). To trap the evaporated liquid products as much as possible, a 15 mL glass tube containing 4 mL DI water at 0 ℃ was connected to the bioelectrolyte reservoir outlet.\nThe FE or ERE of specific product was calculated by the equation:\nn ∗electron transfer number∗F\nx\nFE or ERE = ∗100%\nx x Electrode area∗I∗t\nWhere n is the production amount (mol) of product X, determined by GC or HPLC; F is the Faradaic constant, 96485.33 C/mol; I is the total current density (mA/cm2). Although the equation is same, we use FE and ERE to indicate direct electrochemical products (CO/H ) and indirect microbial products (IPA/EtOH/Acetate).”\n“After accounting the evaporated amount, the average IPA titer reached ~10.8 mM over four days, with a total C oxygenate (EtOH + IPA + Acetate) concentration of ~91.1 mM (Fig. 4c).\n2+\nThe total electron recovery efficiency (ERE) for C oxygenates reached 47.7 ± 6.9% (Fig. 4d).\n2+\nSpecifically, the combined ERE for IPA and EtOH exceeded 26.4% (10.5% for IPA and 15.9% for EtOH).”\n2. Assigning partial current densities to IPA and EtOH (e.g., \"partial current density for IPA\") with the fomula given is fundamentally incorrect for the same reason as point 1. Current density is a property of the electrochemical reaction step (CO2RR to CO/H2).\nResponse: Thanks for pointing this out. Our original goal was to ensure a fair comparison with the prior studies. As shown in Fig. 4e, we hope to emphasize the volumetric and electrode area-normalized production rates at x axis and y axis, respectively, which are two metrics especially relevant for evaluating scale-up potential. In this context, we fully agree that partial current density is not an appropriate metric in this context. Therefore, we directly compare the areal IPA production rate at the unit of mg/cm2/day, in order to avoid any misunderstanding.\nBesides, in the initial version, we mistakenly calculated the “IPA partial current density” in the reference (Science 352.6290 (2016): 1210–1213) by using “electronic energy efficiency” instead of “FE”. We greatly appreciate the reviewer’s insightful observation, which helps us to identify this issue. All related errors have now been corrected in the revised manuscript and supplementary information. Even so, our performance is still the highest reported in the field, and the correction will not affect the overall conclusions of the study.\n“Reports on iMES for IPA production remain scarce, positioning this field as a largely unexplored area. Here, we present the first demonstration of CO-mediated IPA iMES. Due to the sufficient CO supply and superior reducing power relative to H , our system achieved unprecedented areal production rate (3.23 mg/cm2/day) and volumetric production rates (161.3 mg/L/day) for IPA compared to prior studies (Fig. 5e and Supplementary Table S2).”\n3. Attributing the <50% FE (for liquids) solely to a mismatch between CO/H₂ generation and microbial uptake requires stronger evidence. Figure 4b-d shows a decreasing total current density alongside an increasing IPA \"FE\" and concentration over time. This complex trend needs deeper analysis beyond the simple mismatch hypothesis.\nIt is suggested to (1) Quantify CO/H2 concentrations in the headspace of the bioreactor/bioelectrolyte reservoir throughout the 4-day run. This is crucial to confirm accumulation and directly assess the mismatch; (2) Provide post-mortem characterization (XPS, STEM, ICP-MS for Ni leaching - see point 7) of the Ni SAC cathode after the 4-day operation to rule out catalyst degradation as a cause of current density drop; (3) Perform basic kinetic modeling of microbial CO/H2 consumption vs. electrochemical generation rates to substantiate the mismatch claim\nResponse: Thanks for the suggestions. Regarding the suggestion (1), due to the termination of the collaborative project, it is currently difficult for us to restart the operation of the integrated system and retest the headspace CO/H concentrations. However, we have made every effort to address this concern using available indirect evidence and reasonable assumptions to approximate microbial gas uptake rates, as recommended in suggestion (3). Specifically, our gas fermentation system was equipped with a pressure gauge to confirm the headspace pressure of culture bottle could be repressured to 10 psi (Method section). We found that each 24 h fermentation operation resulted in a headspace pressure decrease of >7 psi, indicating that most CO/H2/CO2 were consumed during the 24 h fermentation period, although these results were not recorded in detail as they were not critical to the main scope of the study. Here, we assume that all CO/H2 (7 psi) could be consumed in 24 h, in order to overestimate CO/H2 consumption rate. Based on this assumption, for a 50 mL liquid volume with a maximum OD600 of 1.54 (Method and Supplementary Fig. S1), we can estimate that the CO/H2 consumption rate of our engineered C. ljungdahlii is:\npsi 200 mL\n7 psi∗6984.7 ∗\nPa mL\n1000000\nm3\nSyngas consumption rate in fermenter =\nJ\n8.314 ∗298.15K∗24h\nmol∗K\n= 1.6∗10−4 mol/h\nSo, for the iMES process, the total liquid volume is 20 mL and the maximum OD600 is 1.36, and, if we don’t consider the boosting effect of gas-diffusion electrode, we can approximately estimate that the CO/H2 consumption rate of our engineered C. ljungdahlii in iMES system is about:\n20mL 1.36\nSyngas consumption rate = 1.6∗10−4 mol/h∗ ∗ = 0.6∗10−4 mol/h\n50mL 1.54\nWe can also estimate the CO/H2 (both of them are 2e- products) generation rate based on the average current density (~11.39 mA/cm2, Figure 4b) during the integrated system operation:\nmA A\n11.39 ∗ 1cm2∗10−3 ∗3600s\ncm2 mA\nSyngas generation rate = = 2.1∗10−4mol/h\nC\n2∗96485.33\nmol\nIt is evident that the production rate of CO/H2 is far greater than their consumption rate. Even when considering the enhancement of microbial gas utilization efficiency by gas-diffusion electrodes (e.g. Biotechnology and bioengineering 120.6 (2023): 1465-1477.; Environmental science and ecotechnology 16 (2023): 100261.), we believe that the gas generation rate of the catalyst at our selected potential is still sufficient. Moreover, the operation at -0.75 V vs. RHE was determined not only by the CO/H2 supply but also by the CO/H2 ratio. The relevant discussion can be found in the main manuscript and Supplementary Information:\n“Applying lower overpotentials may improve the FE of C2+ oxygenates but can also decrease the reducing equivalents supply rate and consequently diminish the overall production rate of C2+ oxygenates.60 To ensure sufficient CO supply and a high CO/H ratio, iMES system was operated continuously at -0.75 V vs. RHE (Supplementary Note S1).”\n“Supplementary Note S1 Potential selection for iMES process\nFor the gas fermentation, we employed a gas mixture with a composition of 50%/20%/30% CO/H /CO (CO/H ratio is 5:2). At potentials more negative than -0.75 V vs. RHE, this CO:H ratio could not be maintained due to the increased H production. Our previous studies have shown that a higher CO:H ratio is beneficial for the synthesis of deeply reduced products.5 Therefore, ensuring a sufficient CO:H ratio is essential for optimal IPA production performance, and the operating potential should not be lower than -0.75 V vs. RHE.\nHowever, operating at higher overpotentials also means more energy input and increased waste of CO/H electron donors. In our fermentation experiments with a 50 mL culture volume, we achieved a maximum OD600 of 1.54 (Supplementary Fig. S1). By re-pressurizing the headspace daily, we observed that the pressure dropped by more than 7 psi over 24 hours. To estimate whether the CO/H supply was sufficient during the iMES process, we conservatively assumed that the entire pressure drop was only due to CO/H consumption, thereby overestimating the uptake rate of electron donors. The CO/H consumption rate of engineered C. ljungdahlii can be estimated:\npsi 200 mL\n7 psi∗6984.7 ∗\nPa mL\n1000000\nm3\nSyngas consumption rate in fermenter =\nJ\n8.314 ∗298.15K∗24h\nmol∗K\n= 1.6∗10−4 mol/h\nFor the iMES process, the total liquid volume is 20 mL and the maximum OD600 is 1.36. Without considering the promotion effect of gas-diffusion electrode, we can approximately estimate that the CO/H consumption rate of our engineered C. ljungdahlii in iMES system is about:\n20mL 1.36\nSyngas consumption rate = 1.6∗10−4 mol/h∗ ∗ = 0.6∗10−4 mol/h\n50mL 1.54\nWe can also estimate the CO/H (both of them are 2e- products) generation rate based on the average current density (~11.39 mA/cm2, Figure 4b) at -0.75 V vs. RHE during the integrated system operation:\nmA A\n11.39 ∗ 1cm2∗10−3 ∗3600s\ncm2 mA\nSyngas generation rate = = 2.1∗10−4mol/h\nC\n2∗96485.33\nmol\nAlthough the use of a gas-diffusion electrode can offer enhancements in gas transfer and microbial uptake, operation at -0.75 V vs. RHE can still supply sufficient electron donors for engineered C. ljungdahlii.6,7 By comparing the estimated syngas consumption and generation rates, as well as comprehensively considering both energy input and the CO/H ratio, we conclude that -0.75 V vs. RHE represents the most appropriate operating potential.”\nBesides, although it is currently difficult to restart the iMES experiments, we attempted to address the reviewer’s concerns as much as possible by testing the CO2RR performance of Ni SAC under similar conditions (same reactor, same electrolyte, and same applied potential) for four days. The results showed that the average current density and stability were very similar to those observed in iMES (Supplementary Fig. S22). Post-reaction XPS and STEM characterizations confirmed that the overall structure of the Ni SAC catalyst remained almost unchanged, and ICP analysis of the electrolyte showed that the Ni ion concentration was below the detection limit (Supplementary Fig. S23-S25). Therefore, the decline in current density cannot be attributed to catalyst degradation.\nFrom these observations, we can also conclude that: 1) Inoculation with C. ljungdahlii does not significantly affect the overall electrochemical performance of the electrode. Our previous studies also demonstrated that C. ljungdahlii was difficult to form large-scale biofilms in a flow cell. This highlights the excellent tolerance of Ni SAC to microbial growth media and C. ljungdahlii strains in a gas-diffusion flow reactor. 2) Ni SAC maintained a stable catalyst structure without apparent leaching of toxic Ni ions, ensuring high biocompatibility.\nAll the results and analyses have been updated in the main manuscript and supplementary information:\n“To counteract pH fluctuations that may occur during long-term iMES operation, 100 mM phosphate-buffered saline (PBS) buffer was added to the bioelectrolyte. The introduction of PBS also enhanced the accessibility of alkali metal cations, thereby reducing the overpotential required to achieve similar levels of CO RR activity and selectivity (Supplementary Fig. S21). In addition, the Ni SAC electrode exhibited stable current density and a consistent CO/H ratio for over 100 hours in the bioelectrolyte containing 100 mM PBS (Supplementary Fig. S22). After 100 h operation, post-characterizations (STEM, XPS and ICP, Supplementary Fig. S23-25) demonstrated that the overall structure of the Ni SAC remained stable, and no apparent Ni2+ ion leaching was detected in the electrolyte, indicating the high biocompatibility and stability of Ni SAC. …… Similar electrochemical performance observed before and after inoculating C. ljungdahlii further illustrated the excellent stability and high bioadaptivity of the Ni SAC in the flow cell (Fig. 4b and Supplementary Fig. S22).”\nFurthermore, the mismatch between electron donor supply and microbial consumption is well documented in microbial-electrochemical hybrid systems (Environmental Science and Ecotechnology 19 (2024): 100324; Nature Catalysis 2.5 (2019): 407-414.). The electrochemical process typically generates electron donors in excess of microbial demand. On the one hand, in lab-scale studies, it is necessary to ensure a sufficient supply of electron donors for microbial conversion, which is why relatively high current densities or large electrode areas are often employed. On the other hand, without changing gas transfer characteristics, most microorganisms inherently exhibit relatively low gas utilization efficiency. In the initial version, based on the innovative work by Rodrigues et al. (Nature Catalysis 2.5 (2019): 407-414.), we briefly highlighted the prospect of promoting the mass transfer of electron donors such as CO in order to further enhance the production rate of high-value products and improve electron efficiency, although this is not the main focus of the present study.\nWhile we acknowledge the value of direct headspace CO/H2 measurement, we are confident that the indirect conservative analysis, supported by previous publications and post-reaction characterizations, are sufficient to prove our proposed points and address the reviewer’s concern.\n4. Could substrate inhibition (e.g., high CO/H₂/acetate) or pH shifts contribute to reduced uptake or current? Explain the apparent increase in IPA \"FE\" over time despite decreasing current density. Is this due to lag phase/adaptation, changing microbial metabolism, or accumulation making calculation artifacts?\nResponse: This is a good question. H is a common electron donor for many microorganisms, and the H FE was relatively low, suggesting that H itself was unlikely to induce any inhibition effect. Moreover, our previous work demonstrated that increasing the CO/H2 ratio can promote the formation of deeply reduced products (Biomass and Bioenergy 124 (2019): 95-101.). It has also been reported that engineered C. ljungdahlii was capable of converting C1 chemicals to higher value-added products under CO2/CO gas mixtures without H2 addition (ACS Synthetic Biology 10.10 (2021): 2628-2638.). In our iMES system, under a consistent CO/H2 ratio and syngas production rate (Fig. 4b and Supplementary Fig. S21), the engineered C. ljungdahlii strain grew stably in the bioelectrolyte with 100 mM PBS buffer (for eliminating the pH shift), reaching OD600 comparable to those observed in gas fermentation experiments (Fig. 4b). In our previous iMES work, even operating for a longer time (20 days) in a bigger reactor, we did not observe serious pH shift (Environmental Science and Ecotechnology 19 (2024): 100324). Furthermore, the acetate titer in the iMES experiment did not exceed that of the gas fermentation control (Fig. 1c and 4c). Therefore, based on our previous studies, literature reports, and the experimental evidence presented in this work, we are confident that there was no inhibition effect by CO2/H2/acetate or serious pH shift under the operating conditions used.\nSince the reviewer also mentioned the issue of decreasing current density with increasing ERE (“FE”) in Comment 3, we provide a comprehensive analysis here to address this point.\nFirst, we want to clarify that over the 4-day operating period, both the overall current density and catalyst structure remained stable, as evidenced by Supplementary Fig. S21-S23. Therefore, we do not attribute the observed current behavior to catalyst degradation. In fact, when plotting Fig. 4b, we intentionally set the current density axis to a narrow range (7.5-14 mA/cm2) to better visualize the fluctuations and error bars, which may visually exaggerate the appearance of a “decline”.\nIn addition, current fluctuations can result from various factors, with the dominant one being the loss of the gas-liquid-solid three-phase interface caused by the gradual loss of gas-diffusion later (GDL) hydrophobicity during long-term operation. This leads to a decrease in current density or CO FE. This issue remains a persistent challenge in the field (Accounts of chemical research 51.4 (2018): 910-918.; Energy & Fuels 37.20 (2023): 15283-15308.). Compared to prior literature and our earlier studies, the current density of 10-12 mA/cm2 in this work is relatively moderate, resulting in milder interfacial reactions. Moreover, the use of a pH buffer in the electrolyte helped mitigate excessive local alkalization near the electrode under long-term operation. As a result, electrode flooding was less severe, contributing to the relatively stable current and CO FE observed in our system (Fig. 4e and Supplementary Fig. S21). Over time, the increasingly larger error bars also indicate greater current fluctuations. Nevertheless, the catalyst itself did not observe significant deactivation or degradation. We therefore attribute the slight decrease in average current density to the commonly observed loss of GDL hydrophobicity. Corresponding analysis has been updated in the main manuscript:\n“Over four days of uninterrupted operation, the average current density remained stable at ~11.4 mA/cm2, and the optical density (OD ) of C. ljungdahlii steadily increased over the first three days, peaking at ~1.36 (Fig. 4b). The slightly decreased current density could be attributed to the loss of gas-diffusion layer (GDL) hydrophobicity over long-term operation.56,57”\nFinally, regarding the gradual increase in ERE (\"FE\") over the four-day period, we prefer to attribute this trend to microbial lag phase/adaptation and the effect of accumulation on the calculation method. During the first two days of the growth phase, especially within the initial 24 hours after inoculation, C. ljungdahlii likely needed time to adapt to the electrochemical reactor environment, and the metabolism activity had not yet reached its optimum level, which is a common phenomenon in previous reports (Science 352.6290 (2016): 1210–1213; Nature Catalysis 2.5 (2019): 407-414.). Consequently, the syngas uptake rate was relatively low, leading to a lower ERE in the early period. As C. ljungdahlii continued to grow and adapt, the syngas uptake rate improved correspondingly, resulting in higher ERE values. In addition, as shown in Figure 4d, the ERE values were calculated as cumulative averages, with the value on day n representing the mean ERE across the initial n days of operation. However, when we calculate the daily ERE results without considering the evaporated alcohols (as shown in the table below for reviewer reference only), we can observe that the 1st day ERE of C2+ oxygenates was the lowest at only 20.3%, while the ERE values on the 3rd and 4th days exceeded 48%. This suggests that as C. ljungdahlii reached the growth plateau, the initially low ERE would be averaged with the higher ERE values from the latter days, giving rise to the observed trend of gradually increasing average ERE.\nIn summary, based on the experimental evidence and analyses provided in our responses to comments 3 and 4, we conclude that the 4-day continuous operation did not lead to any serious catalyst degradation or Ni leaching that could compromise the stability of the integrated system. Furthermore, no signs of severe substrate inhibition or pH-induced deactivation of either the microbial strain or the electrocatalyst were observed. The apparent increase in ERE, alongside the modest decline in current density, can be primarily attributed to the microbial lag phase and adaptation during early operation, as well as the use of cumulative averaging in ERE calculations.\n5. The observed current density decay suggests challenges for continuous operation. Discuss the implications explicitly. Acknowledge that the current system configuration appears more suited for batch or fed-batch operation, or propose strategies for enabling continuous operation (e.g., enhanced gas transfer/liquid mixing, cell recycling, product removal, optimized reactor design like membrane bioreactors).\nResponse: Thanks for the suggestion. As suggested, we expanded the discussion about the challenges of iMES process based on the flow system, and proposed the direction of future optimization:\n“The proposed CO-mediated iMES strategy in this study unlocked a new electrochemical route for CO -to-IPA conversion and demonstrated relatively stable current density over a 4-day operation, but the current electrolyzer design will need improvement for large-scale deployment.61 Notably, at -0.75 V vs. RHE, the total FE of liquid products did not exceed 50%, indicating that nearly half of the electrons remain in CO/H . This is primarily because of a mismatch between the generation rate of CO/H and the slower microbial consumption rate.55,62 Looking ahead, future iMES designs employing Ni SAC or other bioadaptive electrodes should prioritize optimizing electron donor mass transfer to microbes, thereby bridging the gap between CO/H production and microbial uptake as well as improving electron utilization efficiency and overall FE toward target products62. Besides, another challenge is the unavoidable flooding of GDL, which can disrupt the gas-liquid-solid triple-phase interface, limit CO/H delivery, and reduce long-term electrochemical efficiency/stability.56,63 Simultaneously, the co-production of IPA, EtOH, and acetate can lead to product mixing in the bioelectrolyte, which will be continuously flushed by flowing media. This not only dilutes the target products but also imposes significant energy cost and financial pressure on downstream separation.10,64 Therefore, future directions should also aim to enhance electrode hydrophobicity to mitigate flooding and explore integrated on-pot separation or microbial recycling strategies to improve product recovery and enable continuous operation, while above mentioned optimizations are not the focus of this current work.”\n6. The stability of Ni SAC in the complex, potentially corrosive bioelectrolyte over long operation (4 days) is paramount but not adequately addressed. Ni leaching could poison microbes or alter catalyst performance. So (ICP-MS) analysis on the bioelectrolyte after the 4-day iMES experiment to quantify dissolved Ni ions is necessary.\nResponse: We thank the reviewer’s concern about the Ni SAC stability and possible Ni ion leaching. Although it’s hard for us to restart the iMES system, we tested the long-term operation again in the same bioelectrolyte (YT+100 mM PBS buffer) without C. ljungdahlii inoculation at -0.75 V vs. RHE and performed comprehensive post-characterizations. ICP results indicated the Ni ions leaking issue over four days was negligible, which is consistent with previous reports about Ni SAC for CO2RR (Journal of Materials Chemistry A 12.14 (2024): 8331-8339.). Indeed, compared to strong basic electrolyte used for conventional CO2 electrolysis, near pH-neutral bioelectrolyte is much less corrosive and therefore can maintain good stability.\n7. The justification for stopping at 4 days is unclear. While FE trends suggest potential improvement with longer runs, other factors (declining current, acetate accumulation, potential cell lysis/decline) might counteract this. Explain why the experiment was run for 4 days. Was microbial growth/productivity plateauing? Was acetate inhibition observed? Was current density becoming too low?\nResponse: Yes, the four days operation was discussed as for the increasing ERE (or “FE”) trend in the response to Comment 4. In fact, after 2-3 days of operation, the OD600 was plateaued, indicating that the cells had ended their natural growth phase. Based on our gas fermentation experiments (Fig. 1b-c and Supplementary Fig. S1), once C. ljungdahlii strains entered the plateaued phase, the concentrations of IPA, EtOH, and acetate would no longer increase substantially. Accordingly, in the iMES system, we terminated the experiment at the end of day 4, when OD600 also ceased increasing.\nMoreover, as detailed in our response to comment 4, a closer examination of the daily ERE values for C2+ oxygenates revealed that the ERE remained almost unchanged between 3rd day and 4th day (as shown below). At this point, the concentrations of IPA, EtOH, and acetate had also already approached the maximum values observed in the gas fermentation system (Fig. 1b-c and Fig. 4c). Therefore, we believe that extending the runtime beyond 4 days would not yield significant improvements in ERE or production rate.\nTotal ERE\nAcetate ERE IPA ERE EtOH ERE\n(not include evaporated amount)\n1st day 16.6 ± 0.7 % 2.2 ± 0.4 % 1.5 ± 1.4 % 20.3 ± 1.7 %\n2nd day 22.4 ± 7.4 % 11.9 ± 1.3 % 7.4 ± 1.9 % 41.7 ± 9.8 %\n3rd day 22.9 ± 5.9 % 8.4 ± 4.7 % 17.5 ± 10.3 % 48.9 ± 20.6 %\n4th day 25.8 ± 5.8 % 11.4 ± 4.4 % 11.8 ± 4.1 % 49.1 ± 12.7 %\nFinally, a 4-day operational period is typical for microbial electrochemical CO2RR studies (Nature Catalysis 2.5 (2019): 407-414.) and is already substantially longer than the stability durations reported in many abiotic CO2RR systems (Angewandte Chemie 134.9 (2022): e202110657.; Journal of the American Chemical Society 142.15 (2020): 6878-6883.). Thus, we consider this testing timeframe sufficient and representative for evaluating the performance and robustness of our iMES system.\n8. Fig.1d&e, The description of bars vs. lines is ambiguous. Please explicitly state in the figure caption of Bar and Line.\nResponse: Thanks, and as suggested all the descriptions now have been clarified in the initial version. “CO2RR FE (left Y axis, bars) and current densities (right Y axis, lines) of d) Ag NPs and e) Ni SAC in bioelectrolyte.” was at the end of Fig. 1 caption.\n9. The term \"ideal electrolyte\" is used without clear definition in the context of each catalyst comparison. Define \"ideal electrolyte\" explicitly for each relevant experiment in the main text or figure captions.\nResponse: We have clarified the “ideal electrolyte” in the Methods section (Electrochemical tests part):\n“Unless otherwise specified, bioelectrolyte refers to the YT medium and Ideal electrolyte only contains 100 mM NaCl without any organics.”\n10. The in situ Raman data suggests organic desorption on Ag NPs at more negative potentials (> -0.8 V vs RHE), correlating with FE recovery. This implies Ag NPs can function in bioelectrolyte but require high overpotentials. Please Briefly discuss this nuance. While Ni SAC performs vastly better across the range, acknowledge that Ag NPs could be driven harder to overcome poisoning, albeit inefficiently.\nResponse: It’s possible if we only consider the Raman results and electrochemical performance trends. However, we believe this still cannot imply that Ag is suitable for integration into the iMES system for IPA production.\n1) First, operating at more negative potential will lead to higher overpotentials and increased energy consumption. Coupled with the significantly higher cost of Ag as a noble metal compared to Ni SAC, this would dramatically increase the capital and operational costs of iMES process; 2) Second, although Ag is widely regarded as a benchmark CO generation catalyst, its maximum CO FE in neutral or near-neutral KHCO3 electrolytes at moderate potential of < -1 V vs RHE is typically ~80%. Even if the poisoning issue could be overcome at higher overpotentials, the fast development of competing H2 evolution reaction at deeper potentials will make it challenging to maintain high CO FE under such conditions (ACS Applied Materials & Interfaces 12.50 (2020): 56642-56649.; Journal of the American Chemical Society 137.43 (2015): 13844-13850.); 3) Finally, and perhaps most importantly, is the issue of metal ion leaching, which has been raised by the reviewer in comment 3&6. We have already confirmed via ICP and XPS that Ni SAC exhibited negligible leaching of Ni2+ due to the strong coordination of Ni-N4 structure. Moreover, the Ni loading in our electrodes is only ~8 μg/cm2, whereas Ag electrode loading amount is ~0.5 mg/cm2 (62.5 times higher), substantially increasing the risk of Ag+ (a well-known antimicrobial agent) leaching (Nature communications 15.1 (2024): 3356.), which could compromise biocompatibility.\nTaking together, although Ag may exhibit improved tolerance to poisoning at deeper potentials, its integration into the iMES system for IPA production remains highly challenging. We have updated the main text to reflect this discussion:\n“This potential-dependent desorption correlates well with the observed recovery of CO RR activity at -0.85 V vs. RHE in our electrochemical measurements, suggesting that the competitive adsorption of organics from the bioelectrolyte and the resulting changes in interfacial properties would be the main reasons for the deactivation of the CO RR on Ag NPs. Nevertheless, due to the rapid development of competing H evolution at higher overpotentials45,46, the higher energy and catalyst costs, as well as the increased risk of Ag+ leaching during long-time operation47, Ag is still a less suitable candidate than Ni SAC for integration into iMES systems targeting IPA production.”\n11. Line 112-117, Adding a schematic figure illustrating the electron flow from CO oxidation via CODH → Fd~red~ → Rnf complex → H+ gradient → ATP synthesis & reduced ferredoxin oxidation → NAD+ reduction → NADH for biosynthesis to enhance clarity. Indicate cellular localization, e.g. membrane for Rnf?\nResponse: We appreciate the reviewer’s suggestion to further illustrate the electron flow in Fig. 1a. In response, we have updated Fig. 1a to include the relevant electron pathways, as well as include the Rnf complex, ATPase, and other associated structures.\n12. Provide a brief stoichiometric comparison. Explain that using H2 as the sole energy source requires diverting more electrons through the energy-conserving Rnf complex solely for ATP generation via H+ gradient, leaving fewer electrons (as reduced Fd or NADH) directly available for the highly reducing IPA biosynthesis pathway compared to when CO, which directly generates Fd~red~ and provides carbon, is utilized.\nResponse: Thanks for the suggestion. Zhu et al (Frontiers in microbiology 11 (2020): 416.) have summarized succinctly the differences between CO and H2/CO2 fermentation by acetogens in the context of acetate or ethanol formation with stoichiometries. We would be happy to include these comparisons or reference this work. In the context of C. ljungdahlii H2/CO2 fermentation, H2 is required for carbon assimilation by both branches of the WLP by generating Fdred. In the methyl branch, CO2 is assimilated via the formate dehydrogenase (Fdh) and H2 is used by a bifurcating hydrogenase (in a complex with the Fdh) to generate Fdred for formate formation. For the carbonyl branch, Fdred is required for CO2 → CO reduction (by the carbon monoxide dehydrogenase). In addition, Fdred is required for ATP generation by the Rnf complex/ATPase, which is essential for autotrophic growth by these organisms. Therefore, when C. ljungdahlii is grown on H2/CO2, the pool of available H2 is split between carbon assimilation and energy generation leading to far less energy available for product formation.\nIn contrast, as the reviewer mentioned, CO can be directly oxidized to form Fdred (via the CODH) which can be utilized directly by the Rnf complex as well as other energy generating enzymes (eg Nfn) to produce NAD(P)H. Therefore, when C. ljungdahlii is grown in the presence of CO/CO2/H2, the available pool of Fdred is far higher to help drive growth as well as IPA formation. The related discussions in the main manuscript have been updated:\n“This CO-dependent metabolic shift arises from its dual role in precursor and energy metabolism (Fig. 1a). Beyond serving as a carbon source for acetyl-CoA formation, CO oxidation via CO dehydrogenase (CODH) generates reduced ferredoxin (Fd ). This electron carrier drives the Rnf complex to establish a transmembrane H+ gradient, thereby generating ATP to sustain growth and NADH to drive IPA biosynthesis.24 H metabolism of C. ljungdahlii is less efficient, although it can also supply reducing equivalents via hydrogenases.25 In H /CO fermentation, H must simultaneously support both branches of the Wood-Ljungdahl pathway (WLP). In the methyl branch, CO is assimilated via the formate dehydrogenase (Fdh) and H is used by a bifurcating hydrogenase (in a complex with the Fdh) to generate Fd for formate formation. While in the carbonyl branch, Fd is required again for CO → CO reduction by CODH. In addition, Fdred is required for ATP generation by the Rnf complex/ATPase, which is essential for autotrophic growth by these organisms. As a result, when C. ljungdahlii grows on H /CO , the available H pool is divided between carbon assimilation and energy conservation, leaving far fewer reducing equivalents available for product (e.g., IPA) formation. In contrast, CO oxidation directly provides Fdred via CODH, which can be efficiently utilized by the Rnf complex and other enzymes (e.g., Nfn) to generate NAD(P)H. Thus, in CO/CO /H fermentation, the pool of Fdred is significantly larger, supporting both robust autotrophic growth and enhanced IPA production.26 Consequently, microbial electrochemical systems targeting highly reduced compounds like IPA must prioritize sustained CO generation for C. ljungdahlii, ensuring both carbon flux and energy conservation requirements are met.”\n13. Line 140-142, cation insensitivity logic chain is problematic. Briefly restate the logic for clarity. linking PZC → field strength → field-dependent intermediate stabilization → reduced cation sensitivity.\nResponse: This is a good suggestion. The statement has been updated in the main manuscript:\n“Notably, recent advances in electrocatalysis revealed that atomically dispersed Ni-N-C configurations surpass conventional Ag catalysts in the abiotic medium.31 In addition, the elevated potential of zero charge (PZC) of Ni single sites sustained increased surface cation charge density than silver, which stabilized the *CO - intermediate and provided unprecedented cation insensitivity, enabling sustained CO FE even in cation-insufficient organic amine electrolytes.32”\nReviewer 2:\nThis is an interesting manuscript describing isopropanol production in an integrated CO2 electrolyzer – bioreactor system using a genetically modified strain of Clostridium ljungdahlii. Advantages of using single-atom nickel catalyst (Ni SAC) for CO2 reduction to CO and its compatibility with the microbial culture are clearly demonstrated. Overall, the reported IPA production rate and especially the current density is one of the highest reported for microbial electrosynthesis cells, although the integrated system used in this study is different from a typical MES cell, which typically uses microbial biofilm at the cathode for CO2 conversion and subsequent biotranformations. Several improvements to the manuscript could be suggested\nResponse: We sincerely thank Reviewer 2 for the positive evaluation of this work and for recognizing this breakthrough in microbial electrosynthesis of isopropanol. We also appreciate the reviewer for the detailed comments and constructive suggestions. Please find our responses to each comment below. All corresponding revisions have been clearly highlighted in both the response letter and the manuscript.\n1. Description of the experimental setup. To my understanding, the experimental setup in ref 20 is different from the one used in this work. In this reference, a gas diffusion cathode was used to facilitate CO2 transport to the cathode active layer and the biofilm. The experimental setup shown in figure 4A shows a CO2 electrolyzer and a bioreactor with liquid recirculation between the two units. A more detailed description of the setup and a better diagram, showing setup technical details rather than a general concept (e.g. a CO2-emitting plant) is needed. Perhaps other references should be verified for accuracy.\nResponse: Thanks for the suggestion. To help illustrate the setup, we a reactor operation scheme in the SI:\n2. Abbreviations: All abbreviations should be explained when used first time, e.g. L346 (PBS buffer, phosphate buffered saline, to my understanding); DFT (density functional theory); XANES (L290) and so on.\nResponse: All the abbreviations mentioned have been explained:\n“To counteract pH fluctuations that may occur during long-term iMES operation, 100 mM phosphate-buffered saline (PBS) buffer was added to the bioelectrolyte.”\n“In-situ Raman and X-ray absorption spectroscopy revealed that the Ni SAC surface resisted organic adsorption and retained its coordination structure during CO reduction in the bioelectrolyte, and density functional theory (DFT) calculations proved the thermodynamic unfavourability of organic adsorption on Ni SAC.”\n“Ex-situ X-ray absorption near edge structure (XANES) analysis revealed that the Ni K-edge absorption energy of the Ni SAC exhibited a shift to higher energy positions compared to Ni foil, resembling the XANES spectrum of NiPc (Fig. 3c).”\n3. Units: line 35 (and further in the text) refers to 161 mg/L/day, which is a volumetric production rate. It is not clear how this value was calculated, based on the fermenter volume or the combined electrolytic cell and fermenter volume? This should be specified in the text. Also, some performance values are given in mg/L and some concentrations in mM (e.g. L109).\nResponse: For the calculation standard, we used the bioelectrolyte volume to normalize the volumetric production rate and provide a fair comparison between our work and previous reports. This standard was further clarified in the Methods section (part of growth of C. ljungdahlii in the biohybrid system):\n“The calculations of volumetric production rate were based on the bioelectrolyte volume in the reservoir. All the results were averaged by at least three independent experiments.”\nBesides, we also provided the mass concentrations to make clearer clarification:\n“The maximum acetate and ethanol titers were constrained to ~42.9 mM (2531.8 mg/L) and ~8.9 mM (409.4 mg/L), respectively (Fig. 1 b-c). In contrast, CO-supplemented fermentation produced ~11.4 mM IPA (681.3 mg/L), representing a 142-times enhancement, which was also accompanied by 1.7 times (73.9 mM, 4359.3 mg/L) increase in acetate and 3.3 times boost (29.3 mM, 1347.8 mg/L) in ethanol production (Figure 1 b-c).”\n4. The duration of the experiments is not mentioned in the discussion and there are no graphs showing concentrations vs time. Perhaps such graphs can be added to the Supplementary Materials.\nResponse: We thank the reviewer’s concern about the concentration trend. Indeed, the concentration change of C2+ oxygenates over four days operation has been shown in Fig. 4c, which can clearly illustrate the products accumulation over time, and related results have been discussed in the main manuscript as well.\n“After accounting the evaporated amount, the average IPA titer reached ~10.8 mM over four days, with a total C oxygenate (EtOH + IPA + Acetate) concentration of ~91.1 mM (Fig. 4c).”\n2+\n5. L132. The term “partial current density” needs to be explained, it is not often used outside of electrochemistry. Also, what other products were formed, H2? Any liquid phase products?\nResponse: Thanks for this question, which is similar to one of the Reviewer 1 questions. Our original goal was to ensure a fair comparison with the prior studies. As shown in Fig. 4e, we hope to emphasize volumetric and electrode area-normalized production rates, respectively, which are two metrics especially relevant for evaluating scale-up potential. Nevertheless, we fully agree that partial current density is not an appropriate metric in this context. Therefore, we directly compare the areal IPA production rate at the unit of mg/cm2/day, in order to avoid any misunderstanding.\nBesides, in the initial version, we mistakenly calculated the “IPA partial current density” in the reference (Science 352.6290 (2016): 1210–1213) by using “electronic energy efficiency” instead of “FE”. All related errors have now been corrected in the revised manuscript and supplementary information. Even so, our performance is still the highest reported in the field, and the correction will not affect the overall conclusions of the study.\n“Reports on iMES for IPA production remain scarce, positioning this field as a largely unexplored area. Here, we present the first demonstration of CO-mediated IPA iMES. Due to the sufficient CO supply and superior reducing power relative to H , our system achieved unprecedented areal production rate (3.23 mg/cm2/day) and volumetric production rates (161.3 mg/L/day) for IPA compared to prior studies (Fig. 5e and Supplementary Table S2).”\nIn all electrochemical tests of Ni SAC, the cathode primarily generated CO with some H2 (Fig. 1e, Supplementary Fig. S2, S4, S5 S6, S21, and S22) and we did not observe any liquid phase electrochemical products like formate above the detection limit. Indeed, Ni SAC has been well explored for CO production without any formate competition due to the favorable CO synthesis pathway (Nature Catalysis 4.12 (2021): 1024-1031.)\n6. L148. Provide the range of current densities (50-500 mA/cm2, based on Figure S2)\nResponse: The range of current densities have been provided and highlighted in the manuscript:\n“For conventional abiotic electrolysis, Ni SAC achieved > 95% of CO FE at industrial current densities (50-500 mA/cm2, Supplementary Fig. S2).”\n7. L149. Perhaps “dramatic” is not the best term.\nResponse: We have revised “dramatic” to a more moderate word (“notable”). The change can still illustrate the strong improvement (~ 25 times enhancement compared to conventional Ag catalyst).\n“In comparison to polycrystalline Ag NPs, Ni SAC exhibited a notable enhancement in CO selectivity and activity in the bioelectrolyte (Fig. 1e and Supplementary Fig. S3). Its CO FE was ~25.3 times that of Ag NPs at -0.85 V vs. RHE and can exceed 92% at higher overpotentials.”\n8. Figure 4a needs to be improved, as mentioned above\nResponse: We thank the reviewer’s suggestion for improving the logical clarity and visual quality of the schematic in Fig. 4a. To balance visual clarity with technical details, we have retained the original illustrative diagram in the main manuscript and added a more detailed process flow diagram in the Supplementary Information.\n9. L476: “anaerobic”\nResponse: Thanks, and we have corrected the typo.\nL481: Specify the number of days and refer to a graph in Supplementary Materials.\nResponse: The operating time has been clarified:\n“After finishing the long-term operation (4 days), all the liquids from the cold trap were collected for further detection.”\nReviewer 3:\nThis manuscript reports a nickel single-atom catalyst (Ni SAC) integrated with Clostridium ljungdahlii for isopropanol (IPA) synthesis from CO₂. The findings that (1) additional CO upregulate IPA production, and (2) Ni SAC shows improved stability in biological buffer, are valuable for advancing the understanding and development of material–microbe hybrid systems. However, this study need to further (1) demonstrate the practical potential of the proposed material–microbe hybrid system; (2) clarify the relationship between IPA synthesis and microbial growth under varying conditions; (3) provide stronger experimental evidence to support the reported hybrid system performance; (4) expand and clarify the experimental methods. Given the current limitations, I would not suggest this manuscript to be accepted by Nature Communication in its present format.\nResponse: We sincerely thank Reviewer 3 for highlighting the significance of our work on CO-mediated isopropanol production and the development of a bioadaptive Ni SAC strategy, which together advances the understanding of material-microbe hybrid systems. Regarding the reviewer’s concerns about insufficient experimental evidence of the integrated system's performance, as well as the relationship between IPA biosynthesis and microbial growth, we have provided point-by-point responses by adding more experiments and analyses. All newly added data and analyses have been clearly highlighted in both the main text and the Supporting Information.\nDetailed Comments:\n1. Since the authors target for large-scale IPA synthesis, please discuss the current industrial process for IPA synthesis. How could your method replace or complement the existing process? You may include considerations of energy consumption and environmental impact.\nResponse: This is a good suggestion. Current industrial IPA production mainly depends on fossil feedstocks and energy-intensive hydrogenation/hydration steps, leading to high carbon emissions. By contrast, syngas fermentation for IPA production has been validated at pilot scale with lower energy demand and net-carbon-negative performance. We propose that, when coupled with renewable electricity, iMES system can utilize distributed and point-source CO2 to provide a cleaner “in-situ” syngas supply. In the main manuscript, we included a brief discussion of the current industrial IPA production and how our proposed iMES process may fit in.\n“Current industrial IPA production predominantly relies on energy-intensive hydration of propylene (two-thirds of supply) or hydrogenation of acetone. Both petro-based routes depend on fossil propylene or H , as well as face challenging separations from IPA/H2O/diisopropylether azeotropes.5”\n“Considering the energy demand and carbon footprint, the iMES process may decouple IPA synthesis from fossil feedstocks by using renewable electricity to generate electron donors. This shifts energy and carbon inputs from fossil feedstocks to electrons and waste CO , avoids 2 steam-reforming emissions, and is operationally attractive for distributed, point-source CO 2 valorization. Notably, fermentation of syngas has already achieved carbon-negative IPA at pilot scale (-1.17 kg CO per kg IPA) when powered by low-carbon energy and waste-gas feeds22, 2 which further highlights the decarbonization potential by interlinking the microbial conversion with renewable syngas supply.”\n2. In the introduction part, it would be better to provide a broader review/perspective of the material-microbe hybrid system, also situate your work more clearly within recent progress in material–microbe hybrid systems, emphasizing its novelty and significance.\nResponse: Per suggestion, we have added the additional discussion as below:\n“Integrated microbial electrochemical systems (iMES) based on gas diffusion electrodes have been shown to enhance kinetic mass transfer during microbial fermentation and potentially overcoming limitations associated with separated abiotic electrolysis and fermenentation.20,21 Moreover, iMES systems enable microbials to convert simple electrode-derived intermediates (e.g. H , CO, and formate) into a wide range of high-value products such as alcohols, carboxylic 2 acids and esters through their native or engineered metabolic pathways22, which are nearly impossible on traditional inorganic catalyst surfaces. This material-microbe synergy thus can expand the accessible electrochemical product space beyond the limits of conventional electrocatalysis.”\n“In this study, we designed a single-site Ni catalyst that can exhibit high tolerance to bioelectrolyte components (i.e. YT medium for microbial growth) and enable selective CO production, thereby significantly enhancing the CO supply rate in biological electrolytes. The synthesized Ni single-atom catalyst (Ni SAC) demonstrated remarkable resistance to complex organic components in the electrolyte. maintaining CO FE nearly identical to that in ideal electrolytes while allowing tunable syngas ratios. When we combined this catalyst with a genetically engineered IPA-producing C. Ljungdahlii biosynthetic platform, we achieved the first-ever CO-mediated CO -to-IPA microbial electrochemical synthesis with a record rate of 2 161.3 mg/L/day. Additionally, we employed multiple in-situ characterization techniques to deeply probe the electrode-electrolyte interfacial properties in the bioelectrolyte and elucidated the deactivation mechanism of conventional electrocatalysts. We discovered that on continuous polycrystalline metal surfaces (e.g., Ag), extensive coverage of organics from the microbial growth medium disturbed the electrode-electrolyte interface and competitively occupied reactive sites, deactivating CO adsorption and conversion. We hope this study will provide 2 new insights for future optimization of interfacial properties and the expansion of material–microbial hybrid systems.”\n3. The claim that the additional of CO would promote the IPA production is interesting. However, there are several questions need to be addressed. (1) In Figure 1 / Figure S1: IPA and acetate concentrations plateau after day 3 under CO/H₂/CO₂, and the OD₆₀₀ also remains unchanged—please clarify the correlation and provide cell viability data; (2) The OD₆₀₀ trend appears unusual; for CO₂/H₂, why is there a drop on day 2? Also, please state the number of replicates in the figure captions; (3) Compare the CO₂/H₂ condition with the CO/H₂/CO₂ condition, the concentrations of IPA, acetate, and EtOH are significantly lower—what are the additional products under CO₂/H₂ condition then since the microbial activity is not significantly weaker from OD600 results? (4) Please explain the rationale for pressurizing the culture tube to 10 psi (Method session, Line 466). Could this low pressure affect microbial growth or shift products toward gaseous species rather than IPA/acetate/EtOH?\nResponse: Regarding the CO effect for promoting IPA production, we prepare the following one-by-one responses:\n(1) We acknowledge that cell viability data may be a direct indicator. Unfortunately, we do not have cell viability data as this would have required regular plating and assessments of CFU/ml of the culture, which could have been detrimental to microbial growth in iMES system. The plateauing that is observed after day 3 is due to the cells ending their natural growth phase which is why OD600 and product formation stop increasing. We chose to continue to monitor growth and product profiles up until day 7 to fully capture any potential residual product formation. However, as the reviewer has noted, most growth and product formation ended on day 3/4.\n(2) As we described in the method section, before performing the comparison of gas fermentation, the C. ljungdahlii strain was cultured in YT medium with a syngas mix of 50%/20%/30% CO/H2/CO2. When the strain was transferred to H2/CO2 mixture, for acetogen growth, it’s often limited or not as robust as growth on CO. We suspect that growth may have slowed down due to adaptation of the cells to a more limited energy source (H2). Drops or slowdowns in growth have been observed in other publications (Frontiers in Microbiology 11 (2020): 724.; Applied and Environmental Microbiology 86.14 (2020): e00730-20.).\n(3) There are no additional products during CO2/H2 growth. Although growth difference was not dramatic, the lack of CO did reflect worse growth and the lack of products. CO can 2 serve as a carbon and energy source, helping to drive product formation as well as growth. With only H2 as the energy source in the CO2/H2 growth, oxidation of H2 for energy is split for carbon assimilation and ATP/NAD(P)H generation. Therefore, we observed much less products when no CO was present. This phenomenon was well characterized in acetogens (Frontiers in microbiology 11 (2020): 416.). We also provided a more detailed discussion with in the main manuscript:\n“This CO-dependent metabolic shift arises from its dual role in precursor and energy metabolism (Fig. 1a). Beyond serving as a carbon source for acetyl-CoA formation, CO oxidation via CO dehydrogenase (CODH) generates reduced ferredoxin (Fd ). This red electron carrier drives the Rnf complex to establish a transmembrane H+ gradient, thereby generating ATP to sustain growth and NADH to drive IPA biosynthesis.24 H metabolism of C. ljungdahlii is less efficient, although it can also supply reducing equivalents via hydrogenases.25 In H /CO fermentation, H must simultaneously support both branches of 2 2 2 the Wood-Ljungdahl pathway (WLP). In the methyl branch, CO is assimilated via the 2 formate dehydrogenase (Fdh) and H is used by a bifurcating hydrogenase (in a complex 2 with the Fdh) to generate Fd for formate formation. While in the carbonyl branch, Fd red red is required again for CO → CO reduction by CODH. In addition, Fdred is required for 2 ATP generation by the Rnf complex/ATPase, which is essential for autotrophic growth by these organisms. As a result, when C. ljungdahlii grows on H /CO , the available H pool 2 2 2 is divided between carbon assimilation and energy conservation, leaving far fewer reducing equivalents available for product (e.g., IPA) formation. In contrast, CO oxidation directly provides Fdred via CODH, which can be efficiently utilized by the Rnf complex and other enzymes (e.g., Nfn) to generate NAD(P)H. Thus, in CO/CO /H fermentation, the pool of 2 2 Fdred is significantly larger, supporting both robust autotrophic growth and enhanced IPA production.26 Consequently, microbial electrochemical systems targeting highly reduced compounds like IPA must prioritize sustained CO generation for C. ljungdahlii, ensuring both carbon flux and energy conservation requirements are met.”\n(4) We pressurized the culture vessels to 10 psi with the input gas mixture primarily to increase gas solubility and ensure a non-limiting supply of CO2/H2/CO. Besides, a pressure of 10 PSI was chosen to reduce potential CO toxicity at high pressures. A pressure of 10PSI is routinely used in our experiments with no effect on growth. We also do not believe this pressure would be insufficient as gas pressure was routinely replenished to 10 PSI to prevent a lack of substrate for the bacterium. This operation is commonly reported in microbial syngas fermentation works (Applied Microbiology and Biotechnology 108.1 (2024): 540.).\n4. The stability of Ni SACs in biological buffer is intriguing. However, it would be better to address the following points. (1) Lines 145–181, please directly compare the electrocatalytic activity of Ni SAC under abiotic and biotic buffer conditions; (2) Line 170, please clarify the full composition of the 100 mM Na⁺ electrolyte. Beyond cation effects, how might buffering alter catalytic activity? (3) I am curious if this Ni SACs catalyst would be applicable in other biological buffers, therefore can be expanded into other systems.\nResponse: We thank the reviewer for concern about the Ni SAC stability. For all the detailed comments, please check the following one-by-one responses:\n(1) Before comparing the performance of Ni SAC under abiotic and biotic buffer conditions, it is important to note that, unless explicitly stated, all performance comparisons between the ideal and bioelectrolyte in this work were conducted without the use of PBS as a pH buffer. We measured the cation concentration, pH, and conductivity of the bioelectrolyte (YT medium), and determined that using 100 mM NaCl as the comparison electrolyte was most appropriate, as it closely matched these parameters while containing no organic components (Supplementary Table S1). The main goal of adding PBS buffer into the 2 bioelectrolyte is to guarantee pH stability for microbials during long-term iMES operation. Although comparing the abiotic test with performance in bioelectrolyte with pH buffer may not be appropriate (different conductivity, different cation concentration, w/ or w/o pH buffer), we also attached the results below for reviewer’s reference.\n(2) The ideal electrolyte composition has been clarified in the Method section:\n“Unless otherwise specified, bioelectrolyte refers to the YT medium and Ideal electrolyte only contains 100 mM NaCl without any organics.”\nIn response to question 1 of comment 4, and as requested by the reviewer, we compared the performance of the ideal electrolyte and the pH-buffered bioelectrolyte for reference. After adding 100 mM PBS into bioelectrolyte (YT medium), the increased total ionic strength led to a higher current density at the same potential compared to that in 100 mM NaCl. Meanwhile, compared to the unbuffered bioelectrolyte (Fig. 1e), the CO FE was significantly improved and became nearly identical to that in 100 mM NaCl. The slightly lower CO FE observed in PBS buffered bioelectrolyte was attributed to the enrichment of organics from the bioelectrolyte within the electrical double layer, which weakened the local electric field strength at the electrode surface. A detailed discussion on this phenomenon has been provided in the main manuscript under the section “Adsorption-Induced Deactivation in Bioelectrolyte.”\nIn fact, if only considering the effect of adding PBS buffer, its influence on CO2RR performance should be positive. In the absence of a pH buffer, the near-electrode pH can increase greatly under high current densities, leading to the formation of bicarbonate/carbonate through reactions with CO2. This in turn reduces CO availability to 2 some extent, even though the operating current density in this work is lower than that typically used in abiotic electrolysis. After adding 100 mM PBS, the cation concentration was further increased, which can help to stabilize the CO2RR intermediates (Science 372.6546 (2021): 1074-1078.). And the buffering capacity of PBS can mitigate bicarbonate/carbonate formation at deeper overpotentials, thereby maintaining near-neutral pH for microbial activity while also preserving CO accessibility at the electrode surface 2 (Advanced Materials 35.13 (2023): 2209590.). However, when we compare the performance in pH buffered bioelectrolyte and in ideal electrolyte, such promotion effects 2 will be mixed with the inhibition effect of organics and quantifying their contribution ratio is very hard, complexing the mechanistic interpretation. This is also why, in our response to question 1 of comment 4, we argued that a direct comparison between these two performances is not appropriate.\n(3) PBS buffer is one of the most commonly used pH buffers in microbial electrolysis. Since we have demonstrated high bioadaptivity by DFT, in-situ characterizations and electrochemical performance, we are confident that Ni SAC can be used in other systems with different biological buffers or mediums. For example, bicarbonate is another common pH buffer in microbial electrosynthesis, and it has been well explored and used in abiotic CO2RR system, with even better CO FE and current density than PBS buffer (Small 19.35 (2023): 2301469; The Journal of Physical Chemistry C 122.7 (2018): 3719-3726. Journal of Materials Chemistry A 12.14 (2024): 8331-8339.). However, we are cautious about directly transferring the Ni SAC findings to other systems. Variables beyond pH or buffer type (e.g., electrolyte hydrodynamics or the ability of the microorganisms to accommodate CO concentrations) can also influence overall performance. A case-by-case assessment is therefore required, but such experiments and analysis are beyond the scope of this work.\n5. Coupling material with microbes for organics synthesis is particularly interesting. However, the experimental data of this part needs to strengthened. (1) Please provide performance comparisons under multiple control conditions (e.g., bare electrode + bio, bare electrode + Ni SAC); (2) Please clearly describe the operational medium. If the microbial inoculation medium (e.g., YTF) is used, would this organic components affect/drive the product formation? Also, it is inappropriate to say it is autotrophic growth with all these organic supplies in the YTF medium (Line 461); (3) When comparing with literature, it would be more appropriate to focus on works targeting IPA as the primary product. Comparisons with studies aiming for different (even higher-value products) are misleading; (4) Please provide some discussions on potential causes of limited overall efficiency; (5) Please address the possibility of biofilm formation at high OD in later days and its impact on Ni SAC activity; (6) Would the Ni SACs be toxic to the microbes? Viability test would be helpful; (7) A photograph or schematic of the experimental setup would be helpful for clarity.\nResponse: We appreciate the reviewer’s suggestion on strengthening the experimental data. For all the detailed comments, please check the following one-by-one responses:\n(1) We thank the reviewer’s suggestion to provide the control group performance. However, due to the termination of the collaborative project, it is currently difficult for us to restart the operation of the integrated system and get the performance of “bare electrode + bio” group. However, the CO2RR performance of bare electrode (gas diffusion electrode) has been previously reported. In better abiotic electrolyte (i.e. 1M KHCO3 solution), bare electrode can only exhibit <5 mA/cm2 without any CO formation at -0.75 V vs. RHE (ACS Energy Letters 6.1 (2021): 33-40.). Under more harsh conditions (i.e. bioelectrolyte), we expect the current density will be further decreased and no CO generation as well. As we showed in Fig. 1b, without any CO supply, IPA cannot be produced. Thus, we expect that the group of “bare electrode+bio” cannot perform good growth and cannot produce any IPA.\nAlthough it’s hard for us to restart the iMES system, we still tested the “Ni SAC + bare electrode” group in the bioelectrolyte with 100 mM PBS at same potential (-0.75 V vs. RHE) for 100 h. As shown below, only CO and H2 were detected, and no other gaseous/liquid products were generated.\n(2) We appreciated the reviewer’s concern of true autotrophy in YT medium. In related studies, C. ljungdahlii growth in the presence of yeast extract and CO2 but without H2 showed no growth (Bioresource Technology Reports 19 (2022): 101156.). It should be also clarified that only YT medium without fructose was used for gas fermentation comparison (Fig. 1b-c and Supplementary Fig. S1) and iMES process (Fig. 4b-d). Therefore, in our conditions we do not believe that the medium (YT) was a significant driver of growth or product formation. Any growth of C. ljungdahlii in our system can be attributed to utilization of gaseous carbon, so it is appropriate to call this growth “autotrophic”.\n(3) Since the deactivation issue of CO2RR has not been resolved before our work, CO-mediated IPA synthesis based on engineered C. ljungdahlii strains has not been reported. For other microbial species, constructing the IPA pathway is also challenging. Therefore, IPA production within iMES systems remained relatively limited. We identified convincing studies published in high-impact journals for comparison. Although those works did not specifically state IPA as their primary target (as iMES processes will inherently produce numerous byproducts, consistent with our own reported data), they all included detailed discussions of IPA formation performance. Thus, we believe selecting the three referenced studies for comparison in our manuscript is appropriate.\nBesides, in the initial version, we mistakenly calculated the “IPA partial current density” in the reference (Science 352.6290 (2016): 1210–1213) by using “electronic energy efficiency” instead of “FE”. All related errors have now been corrected in the revised manuscript and supplementary information. Even so, our performance is still the highest reported in the field, and the correction will not affect the overall conclusions of the study.\n“Reports on iMES for IPA production remain scarce, positioning this field as a largely unexplored area. Here, we present the first demonstration of CO-mediated IPA iMES. Due to the sufficient CO supply and superior reducing power relative to H , our system achieved unprecedented areal production rate (3.23 mg/cm2/day) and volumetric production rates (161.3 mg/L/day) for IPA compared to prior studies (Fig. 5e and Supplementary Table S2).”\nFor the relatively low “FE” (which has been modified to “electron recovery efficiency” in the revised version), we would attribute it to the mismatch between syngas production rate and microbial uptake rate. Although now it’s hard for us to perform the iMES again, we did a simple calculation to illustration of such rate mismatch. Our gas fermentation system was equipped with a pressure gauge to confirm the headspace pressure of culture bottle could be repressured to 10 psi (Method section). We found that each 24 h fermentation operation resulted in a headspace pressure decrease of >7 psi, indicating that most CO/H2/CO2 were consumed during the 24 h fermentation period, although these results were not recorded in detail as they were not critical to the main scope of the study. Here, we assume that all CO/H2 (7 psi) could be consumed in 24 h, in order to overestimate CO/H2 consumption rate. Based on this assumption, for a 50 mL liquid volume with a maximum OD600 of 1.54 (Method and Supplementary Fig. S1), we can estimate that the CO/H2 consumption rate of our engineered C. ljungdahlii is:\npsi 200 mL\n7 psi∗6984.7 ∗\nPa mL\n1000000\nm3\nSyngas consumption rate in fermenter =\nJ\n8.314 ∗298.15K∗24h\nmol∗K\n= 1.6∗10−4 mol/h\nSo, for the iMES process, the total liquid volume is 20 mL and the maximum OD600 is 1.36, and, if we don’t consider the boosting effect of gas-diffusion electrode, we can approximately estimate that the CO/H2 consumption rate of our engineered C. ljungdahlii in iMES system is about:\n20mL 1.36\nSyngas consumption rate = 1.6∗10−4 mol/h∗ ∗ = 0.6∗10−4 mol/h\n50mL 1.54\nWe can also estimate the CO/H2 (both of them are 2e- products) generation rate based on the average current density (~11.39 mA/cm2, Figure 4b) during the integrated system operation:\nmA A\n11.39 ∗ 1cm2∗10−3 ∗3600s\ncm2 mA\nSyngas generation rate = = 2.1∗10−4mol/h\nC\n2∗96485.33\nmol\nIt is evident that the production rate of CO/H2 is far greater than their consumption rate. Even when considering the enhancement of microbial gas utilization efficiency by gas-diffusion electrodes (e.g. Biotechnology and bioengineering 120.6 (2023): 1465-1477.; Environmental science and ecotechnology 16 (2023): 100261.), there is still a big difference between syngas generation and consumption. The related discussions have been updated in supplementary information (Supplementary Note S1).\nIndeed, the mismatch between electron donor supply and microbial consumption was well documented in microbial-electrochemical hybrid systems (Environmental Science and Ecotechnology 19 (2024): 100324; Nature Catalysis 2.5 (2019): 407-414.). The electrochemical process typically generates electron donors in excess of microbial demand. On the one hand, in lab-scale studies, it is necessary to ensure a sufficient supply of electron donors for microbial conversion, which is why relatively high current densities or large electrode areas are often employed. On the other hand, without changing gas transfer characteristics, most microorganisms inherently exhibit relatively low gas utilization efficiency. In the initial version, based on the innovative work by Rodrigues et al. (Nature Catalysis 2.5 (2019): 407-414.), we briefly highlighted the prospect of promoting the mass transfer of electron donors such as CO in order to further enhance the production rate of high-value products and improve electron efficiency, although this is not the main focus of the present study.\n(4) As previous work reported, although C. ljungdahlii strain can directly take some electrons from the electrode, CO and H2 serve as the primary electron donors and dominate the energy flow from electrochemical system to microbials. (Green Chemistry 25.11 (2023): 4375-4386.; Applied and environmental microbiology 79.4 (2013): 1102-1109.). In our group’s previous research, we have already demonstrated that the C. ljungdahlii strain cannot form thick biofilm on carbon-based gas-diffusion electrodes in flow electrolyzers as well. This is likely because electrolyte flow and gas purging would increase the difficulty of strain attachment to the electrode surface. We can further compare whether inoculating the C. ljungdahlii strain, under the same electrolyte and operating conditions, has no significant effect on the operating current density over a four-day period (Fig. 4b and Supplementary Fig. S22). Thus, we can conclude that C. ljungdahlii strain should be difficult to form substantial biofilm on the electrode surface in the flowed iMES system and has little impact on the overall electrochemical performance.\n(5) Similarly, since the cell viability test will be detrimental to microbial growth in iMES system, it’s hard for us to provide this data. However, for the Ni toxicity, we acknowledge that Ni2+ ion is toxic for microbials could be accumulated in the bioelectrolyte if the leaking issue happened. Different from free Ni ions, Ni sites on Ni SAC were stably coordinated by four nitrogen atoms and anchored on the carbon substrate. To provide more convincing evidence, we used ICP-OES to detect the Ni concentration in the bioelectrolyte (with 100 mM PBS) after 100 h operation at -0.75 V vs. RHE. As shown below, even though we performed the long-time test, the accumulated Ni ion concentration in the bioelectrolyte was still ignorable, which is consistent with previous reports about Ni SAC for CO2RR (Journal of Materials Chemistry A 12.14 (2024): 8331-8339.). Indeed, compared to strong basic electrolyte used for conventional CO2 electrolysis, near pH-neutral bioelectrolyte is much less corrosive and therefore can maintain good stability.\n(6) To balance visual clarity with technical details, we have retained the original illustrative diagram in the main manuscript and added a more detailed process flow diagram in the Supplementary Information.\n6. Wikipedia1 says that C. ljungdahlii can produce g/L ethanol, how would this correlate to the mg/L IPA production in this work?\nResponse: The study cited in Wikipedia (Applied biochemistry and biotechnology 39.1 (1993): 559-571.) achieved an ethanol titer of up to 1.8 g/L. To fairly compare our system with previous works that mainly produced acetate/ethanol, both energy and carbon distribution perspectives should be considered. IPA production is much more challenging. IPA is an 18-electron CO2RR product and three-carbon product, compared to 12 electrons and required for two-carbon ethanol. As shown in Fig. 1a, C. ljungdahlii requires more ATP and NADH to drive the conversion of CO into IPA, indicating a higher energetic barrier.\nImportantly, since our engineered pathway can deliver more reducing equivalents toward IPA biosynthesis, it is reasonable that ethanol titers did not reach previously reported maximum results. Nevertheless, our system still achieved acetate and ethanol titers of ~3.3 g/L (16.5 mg/cm2/day, 0.825 g/L/day) and ~1.1 g/L (5.5 mg/cm2/day, 0.275 g/L/day), respectively. These values are comparable to, or even exceeding, some results in literature (Nature Catalysis 2.5 (2019): 407-414.; Environmental science and ecotechnology 16 (2023): 100261.; Applied biochemistry and biotechnology 39.1 (1993): 559-571.). This contrast highlights the energetic and production advantages of our proposed CO-mediated iMES system for high-value chemical biosynthesis.\nReviewer #1 (Remarks to the Author):\nThis is a revised manuscript and the authors have carefully answered all the questions raised by reviewers, so this manuscript can be accepted for publication at present form.\nResponse: We are grateful to Reviewer #1 for the careful evaluation and insightful comments in earlier rounds, which have greatly improved the manuscript. We are pleased that the current revision is satisfactory for publication.\nReviewer #2 (Remarks to the Author):\nThe manuscript can be accepted for publication at present form.\nResponse: We sincerely thank Reviewer #2 for the constructive feedback provided throughout the review process. We are pleased that the revisions have satisfactorily addressed all concerns and appreciate the recommendation for publication.\nReviewer #3 (Remarks to the Author):\nThe authors provided detailed responses but bypassed the critical experimental evidence of cell viability and performance in control groups, which are necessary for evaluating a material-microbe hybrid system for chemical synthesis or energy production1. The performance data in the provided manuscript is only necessary but not sufficient to support that functionality of the proposed hybrid system; while the cell viability tests and all the control groups are crucial to eliminate any potential false positive results. In details,\nResponse: We understand Reviewer #3’s perspective in requesting cell viability data and consider this suggestion very carefully. Most of the reviewer’s concerns are related to cell viability, so we performed additional experiments accordingly. The lab results, together with published literature review and detailed analysis, do support that the factors that may lead to false positives can be ruled out. We provided detailed discussions below. Please check the one-by-one responses as shown below.\n1. As the authors admitted, the performance of isopropanol (IPA) production in the control groups is crucial (Response to Reviewer 3’s Comment 5.1). When observing the performance of the microbe–nickel catalyst system for IPA production, it is critical to eliminate other potential contributors to the observed performance. Although the authors provided a previous publication showing the CO₂ reduction performance of a bare electrode is not as promising as one decorated with a nickel catalyst, we cannot conclude if the bare electrode + microbe system would perform much worse than the proposed microbe–nickel catalyst system as there are potential synergies at the electrochemically active material– microbe interfaces for chemical synthesis in the previously reported works. Moreover, since cell viability is unknown, it is uncertain whether the performance stems from microbial metabolites or extracellular components rather than from living and active microbial cells. Experimental groups in the exact same setup with precise control of variables are critical for supporting the observed performance in the designed system.\nResponse: We understand the reviewer’s concern regarding the performance of the bare electrode + cells group may outperform the catalyst decorated electrode + cells, though as the reviewer indicated that previous studies and general understanding indicated that this is unlikely the case. To make sure we don’t rule out any possibilities as the reviewer insisted, we performed additional experiments of CO2 electrolysis in YT medium buffered with 100 mM PBS using a bare gas-diffusion electrode without any catalysts loading. The relevant data are shown below.\nIn the absence of the Ni SAC, the current densities at same potentials were remarkably lower than those of the Ni SAC electrode. Due to the low conductivity of microbial growth medium, the total current density was also lower than previous report (ACS Energy Letters 6.1 (2021): 33-40). More importantly, no CO signals above the GC detection limit were detected at all applied potentials, indicating that the bare gas-diffusion electrode that we used in this work has no obvious CO2RR ability.\nFirst, as shown in the above figure, the bare electrode yielded a much lower current density, indicating fewer available electrons for microbial growth. For example, at -0.75 V vs. RHE the current density is only 1.58 mA/cm2. Based on Supplementary Note S1, this corresponds to an electron production rate of only ∼0.29×10-4 mol/h. Even with a gas-diffusion layer, this low supply cannot meet the ∼0.6×10-4 mol/h required for robust growth of C. ljungdahlii. Moreover, since we have shown that no IPA is produced without CO feeding (Figure 1b), a bare gas-diffusion electrode alone cannot generate CO and thus cannot support the IPA pathway. Therefore, the combination of insufficient electron supply and the lack of CO confirms that the bare electrode + microbe system cannot outperform the Ni SAC + microbe system. This inherent limitation of electrochemical rate is independent of any potential synergies at the electrochemically active material-microbe interface. In other words, even if such synergies exist, they cannot enable the bare electrode + microbe setup to overcome these barriers to achieve superior growth or IPA production.\nMoreover, we think the possibility of false positives resulting from microbial metabolites or extracellular components should be negligible. For the engineered C. ljungdahlii strain, the principal metabolites in addition to IPA are acetate and ethanol. We are not aware of any abiotic electrocatalyst that can convert ethanol or acetate into IPA. In abiotic CO2 reduction pathways, acetate and ethanol are terminal products that would be desorbed from the catalyst surface (Nature Communications 13.1 (2022): 5482.) and are mechanistically different from the IPA path (indeed, IPA is an extremely rare C3 product in abiotic electrolysis. Ref: J. Am. Chem. Soc. 2024, 146, 46, 31788–31798; Nature catalysis 6.4 (2023): 319-331.). Regarding other extracellular components, we provided more detailed discussions in response to Minor Question 2. We acknowledge that some macromolecules like amino acids, vitamins, purines/pyrimidines and fragments of nucleotides/polypeptides may reside near the electrode surface (e.g., within the electrical double layer). However, our in-situ XAS and in-situ Raman measurements, together with DFT calculations, showed that Ni SAC lacks the ability to polarize or chemisorb such macromolecular species. As a result, the Ni SAC can maintain its CO2RR activity but cannot convert these species. To our best knowledge, there are no reports of Ni SAC reducing extracellular biomolecules to IPA as well. Our control experiments confirmed that CO was the only carbon product at negative potential by using Ni SAC in YT medium (Fig. 1e), and the C. ljungdahlii strain cannot grow in YT medium alone in the absence of CO and H (please see the detailed discussions and results in later response to Comment 2). These results demonstrated that the use of YT medium would not introduce false positives.\n2. The cell viability tests present the functionality of the microbes under testing conditions, whether in different growth conditions (Figure S1) nor electrochemical material-microbial conditions (Figure 4). The optical density (OD) tests themselves only represent the turbidity of the microbial culture (including living cells and all other large particles/aggregates), not the concentration of living cells nor their activity. Particularly, the OD at 600 nm shows large deviations when reaching a high value (often > 1 in the work, Figure S1 and Figure 4b), which introduces further errors in estimating the cell densities.\nResponse: Although OD is an optical measurement, it remains one of the most common 600 metrics in gas fermentation and microbial electrosynthesis studies. During the logarithmic growth phase, an increase in OD directly reflects effective microbial growth. Besides, several 600 pioneer works of microbial synthesis cited by the reviewer (Science 352.6290 (2016): 1210-1213; Nature Catalysis 5.7 (2022): 633-641.) only used OD600 (much bigger than 1) or even products generation rate/electrochemical parameters to directly reflect the viability. That said, we agree with the reviewer that during the stationary phase or at high cell densities, the correlation between OD and actual cell activity may have higher deviation.\n“For the cell viability assays, C. ljungdahlii was grown in 250-mL Duran pressure plus bottles in a syngas mix of 50%/20%/30% CO/H /CO as previously described. Colony forming units 2 2 (CFU) per mL was assessed by taking 1 mL of culture from the bottles on selected days (day 3, 5 and 7), serially diluting the culture to provide sufficient resolution to determine colonies and plated on YT + 5g/L fructose medium. Colonies were counted after 5 days of growth on plates.”\nAs shown in the Supplementary Fig. S2, the engineered C. ljungdahlii strain was unable to grow in the absence of CO/H . The non-zero viability on day 0 could be attributed to living 2 cells from the inoculum, and the cell viability quickly declined to 0 before the second sampling. These results demonstrated that the growth of the engineered C. ljungdahlii strain should be autotrophic, directly addressing a key question raised in the first round of review (Reviewer 3 Comment 5(2)). It also proved that the organic components in YT medium alone should be insufficient to support IPA, acetate, or ethanol production without CO/H . In contrast, under 2 CO/H /CO feeding, cell viability increased significantly during the logarithmic growth phase 2 2 and then gradually declined from day 3 to day 5, corresponding to the plateau observed in OD 600 and metabolite production. These findings also confirmed that cell viability results are fully consistent with the other experimental observations. All the control groups did not have live cells on last sampling day (day 7) and thus the results were not shown in the figure below. Relative discussions have been updated in the main manuscript and supplementary information:\nSupplementary Fig. S2. Cell viability during gas fermentation process. For the groups of CO/H2/CO2 and Ni SAC + CO/H2/CO2, the feeding gas ratio is 50%/20%/30%. For the group of Ni SAC + CO/H2/CO2, Ni SAC electrodes were autoclaved and immersed into YT medium to check its effect on the cell viability. Clearly, the engineered C. Ljungdahlii cannot grow without CO/H2/CO2, and the addition of Ni SAC electrode has no significant effect on the cell viability. All the control groups did not have live cells on last sampling day (day 7) and thus the results were not shown in the figure.\n“The cell viability results showed that the engineered C. Ljungdahlii strain cannot grow without CO/H /CO feeding (Supplementary Fig. S2), indicating that the organic components in YT 2 2 medium alone were not able to support IPA, acetate, or ethanol production. But the introduction of CO/H /CO promoted viability during the logarithmic phase in the fermentation experiments, 2 2 which gradually decreased from day 3 to day 5, corresponding to the plateau observed in both metabolite concentrations and OD .” The response letter mentioned two major challenges 600 regarding further experimental results:\n1. The authors claimed that the termination of the collaborative project prevents them from conducting further experiments related to material-microbial hybrid system (Response to Reviewer 3’s Comment 5.1 and 5.3). However, for any unpublished work like this manuscript, it is common to request further experiments to support the original claims or to clarify ambiguities. Even for published papers, researchers have a responsibility to be able to repeat their published results and to provide additional experimental support when questioned by the public. If the collaborators are listed as co-authors, it is difficult to understand why further experiments cannot be conducted.\nResponse: We fully agree with Reviewer #3’s general principle that authors should provide data to support reasonable requests and ensure the reproducibility of the findings. We have indeed done our best to accommodate these comments. We have added new experiments and results in this newest version as suggested by the reviewer to strengthen the manuscript.\nIf reviewer is aware of the current situation in the U.S., he/she may be a little empathetic and understanding – funding was cut abruptly, which resulted in the project to end, key NREL collaborator to leave, and student lost access to NREL facilities. Re-gaining student access is difficult with the justification of small additional experiments, and even being considered, it takes several months to review. This is beyond our control, and we hope reviewer understands this unusual and very tough situation for us, and to be honest, many scientists in the U.S. right now. We are not trying to avoid repeating experiments, in fact, we wanted to continue the research.\nBut more importantly, we have worked closely with the remaining NREL staff and added experiments and generated new data that we believe sufficiently addresses the reviewer’s concerns. Given that the other two reviewers have already recommended publication, we hope this revised version - with the additional results and expanded discussion - meets Reviewer #3’s expectations.\nWe have repeated the gas-fermentation experiments and assessed cell viability tests. Please see the details in the responses to Comment 1-2 and Major challenge 2. Combining cell viability results with fermentation results, iMES performance, ICP-OES and other experimental results, we believe all the reviewer’s questions like stability, cell viability and the effects of Ni SAC on microbial activity have been addressed.\n2. The authors claimed that the cell viability would ‘be detrimental for the microbial growth’ in their integrated microbial electrochemical systems (iMES) involved experiments (Response to Reviewer 3’s Comment 3.1 and 5.5). However, (1) the cell viability tests have been demonstrated as feasible and noninvasive in all kinds of electrochemical/photoelectrochemical material-microbial hybrid systems for chemical synthesis2-4 or energy production5,6 in both steady and flow systems. These cell viability tests can be straightforwardly test through standard plate counting on agar plates (a common and basic technique in biological studies) or live/dead cell viability assays (e.g., using the commercially available Syto-9 dye) on sampled microbial cultures; (2) Especially, the authors claimed that biofilm is not an issue in their system (Response to Reviewer 3’s Comment 5.4), so it should not be difficult to obtain planktonic microbial cells from the electrolyte for various microbial characterizations. It is difficult to envision how the sampling process would be detrimental to microbial growth, especially since sampling is already conducted for determining reaction products (Method - Growth of C. ljungdahlii in the biohybrid system).\nResponse: Thanks for providing relevant literature on cell viability testing in microbial electrochemical systems. We agree with the reviewer’s assessment that plate counting is a feasible method for evaluating cell viability. However, after carefully reading the references mentioned by the reviewer, we are afraid that some of these studies may not actually match the point the reviewer is trying to make, though keywords seem to match.\nAmong those five works cited by reviewer 3, only Ref. 4 (Science 351.6268 (2016): 74-77.) and Ref. 5 (Science 373.6561 (2021): 1336-1340.) performed direct viability test by plate counting or live/dead cell dyeing to illustrate the materials effect on the cell viability in the microbial electrochemical system. For other cited works:\na) Ref. 2 (Science 352.6290 (2016): 1210-1213.) is the leading work in the field of microbial electrosynthesis. However, the cell viability tests were not performed for the iMES system. In the main manuscript, OD was the major indicator. Indeed, the author clearly claimed 600 in the SI that cell viability tests (spot assays) were used to show the microbial response to different conditions (ROS, metabolite concentration, ion concentration, etc.), instead of the metabolic activity or viability during the iMES process as the reviewer suggested.\nb) Ref. 3 (Nature Catalysis 5.7 (2022): 633-641) is an excellent work about photocatalytic microbial CO2 conversion. The author only used the products generation rates to illustrate the microbial viability, instead of the plate counting numbers (CFU/mL) or live/dead cell dyeing. They also demonstrated the catalyst safety for microbials by immersing the catalyst sheet into the microbial growth solution without illumination, which inspired us to do the indirect cell viability test and check the effects of Ni SAC itself.\nc) Similarly, Ref. 6 (Nature Communications 13.1 (2022): 4369.) only used the electrochemical response (voltage/current change), instead of plate counting results (CFU/mL) or live/dead cell dyeing to show the cell viability was maintained.\nRegarding the potential impact of the Ni SAC on cell viability, we conducted indirect cell viability test following the methods cited in the reviewer’s referenced literature (Ref. 3: Nature Catalysis 5.7 (2022): 633-641.). Specifically, Ni SAC electrodes were fabricated by the same process described in the Methode section (Electrode Fabrication) and then were totally immersed into microbial growth medium (YT medium) with C. ljungdahlii inoculated and CO/H /CO feeding. Other operational parameters were exactly the same as the fermentation 2 2 and cell viability tests without Ni SAC electrode added. Based on the cell viability results of fermentation with and without Ni SAC electrode, we can conclude that Ni SAC electrode (including gas-diffusion layer, catalyst and ionomer binder) should not have a significant effect on the cell viability (As shown in below figure).\nIn response to the first-round review request to verify that Ni SAC is non-toxic to microbes, we conducted the indirect cell viability test as described above. Together with the ICP-OES data (Supplementary Fig. S26) added in the previous revision, these results confirm no significant toxicity from Ni SAC.\nRelative discussions and results have been updated in the main manuscript and supplementary information:\n“The introduction of Ni SAC electrode into hybrid system will not have a significant effect on the cell viability of C. ljungdahlii strain (Supplementary Fig. S2). In addition, the Ni SAC electrode exhibited stable current density and a consistent CO/H ratio for over 100 hours in 2 the bioelectrolyte containing 100 mM PBS (Supplementary Fig. S23). After 100 h operation, post-characterizations (STEM, XPS and ICP, Supplementary Fig. S24-26) demonstrated that the overall structure of the Ni SAC remained stable, and no apparent Ni2+ ion leaching was detected in the electrolyte, indicating the high biocompatibility and stability of Ni SAC.”\nSupplementary Fig. S2. Cell viability during gas fermentation process. For the groups of CO/H2/CO2 and Ni SAC + CO/H2/CO2, the feeding gas ratio is 50%/20%/30%. For the group of Ni SAC + CO/H2/CO2, Ni SAC electrodes were autoclaved and immersed into YT medium to check its effect on the cell viability. Clearly, the engineered C. Ljungdahlii cannot grow without CO/H2/CO2, and the addition of Ni SAC electrode has no significant effect on the cell viability. All the control groups did not have live cells on last sampling day (day 7) and thus the results were not shown in the figure.\nSupplementary Fig. S26. ICP-OES standards and results of bioelectrolyte after long-term operation. The Ni ion concentration after 100 h operation is ignorable and below detection limit.\nOther than that, there are also some minor questions regarding the response that will be briefly mentioned here:\n1. Response to Reviewer 3’s Comment 3.4: During microbial inoculation, is the inner pressure of the microbial vessels ‘10 psi’ or ‘14.7 psi + an additional 10 psi’? Please clarify. Otherwise, 10 psi does not seem sufficient to aid higher gas solubility.\nResponse: Indeed, we pressured our fermentation bottles to “14.7 psi + an additional 10 psi” for the sufficient gas supply. The descriptions in the Method section have been clarified:\n“Frozen stocks of the IPA producing strain of C. ljungdahlii were inoculated into 10 mL of YTF containing fructose in a 15 mL falcon tube. Growth overnight was at 37 °C inside a COY (Grass Lake, MI) anaerobic growth chamber, flushed with 95% N and 5% H2 and maintained 2 anaerobic by palladium catalyst. Overnight cultures were added to 50 mL of YT (no fructose) in a 250-mL Duran pressure plus bottle (DWK Life Sciences, USA), pressurized to an additional 10 PSI above ambient with a syngas mix of 50%/20%/30% CO/H /CO and left to 2 2 grow shaking at 200 RPM at 37 °C. Bottles were regularly pressurized back until the OD 600 reached 1. Afterwards, C. ljungdahlii was transferred to fresh 50 mL YT medium in triplicate and grown either in a syngas mix of 50%/20%/30% CO/H /CO or 20%/80% CO /H 2 2 2 2 pressurized to an additional 10 PSI above ambient again in 250-mL Duran pressure plus bottles. Bottles were routinely repressurized until growth ceased.”\n2. Response to Reviewer 3’s Comment 4: The major killers of catalysts in material-microbe hybrid systems are often extracellular metabolites from the microbes, not the components in the biological buffer. The catalyst stability referred to in this comment is the stability under such a scenario.\nResponse: Yes, we agree that electrolyte and metabolites both impact the system performance. One of the major focuses of this work is to address the deactivation of abiotic catalysts in microbial growth medium, which is a long-standing challenge in MES systems that attempt to use CO RR products (e.g., CO, formate) as electron donors. This issue has been highlighted in 2 prior studies but remains unresolved (Resources, Conservation and Recycling 184 (2022): 106395; ACS Sustainable Chemistry & Engineering 10.13 (2022): 4114-4121; ChemElectroChem 10.23 (2023): e202300311.). In response to Reviewer 3’s statement that catalyst deactivation may be primarily caused by extracellular metabolites from the microbes, we provide the following analysis and summary. Specifically, we show that in the Ni SAC-C. ljungdahlii hybrid system, the metabolites or extracellular components present are unlikely to cause significant catalyst deactivation.\na) As shown in Fig. 4b–d, the engineered C. ljungdahlii continuously grew and produced IPA over four days of operation in the iMES system, which provides direct evidence that the Ni SAC catalyst remained stable. Furthermore, as discussed in our response to Reviewer 3’s question 1, the Ni SAC (which produces CO) cannot convert microbial metabolites (ethanol and acetate) into IPA due to fundamentally different reaction pathways (Nature Communications 13.1 (2022): 5482; J. Am. Chem. Soc. 2024, 146, 46, 31788–31798; Nature catalysis 6.4 (2023): 319-331.). And the possible existence of macromolecules like amino acids, vitamins, purines/pyrimidines and fragments of nucleotides/polypeptides cannot have a significant effect on CO production activity of Ni SAC (Fig. 2-3).\nb) Previous studies have reported that some microbial redox mediators such as phenazine, flavins and quinones can interfere with electrochemical H production. These mediators 2 may bypass the H evolution pathway by directly accepting electrons from the electrode, 2 or they may reduce O₂ to generate H O , leading to a drop in FE and even catalyst 2 2 degradation (Advances in microbial physiology. Vol. 78. Academic Press, 2021. 317-390.; Frontiers in Microbiology 14 (2023): 1149110; J. Chem. Soc., Faraday Trans., 1996,92, 1913-1920). However, unlike Pseudomonas aeruginosa or electroactive microbes (e.g. Shewanella spp.), the C. ljungdahlii strain lacks the biosynthetic genes required to export such mediators (Scientific reports 7.1 (2017): 13135; Applied and Environmental Microbiology 81.24 (2015): 8379-8391.). Moreover, our iMES system maintained a strictly anaerobic environment in the cathodic chamber, which was also physically separated from the anodic chamber by a membrane. As a result, the conditions required for such interference or degradation mechanisms were unlikely in our system.\nc) Since we added PBS buffer in the iMES system, the produced acetic acid will be quickly neutralized and could not have a significant effect on the interfacial or bulk pH.\nIn summary, as shown in Fig. 1d-e, microbial growth medium themselves can impair conventional CO RR performance (e.g., no CO RR activity on Ag electrodes before -0.75 V vs. 2 2 RHE). In addition, based on experimental results and comprehensive analysis, it appears these above potential deactivating factors resulting from microbial metabolites or extracellular components are not widely present in our system or have no significant impacts on the CO RR 2 activity of the Ni SAC catalyst.\n3. Response to Reviewer 3’s Comment 5.2: YT medium typically refers to a nutrient-rich medium. If the YT medium does not provide any organics/energy for microbial use, why not just use a defined, non-nutrient medium?\nResponse: As shown in Fig. 1d–e and Supplementary Fig. S5, the Ni SAC electrode exhibited excellent CO selectivity in both nutrient-rich (YT medium) and non-nutrient medium (PETC medium). However, due to its higher organic content, YT medium presents a more challenging electrode–electrolyte interface for abiotic catalysts. One of our major goal in this work is to evaluate catalyst stability under such realistic and harsh microbial growth conditions. Therefore, we believe that demonstrating long-term Ni SAC performance in YT medium provides a more stringent and meaningful test; if it remains stable here, it will perform even more reliably in milder, non-nutrient environments (Supplementary Fig. S5).\nIn addition, our collaborators previously observed that the engineered C. ljungdahlii strain grew less effectively in non-nutrient media (i.e., PETC medium) than in YT medium. As a proof of concept for iMES, it is important not only to challenge the catalyst under harsher interfacial conditions, but also to support robust strain growth to enable high IPA production rates and electron recovery efficiency. Moreover, our control experiments confirmed that Ni SAC cannot convert organics in YT medium into IPA, acetate, or ethanol (Fig. 1e), and that the C. ljungdahlii strain doesn’t grow in YT medium alone in the absence of CO and H (Supplementary Fig. S2). 2 These results confirm that the use of YT medium would not introduce false positives. Therefore, we believe that YT medium is the more appropriate choice for this system.\n4. Response to Reviewer 3’s Comment 5.3: The Science paper cited by the authors is a leading work in the field for synthesizing high-value, multi-carbon products like polyhydroxyalkanoates with high efficiency. It is quite misleading to cite it here, as this work targets IPA, which was merely a byproduct in that study.\nResponse: We thank the reviewer for pointing this out, and accordingly we removed the study by Liu et al. published in Science from Fig. 4e to avoid any potential misunderstanding. Additionally, we identified a more recent study that specifically targets IPA as the product. Both its areal and volumetric production rates are significantly lower than those achieved in our work, further highlighting the advantages of CO-mediated microbial electrosynthesis.\n1\nMeanwhile, we do believe the Science paper by Liu et al., provided valuable guidance for both the design of this study and future research, so we have added discussion in the main text:\n“It is worth noting that a recent work by Liu et al. reported H -mediated CO reduction using a 2 2 different microbial strain and metabolic pathway.65 The system achieved high rates of PHB generation (~116.7 mg/L/day) along with C alcohols coproduction, and a byproduct of IPA 4-5 (~100 mg/L/day), confirming the direction of combining alternative strains and metabolic pathways with bioadaptive catalysts to access higher-value products.”\nReviewer #3 (Remarks to the Author):\nThe authors have made efforts to address the reviewers' previous concerns by providing additional experimental data. While this is appreciated, my major concern comes from the incomplete set of experimental groups for conclusions related to the system performance and cell viability. It seems like the authors are suffering from the funding cut and lost access to important facilities. Given that, I recommend a minor revision before it can be considered for publication.\nResponse: We deeply appreciate the reviewer’s recognition of our efforts and the time spent on reviewing this manuscript. We have carefully considered the three minor comments and made the responses and revisions below.\nDetailed comments:\n1. The authors' response to Reviewer 3 claims that cited pioneering works \"only used OD600 or product generation rate to directly reflect viability.\" This is incorrect. If the authors read these references carefully as suggested, these works applied methods like plate counting or live/dead assays to determine the cell viability.\nResponse: We apologize for the earlier ambiguity. Our intended meaning was that “these pioneering works used OD600 or product generation rate to directly reflect the cell viability of the iMES or integrated material-microbe system”. We fully agree that cell viability tests by plate counting or live/dead assays are valuable.\nIn reading again carefully the cited refences, we confirm that For Ref. 2 (Science 352.6290 (2016): 1210-1213), the authors did perform the cell viability tests by plate counting to show the effects of toxic ions, ROS and IPA (Fig. S4, S11, S14 from the reference). However, these measurements were carried out outside the MES system to isolate the environmental effects rather than reflecting in-situ viability within an operating iMES setup. For the iMES system, they did not provide cell viability data and used OD600 as the indicator (Fig. 3 from reference 2). Likewise, Ref. 3 (Nature Catalysis 5.7 (2022): 633-641) used the products amount to reveal the viability of integrated microbial-material hybrids (Fig. 4 and supplementary Fig. S12 from reference 3), which was clearly stated in the main manuscript and method section. And the colored images (Fig. 3 from reference 3) were inorganic elemental mapping by scanning electron microscopy instead of live-dead cell staining.\nThanks to reviewer pointing out these studies again, which allowed us to examine and clarify where and how viability measurements were done that give us support on the connections between the different methods. We will take this suggestion and incorporate direct viability assessments into follow-up iMES studies for more comprehensive and rigorous evaluation.\n2. A significant discrepancy exists between the OD600 readings and the colony-forming unit (CFU) data. While OD600 is reported to plateau between Day 3 and Day 5, the CFU drops by three orders of magnitude (from 10⁷ to 10⁴). The authors should provide a explanation for how a 1000-fold loss in cultivable cells is consistent with a stable OD600, as this is counterintuitive and undermines the use of OD as a proxy for viability in this context.\nResponse: This is a good question. The discrepancy indeed stems from the inherent differences between OD and CFU measurements, rather than an inconsistency in our data. OD is a 600 600 measure of optical density, including all the particle concentrations such as live and dead cells and cellular debris, so it doesn’t differentiate live and dead cells.\nDifferently, the cell viability detected by plate counting method directly indicates the number of active cells in the solution. Although the cell viability started to decline between Day3 and 5, the majority of dead cells remain in the solution and contribute the OD600 values. At that stage, OD600 results can no longer reflect cell viability.\nWe appreciate the reviewer’s insightful comment and have updated the relevant discussion in the main manuscript accordingly:\n“The cell viability results showed that the engineered C. Ljungdahlii strain cannot grow without CO/H /CO feeding (Supplementary Fig. S2), but the introduction of CO/H /CO apparently 2 2 2 2 promoted viability during the logarithmic phase in the fermentation experiments, indicating that the organic components in YT medium alone should be insufficient to support IPA, acetate, or ethanol production. A drop in cell viability from day 3 to 5 was also observed, signifying that biological growth and metabolism had ceased and cells were entering apoptosis, which corresponds to the plateau observed in both metabolite concentrations and OD . During this 600 period, dead cells remained in suspension and continued to contribute to the optical density. As a result, the OD was distorted and could not serve as a reliable indicator of actual cell 600 viability.”\n3. The conclusions regarding cell viability are currently unsupported, as the provided data appears to be for control groups only. To substantiate their claims, the authors must present viability data (e.g., CFU) for cells under the relevant material-microbe operating conditions. A direct comparison between experimental and control groups is essential for drawing any meaningful conclusions.\nResponse: We agree that direct viability measurements under full material–microbe iMES operation are valuable. In this study, we established a complete set of experimental and control groups that together allow us to rigorously assess biological activity, electrode biocompatibility, and rule out false positives. As the reviewer indicated we have made the best effort to evaluate these conditions, and per suggestion we have summarized all the experimental and control groups used in this study:\nComparison of different conditions\nComponents\nGroup IPA production/Cell growth\nC. ljungdahlii External CO Ni SAC Electricity\n1 ○ - ○ ○ Yes\n2 ○ ○ - - Yes\n3 ○ ○ ○ - Yes\n4 ○ - - - No\n5 - - ○ ○ No\n“○”indicates “included”, “-” indicates “not included”\nAs shown in the above table, group 1 is the iMES experimental group, which used C. ljungdahlii strain and electrochemically produced CO by Ni SAC under certain applied potentials. And group 2 is the biofermentation experimental group, which used C. ljungdahlii strain with external CO (CO/H2/CO2 mixture) introduced. Both two experimental groups can produce IPA and obviously increased cell viability (Fig. 1b-c, Fig. 4b-d, Supplementary Fig. S2). Other three groups are the control groups for illustrating the effects of different factors.\nAs the reviewer highlighted, the Science (2016) and Nature Catalysis (2022) studies demonstrate that the combination of system performance metrics with indirect viability indicators provides robust evidence of biological activity, biocompatibility, and the exclusion of false positives. These widely accepted approaches published in leading journals give us confidence in applying similar comparisons in the present study, especially under our practical constraints that limit direct in-situ viability testing.\nTherefore, to address the question regarding the potential effects of Ni SAC electrodes, we adopted the method reported in Reference 3 (Nature Catalysis 5.7 (2022): 633-641): Ni SAC electrodes were immersed in the fermentation medium and cell viability was assessed. The results showed that the presence of the Ni SAC electrode had no significant impact on cell viability (group 3), indicating that the combined inorganic components (Ni SAC, carbon paper, and Nafion binder) are non-toxic to C. ljungdahlii. In addition, ICP-OES, HAADF-STEM, and elemental mapping analyses confirmed the structural stability of the Ni SAC electrode and the absence of detectable Ni2+ ion leaching during electrolysis in YT medium (Supplementary Fig. S24-S26). Taken together with the strong performance of Group 1, these findings provide consistent and convincing evidence that the Ni SAC electrode is biocompatible within the iMES platform.\nFurthermore, by comparing the performance of Control Group 4 with Experimental Groups 1 and 2, we observe that C. ljungdahlii was unable to grow effectively or produce IPA in the absence of exogenous CO (whether generated electrochemically or directly supplied). These results (Fig. 1b-c, Fig. 4b-d, Supplementary Fig. S2) can address the reviewer’s initial concern regarding the autotrophic nature of C. ljungdahlii and simultaneously rule out the possibility of false-positive IPA production within the iMES system. Specifically, IPA cannot be synthesized by Ni SAC alone via electrochemical conversion, nor can it be produced through fermentation by C. ljungdahlii without CO provided by the Ni SAC electrode. Therefore, the observed IPA formation in the iMES system is attributable to true biological conversion supported by the electrochemical CO supply, consistent with the controls and prior literature."}, {"round": 2, "reviewer_comments": {"reviewer_1": "This is a revised manuscript and the authors have carefully answered all the questions raised by reviewers (including 1 and 2), so this manuscript can be accepted for publication at present form."}, "author_response": ""}, {"round": 3, "reviewer_comments": {"reviewer_3": "The authors have answered my previous questions, no further comments."}, "author_response": ""}]

Decision letter